Influence of Polymorphism (-G308A) TNF-α on the Periportal Fibrosis Regression of Schistosomiasis After Specific Treatment

Abstract

Aims: Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine and important mediator of severity for periportal fibrosis (PPF). We hypothesized that the (-G380A) polymorphism in the TNF-α gene is associated with regression of PPF after treatment for schistosomiasis mansoni. Methods: This is a retrospective cohort study, involving 124 Brazilian patients infected with Schistosoma mansoni, who were followed for 2 years after treatment to estimate the likelihood of PPF regression. Sociodemographic and clinical factors were also identified, with emphasis on specific treatment. Results: No statistical difference was observed between sociodemographic and clinical factors among the exposed groups. Genotypes (-308) GA/AA were positively associated with the degree of PFF regression (relative risk [RR] = 0.52; ρ = 0.025), as well as in the image pattern of PPF (RR = 0.56; ρ = 0.048), when compared with the genotype (-308) GG. There was no statistical difference in TNF-α serum levels between the exposed groups. Conclusions: These results suggest that the (-G308A) polymorphism of the TNF-α gene may be one of the factors that prevents the regression of the degree and pattern of PPF in the Brazilian population, and thus it may potentially be a predictive factor of PPF intensity in schistosomiasis.

Introduction

Schistosomiasis is an endemic chronic parasitic disease that affects ∼240 million people in the world and, therefore, constitutes a significant public health problem (WHO, 2014). In Brazil, it is estimated that close to 6 million people are infected by Schistosoma mansoni, mainly in the Northeast region of the country with emphasis on the state of Pernambuco, which occupies third place in prevalence and is responsible for the greatest number of deaths by this illness in the country (Pordeus et al., 2008).

The pathology resulting from infection is predominantly caused by the host's immune response to parasite eggs, which are deposited in the liver triggering a chronic granulomatous inflammation, with consequent periportal fibrosis (PPF) and obstruction of the blood flow of the intrahepatic portal vein (PV) branches (Magalhães et al., 2004; Pordeus et al., 2008). This process can lead to the development of portal hypertension, with consequent splenomegaly and appearance of varicose veins in the esophagus. Upper gastrointestinal bleeding caused by rupture of the esophageal varices occurs in 12-15% of the patients and it is the cause of death in around 20% of these cases (Andrade, 2009).

The development and severity of PPF depends mainly on several immunogenetic factors, but may also be influenced by environmental aspects. Among these factors the most cited are gender, age, parasite burden, alcoholism, and frequency and duration of exposure (Silva et al., 2013). By contrast, specific treatment for schistosomiasis may allow partial or complete reversal of PPF, and immune mechanisms may be involved in this process (Andrade, 2005; Guevara et al., 2007).

In addition, the study showed that the hepatic granulomas and the resulting PPF depended on the action of cytokines, and that the tumor necrosis factor alpha (TNF-α) played a central role (De Jesus et al., 2002; Henri et al., 2002). This cytokine is an important mediator of PPF severity. TNF-α plays a key role in the formation of granulomas through schistosomiasis infection and high levels of cytokines were significantly associated with the presence of hepatosplenomegaly and increased risk of developing severe PPF (De Jesus et al., 2002; Henri et al., 2002; Booth et al., 2004).

The TNF-α gene is located in the human leukocyte antigens class III on chromosome 6 p 21.3 (Bouzgarrou et al., 2010). Polymorphism at position -308 of the promoter region of the TNF-α gene—which involves the replacement of the guanine (G) nitrogenous base by adenine (A), the creation of two alleles (-308G and -308A) and three genotypes (GG, GA, and AA)—has been associated with an increase in the expression of TNF-α (Jrad et al., 2007; Bouzgarrou et al., 2010; Qin et al., 2010; Radwan et al., 2012).

The -308A allele is associated with increased levels of the TNF-α constituent in comparison with allele-308G (Dai et al., 2006; Jeng et al., 2007). This association is a possible explanation for the progression of hepatic fibrosis (Wilson et al., 1997). A relationship of causality between polymorphic variations that influence the expression of TNF-α and its effect on the severity of PPF in schistosomiasis needs to be better clarified (Dessein et al., 2009).

A study carried out in a Sudanese population evaluated the influence of polymorphism (-G308A) TNF-α in the severity of schistosomotic PPF, showing no evidence of major effects on the PPF (Moukoko et al., 2003). In addition, there has been no report to date of cohorts evaluating concurrent influence of polymorphism (-G308A) TNF-α on PPF regression. This study investigates the association between polymorphism (-G308A) TNF-α and PPF regression in patients with S. mansoni, after specific treatment, in endemic populations in the state of Pernambuco, Northeast of Brazil.

Material and Methods

Subjects

This is a retrospective cohort study retrieved from a previous study of 193 patients infected with S. mansoni and subjected to specific treatment for schistosomiasis, which determined polymorphism (-G308A) TNF-α and serum concentrations.

In this cohort study 124 patients were listed as being infected with S. mansoni. They were recruited in 2012 and 2013, and were followed up retrospectively for 2 years after specific treatment for schistosomiasis mansoni (SM) to estimate the probability of PPF regression. Sociodemographic and clinical factors were also identified, with emphasis on specific treatment for SM.

These patients were divided into two groups: group 1 (exposed), including 71 patients with genotypes (-308) GA or AA; and group 2 (not exposed), which included 53 patients with genotype (-308) GG. These patients were over 18 years of age and were treated in the Gastroenterology Clinic of the Hospital das Clínicas at the Federal University of Pernambuco (HC-UFPE), Recife, Brazil. All patients of this study came from endemic areas for schistosomiasis, in the state of Pernambuco, Northeast of Brazil.

This study only included patients who had undergone abdominal ultrasound, including a PPF evaluation, following the protocols of Niamey (Richter et al., 2001) and Cairo (WHO, 1993)—before treatment of schistosomiasis and also 2 years after treatment.

The outcome of interest was the regression of the ultrasound assessment of identified cases with PPF in the upper abdomen. The main exhibition was polymorphism (-G308A) TNF-α in the context of the study hypothesis, as a factor associated with PPF severity. The information about these genotypes and the serum levels of TNF-α were obtained for the years 2012 and 2013.

To determine the genotypes GG, GA, and AA, biological samples from all patients were previously subjected to polymerase chain technique for polymorphism analysis by restriction fragment (PCR-RFLP), followed by digestion through restriction of enzyme NcoI to detect the Single nucleotide polymorphism (SNP) in the promoter region of the TNF-α gene (rs1800629). This consisted of the replacement of the nitrogenous base guanine (G) by adenine (A), at position -308 (Cabrera et al., 1995), to define the exhibition groups mentioned above. Serum levels of TNF-α were measured using Enzyme Linked Immunosorbent Assay-the commercial ELISA kit (Biosource, Invitrogen Corporation, Carlsbad, CA), according to the manufacturer's instructions. The results were expressed as pg/mL, based on standard curves (sensitivity < 1.7 pg/mL).

Other explanatory variables were sex, age, contact with contaminated water, and clinical factors (caliber esophageal varices in first endoscopy, presence of gastric varices, red spots, and degree and pattern of PPF). Diameter of the spleen, PV, and splenic vein (SV) were compared before and 2 years after specific treatment for SM.

Information about these clinical variables was abstracted from medical records using a specific form, after the declaration of informed consent by the patient.

Ultrasound evaluation

The diagnoses of the clinical forms of the disease were determined by the patient's medical history and through physical examination, in addition to an evaluation by ultrasound of the upper abdomen through a single operator at the Endoscopy Unit of HC-UFPE, using a Siemens Acuson X150^® equipment, with a convex transducer of 3.5 mHz—to confirm the diagnosis and exclude other liver diseases. The parameters used to define the pattern of PPF were based on the classification of Niamey (Richter et al., 2001): A, absence of fibrosis; B, dubious fibrosis; C, light; D, moderate; E, advanced; and F, very advanced. The classification of Cairo standardizes three degrees for PPF according to the average obtained from measurements of external-external diameter of three peripheral branches of the PV after the second branch: grade I > 3 the 5 mm; grade II > 5 the 7 mm; and grade III >7 mm (WHO, 1993). The parameters adopted as normal and abnormal measures for the diameters of PV, SV, and spleen size (longitudinal axis) in this study were the following: PV-normal ≤ 12 mm, abnormal > 12 mm; SV-normal ≤ 9 mm, abnormal >9 mm; spleen size longitudinal-normal ≤12 cm and abnormal > 12 cm (WHO, 1993; Richter et al., 2001).

Ethical aspects

This retrospective cohort study was part of a study that was approved by the Ethics Committee of the Center for Health Sciences at the Federal University of Pernambuco and informed consents were obtained from all patients before blood sampling (CAAE 03161512.6.0000.5208).

Statistical analysis

The follow-up period considered started from the moment in which the specific treatment was first recorded until 2 years after the treatment. Relative risk (RR) and 95% confidence interval (CI) were used by bivariate analysis to verify the association between genotype polymorphism frequencies of (-G308A) TNF-α and the regression of the degree and standard of PPF of the exposed groups—considering the regression of the degree and pattern of fibrosis as dependent variables, and the variables selected as independent. The association was considered significant when p < 0.05. For these analyses Epi-Info software version 3.5.5 (CDC, Atlanta, GA) was used. To compare the variation of TNF-α levels between the groups, we used the Kruskal-Wallis test.

Results

In a previous study, the polymorphism (-G308A) in the promoter region of the TNF-α gene and serum concentrations were determined from 193 patients. Of these, 12 were excluded because they presented Pattern A (non PPF) on the first abdominal ultrasound, and another 57 of them did not go through the 2 year follow-up after the first specific treatment for SM. Therefore, the final analysis included only 124 patients (Fig. 1).

FIG. 1.

Diagram of patient eligibility.

The distribution of frequencies of the demographic and clinical variables among the exposure groups are illustrated in Table 1. In this study, the average age found among patients was 57 years (±13 years). No statistically significant difference of these factors of exposure (Table 1) was observed between the groups.

Table 1.

Distribution of Sociodemographic and Clinical Variable Frequency According to Polymorphism (-g308a) TNF- α in Patients with Schistosomiasis Mansoni, Pernambuco, Brazil

	Exposure groups (n = 124)
	GA or AA		GG
	n	%	n	%	χ²	ρ-Value
Sex
Male	28	39.4	27	50.9	2.216	0.330
Female	43	60.6	26	49.1
Total	71	100	53	100
HDA
Yes	35	49.3	33	62.3	2.591	0.273
No	36	50.7	20	37.7
Total	71	100	53	100
Treatment of the HDA^a,b
Splenectomy	4	11.4	1	3	1.950	0.582
EDA^c	10	28.6	12	36.4
Splenectomy +EDA	20	57.1	19	57.6
No treatment	1	2.9	1	3
Total	35	100	33	100
Caliber of esophageal varices^d
No varicose veins and fine	35	50.7	19	35.8	2.120	0.145
Medium and thick	34	49.3	34	64.2
Total	69	100	53	100
Red spots
Yes	14	19.7	20	37.7	5.505	0.063
No	57	80.3	33	62.3
Total	71	100	53	100
Gastric varices
Yes	14	19.7	14	26.4	1.460	0.481
No	57	80.3	39	73.6
Total	71	100	53	100
Contact focus after TTO
Yes	16	22.5	13	24.5	0.799	0.670
No	55	77.5	40	75.5
Total	71	100	53	100

HDA, upper gastrointestinal bleeding.

Sixty-eight patients were evaluated who presented HDA.

EDA, endoscopic treatment.

Two patients from the exposed group were excluded because there was no information.

TNF-α, tumor necrosis factor alpha; TTO, treatment; n, number of individuals studied; χ², chi-square.

Table 2 illustrates the distribution of frequencies in the two groups according to estimates of the diameters of the spleen, SV, and PV, before the specific treatment for SM and after the follow up of 2 years. The results showed no statistically significant difference between the diameters of the spleen, SV, and PV, when comparing pre and posttreatment periods.

Table 2.

Distribution of Diameters of the Spleen, SV, and PV Before and 2 Years After Treatment

	Pre-TTO		2 Years after TTO
	n	%	n	%	ρ-Value
Measurements of spleen^a
Normal (≤12 cm)	25	34.7	27	37.5	0.862
Abnormal (>12 cm)	47	65.3	45	62.5
Total	72	100	72	100
Measurements of SV^b,c
Normal (≤9 mm)	42	58.33	49	68.05	0.299
Abnormal (>9 mm)	30	41.67	23	31.95
Total	72	100	72	100
Measurements of PV^d,e
Normal (≤12 mm)	81	70.43	92	80	0.126
Abnormal (>12 mm)	34	29.57	23	20
Total	115	100	115	100

Fifty-two patients splenectomized.

VE not displayed during examination in 8 patients and in 42 splenectomized in pre-TTO.

SV not displayed during examination in 1 patient and in 51 splenectomized in 2°years after TTO.

PV not displayed during examination (excess gases) in 8 patients in pre-TTO.

PV not displayed during examination (excess gases) in 9 patients in 2°years after TTO.

ρ-value, Chi-square; SV, splenic vein; PV, portal vein.

The association between polymorphism (-G308A) TNF-α and the regression of the degree and pattern of PPF are illustrated in Tables 3 and 4. The genotypes (-308) GA/AA showed a protective association (RR = 0.52; ρ = 0.025) for the regression of the degree of PPF and for the regression of the PPF standard (RR = 0.56; ρ = 0.048), when comparing the two groups.

Table 3.

Bivariate Analysis of the Association Between Polymorphism (-G308a) TNF-α and Degree of Periportal Fibrosis Regression in Patients with Schistosomiasis Mansoni, Pernambuco, Brazil

	Periportal fibrosis regression by degree (n = 123)
	Regressed		Not regressed
	N	%	n	%	RR	CI 95%	p-Value
Genotypes (-308) TNF-α
GA or AA (exposed)^a	16	41	54	64.3	0.526	0.31-0.89	0.025
GG (not exposed)	23	59	30	35.7
Total	39	100	84	100

One patient was excluded due to lack of information in the exposed group.

RR, relative risk; CI, confidence interval.

Table 4.

Bivariate Analysis of the Association Between Polymorphism (-G308a) TNF- α and the Standard of Periportal Fibrosis Regression in Patients with Schistosomiasis Mansoni, Pernambuco, Brazil

	Periportal fibrosis regression by standard (n = 122)
	Regressed		Not regressed
	N	%	N	%	RR	CI 95%	p-Value
Genotypes (-308) TNF-α
GA or AA (exposed)^a	16	42.1	53	63.1	0.56	0.33-0.95	0.048
GG (not exposed)	22	57.9	31	36.9
Total	38	100	84	100

Two patients were excluded due to lack of information in the exposed group.

There was no significant statistical difference of average TNF-α levels between the exposure groups (Table 5).

Table 5.

Analysis of the Medians of the Serum Levels of TNF-α in Patients with Schistosomiasis Mansoni According to Polymorphism (-g308a) TNF-α, Pernambuco, Brazil

Cytokine (pg/mL)	GA+AA (n = 50)	GG (n = 32)	ρ^a
Median (minimum-maximum) TNF-α	22 (7-88)	23 (13-62)	0.080

Kruskal-Wallis test.

Discussion

This study assessed the influence of polymorphism (-G308A) TNF-α on PPF regression. This is the first article that analyzes the influence of genetic factors on PPF regression in a group of patients with SM in the state of Pernambuco, Northeast of Brazil.

Although it is well established in the literature that a number of environmental factors, including age, frequency of exposure, and parasitic load can influence the natural history of PPF (Abath et al., 2006; Arnaud et al., 2008), in this sample no statistical difference was observed among the sociodemographic and clinical factors between the exposure groups. Silva et al. (2013) evaluated 178 patients in the state of Pernambuco and stated that environmental factors, such as years of education and specific treatment, had an influence on the development of PPF. However, it is believed that the immune response, which is governed by the genetics of the host, plays a central role in the natural history of the disease.

This study involving Brazilian patients infected with S. mansoni, showed that patients with genotypes (-308) GA/AA had a protective association for regression of the degree and standard of PPF when compared with genotype (-308) GG.

The association between polymorphism (-G308A) TNF-α and hepatic fibrosis has already been extensively studied in other liver diseases (Kusumoto et al., 2006; Jeng et al., 2007; Bouzgarrou et al., 2010; Radwan et al., 2012). However, only one single study has evaluated the influence of polymorphism on the severity of PPF in schistosomiasis (Moukoko et al., 2003). Some studies found no association between this polymorphism and the severity of liver fibrosis (Moukoko et al., 2003; Kusumoto et al., 2006; Bouzgarrou et al., 2010).

Moukoko et al. (2003), observed that within the Sudanese population high levels of TNF-α were associated with higher risk of PPF, but found no evidence of association between polymorphism (-G308A) TNF-α and the severity of PPF. These authors stated that this result does not exclude the possibility that this polymorphism has an effect on the development of PPF.

Bouzgarrou et al. (2010) studied 53 Tunisian patients with chronic hepatitis C and 103 randomly selected healthy blood donor controls, matched by ethnicity and geographical area. In this study, no association was found between polymorphism (-G308A) TNF-α and liver fibrosis severity. The authors concluded that these results, although based on a small cohort, are consistent with other major studies on different ethnic populations (Powell et al., 2000; Rosen et al., 2002; Bahr et al., 2003; Goyal et al., 2004; Abbas et al., 2005), which excluded the influence of this polymorphism on the severity of Hepatitis C virus-related liver fibrosis.

Kusumoto et al. (2006) evaluated 460 patients with positive HCV and 63 healthy controls, from a hyperendemic area for HCV in Japan, and also found no significant association between this polymorphism and the severity of hepatitis. These authors concluded that polymorphism (-G308A) TNF-α can be associated with hepatic fibrosis and that these results may have been influenced by ethnic variations.

On the other hand, some studies involving patients with chronic hepatitis C confirmed the association between polymorphism (-G308A) TNF-α and liver fibrosis severity (Ho et al., 2004; Richardson et al., 2005; Feld and Liang, 2006; Schwabe and Brenner, 2006).

Jeng et al. (2007) studied 108 patients with hepatocellular carcinoma (HCC) and 108 healthy controls, matched by gender and age (±5 years), and concluded that the allele -308A TNF-α was associated with more advanced liver fibrosis, which can contribute to greater risk of HCC.

Radwan et al. (2012) studied 280 Egyptian patients with HCV (152 with cirrhosis and 128 with HCC) and 160 healthy controls, and found that polymorphism (-G308A) TNF-α was associated with risk of liver cirrhosis and HCC in patients with chronic hepatitis C.

A consensus about the influence of this polymorphism on the expression of TNF-α has not yet been established in the literature. Some studies have shown that the allele-308A was associated with increased levels of the TNF-α constituent in comparison with the allele -308G, while others failed to associate the presence of the -308A allele with different levels of TNF-α production (Wilson et al., 1997; Bayley et al., 2004; Jrad et al., 2007). This data may reinforce the hypothesis of this current study, which showed that the tested genotypes (-308) GA/AA can play an important role in the prevention of PPF regression in schistosomiasis, in the Brazilian population.

In the current study, although there was no difference of TNF-α serum levels between the exposure groups, one possible explanation for these results is that individuals with the genotypes (-308) GA/AA, possibly maintain a production of TNF-α residue after the specific treatment, contributing to less reabsorption of fibrosis in this group.

The results found in this study do not exclude the possibility of influence from other cytokines and other genes on PPF intensity. Therefore, future studies with larger samples are needed to better analyze these genotypes and their respective doses of TNF-α serum, to be able to better assess whether there is a connection between polymorphism (-G308A) TNF-α and the expression of TNF-α and PPF intensity, which may influence the regression of PPF after specific treatment of schistosomiasis. To conclude, these results suggest that polymorphism (-G308A) TNF-α can be a factor that prevents the regression of the degree and pattern of PPF in the Brazilian population, and can, therefore, potentially be a predictive factor in the severity of PPF in schistosomiasis.

Footnotes

Acknowledgments

The authors are especially thankful to the patients of the Gastroenterology Clinic of the Hospital das Clinicas/Federal University of Pernambuco, who accepted to participate, contributing with samples without direct benefit from the results, and to the laboratories of Human Molecular Genetics (University of Pernambuco) and of Virology-Immunology Laboratory Keizo-Asami (LIKA) (Federal University of Pernambuco-UFPE), where the analyses were carried out. This research study was supported by the Federal University of Pernambuco (UFPE), Brazil.

Author Disclosure Statement

No competing financial interests exist.

References

Abath

FGC

, Morais

CNL

, Montenegro

CEL

, et al. (2006) Immunopathogenic mechanisms in schistosomiasis: what can be learnt from human studies?. Trends Parasitol, 22:85-91.

Abbas

, Moatter

, Hussainy

, et al. (2005) Effect of cytokine gene polymorphism on histological activity index, viral load and response to treatment in patients with chronic hepatitis C genotype 3. World J Gastroenterol, 11:6656-6661.

Andrade

(2005) Regression of hepatic fibrosis. Rev Soc Bras Med Trop, 38:514-520.

Andrade

(2009) Schistosomiasis and liver fibrosis. Parasite Immunol, 31:656-663.

Arnaud

, Li

, Wang

, et al. (2008) Regulatory role of interleukin-10 and interferon in severe hepatic central and peripheral fibrosis in humans infected with Schistosoma japonicum. Infect Dis, 198:418-426.

Bahr

, El Menuawy

, Boeker

, et al. (2003) Cytokine gene polymorphisms and the susceptibility to liver cirrhosis in patients with chronic hepatitis C. Liver Int, 23:420-425.

Bayley

, Ottenhoff

, Verweij

(2004) Is there a future for TNF promoter polymorphisms?. Genes Immun, 5:315-329.

Booth

, Mwatha

, Joseph

, et al. (2004) Periportal fibrosis in human Schistosoma mansoni infection is associated with low IL-10, low IFN-γ, high TNF-α, or low RANTES, depending on age and gender. J Immunol, 172:1295-1303.

Bouzgarrou

, Hassen

, Gabbouj

, et al. (2010) Lack of effect of tumor necrosis factor-alpha -308 G/A polymorphism on severity of liver fibrosis in Tunisian hepatitis C virus (HCV)-infected patients. Gastroenterol Clin Biol, 34:297-304.

10.

Cabrera

, Shaw

, Sharpies

, et al. (1995) Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis. J Exp Med, 182:1259-1264.

11.

Dai

, Chuang

, Chang

, et al. (2006) Tumor necrosis factor-α promoter polymorphism at position -308 predicts response to combination therapy in hepatitis C virus infection. J Infect Dis, 193:98-101.

12.

De Jesus

, Silva

, Santana

, et al. (2002) Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni. Infect Dis, 185:98-105.

13.

Dessein

, Chevillard

, Arnaud

, et al. (2009) Variants of CTGF are associated with hepatic fibrosis in Chinese, Sudanese, and Brazilians infected with schistosomes. J Exp Med, 12:1-8.

14.

Feld

, Liang

(2006) Hepatitis C—identifying patients with progressive liver injury. Hepatology, 43:S194-S206.

15.

Goyal

, Kazim

, Sakhuja

, et al. (2004) Association of TNF-beta polymorphism with disease severity among patients infected with hepatitis C virus. J Med Virol, 72:60-65.

16.

Guevara

, Noya

, Valero

, et al. (2007) Clinical and ultrasound findings before and after praziquantel treatment among Venezuelan schistosomiasis patients. Rev Soc Bras Med Trop, 40:505-511.

17.

Henri

, Chevillard

, Mergani

, et al. (2002) Cytokine regulation of periportal fibrosis in humans infected with Schistosoma mansoni: IFN-γ is associated with protection against fibrosis and TNF-α with aggravation of disease. J Immunol, 169:929-936.

18.

, Wang

, Chen

, et al. (2004) Increased risk of developing hepatocellular carcinoma associated with carriage of the TNF2 allele of the -308 tumor necrosis factor alpha promoter gene. Cancer Causes Control, 15:657-663.

19.

Jeng

, Tsai

, Chuang

, et al. (2007) Tumor necrosis factor-α 308.2 polymorphism is associated with advanced hepatic fibrosis and higher risk for hepatocellular carcinoma. Neoplasia, 9:987-992.

20.

Jrad

BBH

, Chatti

, Laatiri

, et al. (2007) Tumor necrosis factor promoter gene polymorphism associated with increased susceptibility to non-Hodgkin's lymphomas. Eur J Haematol, 78:117-122.

21.

Kusumoto

, Uto

, Hayashi

, et al. (2006) Interleukin-10 or tumor necrosis factor-α polymorphisms and the natural course of hepatitis C virus infection in a hyperendemic area of Japan. Cytokine, 34:24-31.

22.

Magalhães

, Miranda

, et al. (2004) Cytokine profile associated with human chronic schistosomiasis mansoni. Mem Inst Oswaldo Cruz 99 Suppl, I:21-26.

23.

Moukoko

, Wali

, Saeed

, et al. (2003) No evidence for a major effect of tumor necrosis factor alpha gene polymorphisms in periportal fibrosis caused by Schistosoma mansoni infection. Infect Immun. 5456-5460.

24.

Pordeus

, Aguiar

, Quinino

LRM

, et al. (2008) A ocorrência das formas aguda e crônica da esquistossomose mansônica no Brasil no período de 1997 a 2006: uma revisão de literatura. Epidemiol Serv Saúde, 17:163-175.

25.

Powell

, Edwards

SCJ

, Hay

, et al. (2000) Host genetic factors influence disease progression in chronic hepatitis C. Hepatology, 31:828-833.

26.

Qin

, Liu

, Shi

, et al. (2010) Tumour necrosis factor-α polymorphisms and hepatocellular carcinoma: a meta-analysis. J Int Med Res, 38:760-768.

27.

Radwan

, Pasha

, Mohamed

, et al. (2012) Influence of transforming growth factor-β1 and tumor necrosis factor-α genes polymorphisms on the development of cirrhosis and hepatocellular carcinoma in chronic hepatitis C patients. Cytokine, 60:271-276.

28.

Richardson

, Powell

, Barrie

, et al. (2005) A combination of genetic polymorphisms increases the risk of progressive disease in chronic hepatitis C. J Med Genet, 42, e45.

29.

Richter

, Domingues

ALC

, Barata

, et al. (2001) Report of the second satellite symposium on ultrasound in schistosomiasis. Mem Inst Oswaldo Cruz, 96:151-156.

30.

Rosen

, McHutchison

, Conrad

, et al. (2002) Tumor necrosis factor genetic polymorphisms and response to antiviral therapy in patients with chronic hepatitis C. Am J Gastroenterol, 97:714-720.

31.

Schwabe

, Brenner

(2006) Mechanisms of liver injury. I. TNF-α-induced liver injury: role of IKK, JNK, and ROS pathways. Am J Physiol Gastrointest Liver Physiol, 290:583-589.

32.

Silva

PCV

, Leal

, Domingues

ALC

(2013) Treatment and education reduce the severity of schistosomiasis periportal fibrosis. Rev Soc Bras Med Trop, 46:472-477.

33.

Wilson

, Symons

, McDowell

, et al. (1997) Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A, 94:3195-3199.

34.

World Health Organization (1993) Proposal for a practical guide to standardized use of ultrasound in the assessment of pathological changes. In: Meeting on Ultrasonography in Schistosomiasis, October 1-4, 1990, Cairo.

35.

World Health Organization (2014) Programs and projects: schistosomiasis. A major publichealth Available at: www.who.int/schistosomiasis/en/, accessed December 15, 2014.