Journal of Investigative Medicine

Erythropoietic protoporphyria (EPP) results from an inherited deficiency of the last enzyme of the heme biosynthetic pathway, ferrochelatase (FC). EPP is usually inherited in an autosomal dominant fashion, and the mutations in the FC gene on chromosome 18q21.3 detected in EPP patients are heterogeneous.

In this study, we screened the FC gene for mutations in 12 patients from 10 unrelated families with EPP and their familiy members using heteroduplex analysis, automated sequencing, and restriction enzyme digestion.

We detected 8 different mutations in these patients, including 1 missense mutation, 5 frameshift mutations, and 2 splice site mutations, 6 of which are previously undescribed.

We have established the molecular basis of EPP in 10 unrelated families, thereby providing further evidence for the heterogeneity in this disorder. Importantly, molecular diagnosis allowed revisions in the status of several clinically unaffected silent mutation carriers within the families. We compare the value of genetic research strategies with the combination of biochemical data and clinical phenotype as diagnostic tools to confirm a putative diagnosis in EPP.

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. Interleukin (IL)-4 reduces the production of many proinflammatory cytokines, particularly by activated macrophages. Therefore, we examined the ability of adenovirally delivered IL-4 for the treatment of human RA to reduce the secretion of proinflammatory molecules.

Adenoviral vectors encoding the genes for human IL-4 (AxCAIL-4) and bacterial β-galactosidase (AxCAlacZ) were generated and examined for appropriate production and biological activity. RA synovial tissue (ST) explants or fibroblasts were infected with AxCAIL-4 or a β-galactosidase producing vector, as a control, and conditioned medium (CM) was collected for ELISA analysis.

AxCAIL-4 decreased the production of the inflammatory cytokines IL-1β and tumor necrosis factor-α in RA ST explant CM. IL-8 levels were significantly reduced by 71%, 88%, and 82% at 24, 48, and 72 hours, respectively, in RA ST explant CM. In the same CM, monocyte chemotactic protein-1 (MCP-1) levels decreased 60% at 48 hours. In contrast, RA synovial fibroblast CM levels of MCP-1 were increased by AxCAIL-4. Epithelial neutrophil activating peptide-78 levels produced by RA ST explants were significantly decreased by AxCAIL-4 by 88%, 92%, and 93% at 24, 48, and 72 hours, respectively. Growth related gene product-a levels were likewise decreased in RA ST explant CM. In ST explants as well as RA synovial fibroblasts, IL-4 treatment decreased prostaglandin E₂ (PGE₂) production.

Increased expression of IL-4 via gene therapy may decrease RA-associated inflammation by reducing proinflammatory cytokines and PGE₂.

Verapamil is an effective drug to slow ventricular rate in atrial fibrillation (AF). Clinically, however, IV verapamil enhances AF despite experimental evidence suggesting favorable effects of the drug on AF-induced electrical remodeling of the atria.

To clarify this controversy, IV verapamil's effects were determined in 41 anesthetized dogs, including 6 after beta-blockade. Intravenous verapamil (0.20 mg/kg, bolus, and 0.20 mg/kg/h, infusion) increased the duration of AF (induced by a single extrastimulus), from 19 ± 6 to 130 ± 24 s, P<0.001, and slowed its ventricular response, from 246 ± 25 to 110 ± 15 min¹, P<0.001. Mean aortic pressure, P=0.002, and systemic vascular resistance, P<0.035, decreased, and mean right atrial pressure increased, P<0.001. Plasma norepinephrine concentration increased by 502 ± 83 pg/mL, P<0.001, plasma epinephrine concentration by 804 ± 206 pg/mL, P=0.002, and plasma total catecholamine concentration by 1606 ± 366 pg/mL, P<0.001. Prolongation of AF was related to an increase in mean right atrial pressure, R=0.49, P=0.014, right atrial wall tension, R=0.45, P=0.044, and plasma norepinephrine concentration, R=0.83, P<0.001, with plasma norepinephrine concentration remaining as an independent predictor of AF lengthening on multivariable analysis. In the presence of beta-blockade, verapamil produced comparable or more exaggerated hemodynamic effects, but it did not promote AF.

The prolongation of AF by verapamil can be related directly to the intense sympathetic neurohumoral effect that occurs following the drug's administration.

Stent thrombosis and restenosis are of great clinical significance. We constructed a closed loop in vitro heparinized whole human blood circulation model for testing hemocompatibility of coronary stents. This model allows evaluation of human blood activation by blood-stent interaction in a well-controlled setting. Until now these interactions were studied in the highly coagulable pig coronary artery model.

We evaluated activation of the coagulation system and blood components by uncoated, heparin-coated, and silicon-carbide coated tantalum stents. The effects, measured by biochemical assays, were compared with stainless-steel stents. Also the inhibitory effect on platelet activation by indomethacin equal to the oral effect of 325 mg acetylsalicylic acid daily, was measured and visualized by scanning electron microscopy.

Both activation of the coagulation system and platelets were counteracted by indomethacin, suggesting an important role for platelets in activation of the coagulation system in this model. Despite platelet activation by all stents, the SiC-coated tantalum stent demonstrates a significantly lower GpIIIa receptor-mediated platelet adhesion at the stent surface (21.7×10³ counts per second/mg stent weight) compared to all other stents (stainless-steel 54.0, heparincoated 95.7 and uncoated 76.2×10³ cps/mg). Also activated leukocytes demonstrated a significantly lower CD11b receptor-mediated adhesion at the SiC-coated stent (37.0×10³ cps/mg) than at the stainless-steel stent (114.5×10³ cps/mg).

Data from this in vitro circulation study show a significantly lower platelet and leukocyte adhesion at the surface of the SiC-coated tantalum stent than at the surface of stainless-steel stents or uncoated and heparin-coated tantalum stents.

We have identified 36 human sera sent for autoantibody analyses that produce a unique vesicular staining pattern of the cytoplasm of tissue culture cells. The purpose of this study was to identify the autoantigens that are recognized by the sera that produce this staining pattern and determine if the patients have common clinical features.

A serum from one of the patients (MS) with rapidly progressive demyelinating polyneuropathy was used to isolate a ∼4.5 kb cDNA insert from a HeLa expression library. The purified cDNA (MS-5.1) was characterized by a poly A tail and an open reading frame that encoded 1329 amino acids. The derived amino acid sequence was found to be 99% identical to a 180 kd peripheral endosomal protein named early endosome antigen (EEA1).

Antibodies from rabbits immunized with the recombinant protein and the prototype human serum produced an identical distinctive speckled cytoplasmic staining pattern. These sera also precipitated the in vitro translated recombinant protein and reacted with the isolated recombinant protein in a Western immunoblot. Of the 36 sera that produced an identical staining pattern as the prototype and immune rabbit sera, 8 (22%) had IgG antibodies that recognized the recombinant EEA1 protein when tested by immunoblotting and immunoprecipitation assays. Of the 8 patients with anti-EEA1 antibodies 4 were females, 4 were males, and the mean age was 69 years (range 48 to 86 years).

Diagnoses included: polyneuropathy, lower motor neuron disease, pigmented retinitis, seronegative polyarthritis, interstitial pulmonary fibrosis, Raynaud's phenomenon, Wegener's granulomatosis, and proteinuria. Three of the eight patients with EEA1 autoantibodies died within 1 year after EEA1 antibodies were identified.

A partial or complete steroid resistance, whose cause is not yet clarified, has been documented in many patients with long-standing Crohn's disease (CD). The primary aim of this study was to evaluate the number and affinity of serum protein steroid-binding sites in steroid-resistant patients with Crohn's disease. A secondary goal was to measure insulin sensitivity, an indirect index of steroid effectiveness, in these patients.

The study included 8 male steroid-resistant patients with active ileal CD and 6 healthy male volunteers. Corticosteroid binding globulin (CBG), binding capacity and affinity for cortisol were measured. The binding of cortisol to normal human serum and to serum of patients with CD was also determined. Whole body glucose uptake and oxidation were assessed by euglycemic hyperinsulinemic clamp and indirect calorimetry.

Crohn's patients showed a significantly greater capacity of serum albumin for cortisol than controls (by about 40%, or about 0.15 moles per mole). Conversely, the binding of cortisol to CBG did not show significant differences between groups. Glucose uptake was higher in Crohn's patients than in normal controls (8.82 ± 2.50 vs 7.01 ± 2.24 mg/kg_FFM/min; P = 0.036). Basal serum nonesterified fatty acid (NEFA) levels were lower in patients than in controls (459.64 ± 69.95 vs 1026.48 ± 112.58 µmol/L; P = 0.002).

The observed increase in albumin binding might limit the bioactivity of cortisol in patients with Crohn's disease and contribute to the decreased effectiveness and weaker side effects of glucocorticoid therapy in these patients. The increased number of cortisol binding sites on albumin from patients with CD might be correlated with the significant decrease in serum NEFA, which may compete with steroids for the same sites.

Autofluorescence can be used to detect neoplasia in the colon. Two known fluorophores, collagen and elastin, are probably partly responsible for colonic emission spectra. Their contribution to colonic autofluorescence was investigated.

Autofluorescence spectra of normal, dysplastic, and malignant colonic tissue were studied by using excitation wavelengths from 280 nm to 350 nm. The wavelengths of peak emission and their widths at half maximum intensity were measured. Similar measurements were performed on collagen types I, III, IV, V, IX, and elastin. Colonic spectra were compared to those of collagen and elastin. Spectral differences between collagen types IV (basement membrane) I, III, V, and IX were studied.

Four major emission peaks were noted whose wavelength of peak emission and full widths at half maximum intensity were independent of tissue histology. The emission spectra of type IV collagen differed markedly from that of nonbasement membrane collagens and elastin.

Type IV (basement membrane) collagen is most likely responsible for the emission peak at 365 nm. The spectra of basement membrane collagen and not other types of collagen should be used in studies of epithelial tissue spectra. Elastin did not appear to be responsible for any of the four autofluorescence peaks observed in colonic tissue.