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.

Frequency of hepatitis C and D super infection in patients with hepatitis B related complex liver disorders.

OBJECTIVE: To determine the frequency of super infection of hepatitis C and D in patients with hepatitis B related complex liver disorders and the distribution of HBV genotypes in these patients. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: The Gastroenterology Unit of PMRC in JPMC, Karachi, from July 2006 to June 2007. METHODOLOGY: All patients registered for HBV associated infections were selected. Blood was drawn from 180 patients who fulfilled the inclusion criteria. Those with an incomplete test profile were excluded. All clinical conditions were investigated through liver function tests, coagulation profile, and findings at abdominal ultrasonography, upper gastrointestinal endoscopy and liver biopsy. Liver cirrhosis and hepatocellular carcinoma (HCC) were diagnosed either on the basis of histology, or on a combination of radiological, endoscopic and laboratory data. Hepatitis B virus DNA was extracted from serum, and subjected to a nested PCR using the type specific primers for HBV genotype. Descriptive statistics were used for frequency and mean determination. RESULTS: The 129 patients finally selected for statistical analysis included 108 (84%) males and 21 (16%) females. The age ranged from 6- 68 years (mean=31.5 +/-12.39 years). There were 70 (54.2%) patients of non-cirrhotic, chronic hepatitis (CLD), 38 (29.4%) carriers, 12 (9.3%) cirrhotics and 9 (6.9%) HCC patients. Among the 129 patients, 45 (34.9%) were positive for double infection with HDV. These included 35 CLD cases, 7 cirrhotic and 3 carriers, 4 (3.1%) patients were positive for double infection with HCV including one with CLD, 2 with cirrhosis and one with HCC. Triple infection with HBV/HDV/HCV was present in 4 (3.1%) patients who had CLD. Approximately 59% (n=76) patients were not coinfected, though 9 had developed HCC. The genotype distribution of HBV was observed as D in 98 (76%) patients, A in 24 (18.6%), and AD mix in 7 (5.4%). Genotypes B, C, E or F were not found. Accordingly, genotype D strains were the predominant strains among all categories. CONCLUSION: The frequency of super infection of hepatitis C and D was found to be highest in HBV cirrhosis patients compared to patients having chronic liver disease (non-cirrhotics) and carriers. Genotype D of hepatitis B virus was found dominant in all hepatitis B related complex liver disorders.

Phylogenetic analysis of hepatitis B virus in Pakistan.

OBJECTIVE: To identify the distribution pattern of Hepatitis B Virus (HBV) genotype in a group of patients and to study its phylogenetic divergence. STUDY DESIGN: An observational study. PLACE AND DURATION OF STUDY: The clinics of Gastroenterology Unit, Ziauddin University, from January to December 2006. METHODOLOGY: Two hundred and one HBV infected patients were genotyped for this study. All HbsAg positive individuals, either healthy carriers or suffering from conditions such as acute or chronic hepatitis, cirrhosis and hepatocellular carcinoma were included. Hepatitis B patients co-infected with other hepatic viruses were excluded. Hepatitis B virus DNA was extracted from serum, and subjected to a nested PCR, using the primers type-specific for genotype detection. Phylogenetic analysis was performed in the pre-S1 through S genes of HBV. The divergence was studied through 15 sequences of 967 bp submitted to the DBJ/EMBL/GenBank databases accessible under accession number EF584640 through EF584654. RESULTS: Out of 201 patients tested, 156 were males and 45 were females. Genotype D was the predominant type found in 128 (64%) patients followed by A in 47 (23%) and mixed A/D in 26 (13%). Phylogenetic analysis confirmed the dominance of genotype D and subtype ayw2. CONCLUSION: There was dominance of genotype D subtype ayw2. It had a close resemblance with HBV strains that circulate in Iran, India and Japan.

First successful autologous chondrocyte implantation in Pakistan.

Osteochondritis dissecans entails a hyaline cartilage defect of the articular surface causing pain and functional restriction in young adults, sometimes resulting in early degenerative arthritis. Conventional treatment methods such as abrasion chondroplasty and mosaicplasty have limitations in terms of quality of the resultant cartilage and donor site morbidity. A more recent technique, autologous chondrocyte implantation (ACI) results in hyaline cartilage formation and gives good long-term outcome, but requires a high-level cell culture facility and two surgical procedures. The patient was a young female with knee pain, intermittent locking and feeling of "joint mouse". MRI scan and arthroscopy showed a 2 x 2 cm full thickness osteochondral defect in the medial femoral condyle. A free fragment of articular cartilage was found, which was extracted arthroscopically, and chondrocytes were cultured from it in the Juma laboratory. Subsequently, patient underwent surgery whereby the chondrocytes were injected under a periosteal patch sewn over the defect. Over six months, patient's symptoms completely resolved and she returned to full function. A repeat arthroscopy after one year revealed complete filling of the previous defect with normal appearing cartilage indicating success of the procedure. This technology can be utilized for treating patients with a variety of conditions affecting hyaline cartilage of joints.

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

BACKGROUND: Genotyping 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 needed METHODS: A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's (PMRC) out patient clinics. Two hundred and twenty six (77%) were males, sixty nine (23%) were females (M to F ratio 3.3:1). RESULTS: Out 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. CONCLUSION: Mixed 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.

Identification of myeloperoxidase, alpha-defensin and calgranulin in calcium oxalate renal stones.

BACKGROUND: In order to understand the mechanism of stone genesis, it is essential to determine the characteristics of macromolecules constituting the urinary stones. We characterized proteins from the inner core and outer matrix of calcium oxalate (CaOx) renal stones. METHODS: Inner core and outer matrix of CaOx renal stones were separated and proteins were extracted with a buffer containing SDS and beta-mercaptoethanol. Proteins were analyzed and purified by SDS-PAGE and RP-HPLC respectively. The protein bands from gel and protein fractions were sequenced by MALDI TOF mass spectrometry. ELISA, western and slot blot immunoassays were performed to confirm the identity of the proteins in stones and urine of the stone formers. The potential of the identified protein as an effective promoter or inhibitor was assessed by observing their effects on CaOx crystallization using aggregometer. RESULTS: The inner core extract predominantly exhibited protein species in the molecular weight range of 12-14 kDa. However, a 66 kDa band, identified as osteopontin was also detected in the inner core along with outer matrix and in the urine of stone formers and non stone formers. Purification of low molecular weight proteins was carried out by reversed phase HPLC. Tandem mass spectrometry analysis identified them as myeloperoxidase chain A (MPO-A), alpha-defensin, and calgranulin. ELISA, western blot and slot-blot immuno-assays further confirmed their presence restricted to the inner core and not in the outer matrix. Turbidity assays showed that low molecular weight renal stone proteins promoted the aggregation of CaOx crystals. CONCLUSIONS: Persistent hyperoxaluria leads to tubular epithelial injury, resulting in the release of these anti-inflammatory proteins. These proteins could have been first adsorbed on CaOx crystals thereby become a part of nucleation process leading to inner matrix formation.

Expression of the Androgen Receptor, pAkt, and pPTEN in Breast Cancer and Their Potential in Prognostication.

BACKGROUND: Importance of androgen receptor (AR) as an independent prognostic marker in Pakistani women with breast cancer (BCa) remains unexplored. Our aim was to identify the expression and potential prognostic value of AR, its upstream regulator (pAkt) and target gene (pPTEN) in invasive BCa. METHODS: This study used a cohort of 200 Pakistani women with invasive BCa diagnosed during 2002-2011. Expression of AR, pAkt and pPTEN was determined on formalin fixed paraffin embedded tissue sections by immunohistochemistry. The association of AR, pAkt and pPTEN with clinicopathological parameters was determined. Survival analyses were undertaken on patients with ≥5years of follow-up (n=82). RESULTS: Expression of AR, pAkt and pPTEN was observed in 47.5%, 81.3% and 50.6% of patients, respectively. AR-expressing tumors were low or intermediate in grade (P<.001) and expressed ER (P=.002) and PR (P=.001). Patients with AR+ tumors had significantly higher OS (Mean OS=10.2±0.465years) compared to patients with AR- tumors (Mean OS=5.8±0.348years) (P=.047). Furthermore, AR-positivity was associated with improved OS in patients receiving endocrine therapy (P=.020). Patients with AR+ /pAkt+ /pPTEN- tumors, had increased OS (Mean OS=7.1±0.535years) compared to patients with AR-/pAkt+/pPTEN- tumors (Mean OS=5.1±0.738years). CONCLUSION: AR-expressing tumors are frequently characterized by low or intermediate grade tumors, expressing ER and PR. In addition, expression of AR, pAkt and pPTEN, could be considered in prognostication of patients with invasive BCa.

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.

Gallbladder intestinal metaplasia in Pakistani patients with gallstones.

The gallbladder specimens of patients who underwent cholecystectomy for symptomatic gallstones between 2003 and 2005 were evaluated for the presence of Intestinal Metaplasia. (IM) and its risk factors. IM was positive in 39% of 293 patients tested, and in the comparative analysis of 114 metaplasia positive versus 179 negative patients, a high risk was found in patients who were 60 years or older [adjusted odds ratio (aOR) = 3.0, 95% confidence interval (CI): 1.5, 6.2]. Other factors with aOR greater than 1 were moderate to excessive use of chilies (1.8) and ethnic origin of North India (1.7). Screening method has yet to be devised for early detection of gallbladder cancer by identifying metaplastic lesions early in life. We believe that large geographic variation and lifestyle environmental factors associated with the development of gallbladder metaplasia and cancer mortality are concealed in our study that needs to be further explored.

Genetic variations in a well conserved 5'-untranslated region of hepatitis C virus genome isolated in Pakistan.

The diversity and extent of sequence variations between hepatitis C virus (HCV) isolates from Pakistan were studied and the probable effects of these variations were assessed on secondary viral structures. Sequencing and phylogenetic analysis was performed on 33 samples, of which 25 were typed as genotype 3 by RFLP (restriction fragment length polymorphism) and 8 remained unresolved. Rooted neighbour-joining (NJ) tree revealed that 28 isolates were HCV type 3a and 5 isolates were typed as 3b. The majority of unresolved samples clustered in a different branch of genotype 3, supported by a bootstrap value of 71%. Another, cluster, cluster I, was found to have a bootstrap value of 81%. Genetic distance values showed significant diversity of isolates in these two clusters compared to the reference sequences. Pair-wise comparison showed the presence of additional restriction sites of HaeIII and RsaI in unresolved isolates. In conclusion, unique sequence variability was observed in the 5'-UTR of HCV type 3 isolates from Pakistan. One of the reasons for this sequence variability is the presence of mutations, which are additional restriction sites in the 5'-UTR. These mutations were also responsible for failure of conventional RFLP to type some of the HCV isolates.