Biomedical subjects

S Govindarajan

Publications and source records attributed to S Govindarajan.

At least 19 recordsLinked to original sources

PubMed2001

Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells.

Hepatitis E virus (HEV) is an enterically transmitted virus that causes acute hepatitis. Expression of recombinant HEV capsid protein in insect cells results in two major proteolytically-processed products of 56 and 53kDa which consist of amino acids (aa) 112-607 and 112-578, respectively. The only neutralization epitope identified to date is located at least partially between amino acids 578 and 607 meaning it should be present only in the 56 and not in the 53kDa protein. Previously, it was shown that vaccination with the 56kDa protein greatly reduced virus shedding and protected Rhesus monkeys from hepatitis E when challenged with a high intravenous dose of homologous or heterologous HEV. To evaluate the immunogenicity and protective efficacy of the 53kDa protein, we vaccinated Rhesus monkeys with this protein and challenged them with a high or low dose of homologous virus. Vaccination with the 53kDa protein greatly reduced virus shedding but did not protect against hepatitis following the high dose challenge. Virus was not detected in the vaccinated animals following the low dose challenge, suggesting that sterilizing immunity may have been achieved.

Alanine Transaminase↗

PubMed2001

Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element.

Hepatitis E virus recombinant genomes transcribed in vitro from two cDNA clones differing by two nucleotides were infectious for chimpanzees. However, one cDNA clone encoded a virus that was attenuated for chimpanzees and unable to infect rhesus monkeys. The second cDNA clone encoded a virus that infected both chimpanzees and rhesus monkeys and caused acute hepatitis in both. One mutation differentiating the two clones identified a cis-reactive element that appeared to overlap the 3' end of the capsid gene and part of the 3' noncoding region. Capping of the RNA transcripts was essential for infectivity.

Animals↗

PubMed2001

Extracellular nicotinamide adenine dinucleotide induces t cell apoptosis in vivo and in vitro.

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3(+), CD4(+), and CD8(+) cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69(+) T cells.

Animals↗

PubMed2001

The adaptive evolution database (TAED).

BACKGROUND: The Master Catalog is a collection of evolutionary families, including multiple sequence alignments, phylogenetic trees and reconstructed ancestral sequences, for all protein-sequence modules encoded by genes in GenBank. It can therefore support large-scale genomic surveys, of which we present here The Adaptive Evolution Database (TAED). In TAED, potential examples of positive adaptation are identified by high values for the normalized ratio of nonsynonymous to synonymous nucleotide substitution rates (KA/KS values) on branches of an evolutionary tree between nodes representing reconstructed ancestral sequences. RESULTS: Evolutionary trees and reconstructed ancestral sequences were extracted from the Master Catalog for every subtree containing proteins from the Chordata only or the Embryophyta only. Branches with high KA/KS values were identified. These represent candidate episodes in the history of the protein family when the protein may have undergone positive selection, where the mutant form conferred more fitness than the ancestral form. Such episodes are frequently associated with change in function. An unexpectedly large number of families (between 10% and 20% of those families examined) were found to have at least one branch with high KA/KS values above arbitrarily chosen cut-offs (1 and 0.6). Most of these survived a robustness test and were collected into TAED. CONCLUSIONS: TAED is a raw resource for bioinformaticists interested in data mining and for experimental evolutionists seeking candidate examples of adaptive evolution for further experimental study. It can be expanded to include other evolutionary information (for example changes in gene regulation or splicing) placed in a phylogenetic perspective.

Adaptation, Physiological↗

PubMed2001

Synthesis of a novel hepatitis C virus protein by ribosomal frameshift.

Hepatitis C virus (HCV) is an important human pathogen that affects approximately 100 million people worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. We report here the discovery of a novel HCV protein synthesized by ribosomal frameshift. This protein, which we named the F protein, is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frameshift into the -2/+1 reading frame. This ribosomal frameshift requires only codons 8-14 of the core protein-coding sequence, and the shift junction is located at or near codon 11. An F protein analog synthesized in vitro reacted with the sera of HCV patients but not with the sera of hepatitis B patients, indicating the expression of the F protein during natural HCV infection. This unexpected finding may open new avenues for the development of anti-HCV drugs.

Amino Acid Sequence↗

PubMed2001

The Adaptive Evolution Database (TAED).

BACKGROUND: Developing an understanding of the molecular basis for the divergence of species lies at the heart of biology. The Adaptive Evolution Database (TAED) serves as a starting point to link events that occur at the same time in the evolutionary history (tree of life) of species, based upon coding sequence evolution analyzed with the Master Catalog. The Master Catalog is a collection of evolutionary models, including multiple sequence alignments, phylogenetic trees, and reconstructed ancestral sequences, for all independently evolving protein sequence modules encoded by genes in GenBank [1]. RESULTS: We have estimated from these models the ratio of nonsynonymous to synonymous nucleotide substitution (Ka/Ks), for each branch in their respective evolutionary trees of every subtree containing only chordata or only embryophyta proteins. Branches with high Ka/Ks values represent candidate episodes in the history of the family where the protein may have undergone positive selection, a phenomenon in molecular evolution where the mutant form of a gene must have conferred more fitness than the ancestral form. Such episodes are frequently associated with change in function. We have found that an unexpectedly large number of families (between 10 and 20% of those families examined) have at least one branch with a notably high Ka/Ks value (putative adaptive evolution). As a resource for biologists wishing to understand the interaction between protein sequences and the Darwinian processes that shape these sequences, we have collected these into The Adaptive Evolution Database (TAED). CONCLUSIONS: Placed in a phylogenetic perspective, candidate genes that are undergoing evolution at the same time in the same lineage can be viewed together. This framework based upon coding sequence evolution can be readily expanded to include other types of evolution. In its present form, TAED provides a resource for bioinformaticists interested in data mining and for experimental evolutionists seeking candidate examples of adaptive evolution for further experimental study.

Animals↗

PubMed2001

Fas-mediated apoptosis causes elimination of virus-specific cytotoxic T cells in the virus-infected liver.

Immunity to allogeneic MHC Ags is weak in rodent livers, raising questions as to the mechanisms that might control responses in this organ. Infection with an adenovirus vector reveals that T cell-mediated immunity to nonself-Ags in the liver is self-limiting. Virus-induced liver injury decreases and coincides with disappearance of virus-specific CTL, concomitant to an increase of apoptotic T cells early after infection. But whereas death in CD4 cells is independent of Fas, perforin, and TNF-alpha, that of CD8 cells requires Fas and not perforin or TNF-alpha pathways. Fas ligand is expressed on liver-infiltrating cells, pointing to death by fratricide that causes almost complete disappearance of virus-specific CTL 4 wk after infection. CTL elimination is virus dose dependent, and high doses induced high alanine aminotransferase values, elevated expression of Fas ligand on CD8 cells, and increased CD8 cell migration into the infected liver.

Adenoviridae Infections↗

PubMed2001

Host range studies of GB virus-B hepatitis agent, the closest relative of hepatitis C virus, in New World monkeys and chimpanzees.

GB virus-B (GBV-B) is a member of the Flaviviridae family of viruses. This RNA virus causes acute resolving hepatitis in experimentally infected tamarins, but its natural host remains unknown. GBV-B and a related virus, GBV-A, were recovered from serum containing the "GB agent," which was believed to have originated from a surgeon (initials: GB) with acute hepatitis. GBV-B has special interest because it is the virus related most closely to hepatitis C virus, which is an important cause of acute and chronic liver disease in humans. In the present study, we found that the host range of GBV-B includes owl monkeys. Tamarins and owl monkeys belong to two different families of New World monkeys. The natural history of GBV-B in the two owl monkeys studied was similar to that previously found for tamarins and was characterized by early appearance of viremia and viral clearance. However, the peak viral titers of GBV-B observed in owl monkeys (10(5) genome equivalents [GE] /ml) were lower than those observed in experimentally infected tamarins (10(7)-10(8) GE/ml) and acute hepatitis was observed in only one animal. If GBV-B were indeed a virus of humans, it would be expected to infect chimpanzees, a surrogate of humans, because all recognized human hepatitis viruses are transmissible to chimpanzees and cause hepatitis. However, in the present study, we failed to transmit GBV-B to a naive chimpanzee. In addition, a second naive chimpanzee transfected intrahepatically with RNA transcripts from an infectious clone of GBV-B did not become infected. Thus, chimpanzees are apparently not susceptible to GBV-B. Finally, we failed to detect GBV-B in acute-phase serum from surgeon GB. Our data suggest that GBV-B is not a human virus and that GBV-B, like GBV-A, is a virus of New World monkeys.

Acute Disease↗

PubMed2001

Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins.

Recent developments in directed evolution technologies combined with innovations in robotics and screening methods have revolutionized protein engineering. These methods are being applied broadly to many fields of biotechnology, including chemical engineering, agriculture and human therapeutics. More specifically, DNA shuffling and other methods of genetic recombination and mutation have resulted in the improvement of proteins of therapeutic interest. Optimizing genetic diversity and fitness through iterative directed evolution will accelerate improvements in engineered protein therapeutics.

Antibodies↗

PubMed2001

Failure to infect rhesus monkeys with hepatitis C virus strains of genotypes 1a, 2a or 3a.

The chimpanzee is the only recognized animal model for the study of hepatitis C virus (HCV). However, recently it was reported that rhesus monkeys were susceptible to HCV and developed hepatitis during infection. In the present study, we inoculated two rhesus monkeys each with HCV strain H77 (genotype 1a), strain HC-J6 (genotype 2a) or strain S52 (genotype 3a). Weekly serum samples were tested for liver enzyme values, HCV antibodies and HCV RNA. We did not find evidence of HCV infection in any of the monkeys during 24 weeks of follow-up. Our study demonstrates that rhesus monkeys are not readily infected with HCV and apparently do not represent a useful animal model for the study of HCV.

Animals↗

PubMed2001

Chronic hepatitis C in ethnic minority patients evaluated in Los Angeles County.

OBJECTIVE: The aim of this study was to compare demographic, clinical, and histological features of hepatitis C in four ethnic groups seen at the Los Angeles County/University of Southern California Hepatitis Clinic. METHODS: We evaluated 256 patients with chronic hepatitis C, with 132 (52%) receiving a liver biopsy as part of their evaluation. We estimated fibrosis progression in 103 patients with known duration of disease. RESULTS: Asians (6%) were underrepresented in the hepatitis C cohort, whereas Latinos (51%) were overrepresented, as compared with the entire county population. A history of injection drug use was more frequent in whites (65%) than in African Americans (45%, p = 0.05), Latinos (47%, p = 0.01), or Asians (0%) and more frequent in Latinos (59%) than in Latinas (26%, p = 0.003). Such a gender difference was not found in African Americans or whites. Baseline laboratory values were comparable. The amount of alcohol consumed daily was higher in African Americans than in Asians (p = 0.0001) and whites (p = 0.10). African Americans (0.077 fibrosis stages/yr) and whites (0.084/yr) had significantly lower mean estimated progression of liver fibrosis than Latinos (0.215/yr) with hepatitis C virus infection (ps = 0.03 and 0.02, respectively): this was likely related to their longer estimated duration of disease. CONCLUSION: Minorities represent the majority of chronic hepatitis C cases in the Los Angeles County Hepatitis Clinic. Asians, Latinas, and African Americans are less likely to report injection drug use as a risk factor for hepatitis C virus. Latinos seem to have faster liver fibrosis progression rates than either African Americans or whites.

Adult↗

PubMed2000

Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees.

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the DeltaHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with DeltaHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The DeltaHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.

Animals↗

PubMed2000

Liver fibrosis progression is related to CD4 cell depletion in patients coinfected with hepatitis C virus and human immunodeficiency virus.

A total of 204 patients with liver biopsy-proven hepatitis C virus (HCV) infection, 84 with and 120 without human immunodeficiency virus (HIV) coinfection, were studied, to evaluate variables possibly associated with the stage of liver fibrosis. All patients were injection drugs users, with a mean age of 32 years and an estimated duration of HCV infection of 12 years. Twenty-four patients (11%) had many fibrous septa with (5%) or without (6%) cirrhosis, 56 (27%) had few fibrous septa, and 124 (60%) had no fibrous septa. In all patients, an association was found between CD4 cell counts <500 cells/mm(3)and the presence of many fibrous septa (odds ratio, 3.2; P=.037), independent of HIV infection and other factors. These results suggest that HIV infection-induced CD4 depletion is independently associated with the severity of liver fibrosis in chronic HCV infection.

Adult↗

PubMed2000

Prospective evaluation of infection with hepatitis G virus in relation to hepatocellular carcinoma in Shanghai, China.

The health consequences of hepatitis G virus (HGV) infection in humans remain to be determined. Inconsistent results of HGV and the risk of hepatocellular carcinoma (HCC) have been found in retrospective case-control studies. No prospective studies have addressed this issue. A prospective study including 18,244 men in Shanghai, China, was conducted to assess the role of HGV infection in HCC development. Serum HGV RNA and hepatitis B surface antigen (HBsAg) were assessed in 127 HCC case patients and 660 matched control subjects drawn from the cohort. Four patients with HCC (3.1%) and 21 control subjects (3.2%) were positive for serum HGV RNA. The presence of HGV RNA in prediagnostic serum was not associated with the risk of HCC (odds ratio, 1.0; 95% confidence interval, 0.3-2.9). No association between HGV RNA positivity and HCC risk was observed in either HBsAg-positive carriers or noncarriers. It is concluded that HGV infection plays no role in HCC development in this high-risk population.

Aged↗

PubMed2000

NK cells cause liver injury and facilitate the induction of T cell-mediated immunity to a viral liver infection.

NK cells are a relatively rare cell population in peripheral lymphoid organs but are abundant in the liver, raising questions as to their function in immune responses to infections of this organ. To investigate this, cell-mediated immunity to viral liver infection induced by a type 5, replication-defective, adenovirus was examined. It is shown that NK cells in the absence of T cells cause hepatocyte apoptosis in virus-infected livers associated with an increase in liver enzymes in the serum. Concomitantly, NK cells induce production of IFN-gamma, inhibitable by their elimination before infection. NK cells are shown to be necessary for optimal priming of virus-specific T cells, assessed by delayed-type hypersensitivity response and CTL activity, consistent with their ability to secrete IFN-gamma. The conclusion is drawn that NK cells mediate two important functions in the liver: they induce cell death in the infected organ and concomitantly stimulate the induction of T cell-mediated immunity by release of IFN-gamma.

Adenoviridae Infections↗

PubMed2000

Fas- and tumor necrosis factor receptor 1-dependent but not perforin-dependent pathways cause injury in livers infected with an adenovirus construct in mice.

Intravenous injection of type 5 adenovirus, deleted in the E1 and E3 regions, is shown to result in expression of viral antigens in the liver, initiating lymphocyte infiltration and liver injury. Following this infection, induction of Fas ligand (FasL), tumor necrosis factor alpha (TNF-alpha), and perforin mRNA are all demonstrable in the liver, pointing to a role of respective pathways in liver injury. Making use of mice in which the genes coding for Fas, FasL, TNF receptors (TNFRs), and perforin are inactivated, as well as recombinant proteins that inhibit Fas- and TNF-alpha-mediated apoptosis, it is shown that a functional perforin-mediated mechanism is not obligatory for cellular infiltration and progression of liver injury. In contrast functional Fas- and TNF-alpha-mediated mechanisms were found to be essential for liver injury to occur. Results are presented demonstrating that signaling through TNFR1, but not TNFR2, is involved in TNF-alpha-mediated liver damage. The conclusion is drawn that although perforin mRNA is induced in the virus-infected liver, Fas- and TNF-alpha-mediated mechanisms constitute the principal pathways by which the cell-mediated immune system causes acute liver injury.

Adenoviridae↗

PubMed2000

Functional inferences from reconstructed evolutionary biology involving rectified databases--an evolutionarily grounded approach to functional genomics.

If bioinformatics tools are constructed to reproduce the natural, evolutionary history of the biosphere, they offer powerful approaches to some of the most difficult tasks in genomics, including the organization and retrieval of sequence data, the updating of massive genomic databases, the detection of database error, the assignment of introns, the prediction of protein conformation from protein sequences, the detection of distant homologs, the assignment of function to open reading frames, the identification of biochemical pathways from genomic data, and the construction of a comprehensive model correlating the history of biomolecules with the history of planet Earth.

Amino Acid Sequence↗

PubMed2000

Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV.

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Development of vaccines to prevent HCV infection, or at least prevent progression to chronicity, is a major goal. In mice and rhesus macaques, a DNA vaccine encoding cell-surface HCV-envelope 2 (E2) glycoprotein stimulated stronger immune responses than a vaccine encoding intracellular E2. Therefore, we used DNA encoding surface-expressed E2 to immunize chimpanzees 2768 and 3001. Chimpanzee 3001 developed anti-E2 after the second immunization and antibodies to hypervariable region 1 (HVR1) after the third immunization. Although chimpanzee 2768 had only low levels of anti-E2 after the third immunization, an anamnestic response occurred after HCV challenge. CTL responses to E2 were not detected before challenge, but a strong response was detected after HCV challenge in chimpanzee 2768. An E2-specific CD4+ response was detected in chimpanzee 2768 before challenge and in both chimpanzees postchallenge. Three weeks after the last immunization, animals were challenged with 100 50% chimpanzee-infectious doses (CID(50)) of homologous monoclonal HCV. As a control, a naive chimpanzee was inoculated with 3 CID(50) of the challenge virus. The vaccine did not generate sterilizing immunity because both vaccinated chimpanzees were infected. However, both vaccinated chimpanzees resolved the infection early whereas the control animal became chronically infected. Compared with the control animal, hepatitis appeared earlier in the course of the infection in both vaccinated chimpanzees. Therefore, DNA vaccine encoding cell surface-expressed E2 did not elicit sterilizing immunity in chimpanzees against challenge with a monoclonal homologous virus, but did appear to modify the infection and might have prevented progression to chronicity.

Animals↗