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The principal neutralization epitope of human immunodeficiency virus 1 is localized in the third variable (V3) domain of the external envelope and has been shown to bind isolate-specific antibodies. Therefore, the extent of variation within the nucleic acid sequence encoding this epitope was studied in DNA directly obtained from peripheral blood mononuclear cells of six children and their plasma donor. This revealed that the quasi-species distribution of sequences obtained after cloning varied from recipient to recipient and that the distance from the donor sequences increased over time. V3 nucleotide evolution rates averaged 9.5 x 10(-3) per site per year for silent sites and 11.4 x 10(-3) per site per year for nonsilent sites (vs. 9.7 and 9.8 x 10(-3) per site per year for a control region 5' adjacent to the V3 region) and, although individual differences were observed, did not correlate with the serum antigen levels or disease progression. Sequences of both the epitope coding region itself (V3) and the control region upstream diverted more from the donor sequence among children not progressing to AIDS than among children progressing to AIDS. The evolution of V3 sequences is apparently host dependent, rapid, and independent of the level of antigen expression.

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A panel of murine monoclonal antibodies (MAbs) to the human immunodeficiency virus type 1 trans-activator tat protein were characterized. The anti-tat MAbs were mapped to the different domains of the tat protein by Western blot (immunoblot) and Pepscan analyses. One-half of the MAbs tested mapped to the amino-terminal proline-rich region, and one-third of the MAbs tested mapped to the lysine-arginine-rich region of tat. The individual MAbs were tested for inhibition of tat-mediated trans activation, using a cell-based in vitro assay system. MAbs which mapped to the amino-terminal region of the tat protein demonstrated the highest degree of inhibition, whereas MAbs reactive to other portions of the molecule exhibited a less pronounced effect on tat function.

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Recently a new technique, the polymerase chain reaction (PCR), has been used for the detection and characterization of HIV-1 proviral DNA and viral RNA. These reports support the notion that the PCR is more sensitive and specific than other established HIV-1 detection techniques. However, due to its extreme sensitivity, the PCR is highly susceptible to contamination, resulting in false positive results. To avoid contamination, strict rules on sample preparation and pre- and post-PCR handling are required. Confirmation of both positive and negative PCR results by independent techniques is not always feasible, and, therefore, optimal PCR conditions, inclusion of control samples, repetition of results, and confirmation of specificity by hybridization are required. The choice of the material from which HIV-1 is amplified, the primers used for amplification as well as the PCR conditions will determine what is actually amplified.

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A thirty amino acid synthetic peptide (HGP30) representing the conserved region of HIV-1 p17 induced high titer antibodies to the native p17 in rabbits. This immune sera neutralized HIV-1 replication in cell culture and one of the high titer antisera also inhibited CD4-dependent cell fusion. Pepscan analysis with overlapping nonapeptides derived from the sequence of HIV-1 p17 identified the sequence (KE) ALDKIEE (EQ) as the major antibody binding site. Sera of 9% of AIDS patients (7/76) and 18% of HIV-1 seropositive healthy homosexuals (40/223) were positive for HGP30 antibodies. Decline in HIV-1 p17 antibodies has been shown to be related to disease progression in both children and adults, suggesting that HIV-1 p17 antibodies may be protective. Hence, a synthetic HIV-1 p17 peptide, representing the immunodominant epitope, could be useful as a candidate vaccine for immunization of HIV-1 seronegative or seropositive healthy homosexuals.

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The authors studied thymus specimens taken at autopsy from eight acquired immune deficiency syndrome (AIDS) patients and compared these with those taken from four patients with congenital immunodeficiency (unrelated to an intrinsic thymus defect) and seven patients after allogeneic bone marrow transplantation. In all cases, histology showed a severely involuted architecture, compatible with a debilitating disease before death. There were no major differences between thymus tissue in AIDS patients and in the other patients studied. This argues against the claim expressed in the literature that the epithelial microenvironment incurs particular HIV-1-induced injury in AIDS. This conclusion is substantiated by immunohistochemistry for HIV-1 gag and env proteins, and by hybridohistochemistry for gag/pol and env mRNA of HIV-1. Positive cells were observed only in low numbers, both inside the epithelial parenchyma and in the (expanded) perivascular areas. An interesting finding was the labeling of subcapsular/medullary epithelium in normal uninvoluted thymus by a number of antibodies to HIV-1 gag p17 and p24 proteins. Compatible with this labeling was the staining of epithelial stalks in hyperinvoluted thymuses irrespective of disease category. The previously reported cross-reactivity between HIV-1 core protein and thymosin alpha 1 cannot fully explain this observation, because the epithelium in the hyperinvoluted state is negative for thymosin alpha 1. This study confirms and extends previous reports on the endogenous presence of epitopes of retroviral antigens in thymic epithelium.

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The presence of proteins (p17 and p24 core proteins, gp41 envelope protein) and mRNA (gag/pol and env gene segments) of human immunodeficiency virus type-1 (HIV-1) was analyzed on frozen tissue sections of lymph nodes from HIV-1 infected individuals. Thirty-one lymph nodes were categorized in the stages of follicle hyperplasia (n = 18), follicle degeneration (n = 5), and total depletion (n = 8). The follicle dendritic cells in germinal centers showed the presence of core proteins and, to a lesser extent, gp41. The staining patterns, being similar to those of immunoglobulins, suggested that they occur in the form of immune complexes. In addition there were solitary cells expressing viral protein, in particular gp41, and mRNA. The number of mRNA-positive cells was very low: about five positive cells were observed in a tissue section with about ten (hyperplastic) follicles. HIV-1-mRNA-positive cells were observed both in follicles and interfollicular areas and showed no differences between various stages. The extent and intensity of distinct HIV-1 proteins and HIV-1-mRNA gene segments in follicles were significantly correlated, as was their presence in interfollicular areas. No significant correlation was found between the presence of HIV-1 components in follicles and in interfollicular areas. This indicates that processes involving HIV-1 components occur in a segregated manner in both lymph node compartments. The presence of HIV-1 components did not correspond to any clinical classification (CDC criteria), nor to other histochemical characteristics. An exception was the correlation between gp41-positive cells and CD1-positive interdigitating cells in the interfollicular areas.

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The tat regulatory protein of HIV-1 was expressed as a fusion protein in E. coli and used as antigen to detect antibodies against HIV-tat (anti-tat) in the serum of HIV-1 infected children and adults. HIV-1-infected children showed a higher frequency (55%) of anti-tat than HIV-1-infected adults (36%). Anti-tat were present in only 15% (3/20) of acutely infected individuals. Forty percent (10/25) of individuals with prolonged HIV-1 infection but without antigen were anti-tat positive. Only 13% (3/23) of HIV-1-antibody-positive individuals with prolonged HIV-1 antigenemia were anti-tat positive and titers of anti-tat antibodies declined with time. Pepscan analysis identified the amino terminus of HIV-tat as the major antibody-binding site. Antibodies to HIV-tat occurred as a harbinger of HIV-1 antigen expression and disappeared thereafter, possibly reflecting the transience of HIV-tat expression. Because of the low antigenicity of HIV-tat, antibodies to this regulatory protein are not a reliable marker for either early HIV-1 infection or subsequent disease progression.

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A national multicentre study was performed to investigate the effects of donorselection and the use of heat-treated plasma products on seroconversion to HIV in 157 Dutch haemophiliacs. All patients included in the study were seronegative for HIV antibodies in 1983. Thirteen percent (20/157) seroconverted between 1983 and 1986. Nineteen of 20 seroconversions could be related to the use of non heat-treated products in the year preceding HIV antibody seroconversion. One seroconversion occurred in a person using heat-treated non donor screened product. Seroconversion rate decreased as a result of the policy to discourage high risk blood donors and no seroconversions were observed following the introduction of donor screening in 1985.

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Using a modified immunoblot procedure we looked for early serological markers of disease progression in sequential serum samples from 30 initially symptomless HIV-infected homosexual men. Sixteen men who did not progress beyond persistent generalized lymphadenopathy (PGL) and did not develop HIV antigenaemia showed persistent strong immunoglobulin G (IgG) reactivity to all initially recognized HIV proteins. Three out of four men who did not progress beyond PGL but did develop HIV antigenaemia showed declining or absent IgG reactivity to the outer HIV core protein p17, whereas reactivity to other initially recognized HIV proteins persisted in all four; in one of these subjects a striking decline in anti-p17 reactivity occurred 1 1/2 months after HIV antibody seroconversion and 7 1/2 months before HIV antigenaemia developed. In nine out of 10 men who developed constitutional disease [Centers for Disease Control (CDC) group IV A] or AIDS (CDC groups IV C1 and IV D), a decline in anti-p17 reactivity was seen preceding or at disease development; in two of these men a concomitant decline in anti-p24 reactivity was seen. In the only individual without HIV antigenaemia who developed CDC group IV disease, anti-p17 reactivity declined 10 months before disease development, whereas no similar decline in anti-p24 reactivity was seen. Decline in IgG antibody reactivity to HIV core protein p17 appears to be an earlier marker of disease progression than the previously reported decline in anti-p24 reactivity, and may be of value in selecting individuals for secondary prevention of HIV-related disease development.

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Human immunodeficiency virus (HIV) is lymphotropic and neurotropic. In vivo clinical and immunological abnormalities develop in a large proportion of long-term HIV antibody seropositive persons. Different stages of HIV infection are marked by expression of HIV genes, production of HIV antibodies, formation of antigen/antibody complexes and clearance of such complexes. Transient HIV antigenemia appearing generally 6-8 wk prior to HIV antibody (HIV-Ab) seroconversion and lasting 3-4 mth is generally seen in acute infection. IgM antibodies predominantly to core proteins may occasionally be detectable when, or just before, IgG antibodies appear. If IgG antibodies to both envelope and core proteins persist in the absence of HIV-Ag the short-term prognosis is relatively good. However, HIV-Ag seroconversion may appear at any time after HIV-Ab seroconversion. Progression to AIDS is strongly associated with declining or absent levels of IgG antibodies to p24. IgG2 and IgG4 antibodies to HIV, which are mainly directed to p24, disappear most dramatically. Titers of antibodies to HIV p24 below 64 are strongly associated with the presence of HIV antigen and a poor clinical outcome. HIV antigen was detected frequently in sera from children in all stages of infection in contrast to adults whose sera were generally HIV-Ag negative when asymptomatic and positive when AIDS was apparent. HIV antigen may be less efficiently detected with the present assays in sera from regions where the prototype strains of HIV (HTLV-III and LAV) are less prevalent, like Central Africa. Persistence of HIV-Ag in cerebrospinal fluid (CSF) appears to be pathognomonic for progressive encephalopathy, particularly in children. Levels of HIV-Ag in serum, and possibly in CSF, can be decreased by nucleoside analogues, such as AZT. This indicates HIV-Ag and possibly antibody to HIV core protein p24 as suitable markers for selecting individuals for antiviral therapy as well as monitoring the efficacy of such therapy.

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Human immunodeficiency virus antigen (HIV-Ag) was detected in the serum of most adult (13/16) and paediatric (6/6) AIDS patients and rarely in the serum of symptomless seropositive controls (1/13). It was present in the cerebrospinal fluid (CSF) of all 5 children and most (5/9) adults with AIDS-related encephalopathy, but not in the CSF of 13 symptomless seropositive controls, of whom 8 had antibody in the CSF. A longitudinal study of 1 of the controls with antibody in the CSF showed that HIV-Ag in CSF was present transiently before the occurrence of antibody in the CSF. In serial samples of serum from 35 men who seroconverted HIV-Ag was detected in 11 persons--in 5 before seroconversion and in 6 after. 3 of the 6 who became antigenaemic after seroconversion remained so for the rest of the follow-up. AIDS was diagnosed in 1 patient, 3 months after HIV-Ag was first detected in serum and 6 months after seroconversion. The findings suggest that HIV-Ag appears early and transiently in primary HIV infection. Antibody production follows, after which HIV-Ag may disappear. Its persistence or reappearance seems to correlate with clinical, immunological, and neurological deterioration.

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Monoclonal antibodies (MAb) directed against different epitopes on the equimolar complex of cloacin and immunity protein (cloacin DF13) were isolated, characterized, and used to study the uptake of cloacin DF13 by susceptible cells. Four MAbs recognized the amino-terminal part, one MAb recognized the central part, and three MAbs recognized the carboxyl-terminal part of the cloacin molecule. Three MAbs reacted with the immunity protein. Five MAbs inhibited the lethal action of cloacin DF13, but none of the MAbs inhibited the binding of cloacin DF13 to its purified outer membrane receptor protein or the in vitro inactivation of ribosomes. Binding of cloacin DF13 to susceptible cells cultured in broth resulted in a specific, time-dependent dissociation of the complex and a fragmentation of the cloacin molecules. Increasing amounts of immunity protein were detected in the culture medium from about 20 min after the addition of cloacin DF13. Cloacin was fragmented into two carboxyl-terminal fragments with relative molecular masses of 50,000 and 10,000. The larger fragment was detected 5 min after the binding of the bacteriocin complex to the cells. The smaller fragment was detected after 10 min. Both fragments were associated with the cells and could not be detected in the culture supernatant fraction. Cells grown in brain heart infusion were much less susceptible to cloacin DF13 than cells grown in broth, although they possessed a similar number of outer membrane receptor molecules. This decreased susceptibility correlated with a decreased translocation, dissociation, and fragmentation of cloacin DF13.

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To establish when lymphadenopathy associated virus or human T lymphotropic virus (LAV/HTLV-III) was introduced into the Netherlands, we studied a cohort of homosexual men who participated in a hepatitis B vaccine efficacy study between 1980 and 1982. On entry into the study (November 1980 to December 1981) five (0.7%) out of 685 participants were found to have antibodies to LAV/HTLV-III, and during follow up 15 seroconversions were detected among the 680 who had been seronegative initially (end point attack rate 3%). LAV/HTLV-III was not transmitted by the heat inactivated hepatitis B virus (HBV) vaccine used. Anal sexual contact and antibodies to cytomegalovirus (CMV) were found to correlate with seropositivity or seroconversion for LAV/HTLV-III. Six out of 15 men who seroconverted reported a mononucleosis like illness, but three of them had other concurrent virus infections. To date, only one of the 20 seropositive men has developed the acquired immune deficiency syndrome (AIDS), three years after his seroconversion. This study shows that the introduction of LAV/HTLV-III into the Dutch male homosexual community took place at the end of the 1970s, a few years before the first case of AIDS in a native Dutchman.

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Longitudinal IgG recognition patterns of viral proteins were studied in 15 men who had seroconverted for lymphadenopathy associated virus/human T lymphotropic virus (LAV/HTLV-III). Antibodies to the major viral core protein p24, which is a cleavage product of the gag gene encoded precursor protein pr55, appeared first. These were soon followed by antibodies to pr55 and more gradually by antibodies to the other gag gene encoded cleavage product p18, the env gene encoded transmembrane glycoprotein gp41, the env gene encoded glycoproteins gp65 and gp110, and the putative pol gene product p33. In 13 subjects who remained healthy the reactivity to the different proteins increased or stabilised with time, while in two men who developed acquired immune deficiency syndrome (AIDS) the reactivity, most noticeably to gag encoded proteins, diminished before or at the onset of symptoms.

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Both Enterobacter cloacae H478 and Klebsiella edwardsii S15 were shown to harbour a relatively large conjugative plasmid that coded for cloacin DF13-susceptibility and the production and uptake of a hydroxamate iron chelator, most probably aerobactin. Protein-blotting experiments with antiserum raised against the purified cloacin DF13/aerobactin receptor protein from Escherichia coli (Co1V-K30) revealed that the corresponding outer membrane receptor proteins of Ent. cloacae H478 and K. edwardsii S15 had apparent mol wts of 85 000 and 76 000, respectively. E. coli transconjugants harbouring either the plasmid from Ent. cloacae H478 or K. edwardsii S15 expressed a cloacin DF13/aerobactin outer membrane receptor protein with a mol wt of 74 000. The receptor protein encoded by the Ent. cloacae and K. edwardsii plasmids were immunologically more related to each other than to the pCo1V-K30-encoded receptor protein.

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A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.

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A DNA fragment derived from the ColV-K30 plasmid and coding for both sensitivity to cloacin DF13 and Fe3+-aerobactin uptake was cloned into pBR322. The cloned fragment coded for two polypeptides with molecular masses of 74,000 (the cloacin DF13/aerobactin receptor protein) and 50,000 daltons, respectively. When grown with sufficient iron, cells harboring pFS8 (with this fragment) possessed about 10 times as many receptor protein molecules as compared with cells of Escherichia coli (ColV-K30). The synthesis of the receptor protein specified by pFS8, however, was independent of the availability of iron, in contrast to strains harboring the intact ColV-K30 plasmid. Aerobactin was taken up but not synthesized by cells harboring pFS8. No growth occurred when iron-starved cultures of these cells were incubated with Fe3+-aerobactin, suggesting that expression of other ColV-K30-encoded genes is necessary to remove the iron from the Fe3+-aerobactin complex.

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