Biomedical subjects
Publications and source records attributed to W Borkowsky.
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Gramicidin, cation channel forming ionophore with antibacterial properties, was studied in vitro for inhibition of human immunodeficiency virus (HIV) infection of MT-4 lymphocytes. Effective antiviral concentrations required for complete HIV inactivation were three orders of magnitude lower than 10 micrograms/ml cytotoxic dose. Gramicidin, routinely used as a contraceptive agent, should be considered for clinical application as a spermicide with antiviral activity.
The antiviral effect of the immunomodulating anti-cancer agent, bestatin, was examined in vitro by exposing MT-4 lymphocytes to HIV in the presence of 10-fold dilutions of drug (range 100 micrograms-100 pg/ml). The reduction in infectivity was measured by p24 ELISA and compared to the effect of established anti-HIV drugs-azidothymidine (AZT) and dextran sulfate. The results indicate that low doses of bestatin (1 microgram/ml) can completely inhibit viral infection resulting either from inoculation with free virus or coculture with infected lymphocytes. Unlike AZT or dextran sulfate, bestatin prevents HIV infection without interfering with the rate of cell growth. No appreciable decrease in HIV production was observed when chronically infected virus-producing T cell lines ie, H9, MOLT-4, HPB-ALL, 8E5 and MT-2 were treated with bestatin. Bestatin appears to act in the early stages of viral penetration, possibly through inhibition of lymphocyte-associated aminopeptidases.
An anthelminthic agent, levamisole, also known as a potent immunomodulator, has been successfully used for adjuvant therapy of malignancies and chronic infections underlined by immunodeficiency. We have tested the effect of this drug on de novo viral infection by exposing MT-4 T lymphocytes to HIV in the presence of serial ten-fold dilutions of levamisole (range 10(-3)-10(-9) M). The results indicate that 50% reduction in viral infectivity (IC50) of levamisole starts from as low as 10(-7) M, whereas even the highest millimolar dose of the drug has not shown any appreciable cytotoxicity. Although the mechanism of levamisole action remains unknown, our observation in vitro suggests that levamisole, a clinically established immunomodulator, can be potentially effective for treatment of HIV infection.
OBJECTIVE: To determine whether maternal transmission of human immunodeficiency virus (HIV) is correlated with increased quantities of HIV, decreased frequencies of CD4+ T cells, or increased levels of CD8+ T cells in the transmitting mother. METHODS: Peripheral blood obtained from HIV-infected women at different times during pregnancy was used to measure quantitative cell-associated HIV-1 and CD3+CD4+ and CD3+CD8+ proportions; the plasma was used to perform measurements of quantitative viremia by culture and subsequently to measure quantitative HIV-1 ribonucleic acid levels. These measurements were analyzed with respect to their association with HIV transmission to the baby, which occurred in one fourth of the cases. The children were also studied to determine whether HIV-1 was detected near birth or not until 1 to 8 weeks of life. RESULTS: Increased clonal frequencies of HIV-1-infected peripheral blood mononuclear cells were found in mothers of infected children; fivefold fewer cells were required for a positive culture result (median cell numbers of 10(4.5) vs 10(5.2); p = 0.008). Higher frequencies of infected cells were seen in mothers of babies with evidence of infection at birth than in mothers of infected babies without evidence of infection at birth (p < 0.05). Plasma viremia was measured in 10% of cultures without regard to whether the mothers transmitted virus to their babies. Increased levels of ribonucleic acid as detected by the branched-chain DNA method were measurable more often (45% vs 17%) in the mothers of infected children than in mothers of uninfected children. Proportions of CD4+ and CD8+ T cells were indistinguishable in these two groups of women. CONCLUSIONS: Increased viremia was present in mothers who transmitted HIV to their offspring. This variable could be used to select women at highest risk of transmitting HIV to their offspring for treatment to decrease the HIV burden five-fold.
To evaluate the efficacy of primary chemoprophylaxis in preventing Pneumocystis carinii pneumonia (PCP) in infants with perinatal human immunodeficiency virus-1 infection during the first year of life, we conducted a retrospective chart review of infants with human immunodeficiency virus-1 infection born at New York University Medical Center-Bellevue Hospital Center, in New York. Between March 1989 and March 1993, 24 infants received primary chemoprophylaxis with trimethoprim-sulfamethoxazole in the first year of life and 24 infants did not receive primary prophylaxis. The CD4+ T-lymphocyte counts in the two groups did not differ during the first year of life. The median age at the time of initiation of prophylaxis was 3 months, and the average duration of prophylaxis was 5.5 months. Among the infants who had not received prophylaxis, five cases of PCP were diagnosed at a median age of 5 months; in contrast, no cases of PCP were observed in the infants receiving prophylaxis (log-rank test, p = 0.017). The probability of surviving after 1 year of age was 92% for the children who received prophylaxis and 74% for those who did not (log-rank test, p = 0.035). These data indicate that chemoprophylaxis is highly effective in preventing primary PCP and improving survival time in infants with human immunodeficiency virus-1 infection.
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The effect of zidovudine therapy on human immunodeficiency virus (HIV)-specific antibody production was studied in 64 HIV-1-infected infants and children > 6 months old. HIV-specific in vitro antibody production (IVAP) was measured in cultures of peripheral blood mononuclear cells (PBMC). IVAP decreased in 85% of children after zidovudine was initiated (mean decline, 1 log within 2 months). Effects were seen as early as 1 week after starting zidovudine. No change in IVAP was seen in children not treated. In comparison, plasma core (p24) antigen levels declined and CD4+ lymphocytes increased in only 42% and 52%, respectively, of treated subjects. Thus, the production of antibody to HIV-1 decreases rapidly after the initiation of antiretroviral therapy. This response to therapy may provide a simple and sensitive method of monitoring antiretroviral therapy.
The purpose of this study was to characterize systemic Streptococcus pneumoniae disease in human immunodeficiency virus type 1 (HIV-1)-infected children. All cases of bacteremia and meningitis caused by S. pneumoniae among children less than 18 years old were collected by review of the Microbiology Laboratory records at the Bellevue Hospital Center during the period August 1, 1978, through July 31, 1993. There were 31 bouts of systemic S. pneumoniae disease in 19 of 235 HIV-1-infected children cared for by the Pediatric Infectious Disease staff and 116 bouts in 113 children not known to be HIV-1-infected. Four of the 19 HIV-1-infected children had multiple episodes of S. pneumoniae bacteremia as compared with 3 of 113 in the general population (P = 0.008). The frequency of serotypes and distribution of infections by season of the year did not differ between the 2 groups. The median ages at the time of the S. pneumoniae infection were 1.8 and 1.1 years for the HIV-1-infected children and the general population of children, respectively, when those children with multiple episodes were included for their initial episode only (P = 0.06). In the HIV-1-infected patients, 10 episodes were associated with pneumonia, 5 with pneumonia and otitis media, 5 with otitis media only, 1 with pneumonia and meningitis, 1 with meningitis only and 1 with periorbital cellulitis; 5 had no apparent focus of infection. One episode of pneumonia was complicated by lung abscess and there were 2 deaths. Most HIV-1-infected patients recovered without significant sequelae, and the clinical course of their systemic infections did not appear to be markedly different than that of healthy children.
The purpose of this study was to determine whether soluble CD8 (sCD8) in serum of perinatally human immunodeficiency virus 1 (HIV-1)-infected children during the first year of life differs from that of HIV-1-uninfected control children. Soluble CD8 concentrations in stored plasma and serum samples of children of HIV-1-infected and uninfected mothers were determined using a sandwich immune assay. In the first month of life significantly greater concentrations of sCD8 occurred in 12 HIV-1-infected infants than in 9 uninfected infants born to infected mothers (mean = 1054, SD 540 vs. 589, SD 370 units/ml, P < 0.05), although the CD8+ T cell proportions were not different (21.7 vs. 21.1, P > 0.5). The difference in sCD8 concentrations was most pronounced in 8 infants who were HIV-1 culture positive on initial testing in the first week of life compared with the remaining 4 patients when virus was first detected on subsequent analysis (mean = 1315, SD 446 vs. 529, SD 231 units/ml, P < 0.01). The concentration of sCD8 was also greater in 26 HIV-1-infected children than in either 26 uninfected children born to infected mothers or 25 seronegative children during the first year of life (mean = 1268, SD 529 vs. 630, SD 290 vs. 553, SD 315 units/ml, P < 0.05). Early and persistent elevation in sCD8 probably reflects immune activation resulting from HIV-1 infection. The occurrence of this increase in the neonatal period may reflect prenatal viral transmission, a higher viral inoculum or coinfection with other agents stimulating immune activation.
Virologic and immunologic studies were performed on five patients presenting with primary human immunodeficiency virus type 1 (HIV-1) infection. CD8+ cytotoxic T lymphocyte (CTL) precursors specific for cells expressing antigens of HIV-1 Gag, Pol, and Env were detected at or within 3 weeks of presentation in four of the five patients and were detected in all five patients by 3 to 6 months after presentation. The one patient with an absent initial CTL response had prolonged symptoms, persistent viremia, and low CD4+ T-cell count. Neutralizing antibody activity was absent at the time of presentation in all five patients. These findings suggest that cellular immunity is involved in the initial control of virus replication in primary HIV-1 infection and indicate a role for CTL in protective immunity to HIV-1 in vivo.
OBJECTIVE: The age-related changes in the proportion of CD4 and CD8 lymphocytes in human immunodeficiency virus (HIV)-seronegative children born to HIV-infected mothers (seroreverters) were compared with the changes in these lymphocyte subsets in children born to seronegative women to assess a possible effect of exposure to HIV without infection. DESIGN: There were 146 seroreverter and 72 seronegative children. The median CD4 and CD8 percentages for each of these two groups of children were compared retrospectively at 3-month intervals from birth through 27 months and at a tenth interval for the time beyond 27 months. The weighted average of the within-subject rate of change of CD4 and CD8 percentages were also compared between the two groups. Finally, for each subject, the proportion of the subject's CD4 percentage assays which were <10th percentile of the entire study population (30%) was calculated, and the distributions of the subject-specific proportions were then compared between the seronegative and seroreverter groups using the Wilcoxon rank sum test. The proportion of CD8 assays <10th percentile (12%) or > 90th percentile (26%) were also computed for each subject, and the distributions of the proportions were compared similarly. . RESULTS: The median CD4 percentage of seroreverter children was lower than that for the seronegative children at every interval from birth through 27 months and for the last interval for values obtained at greater than 27 months, although the comparison was statistically significant only at the 4- to 6-month period. The weighted average of the within-subject rate of change of CD4 percentage was -0.09 and -3.0 per year (P = .04), and of CD8 percentage was 1.3 and 1.0 (P = .67), for the seroreverter and seronegative children, respectively. There were significantly more children in the seroreverter group than in the seronegative group who had repeated assays in which the CD4 percentage was < 10th percentile for age (P < .00005). In addition, there was a subset of 10 seroreverter children (6.8%) who had CD4 percentages < 30% on > 50% of their assays, as compared with only one (1.4%) seronegative child. The proportion of CD8 assays < 10th percentile or > 90th percentile were not significantly different between the two groups of children. CONCLUSIONS: The CD4 proportions were persistently lower in the seroreverter than in the seronegative population, although only reaching statistical significance in 1 of 10 3-month intervals. This finding may be due to a subgroup of seroreverter children who have persistently low CD4 lymphocyte percentages.
Most human megakaryocytes (MGKs) express the CD4 antigen on their surface. Approximately 25% have a CD4 receptor density comparable to that of CD4+ T cells (Basch et al, Proc Natl Acad Sci USA 87:8085, 1990). In these studies, we show: (1) the presence of mRNA for CD4 in human MGKs; (2) the binding of human immunodeficiency virus-1 (HIV-1) to human MGKs; (3) the inhibition of binding by anti-CD4 (Leu3a) antibody or rCD4; (4) the infection of a human MGK line, CHRF-288 with HIV-1; and (5) inhibition of infection with anti-CD4. Human MGKs have mRNA for CD4 as shown by in situ hybridization with an RNA probe synthesized from a 3-kb cDNA sequence of plasmid pSP65.T4.8 containing the full-length CD4 sequence. MGKs (23% +/- 17%) bound HIV-1, as determined by anti-gp120 and anti-CD41 staining. Binding to human MGKs could be inhibited 55% to 75% with anti-CD4 or rCD4, respectively. Infection of a CD4+ MGK line (CHRF-288) could be accomplished with HIV-1, as determined by proviral DNA polymerase chain reaction and p24 production. Preincubation with anti-CD4 inhibited apparent proviral DNA infection by 100% and p24 production by 65% to 70%. Thus, human MGKs have a CD4 receptor capable of binding HIV-1. Using this receptor, HIV-1 can infect cells representative of the MGK lineage.
The early serologic response of infants to infection with human immunodeficiency virus type 1 (HIV-1) is normally obscured by the presence of transplacentally acquired maternal HIV antibody. By measuring HIV antibody produced in vitro by lymphocytes isolated from peripheral blood of infants and children of HIV-1-infected mothers, we have been able to study the natural acquisition of humoral immunity to perinatal HIV-1 infection. One hundred ninety-seven infants of HIV-1-infected women were studied prospectively and longitudinally from birth. In the neonatal period, infected infants produced only small amounts of HIV-specific IgG antibodies to a restricted number of antigens. The amount of immunoglobulin to HIV-1 and the number of HIV-1 antigens recognized increased with age. After 6 months of life 85% of infected infants made detectable antibody to two or more viral proteins. Antibody to gp160 appeared first and was the most frequently found at all ages, followed by antibody to the envelope proteins gp120 and gp41. The amount of HIV antibody produced correlated positively with the percentage of CD4+ T lymphocytes in peripheral blood. This assay provides a method of studying the immunogenicity of vaccines against HIV-1 in HIV-1-infected infants and of assessing the effect of early therapeutic interventions on the humoral response to HIV-1.
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A distinct clinical syndrome of cholestasis and hepatitis occurred during early infancy in seven infants with perinatally acquired human immunodeficiency virus 1 infection. In five infants hepatitis was the first manifestation of human immunodeficiency virus 1 infection. The median age of onset of hepatitis was 7 months (range, 5 to 10 months). The mean total bilirubin concentration at presentation was 7.4 mg/dl (range, 3.9 to 11 mg/dl), the mean aspartate aminotransferase was 1512 IU/liter (range, 782 to 2960 IU/liter) and the mean alanine amino-transferase 512 IU/liter (range, 92 to 1247 IU/liter). The absolute CD4 count at the time of onset of hepatitis ranged from 191 to 2298 cells/mm3 (mean, 766 cells/mm3). Six of the seven children died within 12 weeks of onset of hepatitis, three as a result of complications of Pneumocystis carinii pneumonia, and two died of complications secondary to cytomegalovirus. In only one infant was the cause of death the direct consequence of liver failure. The seventh infant died 17 months after the onset of hepatitis of dilated cardiomyopathy. No specific etiologic agent has been identified as the cause of cholestatic hepatitis in these infants. In situ hybridization studies to detect human immunodeficiency virus 1 messenger RNA was negative in the liver tissue obtained at biopsy and autopsy in five of the samples tested.
This investigation compares the results of a new method of diagnosing HIV-1 infection in infants < 6 months of age with currently employed techniques including cocultivation, the polymerase chain reaction (PCR), serum p24 antigen, and in vitro antibody production (IVAP) measurements. The new method, called in vitro antigen (IVAG), measures p24 antigen released into culture supernatants of peripheral blood mononuclear cells that are incubated with Epstein-Barr virus (EBV). No activated donor lymphocytes or interleukin 2 (IL-2) are added to the culture. Using this technique, HIV-1 infection was detected in 15 of 17 HIV-1-infected infants < 2 months of age, including 3 of 7 infants tested at birth, and 15 of 15 HIV-1-infected infants between 2 and 6 months of age. None of 83 determinations of 15 uninfected infants were positive. These results were found to be comparable to results obtained by the traditional cocultivation technique and the polymerase chain reaction. Because of its simplicity and reduced cost, this sensitive and specific assay could be a valuable addition to the current methods of diagnosis of HIV-1 infection in young infants.
Mutations at the adenosine deaminase (ADA) locus result in a spectrum of disorders, encompassing a fulminant neonatal onset severe combined immunodeficiency (SCID) and childhood onset immunodeficiency, as well as apparently normal immune function. The extent of accumulation of the toxic metabolite, deoxyATP, correlates directly with severity of disease. We have now determined the mutations on both alleles of a child with fulminant, neonatal onset ADA- SCID and accumulation of extremely high concentrations of deoxyATP. The genotype was consistent with the severely affected phenotype. One allele carried a large deletion that arose by non-homologous recombination and included the first five exons and promoter region. The second allele carried a missense mutation (G649A) resulting in replacement of Glu217, an amino acid involved in the catalytic site, by Lys and predicting a major alteration in charge. Expression of the mutant cDNA in Cos cells confirmed that the mutation abolished enzyme activity. We have previously reported that a missense mutation at the preceding codon is similarly associated with neonatal onset ADA- SCID and accumulation of extremely high deoxyATP. These findings suggest that genotype-phenotype correlations may be apparent for ADA- SCID, despite the role that random variation in exposure to environmental pathogens may play in the initial phenotype. Such genotype-phenotype correlations may be important to consider in evaluating results of ongoing trials of "gene" and enzyme replacement therapy.