Genetic diversity of Nipah virus in Bangladesh.

BACKGROUND: Nipah virus (NiV) infection, often fatal in humans, is primarily transmitted in Bangladesh through the consumption of date palm sap contaminated by Pteropus bats. Person-to-person transmission is also common and increases the concern of large outbreaks. This study aimed to characterize the molecular epidemiology, phylogenetic relationship, and the evolution of the nucleocapsid gene (N gene) of NiV. METHODS: We conducted molecular detection, genetic characterization, and Bayesian time-scale evolution analyses of NiV using pooled Pteropid bat roost urine samples from an outbreak area in 2012 and archived RNA samples from NiV case patients identified during 2012-2018 in Bangladesh. RESULTS: NiV-RNA was detected in 19% (38/456) of bat roost urine samples and among them; nine N gene sequences were recovered. We also retrieved sequences from 53% (21 out of 39) of archived RNA samples from patients. Phylogenetic analysis revealed that all Bangladeshi strains belonged to NiV-BD genotype and had an evolutionary rate of 4.64 × 10-4 substitutions/site/year. The analyses suggested that the strains of NiV-BD genotype diverged during 1995 and formed two sublineages. CONCLUSION: This analysis provides further evidence that the NiV strains of the Malaysian and Bangladesh genotypes diverged recently and continue to evolve. More extensive surveillance of NiV in bats and human will be helpful to explore strain diversity and virulence potential to infect humans through direct or person-to-person virus transmission.

Raising Backyard Poultry in Rural Bangladesh: Financial and Nutritional Benefits, but Persistent Risky Practices.

Poultry is commonly raised by households in rural Bangladesh. In 2007, the Government of Bangladesh began a mass media campaign to disseminate 10 recommended precautions to prevent transmission of H5N1 from poultry to humans. This longitudinal study explored the contribution of backyard poultry on household economy and nutrition and compared poultry-raising practices to government recommendations. From 2009 to 2012, we enrolled a nationally representative sample of 2489 primary backyard poultry raisers from 115 rural villages selected by probability proportional to population size. Researchers interviewed the raisers to collect data on poultry-raising practices. They followed the raisers for 2-12 months to collect data on household income and nutrition from poultry. Income from backyard poultry flocks accounted for 2.8% of monthly household income. Return on annual investment (ROI) per flock was 480%. Yearly, median family consumption of eggs was one-fifth of the total produced eggs and three poultry from their own flock. Respondents' reported practices conflicted with government recommendations. Sixty per cent of raisers had never heard of avian influenza or 'bird flu'. Among the respondents, 85% handled sick poultry or poultry that died due to illness, and 49% slaughtered or defeathered sick poultry. In 37% of households, children touched poultry. Fifty-eight per cent never washed their hands with soap after handling poultry, while <1% covered their nose and mouth with a cloth when handling poultry. Only 3% reported poultry illness and deaths to local authorities. These reported practices did not improve during the study period. Raising backyard poultry in rural Bangladesh provides important income and nutrition with an excellent ROI. Government recommendations to reduce the risk of avian influenza transmission did not impact the behaviour of poultry producers. Further research should prioritize developing interventions that simultaneously reduce the risk of avian influenza transmission and increase productivity of backyard poultry.

Bat Hunting and Bat-Human Interactions in Bangladeshi Villages: Implications for Zoonotic Disease Transmission and Bat Conservation.

Bats are an important reservoir for emerging zoonotic pathogens. Close human-bat interactions, including the sharing of living spaces and hunting and butchering of bats for food and medicines, may lead to spillover of zoonotic disease into human populations. We used bat exposure and environmental data gathered from 207 Bangladeshi villages to characterize bat exposures and hunting in Bangladesh. Eleven percent of households reported having a bat roost near their homes, 65% reported seeing bats flying over their households at dusk, and 31% reported seeing bats inside their compounds or courtyard areas. Twenty percent of households reported that members had at least daily exposure to bats. Bat hunting occurred in 49% of the villages surveyed and was more likely to occur in households that reported nearby bat roosts (adjusted prevalence ratio [aPR] 2.3, 95% CI 1.1-4.9) and villages located in north-west (aPR 7.5, 95% CI 2.5-23.0) and south-west (aPR 6.8, 95% CI 2.1-21.6) regions. Our results suggest high exposure to bats and widespread hunting throughout Bangladesh. This has implications for both zoonotic disease spillover and bat conservation.

Evolving epidemiology of Nipah virus infection in Bangladesh: evidence from outbreaks during 2010-2011.

Drinking raw date palm sap is the primary route of Nipah virus (NiV) transmission from bats to people in Bangladesh; subsequent person-to-person transmission is common. During December 2010 to March 2011, we investigated NiV epidemiology by interviewing cases using structured questionnaires, in-depth interviews, and group discussions to collect clinical and exposure histories. We conducted a case-control study to identify risk factors for transmission. We identified 43 cases; 23 were laboratory-confirmed and 20 probable. Thirty-eight (88%) cases died. Drinking raw date palm sap and contact with an infected person were major risk factors; one healthcare worker was infected and for another case transmission apparently occurred through contact with a corpse. In absence of these risk factors, apparent routes of transmission included drinking fermented date palm sap. For the first time, a case was detected in eastern Bangladesh. Identification of new epidemiological characteristics emphasizes the importance of continued NiV surveillance and case investigation.

Integrated cluster- and case-based surveillance for detecting stage III zoonotic pathogens: an example of Nipah virus surveillance in Bangladesh.

This paper explores the utility of cluster- and case-based surveillance established in government hospitals in Bangladesh to detect Nipah virus, a stage III zoonotic pathogen. Physicians listed meningo-encephalitis cases in the 10 surveillance hospitals and identified a cluster when ⩾2 cases who lived within 30 min walking distance of one another developed symptoms within 3 weeks of each other. Physicians collected blood samples from the clustered cases. As part of case-based surveillance, blood was collected from all listed meningo-encephalitis cases in three hospitals during the Nipah season (January-March). An investigation team visited clustered cases' communities to collect epidemiological information and blood from the living cases. We tested serum using Nipah-specific IgM ELISA. Up to September 2011, in 5887 listed cases, we identified 62 clusters comprising 176 encephalitis cases. We collected blood from 127 of these cases. In 10 clusters, we identified a total of 62 Nipah cases: 18 laboratory-confirmed and 34 probable. We identified person-to-person transmission of Nipah virus in four clusters. From case-based surveillance, we identified 23 (4%) Nipah cases. Faced with thousands of encephalitis cases, integrated cluster surveillance allows targeted deployment of investigative resources to detect outbreaks by stage III zoonotic pathogens in resource-limited settings.

Identification and epidemiology of a rare HoBi-like pestivirus strain in Bangladesh.

The genus pestivirus of the family flaviviridae consists of four recognized species: bovine viral diarrhoea virus 1 (BVDV-1), bovine viral diarrhoea virus 2 (BVDV-2), classical swine fever virus and border disease virus. A new putative pestivirus species tentatively named as either 'HoBi-like pestivirus' or BVDV-3 has recently been identified in Brazil, Italy and Thailand. Despite reports of serological evidence of BVDV in Bangladesh, the types of the virus circulating in cattle have not been identified. We conducted surveillance in cattle from May 2009 to August 2010 in three government veterinary hospitals to characterize BVDV in cattle of Bangladesh. We tested serum for BVDV using an antigen-capture ELISA. Of 638 cattle samples, 3% (16/638) tested positive for BVDV antigen. The ELISA-positive samples were selected for further molecular detection and characterization of BVDV. Molecular analysis of the partial 5' untranslated region (UTR) nucleotide sequences of BVDV-positive samples identified the rare HoBi-like pestivirus or BVDV-3 virus circulating in cattle of Bangladesh. The identification of this rare HoBi-like pestivirus or BVDV-3 strain in Bangladesh warrants further surveillance to evaluate its impact on livestock production.

Impact of neighborhood biomass cooking patterns on episodic high indoor particulate matter concentrations in clean fuel homes in Dhaka, Bangladesh.

Exposure to particulate matter (PM2.5 ) from the burning of biomass is associated with increased risk of respiratory disease. In Dhaka, Bangladesh, households that do not burn biomass often still experience high concentrations of PM2.5 , but the sources remain unexplained. We characterized the diurnal variation in the concentrations of PM2.5 in 257 households and compared the risk of experiencing high PM2.5 concentrations in biomass and non-biomass users. Indoor PM2.5 concentrations were estimated every minute over 24 h once a month from April 2009 through April 2010. We found that households that used gas or electricity experienced PM2.5 concentrations exceeding 1000 µg/m(3) for a mean of 35 min within a 24-h period compared with 66 min in biomass-burning households. In both households that used biomass and those that had no obvious source of particulate matter, the probability of PM2.5 exceeding 1000 µg/m(3) were highest during distinct morning, afternoon, and evening periods. In such densely populated settings, indoor pollution in clean fuel households may be determined by biomass used by neighbors, with the highest risk of exposure occurring during cooking periods. Community interventions to reduce biomass use may reduce exposure to high concentrations of PM2.5 in both biomass and non-biomass using households.

Indoor exposure to particulate matter and the incidence of acute lower respiratory infections among children: a birth cohort study in urban Bangladesh.

Approximately half of all children under two years of age in Bangladesh suffer from an acute lower respiratory infection (ALRI) each year. Exposure to indoor biomass smoke has been consistently associated with an increased risk of ALRI in young children. Our aim was to estimate the effect of indoor exposure to particulate matter (PM2.5 ) on the incidence of ALRI among children in a low-income, urban community in Bangladesh. We followed 257 children through two years of age to determine their frequency of ALRI and measured the PM2.5 concentrations in their sleeping space. Poisson regression was used to estimate the association between ALRI and the number of hours per day that PM2.5 concentrations exceeded 100 µg/m(3) , adjusting for known confounders. Each hour that PM2.5 concentrations exceeded 100 µg/m(3) was associated with a 7% increase in incidence of ALRI among children aged 0-11 months (adjusted incidence rate ratio (IRR) 1.07, 95% CI 1.01-1.14), but not in children 12-23 months old (adjusted IRR 1.00, 95% CI 0.92-1.09). Results from this study suggest that reducing indoor PM2.5 exposure could decrease the frequency of ALRI among infants, the children at highest risk of death from these infections.

Backyard poultry raising in Bangladesh: a valued resource for the villagers and a setting for zoonotic transmission of avian influenza. A qualitative study.

INTRODUCTION: Backyard poultry raising is common in rural communities and a valued resource that provides food and income for subsistence farmers. Close contact with infected backyard poultry has been associated with H5N1 human cases in different countries. The emergence of this virus within Bangladesh means that backyard poultry raisers are at risk of avian influenza infections. The aim of this study was to understand why people raise backyard poultry and to characterize people's regular interaction with their poultry. METHODS: In 2008, a qualitative study was conducted in two villages from two districts of Bangladesh. In a social mapping exercise the villagers drew all the households in their village: 115 households in the village in Netrokona and 85 households in the village in Rajshahi District. Selected were 40 households (20 households from each of the two villages) for data collection through in-depth interviews (n=40) and household mapping (n=40), and observation sessions (n=16). RESULTS: In both villages, 92% of households raised backyard poultry. The majority of the owners was female and used the money earned from poultry raising to purchase cooking ingredients, clothing, and agricultural seeds, and pay for children's education expenses. The households consumed poultry meat and eggs. In the village in Netrokona, 80% (85/106) of households kept poultry inside the bedroom. In the village in Rajshahi, 87% (68/78) of households had separate cage/night sheds. During feeding the poultry and cleaning the poultry raising areas, villagers came into contact with poultry and poultry feces. Poultry scavenged for food on the floor, bed, in the food pot and around the place where food was cooked. Poultry drank from and bathed in the same body of water that villagers used for bathing and washing utensils and clothes. CONCLUSION: Although raising poultry provides essential support to the families' livelihoods, it exposes them to the risk of avian influenza through close contact with their poultry. Simple warnings to avoid poultry contact are unlikely to change practices that are essential to household survival. Interventions that help to protect poultry flocks and improve household profitability are more likely to be practiced.

Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007.

In February 2007 an outbreak of Nipah virus (NiV) encephalitis in Thakurgaon District of northwest Bangladesh affected seven people, three of whom died. All subsequent cases developed illness 7-14 days after close physical contact with the index case while he was ill. Cases were more likely than controls to have been in the same room (100% vs. 9.5%, OR undefined, P<0.001) and to have touched him (83% vs. 0%, OR undefined, P<0.001). Although the source of infection for the index case was not identified, 50% of Pteropus bats sampled from near the outbreak area 1 month after the outbreak had antibodies to NiV confirming the presence of the virus in the area. The outbreak was spread by person-to-person transmission. Risk of NiV infection in family caregivers highlights the need for infection control practices to limit transmission of potentially infectious body secretions.

Prevalent high-risk respiratory hygiene practices in urban and rural Bangladesh.

OBJECTIVES: To identify existing respiratory hygiene risk practices, and guide the development of interventions for improving respiratory hygiene. METHODS: We selected a convenience sample of 80 households and 20 schools in two densely populated communities in Bangladesh, one urban and one rural. We observed and recorded respiratory hygiene events with potential to spread viruses such as coughing, sneezing, spitting and nasal cleaning using a standardized assessment tool. RESULTS: In 907 (81%) of 1122 observed events, households' participants coughed or sneezed into the air (i.e. uncovered), 119 (11%) into their hands and 83 (7%) into their clothing. Twenty-two per cent of women covered their coughs and sneezes compared to 13% of men (OR 2.6, 95% CI 1.6-4.3). Twenty-seven per cent of persons living in households with a reported monthly income of >72.6 US$ covered their coughs or sneezes compared to 13% of persons living in households with lower income (OR 3.2, 95% CI 1.6-6.2). In 956 (85%) of 1126 events, school participants coughed or sneezed into the air and 142 (13%) into their hands. Twenty-seven per cent of coughs/sneezes in rural schools were covered compared to 10% of coughs/sneezes in urban schools (OR 2.3, 95% CI 1.5-3.6). Hand washing was never observed after participants coughed or sneezed into their hands. CONCLUSION: There is an urgent need to develop culturally appropriate, cost-effective and scalable interventions to improve respiratory hygiene practices and to assess their effectiveness in reducing respiratory pathogen transmission.

Risk factors for Nipah virus encephalitis in Bangladesh.

Nipah virus (NiV) is a paramyxovirus that causes severe encephalitis in humans. During January 2004, twelve patients with NiV encephalitis (NiVE) were identified in west-central Bangladesh. A case-control study was conducted to identify factors associated with NiV infection. NiVE patients from the outbreak were enrolled in a matched case-control study. Exact odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by using a matched analysis. Climbing trees (83% of cases vs. 51% of controls, OR 8.2, 95% CI 1.25-infinity) and contact with another NiVE patient (67% of cases vs. 9% of controls, OR 21.4, 95% CI 2.78-966.1) were associated with infection. We did not identify an increased risk for NiV infection among persons who had contact with a potential intermediate host. Although we cannot rule out person-to-person transmission, case-patients were likely infected from contact with fruit bats or their secretions.

Activation of protein kinase C alpha and delta by bile acids: correlation with bile acid structure and diacylglycerol formation.

The feedback repression of cholesterol 7alpha-hydroxylase transcriptional activity and mRNA levels by taurocholate (TCA) occurs via a protein kinase C (PKC)-dependent signal. To determine whether bile acids could activate PKC indirectly via generation of diacylglycerol (DG), their effects on DG levels in primary cultures of rat hepatocytes were determined using a DG kinase assay. To determine whether bile acids might activate PKC isozymes more directly, their effects on PKC alpha and delta purified from baculovirus expression systems were examined in phosphatidylserine/phosphatidylcholine/Triton X-100 (PS/PC/TX) mixed micelles. Addition of tauroursodeoxycholate (TUDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) (50 microM) to the cells rapidly (15 min) increased DG content in cultured rat hepatocytes to 105%, 155%, and 130%, respectively, as compared to untreated control cultures. Addition of TCA increased PKC alpha specific activity with EC50 of approximately 400 nM; maximal activity was observed with 5 microM. Other taurine-conjugated bile acids (5 microM) increased PKC alpha specific activity (pmol/min/microg protein) in proportion to their relative hydrophobicity: PS/PC/TX 17 +/- 2; + TUDCA 29 +/- 18; + TCA 68 +/-13; + TDCA 166 +/- 21; and, taurochenodeoxycholate 178 +/- 20 (P vs. PS/PC/TX = 0.54, 0.019, 0.002, and 0.001, respectively); unconjugated bile acids gave similar results (r2 for activity vs. hydrophobicity index 0.59). Taurine-conjugated bile acid interaction enthalpies, as determined by dimyristoyl-phosphatidylcholine chromatography, were more highly correlated (r2 = 0.96) with PKC alpha activation than with the hydrophobicity index. TCA also stimulated the activity of purified PKCdelta with EC50 of approximately 150 nM and maximally (2.7-fold) at 1 microM. Free and taurine-conjugated bile acids (1 microM) increased PKCdelta activity according to their hydrophobicity index (r2 = 0.89) and interaction enthalpies (r2 = 0.96).

Hepatocellular protein kinase C activation by bile acids: implications for regulation of cholesterol 7 alpha-hydroxylase.

We have recently shown that taurocholate (TCA) represses the transcriptional activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of the bile acid biosynthetic pathway, through a protein kinase C (PKC)-dependent mechanism in primary cultures of rat hepatocytes. The present studies sought to determine the mechanisms by which bile acids activate hepatic PKC activity and the consequences of this activation on isoform distribution and cholesterol 7 alpha-hydroxylase mRNA levels. TCA (12.5-100 microM for 15 min) increased membrane-associated "classic" isoenzyme cPKC-alpha and "novel" isoenzymes nPKC-delta, and nPKC by two- to sixfold. Membrane-associated PKC progressively increased, and cytosolic PKC decreased, for 1 h after the addition of TCA (50 microM); after 24 h whole cell cPKC-alpha, nPKC-delta, and nPKC were downregulated by 35-55% compared with untreated controls. In a reconstituted assay system, TCA or taurodeoxycholate (10-100 microM) increased calcium-dependent and -independent PKC activity by three- and fourfold, respectively. Taurine-conjugated bile acids stimulated PKC activity in proportion to their hydrophobicity index (r = 0.99). Finally, cholesterol 7 alpha-hydroxylase mRNA was repressed > 75% by phorbol 12-myristate 13-acetate (100 nM for 3 h), a nonselective activator of PKC isoforms. In contrast, selective cPKC-alpha activation with thymeleatoxin (100 nM for 3 h) had no significant effect on cholesterol 7 alpha-hydroxylase mRNA levels. We conclude that bile acids activate hepatocellular PKC, resulting in sequential redistribution and down-regulation of calcium-dependent and -independent isoforms. The calcium-independent PKC isoforms may mediate the repression of cholesterol 7 alpha-hydroxylase mRNA by TCA.

Regulation of cholesterol 7 alpha-hydroxylase expression by sterols in primary rat hepatocyte cultures.

The importance of cholesterol and "oxysterols" in the regulation of cholesterol 7 alpha-hydroxylase is not clear. Previous in vivo studies suggest that cholesterol may up-regulate cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, but these studies are open to question as they were carried out in whole animals. Therefore, we used primary rat hepatocytes, cultured in serum-free medium, to determine the effects of cholesterol on the regulation of cholesterol 7 alpha-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis in this system. Squalestatin (1 microM) decreased cholesterol 7 alpha-hydroxylase specific activity to undetectable levels and decreased steady-state mRNA and transcriptional activity to 13% and 47% of controls, respectively. Mevalonolactone (2 mM) failed to restore cholesterol 7 alpha-hydroxylase specific activity or steady-state mRNA levels in squalestatin-treated cells. Addition of cholesterol, delivered in beta-cyclodextrin, to squalestatin-treated cells restored cholesterol 7 alpha-hydroxylase specific activity and steady-state mRNA to control levels in a concentration (25 microM to 200 microM) -dependent manner. In contrast, the individual addition of selected "oxysterols" (5-cholesten-3 beta, 7 alpha-diol; 5 alpha-cholestan-3 beta, 6 alpha-diol; cholestan-3 beta, 5 alpha,6 beta-triol; 5-(25R)-cholesten-3 beta,26-diol, all at 50 microM) failed to restore cholesterol 7 alpha-hydroxylase mRNA levels in squalestatin-treated cells. These experiments provide evidence that cholesterol rather than "oxysterols" regulate cholesterol 7 alpha-hydroxylase gene expression. Squalestatin (1 microM) treatment increased HMG-CoA reductase specific activity by 229% of controls. Addition of cholesterol (200 microM), but not mevalonolactone (2 mM), to squalestatin-treated cells decreased HMG-CoA reductase specific activity to 19% of control. The primary rat hepatocyte culture system in conjunction with a specific squalene synthetase inhibitor should be a useful model for elucidating the mechanism of regulation of cholesterol 7 alpha-hydroxylase gene expression by sterols.

Repression of cholesterol 7 alpha-hydroxylase transcription by bile acids is mediated through protein kinase C in primary cultures of rat hepatocytes.

Inhibitors of protein kinases were screened for the ability to prevent the repression of cholesterol 7 alpha-hydroxylase mRNA by taurocholate in primary cultures of adult rat hepatocytes. The addition of taurocholate (25 microM) for 6 h decreased cholesterol 7 alpha-hydroxylase mRNA by 64 +/- 3%. However, after a preincubation with the protein kinase C inhibitors calphostin C or chelerythrine, taurocholate had no significant effect on cholesterol 7 alpha-hydroxylase mRNA, or decreased levels by only 23 +/- 8%, respectively. Protein kinase C activation with phorbol 12-myristate, 13-acetate (100 nM) decreased cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by 71 +/- 5% and 60 +/- 16%, respectively, within 3 h. mRNA levels recovered to control levels by 18-24 h, however, consistent with downregulation of protein kinase C. Furthermore, after depletion of protein kinase C with a 24-h preincubation with phorbol diesters, taurocholate (25 microM) repressed cholesterol 7 alpha-hydroxylase mRNA by only 14 +/- 17%. The addition of taurocholate (50 microM) to the culture medium transiently increased membrane-associated protein kinase C activity by approximately twofold, while causing a concomitant decrease in cytosolic activity. Other bile acids increased membrane-associated protein kinase C activity in approximate proportion to their relative hydrophobicity. Finally, immunoblotting experiments revealed translocation of the alpha isoform of protein kinase C to hepatocyte membranes in response to taurocholate. These data suggest that hydrophobic bile acids repress cholesterol 7 alpha-hydroxylase transcription through a protein kinase C-dependent mechanism.

Hormonal regulation of cholesterol 7 alpha-hydroxylase mRNA levels and transcriptional activity in primary rat hepatocyte cultures.

In primary cultures of adult rat hepatocytes the level of cholesterol 7 alpha-hydroxylase steady-state mRNA markedly decreased by 72 h. However, the addition of L-thyroxine (T4) and dexamethasone synergistically returned cholesterol 7 alpha-hydroxylase steady-state mRNA levels near to that of cholestyramine-fed animals. The maximal responses to T4 and dexamethasone in serum-free medium were at 1.0 and 0.1 microM, respectively. The addition of T4 in combination with dexamethasone resulted in an 11-fold increase in transcriptional activity of the cholesterol 7 alpha-hydroxylase gene as compared to no addition controls. The specific activities of cholesterol 7 alpha-hydroxylase in microsomes prepared from cultures treated with dexamethasone and T4 were 1.56 +/- 1.17 nmol/h/mg protein which is similar to that of intact liver (1.70 +/- 0.062 nmol/h/mg protein), but lower than cholestyramine-fed animals. Cholesterol 7 alpha-hydroxylase activity was not detectable (less than 0.020 nmol/h/mg protein) at 72 h in cultures without the addition of both dexamethasone and T4. In the presence of optimal concentrations of dexamethasone and T4, glucagon (0.2 microM), or dibutyryl cAMP (50 microM) decreased (90%) cholesterol 7 alpha-hydroxylase mRNA within 6 h. Transcriptional activity decreased (62%) in 6 h following the addition of glucagon (0.2 microM) to the culture medium. The results reported in this paper suggest an important role for multiple hormones in the regulation of cholesterol 7 alpha-hydroxylase in the liver.

Effect of hydrophobic bile acids on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and mRNA levels in the rat.

We have previously reported that relatively hydrophobic bile acids, decreased hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (reductase) activity whereas, hydrophilic bile acids had little effect on the enzyme. The purpose of the present study was to determine in more detail the mechanism of down-regulation of hepatic reductase activity by hydrophobic bile salts. Groups of rats were fed bile acids of differing hydrophobicity: ursodeoxycholic, cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), or cholesterol for 14 days. Reductase specific activities and concentrations of reductase protein were determined in hepatic microsomes. Quantitation of "steady state" levels of reductase mRNA was performed using Northern and dot blot hybridization. Reductase gene transcriptional activity (nuclear "run-on") was determined in nuclei isolated from livers of animals fed different bile acids. Hydrophobic bile acids and cholesterol significantly decreased reductase activity: CA (57%), CDCA (77%), DCA (73%), cholesterol (89%), and reductase protein levels as measured by an enzyme-linked immunosorbent assay method were also decreased; CA (27%), CDCA (31%), DCA (42%), and cholesterol (35%). Reductase mRNA levels were also decreased after feeding hydrophobic bile acid: CA (43%), CDCA (47%), DCA (54%), and cholesterol (53%). Ursodeoxycholic, a hydrophilic bile acid, caused a much smaller decrease in reductase activity (18%), protein mass (16%), and mRNA levels (10%). Decreased transcriptional activities were observed in CA- and cholesterol-fed rats. Surprisingly, CDCA- and DCA-fed animals showed transcriptional activities similar to control animals even though steady state mRNA levels were low in CDCA- and DCA-fed animals. We hypothesize a post-transcriptional regulation of reductase mRNA by hydrophobic bile acids.

Regulation of cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat.

Cholesterol 7 alpha-hydroxylase catalyzes the rate-limiting step in the bile acid biosynthetic pathway. Regulation of this pathway is thought to occur solely as a result of a negative feedback control mechanism that is dependent upon the flux and composition of bile salts undergoing enterohepatic circulation. We have used the chronic biliary diverted (CBD) rat model to study the mechanism of regulation of cholesterol 7 alpha-hydroxylase by taurocholate. As compared to nonoperated controls, CBD rats exhibited a 4.2-fold increase in cholesterol 7 alpha-hydroxylase-specific activity, a 4.5-fold increase in enzyme mass, a 10-fold increase in steady-state mRNA levels, and a 3.4-fold increase in transcriptional (nuclear "run-on") activity. Intraduodenal infusion of taurocholate at a rate of 36 mumol/100 g/h for 48 h in CBD rats caused a significant (p less than 0.05) decrease (64%) in cholesterol 7 alpha-hydroxylase-specific activity, mass (72%), steady-state mRNA levels (74%), and transcriptional activity (57%) as compared to CBD controls. Cholesterol feeding increased cholesterol 7 alpha-hydroxylase-specific activity (288%), poly(A) RNA levels (291%), and transcriptional activity (220%) as compared to control animals. These results provide convincing evidence that bile salts, either directly or indirectly, down-regulate in vivo transcription of the cholesterol 7 alpha-hydroxylase gene, which is probably the major mechanism regulating the levels of this enzyme. The results of this study also suggest that the promoter for cholesterol 7 alpha-hydroxylase may have both bile salt- and sterol-responsive elements.

Bile salts in submicellar concentrations promote bidirectional cholesterol transfer (exchange) as a function of their hydrophobicity.

Cholesterol, despite its poor solubility in aqueous solutions, exchanges efficiently between membranes. Movement of cholesterol between different subcellular membranes in the hepatocyte is necessary for assembly of lipoproteins, biliary cholesterol secretion, and bile acid synthesis. Factors which initiate and facilitate transfer of cholesterol between different membranes in the hepatocyte are incompletely understood. It is known that cholesterol secretion into the bile is linked to bile salt secretion. In the present study, we investigated the effects of bile salts of different physicochemical properties at submicellar concentrations (150- 600 microM) on the transfer of [14C]cholesterol from hepatocytes, or crude hepatocellular membranes (donors), to rat high density lipoproteins (acceptor). Bile salts included taurine conjugates of ursodeoxycholic acid (TUDCA), hyodeoxycholic acid (THDCA), cholic acid (TCA), chenodeoxycholic acid (TCDCA), and deoxycholic acid (TDCA). High density lipoprotein (HDL) was separated from hepatocellular membranes and the transfer of [14C]cholesterol from the membranes to HDL was quantitatively determined. In the absence of HDL, [14C]cholesterol remained confined to the membrane fraction. Following addition of HDL, [4-14C]cholesterol in the HDL fraction increased linearly over time. Addition of hydrophilic bile salts (TUDCA and THDCA) increased transfer of [4-14C]cholesterol to HDL only minimally. By contrast, more hydrophobic bile salts stimulated transfer of labeled cholesterol to HDL, and their potency increased in order of increasing hydrophobicity (TCA less than TCDCA less than TDCA). Both for single bile salts and mixtures of bile salts at a total bile salt concentration of 0.30 mM, the rate of cholesterol transfer exhibited a strong linear correlation with a bile salt monomeric hydrophobicity index (r = 0.95; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)