Poster Abstracts

P-002 Coinfection With Human T-Cell Lymphotropic Virus Type 1 and Hepatitis C Virus in a Large HTLV-1 Cohort in

Human T-cell lymphotropic virus type 1 (HTLV-1) causes impairment of host immunity and induces functional disturbance of immune responses. Hepatitis C virus (HCV) has been associated with the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. HCV also has been associated with the occurrence of several extrahepatic diseases, such as mixed essential cryoglobulinemia, porphyria cutanea tarda, polyarteritis nodosa, sicca-like syndrome and lichen planus. Due to the role of cellular immunity in the development and progression of HCV-associated diseases, it is expected that the interaction between HTLV-1 and HCV may modify the natural course of these infections. We observed a frequency of 12.2% of HCV infection among a large cohort of HTLV-1 patients. We present the results of clinical and epidemiological analysis of 51 HTLV-1/HCV coinfected patients (cases) and 50 HTLV-1 single infected patients (controls) randomly selected. 64.7% of coinfected and 42% of single infected were male (p < 0.05), the mean of age was 47 years for cases and 52 years for controls. Neurological involvement was more frequent among coinfected patients (72.5%) versus 50% in single infected group (p < 0.05, OR: 2.64 CI 95%:1.15-6.04), past of drug addiction (21.6%) as well as blood transfusion (29.4%) were more prevalent in coinfected patients (p < 0.05).

No difference in the HTLV-1 proviral load, CD4/CD8 T lymphocytes and blood cells count were observed among the groups. Serum aminotransferases level was higher in coinfected than in single infected patients (p < 0.05). In HTLV-1/HCV patients 53.6% present mixed essential cryoglobulinemia, and no correlation was observed between the presence of cryoglobulinemia and neurological involvement.

Our preliminary results suggest that the presence of both viruses was associated with a higher risk of neurological involvement.

P-003 Comparison Between the Osame (1990) and Castro-Costa Et al (2006) Criteria for the Diagnosis of HAM/TSP

Introduction: The diagnosis of HTLV-I associated Mielopathy (HAM/TSP) is based on the demonstration of slowly progressive spastic paraparesis associated with the presence of anti-HTLV-I antibodies in serum and/or cerebrospinal fluid (CSF) (Osame, 1990) The objectives of this work is to apply the Brazilian criterion (Castro-Costa et al, 2006) for the diagnosis of HAM/TSP and evaluate the level of agreement between the criteria of Osame (1990) and Castro-Costa et al.(2006)

Methods: We prospectively evaluated 30 patients at the Neurologic Infections Clinic of HUGG/UNIRIO, previously diagnosed as HAM/TSP by Osame's criterion (1990). All patients were evaluated by the same neurologist (CMSAS). The additional tests included: complete blood count, biochemistry, evidence of rheumatic function, thyroid hormones, vitamin B12 and folate, serology for HIV, HTLV-I (ELISA and Western-blot), syphilis and hepatitis C. The examination of cerebrospinal fluid (CSF held in Lab/SPC/HUCFF UFRJ) consisted on cytology, microbiology, biochemistry, general immunology (albumin ratio and IgG índex, oligoclonal bands) and specific (search for antibodies to HTLV-I by ELISA method, VDRL, HIV). We performed computed tomography of thoracic and lumbar spine in all patients.

Results: The study included 30 patients, 20 were female. The average age ± SD was 54 ± to 11 years and average time of disease ± SD was 10 ± 7 years. All patients had insidious onset. Proximal weakness was seen as symptom in the moment of presentation in 93.3% of cases, 76.7% had urinary symptoms and 53.33%, paresthesia. Blood examination of all patients was normal, except for anti-HTLV-I presence. The CSF examination revealed pleocytosis (> 4 cells/mm³) in 48% of cases, high CSF protein level (protein > 40 mg/dl) in 52%, IgG index = 0, 7 in 54.17%, albumin ratio >8 × 10 − 3 in 21.74%. All samples of CSF were anti-HTLVI positive (ELISA). Kappa index showed high degree of agreement (K = 1.0). The most part of patients fulfilled the criteria for definite form of HAM/TSP.

Conclusion: Both criteria are comprehensive and have high degree of agreement on results, demonstrating accuracy in its applicability.

P-004 Comprehensive Care of People Living With HTLV: A Challenge for the Brazilian Healthcare System

Issues: Brazil is estimated to have the largest number of men and women living with HTLV worldwide. HTLV seroscreening has been compulsory in the country since 1993 and HTLV-related diseases have been reported among Brazilians since the 1990's. HTLV research is recognized as active in Brazil and has yielded significant scientific contributions to the field. However, several intertwining factors have hampered the provision of a comprehensive care approach to people living with HTLV. This paper intends to summon up challenges to be faced in the country to overcome such barriers. Description. Information about the epidemiology of HTLV infection, as well as on its natural history and clinical outcomes is still limited, not only in the general public, but also within the healthcare community. Access to serodiagnostic tools is not homogeneous throughout the country, and confirmatory tests are particularly lacking in most areas. Alternative algorithms for confirmation of HTLV infection, based on molecular amplification of proviral genome sequences from ex vivo PBMC are available in some laboratories. Nevertheless, they are not standardized nor under routine quality control procedures, what makes comparison of data among different institutions difficult. Operational barriers to the scaling-up of these methods include the frequent use of nested PCR protocols and the need for radioisotopic detection of PCR products. A frustrating “bottleneck” in patient care is the unvailability of a structurized referral system. Most patients are thus prevented from been admitted to healthcare settings where there more sophisticated diagnostic technologies and more skilled professionals are available. Moreover, wherever cohorts of HTLV-infected individuals are under follow-up, secondary prevention is limited by the lack of early laboratory markers of HTLV disease progression. As far as HTLV-disease is concerned, evidence is still needed to support therapeutic interventions. Lessons learned. HTLV infection and disease has long been recognized by several Brazilian research and clinical teams. Technical expertise is available in many areas of the country and could foster training initiatives for healthcare personnel from regions still in need. A comprehensive care approach to people living with HTLV will certainly require multiprofessional and interdisciplinary involvement. Next steps. Synergistic action towards the implementation of an effective national policy for HTLV care requires stronger engagement of stakeholders and officials in charge of developing healthcare programmes. Participation of the civil society, fueled with the activism of men and women living with HTLV is critical to help push this agenda forward.

P-005 Disease Perception and Reproductive Decisions in People Living With Human T-Cell Lymphotropic Virus Type 1 (HTLV-1): The Role of Care in Public Health

Introduction: Few studies regarding the subjective impact of infection and/or TSP/HAM have been done. The risk of Mother-to-child Transmission (MTCT) is especially significant in this infection, allowing questioning how the experience of living HTLV-1 interferes in their reproductive decisions.

Objectives: To know how people living with HTLV-1 attribute meaning to the infection and their reproductive decisions, and, as well, to investigate the role of medical care in addressing patient's subjective difficulties regarding sexuality and reproductive decisions.

Methodology: An ethnography study, based on Interpretative Anthropology, was conducted with participant observation, of qualitative nature, in the period of June 2007 to June 2008, with HTLV-1-infected subjects who were attended at the Institute of Infectious Diseases Emílio Ribas in São Paulo, Brazil. The data were collected using in-depth interviews among 13 people living with HTLV-1, regarding socio-demographic questions, perception of infection/disease, sexuality and reproductive decisions. Data were analyzed from theoretical reflections on gender and care in public health. All the volunteers signed an informed consent.

Results: The patients considered HTLV-1 infection as ‘unknown disease’ and this perception interferes negatively on their reproductive decisions, regardless of the presence of symptoms. Furthermore, health professionals have difficulty in considering those reproductive issues, as the focus of their work is the illness, not the subjective aspects involved in this process. In this case, it is possible to understand the role of moral connotation in the judgment of their decisions, as occurs with HIV/AIDS patients. Patient's speeches patients revealed, still, contradictions and ambivalence, especially about the decision to have a child before MTCT risk. They showed significant conflict about breastfeeding inhibition, understood as a representative of the presence of the virus as an ‘intruder in the family’. There was great difficulty in disclosure of HTLV-1 in the family, evidenced in several cases where family members and partners had not been yet tested for HTLV-1. Similarly, in symptomatic, which may reveal that the dynamics and family conflicts have an important role in care and prevention of transmission. There was also strong influence on gender issues, about the female identity and its social role in the reproductive decision of interviewed women.

Conclusion: The subjective issues have to be included to contribute to Public Heath care to allow recovering a reflection on therapeutic care and attention to people living with HTLV-1. Another key issue is how to approach this group and sensitive strategies to their individualities, as HTLV-1 prevention programs should take into account psychological issues, and patient's emotional needs.

P-006 Epidemiologic Profile of the Pregnant Women Submitted to the Classification of the Virus HTLV-1 and 2, São Luis/Maranhão, 2008

Introduction: HTLV is a retrovirus with tropism by T- lymphocytes; kind 1 is endemic in the Caribbean region, Japan, South America, and parts of Africa; kind 2 is found in some native American groups, rarely in Africa, and in Europe it is associated to the use of injectable drugs, and it prevails more in Brazilian indigenous populations. In Brazil it is estimated that 2.5 million people are infected in all the states researched, with different prevalence. The virus is transmitted through breast feeding, sexual contact without protection, blood transfusion, and by sharing contaminated syringes. The infection though HTLV-1 is associated to T- Cells Lymphoma of the adult and Chronic Degenerative Neurologic Syndrome. The diagnosis of the infection by the virus is made through the identification of anti-bodies anti-HTLV-1 and 2 (ELISA) and confirmatory tests, being Western Blot the most used one, however, PCR is the gold standard.

Objectives: To evaluate the epidemiologic profile of pregnant women submitted to the classification for the virus HTLV-1 and 2, assisted during prenatal care at three public services.

Methodology: Transversal study executed between Feb/11 and Dec/03/2008, with 2044 pregnant women at three public service of prenatal care, São Luis/MA. The patients were guided about the study, included after signing the TCLE and filling in a questionnaire. The pregnant women who took part of it were between 18 and 45 years old without psychiatric diseases background, high blood pressure, nephropathy, diabetes and others that characterize specialized prenatal care needs. The sample was calculated in 2041, test power 95%, absolute mistake of 5%, Stata 9.0 program was used, Chi Square test was taken, and p < 0,05 (95%) was accepted as the limit for significance. The Project was approved by CEP/UFMA (Opinion N°568/2007).

Results: The average age was 25 years old, 35.12% are single with partners, 41.10% have olive skin, 57.19% have finished high school, 54.21% started sexual activity between 15 and 18 years old, 35.08% have never used condom during sexual intercourse; 54.99 are primiparous women; 15.22% were breast fed by their mothers and other people. 42.51% have already had children, out of these, 31.9% have already practiced crossed breast feeding. 1.2% have already used illegal drugs, and 9.4% have/have had relationship with drug users.

Conclusion: The sample revealed that the pregnant women presented characteristics that justify the classification of virus HTLV-1 and 2 during the prenatal care, however more studies are necessary for the evaluation of the real situation in the State of Maranhão.

Key words: human T-lymphotrofic virus; pregnant women; epidemiology

P-007 Evidence of a Higher Prevalence of HPV infection in HTLV-1-Infected Women Compared to Uninfected Women

Background: It is estimated that 50.000 individuals (2%) are currently infected with HTLV-1 in Salvador-Bahia, Brazil. This infection is more frequent among women. HPV, another sexually transmitted disease, causes cervical cancer, specially in immunossupressed individuals. HTLV-1 infected individuals are more susceptible to several infectious diseases, but few studies addresses the co-infection between HPV/HTLV-1.

Aim: To determine the prevalence of HPV infection in HTLV-1-infected patients.

Methods: An outpatient cross-sectional study was carried out between September 2005 to December 2008. This study involved 50 HTLV-1-infected women from the Reference Center of HTLV (ELISA + Western Blot) and 40 uninfected from the gynecology clinic, at Bahian School of Medicine. HPV infection was assessed by hybrid capture. HTLV proviral load was quantified by Real time PCR and CD4T lymphocytes by flow cytometry.

Results: The mean age of HTLV-1-infected women (38 ± 10 yrs) was similar to that of controls (36 ± 13 yrs). There were no differences in schooling, marital status, or smoking habits between the groups HTLV infected and uninfected. However, the mean age of first sexual intercourse of HTLV-1-infected women (17 ± 3 yrs) was lower than HTLV-1 uninfected (19 ± 3 yrs) (p = 0.03), conversely the number of sexual lifetime partners was greater in HTLV-1 infected (4 ± 3 yrs) compared to uninfected group (2 ± 1) (p < 0.01). The prevalence of HPV infection was 44% in the group HTLV-1-infected, while this proportion was only 22.5% in the uninfected women (p = 0.03). There was no difference in the frequency of high (group B) and low (group A) risk HPV subtypes between HTLV-1-infected and uninfected groups. Moreover, in the HTLV-1-infected group there was no association between HPV infection and CD4 T cell count or HTLV-1-proviral load.

Conclusion: The prevalence of HPV infection is higher in HTLV infected women. Gynecological evaluation and HPV screening should be considered essential part of the assessment of HTLV-1-infected woman.

P-008 Abstract Withdrawn

P-010 An Uncommon Association of Adult T-Cell Leukemia/Lymphoma With Hyalohyphomycosis

Adult T-cell leukemia/lymphoma (ATL) is a severe disease, caused by the human T-cell lymphotropic virus type-I (HTLV-I). ATL has been classified into five clinical types: acute, chronic, lymphoma, smoldering and primary cutaneous tumoral. Until now there is no report about occurrence of subcutaneous mycosis associated with HTLV-I infection or ATL. We present a case of a patient with chronic ATL who developed a cutaneous hyalohyphomycosis. Case report: A 40 years-old men, HIV negative, carrier of HTLV-I had chronic ATL since November 2006. In October 2007, multiple verrucous plaques, some of them ulcerated, appeared on the anterior aspect of his left tight. He also had generalized and small papular lesions previously diagnosed as verruciform epidermodisplasia. The ulcero-verrrucous lesions were biopsied more than once. Histologically, an extensive granulomatous process with neutrophil abscesses and many septated non-pigmented hyphae were seen in the dermis and subcutis admixed with lymphomatous cells. The areas of lymphoma were seen mainly in deep dermis and subcutis and consisted of pleomorphic medium and large lymphocytes CD2+, CD3+, CD4+, CD5+, CD7+, CD8−, CD20−, CD25+, CD30−, UCHL-1+, CD79a−. The proliferative index (Ki67) was 50%. No necrosis was observed. The pathological diagnosis was cutaneous involvement by ATL with morphology of peripheral T-cell lymphoma, unspecified associated with hyalohyphomycosis. By flow cytometry the PBMC expressed CD2, CD3, CD4, CD5, CD7, CD25, CD45RO, TCRáâ. No expression of TCRãä, CD8 and CD20 was detected. Unfortunately, the fungus was not isolated. In August 2008, he developed acute ATL with high levels of hypercalcemia. He was treated with chemotherapy (CHOP) for ATL and with itraconazole for the mycosis. Now, he is alive with ATL but without relapse of the mycosis.

Comments: This case represents an association in the same cutaneous lesions and in the same histological slides of hyalohyphomycosis and adult T-cell leukemia/lymphoma with the morphology of peripheral T-cell lymphoma, unspecified. The infection by HTLV-I causes an immunodisregulation and the infected individuals are more susceptible to superficial mycoses but there is no reference in the literature of the association of HTLV-I infection or ATL with subcutaneous mycoses. Moreover, to the best of our knowledge, only one case of subcutaneous mycosis occurring in malignant lymphoma other than ATL has been described and in this case report no skin lymphomatous lesions were observed.

P-012 Case Report: HTLV-I-Associated Myelopathy (HAM)/Tropical Spastic Paraparesis (TSP) with Amyotrophic Lateral Sclerosis-Like Manifestations Associated with Adult T Cell Leukemia/Lymphoma (ATL)

Introduction: Human T cell lymphotropic virus type 1 (HTLV-1) may cause, among other diseases, HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP) [1] and adult T cell leukemia/lymphoma (ATL) [2). HAM/TSP is a severe and incapacitating myelopathy with insidious onset that is more common among female patients. [4]. ATL is a severe and commonly fatal form of leukemia/lymphoma that typically occurs in adults aged > 40 years [2]. Rarely, other neurologic diseases have been associated with the HTLV-I such as Amyotrophic Lateral Sclerosis (ALS) like syndrome. We now describe a patient with ALS-like HAM/TSP associated with ATL and uveitis.

Case report: A 46-year-old woman of African descent with a history of paraparesis for 5 years, initially on the right side, and with an insidious and progressive course. She presented a slight gait limitation, and frequent falls. Two years ago, a distal weakness of the upper limbs and urinary urgency appeared, but without paresthesia. Six months later, she started with a history of skin and ocular lesions, and had the diagnosis of smoldering ATL and uveitis. She had been breastfed and had no history of ever having received blood transfusion. Neurological examination: She presented spastic paraparesis, positive bilateral Babinski signal, atrophy and fasciculation of the distal upper limbs, with positive bilateral Hoffmann signal. The tactile and vibratory sensitivity was preserved, with no alterations in cerebellar function. Dermatological examination: Erythematous infiltrated plaques greater than 15 cms were seen on the buttocks, groins, and in the left popliteal fold and small papules on the limbs and back associated with xerosis. Ophtalmologic examination conjunctival erythema with xerophtalmia, and diagnosed uveitis.

Laboratorial data: The patient was HTLV-1 positive (enzyme-linked immunosorbent assay and Western blot analysis), HIV negative, and VDRL negative. Muscular enzyme titers, vitamin B12, and the tests to evaluate the thyroid function were within normal limits. The cerebrospinal fluid exibited a mild pleocytosis. Electroneuromyographic features were compatible with lesion in motor neuron of the anterior horn of the cervical and lumbosacral somite of the spinal cord. Magnetic Resonance Imaging of the cervical spine cord had no alterations. These findings were in accordance with the El Escorial's criteria for the diagnosis of ALS. with high pro-viral load (300.000 log 5,2, quantified using a real-time TaqMan PCR method). At diagnosis of ATL, blood cells count, blood level of calcium and Computed Tomography scan of thorax and abdomen were all normal. Lactate dehydrogenase (LDH) blood level was slightly elevated. A skin biopsy revealed a lymphoma T CD4⁺/CD8⁺ with small cells, mycosis fungoides-like.

Evolution: Skin lesions were treated with psoralen plus UV light therapy, with clearing of the lesions.

Comments: In this clinical report, the initial signals and symptoms are compatible with the clinical features of HAM/TSP. Progressively, the marked atrophy and fasciculation of the distal upper limbs suggests lesion in anterior horn of the spinal cord, leading to the diagnosis of ALS. Hence, this case represents an ALS-like form of HAM/TSP, corroborated by a high proviral load, which is well known to be significantly higher in HAM/TSP than that in HTLV-I carriers, and even higher in ALS-like HAM/TSP. Another fact that emphasizes this diagnosis is the great improvement of motor functions in response to prednisolone.

The association of HAM/TSP with ATL is considered rare. However, in Bahia, Brazil, this association occurs in 14 % of the ATL patients. The patient was considered as smoldering ATL because there was no extracutaneous involvement, no lymphocytosis, no hipercalcemia, and the LDH was only slightly elevated.

P-013 Molecular Diagnosis of ATLL: The Use of Clonality

Background: Early diagnosis of proliferative disorders, such as leukaemia or lymphoma, associated with HTLV-I infections allows rapid and specific treatment. Leukaemias and lymphomas are monoclonal diseases, therefore detecting a monoclonal set of cells should indicate malignant proliferation. When an individual is infected by HTLV-I the virus randomly integrates into the genome of infected T-cells, and consequently T cells with identical integration sites for the HTLV-I provirus are considered to have originated from the same infected cell, belonging to a unique clone. If it can be shown that a significant number of T-cells have the same integration site, then it can be assumed that there is a clonal proliferation, and an early diagnosis of leukaemia/lymphoma can be made. We aimed to validate a PCR-based analysis of the clonality of HTLV-I infected T cells, for the diagnosis of ATLL. Such a method could also be used to monitor response to therapy, and to provide an early detection of relapse.

Methods: We designed the vectorette unit that is ligated to both ends of the fragments and chose two primers for DNA amplification, one specific for HTLV-I LTR and the other for the vectorette unit. DNA was extracted and digested with the restriction enzyme HindIII. The vectorette was ligated to the restriction digested DNA fragments and then the ligated DNA used as a template in a two step touchdown PCR to allow amplification of DNA between the known sequence of HTLV-I and the vectorette unit. After PCR amplification we analyzed the clonal pattern by electrophoresis of the amplicons on a 2% agarose gel.

We then determined HTLV-1 clonality in 15 HTLV-I infected subjects: 6 with an established diagnosis of ATLL, 3 with HAM/TSP, 3 asymptomatic carriers, 1 patient with polymyositis and 2 patients with Strongyloidiasis. The HTLV-I viral load in these patients ranged from 1.6 to 270 HTLV copies per 100 PBMCs.

Results: All 6 patients with ATLL showed a monoclonal pattern. 1/3 patient with HAM who developed ATLL had an oligoclonal pattern in PBMCs and a monoclonal expansion in a lymph node biopsy. All the other patients displayed a polyclonal pattern.

Conclusions: In these patients, our results correlated well with the clinico-pathological diagnosis. The simplicity of the method allows repeated routine testing, with the possibility for early diagnosis of ATLL, monitoring of response to treatment, and early detection of relapse. Further validation of the method is expected from testing of a wider number of patients. This assay may be of most use in patients with lymphomatous presentations in whom the diagnosis of ATLL can be more difficult to confirm than with leukaemia and in patients with HTLV-1 infection and mild lymphocytosis.

P-015 Detection of intrathecal Synthesis of Antibodies Against ENV (GD21 and RGP46-I) and GAG (P24) HTLV-1 Proteins in HAM/TSP, Using Western Blot Test

Introduction: HTLV-1–associated myelopathy (HAM/TSP) is a chronic neurological disease of the central nervous system (CNS). A clinical and laboratorial criterion for the diagnosis of HAM/TSP has been established (Osame, 1990). The laboratory diagnosis is based on detection of antibodies anti-HTLV-1 in serum and cerebrospinal fluid (CSF). It is important to demonstrate intrathecal synthesis of antibodies anti-HTLV-1 while analyzing the CSF, since it represents a conclusive evidence of the immune response against HTLV-1 in the CNS.

Objective: To adapt the test western blot in sense of to demonstrate intrathecal synthesis of antibodies anti-HTLV-1, as a support for the diagnosis of HAM/TSP.

Material and Methods: Paired CSF and serum samples (diluted to the same IgG concentration of the CSF) were selected from 14 HAM/TSP patients followed up at the outpatient Neuroinfection Unit of the Gaffrée & Guinle University Hospital (HUGG). In addition, samples from 16 HTLV-1 seronegative non-HAM/TSP patients with other neurological diseases were also analyzed. All were screened for the presence of HTLV-1 antibodies by ELISA and western blot in serum. The intrathecal synthesis of antibodies anti-HTLV-1 was evaluated by western blot (HTLV-BLOT 2.4). The presence of reactive bands more intense in CSF, than in serum, or bands visualized only in the CSF was considered indicative of intrathecal synthesis of antibodies anti-HTLV-1.

Results: All 14 paired (serum and CSF) samples from patients with HAM/TSP presented evidence of intrathecal synthesis directed against at least one viral protein of HTLV-1. Concerning patients with other neurological diseases, no reactive bands were observed in the 16 paired studied samples. The test presented sensitivity, specificity, positive and negative predictive values of 100%. Intrathecal synthesis of antibodies anti-HTLV-1 were mainly against the env (GD21 and rgp46-I) and gag (p24) proteins, by demonstrating specific bands in 93% (13/14) of the patients, when analyzed individually.

Conclusions: This was the first study in the medical literature which adapted the western blot in search of to detect intrathecal synthesis of antibodies anti-HTLV-1 in patients with HAM/TSP. Due to its high sensitivity and specificity, the test demonstrated to be a useful tool as a laboratorial support in the diagnosis of HAM/TSP. Likewise the criteria of positivity by using western blot in serum, the authors suggest the demonstration of intrathecal synthesis of antibodies against two viral proteins (env and gag) as an indicator of confirmatory test in CSF.

P-016 Development of a New Technique Using High-Throughput Sequencing to Determine the HLTV Clonality

HTLV-1 infects CD4+ T cells and persists lifelong in the host. In each infected person, the proviral load (i.e. the percentage of peripheral blood mononuclear cells that carry the provirus) reaches a steady state level that we believe is determined by a balance between spontaneous proviral expression and the immune response. In each person, the proviral load is constant to within a factor of about 5, but among different infected patients it can differ by more than 1000-fold. The proviral load is strongly correlated with the risk of the inflammatory and malignant diseases associated with HTLV-1. One of the chief determinants of the spontaneous rate of proviral expression is the genomic context of the provirus, i.e. the nature of the host genome surrounding the integrated provirus in the infected T cell. We have shown that proviral integration in a transcriptionally active region of the genome was significantly associated both with a higher rate of Tax expression and with the disease HAM/TSP (Meekings et al. 2008). We have now developed a new experimental approach based on linker mediated PCR and Solexa high-throughput sequencing. This technique is more sensitive and allows simultaneously the quantification of the number and size of HTLV-1-infected T cell clones and the mapping of the integration site in the human genome. The HTLV clonality of a first TSP patient has been determined so far. In this particular case, 3 major clones totalling 93% of all infected cells have been identified. The remaining 7% of the proviral load is composed of more than 200 different clones. We plan to analyse several tens of samples from different patients and at different time points. The large number of distinct insertion sites generated should allow us to interrogate gene ontology and other genomic annotation databases to characterize more deeply the genomic flanking DNA. Also, we will study the relationship between HTLV clonality and the disease status, the level of proviral load, proviral expression and the immune response.

P-017 Epidemiological and Clinical Review of 109 TSP Cases

HTLV-1 virus is endemic in areas of Asia, Africa and America. Our team documented the first case of TSP/HTLV-1 in Jujuy province in 1989, since then, this disease was considered endemic in two regions of our province. Jujuy province it is located in northern Argentina, in a Subtropical region, bordering with Bolivia and Chile and it has 611,888 inhabitants. The prevalence of HTLV-1 antibodies in blood banks is 1 % and higher prevalences were found in general population.

We present epidemiological and clinical aspects of patients with TSP assisted in our Hospital since 1989.

Method: We review epidemiological and clinical data from 109 TSP patients. For the definition of TSP case the criteria of WHO and Belo Horizonte HTLV workshop 2004 were used. The confirmation of viral infection was done by serology (IFA/WB) and using qualitative PCR for HTLV-1 and HTLV-2. To assess motor neurological involvement Osame's score was applied.

Results: TSP prevalence was 17.7 x 100.000 and TSP incidence was 1.5 x 100.000. The rate of TSP in female/male was 8.5/1 and 89% were women.

The average of age was 51 years for women and 47 for men. Eighty seven percent of the patients had antibodies or were positive by PCR for HTLV-1 in blood (89% of women and 62.5% of men). Only 2.5% were indeterminate by WB. Sixty eigth of the patients has Ab for HTLV-1/2 in CSF.

At the time of diagnosis the 68% of patients have less than 5 years of disease, 13% between 6 to 10 years and 19 % more than eleven years of disease. Urinary incontinence was found in 81%, constipation in 83 %. Abolition cutaneous-abdominal reflex in 86 %, Babinski in 87.5 %. Hyperreflexia of upper limbs in 37% and 99 % in lowers limbs respectively. Ankle clonus in 32 % and patellar clonus in 20 %.

The Osame's score evaluation showed grade I in 16% of patients, II in 5 %, III in 4 %, IV in 11 %, V in 24 %, VI in 9 %, VII in 12%, VIII in 7 %, IX in 9 %, X in 0 %, XI in 4% and score XII 2 %.

Conclusion: We have isolated places in our province were the prevalence of TSP is around ten times more 17.7 x 100.000. Most of the patients are high Lander Amerindian descendents mixed with also European descendants. This native people used to give breastfeeding frequently more than 18 moths and endogamy index rate among them is high. The rate F/M is higher than other reports in Sudamerica. Serorreactivity to HTLV was predominant in females. Osame Score V was most frequent, although we found a high rate of patients with early outcome of TSP.

P-023 Family Studies of HTLV-Positive Candidate Blood Donors in

Background: HTLV-1 transmission from mother to child and through sexual contact supports performing family studies of newly diagnosed cases. There is scarce evidence of the diagnostic yield of family studies in relatives of HTLV-1-positive candidate blood donors (HPCBD) because in most endemic countries the strategy implemented at blood banks is limited to index cases.

Methods: Since 1997, three hospitals in Lima tend to referred HPCBD to the Institute of Tropical Medicine Alexander von Humboldt (IMTAvH) for specialized medical care. Here, we repeat at least one HTLV ELISA, and perform confirmatory testing depending on availability of funds. If these additional tests at the IMTAvH give positive results, we suggest starting a family study. According to the family study algorithm used at IMTAvH, partners, parents, children and sometimes also the siblings of HTLV-1-positive cases are offered HTLV screening. For those relatives who accept, a standardized questionnaire is filled out prior to HTLV testing.

Results: From June 1997 to November 2007, we received 332 HPCBD who tested seropositive for HTLV at IMTAvH. The family study was carried out in 157 (47.2%) of these cases. Overall, 449 relatives were screened, with a median of 2 studied relatives per case (interquartile range: 1–4). Offspring (19.8%), spouses (23.6%), siblings (22.0%), mothers (14.2%) and fathers (3.3%) were most frequently studied. The mean age among studied relatives was 36.1 + 18.2 years, and women comprised 66.0%. Out of 449 tested relatives, 164 (36.5%) were HTLV seropositive, and 285 (63.4%) were HTLV seronegative. Confirmatory testing, performed in 97 out of 164 (59.1%) seropositive relatives, corroborated HTLV-1 infection. One hundred two out of 157 HPCBD with family studies (64.9%) had at least one HTLV-1-positive relative. The number of secondarily detected HTLV-1-positive cases per HPCBD ranged from 1 to 8. The mean age among 97 HTLV-1 confirmed positive relatives was 42.4 + 14.8 years; 5 (5%) were less than 18 years old; and 71 (73%) were women. Fifty out of 77 wives and 16/29 husbands were seropositive (65 % vs. 55 %, p = 0.48), as well as 35/64 (55%) mothers, 8/15 (53%) fathers, 10/89 (11%) children, and 27/99 (27%) siblings. At baseline examination, 14 of 164 (8.5%) HTLV seropositive relatives of HPCBD had signs or symptoms of HTLV-1-associated diseases, including 6 cases of HAM/TSP.

Conclusion: Our preliminary results suggest that routine HTLV-1 family study of HPCBD might stand as a useful additional strategy for HTLV-1 disease control in Peru, facilitating the identification of additional cases, their access to health care and ultimately the prevention of silent HTLV-1 spread. This strategy merits locally a thorough cost-effectiveness assessment.

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P-024 Frequency of Human Lymphotropic Virus Type 1 in Samples Screenad in Lacen-Ba, from 2005 to 2007

The Laboratory of Public Health Gonçalo Moniz - LACEN-BA conducts analysis to support in epidemics and outbreaks, as well as attention to epidemiological surveillance of the Bahia State. Salvador, Bahia, is the city that have the highest HTLV-1 prevalence of HTLV-1, and Brazil has been described as the country with the highest absolute number of HTLV-1/2 infected individuals around the world. HTLV-1 is the causal agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and Adult T-cell Leukemia/lymphoma (ATLL), as well as uveitis. There is no clear association of HTLV-2 with any human disease, however some reports associate this infection with T-cell hairy cell leukemia. The aim of this study was to determinate the frequency of HTLV-1/2 positive samples tested by Central Laboratory of Public Health Gonçalo Moniz - LACEN-BA, from 2005 to 2007. In this period, 62,787 samples, including thirty-one Regional Directories of Health (DIRES), were analyzed. These data are a spontaneous sample request sent to this centre. The general frequency of HTLV-1-positive samples was 1.35% (848/62.787). We found positive samples for HTLV-1 in seventy-five cities from Bahia. In Salvador, the frequency was 1.9% (533/28.075). Among all pregnant women assayed the frequency was 0.74% (80/10.790), and 0.98% in pregnant women from Salvador (53/5929). The cities with the highest frequency of positive results were Salvador, Vitória da Conquista, Ilhéus, Feira de Santana, Itapetinga and Itamaraju. 1^st DIRES (Salvador) presented the highest frequency of positive results. This study demonstrated the presence of HTLV-1-infection in several cities of Bahia that was before this research unknown. In addition, this study supplies important information for control and prevention of HTLV infection in all Bahia State.

P-025 Genetic Analysis of HTLV-1 Isolates from Rio de Janeiro, Brazil

Background: The HTLV-1 infection in Brazil is drive mainly for transcontinental cosmopolitan subgroup 1Aa, where isolates are distributed in Latin American clusters. However, Japanese subgroup HTLV-1aB was sporadic identified. On the other hand, genetic polymorphisms on tax and p12 genes were pointed out as related to HAM/TSP condition, phylogenetic subtype, and geographic origin. The aim of this study was to verified the molecular epidemiology of HTLV-1 isolates from Rio de Janeiro, as well as evaluate genetic polymorphisms correlated with clinical status of patients and genetic subgroup of isolates.

Methodology: Thirty two HTLV-1 DNA samples from HAM/TSP patients and asymptomatic HTLV-1 carriers were submitted to nested-PCR for LTR, tax and p12 target regions. PCR products were directly sequenced on both strands using ABI model 3030 automated DNA sequencer. The sequences analysis was performed using Seqmam, Clustal X, BioEdit, GeneDoc programs. Phylogenetic analysis was performed by PAUP* 4.0 by bootstrap of 1,000 replicates

Results: According to LTR most isolates belongs to transcontinental HTLV-aA subgroup and one sample (27PE) to HTLV-1aB. The isolates have the LTR transcontinental genetic signatures except to sample (27PE) who has HTLV-1aB signatures. We also found a genetic polymorphism, C579T, typical from Rio de Janeiro in seven samples. The results of tax gene analysis showed signatures characteristics of tax HTLV-1aA subgroup in most samples and from Japanese subgroup in sample 27PE. Two isolates have an internal tax deletion of 381 and 349bp, respectively. Most samples from Rio de Janeiro have a particular polymorphism C7951T. The genetic analysis of p12 protein showed the genetic signatures previously associated with HTLV-1aA isolates in South America L34, P63, P91, except of sample 27PE which has P23 signature described in HTLV-1aB from Japanese origin. The provirus polymorphism identified are no correlated with HAM/TSP or asymptomatic carriers condition.

Conclusions: In spite of detecting the worldwide prevalent HTLV-1aA in Rio de Janeiro, the study shows a presence of subtype HTLV-1aB in the state, as well as HTLV-1aA particular signatures in LTR and tax provirus regions.

P-026 Genetic Characterization of HTLV Seroindeterminate Isolates from Brazilian Blood Donors

Background: HTLV seroindeterminate individuals are characterized by incomplete western blot (WB) banding patterns for Gag (p19, p24) or Env (GD21, rgp46) proteins. The prevalence of HTLV-1 soroindeterminate in Brazil in blood donor is from 0.08 to 1.8%. It is essential to conduct a genetic characterization of HTLV seroindeterminate isolates since Brazil is an HTLV endemic area and the outcomes of HTLV-1 and HTLV-2 infection are quite distinct.

Methodology: Fifteen HTLV seroindeterminate DNA samples from asymptomatic blood donnors from Rio de Janeiro, were submitted to nested-PCR for tax (HTLV-1/HTLV-2), LTR and pX (HTLV-2) and LTR5′, LTR3′, gag and pol (HLV-1). PCR products were directly sequenced on both strands using ABI model 3030 automated DNA sequencer (Applied Biosystems). The sequences were edited using Seqmam (DNA Star Lasergene) program and identified using the BLAST/NCBI.

Results: Our analysis revealed the presence of HTLV provirus in 13/15 samples and according to sequences analysis 9 were HTLV-1, and 4 both HTLV-1 and HTLV-2. The following positive results were obtained: 8 samples for HTLV-1 tax, 1 sample for HTLV-1 gag and tax, 1 for HTLV-1 tax and HTLV-2 LTR, 1 for both HTLV-1 (LTR3′ and tax) and HTLV-2 (LTR and tax), 1 for both HTLV-1 (LTR and tax) and HTLV-2 tax, 1 sample for HTLV-1 and HTLV-2 tax. In all samples, PCR or sequence analysis failed to detected HTLV-2pX, HTLV-1LTR5′ and HTLV-2 pol target regions.

Conclusions: The prevalence of HTLV infection of HTLV seroindeterminate, based on criterion of at least one HTLV genome region is of 80%. However, in 69.23% is due to only tax sequences. In spite these infection could reflect the presence of HTLV defective provirus, our data also reveled a significant HTLV dual infection of 30.76%.

P-027 Geographical Distribution of HTLV-1/2 in Mothers of Newborns Tested in the Neonatal Screening in Minas Gerais, Brazil

Background: The HTLV-1 is distributed worldwide and is endemic in various parts of the world, including Brazil. Studies have shown a tendency to grouping of seropositive individuals in different geographical areas of the world. The viral transmission from mother to child, which occurs mainly through breastfeeding, is a major factor maintaining the chain of transmission of these viruses. The present cross sectional observational study evaluated the distribution of HTLV-1/2 for puerperal women of Minas Gerais who had their newborn submitted to HTLV-1/2 neonatal screening. The relationship between seropositivity and determinants of socioeconomic position was also examined.

Methods: Minas Gerais State is located in Southeast Brazil, has 586,528.293 Km² area and 19,273,506 inhabitants. The units of analysis used were Minas Gerais State geographic areas (n = 12). Samples consisting of dried blood spots (DBS) were collected from newborns on filter papers, from September to November 2007. These were tested for IgG maternal anti-HTLV-1/2, by enzyme immunoassay (EIA) (Prime Diagnostic do Brasil, São Paulo), specific for detection of anti-HTLV-1 and/or HTLV-2 in samples of dried blood collected on filter paper. The mothers whose newborns were reactive in DBS, had their venous blood collected and the serum was submitted to EIA (Ortho Clinical Diagnostic, USA). The samples found positive were tested with Western Blot (WB) (MP Diagnostics, Singapore), as a confirmatory test.

Results: Of the 55,293 samples on filter paper included in the analysis, 52 were reactive (9.4/10,000, 95% CI: 6.8 to 12.0). HTLV-1/2 positivity was confirmed by WB in 42 mothers (7.6/10,000, 95% CI: 5.3 to 9.9). Two have been identified as carriers of the HTLV-2 and all the remaining (n = 40) were positive to HTLV-1. In only two Minas Gerais geographic areas, Campo das Vertentes and Central Mineira, no samples tested were reactive to HTLV-1/2. In others areas, the seropositivity for HTLV-1/2 ranged from 1.4 to 55.9/10,000. The highest rates of seropositivity were observed in the Vale do Mucuri (55.9/10,000) and Jequitinhonha (16.0/10,000).

Discussion: This was the first time, in our knowledge, that a neonatal screening program was utilized to detect HTLV positive mothers and to adopt measures to block the virus transmission to the newborn. We have found that the HTLV-1/2 geographical distribution was heterogeneous, but with a tendency to concentrate in the Northern and Northeastern parts of the state. Regions with the highest rates of seropositivity coincided with those that have, on average, the worst indicators of socioeconomic position in the State. Knowledge of areas with higher prevalence of HTLV-1/2 in pregnant women may allow the planning of public health interventions, such as screening in prenatal or neonatal periods, and supplying formula milk for newborns of HTLV positive mothers, may drastically reduce the vertical transmission of the virus.

Financial support: FAPEMIG

P-028 5-Lo Pathway and Leukotrienes Receptors Expression in PBMC from HTLV-1 Carriers

Introduction: Leukotrienes (LTs) are bioactive lipids derived from 5-lipoxygenase(5-LO) pathway of arachidonic acid metabolism, and acts by signaling main through their two high affinity membrane receptors BLT1 and CysLT₁R. They are powerful chemoattractants and pro-inflammatory mediators in several inflammatory conditions. However, the relationship of LTs and HTLV-1 infection and related diseases like HAM/TSP is still unknown.

Objective: The aim of this study was to analyze gene expression profile of enzymes involved in the 5-LO pathway and of the high affinity receptors for LTs in peripheral blood mononuclear cells (PBMCs) obtained from HTLV-1 infected individuals.

Methods and Results: PBMCs were separated from six health donors, eleven subjects with HAM/TSP and six asymptomatic HTLV-1 carriers (HAC) and total RNA was obtained using Trizol^® method. cDNAs were synthesized and a quantitative PCR (qPCR) was performed to 5-LO, FLAP, LTC₄ synthase, BLT1 and cysLT₁R genes to determine the relative expression. Equivalent levels of gene expression were observed in HAC and HAM/TSP groups.

Conclusion: Despite similar levels of RNA transcripts obtained in this work, further studies are necessary to measure LTs synthesis, since another enzymes could be involved in the 5-LO pathway. These results may be useful to understand the involvement of LTs in pathogenesis of HAM/TSP.

Financial support: FAPESP, CNPq, CAPES, FUNDHERP.

P-029 Activation of CD4+ T-Cells from HTLV-1-Infected Individuals is Independent of the Level of HTLV-1 Proviral Load

Introduction: HTLV-1 causes a persistent active infection and a spontaneous PBMC proliferation (SP). HTLV-1-infected individuals are more susceptible to several infectious diseases such as strongiloidiases, Hansen's disease, and tuberculosis. We have recently demonstrated that HTLV-1-infected individuals have an immunosuppression, as reflected by a decrease in stimulation index to recall antigens, even in the absence of SP.

Objective: To evaluate the cellular activation and HTLV-1 proviral load in asymptomatic HTLV-1-infected individuals.

Methods: Asymptomatic HTLV-1-infected individuals were classified according to the presence of spontaneous PBMC proliferation in two subgroups SP+ (n = 10) and SP− (n = 8). Controls were healthy donors (n = 10). Membrane activation markers (CD25, CD28, CD45RO, CD69, CD62L, HLA-DR) and intracellular IFN-gamma production of T cell subsets were assessed of freshly PBMC from HTLV-1-infected individuals and uninfected controls by flow citometry. HTLV-1 proviral load was quantified by Real time PCR.

Results: The frequencies of CD4+CD25+, CD4+45RO+ CD25+; CD4+DR+ and CD4+ CD28+ T cells from SP+ and SP− HTLV-1-infected individuals were higher to those of uninfected controls. These proportions were similar in both SP+ and SP− subgroups. However, an increased proportion of CD4+ CD69+ T cells was observed only in SP− HTLV-1-infected group (median 16%, 1–39%), compared to SP+ HTLV-1-infected individuals (median 4.5%, 1–23%)(p = 0.04) and uninfected controls (median 5.5%, 2–9.7%) (p = 0.02). The frequency of IFN-gamma+ CD4+ T cells of SP+ HTLV-1-infected individuals (6.8%, 0.4 –10.7%) was similar to that of SP− HTLV-1-infected individuals (2.8%, 0.2−35%) (p = 0.19). These proportions were significantly higher compared to those of uninfected controls (1%, 0.4–1.5%), (p = 0.001) and (0.04), respectively. HTLV proviral load was positively correlated with the proportion of CD4+CD25+ (r = 0.14, p = 0.036) and CD4+CD45RO+ (r = 0.7, p = 0.0004). In addition, the proportion of activation markers CD25, DR and CD28 on CD4+ T cells were also increased in HTLV-1-infected individuals, even in the group with HTLV proviral load < 10000 copies/10⁶ PBMC.

Conclusion: Cellular activation and IFN-gama production is independent of spontaneous PBMC proliferation and HTLV-1 proviral load, in asymptomatic HTLV-1-infected individuals.

P-031 An Overview of 20 Years of HTLV-1 Study at the institute of Tropical Medicine Alexander von Humboldt in Lima, Peru

Introduction: Since 1989, we have provided HTLV screening at the Institute of Tropical Medicine Alexander von Humboldt (IMTAvH) in Lima. Structured questionnaires are filled out before blood samples are drawn, and specialized care is given to seropositive people. These routine procedures were the foundation of a formal cohort study which is ongoing since 2003. In this overview, we describe baseline characteristics of the HTLV-seropositive cases.

Methods: The persons here described attended or had been referred to our centre between 1989 and 2008 (1) because of suspected HTLV-1-associated diseases; (2) because they were candidate blood donors who had tested positive for HTLV at a blood bank; or (3) because they are close relatives of HTLV-infected subjects. Over this 20-year period, we successively used different, ever-improving brands of ELISA for the serological diagnosis of HTLV. Confirmation tests (commercial western blot or line immuno-assays, and/or in-house proviral load assay) were not available for all ELISA-positive people because of budget restraints.

Results: A total of 1978 HTLV-seropositive persons were registered. Confirmation test results were available for 1411 (71%). The ratio of HTLV-1 to HTLV-2 was 1401 to 10. For 779 subjects (40%), the motive for HTLV testing was a suspected HTLV-1-associated disease: HTLV-1-associated myelopathy/tropical spastic paraparesis (n = 382), strongyloidiasis (n = 172), infective dermatitis (n = 79), adult T-cell lymphoma/leukaemia (n = 37), uveitis (n = 29), scabies (n = 27), tuberculosis (n = 25) or other diseases (n = 28). In addition, there were 342 (18%) candidate blood donors and 812 (42%) close relatives of HTLV-infected people. We identified 425 families with more than one seropositive subject; in 43 of these families, there were five or more infected subjects. Among all seropositive people, the mean age was 42 years (standard deviation 17); and 1129 (58%) were women. Seven hundred thirty-one subjects (38%) were born in Lima; 789 (41%) in the Central or Southern Andes of Peru and 424 (22%) elsewhere. Since December 2003, 959 subjects (48%) have provided blood samples for research purposes during follow-up visits. Findings based on this cohort have been presented in 12 peer-reviewed publications.

Conclusion: The mere dimension of this population of HTLV-1-infected subjects calls for coordinated action in order to foster prevention and control and to expand research of HTLV-1 in Peru.

P-032 Changes of Tax, Foxp3, CTLA-4, IL-10, TGF-ß and GITR MRNA Levels in HAM/TSP Patients Treated with Betamethasone

Corticoidal therapy has been used successfully to decrease symptoms in HAM/TSP patients. In vivo HTLV-I Tax expression has an inhibitory effect on CD4⁺CD25⁺ T regulatory (Tregs) cells. To clarify the effect of corticoids on the role of tax mRNA in the expression of cellular markers genes associated with Tregs function, we measured load tax, foxp3, IL-10, TGF-ß, CTLA-4, GITR mRNA in PBMC serially collected from 8 HAM/TSP patients treated with betamethasone. RNA was measured by real-time polymerase chain reaction. The real-time PCR quantification revealed a decrease of tax and an increased of Foxp3 mRNA associated with treatment and clinical improvement. CTLA-4, GITR, IL-10, and TGF-ß mRNA levels showed an independent variation. We detected an inverse correlation between levels of tax with foxp3 in all cases. Independent of the initial tax levels, with drug treatment there was a decline in tax mRNA and increased Foxp3 mRNA in all patients. These results confirm the beneficial effect of steroids and the usefulness of simultaneous detection of Foxp3 and tax mRNA levels to assess responses to the treatment of HAM/TSP patients.

P-035 Expression of Activation-Induced Cytidine Deaminase in Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is an aggressive neoplastic disease derived from CD4⁺ T-lymphocytes that is caused by a human T-cell leukemia virus type 1 (HTLV-1) infection. Tax is the most extensively studied viral product among HTLV-1-encoded proteins due to its pleiotropic actions on viral and cellular genes. Tax potently increases the expression of viral genes, and it also prevents cell cycle arrest and apoptosis. Various mutations of tumor suppressor genes have been demonstrated in cancer, and it has been established that multiple changes of such genes are necessary for the emergence of malignant cells. In ATL cells, previous studies have demonstrated mutations of tumor suppressor genes. Although Tax is associated with cellular genetic aberration, the mechanisms have not been fully clarified. Activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as an editor of DNA and RNA, is specifically expressed in germinal center B cells. AID is an enzyme essential for somatic hypermutation and class-switch recombination, in immunoglobulin genes. Studies have shown, however, that inappropriate expression of AID acts as a genomic mutator that contributes to tumorigenesis. Indeed, constitutive expression of AID in transgenic mice induces T-cell lymphomas in association with high mutation frequencies. These findings led us to hypothesize that AID may be involved in the accumulation of nucleotide alterations during development of ATL. To gain insight into the molecular mechanism underlying the emergence of somatic mutations in various genes during development of ATL, we examined expression of AID in HTLV-1-infected T-cell lines and primary ATL cells. These cells expressed high levels of AID compared with uninfected T-cell lines and normal PBMCs at both mRNA and protein levels. Immunohistochemical staining of AID in ATL lymph nodes and skin lesions showed that primary ATL cells were indeed stained strongly positive for AID. In vitro infection of a human T-cell line and normal PBMCs with HTLV-1 induced AID mRNA expression. ATL cells cultured for 3 days exhibited augmented AID expression in parallel with the upregulation of Tax. Tax transcriptionally activated AID gene. We demonstrated that Tax-induced AID expression required the NF-kappaB and CREB signaling pathways. The constitutive expression of AID may promote somatic mutation frequency in ATL.

P-036 FAS -670 Promoter Polymorphism may be Associated with HTLV-1 Infection and Clinical Evolution to TSP/HAM

Introduction: The CD4+ T lymphocytes infected by HTLV-1 can be eliminated by apoptosis through interaction with CD8+ T lymphocytes. The Fas and FasL genes are linked closely to the mechanism of the immune system and several polymorphisms in these genes have been associated with susceptibility to certain diseases including LLcTA. This study investigated polymorphisms at positions -670 and -169 (IVS3nt169) in the Fas and FasL genes, respectively, among HTLV-1/2 infected subjects.

Methodology: The blood samples from 40 asymptomatic and 26 TSP/HAM symptomatic patients were collected at the Department of Tropical Medicine, Federal University of Pará (UFPA). Eighty seronegative subjects were used as control group. Aiming to identify the Fas -670 and FasL -169 polymorphisms DNA samples were extracted from PBMCs and analyzed by polymerase chain reaction (PCR-ACRS) followed by RFLP analysis using MvaI and HincII restriction endonucleases, respectively. The products were visualized after electrophoresis in agarose gel 4%.

Results: The analysis of the FasL -169 polymorphism revealed prevalence of 1.52% of the delT/delT mutant genotype among HTLV-1 infected patients and 1.25% in the control population (x² = 0.019; p = 0.9906). The genotype frequencies of the Fas -670 polymorphism showed higher prevalence of genotype GG among HTLV-1 infected subjects (47%) as compared to the control group (27.5%), and this difference was statistically significant (x² = 9.306; p = 0.0095). The comparative between symptomatic and asymptomatic patient groups showed a higher prevalence (x² = 14.58; p = 0.0007) of the genotype GG among patients with TSP/HAM (50%) as compared to asymptomatic subjects (45%). CD4+ T and CD8+ T lymphocytes levels from HTLV-1 infected and seronegative subjects were compared according to the different -670Fas and -169FasL genotypes, but no significant association was observed. The proviral load values were also compared between individuals carrying genotype -169T/T and -169T/delT, and the results were not significant. Furthermore, no association was observed when compared proviral load values in individuals carrying genotypes -670A/A, -670A/G and -670G/G according to the status of symptomatic and asymptomatic infection.

Conclusion: The present result suggests that the presence of the Fas -670 polymorphism seems to be associated with susceptibility to HTLV-1 and maybe increases the predisposition to the development of TSP/HAM in individuals infected by HTLV-1, but further studies must be develop to confirm or not this preliminary finding.

Key words: HTLV, Apoptosis, Fas/FasL genes.

Financial Support: CNPq and UFPA

P-037 A Comprehensive Analysis of Adult T-Cell Leukemia-Lymphoma of Western African Descent by High Resolution Comparative Genomic Hybridization Array

Acute T-cell leukemia/lymphoma (ATLL) is a highly aggressive malignancy caused by human T-cell lymphotropic virus type-1 (HTLV-I). While no specific chromosome or genetic abnormalities have been proven to contribute to the pathogenesis of ATLL, recent comparative genomic hybridization (CGH) studies on Japanese patients have demonstrated frequent genetic lesions (chromosome gains and losses) involving specific chromosomal regions containing potentially important genes. In this study, using a high resolution Agilent platform we performed a comprehensive CGH analysis of sixty DNA specimens, obtained mostly from ATLL individuals of Western African descent from the United States, the Caribbean, and Brazil. Our preliminary results demonstrate the presence of some alterations already reported by others, such as gains in chromosomes 1p21, 4p16, 7p22, 8q24, 9q33-34, 14q31-q32, 16q16, 18q23, 19p13 and deletions at 6q16, 9p21 and q21 and 14q11. Additionally, we have observed several genetic alterations not previously reported in a significant percentage of our ATLL tumors, such as gains in regions of chromosomes 2, 5, and 17, and deletions in chromosomes 3 and 15. Some of these harbor genes of unknown function but most of them contain potential cancer related genes. Our preliminary results confirm there are common genetic alterations in both ATLL of Japanese and West African origin and perhaps other aberrations contributing to the pathogenesis of ATLL separately in both ethnic groups. A more comprehensive analysis will be presented at the meeting.

P-039 Caveolin-1 Expression in Adult T-Cell Leukemia

Caveolae are plasma membrane invaginations that function as regulators of signal transduction. Caveolins are a class of oligomeric structural proteins that are both necessary and sufficient for caveolae formation. Caveolin-1 has been shown to regulate multiple cancer-associated processes including cellular transformation, tumor growth, cell migration and metastasis, cell death and survival, multidrug resistance, and angiogenesis. Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). We examined expression of caveolin-1 and caveolin-2 in HTLV-1-infected T-cell lines and primary ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal PBMCs at both mRNA and protein levels. However, caveolin-2 mRNA expression was not restricted to HTLV-1-infected T cells. Immunohistochemical staining of caveolin-1 in ATL lymph nodes and skin lesions showed that primary ATL cells were indeed stained strongly positive for caveolin-1. Caveolin-1 was also detected in the plasma from patients with ATL. In vitro infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 mRNA expression. The viral protein Tax transcriptionally activated caveolin-1 gene. We demonstrated that Tax-induced caveolin-1 expression required the NF-kappaB and CREB signaling pathways. TGF-beta is an inhibitor of T-cell proliferation. HTLV-1-infected T-cell lines and ATL cells are known to be resistant to TGF-beta-induced growth inhibition. Caveolin-1 colocalized with TGF-beta type I receptor in HTLV-1-infected T-cell lines. We demonstrated that caveolin-1 is able to suppress TGF-beta signaling at the transcriptional level in T cells. Furthermore, downregulation of caveolin-1 expression in an HTLV-1-infected T-cell line exhibited susceptibility to TGF-beta. Thus, we describe a new function of Tax, as a repressor of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

P-041 Clonal Expansion of HTLV-1+ CD8+ Cells Relies on a Defect in the FAS Pathway and an Overexpression of HIAP-1

By analyzing cloned infected cells from individuals without malignancy we recently demonstrated that the clonal expansion of HTLV-1 positive CD8⁺ and CD4⁺ lymphocytes relied on two distinct mechanisms: infection prevented cell death in the former whereas recruiting the latter into the cell cycle (J Clin Invest. 2006;116:974–983). Here we explored the mechanisms underlying apoptosis inhibition in infected CD8⁺ lymphocytes. Staurosporine triggered cell death in all tested clones while agonistic anti-Fas antibodies and recombinant Fas ligand triggered cell death in all CD4⁺ clones, in uninfected CD8⁺ clones and in 2/6 infected CD8⁺ clones. This suggests that a defect in the Fas pathway might account for preventing cell death in these 2 CD8⁺ infected clones. To investigate this, we next analyzed the expression of the main genes involved in apoptosis in cloned cells deriving from TSP/HAM patients. Microarrays were carried out with infected and uninfected CD4 and CD8 clones (3 clones per category). Data were analyzed through the use of Ingenuity Pathways Analysis (Ingenuity^® Systems, www.ingenuity.com) and confirmed by qRTPCR. Among the deregulated genes, c-FLIP-l and hIAP-1, which have been previously found to be implicated in the survival of HTLV-1 infected cells in vitro, were specifically overexpressed in CD8 cells (Blood. 2006;107:4491–4499; Blood 2006;107:3933–3939). hIAP-1 expression was significantly correlated with Tax expression in CD4 and CD8 clones (p < 0.002). Incubation of cells with the NFKappaB inhibitor LLnL significantly decreased hIAP-1 expression in Tax positive CD8 clones and abolished the correlation between tax and hIAP-1 expressions. Together these results suggest that in vivo, hIAP-1 expression is controlled by Tax via NFKappaB. c-FLIP possesses splicing variants having distinct effect on apoptosis (EMBO, 2002; 14:3704–3714). cFLIP-l, but not cFLIP-s was significantly overexpressed in infected CD8+ clones while infected and uninfected CD4+ cells displayed the same amounts of both splicing variants. In contrast to hIAP-1, there was no correlation between tax and cFLIP expression in cloned cells. These results obtained with naturally infected cells suggest that together with a defect in the Fas pathway, hIAP-1 and cFLIP-l overexpression are involved in the CD8-specific apoptosis resistance, with evidence of clonal diversity.

P-042 Comparative Analysis in the CCR5, CCR2 and SDF-1 Genes Mutations in a Selected HIV-1 Infected and Uninfected Population from Brazil

CCR5 and CCR2 act as major co-receptors for the HIV-1 entry in the target cells. Some mutations in the CCR5 gene have been associated with less susceptibility to R5 HIV-1 strain infection and a delayed disease progression and its allelic frequency varies according to the ethnic group. The CCR2-64I mutation has been associated with partial protection to AIDS and its occurrence is higher in Asians and their descendants. Another mutation, in the SDF-1 gene, described as SDF1-3’A has been reported as prevalent in almost all ethnic groups, except in Africans. Although the role of SDF1-3’A in AIDS outcome is not yet clear, some evidences show that this mutation delays the progression to AIDS. These mutations have been related as important factors in the onset of the disease progression. In the present study we performed a comparative analysis between the prevalence of some polymorphisms in CCR5 (delta 32, 303 G/A, 29 A/G, m303 T/A), CCR2-64I and SDF1-3’A genes in a stratified cohort of HIV-1 infected. These analyses were screened by PCR followed by RFLP, when required. It was observed that the CCR5-delta 32, CCR5-29A/G, CCR2-64I and SDF1-3’A allelic frequencies were higher among the Long Progressors than in Rapid Progressors stratified infected individuals, suggesting that these mutations could be delaying the AIDS progression. However, when we compare the allelic frequencies between the infected and uninfected group we did not find significant difference to the studied mutations. It is of paramount importance to obtain a better knowledge of population genetic background in this country as well as the influence of diverse polymorphisms on progression to AIDS.

P-043 Abstract Withdrawn

P-044 Distinct Composition and Function of Tax1 and Tax2 Ubiquitinated Forms

Permanent activation of the NF-?B pathway through the recruitment of the IKK?/NEMO regulatory subunit is one of the key events in the induction of T-cell proliferation by the HTLV-1 Tax (Tax1) viral transactivator. We previously demonstrated that Tax-IKK?/NEMO interaction relies upon Tax1 ubiquitination and that conjugation of Tax1 to ubiquitin (Ub) allows the redistribution of Tax1 and IKK?/NEMO towards perinuclear cytoplasmic spots. Although encoding a Tax protein, HTLV-2 does not cause leukemia. It has been previously suggested that distinct Tax localizations and transcriptional activities account for these differences in pathogenesis. We therefore compared the ability to recruit IKK?/NEMO as well as the ubiquitination status of Tax2 and Tax1. We first show that Tax2 is ubiquitinated and recruits IKK?/NEMO, but that its ubiquitination pattern differs from that of Tax1. Tax2 is conjugated to K29- or K63-bound Ub chains and only delocalizes these two types of chains towards cytoplasmic spots, while Tax1 is also conjugated to K48- or K63-chains and induces the centrosomal redistribution of all types of Ub chains. Overexpression of K29-Ub selectively increases NF-?B promoter activation by Tax2. Wild-type ubiquitin also dramatically increases Tax2 mediated transactivation of the HTLV-2 promoter, while showing little effect on Tax1 transactivation of the viral promoter. Altogether, our results reveal new significant differences between Tax1 and Tax2 and favor the notion that the molecular determinants involved in the activation of either the NF-?B or CREB pathways by Tax1 and Tax2 are not identical.

P-045 Effect of HTLV-1 ORF I Protein Products on the Immunological Synapse and Actin Remodeling

The Orf-I of the Human T-cell Leukemia/Lymphoma Virus Type I (HTLV-1) encodes a 12 kDa protein that is proteolytic cleaved within the ER to yield an 8 kDa protein. The 12 kDa protein remains in the ER and Golgi, whereas the 8 kDa protein traffics to the cell surface and is recruited to the immunological synapse (IS) upon T-cell activation. Genetic analysis in patients infected with HTLV-1 revealed an amino substitution in 12kD that results the express of mainly the 8 kDa protein. Here we have studied the effect of the 8kD isoform on T-cell activation. T-cell receptor (TCR) engagement with antigen presenting cells triggers actin rearrangements that control receptor clustering of signaling protein complexes to form the IS. We hypothesized that the 8 kDa may down-regulate TCR proximal signaling by directly interfering with proper TCR engagement. Jurkat T-cells expressing the 8 kDa or the 12 kDa form were conjugated to super-antigen (SEE) stimulated Raji B-cells and analyzed by flow cytometry and quantitative confocal microscopy. Expression of the 8 kDa resulted in a significant reduction of Jurkat T-cells and Raji B-cells conjugates. Intracellular trafficking of the TCR measured by CD3 to the IS was reduced compared to control Jurkat T-cells. As expected, Jurkat T-cells expressing the 12 kDa form, which remains in the ER, showed no reduction in T-cell conjugation. We have previously shown that p12I inhibits the phosphorylation of LAT, Vav, and PLC?-1 and decreases NFAT activation1. Therefore, we examined the subcellular localization of TCR proximal signaling molecules in the presence of the 8 kDa or 12 kDa forms in Jurkat T-cells. LAT localizes in recycling endosomes and traffics to the IS upon T-cell activation. We found that the 8 kDa form inhibited LAT recruitment to IS by disrupting the trafficking from the recycling endosomes and redistributing LAT uniformly on the cell surface. In addition, cells expressing the 8 kDa protein, PLC?-1 was no longer localized at the IS upon T-cell activation. PLC?-1 recruits the SLP-76 adaptor to LAT, which leads to SLP-76 dependent N-WASP phosphorylation causing actin polymerization. To examine the effects of actin formation, we plated Jurkat T-cells on CD3-coated cover slips, which lead to rapid cell spreading and rearrangements of F-actin into a circumferential actin-rich ring. The cell diameter and the actin intensity upon T-cell stimulation was reduced in the presence of the 8 kDa protein. Also, a decreased concentration of the phosphorylated form of N-WASP was observed at the contact sites. In cells expressiong the 12 kDa form, the actin polymerization and N-WASP distribution was similar to control cells. Together, our results demonstrate that the 8 kDa protein alters both the trafficking and composition of T-cell signaling molecules and actin networks that in turns affect the immunological synapse formation.

P-046 Establishment and Charactarization of the Cell-Free IKK Activation System Induced by HTLV-1 Tax

Human T-cell leukemia virus type 1 (HTLV-1) is etiological agent of an aggressive T-cell malignancy, called adult T-cell leukemia (ATL). HTLV-1 provirus genome encodes Tax protein, which plays a critical role in the induction and development of T-cell transformation. Tax regulates the expression of cellular genes involved in cell growth and survival through modulating various transcription factors. One of the key transcription factors targeted by Tax to facilitate cell transformation is NF-kappaB. As Tax mutants defective in NF-kappaB activation are unable to immortalize T-cells, Tax-induced NF-kappaB activation is essential for T-cell transformation. NF-kappaB is normally sequestered in the cytoplasm by associating with the inhibitory protein, IkappaB. Activation of NF-kappaB involves the phosphorylation of IkappaB by the IkappaB kinase (IKK) complex, which results in the IkappaB degradation. Previous studies have shown that Tax interacts with IKKgamma, the regulatory subunit of the IKK complex, and induces IKK activation. However, the molecular mechanism of Tax-induced IKK activation remains largely unknown. To elucidate this mechanism, we first produced recombinant Tax and established the cell-free assay system that induces IKK activation in a Tax-dependent manner. Next, to determine whether Tax is sufficient for inducing IKK activation, we performed cell-free assay using recombinant Tax and the purified IKK complex. Tax did not activate the purified IKK complex, indicating Tax requires intermediary factors for IKK activation. Therefore, we addressed whether polyubiquitination, which is critical for the IKK activation by cytokines, is involved in Tax-induced IKK activation. Addition of a lysine-less ubiquitin mutant resulted in the suppression of Tax-induced IKK activation, suggesting polyubiquitination is involved in the IKK activation by Tax. We are currently trying to identify the molecules required for the Tax-IKK signaling pathway.

P-047 10M Timed Walks Deteriorate in Patients with Chronic HAM/TSP by 2 Sec/Y Independent of HTLV-1 Viral Load

Background: HAM/TSP has a variable course. Progression in the first 2 years followed by a plateau has been reported but chronic deterioration is also common. The significant changes between the milestones defined by various disability scales may only occur after several years. Detecting more subtle changes is important especially for monitoring therapeutic interventions. We report the findings from a prospective cohort study.

Methods: Prospectively collected clinical and laboratory data were analysed for 48 patients with HAM followed up between 1993 and 2007.

Results: Most patients were female and Afro-Caribbean. Only five patients had received blood transfusions. The median duration of symptoms was 11.6 years. Common first experienced symptoms were unilateral leg weakness (36%) and back/leg pain (30%). The median HTLV-I viral load was persistently high (14 copies/100 PBMCs, 95% CI 13–28). Progression to walking aid (unilateral, bilateral or frame) took a median of 11 years regardless of type and to wheelchair 18 years. Younger patients progressed to additional mobility aids faster (<50 years v >50 years, p = 0.005).

During a median of 3.8 years clinic follow up 10m timed walk (tw) worsened in 77% of patients. At first visit the mean tw was 21.15 sec/10m (8.2-80, 95%CI: 15.8–26.5, median 17.8) and at last follow up 28.7 sec/10m (8.5–95, 95%CI: 21.6–35.8, median 21.3). An overall deterioration in tw of 2 sec/10m/y. HTLV-I viral load did not predict deterioration of tw but was higher in patients with a younger age of onset (median: 1st visit: 11.2 v 1.7,p = 0.09; last visit:14.9 v 5.5, p = 0.05).

Conclusion: Whilst high viral load is found in patients with HAM/TSP HTLV-1 viral load did not significantly fluctuate during follow up nor correlate with disease progression. This suggests that high HTLV-1 viral load may be associated with, but not causative of HTLV-1 associated chronic inflammation. Tw can be used to detect disease progression over shorter periods than disability scales. Defining the natural history of progression in the absence of disease-modifying therapies will allow future comparison with novel interventions.

P-048 A Dyshidrosis-Like Variant of Adult T-Cell Leukemia/Lymphoma with Clinicopathological Aspects of Mycosis Fungoides. A Case Report

Background Around 30 cases of vesiculo-bullous mycosis fungoides (MF) and recently one case of vesicular adult T-cell leukemia/lymphoma (ATL) have been described. Vesiculo-bullous lymphomas are rare and dyshidrosis-like lymphoma are even more rare. Besides, this is the first case of vesicular ATL in which human T-cell lymphotropic virus type I (HTLV-I) proviral integration was determined.

Case report: A 38 years-old man, diabetic, with a 3,5 years history of disseminated papular lesions and lymphocytosis. Four months after the beginning of disease he had the diagnosis of MF with pulmonary involvement and mild lymphocytosis. The lesions disappeared with 6 cycles of CHOP. After 3 years of follow-up confluent dyshidrosis-like eruption appeared in both palms and plants followed by generalized papules. The patient also had lymphocytosis, and infiltration of the bone marrow, but normal values of LDH and calcium. Serologic study revealed HTLV-I positivity and the patient had the diagnosis of chronic ATL. HTLV-I proviral integration was demonstrated by inverse and long PCR. In spite of treatment, he died one month later due to non-controlled diabetes.

Skin biopsy: marked spongiosis, epidermotropism of atypical lymphocytes, Pautrier abscesses and intra-epidermal vesicles with atypical lymphocytes. Infiltration of small and atypical lymphocytes CD2+, CD3+, CD4+, CD8−, CD5+. CD7−, CD20−, and CD25+ was seen in the dermis and epidermis. The proliferative index (Ki-67) was 50%.

Conclusions: 1. This is the first case of ATL with cutaneous vesicular lesions in which HTLV-I proviral integration was demonstrated, unequivocally indicating the relation between the virus and the lymphoma. 2. ATL may present as a cutaneous vesicular disease mimicking dyshidrosis. 3. The delay in the diagnosis of ATL was due to the fact that MF and ATL may present the same clinical and histopathological aspects. This differentiation is important because the prognosis and treatment are different from other T-cell lymphomas. 4. The median survival time for chronic ATL is around 24 months, but this patient had a survival of 38 months in spite of the initial erroneous diagnosis which did not allowed a better choice of treatment. 5. Serology for HTLV-I should be performed in all mature T-cell lymphomas in Brasil.

P-050 Atypical Neurological Symptoms in Patients infected with Human T-Lymphotropic Virus Types I and II (HTLV-I/II) Attending a Rehabilitation Program: Case Reports

Introduction/Objective: The Human T-Lymphotropic virus types I and II (HTLV-I/II) are endemic in some countries, including Brazil. The classical neurological presentation is spastic paraparesis. The aim of this study was to describe atypical neurological symptoms caused by the HTLV-I/II infection in patients attending a rehabilitation program.

Participants/Material and Methods: A cases report study with major symptoms description of atypical neurological manifestation in patients with HTLV-I/II infection admitted between January 2003 and December 2007 to the Spinal Cord Injury Rehabilitation Program at the Sarah Network Hospitals, São Luis, was conducted. Patients presenting anti-HTLV I/II antibody in serum (ELISA and Western-Blot) and in cerebrospinal fluid were included. Sixteen patients were excluded because presented other neurological disease or had no neurological symptoms. Characteristics of the patients with atypical symptoms were described.

Results: Among the 39 patients included in this study, 35 (10 male, 25 female) presented the classical neurological symptoms (spastic paraparesis). Atypical neurological presentation was seen in 4 patients (10.3%). One was a female, with onset of symptoms by the age of 18 years-old, afflicted for 13 years who developed spastic tetraparesis. She presented lumbar pain, neurogenic bladder and used a walker. In three subjects cerebellar signs were observed. Two presented ataxia, dysmetria, movement decomposition and neurogenic bladder. One was a male, onset of symptoms by the age of 36 years-old, 4 years of disease duration, with slight increase in muscle tone, and the other was a female, onset of symptoms by the age of 45 years-old, 7 years of disease duration and flaccid muscle tone. The forth patient was a female with dysdiadochokinesia, dysmetria and movement decomposition, onset of symptoms by the age of 42 years-old, who had the symptoms for 4 years. This patient was wheelchair restricted. She also presented flaccid muscle tone.

Conclusion: In the studied population, 10.3% of the patients developed atypical neurological symptoms secondary to HTLV-I/II infection, including spinocerebellar syndrome, flaccid muscle tone and spastic tetraparesis. The health care professionals who attend these patients must be aware that atypical manifestations may occur in HLTV-I/II infected patients in order to meet the patients specific needs. Other studies are necessary to evaluate the impact of atypical neurological manifestation in these patients function.

P-051 Cervical Lesions in Patients with HAM/TSP: A Report of Two Cases

Introduction: The HTLV-1 infection is associated with a myelopathy of chronic progressive evolution, characterized by a spastic paraparesis. Cervical spinal cord lesions in MRI images have been found in HAM/TSP patients, even without local clinical manifestations. The meaning of these findings is still unclear. We report two HAM/TSP cases, who have cervical spinal cords alterations in the MRI, with different evolution of the images and symptoms.

Case-Report: Patient 1. A white woman of 40 year-old with the diagnosis of HAM/TSP, developed a spastic paraparesis and a urinary retention. In a few weeks the patient developed tetraparesia. The cervical MRI showed hyperintense signals in the anterior cervical spinal cord. The CSF exam revealed inflammatory reaction. No biopsy of the lesion was made.

Patient 2. A black woman of 69 year-old with the diagnosis of HAM/TSP presented with spastic paraparesia and urinary retention. A MRI of cervical spinal cord showed diffuse hyperintense signals in the anterior cervical spinal cord. The CSF exam showed inflammatory reaction. A follow-up MRI was made and these lesions disappeared. This patient had stable clinical course.

Discussion: We present two HAM/TSP cases with the same initial clinical presentation, similar inflammatory CSF results, and cervical spinal cord lesions. One case had a rapidly progressive form of evolution to tetraparesis. The other had no persistence of alterations in the follow-up MRI and her clinical evolution was more stable. Cervical lesions in the spinal cord are atypical findings in HTLV-1 infection. It still unknown the meaning of these findings and if it could be considered part of the natural history of the disease.

P-052 A Hierarchy of Post-Translational Modifications Controls the Transcriptional and Transforming Activities of the HTLV-1 Tax Oncoprotein

Tax-mediated activation of gene expression via the NF-kappaB pathway depends on phosphorylation of Tax at serines 300/301, then ubiquitination at lysines 280/284 leading to activation of the IkappaB kinase complex in the cytoplasm. Activation of cellular gene expression requires, in addition to these cytoplasmic events, the transport of both phosphorylated Tax and the RelA subunit of NF-kappaB to the nucleus with concomitant deubiquitination and sumoylation of Tax at the same lysine residues 280/284, and subsequent formation of the Tax nuclear bodies. Sumoylated Tax recruits the RelA subunit of NF-kappaB in these nuclear structures as well as components of the transcription and splicing complexes. The Tax nuclear bodies also include the acetyltransferase p300, which acetylates Tax at lysine 346 in a phosphorylation and sumoylation-dependent manner. To better understand the sequence of modifications, the enzymes involved in these modifications and their role in Tax transcriptional and transforming activities, we analyzed wild type Tax and various previously characterized Tax mutants by two dimensional gel electrophoresis. The results support the idea that the formation of the Tax nuclear bodies and the constitutive activation of the NF-kappaB pathway critically depend on a hierarchical sequence of post-translational modifications.

P-053 A Parallel Between Genetic Distances Based on Nucleotide Substitution Models in Primate T-Cell Lymphotropic Viruses and Distances Based on Codon Usage and tRNA Availability in the Host Cells

Genetic distances between related DNA sequences can be estimated by nucleotide substitutions models which are the base of modern phylogenetic inferences. In this work we first investigated whether the mutations differentiating the concatenated genes ENV+ TAX of the human and simian T-cell lymphotropic virus types have a proportional correspondence with a distance derived from their codon usage difference. The obtained results considering HTLV types 1, 2 and 3 and STLV types 1, 2 and 3 were not conclusive. Second, we tested another distance based on the difference of the correlation between the codon frequencies in such viral genes and the free isoacceptor tRNA abundances on both hosts, human and simian. In doing this we realized that the pair-wise distances between sequences in a plane defined by both, the distance given by the virus-host codon usage difference and the distance computed from the correlation between viral codon usage and host tRNA availability have a strong correlation with the pair-wise distances calculated with nucleotide substitution models. This way it was possible to construct a phylogeny-like tree from such codon usage and tRNA availability distances, which is very similar to that obtained by classical phylogenetic inference methods. This finding beyond its potential application in virus evolution studies has a biological background that deserves further analysis.

P-055 An interaction Between the Human T-Cell Leukemia Virus Type 1 Basic Leucine Zipper Factor (HBZ) and the KIX Domain of P300/CBP Contributes to the Down-Regulation of Tax-Dependent Viral Transcription

Activation of Human T-cell Leukemia Virus type 1 (HTLV-1) transcription is dependent on the viral transactivator Tax. Alone, Tax lacks DNA-binding activity and is therefore recruited to the viral promoter as part of a complex with the cellular transcription factor CREB (or other members of the activating transcription factor/cyclic AMP-responsive element (CRE)-binding protein (ATF/CREB)) family. These proteins carry a basic leucine zipper (bZIP) domain that stimulates protein dimerization and subsequent DNA binding. Formation of these complexes on the viral promoter serves as a binding site for the coactivators p300 and CREB-binding protein (CBP), which are recruited to the HTLV-1 promoter. In addition to Tax, other viral proteins have been shown to regulate HTLV-1 transcription, one of which is HTLV-1 bZIP factor (HBZ). Interestingly, this protein is encoded by a gene on the negative strand of the provirus and transcribed from a promoter located within the 3′-long terminal repeat. Indeed, HBZ repress Tax-mediated viral transcription by binding to cellular ATF/CREB factors through their bZIP domains. Since HBZ appears to lack the capacity to associate with the HTLV-1 promoter, formation of these heterodimers inhibits ATF/CREB factors from binding DNA and, consequently, prevents the recruitment of Tax to the viral promoter.

In this study, we show that HBZ mutant lacking the COOH-terminal bZip domain retains the ability to repress HTLV-1 transcription. These observation suggest that an another domain of HBZ play a role in this repression. Here, we show that the coactivators CBP/p300 binds to NH2-terminal domain (called AD for Activation Domain) of HBZ, this binding inhibit the recruitement of coactivators on HTLV-1 promoter. We demonstrate that two LxxLL-like motifs located within the AD domain of HBZ are involved in this interaction, and specifically mediate binding to the KIX domain of CBP/p300. We also provide evidence that HBZ disrupts the interaction between Tax and p300/CBP, thereby inhibiting the association of coactivator with the viral promoter. This mechanism may supplement the negative effect of HBZ on the formation of the Tax-CREB complex on the viral promoter. Moreover, interaction between HBZ and CBP/p300 could activate cellular genes regulated by HBZ/JunD heterodimers. In conclusion, by targeting both CREB and p300/CBP, HBZ may have evolved a bipartite mechanism to achieve sufficient repression of transcription for the maintenance of a persistent infection.

P-056 Abstract Withdrawn

Shuh M. Roberts A. Robins A. Gifford E. Jones E. Blake II R. Macon S. Lanosga S. Evans W. Xavier University of Louisiana

P-057 Biophysical Properties of HTLV-I Tax

Human T cell lymphotropic virus, type I (HTLV-I), is the causative agent of adult T cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy. HTLV-I is a retrovirus which contains a 9 kb ssRNA genome, encoding the classic retroviral genes gag, pol, and env. HTLV-I also encodes several regulatory genes, including tax. Tax is a 353 amino-acid, 40 kiloDalton (kDa) protein, located predominantly in the nucleus although a small percentage is isolated in the cytoplasm. Tax functions to regulate viral gene expression by interacting with the host transcription factor family, CREB/ATF. The Tax/CREB complex is formed on the 5′-LTR CRE site and activates viral gene transcription. In ATL cells (CD4⁺, CD8⁻), Tax also associates with two host transcription factors, NF-□B and SRF (serum response factor), resulting in the over-expression of host genes containing the NF-□B responsive element and the SRE (serum response element). These host genes, such as IL-2 and c-fos, are activated constitutively in the presence of Tax and result in cell proliferation of HTLV-I⁺ T cells, potentially leading to leukemia in the HTLV-I patient. The laboratory is specifically interested in determining the mechanism by which Tax activates SRF-dependent transcription during T cell transformation because genes regulated by SRF are considered immediate early response genes that activate quickly after growth factor receptor engagement. We believe that Tax activation of the SRF pathway serves as the initial transformation event. Thus, we are interested in the biochemical properties of Tax interactions with SRF, SRF/DNA, TCF (ternary complex factor, a binding partner with SRF), SRF/TCF, and SRF/TCF/DNA. Prior to conducting biochemical experiments, however, we need to express and purify Tax which has been difficult to accomplish in the HTLV-I field and has impeded any progress toward studying the biophysical properties, including structure determination, of Tax. We present data showing our initial protein purification techniques that result in the isolation of a 99% pure Tax and preliminary data characterizing the biophysical properties of Tax in vitro.

P-058 Blocking VEGF Signaling Inhibits the Invasive Potential of Adult T Cell Leukemia Cells Through the Endothelium

Metastasis is the colonization of selective, secondary organ sites by dissemination of tumor cells from their primary site. Extravasation is a critical step in the metastatic cascade. It is initiated by the specific adhesion of the tumor cells to vascular endothelial cells followed by trans-endothelial migration. ATL (Adult T cell leukemia) cells extravasate through the endothelial barrier using angiogenesis-like mechanisms that induces transient endothelial cell retraction. We explored the effects of the specific tyrosine kinase inhibitor of VEGF receptors PTK-787/ZK-222584 (PTK/ZK), anti-VEGF antibody (bevacizumab) and 18aGA gap junctions' inhibitor on ATL cell extravasation. We showed that HuT-102 and MT2 cell lines induce in vitro angiogenesis tube formation that was inhibited by both PTK/ZK and anti-VEGF. ATL-derived cell free supernatants induce a rapid and sharp increase in the phosphorylation of AKT and ERK, a moderate increase in FAK and potentiate VE-cadherin-ß-catenin interaction. These effects are attenuated by PTK/ZK treatment. In addition to paracrine stimulation, endothelial cell-tumor cell communication studies showed that ATL derived cell lines invade through endothelial monolayers in a gap junction dependent mechanisms. It induces modifications in gap junctions' complex interactions, Cx43, Cas and Src, and in adhesion molecules expression and interaction, VE-cadherin and ß-catenin. Independent of VEGF and gap junctions and through compensatory signaling pathways, hetero-cellular communication induces FAK Y861 and high activation of ERK and AKT signaling pathways. These results demonstrate a central role of secreted VEGF and hetero-cellular communication in cell extravasation. This cross talk induces intercellular junctional complexes unzipping, leading to endothelial cells retraction. This study supports the potential use of PTK/ZK for the treatment of malignancies with angiogenic, invasive and metastatic properties.

P-059 Characterization of the Complete Genome of a Highly Divergent Simian T-Lymphotropic Virus (STLV) Type 3 Obtained Entirely from Dried Blood Spots of a Cercopithecus mona Monkey

The recent discoveries of novel human T-lymphotropic virus type 3 (HTLV-3) and highly divergent simian T-lymphotropic virus type 3 (STLV-3) subtype D viruses from two different monkey species in southern Cameroon suggest that the diversity and cross-species transmission of these retroviruses are much greater than currently appreciated. We describe here the first full-length sequence of a highly divergent STLV-3(Cmo8699AB) subtype D virus obtained by PCR-based genome walking using DNA prepared entirely from two dried blood spots (DBS) collected from a Cercopithecus mona monkey. The genome of STLV-3(Cmo8699AB) subtype D is 8913-bp long and shares only 77% nucleotide identity to other full-length PTLV-3 genomes. Since only very short STLV-3 subtype C sequences are available at GenBank for comparison, we obtained approximately 1 kB tax sequences from peripheral blood mononuclear cell DNA from two STLV-3c-infected Cercopithecus nictitans (Cni3034 and Cni3038). These new tax sequences showed 95% sequence identity to STLV-3(Cmo8699AB) in this very conserved region. Phylogenetic analyses show that this highly divergent virus is distinct and forms an independent lineage with high bootstrap support within the diversity of PTLV-3. Molecular dating using an alignment of PTLV tax sequences inferred a divergence date of 92,072 – 138,560 years ago for STLV-3(Cmo8699AB), indicating an ancient origin for this new PTLV-3. Major structural, enzymatic, and regulatory gene regions of STLV-3(Cmo8699AB) subtype D are intact suggesting a replication competent viral genome. Our results significantly expand the genetic diversity seen within the PTLV-3 phylogroup. Taken together, the inferred ancient origin of STLV-3(Cmo8699AB), the presence of this highly divergent virus in two primate species from the same geographical region, and the ease with which STLVs can be transmitted across species boundaries, all suggest that STLV-3 subtype D may be more prevalent and widespread. Given the high human exposure to nonhuman primates in this region, increased surveillance for this divergent virus is warranted.

P-060 Phylogenetic Analysis Bovine Leukaemia Viruses Isolated in South America: Evidence for Diversification and GP51 Env Protein Evolutionary Constraints

Bovine leukaemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus of the family Retroviridae. BLV infects cattle worldwide and imposes severe economic impacts in dairy cattle industry. To establish the degree of genetic variation among BLV strains circulating in different geographic regions of the world is extremely important for the development of appropriate anti-viral strategies and vaccine development. Little is known about the degree of BLV strains circulating in the South American region. In order to gain insight into these matters, we obtained full-length gp51 env gene sequences from 8 BLV strains isolated in Uruguay. Using these sequences and all 12 available sequences from BLV strains isolated in the Latin American region, as well as 22 BLV sequences isolated elsewhere, maximum likelihood phylogenetic analysis were performed. The results of this work revealed a diversification of BLV in the Latin American region and a more complex epidemiology than previously anticipated. Although a high degree of genetic variation was observed among BLV strains, analysis of synonymous and nonsynonymous substitutions rates throughout full-length gp51 env protein revealed a strong negative selection pressure against amino acid changes in all protein regions studied. Interesting, Bayesian co-evolution studies showed 10 pairs of interacting co-evolving of amino acid positions in the gp51 env protein, revealing a conditional evolutionary dependency of these sites, probably to conserve function.

P-061 Phase II Trial of Denileukin Diftitox in Adult T-Cell Leukemia/Lymphoma

Background: Overexpression of CD25, the alpha-chain of the interleukin-2 receptor, is characteristic of the malignant cells of patients with adult T-cell leukemia/lymphoma (ATL). CD25 can be used to target receptor-directed therapy to ATL. Previous studies using monoclonal antibodies directed at CD25 (murine anti-Tac and daclizumab), or chelated with yttrium-90 as radioimmunotherapy, demonstrated responses and some cures in patients with ATL. Denileukin diftitox is a CD25-targeted genetically engineered fusion molecule of interleukin-2 and truncated diphtheria toxin. It must undergo internalization for therapeutic benefit and therefore CD25 must be co-expressed with the beta- and gamma-chains of the interleukin-2 receptor for efficient uptake. There are anecdotal reports of its clinical efficacy in ATL.

Methods: We are conducting a phase II trial of denileukin diftitox in patients with acute, lymphomatous and chronic ATL. The FDA approved schedule of administration uses every three-week dosing; however, in an attempt to increase drug delivery because of the high proliferative rate of ATL, an every other week schedule was used. Denileukin diftitox is given at a dose of nine micrograms per kilogram per day for five days. Tumor response is evaluated every two cycles unless disease progression is noted earlier.

Results: Eight patients, 4 male and 4 female, median age 50 (range, 43–58 years) were treated, including 1 chronic, 4 lymphomatous, and 3 with acute ATL. All were previously treated with a median of 1 prior therapy (range, 1-4). All but one patient had received prior chemotherapy. A median of 2 cycles of therapy were administered (range, 1–6). Therapy was in general well tolerated although one patient died with a colonic perforation due to unrecognized Epstein-Barr virus related lymphoma associated with her previous therapy with the T cell depleting monoclonal antibody siplizumab. Toxicities observed associated with denileukin diftitox included vascular leak syndrome (1), hypoalbuminemia (3), peripheral edema (1), hepatic dysfunction (3), hypotension (1) and vomiting (3). Transient neutropenia (4), thrombocytopenia (3), anemia (7) and lymphopenia (6) were also seen. Most toxicities were CTC grade 1 or 2 and resolved following therapy. No objective responses were observed; however, two patients had stable disease for 4 and 6 cycles of therapy. Leukemic blood counts decreased dramatically during therapy; however, they rebounded following the five days of treatment.

Conclusion: This reduction in leukemic blood counts demonstrates the clinical activity of denileukin diftitox, but the rapid leukemic cell recovery suggests that higher doses or a more sustained administration schedule will be required to achieve durable responses. Alternatively, denileukin diftitox has been safely combined with CHOP chemotherapy and provides another potential approach to the treatment of ATL.

P-062 Possible Dysfunction of NMD in ATL Cells

Adult T-cell leukemia (ATL) caused by Human T-cell leukemia virus type 1 (HTLV1) is one of the most intractable diseases. The median survival period is about one year because of resistance to conventional combination chemotherapies. It is mandatory to identify specific target molecules of chemotherapy based on the understanding molecular mechanisms of leukemogenesis to establish an effective treatment against ATL.

ATL is believed to be developed by a mechanism of a multistep leukemogenesis that involves at least 5 genetic events. However, the genes involved in the leukemogenesis have not been elucidated to date. To reveal genetic mechanisms underlying ATL leukemogenesis, we have done expression profiling analyses of peripheral blood samples of ATL patients with microarray technologies. The results revealed up-regulated gene expression of many genes compared with the normal PBMC samples. Among them, we found several genes that are known to be target genes of nonsense-mediated mRNA decay (NMD), suggested dysfunction of NMD in ATL cells.

NMD is an mRNA surveillance system of the cells. NMD targets and degrades mRNA with a nonsense codon, i.e. premature termination codon (PTC) positioning upstream of exon-junction-complex (EJC). Aberrant mRNA having PTCs could produce potentially harmful C-terminally truncated protein, which may contribute to oncogenic transformation of the cells.

Up-regulated expression of several NMD target genes in ATL cells suggests dysfunction of NMD that may be one of the genetic events in the leukemogenesis. To test this hypothesis and understand underlying mechanisms, we first studied expression levels of NMD target genes in HTLV-1-transformed cell lines and primary ATL cells, and examined NMD activity in those cell lines using a ß-globin gene-based reporter system.

Results of quantitative real time PCR revealed significant up-regulation of endogenous NMD target genes, including Clk2 variant 2, Rpl3, MDM2 variant C and p53i□ in HTLV-1-transformed cell lines. The levels of expression were at least 1.5-fold higher in ATL cells and HTLV-1-related cell lines compared with those in the normal PBMC samples. Reporter gene assays demonstrated dysfunction of NMD in HTLV-1-transformed cell lines. In NMD-intact cells, transfected NMD-target ß-globin mRNA should be degraded to a significantly lower level, compared to that of wild type ß-globin, which is not a NMD target. In the present study, the levels of NMD-target ß-globin in HTLV-1-related cell lines were more than 1.5-fold higher than those in the two control cell lines, TIG1 and HeLa cells, which are widely accepted as NMD-intact cell lines.

Taken together, these results suggest that the NMD activity is hampered in HTLV-1-transformed cells, resulting in accumulation of NMD target transcripts, which may provide a molecular basis for cellular transformation. Studies in functional effects of above mentioned NMD target transcripts on cell proliferation and apoptosis are now in progress.

P-063 Retinoid and Adult T-Cell Leukemia

We previously reported that retinoid inhibited growth in human T-cell leukemia virus type I (HTLV-I)-positive T-cell lines and in fresh cells from patients with adult T-cell leukemia (ATL). Here, we confirmed the clinical effects of all-tans retinoic acid (ATRA) in 20 patients with ATL. The 20 patients (n = 20) with a median age of 56 (range, 35–73) years who were diagnosed with ATL received ATRA orally. The efficacy of treatment was as follows: no complete response (CR), a partial response (PR) in 40% of the patients, no change (NC) in 45% of the patients, and a progressive disease (PD) in 15% of the patients. In 7 acute-type ATL patients, a PR was achieved in 2 (28.5%), NC was observed in 2 (28.5%), and a PD was observed in 3 (42.8%). In 3 lymphoma-type ATL patients, a PR (100%) was achieved. Among 4 chronic-type ATL patients, a PR was achieved in 1 (25%) and NC was observed in the remaining 3 (75%). In 6 smoldering-type ATL patients, a PR was achieved in 2 (33.3 %) and NC was observed in 4 (66.6%). These results indicated that ATRA might be a useful agent for the safe treatment of ATL. We also have tried new synthetic retinoid, Am-80, for ATL in clinical stage. The mechanism of retinoid treatment has been considered as both inhibitions of viral reverse transcriptase and of NF-□B transcription. Further, a possibility of premature senescence induction by retinoid has been investigated.

P-064 Hepatitis C Virus (HCV) and Human T-Lymphotropic Virus (HTLV) Coinfection: Epidemiologic, Clinical, Laboratory and Histopathological Features

Co-infection with hepatitis C virus (HCV) and human T-lymphotropic virus types 1 (HTLV-1) and 2 (HTLV-2) is expected, as these viruses share common infection routes. However, particular epidemiologic and clinical features of co-infected individuals have not been thoroughly evaluated. A cohort of 30 HCV/HTLV co-infected patients under follow-up at the Hospital das Clinicas, School of Medicine, University of Sao Paulo was compared to 55 HCV-infected subjects from the same institution, in regard to socio-demographic features, risk factors for viral acquisition, clinical and laboratory data, as well as liver histopathologic findings. Fischer's linear discriminant analysis was applied to define classification functions that better identified the combined effect of variables important for the discrimination of three study groups (HCV/HTLV-1, HCV/HTLV-2; HCV). The discriminating accuracy of the model was evaluated by cross-validation, using the leave-one-out technique. In bivariate analysis, no significant difference was found among groups in regard to socio-demographic features, smoking, and risk factors for viral acquisition, such as blood transfusion, tattooing, acupuncture or number of sexual partners. In contrast, alcohol consumption, use of intravenous drugs or inhaled cocaine and sexual partnership with an intravenous drug user were more frequent in the HCV/HTLV-2 group, whereas patients in the HCV group more often reported a sexual partner with hepatitis. Abdominal pain was also more prevalent in the HCV group. HCV/HTLV co-infected patients presented higher median platelet counts, whereas aminotransferase and GGT levels were higher among HCV-infected subjects. No significant difference was seen in liver histopathologic findings, though HCV liver disease-associated abnormalities, such as fibrosis and necroinflammatory activity, were found in patients from the three groups. Classification functions, defined by discriminant analysis, included as relevant variables: sex, age, intravenous drug use and sexual partner with hepatitis. Cross-validation yielded high (87.3%) and intermediate (66.7%) discriminating accuracies for the HCV and HCV/HTLV-2 functions, but showed the model was not useful to distinguish HCV/HTLV-1 co-infected patients.

P-065 High HTLV-1 Proviral Loads a Marker for HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis is also Detected in Patients with Infective Dermatitis Associated with HTLV-1

Background: Infective dermatitis associated with HTLV-1 (IDH) is a chronic and severe form of childhood dermatitis. It has recently been observed that 30% of IDH patients develop juvenile HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The replication of HTLV-I has already been reported to be greater in adult HAM/TSP patients than in asymptomatic HTLV-1 carriers. In this current paper, the proviral load of 28 children and adolescents with IDH unassociated with HAM/TSP was investigated. These results were compared with those found in 28 HTLV-1 carriers and 28 adult patients with HAM/TSP.

Material and Methods: The 28 IDH patients (18 female and 10 male) were followed-up at the dermatology clinic and neuropediatric clinic for 2 ½ years. The mean age of the IDH patients was 10.8 + 3.6 years. The HTLV-1 carriers were selected consecutively from blood bank donors, while patients with HAM/TSP were being followed up at the HTLV-1 clinic of the Federal University of Bahia's teaching hospital. The diagnosis was made in accordance with well-established criteria. The DNA was extracted from 10⁶ PBMCs using a phenol/chloroform procedure. HTLV-1 proviral load was determined using real-time TaqMan PCR assay. The study was approved by the Ethical Committee of the HUPES/UFBA.

Results: The proviral loads of IDH patients were much higher, with a median of 160.480 (range 26.382–965.825) copies per 10⁶ PBMCs compared to the HTLV-1 carriers which presented a median of 25.337 (range 19.00–133.251) copies per 10⁶ PBMCs (p < 0.0001). Moreover, the proviral loads of adult HAM/TSP patients were also higher than those found in carriers, with a median of 127.055 (range 35.00–691.862) copies per 10⁶ PBMCs (p = 0.0017). However, no statistically significant difference was found between the proviral loads of the IDH and the adult HAM/TSP patients (p = 0.1255). No statistically significant associations were observed between proviral loads and ages, duration of breastfeeding, duration of IDH and the status of activity of the disease.

Comments: As proviral load is associated with neurological disability, these data support that IDH patients are at a high risk of developing HAM/TSP.

P-066 High Level of Depression and Anxiety in Tropical Spastic Paraparesis - HTLV Myelopathy (TSP/HAM) Patients

Introduction: Psychiatric disorders appear to be common in patients with chronic diseases, as in Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). Thus, the depression and anxiety symptoms may contribute to the progressive disability and limitations in daily living activities.

Objective: The purpose this study was to verify the frequency of depression and anxiety in TSP/HAM and asymptomatic HTLV-1-infected carriers.

Material an Methods: A cross-sectional study including 100 patients (50 with TSP/HAM and 50 asymptomatic HTLV-I carriers – control group), using the following instruments: A neurological scale to measure the disability level (Osame's Disability Status Scale) and for anxiety and depression diagnosis, BDI and BAI were performed in the participants. All patients had HTLV-I diagnosis by serological and molecular approaches, in the Institute of Infectious Diseases Emílio Ribas Institute Clinic, from May 2008 to February 2009. Data were analyzed statistically by: frequencies, the X² test, and the Pearson correlation test. Data among groups were analyzed and correlated with functional and gravity aspects.

Results: The mean age of patients was 48.3 years, and 72% were females. Other socio-demographic and epidemiological data of the group - married persons (31%), white (40%), hand workers (40%) and Coinfections: HTLV-HCV (7%), HTLV-HIV (7%), HTLV/HIV/HCV (3%), HTLV-HCB (2%), and HTLV-HIV-HCB (1%). The frequency of moderate/severe depression was 40%/ 20% (60%) in the TSP/HAM group and 16%/10% (26%) in the control group (p = 0.0001). The frequency of moderate/severe anxiety was 34%/26% (60%) and 16%/12% (28%), respectively (p = 0.001). There was a significant correlation between anxiety and depression scores (p < 0.01 e r = 0.714). 50 of 100 patients presentend normal gait; 10 patients had partial incapacity to walk with out any help; 30 patients had partial incapacity to walk, needing hand support; and 10 patients had incapacity to walk.

Conclusions: This analysis confirms that depression and anxiety is common in persons with TSP/HAM and suggests the association with functional disability, while, the asymptomatic HTLV-I carriers, that depression and anxiety are associated with multiple factors, for example: economic problems, family problems and Coinfection. More studies are necessary to prove these associations effectively. Thus, evaluation and psychological monitoring can be of help for the treatment of TSP/HAM, in order to improve the quality of life of these patients.

Financial Support: CCD/SES-SP, FAPESP.

P-067 HTLV-1 Infection: Diagnosis and Proviral Load Level Among Asymptomatic Carriers and Patients with Associated Diseases in Reference Center in the Northeastern of Brazil

The infection for the HTLV-1 causes in the carriers, associated diseases who are related to the level of the HTLV-1 proviral load. The aim of the present study was to diagnose the infection for the HTLV-1 and to investigate the role of proviral load in asymptomatics carriers and patients with associated diseases and to investigate the relation of the proviral load of the HTLV-1 with the clinical manifestations and associated diseases. Serological and molecular tests for qualitative PCR for HTLV and proviral load expressed as a number of copies per 106 PBMCs of HTLV-1 had been carried through. Of the initial population 104 patients had been selected, being 101 HTLV-1 (97.1%) and three HTLV-2 (2.9%). Proviral load was quantified for the HTLV-1 and analyzed the association with the clinical manifestations and laboratories behavior in 101 patients. The results had shown higher frequency of the HTLV-1 in women and with age above of 50 years, being the majority of the patients of low level of study, low income. The clinical status of the patients was: 54.5% asymptomatics, symptomatic 7.9%, 5.9% with ATL, 12.9% with HAM/TSP, HAM/TSP-probable 3.9%, HAM/TSP-possible 14.9%. Co-infection with scabies 5%, 12.9% was observed of STD and strongyloidiasis in 6,9%. Proviral load level was higher than 10.000 copies/106 cells PBMC in 54,5% of patients, being the mean 2 times higher in women that in men. The mean HTLV-1 proviral load was highest in HAM/TSP patients. The mean HTLV-1 proviral load, when compared with asymptomatics, was 2.1-fold higher in HAM/TSP, 1,1-fold higher in probable-HAM/TSP, 1,4-fold higher in possible-HAM/TSP and 1,2-fold higher in ATL, and 1,1-fold in symptomatics. The results of this study showed that the patients presented high proviral load level, and no significant differences between HTLV-1 proviral load in sick people and asymptomatics.

P-068 HTLV-I Proviral Load Quantification Using Real Time PCR Methodology

HTLV-I genomic stability suggest that its amplification occurs priory by clonal expansion of the infected CD4+ T cell, rather than by reverse transcription and integration, for this reason it has been suggested that HTLV-I proviral load could be a disease evolution marker, although its relationship with viral pathogenesis has not been establish yet. In Argentina, HTLV-I infection has been documented in several regions, particularly in the northeast provinces.

The aim of our study was to develop a methodology for the quantification of HTLV-I proviral load from peripheral blood mononuclear cell (PBMC).

A real time quantitative PCR assay using SYBR GREEN technology was developed to quantify HTLV-I DNA in PBMCs obtained by two different methods of DNA isolation: 1) salting out and 2) proteinase K treatment (PK). MT-2 cell line was used as standard, and the pol gene was targeted for HTLV-I specific amplification. This cell line was considered to have 2.1 HTLV-I pol gene copies per cell. Albumin gene calibration curves where also constructed as external DNA input control. For PK standard curves (MT-2 PK) serial dilutions from 1 to 100.000 copies/reaction where construct for each gene and three replicates per experiments where amplified. For salting out DNA standards (MT-2 SO) we used dilutions ranging from 2-100,000 copies/reaction and duplicate for each dilution where run per experiment. Each experiment was repeated at least three times.

Calibration curves showed good correlation coefficient (R2 >0,99), with efficiencies above 90% and slopes ranged from −3.4 to −3.8 for both DNA extracted by the two methods and for both amplified genes. The linear range of the calibration curves for pol MT-2 PK was from 10 to 10,000 copies/reaction, and for MT-2 SO where from 20 to 20,000 copies/reaction. For the albumin gene the linearity range obtained for MT-2 PK was from 100 to 50,000 copies/reaction, and from 200 to 100,000 copies/reaction for MT-2 SO. The detection limit of the assay was 400 copies of HTLV-I/106 PBMC by both methods of DNA extraction. Coefficients of variation were determinated for each calibration curve dilution, and they resulted in less than 3% for both inter and intra assay reproducibility evaluation.

A quantitative real time PCR assay was developed for the measurement of HTLV-I proviral load, using two different DNA extraction methods. Both methods resulted reproducible with low variation in both intra and inter assay, with equal detection limit. This assay will allow us to study of the relationship of HTLV-I proviral load and viral pathogeny.

P-069 Identification and Evaluation of Anti-ATL Effects of the Edible Food-Derived NF-Kappab inhibitors (EFNIS)

The use of extracts from plant or edible foods, to alleviate inflammatory diseases such as asthma or dermatitis, has been a popular folk remedy for centuries in Japan and other eastern Asian countries. To assess the efficacy of such ingredients, which we call as the Edible Food-derived NF-?B Inhibitors (EFNIs) isolated mainly from Japanese mushrooms or citruses, we took advantages of our molecular cellular biological techniques which enable to digitalize the activities of the immunologic or stress responsive transcription factor, nuclear factor kappa B (NF-?B).

Transfections of oncogenes or triggering of proinflammatory cytokines to cells has been employed for induction of NF-?B hyper-activation and we narrowed several EFNIs down from 120 samples with the ability of their suppressive action against NF-?B. Among those selected eight EFNIs, we selected a citrus peel extracts J (named as CJEE) for further investigation. CJEE was fractionated into hydrophilic (CJEE-WS) and hydrophobic (CJEE-OS) portions. Although both of them showed suppressive effects, CJEE-OS was 10 fold more active than CJEE-WS against NF-?B. CJEE induced the degradation of oncoprotein Tax in a dose dependent manner and induced growth arrest and apoptosis to ATL cells. Oral administration of CJEE to mice genetically engineered with NF-?B-SEAP gene, which let cells to secretes the human placenta-derived alkaline phosphatase (SEAP) into sera in a NF-?B-responsive element dependent manner, modulated their total SEAP values in sera and reduced the some of skin inflammation symptoms developed spontaneously among these Tg-mice. Detailed mechanisms will be discussed.

P-070 Immunological Biomarkers Related to Dermatological Lesions in Brazilian Individuals with HTLV-1 infection

The study of immunological mechanisms associated with the presence of dermatological lesions in individuals infected with HTLV-1 transcends the understanding of the establishment of dermatological manifestations associated to this infection, since this approach is able to identify prognostic biomarkers as well as to propose adequate parameters for laboratorial monitoring of clinical status after therapeutic treatment. In order to search biomarkers related to establishment/maintenance of cutaneous lesions associated with HTLV-1 infection, a cross-sectional study included 51 subjects residing in the southeast region of Brazil was performed. The objective of this current study was to evaluate, by flow cytometry, phenotypic aspects of circulating leucocytes subpopulations from four groups of individuals as follow: individuals infected by HTLV-1 with or without cutaneous lesions (HTLV+ L+ n = 18 or HTLV+ L− n = 07, respectively), besides of volunteers with or without cutaneous lesions with coincident etiology (CT L+ n = 13 or CT L− n = 13, respectively). The data demonstrated a lower percentage of B-lymphocytes parallel to elevation of T/B-lymphocytes ratio in the HTLV+ L+ group as compared to CT L+ and HTLV+ L− groups. The analysis of the frequency of activated HLA-DR+ T-lymphocytes revealed higher percentage of this marker in HTLV+ L+ group in comparison to CT L− mainly owing to elevation in the percentage of both activated CD4+ and CD8+ T-lymphocytes. Taken together, the data suggest that the previous biomarkers proposed by our group in Brito-Melo et al. (2002) as possible prognostic biomarker HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) as percentage of B-lymphocytes, analysis of T/B-lynmphocyte ratio and percentage of CD8+HLA-DR+/CD8+ cells could be also used in monitoring the cutaneous manifestations associated with HTLV-1 infection. An additional biomarker identified was percentage of HLA-DR+CD4+ T-lymphocytes. Complementary studies aim to characterize a preferential association between immune response and types of cutaneous lesions associated with HTLV-1 infection proceeding from infectious (INF) or autoimmune (AI) nature was carried out. The dermatological manifestations more common in INF group were dermatophytosis while in AI group were ichthyosis and vitiligo. The data displayed that INF-HTLV+L+ group has increased levels of activated CD4+ T-lymphocytes as compared to AI-HTLV+ L+group. In contrast, AI-HTLV-1+L+ group has increased frequency of B-lymphocytes. In conclusion, the search for changes in the phenotypic features of peripheral blood circulating leucocytes subsets has proved to be a very useful model to point-out putative biomarkers to monitoring disease progression. This approach has also contributed to identification of profitable selective biomarkers for clinical studies regarding dermatological manifestations associated with HTLV-1 infection.

P-072 Oral Administration of Polysaccharide Fucoidan Can Induce a Significant Decrease of Proviral DNA Load in Patients with HAM/TSP

HTLV-1 is an exogenous human retrovirus that causes two different diseases; HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia (ATL). Although the most HTLV-1 infected individuals remain lifelong asymptomatic carriers (AC), the higher virus load in HTLV-1 infected individuals increases the risk for developing HAM/TSP and ATL. In addition, the higher proviral load is associated with clinical progression of HAM/TSP. Therefore, determining the agents to reduce the HTLV-1 infected cells is critical for the prevention, and for the treatment of HTLV-1 associated diseases.

Fucoidan is a sulfated polysaccharide contained in various species of the brown seaweed. Recently, Mori et al. observed in vitro that the fucoidan extracted from Cladosiphon okamuranus Tokida significantly inhibited the growth of peripheral blood mononuclear cells (PBMCs) from ATL patients and HTLV-1-infected T-cell lines, but not that of normal PBMCs.

To evaluate the therapeutic potential of the fucoidan for patients with HAM/TSP, fucoidan was given orally in a dose of 6g once daily for 6–12 months in 15 HAM/TSP patients. During the treatment period, no exacerbation has been observed. Tow HAM/TSP patients showed diarrhea which improved immediately after stopping the fucoidan. We examined the viral DNA load in PBMCs from 13 HAM/TSP patients during the treatment using quantitative PCR method. Oral administration of the fucoidan resulted in about 40% decrease of HTLV-1 infected cells. The serum level of soluble IL-2 receptor showed no significant alteration. Next, to clarify the effect of fucoidan on the immune system in the patients with HAM/TSP, we measured the frequencies of the dendritic cells, natural killer cells, invariant natural killer T cells and HTLV-1 specific cytotoxic T lymphocytes before and after the treatment using the flow cytometory technique. Interestingly, fucoidan administration did not change their frequencies significantly. Therefore, oral administration of fucoidan might be able to decrease virus load without the activation of immune system in the host. Our results indicate that fucoidan is not only a promising preventive agent for AC, but also a potential therapeutic agent for the patients with HAM/TSP.

P-074 Clinical Manifestations in a Large Group of HTLV-1 Infected Patients in Northeastern Brazil

Introduction: HTLV-I is considered a low pathogenic retrovirus, since a small proportion of patients progress to HTLV-1 associated myelopathy (HAM/TSP) and adult T cell leukemia (ATLL). However, infected individuals may have other clinical manifestations. The objective of this study was to determine the prevalence of clinical manifestations in HTLV-1 infected subjects at the time of admission in a HTLV clinic.

Methods: A cross-sectional study, with consecutive HTLV-I patients admitted to HTLV-1 clinic, since 2004. Socio-demographic variables, urinary distress inventory, erectile dysfunction questionnaires, neurologic scales (EDSS and OSAME), oral and rheumatological examination were collected for all patients. HTLV proviral load were done by Real Time PCR. Statistical analysis was performed on SPSS 13 software.

Results: 400 HTLV infected subjects were admitted between May/2004 to April/2009. 58.7% were female. The mean age was 46,4 (±13) and 60% were referred by blood banks. After neurologic scales evaluation, 83 (20,7%) patients were classified as HAM/TSP, 255 (63.8%) as asymptomatic (EDSS = 0) and 62 (15.5%) as oligosymptomatic neurologic or urologic symptoms (EDSS = 1.0–1.5). Hyperactive bladder was present in 114 (28,5%) patients and erectile dysfunction in 74 (44,5%) of male patients. About 196 (49%) of patients complained of joint pain and 46(11,5%) had synovitis. Hyperactive bladder, erectile dysfunction, joint pain and synovitis were more frequent in patients with EDSS = 1.0–1.5 than in patients with EDSS = 0 (p < 0.005). The mean age and proportion of female gender were also higher. The mean proviral load were 80.479 copies/10⁶PBMCs, 139.056 copies/10⁶PBMCs and 153.252 copies/10⁶PBMCs in the asymptomatic, oligosymptomatic and HAM patients, respectively.

Conclusions: A large proportion of HTLV patients presented with neurological and urological abnormalities, despite not fulfilling HAM/TSP criteria. We nominated this group as oligosymptomatic myelopathy patients and verified that this group has demographic and clinical characteristics similar to HAM/TSP patients. A cohort study is ongoing to test the hypothesis that oligosymptomatic individuals have a high risk to progress to more severe neurologic symptoms and are at high risk to develop HAM/TSP

P-076 HLA Class I Binding of HBZ is Associated with Reduced HTLV-I Proviral Load and HAM/TSP Prevalence

Aim: to understand what constitutes a protective HLA class I genotype

We hypothesised that a HTLV-I-infected individual's proviral load and HAM/TSP risk was determined by the epitope binding properties of their HLA class I alleles.

We tested this hypothesis in a large (N = 432) cohort of HTLV-I-infected individuals using experimentally-verified epitope prediction methods. We showed that:

Class I alleles previously associated with reduced proviral load and HAM/TSP prevalence (HLA-A*02 and –Cw*08) were predicted to bind epitopes from the viral protein HBZ significantly more strongly than an allele associated with increased proviral load and HAM/TSP prevalence (HLA-B*54).

We conclude that the targeting of HBZ by HLA class I alleles is beneficial in terms of HAM/TSP risk and proviral load. This strongly suggests that a HBZ-specific CD8+ T cell response plays a significant role in the control of HTLV-I infection.

P-077 HTLV-1 Infection Might be Highly Endemic in Rural Communities of the Southern Andes of Peru

Background: In Peru, HTLV-1 infection is estimated to affect 1–2% of the general population. Our Institute in Lima functions unofficially as a referral centre. At this Institute, >1900 people have been diagnosed with HTLV-1 over the past 20 years. Because many of these HTLV-1-infected people were born in the southern Andes, we proposed to evaluate the HTLV-1 frequency in that region

Methods: We conducted a cross-sectional study in six communities in Ayacucho, a region in the southern Andes. For the selection of study sites, we considered (i) the predominating birth places among the participants of the HTLV-1 cohort at our Institute; and (ii) communities with high frequencies of not polio-related lower limb disability, according to the 1993 National Census. Because this disability was more frequent among women than among men in these communities, we assumed that there could be cases of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We visited Pampa Cangallo (province Cangallo); Vilcashuaman (province Vilcashuaman); and Cochani, Acos, Pinahua and Racchi (province Parinacochas). In each community, volunteers >12 years were included. Clinical and epidemiological data was collected, along with a blood sample. Lab tests included two ELISA (Murex and Ortho), and a serological confirmatory test if at least one ELISA was positive. People were defined to be of Andean origin if both parents were born in the Andes and spoke Quechua. The study protocol was approved by the University Institutional Review Board.

Results: The six communities are located at >3000 m above sea level; are mainly rural; and Quechua is spoken by =85% of the population. We included 397 participants: 164 out of 6909 registered inhabitants (2%) in Pampa Cangallo; 154/7323 (2%) in Vilcashuaman; 35/386 (9%) in Cochani; 28/321 (9%) in Acos; 14/272 (5%) in Pinahua and 2/47 (4%) in Racchi. The median age of the participants was 41 years (interquartile range 31–57); 69% were women; and 99% were of Andean origin. HTLV-1 was diagnosed in 11 people: 0/164 in Pampa Cangallo; 3/154 (2%) in VIlcashuaman; 3/35 (9%) in Cochani; 1/28 (4%) in Acos; 3/14 (21%) in Pinahua; and 1/2 in Racchi. There were no cases of HTLV-2. All the HTLV-1-positive participants were part of different families; were born in Ayacucho and were of Andean origin. The age of the infected people ranged from 29–87 years (median 56); 10/11 were women. Ten were apparently healthy, and one woman was diagnosed with HAM/TSP at the time of the interview. Three of 11 had a relative with lower limb impairment compatible with HAM/TSP.

Conclusion: The fact that HTLV-1 infection was present in five out of six communities suggests that HTLV-1 could be highly endemic in the southern Andes. Because the communities with the highest frequency of HTLV-1 were small (<400 inhabitants), we hypothesize that relative isolation combined with other factors such as breastfeeding customs or genetic susceptibility may sustain endemicity.

P-079 HTLV-1 Transmission Among Sexual Contacts and Proviral Load

HTLV-1 infection is associated with sexual transmission and detection of infection in sex partners is important as part of epidemiological investigation for planning preventive measures. The objective is to study the heterosexual transmission in couples in which one of the partners was detected HTLV-1 positive.

Methods: 18 partners of infected patients were tested for screening the infection for HTLV-1/2 with Elisa and the proviral load was performed.

Results: among 18 couples, 11(61%) were male-female positive, 5(28%) male positive-female negative, 2(11%) female positive-male negative; the relationships had a mean of 22 years (40 to 6 years). The proviral load among the positive couples male-female positive had a mean 22 902 copies(71 252 to 1860), male positive-female negative mean 29 940 copies(49 297 to 1330), female positive only and male negative, were 311 425 and 1313copies, respectively. Among the couples male-female positive, two had the partner with ATLL, and one with HAM/TSP. The highest proviral load was detected in a couple where only the female partner was positive (311 425 copies) and had 26 years of relationship, indicating that the direction of transmission from women is less efficient.

P-080 HTLV-I-Associated Myelopathy Manifested after Renal Transplantation

Human T-cell lymphotropic virus (HTLV) antibody screening is not mandatory for organ transplantation. Few cases of HTLV disease after organ transplantation have been reported, mainly adult T cell leukemia. The rate of HTLV disease development is low and the latency time is a few decades. However, the possible influence of immunosuppression in transplant patient on disease development is unclear.

Objective: to describe a case of a patient who developed HTLV associated Myelopathy (HAM) four years after to receive kidney transplantation.

Case report: a male patient with 33 years old had chronic kidney failure of unclear cause and was on dialysis in the last years. He was previously evaluated for infectious illnesses with negative screening serologies before kidney transplantation. He received a kidney graft from his wife, who wasn't tested for HTLV. The transplant was successful and blood transfusion wasn't necessary. He was on immunosuppressive therapy and approximately 4 years after kidney transplantation developed progressing symmetric myelopathy, with quickly evolution and needing of wheel chair in less than 6 months of the beginning of symptoms. Urinary incontinence and severe erectile dysfunction also developed. Serology and liquor were positive for HTLV, with western blot reagent for HTLV-I. HTLV Proviral load were 21.643, 50 copies/10⁶ mononuclear cells .. Symptomatic treatment was instituted, with improvement of the quality of life. After one year of installed myelopathy, the patient had only improvement of some symptoms like erectile dysfunction and neurogenic bladder . In all this time kidney graft remained with normal function. After the discovery of the etiology, his wife was tested for HTLV, and the test was positive for HTLV-I. She remains an asymptomatic carrier.

Conclusion: We described a clinical case of a patient who developed HAM soon after a solid transplantation. Human T-cell lymphotropic virus antibody screening should be recommended uniformly before transplantation in areas with high prevalence of HTLV.

P-082 Human T-Cell Lymphotropic Virus Type -I: Prevalence and Incidence of Infection, a Systematic Review

Data on the epidemiology of HTLV infections is based on the study of a variety of population types including blood donors, hospital populations, injecting drug users and pregnant women, each with considerable potential for bias. Reporting is not uniformly distributed globally.

Previously, available data on the global epidemiology of HTLV-1 infection has been summarised non-systematically and without a focus on general populations. To assess the implications of HTLV-1 infection for healthcare systems, it is essential to know its prevalence at a given point in time. We conducted a systematic review of all published reports of the incidence and prevalence of HTLV-1 infection in the general population, aimed at informing on the current status on the prevalence of HTLV-1 infection worldwide.

Study Design and Setting: Three bibliographic databases were searched. The same online databases were searched for the Journal of Acquired Immune Deficiency Syndrome and Human Retrovirology for abstracts from conferences of the International Retrovirology Association. It is in this journal that a number of previously unpublished papers were subsequently published. Experts in the field were contacted. Assessment for methodological quality of the reviewed studies was according to pre-set criteria.

Results: Of the 59 relevant papers 17 cross sectional studies met the inclusion criteria, none addressed incidence. These studies estimated prevalence of HTLV-1 in Japan, Latin America and the Caribbean Islands with very limited data from Africa, Asia, Europe and North America. Where HTLV-1 was detected the highest prevalence was reported in Japan 36.40% (95% CI 29.90–42.80) and lowest in Latin America where the rates were between 0.24% (95% CI 0.03–0.51) and 1.00% (95% CI 0.378–1.59).

Conclusion: Few data are available from general populations to inform the epidemiology of HTLV infections globally. Consequently, assessment of the public health impact and changing epidemiology is limited. Regional differences and inadequate standardisation of assays and misleading laboratory results from the use of poorly specific assays in some earlier studies may account for the significant difference in the prevalence estimates noted in this review. The findings of this review underline the importance for further conduct of large epidemiological studies on this very important public health concern. This will in turn allow for accurate assessment of the public health impact of these viruses.

P-083 Molecular Characterization of HTLV-1/2 among Blood Donors in Belem, State of Para: First Description of HTLV-2B Subtype in the Amazon Region

The present study describes the molecular characterization of HTLV-1 and HTLV-2 infections among 79 blood donors seropositives attended at the Foundation Center for Hematology and Hemotherapy Pará (HEMOPA). Peripheral blood mononuclear cell DNA of the samples were subjected to nested PCR and restriction fragment-length polymorphism analysis of the pX and env regions, and nucleotide sequencing of the 5’LTR region. It was observed a higher prevalence of HTLV-1 (71%) as compared to HTLV-2 (29%). Phylogenetic analysis, using the Neighbor-Joining method, clearly demonstrated samples clustered within the HTLV-2c molecular subtype. HTLV-1 samples were classified as belonging to the subtype Cosmopolitan, Transcontinental subgroup. Furthermore, the RFLP and phylogenetics analysis described, for the first time, a sample infected by HTLV-2b in the Brazilian Amazon region, emphasizing the need for ongoing molecular studies which can provide a better understanding on HTLV epidemiology in the Brazilian Amazon region.

P-084 Molecular Evidence of HTLV-1 Horizontal Transmission and Development of Adult T-Cell Leukemia/Lymphoma in 21-Year-Old Man

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-lymphotropic virus type 1 (HTLV-1) infection generally many decades after the infection. It is believed that carriers who have acquired infection early in life are at greatest risk of ATLL, and it occurs only in a few percent of infected population usually after 40–60 years of infection. We report a case of ATLL in a 21-year-old patient that was horizontally infected after two years of relationship with an HTLV-1-asymptomatic young woman. This 31-year-old woman was infected by her first husband, a 41-year-old man with HTLV-1-associated myelopathy, and their 11-year-old son that was infected through breastfeeding developed infectious dermatitis. The ATLL patient presents skin lesions and the diagnosis of cutaneous T-cell lymphoma was done by histopathological analysis. Furthermore, mother and siblings from the ATLL patient were negative for anti-HTLV-1 antibodies. HTLV-1 molecular study was performed by nested-PCR targeting the 5′- and 3′-LTRs and tax gene. All amplified products were sequenced on both strands. The sequences were edited and multiple alignments with LTR from the four individuals were done using Clustal-W algorithm. Neighbor-joining tree was obtained using PAUP program and the reliability of the tree was determined by 1000 bootstrap replicates. All the individuals, except the ATLL patient, were positive for all the HTLV-1 genomic regions analyzed. The ATLL patient presented a defective provirus lacking 300bp from the first part of 5′-LTR. The lack of the 5′-LTR, which characterizes a type 2 defective provirus, has been frequently observed in ATLL. The genetic relationship of sequences was determined by the comparison of a 650bp from 3′-LTR. The four segments had identical sequences. The genetic analysis of this 650bp fragment demonstrated that the isolates belonged to the Transcontinental subgroup A of the Cosmopolitan subtype. High sequence identities are typically seen in both vertical and horizontal linked transmission cases of HTLV-1 what was determined among these individuals. Moreover, they have a strong epidemiologic link. A previous report of a 21-year-old man with ATLL was done in Japan but without evidence of horizontal transmission. Our results indicate that the rapid development of ATLL did not depend on virus genetic characteristics. On the other hand, it has been shown in literature that the risk of HTLV-1 transmission increases with the number of shared types of HLA class I between individuals. Surprisingly, the ATLL patient and the woman presents similarities in HLA-A and B loci. This suggests that HLA class I matching could favor woman-man transmission. Indeed, other genetic background of the four individuals are under analysis.

P-085 Prevalence and Risk Factors of HTLV-1/2 in a Cohort of Patients with HIV-1 infection in Two Cities from the State of São Paulo, Brazil

Introduction: The objective of this study was to define the prevalence and risk factors for HTLV-1/2 in HIV-1 infected patients in the state of São Paulo, Brazil.

Methods: We evaluated 319 HIV-1 infected individuals attending specializated clinics in two cities, Ribeirão Preto and São Paulo. Patients were HTLV-1/HTLV-2 interviewed and tested for antibodies against HTLV-1/2 (ORTHO Ab-Capture enzyme immunoassay). Direct PCR DNA sequencing of HTLV-2 tax and HTLV-1/2 long terminal repeat regions were performed on HTLV seropositive subjects for differentiation and subtyping determination.

Results: The overall prevalence of anti-HTLV-1/2 antibodies was 7,5% (24/319; 95% CI: 5.2–11.5). HTLV-1/2 infection was associated with a history of intravenous drug injections and antibodies to hepatitis C virus (p < 0.001), but not with age (p = 0.2), sex (p = 0.9), sexual behaviours or serological markers for sexual transmitted diseases (anti-T. pallidum, anti-HHV8, or anti-HBV antibodies) (p > 0.05). HTLV DNA was detected in 13 of 24 samples, of them, 12 were characterized as HTLV subtype 2c and one as HTLV subtype 1a.

Conclusion: Of the 12 HTLV-2 samples, 7 were injecting drug users indicating this via as important risk factor for HTLV-2 transmission among our HIV-1 infected population.

P-086 Prevalence of Crossed Breast Feeding in Pregnant Women During Prenatal Care, São Luis - Maranhão, 2008

Introduction: Around 1980 important retrovirus were discovered, HTLV, a lymphotrofic human virus, kinds 1 and 2, and HIV- human immunodeficiency virus – both transmissible through breast feeding. The reality kept by the discourse of many pregnant women during the data survey of the research about HTLV prevalence in pregnant women in São Luis made us aware of the need to evaluate the prevalence of the practice of active and passive crossed breast feeding.

Objectives: To identify the profile of pregnant women, and the frequency of active and passive crossed breast feeding among the pregnant women assisted during prenatal care at three public services in São Luis/MA.

Material and Methods: Transversal study executed between Feb/11 and Dec/03/2008 with 2044 pregnant women in three prenatal care public services - São Luis/MA. The patients were guided about the objective of the study and included after signing a free and clarifying consent term, and after filling in a questionnaire. The pregnant women who took part of it were between 18 and 45 years, without psychiatric disturbances background, high blood pressure, nephropathy, diabetes and others that characterize the need of specialized prenatal care. The sample was considered infinite, calculated in 2041, test power of 95%, absolute mistake of 5%; Stata 9.0 program was used; test c²; was executed and p < 0,05 (95%) was accepted as the limit for significance. The Project was approved by CEP/UFMA (Opinion N°568/2007).

Results: The average age was 25.4 years old, 70.25% were breast fed by their mothers. 18.28% were breast fed by their mother and other people. There was no statistical significance by test c² between passive and active crossed breast feeding, and between age range and active crossed breast feeding. The place of birth showed statistical significance in relation to active crossed breast feeding, 12.08% of the pregnant women born in São Luis and 20.58% from inland towns declared active crossed breast feeding, what could be interpreted as an important cultural context on the maintenance of this practice.

Conclusions: It is important that the health professionals that act directly on the guidance about breast feeding consider the crossed breastfeeding as a frequent practice, and the risk of transmission of HIV and HTLV virus through breast feeding.

Key words: human T-lymphotrofic virus; pregnant women; breast feeding

P-088 Immune Response Influence the Disease Expression Associated with HTLV-1 Infection

Background: Central nervous system damage observed in HAM/TSP is related to immunological abnormalities characterized by increased production of pro-inflammatory cytokines, increased number of T cells expressing Tax, high frequency of Tax-specific CD8 T cells expressing TNF-α and IFN-γ and also a high proviral load.

Objective: The aim of this study is to see whether host immune response are involved in the pathogenesis of HAM/TSP and that high production of pro-inflammatory cytokines correlate with different disease expression in HTLV-1 infection.

Methods: Three groups participated in this study: HTLV-1 asymptomatic carriers, HTLV-1 carrier with neurogenic bladder and HAM/TSP patients. Studies were performed on peripheral blood mononuclear cells (PBMC) and the cytokine profile in supernatants of unstimulated culture was determined by ELISA. The phenotypic analysis and the frequency of CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-17 and IL-10 were determined by flow cytometry and IL-17 mRNA expression was evaluated ex vivo and determined by PCR. Proviral load was determined by real time PCR.

Results: IFN-γ, TNF-α and IL-10 levels were significantly higher in patients with neurogenic bladder and HAM/TSP than in HTLV-1 asymptomatic carriers (p < 0.05). There was a significant predominance of CD8 T cells over CD4 T cells producing cytokines in the three evaluated groups. We observed a high frequency of CD8 T cells expressing IFN-γ, TNF-α and IL-10 in patients with myelopathy compared to HTLV-1 asymptomatic carrier and patients with neurogenic bladder. Cells expressing IL-17 were observed in all groups and the main source of this cytokine was CD8 T cells. There was a tendency of CD8 T cells from patients with neurogenic bladder and HAM/TSP to produce more IL-17 than HTLV-1 carriers. IL-17 mRNA expression did not show differences between HTLV-1 infected groups although there was a tendency of HTLV-1 carrier to show a lowest IL-17 mRNA expression than patients with neurogenic bladder or HAM/TSP. Proviral load was higher in patients with neurogenic bladder and in HAM/TSP as compared to HTLV-1 asymptomatic carriers (p < 0.05). No difference between proviral load in patients with HAM/TSP and in patients with neurogenic bladder was observed.

Conclusion: Immunological abnormalities associated to HAM/TSP were more frequently observed in patients with neurogenic bladder than in HTLV-1 asymptomatic, indicating that immunological response play a role in different disease expression in HTLV-1 infection.

Support: CNPq and FAPESB

P-089 Increased All-Cause and Cancer Mortality Associated with HTLV-II Infection

Background: HTLV-I and -II cause chronic human retroviral infections, but few studies have examined the impact of either virus on survival among otherwise healthy individuals. Although some studies have found increased mortality associated with HTLV-I, there is little data on HTLV-II and mortality.

Methods: We analyzed all-cause and cancer mortality in a prospective cohort of 155 HTLV-I, 387 HTLV-II and 799 seronegative subjects. Vital status was ascertained using death certificates, the U.S. Social Security Death Index, or family report, and causes of death were grouped into 10 categories. We calculated hazard ratios (HRs) and 95% confidence intervals (CIs) using Cox proportional hazards models, adjusting for the following possible confounding variables: age, gender, race/ethnicity, educational attainment, income, blood center, donation type, body mass index, cigarette and alcohol consumption, hepatitis C virus status and injection drug use.

Results: Over a mean follow-up of approximately 12 years, there were 104 deaths in the cohort, including 22 HTLV-I, 41 HTLV-II and 41 HTLV-seronegative deaths. Cancer was the predominant cause of death in all three groups, resulting in 8 HTLV-I, 17 HTLV-II and 15 HTLV-seronegative deaths. HTLV-I status was not associated with increased all-cause mortality, though there was a positive trend (HR 1.7, 95% CI 0.9–3.4). After adjustment for confounding, HTLV-II status was associated with increased all-cause mortality (HR 2.5, 95% CI 1.4–4.3) and cancer mortality (HR 3.9, 95% CI 1.6–9.2). No particular cause of cancer death was overrepresented among HTLV-II subjects.

Conclusions: HTLV-II was strongly associated with increased all-cause and cancer mortality, after controlling for race/ethnicity, socioeconomic status, injection drug use and smoking. An excess of HTLV-II cancer deaths could reflect impaired immune response to tumor cells or worse cancer screening or treatment in HTLV-II infected individuals.

Supported by National Heart, Lung and Blood Institute grants 2R01-HL-62235 and K24-HL-75036 to Dr. Murphy.

P-091 Mapks and CD8+CD28− Regulatory T Cells May Change the Lymphocyte Response to Antigens and Glucocorticoids in Patients with HTLV-I Associated Myelophathy (HAM)

Lymphocytes of HTLV-I-infected patients were found tolerant to mitogenic stimuli as well as glucocorticoid treatment. These data suggest that common signaling events to these two phenomena. The underlying mechanisms of these phenomena may include changes in cellular composition, cytokines millieu and the differential activation of mitogen-activated protein kinases (MAPKs). ERK MAPK could be implicated with changes in cellular proliferation and glucocorticoid resistance, while p38 MAPK may induce anergy and increased glucocorticoid sensitivity. We investigated the role of (i) p38 and ERK MAPKs, (ii) lymphocyte subpopulations, (iii) and cytokines implicated in antigen or glucocorticoid-induced immunomodulation. Twenty-one asymptomatic carriers (AC), 19 patients with HTLV-I-associated myelophathy/ tropical spastic paraparesis (HAM/TSP) and 21 healthy subjects took part in this study. Peripheral blood mononuclear cells were isolated and cultured in vitro to assess lymphocyte proliferation and sensitivity to glucocorticoid dexamethasone. The expression of phospho-MAPKs, lymphocyte subsets and cytokines were assessed by flow cytometry. Patients with HAM/TSP had a higher p38/ERK ratio (p < 0.05) associated with a reduced response to mitogens (phytohaemagglutinin or PMA plus ionomycin) (p < 0.001) and higher sensitivity to dexamethasone (p < 0.05). HAM/TSP patients presented higher levels of activated T cells and CD8⁺CD28⁻ regulatory T cells, being negatively related to the mitogenic response. These data suggest that multiple underlying mechanisms could be involved with HTLV-I-related changes in cellular response to mitogens and glucocorticoids.

P-092 Microarrays Analysis of Gene Expression in CD4+ T Cells Isolated from HTLV-1 Infected Individuals

Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that infects about 20 million people worldwide and causes primarily two distinct clinical pathologies: adult T cell lymphoma/leukaemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The mechanisms involved in HTLV-1 related diseases are not well elucidated, but host factors are an essential part in TSP/HAM pathogenesis, like effectiveness of immune response. CD4⁺ T cells are the predominant subset of cell infected with HTLV-1 and are also important as an effector cell against the infection. We used a microarray platform composed by 44,000 human genes to performed the gene expression profile of CD4⁺ T cells isolated by immunomagnetic separation from four assymptomatic (HAC) and four HAM/TSP individuals. The analysis revealed around 2,682 genes differentially expressed. Of them, 956 genes were up-regulated in HAM/TSP group while 1,726 genes were overexpressed in HAC group. The expression profile demonstrated a high number of genes associated with T cell receptor signaling pathway activation like VAV1, ZAP70, LCK in HAM/TSP group. These facts suggest that there is a strong CD4⁺ T cell activation in HAM/TSP individuals. These results will enable the understanding of cellular and molecular events that occur during the course of infection in CD4⁺ T cells and will give insights to elucidate host factors contributing to the genesis of HAM/TSP.

Key words: HTLV-1, CD4⁺ T cells, gene expression, microarrays, HAM/TSP

Supported: CTC/FUNDHERP

P-093 Morphological and Functional influences of the Contact between Thymic Epithelial Cells and HTLV-1 Infected T Lymphocytes

Human T Lymphotropic Vírus type 1 (HTLV-1) can lead to development of lymphoma and leukemia or still leads to commitment of nervous and muscular systems, because of the inflammatory phenomena that can be risen. Although it is a virus with T lymphocyte tropism, there are only a few number of studies of HTLV directly related to the thymus and to the infection of cells from the immune system, except, of course, the lymphocytes. Considering this lack of information about what occurs during the long time of viral latency, we intend to investigate if thymic epithelial cells (TEC) can be a target for HTLV-1 infection, making possible that thymus becomes a reservoir for the virus. Specifically, we want to study if TEC suffer alterations after the contact with HTLV-infected cells and if the virus can be transmitted to TEC. Experiments confirm that our T cell line (C91PL) is infected by HTLV-1. Our results also show that infected lymphocytes adhere more to TEC than non-infected lymphocytes and that there is syncytium formation between infected lymphocytes and TEC, which, accordingly with the literature, can represent one step for infection. Moreover, TEC and infected lymphocytes express Neuropilin1, one of the receptors involved in HTLV transmission. In addiction, migration experiments indicate that infected lymphocytes tend to migrate more in response to TEC and their newly synthesized products, but not only to their supernatant.

P-094 Natural STLV-1 Infection in Non-Human Primate: Implication for Human Infection and Associated Diseases Progression

STLV-1 naturally infects Old World monkeys and shares virological, immunological, and molecular features with HTLV-1. Thus, natural STLV-1 infection could provide an invaluable in vivo model of studying the pathogenesis of HTLV-1. We used mandrills (Mandrillus sphinx) naturally infected with STLV-1 as a model for evaluating the influence of natural STLV-1 infection on the dynamics and evolution of the immune system during chronic infection. We first showed that the STLV-1 proviral load increased significantly with the duration of infection. We also found that the percentage of CD4+ T cells co-expressing the activation marker HLA-DR and the mean percentage of CD25+ in CD4+ and CD8+ T cells were significantly higher in infected than in uninfected animals. STLV-1 proviral load was correlated positively with T-cell activation. Furthermore, the proliferation marker Ki67 was higher in the STLV-1 infected monkeys compared to other group. Indeed, recently, the role of co-infection with various pathogens was associated with the development of HTLV-1 associated diseases. Thus, dual natural infection of mandrills with SIV and STLV (mandrills are naturally infected also with SIV) could influence STLV-1 viral dynamics, immune system disruption and disease progression. Therefore, we evaluated the role of SIV/STLV co-infection in mandrills and the outcome on the immune response and the development of the associated diseases. Our results showed that the lowest number of CD4+ T cells was found in the co-infected group. Moreover, in the co-infected monkeys, the percentages of activated CD4+ and CD8+ cells were significantly much higher than in the other group. Interestingly, two co-infected animals with the highest STLV-1 proviral loads and high percentages of activated CD4+ and CD8+ cells developed HTLV-1 like associated diseases (infective dermatitis and scabies). These findings suggest that co-infection with SIV might play a role in the disequilibrium of the immune system which leads to high STLV-1 proviral load and eventually to a disease progression. To confirm this hypothesis, we induced experimentally immunosuppression in the STLV-1 infected monkeys. We showed that, during immunosuppression, the percentages of CD8+ expressing HLA-DR and of CD4+ T cells expressing the proliferation marker Ki67 decreased significantly, although the percentage of CD8+ T cells expressing Ki67 increased significantly by the end of treatment. Interestingly, the STLV-1 proviral load increased significantly after immunosuppression in the monkey with the highest load. These findings allow us to reconsider the impact of multiple retroviral infections in NHP demonstrating that naturally infected mandrills could help us to understand the relations between host and virus for the disease progression. Such studies would provide important information for the development of immune-based therapeutic strategies.

P-095 Evaluation of IGG Anti-Tax Reactivity and Proviral Load in Groups of HTLV-1 Infected Individuals and Control

The Human T-lymphotropic virus (HTLV-1) is the etiologic agent of various syndromes as HAM/TSP, ATLL and rheumatologic diseases. The interaction established between the host and the virus directs the clinical status of infected patient for asymptomatic carrier or induce the syndromes related to HTLV-1. Many factors contribute for the development of the diseases, like a vigorous immunologic response, with the presence of the Tax - specific cytotoxic T lymphocytes (CTL), T regulatory cells and anti-Tax antibodies. However, the clinical status of HTLV-1 infected people remains needing novel pathogenesis studies and approaches. The objective of our study was to assess some aspects of the immunologic response (IgG anti-Tax reactivity) and the DNA proviral load of asymptomatic individuals (AS – without signs and symptoms and AS*- with signs and symptoms, however without HAM/TSP defined criteria), with HAM/TSP and rheumatologic diseases. We development an infection/ transfection system using Vaccinia virus and plasmid pLW44/Tax for Tax protein expression in Vero cells and use as solid support in Western assays and immunofluorescence by flow cytometry. The DNA proviral load was evaluated in PBMC of 72 HTLV-1 infected individuals by kinetics PCR. The results of Western assays showed an IgG anti-Tax reactivity in 50% of HTLV-1 infected patients (asymptomatic and HAM/TSP). In the experiments of immunofluorescence by flow cytometry were analyzed the IgG anti-Tax reactivity in 81 patients (nine HTLV-1 non-infected individuals - NI, 11 HTLV-1 positive individuals, but without clinics signals and symptoms – AS, 24 HTLV-1 positives individuals with some signals and/or symptoms, but that don't filling the criteria for HAM/TSP, AS*, seven individuals with rheumatologic diseases – DR and 30 patients with HAM/TSP – HT. The results showed that the HT group presented the stronger IgG anti-Tax reactivity (p = 0.04) in comparison to AS group and in comparison to AS+AS* group (p = 0.02). The DNA proviral load evaluated in PBMCs of HTLV-1 infected individuals was higher in the HT individuals group in comparison to AS* individuals group (p = 0.01), and in comparison to AS+AS* group (p = 0.004). There was no correlation between DNA proviral load and the IgG anti-Tax reactivity. The biological system developed showed to be an important tool in serological evaluation of HTLV-1 infection and chronification.

Key words: HTLV-1, Tax, Recombinant protein, Vaccinia virus.

P-096 Abstract Withdrawn

P-097 Glycogen Synthase Kinase-3 (GSK-3) Inhibition Induces Cytotoxicity in Adult T-Cell Leukemia/Lymphoma (ATLL) Cell Lines

Purpose: Adult T-cell Leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-cell lymphotrophic virus type 1 (HTLV-1). Currently, although combination chemotherapy has improved the patients survival, the prognosis of aggressive ATLL still remains poor and new therapeutic approaches are needed. It has been shown that NF-? B is constitutively activated in ATLL and is considered responsible for cell growth and prevention of cell death. Several studies demonstrated that pharmacologic inhibition of GSK-3 suppresses NF-ƒÈB activity and leads to cytotoxicity in cancer cells. Therefore we evaluated the effect GSK-3 inhibition by 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) has on NF-ƒÈB activity in ATLL cell lines in an attempt to develop a new therapeutic approach.

Methods: We used 6 HTLV-1-infected cell lines: MT-1, TL-om1 and ED-40515(-) are derived from leukemic cells and SLB-1, MT-2 and C5/MJ are from in vitro transformed cells. Cell Titer-Glo assays followed by luminometric acquisition was used to determine growth inhibition of cells. Yopro-1 plus propidium iodide (PI) followed by FACS calibur acquisition was used to assess cytotoxicity. NF-ƒÈB activity was measured by an ELISA-based assay and immunofluorescence staining to subunits of NF-ƒÈB.

Results: The effect on growth inhibition was shown as early as 2 hours after the exposure of ATLL cells to TDZD-8 in a time dependent manner. IC50 values at 24 hours among 6 cell lines were in the low micromolar range (6.3 to 11.4 ƒÊM). There are no differences in IC50 values between Tax positive and negative cell lines. Flow cytometric analysis revealed the cytotoxic effect of TDZD-8 at 24 hours. (IC50: 10uM) In 3 hours, TDZD-8 reduced NF-ƒÈB transcriptional activity.

Conclusions: Targeting GSK-3 inhibition effectively induces cytotoxicity in vitro by reducing NF-ƒÈB activity in ATLL cell lines. Ongoing experiments are looking at the activity of TDZD-8 in murine xenograft models ATLL.

P-098 HTLV in Pre-Columbian Mummies in Bolivia

Background: The prevalence of HTLV-1 and 2 among Amerindians have raised the hypothesis of their spread from Africa to the New World following the ancestral human migrations. Concerning HTLV-1, other routes, in special, the Africans slave trade during centuries, are pointed. The complete scenario about the forces that drive the worldwide distribution of HTLV is still incomplete. Here is presented, by the first time, biological evidences of the presence of HTLV-2 in a human remain from Tiwanaku archaeological site/Bolivia (1.750BP) and, the recovery of ancient DNA (aDNA) sequence corresponding to the segment of tax gene virus type 2. Moreover, we also recovered, from a human teeth obtained in a Chuquisaca archaeological site/Bolivia (800-1200DC), segment of tax gene type 1.

Methodology: The samples were processed following all recommendations concerning aDNA manipulation in order to avoid contemporary contamination.

Results: Initially, aDNA hybridization screening of 29 naturally mummified human tissues from Bolivian archaeological sites, using tax and LTR HTLV as probes, identified two positive samples. A second hybridization using HTLV-1 and 2 specific probes confirmed positiveness to HTLV-1 and HTLV-2. PCR targeting tax gene was applied, and the 219 bp amplicon was sequenced, revealing the presence of HTLV-1 virus in sample 1710, from Chuquisaca and HTLV-2 in sample 1590 from Tiwanaku. The sequence analysis of the human mitochondrial DNA HVS-I region characterized the 1590 sample as belonging to haplogroup D, one of the major Amerindian haplogroups.

Conclusions: A controversial paleonthological study with Chilean mummies from pre-Columbian times had demonstrated the antiquity (1500ya) of human HTLV-1 infection in South America. Here we have new evidences corroborating with their findings. Moreover, supporting the epidemiological hypothesis and linking the virus out of Africa following the ancient human migrations, is demonstrated the ancestral presence of HTLV-2 in Amerindians.

P-099 HTLV-1 BZIP Factor Selectively Suppresses Classical NF-KB Pathway

Human T cell leukemia virus type I (HTLV-1) is an oncogenic retrovirus that is etiologically associated with a T-cell neoplasm, adult T-cell leukemia (ATL). The activation of NF-kB by Tax proposed to play a crucial role in both normal T cell activation and HTLV-1 induced T cell transformation. The HTLV-I bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both suppression of Tax-mediated viral gene transcription and T-cell proliferation, suggesting the close relation with Tax. In this study, we found that HBZ specifically suppressed p65 mediated classical pathway, but without modified the alternative NF-kB signaling pathway. Using coimmunoprecipitation, we demonstrated the physical association between HBZ and p65, and this interaction abrogates the p65 from its DNA binding sites. In the other aspect, HBZ activated the promoter activity of PDLIM2 and induced the upregulation of PDLIM2 ubiquitin E3 ligase expression, which induced p65 degradation through ubiquitination-dependent pathway. Finally, HBZ actually repressed the transcription of some classical NF-kB target genes, such as IL-8, IL2RA, IRF4, VCAM-1 and VEGF. This selective inhibitory effect of HBZ on classical NF-kB pathway modulates Tax mediated cellular transformation and might served as part of a viral strategy of effective replication.

P-100 HTLV-1 Infection Modifies Telomerase Gene Expression Toward Immune Senescence of Infected or Uninfected CD4 and CD8 T-Cells

Antigenic stimulation promotes hTERT expression and telomerase activity allowing alloreactive T-cells to proliferate and persist. Some defects in this pathway have been evidenced during chronic virus infections, eliciting exhaustion of antigen-specific cells and thereby immune senescence and pathogenesis. The interplays between HTLV-1 replication and cellular immune response include the control of proviral load and can elicit immunosuppression, molecular mimicry, mutants escape, and bystander effects. Together with the activated versus resting T-cell state, Tax, HBZ, and possible other viral compounds have been found to positively or negatively interfere with hTERT expression in HTLV-1 positive cells. These alterations are thought to account for the persistent clonal expansion of infected cells as in their malignant transformation. In vivo, infected cells represent only 1–5% of the T cell repertoire of HTLV-1 infected individuals and we hypothesized that whether a replicative senescence-based exhaustion of antigen-specific cells pertained during persistent HTLV-1 infection, then it should necessary affect both infected and uninfected cells. To investigate this, we generated CD4 and CD8 clones from uninfected and HTLV-1 infected individuals, including HAM/TSP and ATLL, and monitored telomerase homeostasis in activated versus resting cells. Uninfected resting CD4 clones from HAM/TSP displayed significantly higher levels of hTERT expression (22-fold) than those from uninfected controls. Upon infection, they overexpressed hTERT but the difference was not significant for the active + A+ B hTERT isoform. In contrast to CD4, uninfected CD8 clones from HAM/TSP displayed roughly identical amounts of hTERT transcripts than CD8 control clones whereas upon infection, CD8 clones significantly underexpressed hTERT (4-fold). As previously described by others, repeated PHA stimulation triggered hTERT overexpression (8-fold) in CD4 cells and hTERT underexpression in CD8 cells (12-fold) deriving from uninfected individuals. In sharp contrast, PHA stimulation strongly decreased hTERT expression in both uninfected and infected CD4 clones from HAM/TSP. For the 4 main hTERT isoforms, the amount of transcripts decreased below the detection threshold of our qPCR assay in the former while it decreased by a factor 4 in the latter. As in control CD8 clones and CD4 clones from HAM/TSP, PHA stimulation decreased hTERT expression in infected CD8 clones from HAM/TSP (2.3-fold). ATLL cells significantly underexpressed hTERT when compared to infected- resting (24-fold) or -activated (6-fold) CD4 clones. In conclusion HTLV-1 infection selects for uninfected and infected CD4 or CD8 T cells displaying the hallmark of immune senescence. In this context, cellular infection possesses opposite effects on hTERT in CD4 versus CD8 clones while among infected cells, ATLL clones harbor the lowest amount of hTERT transcripts. Targeting telomerase gene dysregulation will be of interest for preventing the exhaustion of virus and tumor-specific effectors in infected individuals.

P-101 Abstract Withdrawn

P-102 Abstract Withdrawn

P-103 Abstract Withdrawn

P-104 Identification of Activated Genes in CD8+ T Cells Isolated from HTLV-1 Infected Individuals

Human T cell lymphotropic virus type 1 (HTLV-1) infection is associated with two distinct clinical pathologies: adult T cell lymphoma/leukaemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Most of HLTV infected individuals remain asymptomatic, however about 3% develop the neurologic disease HAM/TSP. Host immune response are thought to influence the outcome of the infection and progression to disease. To identify genes differentially expressed among non-infected individuals, asymptomatic (HAC) and TSP/HAM patients we performed a serial analysis of gene expression (SAGE) using CD8⁺ T cells isolated from these individuals. The expression profile revealed around 900 genes differentially expressed between control group and HAC or TSP/HAM, and 300 genes were identified between HAC and TSP/HAM groups. A remarkable feature of the HAM/TSP up-regulated genes was that a significant number of them encode cell adhesion, chemotaxis and cytolysis proteins. The results revealed that perforin 1 (PRF1), granzyme A (GZMA), chemokine (C-C motif) ligand 5 (CCL5) and paxillin (PXN) were up-regulated in HAM/TSP. Besides, chemokine (C-X-C motif) receptor 4 (CXCR4) was suppressed in this group. These genes were validated by real time PCR in 17 HTLV-1 asymptomatic carriers, 14 patients with HAM/TSP and 24 healthy individuals and the results were confirmed. The data suggests that there is a strong CD8⁺ T cell activation against HTLV-1 infected cells, since HAM/TSP individuals has a higher proviral load. Also, it seems to be a strong inflammation cells migration probably to central nervous system, where CCL5 and CXCR4 seem to be involved. These conditions could contribute to exacerbated inflammation that occurs in HAM/TSP. Our results suggest that CD8⁺ T cell gene expression profile differs among the three populations studied and some molecules could have a central role in development of HAM/TSP.

Key words: HTLV-1, SAGE, CD8 T cells.

Supported by: FAPESP, CTC/FUNDHERP.

P-105 Clinical, Functional and Virologic Findings in Patients with Neurologic Disease Caused by HTLV-1 at Belém-Pará

The Human T cells Lymphotropic Virus (HTLV-1 and HTLV-2) are retrovirus which cause the adult T cell lymphoma/leukemia (ATL) and tropical spastic paraparesis (TSP) or HTLV-associated myelopathy (HAM). Other neurologic presentations are finding include sensory disturbances and hyperactive reflexes. The prevalence of the HTLV-1 infection in Brazil is elevated (0, 8–1, 8%), the HTLV-1 and HTLV-2 are endemic in the Amazon region. The health care professional knows a little bit about the HTLV infection and the diseases related to these virus. This is a transversal description, case-control study with 76 patients' carriers of the HTLV-1 and 2 admitted in the Núcleo de Medicina Tropical at Belém-Pará. The patients were submitted to clinical, functional (OMDS), neurological, laboratorial (TCD4+ lymphocytes count, proviral load quantification) evaluations and nuclear magnetic resonance (NMR). The patients HTLV-1 with abnormal neurologic findings were the cases (n = 19) and the patients asymptomatic with normal neurological evaluation were the cases-control (n = 40). The female sex were 66.1%, the media age was 50.7 years. The distribution of the average TCD4+ lymphocytes count in the two groups was within the normal reference values; the proviral load was more elevated in the cases group. The search of antibody anti-HTLV-1 in the cerebrospinal fluid was positive in 93, 3% of the cases. The neurological evaluation showed 16 patients (84, 2%) with TSP/HAM (p < 0.0001). In fourteen cases (73, 7%) the length of the disease was between 4-9 years. The evaluation of mass muscle strength flexor and extension of the knees showed that 63.2% of the cases had 3 degree and 68.4% had 4 degree, respectively (p < 0.0001). The reflexes were normal MMSS, patellar reflex and Achilles tendon reflex were hyperactive in 78.9% and 73.7%, respectively. Babinski bilateral signal was detected in 73.7% of the cases and Hoffman signal in 26.3%. Bilateral clonus was present in 13 patients. Tactual sensibility was abnormal (31.6%), mass muscle strength of legs was stronger than normal (63.2%) and the cases had also urine symptoms (89.5%). In 17 NMR done, 13 (76.47%) had abnormal image in the thoracic bone marrow. There were not associations between proviral load, OMDS, duration of the disease and NMR findings. The most of the cases with neurological disease related to the HTLV-1 were consistent with TSP/HAM; the elevated proviral load seems to be a marker of the development of the disease.

Key words: HTLV, TSP/HAM, neurological disease.

P-106 Detection and Quantification of Cervical and Thoracic Spinal Cord Atrophy of HAM/TSP Patients Using a 3D Semi-Automatic Technique

Objective: There is paucity in quantification methods for spinal cord atrophy evaluation in neurological disorders. Here we present a novel semi-automatic technique for accurate 3D quantification of spinal cord atrophy from MRI. To evaluate the applicability and sensitivity of the proposed technique we measured the spinal cord volume of HAM/TSP patients.

Background: HAM/TSP is a chronic inflammatory disorder affecting primarily the spinal cord. No clear relationship has been established between thoracic cord atrophy measured by conventional techniques and disability in HAM/TSP.

Design/Methods: Five patients and 5 age, gender, height and weight matched healthy volunteers (HVs) underwent 1.5T MRI of the cervical and thoracic spinal cord under a clinical protocol. Spinal cord volume was measured from consecutive high resolution sagittal T1-W MR images using a semi-automatic technique based on level sets. An unpaired t-test was used to assess statistical significance.

Results: The cervical spinal cord volume was 9720.84+/−796.89 mm³ for HVs and 6588.84+/−896.78 mm³ for HAM/TSP patients (p = 0.0004). The thoracic spinal cord volume was 14049.49+/−980.94 mm³ for HVs and 8882.65+/−2366.19 mm³ for HAM/TSP patients (p = 0.002). These results suggest that atrophy is apparent throughout the spinal cord not usually seen by visual inspection alone of the MRIs.

Conclusion: Here we define an accurate and sensitive detection and quantification method for the evaluation of spinal cord atrophy. In HAM?TSP patients, spinal cord atrophy in was detected and quantified at both the cervical and thoracic levels. Such measurements may improve monitoring of disease progression in HAM/TSP and other neurological disorders including multiple sclerosis.

P-107 Do Glucocorticoids Offer a True Kind of Relief in HAM/TSP?

Human T-cell lymphotropic virus type I (HTLV-I) is a retrovirus with a special tropism for infecting T cells, inducing spontaneous proliferation (SP) in vitro. Clinically, this virus induces HTLV-I-associated myelopathy (HAM). The treatment of HAM is usually based on anti-inflammatory and immunosuppressive drugs such as synthetic corticoids (CCs). Recently we demonstrated that resistance to CC in vitro was related to SP. However, the use of CCs has yielded controversial results in both experimental and clinical studies. Thus, the aim of this work was to assay CCs response in vitro as well as in vivo, linking these data with the cellular proliferation and to the efficacy of other immunosuppressive drugs. Eight asymptomatic carriers (AC), eight patients with HAM and eight controls were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured in vitro to assess cellular sensitivity to dexamethasone (10(−9) to 10(−5) M) by the assessment CC-induced suppression of T-cell proliferation. SP was determined by visual identification of several cellular clusters in unstimulated cells culture. The efficacy of CC therapy in HAM was studied through oral administration of high-dose prednisolone (1mg/Kg/day) by at least 3 months. In some HAM patients that didn't show improvement with CC therapy, we also evaluated the efficacy to another immunosuppressive drug (azathioprine 2 mg/kg/day). Disability scores (EDSS) were evaluated by the same investigator before and after treatments. Cells of controls had normal proliferation and were sensitive to CCs in vitro. Thirty-seven percent (3/8) of the ACs and 75% (6/8) of the individuals with HAM presented SP. Interestingly, 67% (2/3) of the ACs with SP presented some symptoms (oligosymptomatic pattern), but these were not enough for the HAM diagnosis. Fifty percent (4/8) of AC and HAM (4/8) individuals were in vitro CC resistant. Fifty percent (2/4) of theses resistant ACs and 100% (4/4) of resistant HAM patients showed SP. Eighty-five percent (6/7) of the HAM patients didn't show significantly improvements with CC therapy. All in vitro CC resistant patients (4/4) were also resistant to CC therapy. However, two patients sensitive in vitro to these drugs (2/4) didn't show clinical improvement. Interestingly, 75% (3/4) of the patients that used other immunosuppressive drug (azathioprine) presented expressive improvement of the symptoms. The duration time of HAM was similar between subgroups of steroid sensitivity and cellular proliferation. Finally, the in vitro response to corticoids was in agreement with CC treatment in vivo. The SP seems to influence the in vitro CC sensitivity and symptomatology, but it is unclear if it cooperates to these situations. The CC therapy showed low efficiency in HAM individuals but, on the other hand, other immunosuppressive therapy resulted in a large improvement of the patient's clinical status.

P-108 Evaluation of Patients with Tropical Spastic Paraparesis using the Spastic Paraplegy Rating Scale

Objective: To evaluate the effectiveness of the Spastic Paraplegic Rating Scale (SPRS) in clinical assessment of patients with HAM/TSP.

Background: Tropical spastic paraparesis (TSP) is a myelopathy associated to infection of HTLV-I, clinically characterized by a chronic and progressive spastic paraparesis, related to several degrees of sphincter and sensory disturbances, being predominant in tropical areas. The neurological examination generally shows weakness and spasticity in the lower limbs with an adductor pattern. Commonly, the evolution of the motor disability is analyzed by the Kurtzke Expanded Disability Status Scale (EDSS), which was designed for evaluating patients with multiple sclerosis. However, the progression of other important aspects of the disease, sphincteric complaints and pain, is not mentioned. Such symptoms and signals are very similar to those of the Hereditary Spastic Paraparesis uncomplicated form. The Spastic Paraplegia Rating Scale (SPRS) has been developed in order to measure the severity of this disease.

Methods: We have made a cross-sectional study from a cohort of 197 patients with seropositivity to HTLV-I. 79 from this group presented spastic paraparesis and only 39 had defined diagnosis of HAM/TSP, according to new criteria. On those ones, expanded disability status scale (EDSS) and SPRS scales have been applied.

Results: Scores from scales were compared by using Spearman correlation coefficients: SPRS/Kurtze □0,8033 (p < 0,0001).

Conclusions: In orphan diseases, such as HAM/TSP, there are few clinical markers to measure disease severity. Scales like SPRS are a useful instrument to quantify the impairment or improvement of the patient during treatment, because it is practical and does not need any specific tool.

P-111 DC-Sign as a Potential Target to Block HTLV-1 Transmission and Infection

Despite the susceptibility of dendritic cells (DCs) to HTLV-1 infection and the defined role of these cells in disease pathogenesis, the mechanisms of viral binding to DCs have not been fully delineated. Recently, a glucose transporter GLUT-1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) were demonstrated to facilitate HTLV-1 entry into T cells. DCs express their own array of antigen receptors, the most important being the DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) with respect to retrovirus binding. Consequently, the role of DC-SIGN and other HTLV-1 receptors was analyzed in viral binding, transmission, and productive infection using monocyte-derived DCs (MDDCs), myeloid DCs, and B-cell lines expressing DC-SIGN. The relative expression of DC-SIGN, GLUT-1, HSPGs, and NRP-1 was first examined on both DCs and B-cell lines. Although inhibition of these molecules reduced viral binding, HTLV-1 transmission from DCs to T cells was mediated primarily by DC-SIGN, with some indication of GLUT-1 involvement as well. DC-SIGN was also shown to play a role in the infection of MDDCs as well as model B-cell lines. HTLV-1 infection of MDDCs was also achieved in myeloid DCs following the enhancement of virus-induced interleukin-4 production and subsequent DC-SIGN expression in this cell population. This study represents the first comprehensive analysis of potential HTLV-1 receptors on DCs and strongly suggests that DC-SIGN plays a critical role in HTLV-1 binding, transmission, and infection, thereby providing an attractive target for the development of antiretroviral therapeutics and microbicides.

P-112 High Constitutive Expression of ATF-3 in Adult T-Cell Leukemia

ATF-3 is a member of the ATF/CREB family and its overexpression is a molecular hallmark of classical Hodgkin lymphoma that contributes to the malignant growth of Hodgkin/Reed-Sternberg cells. Adult T-cell leukemia (ATL) is a mature CD4⁺ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Although it is a fact that HTLV-1 reaches an oncogenic event and causes ATL, the oncogenic mechanism of HTLV-1 is not fully understood. The viral protein Tax can immortalize primary human T cells, and when expressed as a transgene, it provokes leukemogenesis and lymphomagenesis in animals. To clarify involvement of ATF-3 expression in the pathogenesis of ATL, RT-PCR and Western blot analyses were carried out on a panel of T-cell lines and PBMCs from patients with ATL or healthy volunteers. All HTLV-1-infected T-cell lines and primary ATL cells demonstrated strong overexpression of ATF-3 mRNA and protein compared with uninfected T-cell lines and normal PBMCs. Immunohistochemical staining of ATF-3 in ATL lymph nodes and skin lesions showed that primary ATL cells were indeed stained strongly positive for ATF-3. Induction of Tax by Cd²⁺ in JPX-9, a subline of Jurkat human T-cell line carrying Tax under the control of metallothionein promoter, led to upregulation of ATF-3. A luciferase reporter gene under the control of the ATF-3 promoter was expressed by cotransfection of an expression vector for Tax. Using Tax mutants, we demonstrated that Tax-induced ATF-3 expression required the ATF/CREB signaling pathway. Deletion and mutation analyses of the 5’ flanking sequence of the ATF-3 gene promoter revealed that one of the major elements responsible for the induction by Tax is an ATF/CRE located at −92 to −85 relative to the transcriptional start site. Gel shift assay demonstrated that a complex containing ATF-1, ATF-3, CREB-1, c-Jun, JunB, and JunD increased binding to the ATF/CRE site in the Cd²⁺-treated JPX-9 cells and HTLV-1-infected T-cell lines. Short interfering RNA against ATF-3 inhibited cell growth of HTLV-1-infected T-cell lines, but not an uninfected T-cell line. Thus, our data suggest an important oncogenic role of ATF-3 expression induced by Tax in ATL.

P-113 Abstract Withdrawn

P-115 Identification of E3 Ligases Required for Tax Activation of NF-KB

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein deregulates various cellular signaling pathways including NF-? B. The activation of NF-?B by Tax plays a crucial role in HTLV-1-induced transformation. Previously, we have demonstrated that Tax undergoes lysine 63 (K63)-linked polyubiquitination that requires the ubiquitin conjugating enzyme Ubc13. However, the identity of an E3 ligase that catalyzes K63-linked polyubiquitination of Tax has remained elusive. In this study, we have identified two E3 ligases that are required for K63-linked polyubiquitination of Tax and subsequent Tax-mediated NF-? B activation. Based on these new findings, we propose a model in which Tax hijacks host ubiquitination machinery to activate NF-? B and promote the proliferation and transformation of HTLV-1-infected T lymphocytes.

P-116 Identification of HTLV-1 Tax Amino Acid Signals and Cellular Factors Involved in Secretion of the Viral Oncoprotein

HTLV-1 is the etiologic agent of a number of pathologic abnormalities, including adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The viral oncoprotein Tax has been implicated in the pathogenesis of these diseases. Recently, cell-free Tax was detected in the cerebrospinal fluid of HAM/TSP patients, implying that extracellular Tax is relevant to neurologic disease. Additionally, the presence of a nuclear export signal within Tax and its active secretion has been demonstrated in vitro. However, the mechanism of Tax secretion remains to be established. Studies reported herein elucidate the process of Tax secretion and identify domains of Tax critical to its subcellular localization and secretion. Tax was shown to interact with a number of cellular secretory pathway proteins in both the model cell line BHK (baby hamster kidney)-21 and an HTLV-1-infected T cell line C8166, physiologically relevant to HTLV-1-induced disease. Silencing of selected components of the secretory pathway affected Tax secretion further confirming regulated secretion of Tax. A comparison of the Tax protein sequence with amino acid signals known to target proteins to the secretory pathway revealed a number of putative secretory signals. Mutations in two signals DHE and YTNI resulted in aberrant subcellular localization of Tax and significantly altered protein secretion. Together, these studies demonstrate that Tax secretion is a regulated event facilitated by its interactions with proteins of the cellular secretory pathway and the presence of secretory signals within the carboxy-terminal domain of the protein.

P-118 Kinetic Analysis of Human T-Cell Leukemia Virus Type 2 Expression in Chronically - Infected Cells and Patient PBMCS

Kinetic analysis of Human T-cell Leukemia Virus type 2 expression in chronically- infected cells and patient PBMCs. Cecilia Bendera, Paola Righia, Francesca Rendeb, Paola Ronzic, Claudio Casolic, Vincenzo Ciminaleb and Umberto Bertazzonia a) Dept of Mother and Child, Biology and Genetics, University of Verona; b) Dept. of Oncology, University of Padova; c) Dept of Clinical Sciences, University of Milano, Italy Characterization of HTLV transcript expression during the infection process is essential to obtain novel information on viral gene products, and their influence on proliferation, cell cycle control and signalling. To date, very little information is available on the quantitation and timing of HTLV-2 viral transcription. Using a series of splice-junction-specific primers and probes to quantitative all HTLV-2 transcripts by Taqman^® real time RT-PCR, we measured the kinetics of viral expression in chronically-infected human T- and B-cell lines and in PBMCs from infected subjects. The time course of viral transcripts expression in C344 T-and BJAB-Gu B-cell lines was analyzed after 10 fold dilution of optimally growing cells. Tax/rex, gag/pol, env and p28, p22/p20 rex-2 mRNAs were expressed at highest levels after 48 hours in C344 cells, and declined afterwards, whereas tax/rex and p28, p22/p20 rex-2 showed a biphasic profile in BJAB-Gu cells with an early peak at 24 hours and a second one at 72 hours. Expression of accessory genes was significantly lower than structural and regulatory proteins. The kinetics of gene expression from PBMCs obtained from HTLV-2B infected subjects showed an early transcription of tax/rex followed by a gradual and steady increase of gag/pol, p28, p22/p20 rex−1 and −2, whereas env and accessory transcripts were below our limit of detection. These results indicate that expression of different HTLV-2 genes follows a distinct timing both in infected cell lines and PBMCs isolated from infected patients.

P-119 Molecular and Immunological Characterization of Human T-Cell Lymphotropic Virus infection in Patients with Neurological Disorders and Asymptomatic Carriers

This study aimed to perform the immunological and molecular characterization of HTLV-1/2 infection among 42 asymptomatic carriers and 19 patients with neurological symptoms (16 with TSP/HAM and three with peripheral neuropathy). A control group composed by 100 HTLV-1/2 seronegative subjects from Belém-PA was also analyzed. The blood samples were collected aiming to perform HTLV serologic analysis, to quantify CD4+ and CD8+ T lymphocytes by cytometric analysis, to measure the proviral load and the characterization of types and subtypes of HTLV. Among the 42 asymptomatic carriers it was observed 35 (83.3%) HTLV-1 and 07 (16.7%) HTLV-2 infected subjects (p < 0.0001). Among the 19 symptomatic carriers, 18 (94.7%) samples were infected by HTLV-1 and one subject, with peripheral neuropathy, was infected by HTLV-2 (5.3%). The phylogenetic analysis of 5′LTR regions grouped 34 samples (60%) of HTLV-1 as subtype Cosmopolitan, subgroup Transcontinental, and 05 HTLV-2 samples (72.2%) as HTLV-2c subtype. The mean values of CD4+ and CD8+ T lymphocytes levels was higher among symptomatic, but no significant differences was observed as compared to the asymptomatic and control group. There was a higher mean of the proviral load values among symptomatic individuals when compared to asymptomatic (p = 0.0123). The present results confirmed the tropical spastic paraparesis occurrence associated to HTLV-1 infection in Belém, Pará. Furthermore, the molecular characterization of the virus corroborates previous results which demonstrated the presence of HTLV-1 subtype A (Transcontinental) as the most prevalent among symptomatic and asymptomatic infected subjects in Brazil and shows, for the first time in the Amazon region, the occurrence of HTLV-2 infection associated to peripheral neuropathy. The presence of HTLV-2c confirms this molecular subtype as the highest prevalent in the Brazilian Amazon region. The highest mean of proviral load among symptomatic subjects highlights the importance of this viral marker in the development of TSP/HAM. Continuous studies are being performed in the Amazon region aiming to better understand of epidemiological features of HTLV-1/2 infection in this geographic area as well as to monitoring the emergence of new subtypes and disease associated to HTLV-1 and HTLV-2.

Financial support: PN-DST & Aids/Ministério da Saúde, CNPq.

P-120 Seroprevalence Study of Human T-Lymphotropic Virus Type 1, Human Immunodeficiency Virus, Tripanosoma cruzi and Treponema pallidum Infections in Kollas and Guarani Natives from Argentina

Introduction: HTLV-1/2 is distributed worldwide and is endemic among natives of South America. In Argentina, HTLV-1 infection has been reported as endemic among natives (Kollas) from the Northwest, while HTLV-2 among Tobas and Wichis from the Gran Chaco Region in the North. Tripanosoma cruzi (T. cruzi) was also detected among these two native populations, while Treponema pallidum (T. pallidum) infection was only reported in aboriginals from the Chaco Region. Human Immunodeficiency virus (HIV) has not been reported up to the present in these communities.

Objective: To determine the prevalence of HTLV-1/2, HIV, T. cruzi and T. pallidum infections in natives from Northwest (NW) and Northeast (NE) regions of the country and to perform the molecular characterization of HTLV-1/2.

Materials and Methods: A total of 112 Kollas from Jujuy (NW) and 298 Guarani from Misiones (NE) individuals were studied. An informed consent was signed by all participants. The screening was performed by HTLV-1 PA, Serodia, Fujirebio; HIV-1/2 ELISA Dade Behring; Enzygnost anti HIV ½ plus; SFD HIV ½ PA, Biorad Fujirebio. Reactive samples were confirmed by WB: HTLV Blot 2.4 Genelabs Diagnostics and New Lab Blot-1, Bio-Rad, respectively. Screening of T. cruzi was performed by indirect HA and ELISA (FATALA KIT) and for T. pallidum infection by VDRL test (Wiener Lab.) and HA (TPHA Biosystems). Confirmation of repeatedly reactive/discordant samples was performed by IFI (FATALA KIT for T. cruzi) and IFI (FTA-abs Immunofluorescence Biocientífica SA, for T. pallidum). Phylogenetic analysis was carried out by MrBayes program and tree topology was visualized by TreeView.

Results: Eleven (9.8 %) out of 112 Kollas were confirmed as HTLV-1 positive classified as Transcontinental Subgroup. All Guarani samples were HTLV-1/2 negative. Two Guarani individuals were HIV seroindeterminate. Three (2.3%) Kollas were positive for T. cruzi infection while 11 (9.8%) Kollas and 20 (6.7%) Guaranies were positive for T. pallidum infection. Two (1.8%) Kollas were co-infected with HTLV-1/T. pallidum. None of positive individuals reported previous blood transfusion or illegal drug use.

Conclusions: This study shows that HTLV-1 infection has been maintained among Kollas from the NW with a similar seroprevalence to previous reports, while T. cruzi infection was significantly lower. In opposite to other Guaraní populations of Paraguay or Brazil, no HTLV-1/2 cases were detected in this Guaraní community. Concerning the prevalence of T. pallidum infection among Guaranies, it was higher than in blood donors of the same area. Finally, our data highlights the importance of implementing new public health policies adapted to aboriginal communities focusing on sexual and mother-to-child transmission of these infectious diseases.

P-121 Abstract Withdrawn

P-122 Therapeutic Effects of a Novel NF-Kappa B Inhibitor, BAY65-1942, in a Mouse Model of ATLL

HTLV-I Tax transgenic mice generated using the Lck proximal promoter develop diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute Adult T cell Leukemia/Lymphoma (ATLL). Notably as in human ATLL constitutive activation of NF-kappa B was associated with disease development. In this study, we examined the effect of Bay65-1942, a novel inhibitor of IKKbeta on disease development. In vitro, TUNEL staining indicated that Bay65-1942 treatment sensitizes mouse ATLL cells to apoptosis, and DNA fragmentation resulting from apoptosis could be demonstrated by electrophoresis. NOD-SCID mice which received intraperitoneal inoculation of mouse ATLL cells were subjected to Bay65-1942 treatment in vivo. The mean survival in Bay65-1942-treated mice was 38.6 ± 4.6 days versus 30.5 ± 1.5 days for control-treated mice (p = 0.039, Log rank p-value). Histopathologically, suppression of leukemic cell growth was evident in Bay65-1942 treated mice. These findings demonstrate that Bay65-1942 could be a potentially effective therapeutic agent for ATLL.

P-123 Two Cases of Leukemic Infiltration of Breast in Adult-T-Cell Leukemia/Lymphoma Patients

Adult T-cell leukemia/lymphoma is a lymphoproliferative disorder of mature T lymphocytes associated with infection with human T-cell lymphotrophic virus type I (HTLV-1), characterized by clinical and laboratory polymorphism. Infiltration of mammary glands is a rare event during clinical course of ATLL. We present two rare cases of ATLL with infiltration of mammary glands occurred in the last decade.

Case 1: A 40-year-old male was admitted with long-standing skin lesions and leukocytosis diagnosed in 90's. Peripheral blood lymphocytes presented CD2, CD4, CD25, CD38 membrane surface antigens. Monoclonal expansion of lymphoid cells integrated with HTLV-1 genome was observed, and the diagnosis of ATLL chronic type was made. He underwent a chemotherapy regimen, and skin lesions and leukocytosis improved markedly. He progressed with an indolent clinical course of ATL, when he was admitted with bilateral hyperplasia of breast. Breast biopsy revealed bilateral gynecomasty, extensive leukemic infiltration of typical ATL cells in the mammary glands, and the presence of mammary epithelial cells productively infected with HTLV-1.

Case 2: A 39 years,-old female, from Pernambuco-Brazil, ATLL diagnosed in 2005, presented with leukocytosis with flower cells, CD 25+, the presence of HTLV-1 was confirmed. Chemotherapy with CHOP-6 cycles induced remission. INF+AZT was introduced and maintained remission until January 2007, when presented tumor mass in abdomen, lymph nodes and hypercalcemia. Three cycles of CHOP, induced reduction of mass, and INF+AZT was re-introduced. In May 2007, presented with tumor enlargement of breast bilateral. Mammography identified tumor measuring 4 × 2,9 cm Wright breast and 8,4 × 2,1 in left breast. Biopsy confirmed the presence of CD3+,CD30+,CD25+ with 90% of proliferation index, CA-15-3 45,6U/L(normal up 25), LDH 2090 U/L. After 2 months of INF+AZT daily reduction of masses occurred and disappearance of lymph nodes. In February 2008, the masses grew outside the breast, having bilateral tumors. The progression of disease was out of control, the tumors infected, and did not respond well to antibiotic therapy, and finally 2 months later, the patient died in renal failure. These cases illustrated the diversity of presentation of the diseases associated with HTLV-1 and difficulties in controlling lymphocytes proliferation in tissues.

P-124 Neurological Manifestations in HIV/HTLV-Coinfected Patients

Objective: Determine prevalence of HTLV infection in HIV-individuals. Assess the risk for neurological diseases and laboratorial parameters in coinfected compared with HIV-patients.

Background: HIV neurological diseases affect more than 40% of AIDS patients. In the other hand, HTLV-1- myelopathy, main neurological disease associated with HTLV-1, is estimated to occur in less than 2% infected individuals. However, other HTLV-1 neurological diseases as peripheral neuropathy (PN) have been reported. There is reasonable concern about increased risk for neurological diseases in HIV/HTLV-individuals, but little is know about this issue in patients receiving highly active antiretroviral therapy (HAART).

Design/Methods: From 726 HTLV-individuals, we selected 90 with negative HIV serology. From a cohort of 2678 HIV-individuals, we randomly selected 500 on HAART to HTLV-antibodies determination. All patients were submitted to neurological assessment, HIV viral load (VL), HTLV proviral load (PVL), and CD4/CD8 counts.

Results: 480 HIV-patients were tested and 48 were infected by HTLV (34 HTLV-1, 5 HTLV-1/2, 3 HTLV-2, 6 indeterminate). Prevalence of any neurological disease in HIV/HTLV was much higher than in HIV-patients (26/48 vs 56/376; p < 0.0001). Myelopathy was diagnosed in 12/48 HIV/HTLV and in 2/430 HIV-patients (p < 0.0001) while PN was present in 18/48 HIV/HTLV and in 52/380 HIV-patients (p < 0.0001). There was not difference between groups in laboratorial parameters assessed.

Conclusions/Relevance: 10% of HIV-patients were coinfected with HTLV. These patients presented higher prevalence of PN and myelopathy. Higher prevalence of myelopathic individuals was already expected in coinfected, but higher prevalence of PN was surprisingly since that HIV is associated with it. This suggests that there is a synergistic interaction between HIV/HTLV that results in nervous system diseases. We do not observe laboratorial differences between groups as described in the past. Possible reason for this discrepancy is that our patients were on HAART, while majority published papers were conducted in era pre-HAART.

Study supported by: Programa Nacional DST/AIDS do Ministério da Saúde, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação Oswaldo Cruz (FIOCRUZ).

P-125 Onychomycosis: Another Complication of HTLV-1 Infection?

Background: HTLV-1-infected subjects are at increased risk of developing specific infectious complications, including strongyloidiasis and scabies. Noteworthily, severe onychomycosis has been linked to immune suppression. We prospectively assessed the association between HTLV-1 and onychomycosis.

Methods: Our Institute provides HTLV-1 testing to relatives of HTLV-1-infected people and patients with suspected HTLV-1-associated diseases (mostly neurological disorders and strongyloidiasis). In March 2006, we add a general examination to the routine interview perform on all subjects prior to HTLV testing. The diagnosis of onychomycosis was made clinically; the number and distribution of affected nails was registered. Results here presented excluded subjects with (1) known HIV infection, (2) onychomycosis as the motive of test and (3) prior knowledge of the participant's HTLV-status, to avoid examiner bias. We assessed the association between onychomycosis and HTLV-1 through bi- and multivariable analyses. Logistic regression was used to adjust for age, sex and motive for HTLV-1 testing, using 6 affected nails (90^th percentile) as a cut-off point.

Results: Between March 2006 and April 2008, we included 893 subjects: their mean age was 38 years (standard deviation 19); 527 (59%) were women. Onychomycosis of at least one nail was observed in 323 participants (36%). Two hundred thirty-six subjects (26%) were HTLV-1 positive. The median number of affected nails was higher in HTLV-1-positive than in HTLV-1-negative subjects (Mann-Whitney test: p = 0.0005). Thirty-eight of 97 subjects with 6 affected nails (39%) were HTLV-1 infected, compared to 198 of 796 subjects with <6 affected nails (25%; crude OR 1.9; 95% confidence interval (CI): 1.2–3.1; p = 0.003). This association remained significant in the multiple logistic regression model (adjusted OR 2.1; 95% CI: 1.3–3.4; p = 0.003) and when we used different cut-off points (from 1 to 10 affected nails).

Conclusion: The independent association between HTLV-1 infection and onychomycosis found in this setting suggests that the immune suppression induced by HTLV-1 may have more consequences than already described.

P-127 Relevance of HTLV-1 Proviral Load Quantification by Real-Time PCR in Asymptomatic and Symptomatic Patients Living in Argentina

Background: In Argentina, the prevalence of HTLV-1 greatly varies according to the region with some endemics areas mainly in the north. HTLV-1 infected patients frequently remain asymptomatic but a minority develops HAM/TSP, ATL or other HTLV-1 associated diseases. The magnitude of HTLV-1 proviral load has been proposed as a biological marker of disease progression. The aim of the study was to measure HTLV-1 proviral load in asymptomatic and symptomatic patients.

Methods: Twenty-six HTLV-1 positive individuals were included in the study. Of them, 22 were asymptomatic carriers (15 females) and 4 symptomatic patients (3 females) with HAM/TSP. The median age was 42 ys old for female and 39 ys old for males. Viral infection was confirmed by serology (WB) and a qualitative PCR. HTLV-1 proviral load in PBMCs was quantified using a sensitive SYBR green real-time PCR method. All samples were analyzed for HTLV-1 pol gene and albumin gene in parallel to normalize the DNA input for each reaction. The lower limit of detection for the assay was <2.6 log10 copies/106 PBMCs. Differences among groups were evaluated by Mann-Whitney test.

Results: According to the place of birth, three groups were considered: 1) North: (n = 15) Jujuy and Salta (north provinces of Argentina), Bolivia, Peru and Paraguay, 2) Center: (n = 9) Buenos Aires and Santa Fe and 3) other countries: (n = 2) Chile and Corea. The median proviral load was significantly higher in subjects born in the North (median = 4.69 log10 copies/106 PBMCs, IQR2:4.43–5.00) compared to the Center (median = 3.83 log10 copies/106 PBMCs, IQR: 2.63–3.84, p = 0.02). All the HAM/TSP patients were born in the North and had significantly higher proviral load (median = 5.18 log10 copies/106 PBMCs, range: 5.00–5.73) than asymptomatic carriers (median = 4.18 log10 copies/106 PBMCs, range: <2.6–5.73, p = 0.003). Among asymptomatic carriers higher proviral loads was also observed in subjects born in the North (p = 0.018). Symptomatic patients (median = 54 ys) were significantly older than asymptomatic carriers (median = 38.5 ys, p = 0.02), however no difference was observed among proviral load and age nor gender.

Conclusions: We evaluated, for the first time, to our knowledge, HTLV-1 proviral load in subjects living in Argentina. In this preliminary study, we demonstrated that HTLV-1 proviral load level in PBMCs was associated with the phenotype of the infection, being lower in asymptomatic carriers and higher in symptomatic patients.

P-128 Risk Factors for UTI in Patients with HAM/TSP

Hypothesis: Catheterised HAM patients are at higher risk of developing an UTI than non- catheterised. Other factors may put patients at risk of developing an UTI.

Background: Urinary catheterisation is associated with higher risk of UTIs and is inevitable in long-term catheterised patients.

Definition: For the purpose of this study an UTI is defined as microscopically confirmed urine culture >10⁵ cfu/ml, single or mixed bacteria, WCC>50, with or without systemic symptoms.

Aim: To investigate risk factors for UTIs in patients with HAM: catheters; gender, age, co-morbidity, immunosuppression.

Methods: Retrospective case note evaluation at a tertiary care clinic in London UK 2006–2008. Results 38 patients with HAM were 33 (86.8%) females. 30 Afro- Caribbean, 5 Caucasian and 3 African. 37/38 (97%) had bladder symptoms. Mean age 59 years. Twenty (52.6%) patients used urinary catheters 11 (30%) intermittent self-catheterisation (ISC), 6 (16%) suprapubic catheter (SPC) and 3 (8%) indwelling catheters (IDC). 17(45%) patients experienced one or more UTI's in a year. 13/17 (76.4%) had a catheter associated UTI contributing 29 episodes.Risk factors Average incidence of UTI was suprapubic and intermittent catheterisation 1.3 infection/y; indwelling urethral catheter 0.3 /y; no catheter 0.4 /y. Average age with UTI was 63.5years v 57.2 years if no UTI (T-test p = 0.15) Rate amongst females was 16 (42.1%) v males − 1(2.6%).Duration of the disease. Patients experiencing UTIs has HAM for average 12.9 years v 11.4years if noUTI (p = n.s.) Compared with 2006 in 2008 9 patients had more UTIs, 4 patients had fewer and 13 patients no change. 3/9 patients started or changed to SPC.

Treatment: Two patients were on prophylactic antibiotics.

Co-morbidity: Out of 17 patients who had at least one UTI in the last year 7 were observed to have other inflammatory conditions; 4 had acute or chronic infections; 3-kidney disease; 2-diabetes; 2-hypertension; 1-urogynecologic problem.

Immunosuppressive treatment 6/38 patients had been on immunosuppressive treatment-4/17(23.5%) in UTI group and 2/21(9.5%) in non-UTI group.

Conclusion: UTI's are common in HAM/TSP patients.

There is a high incidence (76.4%) of catheter associated UTIs in HAM patients. Our data showed that SPC and ISC contributed more to the development of UTIs than previously thought. Although patients who developed UTIs were slightly older than the main group, the age difference between UTI group and non-UTI group was not statistically significant.

Despite active management some patients had more UTIs in 2008 than in 2006 despite switching to SPC. Co-morbidity plays a role in the development of UTIs. All 17 patients in the UTI group had one or more other health conditions, such as co-infection, inflammatory or kidneydisease or diabetes. Increased use of immunosuppressive therapy in this cohort may have increased the incidence of UTIs.

P-130 South African individuals with Human T-Lymphotropic Virus Type 1 (HTLV-1) Associated Infective Dermatitis do not have an Atopic Diathesis

HTLV-1-associated infective dermatitis (HAID) is characterised by exudation and crusting around the nostrils, ears and scalp. Like atopic dermatitis (AD), HAID is predominantly a disease of childhood and associated with susceptibility to S. aureus infection. Previous studies have documented elevated IgE levels in the serum of individuals with HAID but it was unclear whether this was related to atopic disease susceptibility. Our aim was to investigate whether HAID associates with an atopic diathesis. We recruited 14 cases with HAID in South Africa, together with a group of 23 controls (including 5 patients with AD). We designed a questionnaire incorporating all clinical criteria for AD that have been published to date looking for evidence to support the possibility that HAID patients may have background atopy/atopic tendencies. We obtained relevant clinical history and examined all cases and controls based on this questionnaire. We also obtained serum, skin, DNA and PBMC samples from this cohort. We documented the total IgE and house dust mite specific (HDM) IgE in the patients with HAID compared to controls. To investigate whether other associations of atopic dermatitis, such as filaggrin (FLG) genotype and expression are linked to HAID we stained paraffin embedded skin sections of cases and controls for FLG protein. Preliminary immunostaining for HTLV-I Tax and Gag in paraffin embedded skin sections of some HTLV-1-positive cases and a group of controls (including some ACs), was also done.

Results: None of the cases with HAID had a history of asthma; however there was a high incidence of dry skin. Our cohort was not anaemic (mean Hb 12.0g/dl for cases, 11.9g/dl for controls) nor had lymphocytosis (mean lymphocyte count 2.9 × 109 for cases and 3.0 × 109 for controls). ESR was elevated (mean of 24.1mm/hr for cases; 16.8mm/hr in controls). The mean total IgE in HAID cases was 652 kUA/L and HDM-specific IgE of 0.76 kUA/L. The mean IgE and HDM-specific IgE levels for the controls was: 659 kUA/L and 7.3 kUA/L (healthy), 464 kUA/L and 1.39 kUA/L (HTLV-1 infection, but no HAID); 5000 kUA/L and 80.2 kUA/L for AD controls. The levels of HDM-specific IgE were not significantly different between cases and HTLV-1 infected (but no HAID) controls. FLG protein expression was downregulated in the epidermis of HAID cases compared to controls. Tax staining was not observed but a moderate Gag labelling within cells present in the dermis of cases of HAID compared to controls.

Conclusions: Our preliminary data show that in South Africa, the presence of HAID does not associate with asthma or a significantly elevated total or allergen-specific IgE compared to non-atopic controls, suggesting that the HAID phenotype is not dependent on atopic susceptibility. Further studies are underway to confirm the possible presence of HTLV-1 Gag protein in the skin of HAID cases. We now plan to investigate the role of T-cells in disease pathogenesis.

P-131 Dizziness, Tinnitus and Hearing Loss Related to HTLV-1 Infection

Introduction: HAM/TSP is related to a predominant damage of the spinal cord motor tract. Abnormalities in this tract may be related to balance disorders due to a late proprioception arc-reflex response. The correlation of HTLV-1 infection and otoneurological manifestations has not been studied.

Objective: To estimate the prevalence of otoneurological complaints in HTLV-1-infected individuals and to compare with the general population.

Type of study: A case-control study.

Methodology: Individuals with and without HTLV-1 infection answered a questionnaire related to hearing-loss, dizziness and tinnitus.

Results: From the 54 individuals of the control group, 11 (20.4%) reported dizziness, 9 (16.7%) hearing loss, 6 (11.1%) tinnitus and 18 (33.3%) at least one of the three evaluated symptoms. From the 96 patients infected by HTLV-1, 39 (40.6%) reported dizziness, 25 (26%) hearing loss, 38 (39.6%) tinnitus and 59 (61.5%) at least one of the three evaluated symptoms. The individuals infected by the HTLV-1 exhibited a higher frequency of dizziness (p = 0.009) and tinnitus (p = 0.000).

Conclusion: A potential association between HTLV-1 infection and the complaints of dizziness and tinnitus can be related to abnormalities in the motor vestibulospinal tract. Reports of postural instability have been shown to precede neurological abnormalities in HTLV-1-infected patients. The follow-up of these patients reporting dizziness and tinnitus will clarify the prognostic value of such symptoms for HAM/TSP.

P-133 Prevalence of HIV I/II-HTLV Coinfection among Blood Donors in São Luís - Maranhão

The human T-lymphotropic virus type I and II (HTLV-I/II) and the human immunodeficiency virus type I and II (HIV-I/II) are the only human retrovirus associated with pathological processes. Despite their distinct biological features, they share similiar epidemiological characteristics, including routs of transmission and high prevalence in certain geographic locations, wich could lead to coinfection. Studies show that HTLV-I/II and HIV-I/II coinfection may result in significant changes in the disease progression and survival rate.

The aim of this study was to detect the prevalence of HTLV-I/II and HIV-I/II infection and the coinfection among blood donors in the "Centro de Hemoterapia e Hematologia do Maranhão" (HEMOMAR) between January and December 2006, by the enzyme immune assay test (ELISA). The social-demographic features of the subjects with confirmatory test Western-Blot were described.

The prevalence of HTLV-I/II infection according to the ELISA test was 0.89/1000 among men, 4.27/1000 among women and 1.56/1000 considering both groups. The prevalence of HIV-I/II infection was 4.74/1000, with similar rates between men and women. The HTLV-I/II and HIV-I/II coinfection rate was 0.06/1000.

The epidemiological profile of the sample showed that HTLV-I/II infected individuals were older when compared with HIV-I/II infected individuals. Low social-demographic indicator was observed at both groups considering occupation.

P-135 Prevalence of HTLV-1/2 and Other Mandatory Detections in a Public Setting of Buenos Aires, Argentina from 2003 to 2008

Prevalence of HTLV-1/2 and other mandatory detections in a public setting of Buenos Aires, Argentina from 2003 to 2008

Introduction: Contrary to the recommended model of recruitment of altruistic or community volunteer blood donations proposed by the World Health Organization, Argentina's blood supply is mainly based on repository or replacement donations. At present, the detection of markers for HIV, HBV, HCV, T. cruzi, Brucella and T. pallidum is mandatory in blood banks in our country including HTLV-1/2 since December 2004. The aim of this study was to estimate the prevalence of and trends in transfusion-related infectious diseases in a public setting of Buenos Aires City.

Materials and methods: Information on epidemiological characteristics of blood donors and serological tests performed by routine screening procedures and confirmation tests were available from donor records from January 2003 to December 2008 at “Juan A. Fernandez” Hospital Blood Bank.

Results: A total of 28,483 blood donations were studied. Of them 7,442 (26.1%) were from females, 14,582 (51.2%) were younger than 35 years-old, and 23,746 (83.4%) were Argentine. The prevalence of EIA repeatedly-reactive samples was 0.8% for HTLV-1/2, 0.3% for HIV, 3.1% for HBV, 1.0% for HCV, 3.2% for T. cruzi, 0.3% for Brucella and 1.2% for T. pallidum. Out of 23,483, 25 samples were confirmed as HTLV-1/2 positive by WB yielding a final prevalence of 0.09%. There was a significant decrease in the prevalence of HBV and T. cruzi over time. The trend was consistent in male and female donors for HBV while for T. cruzi there was a decreasing tendency especially among men.

Conclusion: These results confirm Buenos Aires as a non endemic area for HTLV-1/2 infection. While HBV prevalence was higher the prevalence of HIV, Brucella and T. pallidum was similar when compared to reports from the Ministry of Health among blood donors from this city. These data suggest that blood safety measures may be helping to reduce the risk of some transfusion related infections; however, more screening programs need to be effectively targeted to reduce transfusion transmitted infections.

P-137 Progression of Bovine Leukemia Virus in Argentinean Dairy Cattle

More than 70% of dairy farms in Argentina are infected with Bovine Leukemia Virus (BLV). The individual rate of infection raises up to 100% in more than 30% of the farms. In this work we studied the progression of infection under natural conditions on a highly infected dairy farm since birth until the lactating age, where management practices to prevent the transfer of BLV-infected blood between animals have not been successful to prevent BLV spread. BLV-specific antibodies were found on 84% of the cows with more than one parturition, using an in-house developed ELISA that detects antibodies against the p24 viral protein. Lower reactivity on heifers (60%) evidenced that transmission is favored by time within the herd. Fifty four collostrated calves born from BLV infected dams were followed since birth until the 24 years old. Individual samples of collostrum and blood from their dams were also analyzed. Proviral DNA was detected on 4 out of 54 calves at 3–5 days old by conventional and real-time PCR. All these animals remained as reactive by PCR until the end of the study, evidencing that about 7% of infections are produced early in life, as a consequence of (i) an in utero infection or (ii) BLV horizontal transmission during the delivery or via the oral route by consuming collostrum and/or infected milk before the intestinal closure. Provirus was detected on collostrum of 8 out of 54 BLV-infected dams (14.8%) by nested PCR. Only one of the calves born from these cows was stated as early infected minimizing the impact of oral transmission by collostrum under natural conditions. No association between the proviral load on peripheral blood and the presence of provirus in collostrum was found. During the course of the study the infection rate gradually increased from 7 to 13% on the first year, and up to 20.4% on the second year, since proviral DNA and specific antibodies were detected in 2, 1, 1 and 3 more calves, at 3, 9, 15 months and 21 months old respectively.

Decrease of animal and herd prevalence is the main goal in our country. The development of programs for the control of BLV on dairy herds should be based on selecting an economical and practical strategy to interrupt virus spread. In our natural context, we described the first year of age as a good period to establish this strategy to effectively break the virus cycle of transmission.

P-138 Proviral Load and Clinical Follow-Up of Peruvian Children with HTLV-1 Infection

Background: In HTLV-1-endemic areas, mother-to-child transmission favors the acquisition of this infection during childhood. Except for infective dermatitis (ID), pediatric health effects of HTLV-1 have been scarcely reported.

Methods: At our Institute, HTLV-1 testing is performed among (1) subjects with a suspected HTLV-1-associated disease (including ID); (2) candidate blood donors with seropositive results during screening; and (3) relatives of HTLV-1-infected people. A standardized questionnaire is filled out prior to HTLV testing, and medical care is facilitated to seropositive cases. More recently, proviral load (PVL) is measured with a SYBR Green-based real-time quantitative PCR assay in peripheral blood mononuclear cells (PBMC) using primers targeting the pX region and normalizing the HTLV-1 copy number to the amount of ERV-3. We describe the main clinical events observed in HTLV-1 pediatric cases by medical specialists during subsequent, outpatient consultations at our Infectious Disease Unit. HTLV-1-positive pediatric cases were defined as those children diagnosed with HTLV-1 between 3 and 12 years old with confirmatory testing and/or with a mother proven to be HTLV-1 infected.

Results: Between June 1994 and February 2008, 102 HTLV-1-seropositive children less than 13 years old were evaluated at our Institute. For 84 children (82%), the HTLV-1 status of the mother was known: 72 out of 75 mothers (96%) were HTLV-1 positive. Overall, there were 91 HTLV-1-positive pediatric cases: 61 (67%) had a confirmed diagnosis of HTLV-1 and 30 (33%) lacked confirmatory testing, but their mothers were HTLV-1 infected. At the first visit, the mean age of seropositive pediatric cases was 8 years (standard deviation 3); 52 (57%) were girls. In 50 (57%), HTLV testing was done as part of family studies, whereas in 37 (43%) it was indicated because of a suspected HTLV-1-associated disease, such as ID (28/37; 76%) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP: 3/37; 8%). At baseline, 39/91 (43%) had signs or symptoms suggestive of diseases. Follow-up information was available for 62 (67%): the median time of follow-up was 2.5 years (IQR 6 months-14 years), with a cumulative time of 3.7 years/person. During follow-up, 15 clinical events were observed among 52 cases without symptoms at baseline: 7 episodes of strongyloidiasis, 3 additional cases of HAM/TSP, 2 episodes of keratitis, 1 episode of crusted scabies, 1 of Sjögren syndrome and 1 of ID. PVL results were available in 32 cases (35%): the mean values were significantly higher in the group with clinical events compared to “asymptomatic” cases: 36230 vs. 1220 copies/104 PBMC, p = 0.032).

Conclusion: Despite study limitations, our results suggest that children with HTLV-1 may require close follow-up in order to detect and manage a series of clinical events of uncertain health impact. In this setting, PVL might contribute to better define higher risk groups.

P-140 Selection of HTLV 1 and 2 Virus in Pregnant Women During Prenatal Care, São Luis, Feb/Dec – 2008

Introduction: HTLV is a retrovirus with tropism by T- lymphocytes; kind 1 is endemic in the Caribbean region, Japan, South America, and parts of Africa; kind 2 is found in some native American groups, rarely in Africa, and in Europe it is associated to the use of injectable drugs. In Brazil it is estimated that 2.5 million people are infected in all stages where it was researched, prevailing differently. HTLV-2 prevails more in Brazilian indigenous populations. The virus is transmitted through breast feeding, sexual contact without protection, blood transfusion and by sharing contaminated syringe. The infection by HTLV-1 is associated to T-Cells Lymphoma of the adult and Chronic Degenerative Neurologic Syndrome. The serologic diagnosis of the infection by the virus is made on the classification through the identification of anti-bodies anti-HTLV-1 and 2, and through confirmatory tests or diagnosis, being Western Blot the most used; but PCR is the gold standard.

Objectives: to identify the prevalence of HTLV-1 and 2 on the classification of pregnant women assisted during prenatal care at three public services.

Methodology: Transversal Study, executed between Feb/11 and Dec/03/2008, with 2044 pregnant women at three prenatal care public services, São Luis/MA. The patients were guided about the study, and included after signing the TCLE and filling in a questionnaire. The pregnant women who took part of the research were between 18 and 45 years old without psychiatric disease background, high blood pressure, nephropathy, diabetes and others that characterize the need for specialized prenatal care. The sample was calculated in 2041, test power of 95%, absolute mistake of 5%, Stata 9.0 program was used, Chi Square test was executed and p < 0.05 (95%) was accepted as the limit for significance. The project was approved by CEP/UFMA Opinion N°568/ 2007. On the classification, digital blood collection in filter paper was used and processed at NUPAD – MG laboratory, to be submitted to immuno-enzymatic experiment technique. The pregnant women that presented altered result were contacted for new collection of peripheral venous blood for western blot and PCR confirmatory tests.

Results: Out of the 2044 pregnant women who were evaluated, 07 of them presented altered results, 01 undetermined and 06 were positive, showing prevalence of 0.3%.

Conclusion: The sample showed that although high prevalence has not been identified, it is necessary a larger study that contemplates other extracts of the population so that the reality of the presence of the virus in the State of Maranhão can be identified.

Key words: human T-lymphotrofic virus; pregnant women; diagnosis serum

P-142 Societal Breastfeeding Customs in Small, Rural Communities: Can They Contribute to Endemicity of HTLV-1 in the Peruvian Andes?

Background: In a recent cross-sectional study, we found high frequencies of HTLV-1 infection in five rural communities of Ayacucho, a region in the southern Andes. In search of an explanation for the high HTLV-1 endemicity observed in these five communities, we evaluated a series of known risk factors for HTLV-1 transmission.

Methods: The main study was conducted in six rural, Quechua-speaking communities in the region of Ayacucho. We report here information on drug use, blood transfusion, sexual risk behaviour and breastfeeding collected in the five communities where we found HTLV-1 infection: Vilcashuaman (province Vilcashuaman) with 7323 inhabitants; and Cochani, Acos, Pinahua and Racchi (province Parinacochas) with 386, 321, 272 and 47 inhabitants, respectively. We interviewed 233 volunteers >12 years old (154 from Vilcashuaman and 79 from Parinacochas). The information was gathered in Quechua by trained staff prior to blood sampling for HTLV-1 tests. The frequency of HTLV-1 infection was 2% among the volunteers from Vilcashuaman and 10% among the volunteers from Parinacochas.

Results: None of the 233 volunteers had ever used intravenous drugs. Nine (4%) reported the history of a blood transfusion: 8/9 were women; and among these women, 6/8 had received the transfusion because of gynaecological or obstetrical pathology. None of the participants declared to be sex workers. The median number of lifetime sexual partners was 1 (interquartile range [IQR] 1–2); and the median age at the first sexual relationship was 18 years (IQR 16-20). 222 out of the 226 participants (98%) stated they had received breastfeeding. Among the volunteers from Vilcashuaman, 7% had been breastfed for <12 months; 76% for 12–24 months; and 18% for >2 years. Among the volunteers from Parinacochas, 5% had been breastfed for <12 months; 89% for 12–24 months; and 7% for >2 years. Eleven out of 202 (5%) reported that they had received breastfeeding at least once by someone who was not their biological mother. Five out of seven participants who knew the person who had given them breastfeeding beside their mother, reported that this person was a relative. Thirty out of 141 (21%) women stated that they had given breastfeeding at least once to a child not of their own; 15 out of 23 women (65%) reported that they were related to this child. Nineteen women explained why they had given breastfeeding to another child: in 8/19 cases, the biological mother had not enough breast milk; in 3/19, the biological mother had gone to work; in 2/19, the biological mother had died or was ill; and in 2/19, the children had been abandoned.

Conclusion: In addition to the duration of breastfeeding, specific communitarian customs, such as giving breastfeeding not only to the biological offspring, could contribute to endemicity-sustaining dynamics of HTLV-1 transmission in small, rural communities in Peru.

P-145 NK Cells Constitute a Major Reservoir In Vivo for HTLV-2 Infection

Early studies reported a preferential tropism of HTLV-2 for CD8+ T lymphocytes. Subsequent studies suggested that the virus can also infect CD4+ T-cells and non T lymphocytes such as B cells, particularly in subjects with a high viral burden. Here we show that NK cells are infected in vivo by HTLV-2 and constitute a major reservoir of infection.

PBMCs from HTLV-2 infected individuals were submitted to immune-magnetic isolation of CD4+, CD56+, CD8+ and CD19+ subpopulations. For fluorescence-activated cell sorting (FACS), PBMCs were stained with a combination of fluorescently-labeled antibodies for surface markers. Proviral load (PVL) was measured by a multiplex real-time PCR assay on DNA lysates extracted from total PBMCs and the isolated subsets. Intracellular staining was performed in isolated lymphocyte subsets after short-term cultures, in order to detect HTLV-2 Tax expression.

Six out of the 12 evaluated samples had undetectable PVL. CD8+ T cells showed the highest PVL levels, followed by CD56+ cells, CD19+ cells and CD4+ cells. The possibility that the PVL detected on NK cells was due to contaminants was excluded by detecting proviral sequences in highly purified NK cells isolated by FACS. HTLV-2 Tax protein could be detected in all cellular subsets harboring the provirus, after short-term cultures.

This study provides novel evidence that HTLV-2 naturally infects NK cells, at high proviral load levels. Infected NK cells could be potentially dysfunctional, resulting in viral escape from the innate immune surveillance. A clearer assessment of the in vivo spectra of the infection is essential for understanding virus – host interactions and the mechanisms of virus persistence.

P-147 Quantification of CD8+ T Cell Responses to HTLV-1: Balance of inflammatory and Antiviral Effects

Gene-expression microrarrays and ex-vivo assays of cytotoxic T lymphocyte (CTL) function have shown that the CTL effector function plays an important role in controlling HTLV-1 pathogenesis and proviral load. Our laboratory has previously developed an assay to quantify the rate of lysis of naturally infected CD4+ cells by autologous CTLs in fresh, unstimulated PBMCs. Using this assay, we showed that rate of lysis is inversely correlated with proviral load. We hypothesise that an efficient CTL response is associated with a low proviral load, and hence a low risk of development of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP).

One aim of the laboratory is to identify and quantify the factors responsible for the efficiency of the HTLV-1-specific CTL response at the single-cell level, using a physiologically relevant assay of T-cell function. CTLs possess an array of effector responses which include the release of perforin and granzymes from lytic granules, induction of apoptosis via Fas-FasL ligation, and the production of cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Traditionally, virus-specific CD8+ T-cell frequency has been estimated by enumerating CD8+ T cells which produce IFN-gamma in an antigen specific manner. However, lytic granule release is thought to be the primary method of killing in the lysis assay. CD107a, a lysosomal membrane protein, is exposed on the cell surface after degranulation, therefore may be more closely related to killing than standard IFN-gamma assay.

We plan to quantify degranulation of CTLs by detecting CD107a mobilisation in response to peptides spanning the entire tax protein. With this assay we aim to evaluate the contribution of CD107a+ CTLs to the rate of lysis and to the proviral load. Simultaneously, we plan to detect if a CD8+ antigen-specific cell is also producing either IFN-gamma or TNF-alpha or both. By studying the different effector mechanisms of CD8+ T cells we aim to test the hypothesis that the balance between beneficial (virus-killing) and harmful (inflammatory) mechanisms differs systematically between patients with HAM/TSP and asymptomatic HTLV-1 carriers.

P-148 Sensitivity and Efficiency of HTLV-1-Specific Cytotoxic T Lymphocytes

In human T-lymphotropic virus type 1 (HTLV-1) infection, a high frequency of HTLV-1-specific cytotoxic T lymphocytes (CTLs) can co-exist stably with a high proviral load, and the proviral load is strongly correlated with the risk of HTLV-1-associated inflammatory diseases. These observations led to the hypothesis that HTLV-1 specific CTLs are ineffective in controlling HTLV-1 replication but instead contribute to the pathogenesis of the inflammatory diseases. However, evidence from host and viral immunogenetics and gene expression microarrays suggest that robust expression of CTL effector functions are associated with a low proviral load and a low risk of HAM/TSP.

Using the well recognised Tax protein as an antigen, we quantified the frequency, lytic activity and functional avidity of HTLV-1-specific CD8+ cells in fresh PBMCs from individuals with natural HTLV-1 infection. Lytic efficiency was determined by estimating the rate of killing of HTLV-1-expressing infected CD4+ T cells by autologous CTL. Avidity and frequency of CTL were quantified using an interferon-gamma Elispot assay.

The lytic efficiency of the CD8+ T-cell response was inversely correlated with both the proviral load and the rate of spontaneous proviral expression. The functional avidity of HTLV-1-specific CD8+ cells was strongly correlated with their lytic efficiency. We conclude that efficient control of HTLV-1 in vivo depends on the CTL lytic efficiency, which depends in turn on CTL avidity of antigen recognition. CTL ‘quality’ determines the position of virus-host equilibrium in persistent HTLV-1 infection.

We are currently investigating the role of HLA type in determining functional avidity, and hence, lytic efficiency. In addition, we are characterising the frequency and functional avidity of HBZ specific CTL, in order to determine their contribution to the dynamic equilibrium between viral expression and host response.

P-150 The HTLV-1 Immune Signature: Identification of an HTLV-1 Specific Transcriptional Fingerprint by Microarray

The Human T Lymphotropic Virus Type 1 (HTLV-1) particle represents a unique combination of molecular patterns which are recognized by the pattern-recognition and antigen-specific receptors on peripheral blood immune cells. The subsequent signalling process as well as the action of viral proteins themselves result in distinct patterns of gene expression that reflect the interaction between HTLV-1 and the host immune system (“HTLV-1 immune signature”).

The aim of this project is to identify an HTLV-1 specific transcriptional profile in peripheral blood cells by performing microarrays on whole blood samples from infected patients. Data analysis follows a modular framework approach to facilitate the detection of the immune signature against background noise: co-ordinately up- or down-regulated gene clusters are assigned to previously objectively identified transcriptional units (modules). These modules are associated with functional characteristics, e.g. cytolytic genes, inflammatory genes or genes commonly expressed in specific cell types. Mapping changes in gene expression at the module level in patients in comparison to uninfected controls and data of other diseases then generates the HTLV-1-specific transcriptional fingerprint.

The microarray data is complemented with an extensive phenotypic characterisation of blood monocytes, dendritic cells and lymphocyte populations by 9-colour flow cytometry. Correlation of these data sets with proviral load, Tax expression and disease status will suggest new hypotheses as to the molecular mechanisms of HTLV-1 pathogenesis and persistence.

This study is funded by the Wellcome Trust (UK).

P-151 The Molecular Mechanism in the Differentiation of HTLV-1 Infected CD4+CD25+CCR4+ T-Cells through HTLV-1 Tax

Human T-lymphotropic virus type I (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is known as HTLV-1 infected T-cells triggered hyper immune response, which leads to inflammation of the central nervous system. Recently, we demonstrated that the majority of CD4⁺CD25⁺CCR4⁺ T-cells were infected with HTLV-1 in HAM/TSP- as well as ATL patients. When we compared these HTLV-1 infected CD4⁺CD25⁺CCR4⁺ T-cells between ATL and HAM/TSP, CD4⁺CD25⁺CCR4⁺ T-cells of ATL showed high Foxp3 expression (a marker of regulatory T-cells) and low proinflammatory cytokines expression. On the other hand, CD4⁺CD25⁺CCR4⁺ T-cells of HAM/TSP showed low Foxp3 expression and significantly high levels of Th1 type cytokine IFN-?. Importantly, the levels of HTLV-1 tax expression in CD4⁺CD25⁺CCR4⁺ T-cells of HAM/TSP was extremely higher than that of ATL. Therefore, we hypothesized that HTLV-1 tax expression may influence the differentiation and function of CD4⁺CD25⁺CCR4⁺ T-cells toward IFN-? producing Th1-like T-cells.

To investigate whether HTLV-1 tax can effect the differentiation of T-cells, we analyzed the expression of IFN-? using JPX-9 cells which can be induced the HTLV-1 tax expression by CdCl₂. Furthermore, to determine the target molecules of HTLV-1 tax in inducing the IFN-? expression, we initially compared the gene expression pattern of CD4⁺CD25⁺CCR4⁺ T-cells between HAM/TSP-, ATL- patient and healthy donor using DNA microarray analysis. Within the differentially expressed genes, we identified the molecule which is regulated by HTLV-1 tax in controlling the IFN-? expression and Th1 differentiation.

P-152 The Rate of T Cell Clonal Expansion and Proviral Load Levels among Human T Cell Lymphotropic Virus Type 1 (HTLV-1) Asymptomatic Carriers

Introduction: Early prediction of Adult T Cell Leukemia (ATL) in HTLV-1 carriers can be difficult before a disease becomes clinically evident. In this study we aimed to determine the rate of T cell clonality among HTLV-1 asymptomatic carriers with different titers of proviral load (PL).

Methods: We first applied a sensitive SYBR Green real time PCR on mononuclear cells from cohort of 213 HTLV-1 serological positive samples of asymptomatic subjects. PL was classified into three categories; low titer (=50 copies/1000 PBMCs), intermediate titer (50-100 copies/1000 PBMCs) and high titer (=100 copies/1000 PBMCs). All patients were assessed for their T cell receptor multiplex PCR.?rearrangements by TCR.

Results: Of the 213 samples tested for HTLV PL, 72 (33.8%) were classified as low PL, 64 (30.0%) had intermediate PL. and 77 (36.2%) had high PL. Of the 72 samples with low PL, oligoclonal and monoclonal populations were detected in 10 (13,9%) and 4 (5,6%), respectively. Of the 64 samples with intermediate PL, oligoclonal and monoclonal populations were detected in 19 (29,7%) and 13 (20,3%), respectively. Of the 77 samples with high PL, oligoclonal and monoclonal populations were detected in 23 (29,9%) and 36 (46,8%), respectively (p trend < 0.05).

Conclusions: Our results showed higher rate of oligoclonal and monoclonal T cell proliferation in HTLV-1 asymptomatic carriers particularly those who have PL of = 100 copies/1000 PBMCs. Moreover, it provided evidence that both HTLV PL and clonality test are capable to accurately identify HTLV-1 asymptomatic carriers with a low preleukemic clone density who are likely to progress to ATL, hence facilitate disease staging.

P-153 Immunomodulatory Properties of the Tax Protein on Primary Human Dendritic Cells

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense cytotoxic T cell (CTL) response directed against the viral transactivator protein Tax. Additionally, patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DCs) likely contributing to the robust Tax-specific CTL response. In this study, extracellular Tax has been shown to induce maturation and functional alterations in human monocyte-derived DCs, critical observations being confirmed in freshly isolated myeloid DCs. Tax was shown to promote the production of proinflammatory cytokines and chemokines involved in DC activation process in a dose- and time-dependent manner. Furthermore, Tax induced the expression of DC activation (CD40, CD80, and CD86) and maturation (CD83) markers and enhanced the T cell proliferation capability of DCs. Heat inactivation of Tax resulted in abrogation of these effects indicating a requirement for the native structure of Tax. Tax was found to efficiently bind to the DC membrane and was internalized within a few hours suggesting that extracellular Tax may possess an intracellular mechanism of action subsequent to entry. Finally, inhibitors of cellular signaling pathways NF-?B, protein kinase, tyrosine kinase and phospholipase C were shown to inhibit Tax-mediated DC activation. This is the first study reporting the immunomodulatory effects of extracellular Tax in the DC compartment. These results suggest that DCs, once exposed to Tax by uptake from the extracellular environment, can undergo activation providing constant antigen presentation and costimulation to T cells leading to the intense T cell proliferation and inflammatory responses underlying HAM/TSP.

P-159 PIN1 Interacts and Modulates Tax's Activation of NF-KB

NF-□B activity been shown to be critical for multiple biological processes including cell growth, cellular. The current model suggests that IKK complex (IKK □, □, □) is a nexus for NF-□B signaling. In this regard, transformation of cells by the oncoprotéine Tax requires binding to IKK□/NEMO.

Here, we describe a cellular factor that influences Tax-IKK□□?interaction. We show that the peptidyl-prolyl cis-trans isomerase (PPIase) Pin1 regulates Tax activity and contributes to its signaling through NF-□B. An unexpected finding from our study is the over expression of Pin1 in ATL cells. Indeed, Pin1 is an E2F downstream target gene, whose expression is strongly regulated in normal cells. However several studies show that in cancer cells, Pin1 levels are elevated. The prevalent deregulation of E2F/Rb pathways in many human cancers may play a critical role in the up-regulation of Pin1 in human cancers. Indeed, we also show that Tax interacts with Pin1 both in transfected HEK 293T cells and in HTLV-1-transformed cell lines. Moreover, knockdown of Pin1 impair Tax/ IKK□ binding and attenuate for its induction of anchorage independent foci formation of cell growth.

Taken together, our findings suggest that Tax is a Pin1 substrate and that Pin1-mediated isomerization is an important regulator of Tax's protein-protein interaction. Our experiments describe a positive Tax-Pin1 feedback circuit. In HTLV-1 cells, we envision that expression of viral Tax protein first activates E2F to enhance the transcription and subsequent expression of Pin1. In return, Pin1 recognizes phosphorylated Tax to regulate the latter's interaction with IKK□, enhancing Tax's transformation potential through NF-□B. Our current results expand the range of Pin1's oncogenic involvement to include ATLs. Because Pin1 is normally expressed at very low levels in most tissues and Pin1 knockout mice reach adulthood, it could be that there is a reasonable treatment window in which anti-Pin1 therapy might be effective without creating significant general toxicity. This notion may merit further exploration for its ATL-treatment potential.

P-161 Subclassification of Adult T-Cell Leukemia-Lymphoma (ATLL) Using the Novel nCounter Gene Expression Detection System

The nCounter gene expression system captures and counts individual mRNA transcripts from total RNA or crude cell lysate in limited quantities using gene specific fluorescently labeled oligonucleotide probes without performing any enzymatic reactions or amplification steps. We assembled a panel of 156 genes encoding proteins involved in innate immunity, oncogenesis, cell proliferation and apoptosis, in order to assess a cohort of primary adult T-cell leukemia-lymphoma (ATLL) specimens. ATLL is a fatal lymphoid malignancy caused by the human T-cell lymphotrophic virus type I (HTLV-I). This disease is subclassified into four different variants, of which the most common and aggressive ones are the acute and lymphomatous types. A key molecular feature of ATLL is the high constitutive expression of NF-?B, which is known to exert a predominantly anti-apoptotic effect which likely contributes to treatment resistance. In a preliminary analysis of twenty-four primary ATLL tumor samples, we found differential expression of several genes, including well known NF-?B targets, between different disease forms. In order to validate our assay, we compared the performance of the nCounter system with a custom Taqman array and we demonstrate that both systems have high concordance and comparable sensitivities in regards to differential gene expression and fold-change measurements. We have just completed the experiments involving 60 ATLL tumors obtained from the United States, mostly from patients of Caribbean origin, and from Brazil, and we are currently evaluating their data. In conclusion, the nCounter system can help subcharacterize tumors at the molecular level and serve as potential tool for generating gene expression data of clinical or prognostic value. A comprehensive analysis of gene expression data obtained from our ATLL cohort will be presented at the meeting.

P-162 The Role of Tax-2 in Modulation of CCL3L1 Expression, the Most Potent HIV-1 Suppressive CC-Chemokine

Background: A substantial percentage (10–20%) of IDUs dually infected with HIV-1 and HTLV-2 are associated with a LTNP condition. In co-infected patients, the delayed progression to AIDS was related to an observed up-regulation of CCL3L1, the most potent CCR5 ligand and HIV-1-inhibitory chemokine. Recently, we demonstrated that in HTLV-2/HIV-1 multiply exposed-uninfected seronegative and LTNP co-infected subjects, the up-expression of CCL3L1 is independent by CCL3L1 genotype but triggered by HTLV-2 infection via a post-genomic mechanism. As Tax-2 transcriptional activator is known to transactivate the promoters of MIP-1beta and RANTES, here, we investigated Tax-2 function on CCL3L1 induction.

Methods: In order to transfect tax-2 in an inducible system, the Jurkat Tet-On cells, showing 1 copy number pdg of CCL3L1 gene and no expression of its mRNA, were selected. After cloning the tax-2 gene, derived from HTLV-2 Gu isolate, into a responsive bidirectional tight expression plasmid, expressing the reporter gene AcGFP, the vector was transiently transfected in proliferating cells by nucleofection. Genes' expression was turned on by the addition of doxycycline (Dox) and transfection efficiency was calculated based on FACS results. Once induced, tax-2 as well as CCL3L1 mRNA expression were evaluated by real-time PCR. In addition, cellular distribution of Tax-2 protein was visualized by indirect immunofluorescence.

Results: Relative mRNA level analysis showed that tax-2 expression peaks already at 8 hours post-Dox activation (14-fold with respect to unstimulated control) and decreases after 24 hours of culture. In addition, CCL3L1 mRNA expression peaks at 24 hours, exhibiting an increased level of 700-fold relative to unstimulated culture. Thus, CCL3L1 promoter is quickly responsive to Tax-2, and although expression of Tax-2 is present at low levels, its efficiency of transactivation is very high. Immunofluorescence displays both nuclear and cytoplasmatic localization of Tax-2 protein.

Conclusions: Taken together, our findings confirm a crucial protective role of CCL3L1 from both HIV infection and disease progression, and clearly demonstrate that Tax-2, being able to activate CCL3L1 promoter, acts as key factor in the inhibition of HIV-1 infection sustained by HTLV-2, a virus not yet disease associated. Studies are ongoing to identify the mechanisms by which Tax-2 controls CCL3L1 expression.

P-163 HTLV-1-Associated Myelopathy: Impact of the Neurological Deficit in the Quality of Life

Introduction: The human T-cell lymphotropic virus type 1-associated myelopathy (HAM/TSP) is caused by HTLV-1 infection. The predominant clinical feature is the spastic paraparesis usually accompanied by sphincters' disturbances. Functional impairment compromises the patient's quality of life. Assessment measures for activities of daily life and disabilities turns to be important to define specific and effective goals and interventions for these patients.

Objectives: 1. To assess functional capacity of patients with HAM/TSP; 2. To describe the functional capacity of these patients using the Functional Independence Measurement (FIM); 3. To demonstrate the application of the Expanded Disability Status Scale in gait assessment; 4. To determine the prevalence of gait disturbances using Osame's scale; 5. To compare the EDSS, FIM and Osame's scores in relation to WHO's quality of life indicator; and, 6. To propose an assessment protocol to measure functional capacity in HAM/TSP.

Methodology: Descriptive, transversal study with 30 patients with HAM/TSP attending the Neuroinfection outpatient clinic of the Gaffree Guinle Hospital, between February 2008 and February 2009. Patients were submitted to neurological exam and physiotherapeutic evaluation. EDSS, FIM and Osame's scale and the SF-36 survey were applied. Concordance level among the instruments was evaluated.

Result: The studied group presented mean ± SD evolution time of 10.6 (+7.6) years and represented 66% of the group. Dependence condition was the result from the FIM score (70%), EDSS score (67%) and Osame score (67%). Coefficient kappa demonstrates that there is concordance among the instruments' scores. The domains “functional capacity” and “limitation because physical condition” of the quality of life measure suggests that the lack of functional independence may compromise the patient's quality of life.

Conclusion: The progressive, invalidating gait disturbance compromises the functional independence for the activities of daily life. The measurements used in this study describe the majority of the patients as dependent, and the impact of this dependency is showed in the quality of life score. The concordance among the instruments suggests the importance in the elaboration of a protocol based on a serial assessment using these scales.

P-164 Methylprednisolone in Subacute HAM/TSP

Introduction: HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic disabling disease caused by HTLV-I. It is estimated that between 10 and 20 million people are infected worldwide but only a small proportion will develop neurologic complications. HAM/TSP is usually described as myelopathy with an insidious onset and a slow evolution over the years. However, there are reports of patients with an aggressive course and early disability. The factors implicated in this subacute evolution as well as the response to treatment with high-dose methylprednisolone are not known to date.

Methods: We enrolled six patients with subacute HAM/TSP, defined as the requirement of a wheel chair during the first 2 years after the onset of symptoms, followed at the HTLV outpatient clinic at the Instituto de Pesquisa Clinica Evandro Chagas- FIOCRUZ, Rio de Janeiro, Brazil. Participants underwent bimonthly interviews which included complete neurological examination and determination of HTLV-I proviral load. Neurological disability was determined trough the IPEC disability scale, which was developed exclusively for the prospective assessment of HAM/TSP and the Extended Disability Status Scale (EDSS). A thoracic spinal cord MRI was perfomed in 4/6 individuals. The 6 patients received methylprednisolone 1g/day for three consecutive days every month for a total of six months.

Results: There were three men and three women and the mean age at the onset was 46.4 years. The HTLV-I proviral load before treatment was 9.17 copies/100 cells (range 0.4–14.82) and there was no significant decline after. The findings at the neurological examination were typical of HAM/TSP but a thoracic sensory level was present in 2/6 participants. The disability remained unchanged in all but one patient, who was able to walk with bilateral support after six months of treatment. The adverse effects were mild and transient except for disseminated candidiasis in one patient which led to the interruption of treatment. Thoracic spinal cord MRI was normal in all cases studied.

Conclusions: there is a small group of patients with HAM/TSP who have an aggressive evolution of disability despite a relative low HTLV-I proviral load. In two of our patients, we observed a sensory level, which is uncommon in patients with typical HAM/TSP, but we were not able to determine any radiological sign associated with this subacute evolution. High-dose methylprednisolone is well tolerated but is inefective in this subgroup of patients.

P-167 Vestibular-Evoked Myogenic Potential (VEMP) to Evaluate Cervical Myelopathy in Human T Cell Lymphotropic Virus Type I infection

Introduction: Vestibular-Evoked Myogenic Potential (VEMP) is generated by acoustic or galvanic stimuli, passing through the vestibulo-spinal motor tract, the spinal nerves and recorded from surface electrodes on the sternocleidomastoid muscle. This test may be of value to investigate sub-clinical cervical mielopathy. HAM/TSP is a progressive inflammatory myelopathy with predominant lesions at the thoracic spinal cord level, although cervical spine can be affected.

Objective: To define clinical usefulness of VEMP to detect cervical medular involvement related to human T cell limphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Results: VEMP had normal values in the seronegative group (30 controls). In the seropositive, abnormal VEMP was seen in 11/22 (50%) of the HTLV-1 asymptomatic carriers, 7/10 (70%) of those with complaints of walking difficulty and 8/10 (80%) of the HAM/TSP patients. In this last group, the pattern of response was different. No VEMP response was more frequent when comparing to the asymptomatic carriers (2-tailed P value = 0.001).

Conclusions: VEMP may possibly be useful to identify patients with subclinical cervical myelopathy and to distinguish variable degrees of functionalinjury. Minor injury would be related to latency prolongation and major injury to no VEMP response.

P-169 Presentation of HTLV-1 Tax Protein by Dendritic Cells: The Underlying Mechanism for the HTLV-1-Associated Neuroinflammatory Disease

HTLV-1 is the etiologic agent of a debilitating neurologic disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease features a robust immune response including the oligoclonal expansion of CD8+ CTLs specific for the viral oncoprotein Tax. The key pathogenic process resulting in the proliferation of CTLs and the presentation of Tax peptide remains uncharacterized. We have investigated the role of APCs, particularly dendritic cells (DCs), in the priming of anti-Tax CTL response under both in vitro and in vivo conditions. We investigated 2 routes (direct verus indirect) of Tax presentation using live virus and CD4+ T-cell lines (MT-2, an HTLV-1-infected cell line, and C8166, an HTLV-1-mutated line that only expresses Tax), respectively. Our results indicated that DCs are capable of priming a pronounced Tax-specific CTL response in cell cultures consisting of naive PBLs as well as in HLA-A*0201 transgenic mice (line HHD II). DCs were able to successfully direct the presentation of Tax through infected T cells, live virus, and cell-free Tax. These observations were comparable to those made with a known stimulant of DC maturation - a combination of CD40L and IFN-?. Our studies clearly establish a role for this important immune cell component in HTLV-1 immuno/neuropathogenesis and suggest that modulation of DC functions could be an important tool for therapeutic interventions.

P-171 Regulation of HTLV-1 Promoter Formatted in the Context of Chromatin in T Cells

Activation of human T cell leukemia virus type 1 (HTLV-1) promoter or long terminal repeat (LTR) is critical for the expression of viral genes and subsequent infectious virus production. Like eukaryotic DNA, the integrated proviral DNA is coiled with histone and nonhistone proteins to form nucleosomal structures that comprise the basic unit of chromatin; thus the proviral genome functions as a surrogate cellular transcriptional unit. Most studies concerning HTLV-1 LTR regulation have been performed with the transiently transfected promoter, which has been shown to differ physically from the integrated proviral DNA. Herein, the effect of the HTLV-1 transactivator protein Tax on the chromosomally integrated viral LTR was examined in the Jurkat CD4+ T cell line, a widely used model for the primary infected cell population during the course of human infection. This cell line was stably integrated with the HTLV-1 LTR driving the indicator gene luciferase and propagated in multiple single-cell clones. Comparative transcription factor profiling of these stably integrated clones with transiently transfected T cells in the presence of Tax indicated specific activation of substrates and factors associated with chromatin remodeling complexes. Using microRNA microarray and bioinformatics tools, Tax was demonstrated to down-regulate the expression of microRNAs associated with the regulation of factors required for chromatin remodeling. These results demonstrate that Tax modulates the cellular microRNA pathway to control viral transcription by down-regulating microRNAs responsible for regulating expression of chromatin remodeling factors. These observations offer insight into activation and transcriptional regulation of HTLV-1 by the host RNA interference pathway.

P-172 Tax Expression in the Early Pre-Tumoral Clone: Observations in the BLV Ovine Leukemia Model

Although targeting different lymphocyte subsets, BLV and HTLV-1 share a multitude of structural and functional similarities, and in both cases, mechanisms of malignant progression are dependent on the viral Tax protein. In HTLV-1-associated diseases the latency period might extend over several decades and early samples are rarely available for analysis. The adequacy of the BLV-associated ovine leukemia as a model is supported by the complete penetrance of disease in BLV-infected sheep, the relatively short period before tumor onset, and the possibility of exploring transformation-initiating events that precede aggressive malignancy in vivo. Although Tax plays a critical role in transformation, its expression is frequently abolished in malignant cells. Tax silencing results from either genetic or epigenetic changes and is believed to contribute to leukemia progression possibly through immune escape mechanisms. Here, we examined pre-tumoral cell populations isolated from BLV-infected sheep using IPCR techniques and clonotypic amplification of tumor-specific BLV integration sites. We found that a genetic defect previously shown to be responsible for Tax inactivation in leukemic B-cells was present in the early pre-leukemic clone, indicating that silencing occurred prior to leukemia onset. These observations suggest that the Tax oncoprotein can be switched off at initial stages of the oncogenic process. Our findings might be of interest for understanding how the subtle equilibrium between Tax expression, Tax silencing, and host immune responses impact on tumor progression.

P-173 Abstract Withdrawn

Iha H. Nishizono A. Ikebe E. Peloponese J. Jeang K. Nakagawa-Motohara M. Ogata M. Yedavalli V.R. Oita University Faculty of Medicine

P-174 TAX1BP1 in ATL Cells

Nuclear factor kappa B (NF-?B) is a key mediator of immune or anti-apoptotic responses against multiple outer cell stimuli. Unchecked NF-?B signaling, however, can engender autoimmune pathologies and cancers. Tax oncoprotein of human T-lymphotropic virus type-1 (HTLV-1) persistently activates NF-?B and immortalizes cells and eventually develops a recalcitrant adult T-cell leukemia/lymphoma (ATL). Tax1-binding protein 1 (TAX1BP1) has originally isolated by its ability to bind with Tax and we have demonstrated that TAX1BP1 is a negative regulator of TNF-a- and IL-1ß-induced NF-?B activation and that binding to mono- and polyubiquitin by a ubiquitin-binding zinc finger domain in TAX1BP1 is needed for TRAF6 association and NF-?B inhibition. We have also demonstrated that mice genetically knocked out for TAX1BP1 are born normal, but develop age-dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF-a and IL-1ß. Mechanistically, TAX1BP1 acts in NF-?B signaling as an essential adaptor between A20 and its targets in normal cells. To know how Tax disturbs TAX1BP1's normal function in ATL cells, the cytoplasmic and nuclear fractions of ATL cells were probed with anti-Tax antibodies. Although TAX1BP1 is intrinsically a cytoplasmic protein, significant amount of TAX1BP1 in nucleus was detected in all ATL cells and the whole protein expression level of TAX1BP1 was strongly enhanced. We are in the process of proving its detailed mechanism.

P-176 The 8 kDa Form Encoded by the HTLV-I ORF-I is Associated with Low Provirus Levels in a Subset of HTLV-I Infected Individuals

Human T-Leukemia/Lymphoma virus type I (HTLV-I) is etiologically associated with a neoplastic malignancy of T-lymphocytes Adult T-cell Leukemia/Lymphoma (ATL/L) and a neurodegenerative disorder HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-I open reading frame I (orf-I) encodes highly hydrophobic non-structural proteins. Expression of the singly or doubly spliced orf-I cDNA results in the production of the 12 kDa and 8 kDa protein isoforms. The uncleaved 12 kDa form resides in the ER and likely affects MHC-I and chains of the IL-2R in this locale. In contrast, the cleaved 8 kDa isoform traffics to the cell surface, is recruited to the immunological synapse upon T-Cell Receptor (TCR) stimulation and decreases TCR signaling and viral replication. Genetic analysis of orf-I from ex vivo samples of HTLV-I infected individuals reveals predominant amino acid substitutions within orf-I that affects proteolytic cleavage, suggesting that ER or membrane associated functions of the orf-I protein products may contribute to the survival and persistence of HTLV-I infected T-cells in the host. To investigate whether there is a relationship between p12 isoforms and virus level, which is the best predictor of disease development, we performed a study whereby the provirus level in blood was measured and its level associated with the presence of the 8 kDa or the 12 kDa isoforms or both. We obtained samples from 100 HTLV-I infected individuals from the Caribbean, France, Brasil and Africa. The provirus levels in these individuals ranged from 0.2 to 254 copies of proviral DNA per 10^6 PBMCs. DNA sequencing and reverse genetics demonstrated mutations at or in the vicinity of both cleavage sites within orf-I that affected cleavage. We found rare mutations at position 26 (D to N/E) in orf-I that resulted mainly in the presence of the 8 kDa isoform. Importantly, patients that carried this mutation had significantly lower provirus level that those that carried either the 12 kDa isoform or both isoforms. Analysis on a larger cohort of individuals will be required to unambiguously demonstrate that the 8 kDa form truly affects proviral load in HTLV-I infection.

P-177 The Effect of the Histone Deacetylase Inhibitor Valproic Acid on Tax Expression, Spontaneous Degranulation and Cell Survival in HTLV-I Infected Peripheral Blood Mononuclear Cells

The effect of the histone deacetylase inhibitor valproic acid on Tax expression, spontaneous degranulation and cell survival in HTLV-I infected peripheral blood mononuclear cells. Jhanelle Graham, Unsong Oh, Yoshimi Akahata, Steven Jacobson.

Background: Recent studies aimed at exposing transcriptionally silent HTLV-I infected lymphocytes by epigenetic modification suggest that the use of histone deacetylase inhibitors such as valproic acid might decrease proviral loads in subjects with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Objective: We examined the effect of valproic acid on key mechanisms determining proviral loads in HTLV-I infected cells including viral gene expression, HTLV-I-specific cytolysis and cell toxicity to analyze the relative impact of each on HTLV-I proviral load during treatment.

Methods: Peripheral blood mononuclear cells (PBMC) from subjects with HAM/TSP were cultured in the presence or absence of valproic acid. After 18 hour culture, cells were assayed for Tax protein expression, spontaneous CD107a degranulation and cell survival by flow cytometry.

Results: Tax protein expression was increased in HAM/TSP PBMC treated with valproic acid (p = 0.008) in a dose dependent manner. CD8 T cell-depleted PBMC showed increased Tax expression with valproic acid, but to a lesser extent. In contrast, purified CD4+ T cells from HAM/TSP subjects showed decreased Tax expression in the presence of valproic acid (p = 0.02). Spontaneous CD107a mobilization, a marker of cytolytic degranulation, was inhibited by valproic acid in a dose dependent manner in HAM/TSP PBMC. Significantly decreased cell survival of monocytes (14% survival) was observed in the presence of valproic acid (p = 0.017).

Conclusion: The impact of valproic acid on HTLV-I proviral load cannot be explained by increased viral gene transcription alone. The effect of valproic acid on inhibition of spontaneous degranulation and impaired cell survival of monocytes suggest the need to further characterize the role of valproic acid in HTLV-I infection.

P-178 The Receptor Complex Associated with HTLV-3 ENV-Mediated Binding and Entry is Distinct from, but Overlaps with, the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus HTLV-3. We have therefore examined the entry requirements of HTLV-3. We observed that HTLV-3 SU protein binds efficiently to both activated CD4+ and CD8+ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4+ T cells, and HTLV-2 SU, which primarily binds to activated CD8+ T cells. Neuropilin 1 (NRP-1) and the glucose transporter GLUT1, are involved in both HTLV-1 and HTLV-2 entry. For HTLV-1, but not HTLV-2, efficient binding and entry also requires heparan sulfate proteoglycans (HSPGs). Binding studies with HSPGs and NRP-1 revealed that these molecules enhance HTLV-3 SU binding. However, unlike HTLV-1, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, as for HTLV-1, the glucose transporter GLUT-1 functions at a post-binding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4+ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1 and GLUT-1. Our results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.