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Biomedical subjects

F K Askari

Publications and source records attributed to F K Askari.

11 recordsLinked to original sources
PubMed1997

Immunization.

Immunization is undergoing important changes, with improved vaccines replacing less immunogenic or less safe vaccines, new vaccines for common diseases such as chickenpox and hepatitis A infection, and improved immunization schedules. Immunization is also being transformed by basic work in molecular medicine. Vaccines made of DNA are being developed as a form of gene therapy that use the patient's own cellular machinery to make foreign proteins that stimulate an immune response. Currently immunization is used to protect patients prior to exposure to an infectious agent or during the incubation phase after exposure, but before disease has occurred. New technologies are being investigated to induce the immune system to fight infections that have already produced chronic disease such as acquired immunodeficiency syndrome and chronic hepatitis B virus infection.

Humans↗
PubMed1997

Augmentation of ouabain sensitivity of rat liver Na/K-ATPase by in vivo adenovirus-mediated expression of the Na/K-ATPase alpha2 subunit.

These are the first experiments to study the effect of in vivo expression of the Na/K-ATPase alpha2 subunit which serves as a receptor for cardiac glycosides. The alpha2 subunit is not normally expressed in rat liver, so hepatocytes which lack endogenous alpha2 protein are a logical first target to study the effects of alpha2 expression on membrane Na/K-ATPase activity. At 3 days after alpha2 adenovirus vector infusion, Wistar rat livers contained alpha2 DNA, alpha2 mRNA, and alpha2 protein. Rat liver membrane ouabain binding activity and the sensitivity of Na/K-ATPase activity to ouabain significantly increased. Total membrane Na/K-ATPase was regulated at a constant level while expressed alpha2 activity represented 10% of the total active Na/K-ATPase sites in alpha2 transduced rat liver. These studies are the first to establish a paradigm in which an endogenous drug receptor is expressed to alter cellular pharmacologic sensitivity.

Adenoviruses, Human↗
PubMed1996

Identification of bilirubin UDP-GTs in the human alimentary tract in accordance with the gut as a putative metabolic organ.

The initial identification of traditionally hepatic enzymes expressed in the gut has led to the hypothesis that the gut may function as a metabolic organ. The UDP glucuronosyltransferases (UDP-GTs) play an important role as phase II metabolizing enzymes. Previously members of this family have been identified in the gut by non-isoform specific immunoreactivity, and a small amount of bilirubin glucuronosyltransferase activity was detected in the colon. Recent reports of gut transplantation to reverse the metabolic defect in Gunn rats raised further interest in the expression and distribution of human bilirubin (UDP-GTs (HUG Br 1 and HUG Br 2) in the human alimentary tract. The availability of molecular genetic probes for HUG Br 1 and HUG Br 2 permits the screening of the alimentary tract for the presence of isoform specific message. RNA samples extracted from pinch biopsy specimens of buccal mucosa, esophagus, stomach body, antrum, duodenum, and colon were analyzed for expression of HUG Br 1 and HUG Br 2. HUG Br 1 hybridization was detected in duodenum > colon, whereas HUG Br2 hybridization was detected in duodenum > esophagus > colon. Immunoreactivity data confirmed the presence of HUG Br 1 protein at low levels in the duodenum, whereas the less abundant HUG Br 2 protein was below the limits of detection of isoform specific anti-peptide antibodies. Bilirubin specific reactivity was demonstrated in duodenal samples but not antrum samples, consistent with the molecular genetic data. The presence of functional bilirubin UDP-GT isoforms in human alimentary tract supports the notion that the gut may function as a metabolic organ and may have diagnostic and therapeutic implications for disorders of bilirubin metabolism.

Adult↗
PubMed1996

DNA vaccines.

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DNA↗
PubMed1996

Complete correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome type I following transient in vivo adenovirus-mediated expression of human bilirubin UDP-glucuronosyltransferase.

Recombinant adenoviral vectors are useful for the in vivo expression of genes in hepatocytes. Adenoviral vectors deleted in E1a, E1b, and E3b were constructed and used to study in vivo expression of the major human bilirubin UDP-glucuronosyltransferase isoform (HUG Br1) under the transcriptional control of the cytomegalovirus (CMV) immediate-early promoter-enhancer (H5.010CMV hugBr1). As a control, a recombinant adenoviral vector containing the beta-galactosidase reporter gene driven by the CMV promoter-enhancer was employed (H5. 010CMVlacZ). Recombinant virus was expanded following exposure to E1 transcomplementing (293) cells and concentrated to t titer of approximately 10(13) particles per milliliter. A rat model for Crigler-Najjar syndrome type I deficient in HUG Br1 (ie the Gunn rat) was injected with 5 X 10(9) plaque-forming units (p.f.u.) via the portal vein of either H5.010CMVhugBr1 or H5. 010CMVlacZ. Rats from each set were killed at 3 days, 11 days and 22 days after infusion. Liver total cellular DNA, RNA and protein were analyzed for the transgene and the transgene product at the specified times. Analysis of livers by Southern blot hybridization demonstrates sequence-specific hybridization to adenoviral vector DNA, and Northern blot hybridization demonstrates sequence-specific hybridization to transgene-derived RNA. DNA levels peak at approximately one copy number at 3 days and decline over 22 days. RNA and Western blot analyses demonstrate overexpression of message and protein at 3 days, declining over 22 days. In virto functional assay for bilirubin glucuronosyl-transferase activity demonstrates overexpression of bilirubin UDP-glucurosyltransferase function. In situ hybridization of frozen sections to detect expressed mRNA using beta-galactosidasederived 35S-labeled riboprobes demonstrates adenovirus-derived transgene expression in hepatocytes. Significant drops in serum bilirubin levels were noted following expression of HUG Br1 but not beta-galactosidase. The drop in serum bilirubin correlates with the appearance of bilirubin glucuronides in bile. In summary, recombinant adenoviral vectors were used to demonstrate in vivo complementation of the genetic defect in Gunn rat livers with the HUG Br1 cDNA leading to a resolution of hyperbilirubinemia lasting approximately 7 weeks. These studies suggest that delivery of the HUG Br1 cDNA might provide a reasonable therapeutic benefit for Crigler-Najjar syndrome type I patients, as safe and efficacious gene delivery systems are developed.

Adenoviridae↗
PubMed1995

High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice.

Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.

3T3 Cells↗
PubMed1995

Sequence analysis of the hepatitis C virus (HCV) core gene suggests the core protein as an appropriate target for HCV vaccine strategies.

Hepatitis C virus (HCV) is a major healthproblem with a prevalence of 1% in the United States population, and a significant percentage of infected patients progress to chronic liver disease and cirrhosis. Interferon therapy has demonstrated that the immune system can be modulated to alter the acute course of the disease, but long-term treatments remain elusive. Prevention of hepatitis C infection is therefore an important strategy to mitigate the impact of this disease. Initial attempts at vaccination have focused on recombinant envelope vaccines, which have shown an ability to protect against very low titre challenges of HCV in chimps. The need for vaccines capable of protecting against higher titre challenges has led to the search for alternative vaccine strategies. The most highly conserved structural protein in the HCV genome is the core protein, and vaccine strategies targeting the core protein have been proposed to increase vaccine efficacy. The variability of HCV core sequences and genotypes in the Ann Arbor patient population are not known, and the present study was undertaken to assess the theoretical feasibility of developing a HCV core vaccine by excluding promiscuous core (C) gene variability as a mechanism of vaccine failure. Results of nucleotide and deduced amino acid sequence analysis from 13 of 14 patients studied reveal a 93% nucleotide and 96.4% amino acid core sequence homology in the C gene regions studied. Genotype analysis revealed four of 14 to be type 1a and nine of 14 to be type 1b with one infection not being sufficiently characterized to determine genotype. These results demonstrate a sufficiently high degree of conservation of HCV core sequences in our patient population to permit design of a vaccine directed against core protein.

Amino Acid Sequence↗
PubMed1992

Targeted delivery of antisense oligonucleotides by molecular conjugates.

Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly(L-lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 microM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 microM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.

3T3 Cells↗