Haji Akbar

The changing epidemiology pattern and frequency distribution of hepatitis C virus in Pakistan.

Information regarding the changing pattern in hepatitis C virus (HCV) genotypes/subtypes and resulting disease outcome is not well known. The specific objective of this study was to find out the frequency distribution of HCV genotypes and changing pattern of various HCV genotypes overtime in well-characterized Pakistani HCV isolates. The genotype distribution of HCV from all the four provinces of Pakistan was tracked for a period of 10 years (2000-2009) on total 20,552 consecutive anti-HCV and HCV RNA positive patients sample using type-specific genotyping assay. Of these, 16,891 (82.2%) samples were successfully genotyped. Of these 11,189 (54.4%) were males and 9363 (45.55%) were females. Of the successfully genotyped samples, 12,537 (74.2%) were with 3a, 1834 (10.9%) with 3b, 50 (0.24%) with 3c, 678 (3.3%) with 1a, 170 (0.83%) with 1b, 49 (0.24%) with 1c, 431 (2.1%) with 2a, 48 (0.23%) with 2b, 3 (0.01%) with 2c, 13 (0.06%) with 5a, 12 (0.06%) with 6a, 101 (0.49%) with 4, and 965 (4.7%) were with mixed-genotype infection. A changing pattern of HCV genotypes prevalence was observed in Pakistan overtime, with an increase in the relative proportion of genotype 3a and mixed genotypes and a decrease of genotypes 3b, 2b, 4, 5a and 2a. This changed HCV genotype pattern might have direct impact on HCV disease outcome and new therapeutic strategies may be needed.

Body condition score at calving affects systemic and hepatic transcriptome indicators of inflammation and nutrient metabolism in grazing dairy cows.

Calving body condition score (BCS) is an important determinant of early-lactation dry matter intake, milk yield, and disease incidence. The current study investigated the metabolic and molecular changes induced by the change in BCS. A group of cows of mixed age and breed were managed from the second half of the previous lactation to achieve mean group BCS (10-point scale) that were high (HBCS, 5.5; n=20), medium (MBCS, 4.5; n=18), or low (LBCS, 3.5; n=19). Blood was sampled at wk -4, -3, -2, 1, 3, 5, and 6 relative to parturition to measure biomarkers of energy balance, inflammation, and liver function. Liver was biopsied on wk 1, 3, and 5 relative to parturition, and 10 cows per BCS group were used for transcript profiling via quantitative PCR. Cows in HBCS and MBCS produced more milk and had greater concentrations of nonesterified fatty acids and β-hydroxybutyrate postpartum than LBCS. Peak concentrations of nonesterified fatty acids and β-hydroxybutyrate and greater hepatic triacylglycerol concentrations were recorded in HBCS at wk 3. Consistent with blood biomarkers, HBCS and MBCS had greater expression of genes associated with fatty acid oxidation (CPT1A, ACOX1), ketogenesis (HMGCS2), and hepatokines (FGF21, ANGPTL4), whereas HBCS had the lowest expression of APOB (lipoprotein transport). Greater expression during early lactation of BBOX1 in MBCS and LBCS suggested greater de novo carnitine synthesis. The greater BCS was associated with lower expression of growth hormone/insulin-like growth factor-1 signaling axis genes (GHR1A, IGF1, and IGFALS) and greater expression of gluconeogenic genes. These likely contributed to the higher milk production and greater gluconeogenesis. Despite greater serum haptoglobin around calving, cows in HBCS and MBCS had greater blood albumin. Cows in MBCS, however, had a higher albumin:globulin ratio, probably indicating a less pronounced inflammatory status and better liver function. The marked decrease in expression of NFKB1, STAT3, HP, and SAA3 coupled with the increase in ALB on wk 3 in MBCS cows were consistent with blood measures. Overall, results suggest that the greater milk production of cows with higher calving BCS is associated with a proinflammatory response without negatively affecting expression of genes related to metabolism and the growth hormone/insulin-like growth factor-1 axis. Results highlight the sensitivity of indicators of metabolic health and inflammatory state to subtle changes in calving BCS and, collectively, indicate a suboptimal health status in cows calving at either BCS 3.5 or 5.5 relative to BCS 4.5.

Hepatitis C virus genotype 3a with phylogenetically distinct origin is circulating in Pakistan

BackgroundHepatitis C virus (HCV) is one of the leading causes of viral hepatitis worldwide and its genotype 3a is predominant in vast areas of Pakistan.FindingsThe present study reports the first full sequence of HCV 3a isolate PK-1 from Pakistan. This nucleotide sequence was compared with six other HCV genotype 3a full length sequences from different regions of the world by using statistical methods of phylogenetic analysis.ConclusionThe nucleotide difference of these seven sequences shows that HCV genotype 3a of phylogenetically distinct origin is circulating in Pakistan.

Feed restriction, but not l-carnitine infusion, alters the liver transcriptome by inhibiting sterol synthesis and mitochondrial oxidative phosphorylation and increasing gluconeogenesis in mid-lactation dairy cows

Abomasal carnitine infusion during acute feed restriction increases hepatic fatty acid oxidation and decreases liver lipid in dairy cows. Eight mid-lactation Holstein cows were used in a replicated 4×4 Latin square design with 14-d periods. A 2×2 factorial arrangement was used to determine the effects of water infusion+ad libitum dry matter intake (DMI), water infusion+restricted DMI (50% of previous 5-d average), l-carnitine infusion (20 g/d)+ad libitum DMI, or l-carnitine infusion+restricted DMI. Liver RNA from 7 healthy cows was used for transcriptome profiling using a bovine microarray. An ANOVA with a false discovery rate was used to identify treatment and interaction effects. A substantial transcriptome change was observed only with DMI restriction, resulting in 312 (155 downregulated, 157 upregulated) differentially expressed genes. Quantitative PCR was performed to verify microarray data and measure expression of additional genes not present on the microarray. The quantitative PCR data confirmed the effect of feed restriction but not of l-carnitine treatment. Feed restriction increased expression of GPX3 and of genes associated with gluconeogenesis (PC, PDK4), inflammation (SAA3), and signaling (ADIPOR2). In contrast, feed restriction downregulated BBOX, a key for l-carnitine biosynthesis, and the transcription factor HNF4A. The bioinformatics functional analysis of genes affected by DMI restriction uncovered biosynthesis of cholesterol and energy generation by mitochondrial respiration as the most relevant and inhibited functions. The data also indicated an increase of flux toward gluconeogenesis. We interpreted those results as a likely response of the liver to spare energy and provide glucose for the lactating mammary gland during feed deprivation.

Pattern and molecular epidemiology of Hepatitis B virus genotypes circulating in Pakistan.

The continuously mutating nature of Hepatitis B virus (HBV) is responsible for the emergence of varying genotypes in different regions of the world affecting the disease outcome. The objective of the current study was to find out the pattern of HBV genotypes circulating in Pakistan. HBV genotypes were determined in HBV chronic patients of different age and gender from all the four different geographical regions (provinces) of Pakistan for a period of 2 years (2007-2009). Out of the total 3137 consecutive patients, 300 (175; 58.3% males and 125; 41.7% females) were randomly selected for HBV genotype A through H determination using molecular genotyping methods. Total 269 (89.6%) isolates were successfully genotyped where as 31 (10.3%) samples failed to generate a type-specific PCR band and were found untypable. Out of the successfully genotyped samples, 43 (14.3%) were with type A, 54 (18%) were with type B, 83 (27.6%) were with type C, 39 (13%) were with type D, 2 (0.6%) were with type E, 4 (1.3%) were with genotype F and total 44 (14.6%) were with mixed HBV infections. Of the mixed genotype infection cases, 16 were with genotypes A/D, 9 were B/C, six were A/D/F, five were with genotypes A/F, two were with A/B/D and B/E and one each for A/C as well as A/E genotypes. Four common genotypes of HBV found worldwide (A, B, C & D) were isolated from Pakistan along with uncommon genotypes E and F for the first time in Pakistan. Overall Genotype C is the most prevalent genotype. Genotypes B and C are predominant in Punjab & Balochistan and Khyber Pakhtoonkhwa, respectively whereas genotype A in Sindh.

High baseline interleukine-8 level is an Independent risk factor for the achievement of sustained virological response in chronic HCV patients

Hepatitis C virus (HCV), a major cause of liver disease throughout the world, is difficult to treat with interferon (IFN) (and various formulations and combinations thereof) being the only approved molecule available. It has been investigated recently that proinflammatory chemokine interleukin-8 (IL-8) induced by HCV partially inhibits the antiviral IFN-α therapy. Therefore, the current study was aimed to prospectively utilize the baseline IL-8 levels in the HCV infected serum and predicts its role in sustained virological response (SVR) to IFN-α+ribavirin therapy, in chronic HCV patients in Pakistan. One hundred and ten hepatitis C patients without any other infections underwent IFN-α+ribavirin combination treatment. Baseline IL-8 levels were determined before starting of the therapy for all these patients. Fifteen normal volunteers negative for HCV were kept as control. The baseline IL-8 levels were found significantly higher in all HCV positive patients as compared to normal healthy volunteers (1083.54 ± 85.72 pg/ml versus 6.99 ± 1.05 pg/ml [mean ± SEM], p<0.01) and were also significantly higher in non-responders than responders (p<0.05). Comparatively higher mean baseline IL-8 levels were observed in non-responders (2442.02 ± 159.92 pg/ml), than late (1009.31 ± 45.31) and rapid (540.91 ± 27.06 pg/ml) responders. Significant relation was observed between baseline IL-8 level and response to IFN therapy (p<0.01). Results of this study suggest that increased levels of IL-8 in HCV infection might be involved in pathogenesis, persistence and resistance to IFN-α+ribavirin combination therapy.

Occult hepatitis C virus infection and associated predictive factors: The Pakistan experience

The aim of the present study was to determine the presence of HCV RNA in the liver biopsies of patients with abnormal liver tests but without detectable serum HCV RNA and anti-HCV antibodies in sera. Liver biopsies and whole blood of total 31 patients who were negative for anti-HCV antibodies with elevated liver function tests were received at Division of Molecular Diagnostics, University of the Punjab Pakistan from January 2002 to June 2009 for the detection of HCV RNA. HCV RNA status of the subjects was tested by reverse-transcription PCR and quantified using SmartCycler II real-time PCR. HCV genotyping was carried out in HCV RNA positive samples using molecular genotyping method. HCV RNA was found in liver-biopsy specimens from 23 (74.2%) of the total 31 patients negative for anti-HCV antibodies and undetectable serum HCV RNA. HCV RNA of both negative and positive polarity was found in the livers of 8 (25.8%) patients. Genotyping analysis showed that 65% patients were infected with HCV 3a, 17% with 3b, 13% with 1a and 4% patients were found with untypable genotype. In a multivariate logistic regression model, patients having previous history surgeries, male sex and age above 30 years were significantly associated with the presence of occult HCV infection (p<0.05). In conclusion, patients with elevated liver enzymes and negative HCV antibodies and negative serum RNA may have occult HCV infection and its chance increases with previous history of surgeries, in male sex and above 30 years of age.

The prevalence of HBV infection in the cohort of IDPs of war against terrorism in Malakand Division of Northern Pakistan

BackgroundHepatitis B is an important public health problem in the Pakistani population and is the major cause of chronic hepatitis, cirrhosis, fibrosis and hepatocellular carcinoma. High prevalence of HBV infections has been observed especially in areas of low economic status. In spite of effective immunization programs, no significant change has been observed in the epidemiology of HBV in the rural areas of Pakistan (~67.5% of the total population) mainly due to lack of interest from government authorities and poor hygienic measures. The current study was aimed at estimating the prevalence and risk factors associated with HBV infection within internally displaced persons (IDPs) due to war against terrorism in the Malakand Division of Northern Pakistan.MethodsBlood samples from 950 IDPs suspected with HBV infection (including both males and females) were collected and processed with commercial ELISA kits for HBsAg, Anti HBs, HBeAg, Anti HBe antibodies. The samples positive by ELISA were confirmed for HBV DNA by real-time PCR analysis.ResultsThe overall prevalence of HBV observed was 21.05% of which 78.5% were males and 21.5% were females. Most confirmed HBV patients belong to the Malakand and Dir (lower) district. High-risk of infection was found in the older subjects 29.13% (46-60 years), while a lower incidence (11.97%) was observed in children aged <15 years. Lack of awareness, socioecomic conditions, sexual activities and sharing of razor blades, syringes and tattooing needles were the most common risk factors of HBV infection observed during the cohort of patients.ConclusionThe present study, revealed for the first time a high degree of prevalence of HBV infection in rural areas of Northern Pakistan. The noticed prevalence is gender- and age-dependent that might be due to their high exposures to the common risk factors. To avoid the transmission of HBV infection proper awareness about the possible risk factors and extension of immunization to the rural areas are recommended.

Dietary Lipid During Late-Pregnancy and Early-Lactation to Manipulate Metabolic and Inflammatory Gene Network Expression in Dairy Cattle Liver with a Focus on PPARs

Polyunsaturated (PUFA) long-chain fatty acids (LCFAs) are more potent in eliciting molecular and tissue functional changes in monogastrics than saturated LCFA. From −21 through 10 days relative to parturition dairy cows were fed no supplemental LCFA (control), saturated LCFA (SFAT; mainly 16:0 and 18:0), or fish oil (FISH; high-PUFA). Twenty-seven genes were measured via quantitative RT-PCR in liver tissue on day −14 and day 10. Expression of nuclear receptor co-activators (CARM1, MED1), LCFA metabolism (ACSL1, SCD, ACOX1), and inflammation (IL6, TBK1, IKBKE) genes was lower with SFAT than control on day −14. Expression of SCD, however, was markedly lower with FISH than control or SFAT on both −14 and 10 days. FISH led to further decreases in expression on day 10 of LCFA metabolism (CD36, PLIN2, ACSL1, ACOX1), intracellular energy (UCP2, STK11, PRKAA1), de novo cholesterol synthesis (SREBF2), inflammation (IL6, TBK1, IKBKE), and nuclear receptor signaling genes (PPARD, MED1, NRIP1). No change in expression was observed for PPARA and RXRA. The increase of DGAT2, PLIN2, ACSL1, and ACOX1 on day 10 versus −14 in cows fed SFAT suggested upregulation of both beta-oxidation and lipid droplet (LD) formation. However, liver triacylglycerol concentration was similar among treatments. The hepatokine FGF21 and the gluconeogenic genes PC and PCK1 increased markedly on day 10 versus −14 only in controls. At the levels supplemented, the change in the profile of metabolic genes after parturition in cows fed saturated fat suggested a greater capacity for uptake of fatty acids and intracellular handling without excessive storage of LD.

Postpartal Subclinical Endometritis Alters Transcriptome Profiles in Liver and Adipose Tissue of Dairy Cows

Transcriptome alterations in liver and adipose tissue of cows with subclinical endometritis (SCE) at 29 d postpartum were evaluated. Bioinformatics analysis was performed using the Dynamic Impact Approach by means of KEGG and DAVID databases. Milk production, blood metabolites (non-esterified fatty acids, magnesium), and disease biomarkers (albumin, aspartate aminotransferase) did not differ greatly between healthy and SCE cows. In liver tissue of cows with SCE, alterations in gene expression revealed an activation of complement and coagulation cascade, steroid hormone biosynthesis, apoptosis, inflammation, oxidative stress, MAPK signaling, and the formation of fibrinogen complex. Bioinformatics analysis also revealed an inhibition of vitamin B3 and B6 metabolism with SCE. In adipose, the most activated pathways by SCE were nicotinate and nicotinamide metabolism, long-chain fatty acid transport, oxidative phosphorylation, inflammation, T cell and B cell receptor signaling, and mTOR signaling. Results indicate that SCE in dairy cattle during early lactation induces molecular alterations in liver and adipose tissue indicative of immune activation and cellular stress.