relationship between expression of Toll-like receptor 4 in chronic hepatitis C patients and different stages of liver fibrosis

Aim: The objective of this work is to find out whether there is a relation between the expression of TLR4 and fibrosis progression in chronic HCV patients

Background: Toll-like Receptor 4 [TLR4] is a pattern recognition receptor whose activation results in the production of several pro-inflammatory cytokines

Methods: Fifty patients with chronic HCV were included. They were divided into group A: 40 patients [F1-F4] and group B [control group] which included ten patients [F0] based on fibroscan value. All patients were exposed to clinical and laboratory evaluations preliminary to antiviral therapy, assessment of TLR4 mRNA by Real Time- PCR

Results: Twenty-eight males and 22 females with a mean age 28.9 +/- 6.1 years. The mean TLR4 expression is 11.2 +/- 7.4 folds, TLR4 expression in F0 group is 2.8 +/- 1.9, in F1 group 4.8 +/- 1.5, F2 group 10.2 +/- 2.5, F3 group 16.8 +/- 1.5 and in F4 21.3 +/- 3.6 folds [p<0.001]. TLR4 showed a positive correlation with age, fibrosis stage, HCV RNA, serum transaminases, total bilirubin and prothrombin time, a negative correlation with platelet count and serum albumin. Fibrosis progression was independently associated with TLR4 expression [beta=0. 648, P<0.0001], RNA [beta= 0.160, P =0.001] and platelet count [beta= -0.248, P = 0.004]

Conclusion: The expression of TLR4 is highly correlated with the fibrosis progression; TLR4 may be a potential target for drugs to limit the progression of fibrosis

ESAT-6-ELISpot and interferon gamma in the diagnosis of pleural tuberculosis

Appropriate diagnostic methods for tuberculous pleural effusion are vital. The IFN-gamma tests using specific Mycobacterium Tuberculos is antigens in samples from the site of infection may be promising in diagnosis of tuberculosis. Objective we examined the ability of ELISpot test using circulating peripheral blood mononuclear cells [PBMC] and compartmentalized pleural fluid mononuclear cells [PFMC] for diagnosis of active TB infection in patients with tuberculous pleural effusion. Methods PBMC and PFMC-based ELISpot test for IFN-gamma test using specific M. tuberculosis antigen: Early Secretory Antigen Target-6 protein [ESAT-6] was used for diagnosis of active TB infection. Thirty-five patients with clinically suspected tuberculous pleural effusion were enrolled over a 12-month period. Results 11 patients out of 35 were positive by culture and PCR [31.4%]. Incubation of PBMC with ESAT-6 for 8 h showed sensitivity and specificity of 82% and 92%, respectively, for the PBMC-ELISpot as compared to PFMC-ELISpot that was 54% and 96% respectively. With 24 h incubation of ESAT-6 there was around 2.5 fold increase in the median number of spot forming cells [SFCs] in PFMC from 30 to 74, whereas there was minimal increase of median number of SFCs in PBMC from 55 to 60. Conclusion ESAT-6 - ELISpot using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-gamma in pleural effusion [PE] is another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap

Interferon gamma as a diagnostic marker for pleural tuberculosis

Background appropriate diagnostic methods for tuberculosis pleural effusion [TBPE] are vital. The IFN-gamma tests using specific M. Tuberculosis antigens in samples from the site of infection may be promising in diagnosis of tuberculosis

Objective we examined the ability of ELISpot test using circulating peripheral blood mononuclear cells [PBMC] and compartmentalized pleural fluid mononuclear cells [PFMC] for diagnosis of active TB infection in patients with TBPE

Methods PBMC and PFMC-based ELISpot test for IFN-gamma test using specific M. Tuberculosis antigen: Early Secretory Antigen Target-6 protein [ESA T-6] was used for diagnosis of active TB infection. Thirty-five patients with clinically suspected TBPE were enrolled over a 12-month period

Results eleven patients out of 35 were positive by culture and PCR [31.4%]. Incubation of PBMC with ESAT-6 for 8 hrs showed sensitivity and specficity of 82 % and 92 % respectively .for the PBMC-ELlSpot as compared to PFMC- ELlSpot that was 54% and 96 % respectively. With 24 hrs incubation of ESAT- 6 there was around 2.5 fold increase in the median number of spot forming cells [SFCs] in PFMC from 30 to 74, whereas there was minimal increase of median number of SFCs in PBMC from 55 to 60

Conclusion ESAT-6 - ELlSpot using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-gamma in pleural effusion [PE] is another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap

Pro-inflammatory CD14+ CD16++ monocytes in HCV persistent and spontaneously cleared infections

In human blood, two Monocytes [Mo] subpopulations have been distinguished: the classical CD 14+'DR' Mo and pro-inflammatory CD14'CD16-'DR' Mo. In HCV antibody positive with persistent HCY [positive HCV RNA], we found decreased numbers of the pro-inflammatory Mo in 20 patients with persistent HCV with an average of [34.1 f 22 cells/pl] in comparison to the numbers in the cleared patients [positive HCV Ab with negative HCV MA] [85 + 35 cells/d]. Intracellular TNF was measured by three-color immunofluorescence and flow cytometry after stimulation with Lipopolysaccharide [LPS]. In patients with persistent HCV infection, pro-inflammatory Mo showed one and half fold higher level of intracellular TNF staining. This indicates that the minor population of pro-inflammatory Mo plays pronounced roles in the HCV clearance or persistence

Caspase 3 activity in monocytes and liver cell injury in hepatitis C patients

Hepatic cirrhosis is a frequent complication in chronic hepatitis C virus [HCV] infection which occurs via an unclear mechanism. Although apoptosis has been claimed to be associated in liver diseases, its relation to HCV-associated fibrosis is largely unexplored. We have studied the role of caspase 3 in monocytes in hepatitis patients with HCV infection using two caspase assays: blot assay and flow cytometry assay. Purified monocytes demonstrated caspase 3 activity by western blot at 0 timepoint in 12 patients with HCV infection [10 HCV patients with elevated liver enzymes and 2 out of 10 HCV patient with normal liver enzymes]. At the same time, purified monocytes from normal healthy controls showed caspase 3 activity only after 16 hr culture. Caspase 3 activation was also confirmed by flow cytometry analysis using the PhiPhiLux assay where in 12 hepatitis patients' samples caspase 3 activation was detected at 0 time before culture. Using the PhiPhiLux assay the differences were highly significant for both hepatitis patients groups in comparison to the normal healthy control [p** < 0.001]. The average mean fluorescence intensity was 222 +/- 63 in the hepatitis with elevated liver enzymes, 52 +/- 34 in patients with normal liver enzymes and 8.6 +/- 2.8 for the normal healthy controls. Moreover, there was a significant correlation between caspase 3 activity and liver enzyme levels in the hepatitis patients. This results show that caspase activation is associated with inflammatory reactions in chronic HCV infection and caspase 3 activation levels, especially in monocytes, might be reliable tools to detect high levels of liver injury in chronic HCV infection