Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.
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PMID:R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens. 157 Nov 53

In this review B cell responses in HIV-infected individuals are summarized together with the techniques used to date to produce human monoclonals to HIV and the properties of these antibodies. Profound disturbances in B cell responses are apparent both in vivo and in vitro. While there is evidence in vivo of marked polyclonal B cell activation, primary and secondary antibody responses are impaired. Similarly these cells exhibit spontaneous immunoglobulin secretion upon in vitro culture but do not readily respond to B cell mitogens and recall antigens including HIV. Furthermore, certain of these defects can be reproduced in normal B cells in vitro by incubation with HIV or HIV coded peptides. Individuals infected with HIV develop antibodies to HIV structural proteins (e.g. p17, p24, gp41 and gp120) and regulatory proteins (e.g. vif, nef, RT). Autoantibodies against a number of immunologically important molecules are also frequently observed. The anti-HIV antibodies are predominantly of the IgG1 isotype and exhibit a variety of effects on the virus in vitro. To date, using conventional immortalization strategies, an appreciable number of human monoclonals to HIV have been developed. These have been specific for gp41, gp120 and gag with antibodies of the former specificity predominating. The majority of these antibodies have been of the IgG1 isotype. Only a small number of the antibodies neutralize virus in vitro and most of these react with gp120. The neutralizing antibodies recognize conformational and carbohydrate epitopes or epitopes in amino acid positions 306-322. The predominant epitopes recognized by the anti-gp41 antibodies were in amino acid positions 579-620 and 644-662. A high percentage (congruent to 25%) of these antibodies enhance viral growth in vitro. The problems relating to the production of human monoclonals to HIV are discussed together with strategies that could be used in the future.
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PMID:B cell responses to HIV and the development of human monoclonal antibodies. 157 84

The nervous system is frequently affected in patients with the acquired immune deficiency syndrome (AIDS). In addition to opportunistic CNS infections and cerebral lymphomas, approx. 20% of the patients develop HIV-associated encephalopathies. Two major histopathological manifestations are observed. HIV leukoencephalopathy (progressive diffuse leukoencephalopathy) is characterized by a diffuse loss of myelin in the deep white matter of the cerebral and cerebellar hemispheres, with scattered multinucleated giant cells and microglia but scarce or absent inflammatory reaction. HIV encephalitis (multinucleated giant cell encephalitis) is associated with accumulations of multinucleated giant cells, inflammatory reaction and often focal necroses. In some patients, both patterns may overlap. In order to identify the HIV genome in the CNS, brain tissue from 27 patients was analyzed for the presence of HIV gag sequences using the polymerase chain reaction (PCR) and primers encoding a 109 base pair segment of the gag gene. Amplification of HIV gag succeeded in all 5 patients with clinical and histopathological evidence for HIV encephalopathy but was negative in the 20 AIDS patients with opportunistic bacterial, parasitic and/or viral infections or with cerebral lymphomas. These results strongly suggest that the evolution of histopathologically recognizable HIV-encephalopathies closely correlates with the presence and/or tissue concentration of HIV. Since there were no cases with amplified HIV DNA in the absence of HIV-associated tissue lesions, we conclude that harboring and replication of HIV in the CNS rapidly causes corresponding clinical and morphological changes of HIV-associated encephalopathies. In two children with severe HIV encephalomyelitis, large amounts of HIV gag and env transcripts were detected in affected areas of the brain and spinal cord by in situ hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neuropathology and pathogenesis of HIV encephalopathies. 158 55

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.
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PMID:Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation. 158 12

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.
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PMID:Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products. 159 13

Among 770 Western blots for HIV-1 confirmation on sera from subjects at high risk for HIV infection, 4.3% (33 cases) were indeterminate. Isolated, stable, reproducible anti-gp 160 reactivity, highly suggestive of a nonspecific reaction, was found in 16% of cases. There were three other probably nonspecific patterns with anti-gp 160 and either anti-gp 41 or anti-gp 120 reactivities and thin, atypical bands. Two patterns with anti-p24 and either anti-gp 160 or anti-gp 120 reactivities were consistent with HIV-1 seroconversion. Reactivity directed solely against gag products was seen in 18% of cases. The repeat test, performed in 16 cases, showed an identical pattern in 3 cases, a modified pattern in 3 cases, negative results in 9 cases, and seroconversion in 1 case. No case of HIV-2 infection was detected. Indeterminate Western blot results reflect nonspecific reactivity in most instances but should nevertheless lead to the exclusion of technical artefacts, seroconversion, and HIV-2 infection.
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PMID:[Serological confirmation of HIV infections in hospital practice: analysis of indeterminate western blot results]. 160 65

Replication competent chimeric viruses that express the gag and pol proteins of SIVmac and the env proteins of HIV-1 were made. One such chimeric virus, SHIV-4, that expresses the vif, vpx, vpr, and nef regulatory genes of SIV and the tat and rev regulatory genes of HIV-1 replicated efficiently in cynomolgus monkeys. This model system can be used to evaluate the efficacy of anti-HIV-1 vaccines directed at the envelope glycoproteins, anti-HIV-1 envelope glycoprotein antiserum or monoclonal antibodies, and anti-HIV-1 drugs designed to inhibit the tat, rev, or env functions.
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PMID:Infection of cynomolgus monkeys with a chimeric HIV-1/SIVmac virus that expresses the HIV-1 envelope glycoproteins. 161 62

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.
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PMID:Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors. 162 70

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev stimulates expression of structural viral proteins via a target response element (RRE) located within gag-pol and env mRNAs. To analyse the HIV-2 Rev trans-activation effect on the expression of the envelope protein, we cloned a functionally active HIV-2 rev cDNA and showed that it contained four exons. Using transient expression assays, we mapped a 353 bp RRE fragment within the env gene of HIV-2 on which both HIV-1 and HIV-2 Rev could act. Interestingly, smaller fragments suppressed the use of additional splice sites within the env gene and caused envelope protein expression independent of Rev.
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PMID:Functional mapping of the rev-responsive element of human immunodeficiency virus type 2 (HIV-2): influence of HIV-2 envelope-encoding sequences on HIV-1 gp120 expression in the presence or absence of Rev. 162 2

We have established a genetic assay for the multimerization of retroviral gag polyproteins. This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S. Fields and O. Song, Nature (London) 340:245-246, 1989). In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55gag. The coexpression of these two hybrid proteins in S. cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene. Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins. In contrast, the heterologous GAL4-HIV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression. The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.
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PMID:Genetic assay for multimerization of retroviral gag polyproteins. 162 70


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