Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low‑speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK‑iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low‑speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low‑speed centrifugation significantly enhances the transfection efficiency of BLOCK‑iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time‑saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay.

Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [ 3 H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher ( P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.

Prolonged jaundice attributed to super infection of hepatitis E virus in a case of resolving leptospirosis.

India is endemic for both Leptospira and hepatitis E virus (HEV). The clinical presentations of these diseases have overlapping features. We report a case of superinfection of HEV in a patient with resolving leptospirosis with underlying Hodgkin lymphoma. The diagnosis of HEV in our case was established by HEV-RNA PCR as our patient was immunosuppressed. The present study highlights the need for molecular diagnosis in the case of HEV infection with strong clinical suspicion and negative serological results.

Benign recurrent intrahepatic cholestasis (BRIC) in an adult.

Benign recurrent intrahepatic cholestasis is a rare hereditary disorder characterised by recurrent episodes ofcholestasis. We report the case of a young male patient with benign recurrent intrahepatic cholestasis who presented to us with recurrent cholestatic jaundice and pruritus with negative work up for all possible aetiologies and a liver biopsy consistent with intrahepatic cholestasis. He improved on treatment with ursodeoxycholic acid and ondansterone and is doing well on follow up.

Insulin tolerance test is comparable to homeostasis model assessment for insulin resistance in patients with nonalcoholic fatty liver disease.

INTRODUCTION: Insulin resistance (IR) is common in patients with nonalcoholic fatty liver disease (NAFLD). We compared the performance of insulin tolerance test and the homeostasis model assessment (HOMA) for measuring IR in such patients. METHODS: In a prospective study, IR was determined using both insulin tolerance test and HOMA in 22 patients with NAFLD. Rate constant for insulin tolerance test (KITT) was calculated using the formula KITT (%/min) = 0.693/t(1/2), where t(1/2) was calculated from the slope of plasma glucose concentration during 3-15 minutes after administration of intravenous insulin. IR was assessed using HOMA as the product of fasting insulin (microU/L) and fasting plasma glucose (mmol/L) levels divided by 22.5. RESULTS: All the 22 patients had IR. Results of KITT and HOMA-IR for determining IR showed a fair correlation (r = 0.55; p = 0.03). CONCLUSIONS: Insulin tolerance test may be a useful method for assessing IR in patients with NAFLD.

Prevalence & significance of hepatitis C virus (HCV) seropositivity in blood donors.

BACKGROUND & OBJECTIVES: The clinical significance of anti HCV antibodies in healthy blood donors remains uncertain. These donors are usually asymptomatic and it is difficult to elicit risk factors of acquiring HCV infection during pre-donation questioning. Limited information on donor recall and follow up studies on anti HCV positive blood donors have been reported from India. Paucity of data which is likely to have an impact on safe blood transfusion programme has prompted us to undertake this study to assess the significance of HCV seropositivity in blood donors with respect to their clinical, biochemical and virological profile. METHODS: A total of 16,250 blood units were screened for the mandatory tests using third generation ELISA (anti HIV 1&2, anti HCV, HBsAg), VDRL and peripheral smear for malaria. Donors reactive for anti HCV were informed. Repeat anti HCV reactive donors were subjected to detailed clinical history focusing on risk factors for HCV transmission. The blood tests included liver function tests (LFT), coagulation and autoimmune profile, qualitative serum cryoglobulins and HCV RNA detection. These donors were followed at 2-3 monthly intervals for a minimum period of six months by LFT. RESULTS: An overall seropositivity of 0.44 per cent (72/16,250) was observed in our donors which was significantly lower in first time, young voluntary donors as compared to replacement donors (0.27 vs. 0.60%). In contrast to drug abuse (6.4%) we found minor percutaneous routes like sharing of shaving kits or visit to a road side barber (32%) as the major risk factor for HCV transmission. There was no prior history of blood transfusion in any of these donors; however history of some surgical procedures was present in 25.8 per cent. Raised transaminases and HCV viraemia were observed in 87 and 71 per cent donors respectively. An association was observed between HCV RNA when the ELISA ratio was >5. INTERPRETATION & CONCLUSION: Voluntary donors form a safe source of blood supply and efforts should be made to increase this precious source to 100 per cent. Abbreviated behavioural donor screening questionnaire for repeat donors is not advisable. Awareness and education of donors is required regarding modes of HCV transmission. HCV positive donors should be informed about their disease, counselled and referred to hepatologist, and permanently deferred for future donations.

Gross anatomy of the retrohepatic segment of the inferior vena cava in northwest Indians.

BACKGROUND AND OBJECTIVES: Information regarding the size and position of the ostia of veins opening into the retrohepatic segment of inferior vena cava (HIVC) in northwest Indians is not available. Knowledge of gross anatomy of this segment is of importance in cases of segmental resection of the liver involving the groove for inferior vena cava (IVC) and when performing selective hepatic venography. We carried out this study to provide information on gross anatomy of HIVC in northwest Indians. METHODS: Livers were obtained from 500 adult autopsy subjects. The HIVC was opened posteriorly by a vertical cut and its circumference at the upper and lower cut ends was measured. To study the position of the ostia of the hepatic veins, HIVC was divided transversely into upper, middle and lower thirds. The anterior and anterolateral walls of HIVC were also divided into four equal parts longitudinally. The venous ostia were classified according to the size of their openings. In addition, in 100 livers the openings were injected with a 20 per cent solution of cellulose acetate butyrate (CAB) in acetone and veins were dissected. RESULTS: The HIVC extended upwards and to the left either obliquely (66.4%) or by describing a gentle curve (33.6%) in its upper half or upper third. Its average length was about 71 mm. Mean diameter at the upper cut end was about 19 mm. The posterior aspect of the upper half or upper one third of HIVC was covered by an extension of the caudate lobe completely (4%) or incompletely (7.4%). The ostia of the left, middle and right hepatic veins were large (>10 mm) and were located in the upper third segment of HIVC. In 87 per cent of specimens the left and middle hepatic veins had a common opening on the left anterior area. The ostium of the right hepatic vein was present in the right anterior area. INTERPRETATION AND CONCLUSION: In conclusion, our study provided gross measurements of HIVC in northwest Indians. A knowledge of the anatomy of HIVC and hepatic venous ostia will help the clinician interventional operator in planning the treatment by choosing a balloon of correct size and at correct site. The measurements helps in determining the fall in portal pressure with pharmacotherapy given for the prevention of variceal bleed.

Is vaccination against hepatitis A virus required in patients with cirrhosis of the liver?

Hepatitis A virus (HAV) superinfection can cause decompensation in patients with chronic liver disease. The risk of HAV superinfection depends on the endemicity of HAV in that particular population. Recommendations of vaccination against HAV in patients with cirrhosis of the liver in India are lacking. Our aim was to assess the seroprevalence of anti-HAV antibodies in our population of patients with cirrhosis of the liver. In a retrospective analysis, the serum of 55 patients with cirrhosis of the liver was tested for total anti-HAV antibodies by ELISA. In the 35 patients who presented with acute decompensation, the serum was also analysed for IgM anti-HAV antibodies. Fifty-four of the 55 cirrhotics (99%) were positive for total anti-HAV antibodies. All 35 patients with decompensated liver disease were also positive for total anti-HAV antibodies, and none of them tested positive for IgM anti-HAV antibodies. Most of the patients with cirrhosis of the liver in the population studied were already exposed to HAV. Routine vaccination against HAV cannot be recommended in these patients.

Minimal hepatic encephalopathy: natural history, impact on daily functioning, and role of treatment.

Cirrhosis of liver is often complicated by minimal hepatic encephalopathy (mHE), which is detected by neuropsychiatric and neurophysiological tests. mHE develops more commonly in cirrhotics with severe liver disease and in those with esophageal and gastric varices. On follow up, these patients more often develop overt encephalopathy as compared to cirrhotics without mHE. mHE may affect daily activities like sleep, driving ability, alertness, social interaction, and communication. It is probably also an independent predictor of survival. The most practical treatment strategy for mHE has not been established; however, it can be treated as effectively as overt encephalopathy with similar agents. Treatment improves mHE in terms of psychometric tests, but improvement in daily functioning has not been well documented.

Helicobacter pylori and hepatic encephalopathy.

Several extra-intestinal diseases have been associated with Helicobacter pylori infection. Hepatic encephalopathy has been linked to H. pylori infection because of the ammonia produced by the organism in the stomach. H. pylori infection is commoner in cirrhotic patients with hepatic encephalopathy than in those without. Increased ammonia levels have been observed in the gastric juice and blood more commonly in cirrhotics with H. pylori infection than in those without. Though the amount of ammonia produced by H. pylori may be too small to contribute to hepatic encephalopathy, eradication of H. pylori has been shown to improve the blood ammonia levels and hepatic encephalopathy.

Occult hepatitis B virus (HBV) infection in healthy blood donors.

Blood transfusion is an important route of transmission of hepatitis B virus (HBV). Occult HBV infection can exist in the absence of HBsAg and can be detected by determining HBV DNA. To determine the occult HBV infection in healthy blood donors. One hundred adult healthy blood donors, negative for HBsAg, anti HCV, HIV-1 and other risk factors were screened for HBV DNA by PCR. All the healthy blood donors were negative for HBV DNA by PCR. Occult HBV infection does not occur in the healthy blood donors in the population studied.

Paucity of anti-hepatitis C virus antibodies in the serum of Indian patients with Sjogren's syndrome and inflammatory myositis.

Several extrahepatic manifestations have been associated with infection with Hepatitis C virus (HCV) infection. It has been associated with Sjogren's syndrome (SS) and inflammatory myositis (IM). The objective was to look at the prevalence of anti-HCV antibodies in the serum of SS and IM patients of Indian origin. Individuals satisfying the European Economic Community criteria for the diagnosis of SS and those satisfying the criteria of Bohan and Peter for the diagnosis of IM were recruited in the study. Routine evaluation for liver functions was made. Anti-HCV antibodies were tested by a third generation ELISA, using microplate HCV3.0 ELISA. Of the 23 patients with SS studied, 14 had extraglandular features. The commonest were anaemia and arthritis in six each, followed by in lymphopenia in two. One patient each had interstitial lung disease, hypothyroidism and chronic active hepatitis. Twenty-two patients with IM were studied alongside. None of the patients had abnormal liver functions. One patient with primary SS tested positive for anti-HCV antibodies. None of the patients with inflammatory myositis tested positive for anti-HCV antibodies. The presence of anti-HCV antibodies in our cohort of patients with SS and IM is low and more in keeping with the generally low prevalence of the infection in the Indian population.

Hepatic veins in northwest Indian subjects.

Formation, position, course and areas of drainage by hepatic veins were studied in 153 adult autopsy specimens. There were three major hepatic veins; left, middle and right. The middle hepatic vein (MHV) joined the left hepatic vein (LHV) in 88.2% specimens before the latter opened into inferior vena cava (IVC). Each major hepatic vein was joined by a superior vein. An intersegmental vein was present in the septum between the medial and lateral segments; it either joined the LHV or MHV. Formation and course of posterior hepatic veins (small veins) have been described and a nomenclature has been suggested. The right suprarenal vein either opened directly into IVC or joined the superior or inferior posterior hepatic veins.

Hepatitis B and C virus--prevalence and prevention in health care workers.

BACKGROUND: Hepatitis B and C viruses are important causes of liver related morbidity and mortality. We aimed at determining the presence of hepatitis B and C virus infections in the health care workers (HCWs) and their compliance for the HBV vaccination. METHODS: Three thousand five hundred and fifty six health care workers were screened for HBsAg and 115 for anti-HCV by ELISA. HBsAg negative individual were offered HBV vaccination and record of their compliance was kept. Anti-HBs titers were determined one month after 2nd or 3rd dose of vaccine in 273 subjects. RESULTS: Out of 3556 health care workers, 61 (1.7%) were found to be positive for HBsAg. One out of 115 HCWs (0.87%) was found to be positive for anti-HCV. Fifteen percent of HCWs received only one dose, 26% received two doses 59% received three doses and 2.5% also received the booster dose of the HBV vaccine. All those tested had anti-HBs titers more than 10 mUI/ml. CONCLUSION: In HCWs, HBsAg and anti-HCV prevalence was found to be 1.7% and 0.87% respectively. HCWs in our hospital, despite the awareness on HBV and HCV infection are noncompliant for HBV vaccination.