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Since the 1918 influenza pandemic, non-randomised studies and small clinical trials have suggested that convalescent plasma or anti-influenza hyperimmune intravenous immunoglobulin (hIVIG) might have clinical benefit for patients with influenza infection, but definitive data do not exist. We aimed to evaluate the safety and efficacy of hIVIG in a randomised controlled trial.

Influenza causes substantial morbidity and mortality despite available treatments. Anecdotal reports suggest that plasma with high antibody titres to influenza might be of benefit in the treatment of severe influenza.

Idiopathic CD4+ lymphocytopenia (ICL) is a rare non–HIV-related syndrome with unclear natural history and prognosis. This prospective natural history cohort study describes the clinical course, CD4 T lymphocyte kinetics, outcome, and prognostic factors of ICL. Thirty-nine patients (17 men, 22 women) 25 to 85 years old with ICL were evaluated between 1992 and 2006, and 36 were followed for a median of 49.5 months. Cryptococcal and nontuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In 32, CD4 T-cell counts remained less than 300/mm3 throughout the study period and in 7 normalized after an average of 31 months. Overall, 15 (41.6%) developed an opportunistic infection in follow-up, 5 (13.8%) of which were “AIDS-defining clinical conditions,” and 4 (11.1%) developed autoimmune diseases. Seven patients died, 4 from ICL-related opportunistic infections, within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4 but not CD8 T lymphocytes. CD8 T lymphocytopenia (< 180/mm3) and the degree of CD4 T cell activation (measured by HLA-DR expression) at presentation were associated with adverse outcome (opportunistic infection-related death; P = .003 and .02, respectively). This trial is registered at http://clinicaltrials.gov as #NCT00001319.

Idiopathic CD4+lymphocytopenia (ICL) is a rare non–HIV-related syndrome with unclear natural history and prognosis. This prospective natural history cohort study describes the clinical course, CD4 T lymphocyte kinetics, outcome, and prognostic factors of ICL. Thirty-nine patients (17 men, 22 women) 25 to 85 years old with ICL were evaluated between 1992 and 2006, and 36 were followed for a median of 49.5 months. Cryptococcal and nontuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In 32, CD4 T-cell counts remained less than 300/mm3throughout the study period and in 7 normalized after an average of 31 months. Overall, 15 (41.6%) developed an opportunistic infection in follow-up, 5 (13.8%) of which were “AIDS-defining clinical conditions,” and 4 (11.1%) developed autoimmune diseases. Seven patients died, 4 from ICL-related opportunistic infections, within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4 but not CD8 T lymphocytes. CD8 T lymphocytopenia (< 180/mm3) and the degree of CD4 T cell activation (measured by HLA-DR expression) at presentation were associated with adverse outcome (opportunistic infection-related death; P= .003 and .02, respectively). This trial is registered at http://clinicaltrials.govas #NCT00001319.

ABSTRACTAn accurate and reproducible CD4 count is a fundamental clinical tool for monitoring and treating human immunodeficiency virus infection and its complications. Two methods exist for calculating absolute CD4 counts: dual-platform technology (DPT) and single-platform technology (SPT). Numerous studies have documented the unacceptably wide range of variation in absolute CD4 counts between laboratories. SPT was introduced in 1996 to reduce the interlaboratory variation in absolute CD4 counts. The aim of this study was to compare DPT with the BD Biosciences Trucount method (an SPT method). Both the percentages of CD4 (r= 0.986; P= 0.0541) and the absolute CD4 counts (r= 0.960; P= 0.0001) had very good correlation between the two methods. However, poor correlation was observed for the CD8+RO-(r= 0.314; P= 0.0002), CD8+DR+(r= 0.666; P= 0.0138), CD3+CD38+(r= 0.8000; P= 0.0004), CD3+CD25+(r= 0.464; P= 0.0082), and CD4+CD38+(r= 0.357; P= 0.0127) measurements.

ABSTRACTThis study compared two commercially available assays for the measurement of hepatitis C virus (HCV) RNA levels, the Bayer HCV RNA (version 3.0) branched DNA assay and the Abbott HCV analyte-specific reagent real-time PCR assay, to assess their quantitative relationships, ease of performance, and time to completion. The study group consisted of randomly selected patients from the NIAID human immunodeficiency virus (HIV) outpatient clinic who were infected with HIV type 1 and HCV. One hundred eighty-four samples from 66 patients coinfected with HIV and HCV receiving treatments under various protocols were analyzed for the correlation and agreement of the results. The results indicated that the two assays correlate well in the overlapping linear ranges of the assays and show good agreement. From the results obtained, we have derived a mathematical formula to compare the viral load results between the two assays, which is given as log10Abbott assay measure = 0.032 + 1.01 log10Bayer assay measure. Although it is preferable to use the same quantitation assay throughout the course of a patient's treatment, valid comparisons of the HCV RNA levels may be made between the results obtained by either of these assays in the overlapping linear range (615 to 7,700,000 IU/ml).

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

The present study examined the safety and relative in vivo survival of genetically engineered CD4+ T lymphocytes in human immunodeficiency virus (HIV)-infected individuals. Ten pairs of identical twins discordant for HIV infection were recruited, with the uninfected twin serving as the lymphocyte donor. Ten subjects were treated with a total of 19 separate infusions of retroviral vector-transduced CD4+ enriched T cells. Control (neo gene) or anti-HIV gene (antisense trans-activation response [TAR] element and/or trans-dominant Rev)-engineered lymphocytes were monitored in peripheral blood for 3 years, using a vector-specific PCR assay. Data from 9 of the 10 patients (15 of the 19 infusions) demonstrated preferential survival of CD4+ lymphocytes containing the anti-HIV gene(s) in the immediate weeks after infusion. In six of six patients studied long term (>100 weeks), only T cells containing the anti-HIV genes were consistently detected. In addition, a marked survival advantage of anti-HIV gene-containing T cells was observed in a patient treated during a period of high viral load. Thus, these data strongly support the hypothesis that anti-HIV genes afford a survival advantage to T cells and potential benefit to HIV-1+ individuals.

ABSTRACTTo investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis.

Administration of interleukin 2 (IL-2) leads to selective and sustained CD4+ T-cell expansions in patients infected with HIV. It has been hypothesized that persistent CD4+ T-cell proliferation is the primary mechanism maintaining these expansions. T-cell proliferation was studied by ex vivo bromodeoxyuridine (BrdU) incorporation and intracellular Ki67 staining in HIV-infected patients treated with antiretroviral therapy (ART) with or without IL-2. In contrast to the tested hypothesis, HIV-infected patients treated with IL-2 had lower CD4+ T-cell proliferation compared to patients treated with ART alone. Independently of viral load changes, administration of IL-2 led to a decrease in basal CD4+ T-cell proliferation. Total numbers of CD4+ T cells with naive and recall, but not effector, memory phenotype were increased. The degree of CD4+ T-cell expansion correlated with the decreases in proliferation and a strong association was seen between these decreases and the expansion of the CD4+/CD25+ subset. Intermittent IL-2 in HIV-infected patients leads to expansions of CD4+/CD25+ T cells with naive and recall memory phenotypes that strongly correlate with decreases in proliferation. These data suggest that decreased T-cell proliferation is central in the CD4+ T-cell expansions induced by IL-2.

Administration of interleukin 2 (IL-2) leads to selective and sustained CD4+T-cell expansions in patients infected with HIV. It has been hypothesized that persistent CD4+T-cell proliferation is the primary mechanism maintaining these expansions. T-cell proliferation was studied by ex vivo bromodeoxyuridine (BrdU) incorporation and intracellular Ki67 staining in HIV-infected patients treated with antiretroviral therapy (ART) with or without IL-2. In contrast to the tested hypothesis, HIV-infected patients treated with IL-2 had lower CD4+T-cell proliferation compared to patients treated with ART alone. Independently of viral load changes, administration of IL-2 led to a decrease in basal CD4+T-cell proliferation. Total numbers of CD4+T cells with naive and recall, but not effector, memory phenotype were increased. The degree of CD4+T-cell expansion correlated with the decreases in proliferation and a strong association was seen between these decreases and the expansion of the CD4+/CD25+subset. Intermittent IL-2 in HIV-infected patients leads to expansions of CD4+/CD25+T cells with naive and recall memory phenotypes that strongly correlate with decreases in proliferation. These data suggest that decreased T-cell proliferation is central in the CD4+T-cell expansions induced by IL-2.

ABSTRACTMore sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 106per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV+) patients receiving highly active antiretroviral therapy (HAART), and HIV+ patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4+/CD25+ T cells. Increased CD25 expression, especially in CD4+ T cells but also in CD8+ T cells, without increases in expression of the ß and ? chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4+ T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4+/CD25+ T cells correlated positively with both the total and naive CD4+ T-cell counts. A discrete population of CD45 double intermediate RA+/RO+CD4+ cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4+ count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4+/CD25+ and CD4+/CD25- cells from IL-2–treated HIV+ patients proliferated in response to mitogens, specific antigens, and T-cell-receptor–mediated stimuli. Thus, intermittent administration of IL-2 in HIV+ patients leads to preferential expansion of a unique subset of CD4+ T cells that may represent a critical population in T-cell homeostasis.

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV+) patients receiving highly active antiretroviral therapy (HAART), and HIV+patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4+/CD25+T cells. Increased CD25 expression, especially in CD4+T cells but also in CD8+T cells, without increases in expression of the β and γ chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4+T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4+/CD25+T cells correlated positively with both the total and naive CD4+T-cell counts. A discrete population of CD45 double intermediate RA+/RO+CD4+cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4+count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4+/CD25+and CD4+/CD25−cells from IL-2–treated HIV+patients proliferated in response to mitogens, specific antigens, and T-cell-receptor–mediated stimuli. Thus, intermittent administration of IL-2 in HIV+patients leads to preferential expansion of a unique subset of CD4+T cells that may represent a critical population in T-cell homeostasis.

Treatment advances have led to dramatic clinical improvements for patients with HIV-1 infection. These clinical improvements reflect treatment-related improvements in immune function, which are most striking in individuals who develop exaggerated immune inflammatory responses to occult opportunistic infections. The mechanisms accounting for these exaggerated immune responses are unknown. To gain insight into these mechanisms, we intensively studied a subject untreated for disseminated tuberculosis and HIV-1 coinfection who then began treatment for both diseases. We examined the changing frequencies of Mycobacterium tuberculosis (MTB)-specific CD4+ T cells that produced interferon γ (IFN-γ) after short-term stimulation with MTB antigen, and we compared these frequencies with those in HIV-1-seronegative subjects with and without prior exposure to MTB antigens. For the HIV-1/MTB-coinfected subject, the proportion of peripheral blood CD4+ T cells expressing MTB-specific IFN-γ was 8.6% at 11 days, 11% at 33 days, and 33% at 95 days after starting treatment for HIV-1. CD4+ IFN-γ+ T cells had a CD45RA-CD62L- (effector memory) phenotype and most coexpressed interleukin 2. Median frequencies of CD4+ IFN-γ+ T cells from six subjects without and nine subjects with prior exposure to MTB antigens were 0.06 and 0.46%, respectively. We conclude that individuals starting treatment for disseminated tuberculosis and HIV-1 coinfection can accumulate remarkably large numbers of MTB-specific CD4+ T cells in the peripheral blood. The rapid expansion of antigen-specific effector CD4+ T cells is one mechanism to explain immediate improvements in clinical immunity after HIV-1 treatment. This mechanism provides a theoretical framework to understand the unusual inflammatory responses recently reported to occur after starting HIV-1 treatment.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = −0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

To characterize the immunological effects of intermittent IL‐2 therapy, which leads to selective increases in CD4+T lymphocytes in HIV‐infected patients, 11 patients underwent extensive immunological evaluation. While IL‐2 induced changes in both CD4+and CD8+cell number acutely, only CD4+cells showed sustained increases following discontinuation of IL‐2. Transient increases in expression of the activation markers CD38 and HLA‐DR were seen on both CD4+and CD8+cells, but CD25 (α chain of the IL‐2 receptor) increased exclusively on CD4+cells. This increase in CD25 expression was sustained for months following discontinuation of IL‐2, and was seen in naive as well as memory cells. IL‐2 induced cell proliferation, but tachyphylaxis to these proliferative effects developed after 1 week despite continued IL‐2 administration. It thus appears that sustained CD25 expression selectively on CD4+cells is a critical component of the immunological response to IL‐2, and that intermittent administration of IL‐2 is necessary to overcome the tachyphylaxis to IL‐2‐induced proliferation.

The ability of IL-2 to induce expansion of the CD4+T lymphocyte pool has made it the most studied cytokine in the treatment of HIV infection. The majority of trials have used an empirical regimen of 5-day IL-2 cycles given every 8 weeks—a regimen based upon early pharmacodynamic studies and patient preference. To better define optimal duration and frequency of cycles, a randomized trial was conducted in which patients who received this “standard” regimen were compared to patients who received cycles of variable duration (based on individual patterns of cell cycle progression) and to patients who received cycles of variable frequency (based on individual CD4+T lymphocyte responses to previous cycles). Twenty-two patients with HIV-1 infection and CD4+T lymphocyte counts > 200 cells/mm3were randomized to one of three treatment groups for 32 weeks of study. Eight participants received four 5-day IL-2 cycles (controls) every 8 weeks; 7 participants received four cycles of longer duration (mean 7.7-days); and 7 participants received an increased frequency of 5-day cycles (every 4.1 weeks on average). All three groups experienced significant increases in mean CD4+T lymphocytes. However, there were no statistically significant differences in CD4+T lymphocyte increases between the group that received longer cycles (median increase 239 cells/mm3, P= 0.78) or between the group that received more frequent cycles (median increase 511 cells/mm3, P= 0.54) and the control group (median 284 cells/mm3). HIV-1 viral loads decreased during the study period in all three groups. Our inability to demonstrate a significant advantage of increased frequency or duration of IL-2 administration provides corroborating experimental evidence for the use of an IL-2 regimen consisting of 5-day cycles administered no more frequently than every 8 weeks in future clinical trials aimed at expanding the CD4+T lymphocyte pool.

ABSTRACTThe combination of an amino acid deletion at codon 67 (?67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the ?67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3'-azido-3'-deoxythymidine (AZT)-resistant variant containing ?67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without ?67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the ?67+T69G/K70R/L74I/K103N/T215F/K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the ?67 deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2',3'-dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the ?67 deletion led to a virus with improved replication and high-level AZT resistance.

To study human immunodeficiency virus (HIV)–specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-ζ) between identical twins discordant for HIV infection. Single and multiple infusions of 1010 genetically modified CD8+ T cells resulted in peak fractions in the circulation of approximately 104 to 105modified cells/106 mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4+ and CD8+ T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 103 to 104 modified cells/106 total CD4+ or CD8+cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen–specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4+ and CD8+ T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4+ T cells in providing “help” to cytotoxic effectors.

Disturbances in inflammatory cytokine production and immune regulation coupled with human herpesvirus-8 (HHV-8) infection underlie the current understanding of the pathogenesis of Kaposi's sarcoma (KS), the most common HIV-associated malignancy. The low affinity Fc gamma receptors (Fc?R) for IgG link humoral and cellular immunity by mediating interaction between antibodies and effector cells, such as phagocytes and natural killer cells. We examined the frequency of polymorphic forms of the low affinityFc?Rs, Fc?RIIA,Fc?RIIIA, and Fc?RIIIB in 2 cohorts of HIV-infected men with KS and found that theFc?RIIIA genotype exerts a significant influence on susceptibility to or protection from KS. The FF genotype was underrepresented in patients with KS, whereas the VF genotype was associated with development of KS. A similar association was observed between Fc?RIIIA genotypes and HHV-8 seropositivity. These observations suggest a possible role forFc?RIIIA in the development of KS during HIV infection.

Disturbances in inflammatory cytokine production and immune regulation coupled with human herpesvirus-8 (HHV-8) infection underlie the current understanding of the pathogenesis of Kaposi’s sarcoma (KS), the most common HIV-associated malignancy. The low affinity Fc gamma receptors (FcγR) for IgG link humoral and cellular immunity by mediating interaction between antibodies and effector cells, such as phagocytes and natural killer cells. We examined the frequency of polymorphic forms of the low affinityFcγRs, FcγRIIA,FcγRIIIA, and FcγRIIIBin 2 cohorts of HIV-infected men with KS and found that theFcγRIIIAgenotype exerts a significant influence on susceptibility to or protection from KS. The FF genotype was underrepresented in patients with KS, whereas the VF genotype was associated with development of KS. A similar association was observed between FcγRIIIAgenotypes and HHV-8 seropositivity. These observations suggest a possible role forFcγRIIIAin the development of KS during HIV infection.

Study Objective. To evaluate the effect of interleukin‐2 (IL‐2) infusions on the pharmacokinetics of indinavir in patients infected with the human immunodeficiency virus.

A novel model was developed to characterize the time-varying clearance of recombinant interleukin-2 (IL-2). Sixty-eight patients with human immunodeficiency virus infection received 83 cycles of IL-2 either by continuous infusion or by subcutaneous injection for 5 days. IL-2 concentrations after intravenous infusions peaked at 24 hours and then declined by 55% to 78% during the remainder of the infusion. Soluble IL-2 receptors increased greater than 10-fold before gradually returning to baseline. Subcutaneous administration showed a dose-dependent decrease in area under the concentration-time curve (AUC) between days 1 and 5. A model was developed in 9 patients who had IL-2 concentrations and soluble IL-2 receptors determined by ELISA. Concentrations were fitted by an indirect stimulatory pharmacodynamic model. An additional 59 patients with only IL-2 concentrations were fitted to a simplified empiric model. Both models provided an overall r2of 0.99 for the plot of observed versus fitted concentrations. The time-dependent increase in IL-2 clearance, likely receptor-mediated, was well described with use of an indirect-effects pharmacokinetic-pharmacodynamic model.

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs) has been demonstrated to participate in the oxygen dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied—sodium azide, potassium cyanide, and 3-aminotriazole—differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.

The CD4+T-cell pool in HIV-infected patients is in a constant state of flux as CD4+T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+T-cell population relative to the CD4/CD45RO+population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+T cells persist in vivo for weeks to months and that the CD4+T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.

Chronic granulomatous disease (CGD) is a genetically heterogeneous syndrome characterized by a microbial killing defect of polymorphonuclear leukocytes (PMNs) due to lack of superoxide O2generation. Recent studies indicate that the neutrophil O2-generating system consists of at least two components, flavoprotein—flavin adenine dinucleotide (FAD)— and cytochrome b. We evaluate the cytochrome b and FAD content in PMN from 30 CGD patients. The method for quantitating cytochrome b was modified by using PMN sonicates incubated with azide plus hydrogen peroxide. With this approach, several absorption peaks corresponding to myeloperoxidase and eosinophil peroxidase, which overlap with peaks of cytochrome b, were obliterated from reduced-minus-oxidized spectra, whereas the peaks of cytochrome b were not and could be readily quantitated. Cytochrome b was detected in PMNs from all 24 normal adults (47.4 ± 2.9 pmol/7.5 × 106cells), was absent in PMNs from 11 male CGD patients and one female CGD patient but was present in normal amounts in PMNs from nine male and nine female CGD patients. Stimulated nitroblue tetrazolium (NBT) tests performed on PMNs from mothers of CGD patients indicated that cytochrome b deficiency was associated with X-linked inheritance, except in one case in which probable autosomal recessive inheritance was demonstrated. The PMN NBT test of the mother of another male patient without cytochrome b deficiency suggested an X-linked form of inheritance. In related studies, the FAD content in PMN particulate fractions was reduced in 4 of 28 CGD patients studied. All four CGD patients with reduced FAD lacked cytochrome b. However, three patients with cytochrome b deficiency had normal FAD. Thus, the results indicate that PMN cytochrome b deficiency is observed in most X-linked and in some autosomal recessive CGD, that cytochrome b deficiency may be associated with FAD deficiency, and that cytochrome b and FAD are normal in most patients with non-X-linked CGD.

To examine the relationship between binding of surface-active stimuli to human neutrophils and activation of the superoxide (O2. ) and peroxide (H2O2) forming oxidase(s) of these cells, oxygen (O2) consumption and O2. and H2O2release were measured following stimulation by plant lectins. Both concanavalin A (Con-A) and phytohemagglutinin (PHA) were more active than other lectins in stimulating the respiratory activity of neutrophils. When the cells were pretreated with cytochalasin B (Cyto-B), recovery of extracellular O2and H2O2was increased by 3-10-fold, as found previously with particulate stimuli. Whereas cytochalasin B reduced O2consumption by cells triggered with particulate stimuli, cytochalasin B treatment of cells produced more than a threefold increase in 02consumption by cells triggered with plant lectins. The stimulatory effect of cytochalasin B on the respiration of Con-A- or PHA-treated cells was rapid, with an enhancement observed within 60 sec. With Con-A-stimulated cells, it could be inhibited by alpha-methyl-D-mannoside (AMM), indicating that it was specifically due to an activity mediated by the lectin as opposed to some other action of cytochalasin B. To correlate these events with binding of the lectin to the cells, 3H-Con-A was employed. Pretreatment of cells with Cyto-B increased 3H-Con-A binding in direct parallel to the stimulation of respiration. Similar results were found when the Cyto-B was added in the middle of an incubation period. Both the increased binding and respiratory activity of the cytochalasin-B-treated cells could be inhibited by the simultaneous addition of AMM. These findings support the concept that the degree of activation of the neutrophil oxidase(s) is a direct function of the extent of binding of the initial stimulus. They also suggest that on-going stimulus binding is a prerequisite for continued respiratory activity. The precise nature of the lectin binding sites “uncovered” in Cyto-B-treated cells and their potential relationship to sites of oxidase activity or regulation remains to be determined.

Changes in CD4+T-cell surface marker phenotype and antigen receptor (TCR) repertoire were examined during the course of HIV infection and following therapy. A preferential decline in naive CD4+T cells was noted as disease progressed. Following protease inhibitor therapy, naive CD4+T cells increased only if they were present before initiation of therapy. Disruptions of the CD4+TCR repertoire were most prevalent in patients with the lowest CD4+T-cell counts. Antiviral or IL-12 therapy-induced increases in CD4+T-cell counts led to only minor changes in previously disrupted repertoires. Thus, CD4+T-cell death mediated by HIV-1 infection may result in a preferential decline in the number of naive CD4+T cells and disruptions of the CD4+T-cell repertoire that are not immediately corrected by antiviral or immune-based therapies.

Our objective was to map serial patterns of Western blot reactivity over time of a cohort of initially ELISA-negative, Western blot-indeterminate individuals from a high-risk group and to determine if these individuals were at increased risk of harboring occult HIV-1 infection. A 2-year prospective study used serial ELISA, two types of Western blot, immunologic profiles, HIV-1 culture, and analysis by polymerase chain reaction. Subjects were 20 ELISA-negative, Western blot indeterminate homosexual volunteers and 20 matched seronegative controls. Results showed that 19 of 20 study subjects completed a mean of 17.0 months of clinical and laboratory follow-up. Reactivities with p24 and/or with p55 were the two most commonly observed Western blot patterns, occurring in 70% of individuals. Specific Western blot reactivity was dependent upon the particular immunoblot preparation being used and varied considerably on a longitudinal basis. No individual pattern appeared predictive of an increased likelihood of subsequent seroconversion to HIV-1 relative to controls. By all other criteria including polymerase chain reaction analysis, samples from 17 of 19 individuals remained negative for HIV-1 at each time point. Two individuals evolved from an indeterminate to a positive Western blot and, simultaneously, from a negative to a positive polymerase chain reaction analysis, during follow-up. Our conclusions were as follows. ELISA-negative, Western blot-indeterminate individuals from a high-risk group show marked variability in immunoblot findings over time, and these patterns do not appear predictive of an increased likelihood of infection. Polymerase chain reaction analysis is clearly of value in providing confirmation of the low probability of infection in this group, although in patients who do become infected, detection by this test may not always precede diagnosis by serologic methods.

An lgG1monoclonal antibody, 31D8, that recognizes normal neutrophil (PMN) membranes, was used to study PMN from patients with chronic myelogenous leukemia (CML). Nineteen patients with Philadelphia chromosome positive CML were followed over a ten-month period and compared with 23 normals, six patients with leukemoid reactions, and eight patients with phagocytic cell defects. The percentage of PMN binding of 31D8 among normal subjects was variable about a normal distribution with an average of 95 ± 2% of cells binding 31D8. In contrast, there were two groups of CML patients: in 14 patients 88 ± 3% PMN bound 31D8 while in the remaining five patients only 6 ± 6% PMN bound 31D8. PMN 31D8 binding was normal in the control patient groups. Control antibodies 7C3 (binds to PMN precursors) and OKM1 (binds to the CR3 (iC3b) receptor) bound normally to CML neutrophils. Functionally, CML cells had normal chemotaxis to several stimuli and normal superoxide generation to phorbol myristate acetate. However, superoxide production in response to fmet-leu-phe was significantly less in 31D8 negative CML PMN than both 31D8 positive CML PMN and normal PMN which contained 85% 31D8 positive and 15% 31D8 negative PMN. Clinically, 2 of 14 CML patients with 31D8 positive PMN were in blast crisis (one extramedullary) at the time of study and the other 12 patients remained clinically stable in the chronic phase during the ten months of study. In contrast, one of five patients with 31D8 negative PMN was in blast crisis at the time of study and all four of the remaining patients progressed to either the accelerated phase or blast crisis. Three of these patients died of their disease eight to ten months after their initial study. Thus, failure of CML cells to bind 31D8 may be useful for predicting which patients are likely to progress to the accelerated phase or blast crisis.

ABSTRACTDelayed processing of peripheral blood or peripheral blood mononuclear cell isolation and cryopreservation can lead to the detection of somewhat higher levels of CD31 expression on nai¨ve CD4 T cells by flow cytometry. These observations should be considered in the planning of multicenter clinical trials and in the interpretation of the results of functional studies.

ABSTRACTDecreases in the detection of CD127 expression on T cells of human immunodeficiency virus-infected patients by flow cytometry can occur by delayed processing or by peripheral blood mononuclear cell isolation and cryopreservation. These observations should be considered in the interpretation of functional studies and the planning of multicenter clinical trials.

ABSTRACTThis study evaluated correlation and agreement between version 3 of the Quantiplex human immunodeficiency virus type 1 (HIV-1) RNA assay (v3 branched DNA [bDNA]) and a sensitized Amplicor HIV-1 Monitor assay (reverse transcription [RT]-PCR) for the measurement of HIV RNA. Three hundred eighteen samples from 59 randomly selected, HIV-1-seropositive persons on various drug protocols from the National Institute of Allergy and Infectious Diseases HIV outpatient clinic were studied. The results indicate that v3 bDNA and RT-PCR are highly correlated (r= 0.98) and are in good agreement (mean difference in log10copies/ml ± 2 standard deviations = 0.072 ± 0.371). The relationship between values obtained by both assays is given by the following equation: log10v3 bDNA = -0.0915 + 1.0052 · log10RT-PCR. This represents a 1.026-fold difference between log10RT-PCR values and log10v3 bDNA values.

Random migration, chemotaxis, phagocytosis, and bactericidal ability of neutrophils from 5 patients receiving lithium carbonate were compared with those of neutrophils from healthy donors. These cells functioned normally in all respects. Neither sera from patients receiving lithium carbonate nor the addition of lithium chloride to control cells in vitro significantly altered their functional capacity. These findings suggest that neutrophil function in patients receiving lithium therapy is preserved, and they support the potential utility of this drug as a leukopoietic agent in neutropenic states.

The safety and efficacy were assessed of 5-day cycles of subcutaneous (sc) interleukin-2 (IL-2) every 8 weeks in human immunodeficiency virus type 1-infected outpatients with > 200 CD4 cells/ mm3. Immunologic, virologic, and toxicity parameters were measured in 18 patients receiving standard antiretrovirals plus 5-day courses of sc IL-2 (3–18 MIU/day) every 2 months. Systemic toxicities established the maximally tolerated dose (MTD) of IL-2 as 15 MIU/day. CD4 cell responses appeared to correlate directly with baseline CD4 cell counts, with several patients experiencing a dramatic rise after 3 cycles. Virus load increased only transiently in the peri-injection period. It was concluded that serial cycles of outpatient sc IL-2 can be administered safely, with an MTD of 15 MIU/day. Patients with higher baseline counts appear to have a greater CD4 cell response to sc IL-2 therapy.

Single and multiple doses of sCD4-PE40, a soluble recombinant fusion toxin selectively toxic to gp120-expressing cells, were evaluated in persons infected with human immunodeficiency virus type I (HIV-I). Seventeen of 24 patients who completed a single-dose safety trial were given either 1,5, 10, or 15 µg/kg of sCD4-PE40 by intravenous bolus once a month for 2 months, then weekly for 6 weeks. The weekly maximally tolerated dose was 10 µg/kg. The major toxicity was a transient dose-dependent elevation in hepatic aminotransferases peaking 48 h after infusion. Anti-Pseudomonas exotoxin antibody developed in 58% of recipients, and sera from 13 of 17 showed neutralizing activity against sCD4-PE40. No consistent changes in immunologic or virologic markers were observed. Weekly infusions of ? 10 µg/kg of sCD4-PE40 are generally well tolerated, but additional studies correlating optimal dosing and frequency of administration with efficacy will be needed to define the role of this novel agent in the management of HIV-1-infected patients.

Twenty-four patients infected with human immunodeficiency virus type 1 (HIV-1) who had CD4+ counts of O.2–0.5 × 109 cells/l received granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with zidovudine plus escalating doses of daily subcutaneous interferon-α. Mean neutropenia-inducing doses of interferon-α were 9.4 × 106 and 10.6 × 106 IU/day for groups receiving 100 or 200 mg zidovudine every 4 h, respectively. Mean GM-CSF doses used to reverse neutropenia were 0.64 and 0.63 µg/kg/day for these two groups, respectively, although the mean minimum effective GM-CSF dose for both was only 0.30 µg/kg/day. Serum p24 antigen declined >70% in all 5 antigenemic patients. Toxicities included a dose-dependent increase in lymphokine-like side effects (100%), anorexia and weight loss (42%), fatigue (42%), and anemia (50%). While toxicities of the combination can be significant, low-dose GM-CSF readily ameliorated neutropenia associated with zidovudine and interferon-α therapy without adversely affecting the antiviral properties of the combination.

A nonrandomized trial was undertaken to evaluate the combination of didanosine and interferon-α (IFN-α) in human immunodeficiency virus (HIV)-infected patients. Thirty-six volunteers with >200 × 106 CD4 cellslL received didanosine (one 100-,250-, or 375-mg sachet twice daily) for at least 6 weeks, following which IFN-α (1, 5, 10, or 15 MU/day) was begun. Didanosine (one 375-mg sachet twice daily) was substituted for zidovudine in 14 additional patients who had received IFN-α and zidovudine for 7-45 months. Thirty-five patients completed the 34-week study. Clinical or chemical pancreatitis was the most common (6 patients) dose-limiting toxicity. CD4 cell counts increased with didanosine but declined following the addition of IFN-a; CD4 cell percents tended to increase and remain elevated. Thus, combination therapy with didanosine and IFN-α can be safely administered to patients with HIV infection. The clinical benefit of this combination therapy will require further evaluation.