The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells requires both CD4 (ref. 1, 2) and one of a growing number of G-protein-coupled seven-transmembrane receptors. Viruses predominantly use one, or occasionally both, of the major co-receptors CCR5 or CXCR4, although other receptors, including CCR2B and CCR3, function as minor co-receptors. CCR3 appears critical in central nervous system infection. A 32-base pair inactivating deletion in CCR5 (delta 32) common to Northern European populations has been associated with reduced, but not absolute, HIV-1 transmission risk and delayed disease progression. A more commonly distributed transition causing a valine to isoleucine switch in transmembrane domain I of CCR2B (64I) with unknown functional consequences was recently shown to delay disease progression but not reduce infection risk. Although we confirm the lack of association of CCR2B 64I with transmission, we cannot confirm the association with delayed progression. Although subjects with CCR5 delta 32 defects had significantly reduced median viral load at study entry, providing a plausible explanation for the association with delayed progression, this association was not seen with CCR2B 64I. Further studies are needed to define the role of CCR2B64I in HIV pathogenesis.

The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression.

Cellular entry of human immunodeficiency virus type 1 (HIV-1) requires binding to both CD4 (ref, 1, 2) and to one of the chemokine receptors recently discovered to act as coreceptors. Viruses that infect T-cell lines to form syncytia (syncytium-inducing, SI) are frequently found in late-stage HIV disease and utilize the chemokine receptor CXCR-4; macrophage-tropic viruses are non-syncytium-inducing (NSI), found throughout disease and utilize CCR-5 (ref. 3-11). We postulated that CCR-5 gene defects might reduce infection risk in seronegative subjects and prolong AIDS-free survival in seropositive subjects with NSI but not SI virus. Homozygous (delta ccr5/delta ccr5) and heterozygous (CCR5/delta ccr5) CCR-5 deletions (delta ccr5) were found in 7 (2.7%) and 51 (19.5%), respectively, of 261 seronegative subjects from the San Francisco Men's Health Study. CCR-5/delta ccr5 genotype was identified in 33 of 172 (19.2%) nonprogressors and 25 of 234 (10.7%) progressors from the seropositive arm of this cohort. The delta ccr5 allele conferred a significant protective effect against HIV-1 infection (P = 0.001) and a survival advantage against disease progression (P = 0.02). Although both progressing and nonprogressing CCR5/delta ccr5 subjects were identified, a distinct survival advantage was shown for those with NSI virus (P < 0.0001). Thus, the protective effect of delta ccr5 against disease progression is lost when the infecting virus uses CXCR-4 as a coreceptor.

In vitro studies of the rabbit immune system. V. Suppressor T cells activated by concanavalin A block the proliferation, not the induction of antierythrocyte plaque-forming cells.

The late B-cell proliferative phase of the in vitro antibody response by rabbit spleen cells is highly susceptible to suppression by activated T cells. The in vitro antisheep erythrocyte plaque-forming cell (PFC) response by spleen cells from normal or primed rabbits can be suppressed by adding concanavalin A (Con A), Con A-prestimulated peripheral blood or spleen lymphocytes, or supernates from Con A-prestimulated peripheral blood lymphocytes. The suppression is not mediated by a direct interaction of Con A with responding cells as shown by the effectiveness of prestimulated cells. Primed spleen cultures remain sensitive to Con A suppression as late as 72 h after initiation, and the addition of Con A after 24-72 h rapidly stops the increase in the number of PFC. T cells are required for Con A addition to be effective but the suppression can be induced at a time when T-helper cells are no longer necessary. Further, the suppressive effect of Con A addition is abrogated by specific antisera to rabbit T cells. We propose that Con A activates suppressor T cells which then exert their effects on proliferating PFC or their immediate precursor B cells. The early inductive or recruitment phase of the response is probably not blocked by suppressor cells. Also, there is an apparent relationship between the number of proliferating B cells and the number of suppressor cells required. Finally, the difficulties in inducing a stimulatory effect by Con A and the prolonged period that Con A addition is suppressive suggests that the rabbit has relatively more and/or longer-lived suppressor cells than the mouse and may be a particularly useful species for studying suppressive phenomena and their mechanisms.

Rabbit blood lymphocytes may be T cells with surface immunoglobulins.

Rabbit peripheral blood lymphocytes and thymus cells do not respond to lipopolysaccharide mitogen in vitro, whereas spleen cells do. Soluble concanavalin A consistently stimulates 80 to 90 percent of rabbit peripheral blood lymphocytes, and the morphologic changes associated with such transformation may be observed within 18 hours after stimulation. Approximately 80 percent of rabbit peripheral blood lymphocytes have demonstrable immunoglobulin markers. These and other observations suggest that most rabbit peripheral blood lymphocytes are T cells with surface immunoglobulins.

Analysis of the steroid receptor coactivator 1 (SRC1)-CREB binding protein interaction interface and its importance for the function of SRC1.

The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two alpha-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligand-induced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.

New insights into the mechanism of inhibition of p53 by simian virus 40 large T antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) has been shown to inhibit p53-dependent transcription by preventing p53 from binding to its cognate cis element. Data presented in this report provide the first direct functional evidence that T antigen, under certain conditions, may also repress p53-dependent transcription by a mechanism in which the transactivation domain of p53 is abrogated while DNA binding is unaffected. Specifically, p53 purified as a complex with T antigen from mouse cells was found to bind DNA as a transcriptionally inactive intact complex, while that purified from human cells was found to bind DNA independently of T antigen and could activate p53-dependent transcription. This difference in activity may be dependent on a different interaction of T antigen with mouse and human p53 and, in addition, on the presence of super T, which is found only in transformed rodent cells. These results suggest that subtle yet important differences exist between the inhibition of p53 by T antigen in mouse and human cells. The implications of this finding with respect to SV40-associated malignancies are discussed.

p53 Stimulates TFIID-TFIIA-promoter complex assembly, and p53-T antigen complex inhibits TATA binding protein-TATA interaction.

Simian virus 40 large T antigen has been shown to inhibit p53-mediated transcription once tethered to p53-responsive promoters through interaction with p53. In this study we report that p53 stimulates transcription by enhancing the recruitment of the basal transcription factors, TFIIA and TFIID, on the promoter (the DA complex) and by inducing a conformational change in the DA complex. Significantly, we have demonstrated that T antigen inhibits p53-mediated transcription by blocking this ability of p53. We investigated the mechanism for this inhibition and found that DA complex formation was resistant to T-antigen repression when the TFIID-DNA complex was formed prior to addition of p53-T antigen complex, indicating that the T antigen, once tethered to the promoter by p53, targets TFIID. Further, we have shown that the p53-T antigen complex prevents the TATA binding protein from binding to the TATA box. Thus, these data suggest a detailed mechanism by which p53 activates transcription and by which T antigen inhibits p53-mediated transcription.

Effect of anti-alpha1-fetoprotein on alpha1-fetoprotein-producing rat tumors in vivo and in vitro.

Active or passive immunization of rats to alpha1-fetoprotein (AFP) does not consistently inhibit the growth of AFP-producing transplantable hepatomas in vivo, and anti-AFP does not kill these hepatomas in vitro. However, 3 of 14 rats in 1 experiment responded to passive immunization by reversal of tumor growth as evidenced by normalization of elevated AFP serum concentrations, and 1 of 9 rats actively immunized with rat AFP in complete Freund's adjuvant had suppressed growth of transplantable hepatoma 7777.

Transcription by RNA polymerase II in DNA-PK deficient scid mouse cells.

DNA-dependent protein kinase (DNA-PK) is involved in DNA repair but there is some evidence to suggest that it is also involved in regulating transcription. We used a pair of cell lines, SCVA2 and SC(8)-10, which are DNA-PK negative and positive respectively, in order to examine the effect of DNA-PK upon transcription. Initial experiments were performed using p53 as an activator of transcription because DNA-PK has been proposed as a candidate upstream activator of p53. It was found both in vivo and in vitro that efficient p53-dependent transcription required the presence of DNA-PK. However, phosphorylation of p53 by DNA-PK did not affect the DNA-binding ability of p53 nor its transcriptional activity when tested in vitro. Subsequent in vivo experiments suggested that a number of transcription activators functioned more efficiently in the presence of DNA-PK. Therefore DNA-PK may play a general role in regulation of transcription driven by RNA polymerase II. In addition, DNA-PK is shown to have no specific effect on p53-dependent transcription.

The characterization of non-progressors: long-term HIV-1 infection with stable CD4+ T-cell levels.

OBJECTIVE: To identify and characterize individuals with long-term HIV-1 infection who have experienced little or no progressive CD4+ T-cell loss during follow-up. PATIENTS AND METHODS: The rate of CD4+ T-cell loss (CD4 slope) was determined for each of the 290 participants in the San Francisco Men's Health Study who were seropositive at study entry in 1984. The study population was stratified, by CD4 slope, into 10 groups of 29 individuals and each group was characterized using a variety of cross-sectional and longitudinal laboratory measurements. RESULTS: Approximately 10% of the HIV-1-infected men experienced no net CD4+ T-cell loss during 78 months of follow-up. Compared with all other seropositive subjects, these 'non-progressors' were the extreme cases in a relatively continuous distribution of CD4 slopes, rather than a discrete subpopulation. Although they had no net cell loss, their mean CD4+ cell count was approximately 400 x 10(6)/l lower and their mean CD8+ cell count approximately 250 x 10(6)/l higher than seronegative subjects, suggesting early changes followed by stabilization. The CD4 slope was associated with the levels of beta 2-microglobulin, neopterin, p24 antibody, erythrocyte sedimentation rate, viral burden, and the proportion of HIV-1 isolates with tropism for the MT-2 T-cell line. Multivariate cluster analysis of these laboratory markers did not distinguish the non-progressors as a distinct subgroup. CONCLUSIONS: These findings support both a biphasic natural history and the suggestion that the broad range in HIV disease progression rates may be the result of several independent factors interacting in a variety of combinations. Recent changes in laboratory markers, known to predict both CD4+ cell loss and AIDS, suggest that non-progressors are undergoing slow HIV disease progression.

Home collection for frequent HIV testing: acceptability of oral fluids, dried blood spots and telephone results. HIV Early Detection Study Group.

OBJECTIVE: To assess the feasibility and acceptability of bimonthly home oral fluid (OF) and dried blood spot (DBS) collection for HIV testing among high-risk individuals. DESIGN: A total of 241 participants [including men who have sex with men (MSM), injecting drug users (IDU), and women at heterosexual risk] were recruited from a randomly selected subset of study participants enrolled at four sites in the HIV Network for Prevention Trials (HIVNET) cohort, and assigned at random to bimonthly home collection of OF or DBS specimens over a 6 month interval. Participants could select telephone calls or clinic visits to receive HIV test results. METHODS: Bimonthly specimens were tracked for adherence to the schedule, were evaluated for adequacy for testing, and tested using antibody assays and polymerase chain reaction (PCR) for DBS. The acceptability of bimonthly home OF and DBS collection and telephone counseling was assessed in an end-of-study questionnaire. RESULTS: The laboratory received 96 and 90% of expected OF and DBS specimens, respectively; 99% of each specimen type was adequate for testing. Almost all (95%) participants chose results disclosure by telephone. The majority of participants (85%) reported that bimonthly testing did not make them worry more about HIV, and almost all (98%) judged that with bimonthly testing their risk behavior remained the same (77%) or became less risky (21%). CONCLUSION: Bimonthly home specimen collection of both OF and DBS with telephone counseling is acceptable and feasible among study participants at high risk. These methods will be useful for the early detection of HIV infection and remote follow-up of research cohort participants in HIV vaccine and prevention trials.

Effect of hydroxy-vitamin D3 derivatives on 45Ca release from rat fetal bones in vitro.

Several hydroxy vitamin D3 (OH-D3) derivatives were tested for biological activity by measuring 45Ca release from prelabeled rat fetal bones, in vitro. In a 72 h continuous culture, 1 alpha,25-(OH)2-D3 produced a significant effect at 10 pg/ml. A similar effect was produced by 50 ng/ml of either 25-OH-D3 or 5,6-trans-25-OH-D3, and by 500 pg of 3 deoxy-1 alpha,25-(OH)2-D3. 24R,25-(OH)2-D3 was more active than 24S,25-(OH)2-D3, and the R isomer had activity that more closely resembled the biosynthetic compound. 3-deoxy-1 alpha-OH-D3 was inactive at a concentration of up to 1 microgram/ml. Using a 24 h preincubation period with no added vitamin D3 derivative, a steep dose-response curve could be obtained with 1 alpha,25-(OH)2-D3 over a range of 2-10 pg/ml during a subsequent 96 h incubation period, and 1 alpha,25-(OH)2-D3 was found to have about 5000 times the activity of 25-OH-D3.

The relationship between AIDS and immunologic tolerance.

A hypothesis is presented in which the immunodeficiency and cell loss leading to acquired immune deficiency syndrome and the clonal deletion associated with immunologic tolerance occur through a common mechanism. In a previous publication we proposed that the interaction of human immunodeficiency virus (HIV) with CD4 delivers activation signals that disrupt immune system regulation. In this article, we compare the biology of HIV infection with recent discoveries concerning the "two-signal" molecular mechanism for thymic selection. We propose that two-signal activation is normally followed by clonal expansion and the programmed death of most or all daughter cells through mechanisms that are proportional to the strength and duration of the activation signals. In the thymus, self-reactive cells are trapped in the continuous presence of both antigen signals (signal 1) and costimulatory signals (signal 2), leading to clonal deletion. In mature lymphocytes, we propose that HIV infection contributes a chronic high-affinity signal 2, which shifts the equilibrium of antigen-activated T-cell populations further toward programmed death. This leads to incremental memory cell deficits and gradual clonal deletion at a rate dependent on the frequency of antigen exposure and the ability of an HIV quasispecies to induce signal 2.

AIDS as immune system activation. II. The panergic imnesia hypothesis.

A hypothesis is presented in which HIV infection leads to immunodeficiency through indirect subversion of critical T cell regulatory mechanisms. Acting at the T cell receptor complex (TCR), viral components (gp120) mimic the natural ligands of CD4, molecules of the major histocompatibility complex (MHC), and deliver physiologically active, inappropriate signals resulting in generalized, uncontrolled lymphocyte activation, or "panergy." Clinical manifestations of panergy include autoimmune phenomena, lymphadenopathy, hyperglobulinemia, and symptoms mediated by lymphokines. Immunologic unresponsiveness occurs early in HIV infection prior to T cell depletion because activated cells do not respond to further stimulation. Ultimately, activation disrupts T cell homeostasis by interference with the generation of memory cells ("imnesia") and leads to net T cell loss, clonal deletion, and the development of AIDS. The clinical and immunologic features of HIV disease and AIDS are reviewed from this perspective. This hypothesis is consistent with the paucity of infected T cells, the clinical findings of both AIDS-related complex (ARC) and frank AIDS, the prolonged "incubation period," and a role for antigen-specific cofactors. Based on this view of HIV pathophysiology, therapeutic modalities should avoid immune stimulation and seek to block aberrant gp120 signals at CD4 and eliminate HIV-infected cells.

An evaluation of the polymerase chain reaction in HIV-1 seronegative men.

The apparent detection of human immunodeficiency virus (HIV-1) DNA by the polymerase chain reaction (PCR) in seronegative individuals has been the subject of great concern. In this study, 324 seronegative participants in the San Francisco Men's Health Study were evaluated for evidence of infection using a PCR testing algorithm with multiple amplifications targeting different regions of the HIV-1 genome. While most PCR reactions were negative, 8.6% of the specimens showed weak reactivity with one or more primer sets. However, all were negative with at least one primer set and no definitively positive specimens were identified. This study addressed the possibility that some of these PCR reactions might represent latent infection or abortive exposure, leaving residual integrated DNA, rather than false-positive reactions. The frequency of such reactions was determined in homosexual men who have been at risk for HIV-1 infection and in exclusively heterosexual men who have little or no past exposure. The results demonstrate an identical frequency and distribution of equivocal PCR reactions in both populations. Assuming that there is minimal HIV-1 infection among seronegative heterosexual men in San Francisco, we conclude that PCR testing does not provide evidence for a reservoir of occult HIV-1 infection in seronegative homosexual men.

Neutralizing antibody responses to autologous and heterologous isolates of human immunodeficiency virus.

Although laboratory-adapted strains of human immunodeficiency virus (HIV) are generally highly sensitive to neutralization by HIV-positive patient sera, we have found a more complex pattern of cross-neutralization and neutralization resistance among low-passage clinical isolates. These HIV isolates, like many other lentiviruses, resisted neutralization by the patient's own (autologous) antibodies. We assessed the degree of antigenic relatedness between different patient isolates of HIV through cross-neutralization with heterologous sera and virus isolates. Complicated patterns emerged, with variation in breadth of neutralization among individual plasmas and variation in frequency of neutralization among isolates. In longitudinal studies of individuals, we found that some but not all such patients develop a neutralizing response that "catches up" with their earlier isolates after a lag period. Taken together, these data suggest that an individual's immune response broadens with time because of cumulative exposure to multiple antigenic variants that arise throughout HIV disease.

Circulating HIV-1-infected cell burden from seroconversion to AIDS: importance of postseroconversion viral load on disease course.

The purpose of this study was to characterize quantitative changes in circulating infected cells over the natural history of human immunodeficiency virus (HIV) disease in relation to clinical/immunological outcome. HIV-1 gag DNA polymerase chain reaction (PCR) and peripheral blood mononuclear cell (PBMC) co-cultures were performed on limiting dilutions of cryopreserved PBMC from specimens collected at enrollment and after 5 years of follow-up from nine seropositive subjects classified as rapid progressors, nine intermediate progressors, and 10 nonprogressors. Limiting dilution PCR was also performed on serial pre/postseroconversion specimens from 18 seroconvertors. By quantitative DNA PCR analysis, the infected cell burden was significantly higher at enrollment in the RP [mean of 330 PCR units (PCRU)/10(6) PBMCs] than in the IP (160 PCRU/10(6) PBMCs) and NP (73 PCRU/10(6) PBMCs) groups (p = 0.05). When results were analyzed on an individual level with proportional hazard regression, baseline PCRU (p = 0.05) and CD4 slope (p = 0.0007) were significantly associated with developing acquired immune deficiency syndrome (AIDS) in 5 years, but baseline tissue culture infectious units (TCIU) was not. The increase in PCR-positive cells after 5 years was modest in all three groups (two- to fivefold), whereas the proportion of PCR-positive cells that yielded virus in culture increased significantly (21- to 31-fold) over time in all three groups. Infected cell burden in postseroconversion specimens was relatively stable within each subject, but varied greatly (from 1.6 to 1,024 PCRU/10(6) PBMCs) among subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-1 PCR and isolation in seroconverting and seronegative homosexual men: absence of long-term immunosilent infection.

The presence of detectable HIV-1 prior to the appearance of HIV-1-specific antibody was assessed in 41 incident infections that occurred during a 6-year prospective cohort study. All available antibody-negative samples (n = 138) and the first antibody positive sample (n = 41) were tested, under code, by the polymerase chain reaction (PCR) in two laboratories and by HIV-1 isolation in a third laboratory. Samples were available as long as 66 months and at least 18 months before seroconversion for 24/41 subjects. An equal number of time-matched control specimens from persistently seronegative homosexual men and 103 samples from normal blood donors were also tested under code. Samples with discordant results were subjected to coded repeat analysis along with appropriate controls. All but one of the 41 first antibody-positive specimens (97.6%) were PCR positive and 65% were isolation positive. Two control specimens from seronegative homosexual men were PCR positive and one was culture positive, but HLA typing provided clear evidence of specimen mix-up in the specimen archive. For 37/41 seroconvertors, all available antibody negative specimens were negative by both PCR and virus isolation. In three cases, the specimen obtained 6 months before seroconversion was PCR and isolation positive. One specimen, obtained 12 months before SC, was PCR positive and isolation negative but was determined to be the result of sample contamination. Both PCR and isolation were negative in this subject 6 months before SC. In conclusion, we were unable to detect immunosilent infection > 6 months before seroconversion in high-risk homosexual men.

The initial immune response to HIV and immune system activation determine the outcome of HIV disease.

A study was conducted to assess the relative contribution of the HIV-1-specific immune response and -nonspecific immune activation to HIV disease progression. The titer of antibody to the p24 core protein and the concentration of serum neopterin were measured in 238 HIV-1-seropositive subjects in a prospective cohort study of homosexual men. Antibody titers were extremely variable among cohort participants but relatively stable over time, suggesting inherent differences in the initial immune response capacity. Neopterin concentrations were also variable at cohort entry but generally increased over time. These two markers, measured at cohort entry, had powerful and independent predictive value for the development of AIDS up to 54 months before diagnosis. Subjects with low antibody titers and high levels of neopterin, had the highest incidence of AIDS (60% over 54 months). Patients with low antibody or high neopterin alone had an intermediate risk (34% incidence) and less than 10% of those with high antibody and low neopterin developed AIDS. We propose that the initial immune response to HIV and virus-mediated immune system activation are independent and innately variable components of an individual's response to HIV infection that interact to determine the clinical outcome.

Use of T lymphocyte subset analysis in the case definition for AIDS.

Infection with HIV-1 and < 200 CD4+ lymphocytes/mm3 has been proposed as an AIDS-defining condition. We have evaluated the effects of using this and other T-cell subset measurements, in the diagnosis of AIDS in two cohorts of homosexual/bisexual men in San Francisco. Among 762 HIV-1 infected men, 200 CD4+ lymphocytes/mm3 corresponded to 13 percent CD4+ lymphocytes and a CD4+/CD8+ ratio of 0.23. If these AIDS-defining criteria had been implemented in mid-1991, the number of living AIDS cases would have increased by 106 (212%), 133 (266%), and 136 (272%), respectively. When these criteria were first met, either before or in the absence of a clinical AIDS diagnosis, about half of the subjects were asymptomatic and the median clinically AIDS-free interval was approximately 2 years. Using two consecutive tests or pair-wise combinations of criteria reduced the number of cases identified by testing error or transient biological variation, but the number of living AIDS cases would still be increased more than twofold. Finally, any AIDS case definition using a specific T-cell subset value will be compromised by the inherent variability in these measurements and the substantial overlap in the results for those with and without clinical manifestations of HIV infection.