Role of fibrogenic markers in chronic hepatitis C and associated hepatocellular carcinoma.

Detection and follow up of fibrogenesis in chronic hepatitis C (CHC) is mandatory for early treatment and risk stratification. The current study included 120 patients with CHC, of whom 30 had liver cirrhosis (LC) and 30 had hepatocellular carcinoma (HCC). 15 wedge liver biopsies, taken during laparoscopic cholecystectomy, were included as normal controls. Cases were subjected to laboratory investigations, serologic markers for viral hepatitis and assessment of circulating levels of hyaluronic acid (HA) and platelet-derived growth factor (PDGF). Immunohistochemical expression of connective tissue growth factor (CTGF), PDGF and transforming growth factor-ß1 (TGF-ß1) was also carried out. A significant increase (p < 0.01) in serum HA was noticed in CHC, LC and HCC compared to controls. Although, a significant decrease in serum PDGF was detected in CHC and LC compared to controls, HCC values were comparable. A significant up-regulation of CTGF was detected in CHC, LC and HCC (p < 0.01) in contrast to its limited mild expression in normal livers. Intense PDGF positive staining was noticed in CHC, LC and HCC compared to scattered faint expression in controls. The significant expression and marked intensity of PDGF staining matched the progress to tumorigenesis. A positive TGF-ß1 immunostaining was also noticed in CHC, LC and HCC. An intense and extensive cytoplasmic expression of TGF-ß1 was encountered in patients with LC revealing that CTGF, PDGF and TGF-ß1 act synergistically in LC. Data revealed that HA and CTGF may be implicated as important diagnostic parameters for assessment of hepatic fibrosis and PDGF for monitoring malignant transformation in CHC.

The coagulation profile in hepatosplenic schistosomiasis.

The biological activity of blood coagulation factors II, V, VII, VIII, IX, X, XI and XII, fibrinogen and prekallikrein was assessed in 15 healthy subjects and 60 patients with endemic Egyptian hepatosplenomegaly. The degree of liver disease was graded according to the Child-Pugh classification, the intensity of S. mansoni infection was monitored by determination of circulating schistosome immune complexes (CSIC) level using a monoclonal antibody and hemostasis activation was detected by measurement of hemostatic markers D-dimer and prothrombin fragment 1 + 2 (F1+2). Functional activity of antithrombin III, alpha2-antiplasmin and protein C as well as quantitative determination of plasma concentrations of alpha1-antitrypsin, C1 activator inhibitor and alpha2-macroglobulin were also carried out. The progressive deterioration of liver function which matched the severity of the disease and the intensity of schistosomal infection led to a reduction in anticoagulant proteins (decreases in antithrombin III and protein C) resulting in hypercoagulability and thrombin generation (increased F1+2) subsequently followed by consumption (prolongation of coagulation screening tests, thrombocytopenia, hypofibrinogenemia and decreased factor VIII resulting in hypocoagulability and secondary fibrinolysis (increased D-dimer and decreased alpha2-antiplasmin). A significant decline in fibrinogen and factors VII, XII and prekallikrein was detected in bleeders compared with ascitic patients. The decline in factor XII was closely related to CSIC high titers in all disease groups, but was not correlated to D-dimer or F1+2 concentrations. This suggests that circulating schistosome immune complexes may exert an inhibitory effect on contact factor XII which should be taken into account when considering the reasons for schistosomal coagulopathy and bleeding in hepatosplenic schistosomiasis.

Fibronectin, platelet factor 4 and beta-thromboglobulin in endemic hepatosplenic schistosomiasis: relation to acute hematemesis.

Some platelet alpha-granule contents were assessed in parallel with other markers of hemostatic imbalance in 50 patients with hepatosplenic schistosomiasis (15 patients with compensated hepatosplenomegaly, 15 patients with advanced hepatic fibrosis and ascites and 20 patients during an acute attack of hematemesis from ruptured esophageal varices). Platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), fibronectin (FN), prothrombin fragment 1 + 2, thrombin-antithrombin (TAT) complexes, fibrin degradation products (FbDP) and D-dimer were assessed in schistosomal patients compared to controls (15 healthy subjects). A significant increase in both thrombin (high TAT and prothrombin fragment 1 + 2 levels) and plasmin (high FbDP and D-dimer levels) generation was detected in decompensated patients establishing the presence of a steady state of low-grade disseminated intravascular coagulation, with and without overt bleeding, in these patients. A decrease in plasma FN concentration was found in diseased groups compared to controls. The reduction in plasma levels of FN paralleled the defective liver function and matched the relative decrease in tissue FN in liver specimens of decompensated patients suggesting that FN levels can be used to evaluate the pathological staging of the disease. A significant increase in beta-TG and PF4 levels was noted in decompensated patients with ascites and/or acute hematemesis compared both to controls and compensated patients reflecting platelet alpha-granule release and consequently increased in vivo platelet activation which may initiate and/or perpetuate the pathophysiological mechanisms of the hemostatic imbalance underlying the hemorrhagic diathesis in hepatosplenic schistosomiasis.

Hyperfibrinolysis in hepatosplenic schistosomiasis.

AIM: To evaluate the nature of accelerated fibrinolysis in hepatosplenic schistosomiasis. METHODS: The biological activity of plasminogen (Plg), plasminogen activators (PA), alpha 2-antiplasmin (alpha 2-AP) and plasminogen activator inhibitor-1 (PAI-1) was determined by photometric analysis in 15 compensated and 35 decompensated patients with endemic Egyptian hepatosplenomegaly. Quantitative measurement of plasma concentrations of tissue t-PA, t-PA-PAI-1 complex, alpha 2-antiplasmin-plasmin complex (alpha 2-APP), fibrinogen degradation products (FbDP), D-dimers (D-D), thrombin-antithrombin complex (TAT) and prothrombin fragment (F 1 + 2) complexes, using double antibody sandwich enzyme linked immunosorbent assays and grading of the degree of hepatic insufficiency according to the Child-Pugh classification, were also carried out. RESULTS: The progressive deterioration of liver function in schistosomal patients, which matched the severity of the disease, led to simultaneous defects in profibrinolytic (decreased Plg and increased PA and t-PA) and antifibrinolytic (decreased alpha 2-AP and PAI-1) factors-the latter defects being the most prominent-resulting in significant generation of plasmin (increased APP complexes) and therefore enhanced fibrinolysis (increased FbDP and D-dimer). The raised concentrations of FbDP, D-D, TAT and F 1 + 2 established its secondary nature. CONCLUSION: These findings suggest that the amount of PAI-1 available to bind and neutralise circulating t-PA may be a critical factor in the progress of hyperfibrinolysis observed in hepatosplenic schistosomiasis, and that the pronounced reduction in its plasma concentration may be regarded as a potential warning indicator of haemostatic imbalance in decompensated schistosomal patients at high risk of variceal bleeding.

Isolation and concentration of microfilariae from peripheral blood of Wuchereria bancrofti infected patients by density gradient centrifugation.

A Ficoll-Hypaque gradient centrifugation technique was used for isolation and concentration of microfilariae from peripheral blood of 30 subjects with clinically and parasitologically diagnosed Wuchereria bancrofti infections. 86% of the microfilariae were found in the Ficoll-Hypaque layer. None were detected in the plasma, leucocyte layer or lower erythrocyte layer. 14% of microfilariae were identified on the top part of the erythrocyte layer. A 35 fold concentration and 88% quantitative recovery of parasites was achieved by conventional centrifugation of microfilariae-rich Ficoll-Hypaque layer. Following the centrifugation procedures, living motile microfilariae were separated. These results indicate that Ficoll-Hypaque centrifugation technique could be an effective method for the detection of low levels of microfilaraemia, and for obtaining relatively pure suspensions of living microfilariae for metabolic studies, production of antigen-rich excretory-secretory products and antigen analysis.

Antifilarial IgM versus IgG antibody determination in the diagnosis of Wuchereria bancrofti infection in Egyptians.

Circulating antifilarial IgM and IgG antibodies were assessed by indirect ELISA in 184 serum specimens from 80 patients with clinically and parasitologically diagnosed filarial infections (20 with acute filariasis 40 with chronic filariasis & 20 asymptomatic microfilaraemic subjects), 64 individuals with other parasitic infections, 20 parasitologically-free subjects from filariasis endemic areas and 20 normal healthy controls. A soluble surface membrane extract from Dirofilaria immitis worms was used as the antigen. Using a single serum dilution of 1:128 and optical densities (OD) at 492 nm, the respective cut off values for IgM and IgG were found to be 0.24 and 0.22. All healthy non-endemic controls were seronegative by IgM and IgG ELISAs. The highest antifilarial IgM OD492 values were obtained in 20 patients with acute filariasis (95% sensitivity), while the highest antifilarial IgG OD492 values were observed n 40 patients with chronic filariaisis (97.5% sensitivity). Asymptomatic microfilaraemic subjects gave IgM and IgG OD492 values which were significantly lower than those of other forms of clinical disease and endemic control subjects. The antifilarial IgM and IgG respective sensitivities in asymptomatic subjects were 75% and 70%. Endemic controls had positive antifilarial IgM (65%) and IgG (75%) levels. Of 64 subjects with other parasites only one with Ancylostoma duodenale had positive IgM level (98.4% specificity); while 9 patients with nematodal infections mainly had false positive antifilarial IgG antibody levels (85.9% specificity). These results suggest that measuring circulating antifilarial IgM antibody level may have some diagnostic advantage over measuring IgG antibody level for the detection of active filarial infection and consequently better management of the disease.

Effect of praziquantel on certain immune responses of schistosomal Egyptian patients. II. Cell-mediated lymphoproliferative response.

The lymphoproliferative blastogenic responses to mitogens, PHA and Con A, and to schistosome-derived antigens. S. mansoni worm and egg, were tested in 35 schistosomal patients and 10 healthy controls. Of the former group, 18 patients had intestinal mansoniasis and 17 had mansoniasis with hepatosplenomegaly. The test was repeated 2 weeks and 1 and 2 months after treatment with praziquantel. The delayed intradermal test for schistosomiasis was performed on 25 of the schistosomal patients and was repeated 1 month after treatment. Statistical analysis of results of lymphoproliferative blastogenic responses showed no significant differences between the control and the two schistosomal groups in response to mitogens. The group with intestinal mansoniasis responded significantly to both schistosomal antigens, compared to the control and hepatosplenic groups. Their proliferative responses showed a significant rise 2 weeks after treatment, then a gradual drop at 1 and 2 month intervals. The hepatosplenic group responded significantly to worm antigen before treatment; their proliferative responses to both schistosomal antigens showed a significant rise 2 weeks after treatment and remained raised thereafter. No relationship was established between either of the two schistosomal groups for age, intensity of infection or positive delayed intradermal reaction.