Anwar Ali Siddiqui

Hepatitis B virus subgenotypes D1 and D3 are prevalent in Pakistan

BackgroundAs the hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis. In Pakistan, there is no reported data on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of mutations present in the strains prevalent here.Findingsto confirm specificity of PCR typing, phylogenetic analysis of the pre-S1 region and the divergence was studied through 13 sequences of 362 bp (accession number EF432765 – EF432777). A total of 315 serum samples, selected from HBsAg positive patients representing the major ethnic groups, residing in Karachi, Sindh were tested for genotyping. Genotype D (219/315) was found to be the most prevalent (70%) amongst our patients. The rest of the genotypes A and a mixture of A and D (AD) were distributed as 20%, and 10% respectively. Phylogenetic tree demonstrated clustering of 11 samples with subgenotype D1 sequences and the remaining two strains on a branch within D3 samples. All samples intermixed with strains from other countries and were found to be closely related to Indian, Iranian and Egyptian HBV strains with 98.7 – 99.0% homology.ConclusionThis study confirms the predominance of genotype D in southeastern Asia and presence of subgenotypes DI and D3 in the Pakistani infected patients. More studies are required to investigate the reason for fewer inclusions of D3 compared to the D1 in Pakistani HBV strains.

Risk of transmission and features of hepatitis C after needlestick injuries.

The rate of transmission and management of needlestick injuries from hepatitis C virus (HCV) patients to healthcare workers is still a matter of debate. We used a stringent protocol using monthly transaminase levels and polymerase chain reaction for HCV RNA to monitor 53 healthcare workers prospectively for up to 6 months following needle injuries from HCV-positive patients. Evidence of transmission of HCV was found in only 2 workers (4%) with mild asymptomatic infection, one of which resolved spontaneously. Based on our experience, we now use a less-intensive follow-up protocol. Further investigation is required to determine the most cost-effective method to monitor individuals who suffer a needlestick injury from an HCV-positive patient.

The association of complex liver disorders with HBV genotypes prevalent in Pakistan

BackgroundGenotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depends largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from south East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much neededMethodsA total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Councils (PMRC) out patient clinics. Two hundred and twenty six (77%) were males, sixty nine (23%) were females (M to F ratio 3.3:1).ResultsOut of 295 patients, 156 (53.2%) had Acute(CAH), 71 (24.2%) were HBV Carriers, 54 (18.4%) had Chronic liver disease (CLD) Hepatitis. 14 (4.7%) were Cirrhosis and HCC patients. Genotype D was the most prevalent genotype in all categories of HBV patients, Acute (108), Chronic (39), and Carrier (53).Cirrhosis/HCC (7) were HBV/D positive. Genotype A was the second most prevalent with 28 (13%) in acute cases, 12 (22.2%) in chronics, 14 (19.7%) in carriers and 5 (41.7) in Cirrhosis/HCC patients. Mixed genotype (A/D) was found in 20 (12.8%) of Acute patients, 3 (5.6%) of Chronic and 4 (5.6%) of carriers, none in case of severe liver conditions.ConclusionMixed HBV genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D) did not significantly appear to influence the clinical outcome.

Health care risk factors among women and personal behaviours among men explain the high prevalence of hepatitis C virus infection in Karachi, Pakistan

Summary.  To estimate the prevalence and identify factors associated with hepatitis C virus (HCV) infection among men and women in Karachi, Pakistan. We conducted a cross‐sectional study of adult men and women in a peri‐urban community of Karachi (Jam Kandah). Households were selected through systematic sampling from within all villages in the study area. All available adults within each household were interviewed about potential HCV risk factors. A blood specimen was collected to test for anti‐HCV antibodies by enzyme immunoassay. We used generalized estimating equations while accounting for correlation of responses within villages to identify the factors associated with HCV infection. Of 1997 participants, 476 (23.8%) were anti‐HCV positive. Overall, HCV infection was significantly associated with increasing age, ethnicity, and having received ≥2 blood transfusions, ≥3 hospitalizations, dental treatment and >5 injections among women. Among women, ≥2 blood transfusions [adjusted odds ratio (aOR) = 2.32], >5 injections during the past 6 months (aORs = 1.47), dental treatment (aOR = 1.31) and increasing age(aOR = 1.49), while among men, extramarital sexual intercourse (aOR = 2.77), at least once a week shave from barber (aOR = 5.04), ≥3 hospitalizations (aOR = 2.50) and increasing age (aOR = 1.28) were associated with HCV infection. A very high prevalence of HCV was found in the study population. Among women, unsafe health care practices, while among men extramarital sex, shaving from a barber and hospitalizations were associated with HCV infection. Efforts are needed to improve the safety of medical procedures to reduce the transmission of HCV in Pakistan [Corrections made in Summary after initial online publication.].

Berberis vulgaris Root Bark Extract Prevents Hyperoxaluria Induced Urolithiasis in Rats

Berberis vulgaris is a widely used plant for the treatment of urolithiasis. To evaluate its antiurolithic potential, the crude aqueous‐methanol extract of Berberis vulgaris root bark (Bv.Cr) was tested in an animal model of urolithiasis, developed in male Wistar rats by adding 0.75% ethylene glycol in drinking water. Bv.Cr (50 mg/kg) inhibited CaOx crystal deposition in renal tubules and protected against associated changes including polyuria, weight loss, impaired renal function and the development of oxidative stress in kidneys. Activity‐guided fractionation revealed the concentration of antiurolithic constituent(s) mainly in the aqueous fraction. These data, indicating the presence of antiurolithic activity in Berberis vulgaris root bark, rationalize its medicinal use for the treatment of urolithiasis. Copyright

Comparison of high performance liquid chromatography, radio immunoassay and electrochemiluminescence immunoassay for quantification of serum 25 hydroxy vitamin D

OBJECTIVE To compare the performance of radioimmunoassay (RIA) with high-performance liquid chromatography (HPLC) and electrochemiluminescence (ECLIA) for the quantification of vitamin D (25OHD). METHODS HPLC method for the determination of 25OHD in human biological samples was developed and compared in terms of accuracy and precision with a commercially available RIA assay. Performance of RIA assay with ECLIA technology for 25OHD analysis was further compared. RESULTS Median 25OHD levels with HPLC vs. RIA were 50.1nmol/L (IQ=17.7-199.4nmol/L) and 51.1nmol/L (IQ=12.5-187.2nmol/L) respectively, whereas median 25OHD concentration with RIA vs. ECLIA was 32.4nmol/L (9.98-199.7nmol/L) and 29.9nmol/L (4.9-214.6nmol/L), respectively. Comparison data for HPLC vs. RIA showed RIA=-1.13+1.01 (HPLC) (RMSE=11.2nmol/L) and for RIA vs. ECLIA revealed, ECLIA=3.21+0.9 (RIA) (RMSE9.6nmol/L). CONCLUSION Acceptable correlation was observed among HPLC and RIA and also with RIA and ECLIA in quantification of 25OHD.

Proteins in renal stones and urine of stone formers.

Abstract Knowledge of the essential characteristics of macromolecules constituting the organic matrix of the nidus of urinary stones is required to understand the mechanism of urolithogenesis. The aim of this study was to isolate and characterise those stone nidus proteins. Using an extraction buffer containing SDS and β-mercaptoethanol, we were able to overcome known problems of protein isolation from urinary stone matrix. These proteins were characterised by a strong tendency to aggregate under reducing and denaturing conditions. On SDS-PAGE, their molecular weights range from ≤12 to 66 kDa. Antisera raised against stone matrix proteins showed a cross-reactivity between proteins isolated from different stones irrespective of their origin or mineral composition. Moreover, urinary proteins from stone formers also cross-reacted with these whereas there was no reaction with urinary proteins of non-stone formers. Western blotting confirmed these findings. Given the above summarised properties, it can be safely concluded that these proteins are prevalent in urines of stone formers, that they are selectively incorporated into renal stones of all aetiologies, and that they most likely have a role in nidus and, therefore, early stone formation.

Altered expression of cell cycle and apoptotic proteins in chronic hepatitis C virus infection

BackgroundA disrupted cell cycle progression of hepatocytes was reported in chronic hepatitis C virus (HCV) infection, which can contribute significantly in the associated pathogenesis. The present study aimed to further elaborate these disruptions by evaluating the expression of key cell cycle and apoptotic proteins in chronic HCV infection with particular reference to genotype 3. Archival liver biopsy specimens of chronic HCV-infection (n = 46) and normal histology (n = 5) were analyzed by immunohistochemistry using antibodies against proliferation marker Mcm-2, G1 phase marker Cyclin D1, S phase marker Cyclin A, cell cycle regulators p21 (CDK inhibitor) and p53 (tumor suppressor protein), apoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2.ResultsElevated Mcm-2 expression was observed in hepatocytes in chronic HCV infection, indicating increased cell cycle entry. Cyclin D1 expression was higher than cyclin A, which suggests a slow progression through the G1 phase. Expression of cell cycle regulators p21 and p53 was elevated, with no concordance between their expressions. The Mcm-2 and p21 expressions were associated with the fibrosis stage (p = 0.0001 and 0.001 respectively) and that of p53 with the inflammation grade (p = 0.051). Apoptotic marker, Caspase-3, was mostly confined to sinusoidal lining cells with little expression in hepatocytes. Anti-apoptotic protein, Bcl-2, was negligible in hepatocytes and detected principally in infiltrating lymphocytes. Expression of all these proteins was unrelated to the HCV genotype and were detected only rarely in the hepatocytes of normal liver.ConclusionThe results showed an arrested cell cycle state in the hepatocytes of chronic HCV infection, regardless of any association with genotype 3. Cell cycle arrest is characterized by an increased expression of p21, in relation to fibrosis, and of p53 in relation to inflammation. Furthermore, expression of p21 was independent of the p53 expression and coincided with the reduced expression of apoptotic protein Caspase-3 in hepatocytes. The altered expression of these cell cycle proteins in hepatocytes is suggestive of an impaired cell cycle progression that could limit the regenerative response of the liver to ongoing injury, leading to the progression of disease.

Cytotoxic effects of aflatoxin B1 on human brain microvascular endothelial cells of the blood-brain barrier.

Aflatoxins are mycotoxins produced by Aspergillus spp. Although AFB1 is implicated as a carcinogen in hepatocellular carcinoma, brain autopsies in affected areas have revealed its presence in 81% of cases. Given its haematogenous spread, here we determined the cytotoxic effects of AFB1 on primary human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, human umbilical vein endothelial cells (HUVEC) as well as immortalized epithelial cells of human hepatocellular carcinoma (Huh7). The cell types were exposed to AFB1 (3-32 nM) for 24 h and release of lactate dehydrogenase was measured as cell cytotoxicity marker. Furthermore, DNA was collected from both cell types and DNA adduct formation was determined by immunoblot using anti-AFB1-DNA adduct antibody. At 32 nM, AFB1 killed >85% HBMEC, while controls showed minimal effects (P < .05). Similar concentrations of AFB1 showed 22% cell death of HUVEC, while the same concentration did not kill Huh7. At low concentrations, in other words, 3.2 nM, AFB1 produced DNA adduct formation in HBMEC, while high concentration (32 nM) did not form DNA adducts. For HUVEC, 16 nM and 32 nM exhibited DNA adduct formation. For Huh7, 3.2 nM did not form DNA adducts, while 32 nM exhibited DNA adduct formation. For the first time, we report that AFB1 affected the viability of primary endothelial cells but not immortalized Huh7 cells. Cytotoxicity of brain endothelial cells suggests extra-hepatic complications post-AFB1 exposure.

Albumin precursor and Hsp70 modulate corneal wound healing in an organ culture model

In order to investigate the role of albumin precursor and Hsp70 in corneal wound healing, we have analyzed the distribution of these proteins in wounded and non-wounded corneas of rabbits and the effects of topical applications of anti-albumin precursor and anti-Hsp70 antibodies on wound healing. Anti-albumin precursor and anti-Hsp70 antibodies were topically applied in healing corneal epithelium of rabbit eyes in organ culture. Corneas were allowed to heal in vitro for up to 120 h in serum-free medium with 5 and 10 μg/ml or without (migrating control) anti-albumin precursor/ or anti-Hsp70 antibodies. Fibronectin (Fb) (5 μg/ml) was used as a positive control. Immunofluorescence labelling was used to detect proteins in corneal epithelium at various time intervals following an epithelial defect. Delay in wound healing (p<0.005) was observed with 10 μg/ml anti-albumin antibody labelling. A similar pattern was observed when anti-fibronectin antibody (5 μg/ml) alone and in combination with anti-albumin (10 μg/ml) was ectopically added with wound closure occurring at 120 h. However with anti-Hsp70 antibody (5 μg/ml) slightly delayed (p<0.005) wound closure was observed at 96 h and considerable retardation >120 h with 10 μg/ml. Additionally, immunofluoresence showed a strong co-localization of Hsp70 and albumin precursor during the active phase of wound healing. The presence of albumin precursor and Hsp70 in the epithelial compartment of the cornea indicates a role for these proteins in modulating cell behavior such as epithelial growth, adhesion or regeneration, thus contributing to corneal epithelial wound healing.