Impaired macrophage phagocytosis of apoptotic neutrophils in patients with human immunodeficiency virus type 1 infection

Dysfunction of neutrophils (polymorphonuclear leukocytes [PMNL]) and macrophagic cells occurs as a consequence of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages contribute to the resolution of early inflammation ingesting PMNL apoptotic bodies. This study investigated macrophage ability to phagocytose PMNL apoptotic bodies in patients with HIV-1 infection in comparison with uninfected individuals and the effect of HIV Nef protein on apoptotic body phagocytosis to determine if phagocytic activity is impaired by HIV infection. Monocytes/macrophages were isolated from 10 HIV-1-infected patients and from five healthy volunteers, whereas PMNL were isolated from healthy volunteers. Macrophage phagocytosis of apoptotic PMNL was determined by staining of apoptotic bodies with fluorescein-conjugated concanavalin A or with fluorescein-labeled phalloidin. Our data show significant impairment of PMNL apoptotic body macrophage phagocytosis in subjects with HIV-1 infection presenting a concentration of CD4(+) T lymphocytes of >200/mm(3) and in particular in those with <200 CD4(+) T lymphocyte cells/mm(3). In addition, HIV-1 recombinant Nef protein is able to decrease phagocytosis of apoptotic PMNL from normal human macrophages in a dose-dependent manner. The results of our study suggest that impaired macrophage phagocytosis of PMNL apoptotic bodies may contribute to the persistence of the inflammatory state in HIV-infected subjects, especially during opportunistic infections that are often favored by defective phagocytic activity.     

Correlation of CD4+ T-Cell Counts Estimated by an Immunocapture Technique (Capcellia) with Viral Loads in Human Immunodeficiency Virus-Seropositive Individuals

As antiretroviral therapy becomes more affordable, valid, reliable, and inexpensive laboratory tests are also needed to monitor the progression of disease in people with human immunodeficiency virus (HIV) infection. The CD4⁺ T-cell counts estimated by Capcellia, an immunocapture method, and flow cytometry were compared and were correlated with HIV type 1 (HIV-1) load. There was a significant negative correlation between the HIV-1 load and CD4⁺ T-cell counts estimated by flow cytometry (r = −0.63, P = <0.001) as well as between the HIV-1 load and CD4⁺ T-cell counts estimated by Capcellia (r = −0.61, P = <0.001). Capcellia is a cost-effective, user-friendly assay that correlated well with HIV-1 load determinations for individuals both with and without treatment.     

Immunological responses and long-term treatment interruption after human immunodeficiency virus type 1 (HIV-1) lipopeptide immunization of HIV-1-infected patients: the LIPTHERA study

We studied the time course of immunological and virological markers after highly active antiretroviral therapy (HAART) interruption in chronically human immunodeficiency virus type 1 (HIV-1)-infected patients immunized with an HIV lipopeptide preparation. In a prospective open pilot study, 24 HIV-1-infected HAART-treated patients with undetectable plasma viral loads (pVLs) and CD4⁺ T-cell counts above 350/mm³ were immunized at weeks 0, 3, and 6 with a candidate vaccine consisting of six HIV lipopeptides. At week 24, patients with pVLs of <1.7 log₁₀ copies/ml were invited to stop taking HAART. Antiretroviral therapy was resumed if the pVL rose above 4.47 log₁₀ copies/ml and/or if the CD4⁺ cell count fell below 250/mm³. Immunological and virologic parameters were studied before and after HAART interruption. The median baseline and nadir CD4⁺ cell counts were 482 (interquartile range [IQR], 195 to 826) and 313 (IQR, 1 to 481)/mm³, respectively. New specific CD8⁺ cell responses to HIV-1 epitopes were detected after immunization in 13 (57%) of 23 assessable patients. Twenty-one patients were evaluated 96 weeks after HAART interruption. The median time to pVL rebound was 4 weeks (IQR, 2 to 6), and the median peak pVL was 4.26 (IQR, 3 to 5) log₁₀ copies/ml. Thirteen of these 21 patients resumed HAART a median of 60 weeks after immunization (IQR, 9.2 to 68.4 weeks), when the median pVL was 4.8 (IQR, 2.9 to 5.7) log₁₀ copies/ml and the median CD4⁺ cell count was 551 (IQR, 156 to 778)/mm³. Eight patients were still off therapy at 96 weeks, with a median pVL of 4 (IQR, 1.7 to 4.6) log₁₀ copies/ml and a median CD4⁺ cell count of 412 (IQR, 299 to 832)/mm³. No clinical disease progression had occurred. Despite the lack of a control arm, these findings warrant a randomized study of therapeutic vaccination with HIV lipopeptides followed by long-term HAART interruption in AIDS-free chronically infected patients.     

The selective engulfment of apoptotic bodies by dendritic cells is mediated by the αvβ3 integrin and requires intracellular and extracellular calcium

Dendritic cells derived in vitro from monocytes are known to be poor phagocytes. Here we show that, unlike macrophages, monocyte-derived dendritic cells indeed fail to take up opsonized particles or necrotic cells; however, apoptotic bodies are efficiently engulfed by dendritic cells. The temperature dependence and the sensitivity to cytochalasin D indicate that the apoptotic body engulfment is representative of early stages of phagocytosis. Inhibition studies with ligands for surface molecules involved in recognition of apoptotic bodies, such as vitronectin receptor, CD36 and phosphatidylserine receptor, revealed that apoptotic body engulfment by dendritic cells is mediated preferentially by the vitronectin receptor αvβ3, while all the receptors, with different efficiency, are engaged in phagocytosis of apoptotic bodies by macrophages. The interaction between apoptotic bodies and dendritic cells elicits a rise in intracellular free calcium concentration ([Ca²⁺]_i) which is essential for the process of engulfment. Either intra- or extracellular Ca²⁺ buffering inhibits apoptotic body engulfment by dendritic cells and [Ca²⁺]_i increases, indicating the involvement of both intra-and extracellular Ca²⁺. In contrast, Ca²⁺ mobilization is dispensable for macrophage phagocytosis of apoptotic bodies. The different requirements of Ca²⁺ in macrophages and dendritic cells is possibly due to the differential usage of phagocytic receptors (CD36 vs. αvβ3) and might reflect different fates of apoptotic bodies in the two cell types.     

Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk

To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 ± 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-¹⁴C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-³H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.     

Effect of Pregnancy and Human Immunodeficiency Virus Infection on Intracellular Interleukin-2 Production Patterns

Human immunodeficiency virus type 1 (HIV-1) infection decreases the production of interleukin-2 (IL-2) from CD4⁺ and CD8⁺ T cells. Recombinant IL-2 (rIl-2) has been given to HIV-infected individuals to generate significant increases in CD4⁺ T-cell counts. There are limited data regarding the effects of pregnancy and HIV infection on IL-2 production in humans. To investigate the effects of human pregnancy, HIV infection, and HIV therapy on IL-2 production, we evaluated 61 women. Intracellular IL-2 production by CD4⁺ T cells from nonpregnant HIV-infected women was significantly lower than in that in uninfected women (45% ± 8% versus 52% ± 8%, P = 0.04). In contrast, there was no difference in levels of intracellular IL-2 production between HIV-infected and uninfected pregnant women. These observations suggest that pregnancy may down-regulate IL-2 production regardless of HIV infection status. Future studies should evaluate IL-2 production patterns in larger cohorts of women so that the physiological significance of IL-2 down-regulation in pregnancy can be further evaluated. This information is essential to assess the possible use of IL-2 supplementation therapy as a means of enhancing immune responses among HIV-infected pregnant women.     

Macrophage-Mediated Responses to Candida albicans in Mice Expressing the Human Immunodeficiency Virus Type 1 Transgene

The critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicans in transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4⁺ T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIV^MutA Tg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicans and displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicans and enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H₂O₂ by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicans was moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicans or cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H₂O₂ and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.Oropharyngeal candidiasis (OPC) is the most frequent opportunistic fungal infection among human immunodeficiency virus (HIV)-infected patients (64). Although the incidence of OPC in HIV infection is sharply reduced by highly active antiretroviral therapy (45), it remains a common coinfection worldwide. The critical impairments of innate and adaptive immunity that are responsible for the onset and maintenance of mucosal candidiasis in HIV infection have not been fully determined (15, 25). A correlation has been established in HIV infection between symptomatic OPC and reduced CD4⁺ cell count (6, 46, 55), HIV viral load (6, 46), and the development of AIDS (55). Studies conducted with experimentally infected normal, nude, and cytokine-specific gene knockout mice indicated that host defense against OPC requires intact Th1- and Th17-mediated immune responses to Candida albicans, including production of interleukin 12 (IL-12), CD4⁺ T-cell augmentation of monocyte and polymorphonuclear leukocyte (PMN) functions, and mucosal production of nitric oxide (NO) (1, 7, 11, 17-21, 34, 61). Using a model of mucosal Candida infection in transgenic (Tg) mice expressing HIV-1 Nef in CD4⁺ T cells, dendritic cells, and macrophages which closely mimics the clinical and pathological features of candidal infection in human HIV infection (14), we have previously shown that altered CD4⁺ T-cell phenotype and function determine the susceptibility to chronic carriage of C. albicans in these Tg mice (37). However, PMNs from the Tg mice were unimpaired in their capacity to produce an oxidative burst and to phagocytose and kill C. albicans in vitro, and depletion of PMNs in these Tg mice did not alter the oral or gastrointestinal burdens of C. albicans or cause systemic dissemination (42). Accordingly, the defective anti-Candida effector mechanisms that render these Tg mice susceptible to mucosal candidiasis have not yet been identified.Oral colonization and infection of mice with C. albicans trigger macrophage recruitment to the mucosa of the oral cavity (9), stomach (10, 71), and cecum (12), suggesting that these cells play a role in resistance to mucosal candidiasis (68). Activated macrophages have the capacity to kill C. albicans by their production of the reactive oxygen intermediates (ROIs) O₂⁻ and H₂O₂, by the formation of peroxynitrite from O₂⁻ and the reactive nitrogen intermediate NO, and by oxygen-independent candidacidal mechanisms (68-71,73). The participation of macrophages in host resistance has been demonstrated by the enhanced susceptibility of severe combined immunodeficiency (SCID) mice to disseminated candidiasis of gastrointestinal origin after treatment with poly(I-C), an inhibitor of macrophage candidacidal activity (33).Treatment of human monocyte-derived macrophages with HIV-1 Nef protein or infection of these cells with HIV-1 alters cellular signal transduction pathways and specifically activates NF-κB, STAT1 and STAT3, mitogen-activated protein kinases, and genes for several inflammatory factors, including macrophage inflammatory protein 1α, macrophage inflammatory protein 1β, IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) (5, 24, 40, 59). Therefore, the anticandidal properties of macrophages could be altered either directly by the expression of HIV-1 gene products within this cell population or indirectly by inadequate cytokine signaling from defective CD4⁺ T cells. In several investigations producing conflicting results, phagocytosis and killing of C. albicans by blood monocyte-derived macrophages from HIV-infected patients have been found to be either normal (56, 57) or reduced (13), possibly by HIV Nef (35, 62).With the recognition that classically activated (M1) macrophages (27, 44) primarily mediate the effector arm of a CD4⁺ T-cell-dependent protective Th1 adaptive immune response by their production of ROIs and reactive nitrogen intermediates which kill C. albicans (8, 43, 52, 54, 63), we asked whether a defective mucosal macrophage response to C. albicans contributes to the phenotype of chronic oral candidiasis in these Tg mice expressing HIV-1. The likelihood of such a defect was considered significant because CD4⁺ T cells are quantitatively and functionally defective in these Tg mice, and these alterations of CD4⁺ T cells determine at least in part the susceptibility of these animals to chronic carriage of C. albicans (37). We found that macrophages from these Tg mice display a polarization toward an alternatively activated phenotype and are successfully recruited to the mucosa in response to C. albicans. Although the production of H₂O₂ was modestly reduced, the production of NO and the killing of C. albicans by macrophages were both unaltered by expression of the HIV-1 transgene, and no further augmentation of oral burdens of C. albicans was found in NOS2^−/− gene-deficient Tg mice. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine susceptibility to mucosal candidiasis.     

Tetraspanin CD63 is a regulator of HIV-1 replication

Macrophages and CD4⁺ T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4⁺ T-cells, dendritic cells, and a CD4⁺ cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4⁺ cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4⁺ T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2).     

Low CD4+ T-Lymphocyte Values in Human Immunodeficiency Virus-Negative Adults in Botswana

CD4⁺-lymphocyte counts (LCs) play a crucial role in the management and monitoring of HIV infection. Variability in CD4⁺ LCs has been reported to occur as a result of measurement techniques and/or biological variations. We report on the CD4⁺ LCs of healthy human immunodeficiency virus (HIV)-seronegative adults in Botswana. Samples were obtained from HIV-seronegative blood donors. The median CD4⁺ LC was 726 cells/mm³ (for females, 782 cells/mm³; for males, 698 cells/mm³). The median CD8⁺ LC was 488 cells/mm³ (for females, 494 cells/mm³; for males, 485 cells/mm³). The median CD4⁺-to-CD8⁺ ratio was 1.57 (for females, 1.66; for males, 1.51). Our findings of low CD4⁺ LCs among HIV-negative adults in Botswana are significant and have important implications for the management of HIV disease in the population of this sub-Saharan African country.     

Expression of Regeneration and Tolerance Factor Correlates Directly with Human Immunodeficiency Virus Infection and Inversely with Hepatitis C Virus Infection

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3⁺ T cells of HIV-seropositive (HIV⁺) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV⁺ HCV-seropositive (HCV⁺), HIV- and HCV-seropositive (HIV⁺ HCV⁺), and HIV- and HCV-seronegative (HIV⁻ HCV⁻) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV⁺ individuals is upregulated and that its expression on T cells from HCV⁺ individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3⁺ T cells was seen in both HIV⁺ and HIV⁺ HCV⁺ individuals compared to HIV⁻ HCV⁻ individuals. HCV⁺ individuals had lower levels of RTF expression than HIV⁻ HCV⁻ individuals (P < 0.005 for CD4⁺; P < 0.0005 for CD8⁺). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV⁺ > HIV⁺ HCV⁺ > HIV⁻ HCV⁻ > HCV⁺. The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.     

Apoptosis in Cord Blood T Lymphocytes from Infants of Human Immunodeficiency Virus-Infected Mothers

Apoptosis continues to be controversial in human immunodeficiency virus (HIV)-induced pathogenesis. To investigate whether apoptosis occurs with HIV exposure with or without subsequent infection, levels of apoptosis were measured in cord blood lymphocytes (CBL) from seven newborns delivered to HIV-infected mothers and seven normal, unexposed newborns. Live cells were costained with antibodies to cell surface markers and the DNA dye 7-amino actinomycin D to immunophenotype apoptotic CBL subsets. Apoptosis was measured in fresh and cultured CBL in the presence and absence of CD3 T-cell receptor stimulation. Compared to the CD4⁺ CBL from HIV-unexposed newborns, CD4⁺ CBL from six HIV-exposed, noninfected newborns demonstrated significantly greater apoptosis after overnight culture even in the absence of CD3 stimulation. Compared to HIV-unexposed controls, CD8⁺ CBL from the six HIV-exposed newborns also demonstrated increased levels of apoptosis after overnight culture without stimulation. The one HIV-infected newborn in this study showed the highest levels of CD4⁺ and CD8⁺ apoptotic CBL. The data suggest that levels of apoptosis are increased in infants in association with HIV infection. Furthermore, CD4⁺ and CD8⁺ cord blood lymphocytes from HIV-exposed infants behaved differently than T lymphocytes from either normal, unexposed infants or an HIV-infected infant. These results suggest that exposure to HIV or HIV-induced factors increases the levels of apoptosis in CBL.     

Defective phagocytosis by human monocyte/macrophages following HIV-1 infection: underlying mechanisms and modulation by adjunctive cytokine therapy.

Defective immunological function of cells of the macrophage lineage contributes considerably to the pathogenesis of HIV-1 infection. Impairment of phagocytosis of opportunistic pathogens such as Mycobacterium avium complex (MAC), Pneumocystis carinii, Toxoplasma gondii or Candida albicans by peripheral blood monocytes, tissue macrophages and monocyte-derived macrophages following in vivo and in vitro HIV-1 infection is well documented. The development of opportunistic infections due to these pathogens in HIV-infected individuals at late stages of disease is attributed to defective monocyte/macrophage function. The mechanisms whereby HIV-1 impairs phagocytosis are not well known. A number of phagocytic receptors normally mediate engulfment of specific opportunistic pathogens by cells of macrophage lineage; distinct mechanisms are triggered by pathogen-receptor binding to promote cytoskeletal rearrangements and engulfment. This review focuses on the signalling events occurring during Fcgamma receptor- and complement receptor-mediated phagocytosis, and considers the mechanisms by which HIV-1 inhibits those signalling events. Since macrophage function is enhanced by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma), the use of these immunomodulators is of potential interest as adjunctive immunotherapy in immunosuppressed individuals. In this review we present examples of clinical applications of GM-CSF and IFN-gamma therapy for the treatment of opportunistic infections in HIV-infected individuals receiving antiretroviral drugs.     

Expression of Adhesion Molecules and CD28 on T Lymphocytes during Human Immunodeficiency Virus Infection

Adhesion molecules, which play a major role in lymphocyte circulation, have not been well characterized in human immunodeficiency virus (HIV) infection. T-lymphocyte populations, including CD3, CD4, CD28, and adhesion molecules (L selectin, LFA-1, VLA-4, and ICAM-1) were measured by flow cytometry in a cross-sectional study of 100 HIV-infected and 49 HIV-seronegative adults. HIV-infected adults had lower numbers of CD3⁺ lymphocytes expressing L selectin (P < 0.0001) and VLA-4 (P < 0.01) and higher numbers of CD3⁺ lymphocytes expressing LFA-1^bright (P < 0.002) than did HIV-negative adults. By CD4⁺-lymphocyte count category (>500, 200 to 500, or <200 cells/μl), HIV-infected adults with more advanced disease had lower percentages of CD3⁺ lymphocytes expressing L selectin and VLA-4 and higher percentages of CD3⁺ lymphocytes expressing LFA-1. The percentages of CD3⁺ CD28⁺ lymphocytes and of CD3⁺ L selectin⁺ lymphocytes were positively correlated (Spearman coefficient = 0.86; P < 0.0001), and the percentage of CD3⁺ CD28⁺ lymphocytes and the CD3⁺ LFA-1^bright lymphocyte/CD3⁺ LFA-1^dim lymphocyte ratio were negatively correlated (Spearman coefficient = −0.92; P <0.00001). The results of this study suggest that HIV infection is associated with altered expression of adhesion molecules.     

Production of a Dendritic Cell-Based Vaccine Containing Inactivated Autologous Virus for Therapy of Patients with Chronic Human Immunodeficiency Virus Type 1 Infection

In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8⁺ and CD4⁺ T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4⁺ T cells with the virus; (iii) inactivation of the virus in CD4⁺ T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4⁺ T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4⁺ T cells. CD4⁺ T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID₅₀; which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4⁺ T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID₅₀ of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 μg/ml) and UVB irradiation (312 nm) reduced the TCID₅₀ of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4⁺ T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).Antiretroviral therapy (ART) has been widely used to suppress human immunodeficiency virus type 1 (HIV-1) replication and increase the number of CD4⁺ T cells in patients with HIV-1 infection. However, in most of these patients, the recovery of anti-HIV-1-specific T-cell function is incomplete. As the complete restoration of T-cell immune function is considered to be necessary for effective control of the viral infection, additional measures aimed at the bolstering of the HIV-1-specific adaptive immunity in patients treated with ART are being evaluated.Dendritic cells (DCs) are the most potent antigen-presenting cells that can both prime and sustain memory responses (24, 28). DCs have been used increasingly frequently in vaccines against cancer and viral infections (4, 13, 20). Previous studies from our group showed that DCs derived from the blood of subjects with chronic progressive HIV-1 infection and not receiving ART were able to stimulate anti-HIV-1 reactivity (5). HIV-1-reactive CD8⁺ T cells are detectable in the peripheral circulation of subjects receiving ART following in vitro activation with many types of HIV-1 antigens, including HIV-1 proteins, HIV-1 peptides, and virus-infected apoptotic cell-loaded matured DCs (6, 10, 14, 22, 23, 31). We hypothesized that it may be possible to reconstitute the reactivity of naïve and memory virus-specific T cells by delivering to patients autologous DCs engineered ex vivo to express and present known immunodominant peptides of HIV-1. To this end, we have recently completed a phase I clinical protocol in which autologous monocyte-derived DCs were pulsed with a mix of three HIV-1 peptides (Gag, Pol, and Env) and one influenza A virus (matrix) major histocompatibility complex class I supertype peptide and delivered as vaccines to 18 HIV-1-infected, ART-treated subjects (5). This vaccination strategy was found to be safe and feasible and resulted in a transient but significant increase in the frequency of CD8⁺ T cells specific for HIV-1 peptides present in the vaccine (5). On the basis of the results of this trial, we have been considering a strategy of stimulating HIV-1-specific, naïve CD8⁺ and CD4⁺ T cells by priming them with DCs engineered to express autologous HIV-1 (19). The rationale for this strategy is that autologous virus represents a large repertoire of the host''s diverse HIV-1 antigen pool and offers the potential to elicit the most specific, broadest, and most effective immune responses for each subject''s quasispecies of HIV-1, thus increasing vaccine efficacy.In this report, we provide evidence that the production of an antiviral vaccine containing autologous DCs fed with inactivated HIV-1-infected, autologous, apoptotic CD4⁺ T cells is feasible, can be successfully accomplished in a good manufacture practice facility, and can be scaled up for therapeutic delivery to HIV-positive (HIV-1⁺) patients. The production process consists of several steps: (i) isolation of autologous virus from the peripheral blood of HIV-1-infected subjects; (ii) superinfection of autologous enriched CD4⁺ CD8⁻ T cells with viral supernatants; (iii) virus inactivation by psoralen and UVB irradiation; (iv) testing for p24 levels and the residual HIV-1 load by determining the 50% tissue culture infective doses (TCID₅₀) for apoptotic CD4⁺ T cells; and (v) loading of autologous DCs with apoptotic, HIV-1-infected CD4⁺ T cells. Although this process is complex, it has been successfully scaled up for therapeutic vaccine production.     

The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3^−/− mice had a higher mortality rate than C3^+/+ mice. Factor B^−/− mice, but not C4^−/− mice, infected with P. aeruginosa had a mortality rate similar to that of C3^−/− mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3^−/− mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3^+/+ mice at 24 h postinfection. In vitro, phagocytic cells from C3^+/+ or C3^−/− mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3^−/− mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1β (IL-1β), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3^+/+ mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.     

Comparison of Human Immunodeficiency Virus Antigens as Stimulants for Lymphocyte Proliferation Assays

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (gag) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm³, and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.     

Association of Selected Phenotypic Markers of Lymphocyte Activation and Differentiation with Perinatal Human Immunodeficiency Virus Transmission and Infant Infection

This study of a subset of women and infants participating in National Institutes of Health Pediatric AIDS Clinical Trials Group protocol 185 evaluated lymphocyte phenotypic markers of immune activation and differentiation to determine their association with the likelihood of human immunodeficiency virus (HIV) transmission from the women to their infants and the potential for early identification and/or prognosis of infection in the infants. Lymphocytes from 215 human immunodeficiency virus type 1 (HIV)-infected women and 192 of their infants were analyzed by flow cytometry with an extended three-color panel of monoclonal antibodies. Women who did not transmit to their infants tended to have higher CD4⁺ T cells. Most notably, levels of total CD8⁺ T cells and CD8⁺ CD38⁺ cells made significant independent contributions to predicting the risk of mother-to-child transmission. Adjusting for HIV-1 RNA level at entry, a one percentage-point increase in these marker combinations was associated with a nine percent increase in the likelihood of maternal transmission. Total as well as naïve CD4⁺ T cells were significantly higher in uninfected than infected infants. Total CD8⁺ cells, as well as CD8⁺cells positive for HLA-DR⁺, CD45 RA⁺ HLA-DR⁺, and CD28⁺ HLA-DR⁺ were elevated in infected infants. Detailed immunophenotyping may be helpful in predicting which pregnant HIV-infected women are at increased risk of transmitting HIV to their infants. Increasing differences in lymphocyte subsets between infected and uninfected infants became apparent as early as six weeks of age. Detailed immunophenotyping may be useful in supporting the diagnosis of HIV infection in infants with perinatal HIV exposure.     

Cigarette smoke exposure attenuates cytokine production by mouse alveolar macrophages

HIV-1 infection impairs alveolar macrophage immune function and renders patients susceptible to pneumonia by poorly understood mechanisms. Alveolar macrophage maturation and function depends on granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced and secreted by the alveolar epithelium. Macrophages respond to GM-CSF through the GM-CSF receptor (GM-CSFR), which has a binding subunit (GM-CSFRalpha) and a signaling subunit (GM-CSFRbeta). In this study, we measured GM-CSFR expression and alveolar macrophage function in a transgene HIV-1 rat model (NL4-3Delta gag/pol); this construct bears a pro-virus with gag and pol deleted, but other HIV-1-related proteins, such as gp120 and Tat, are expressed, and the rats develop an AIDS-like phenotype as they age. We first determined that HIV-1-transgenic expression selectively decreased alveolar macrophage expression of GM-CSFRbeta and impaired bacterial phagocytosis in vitro. Next, we examined the role of zinc (Zn) deficiency as a potential mechanism underlying these effects, and determined that HIV-1-transgenic rats have significantly lower levels of Zn in the alveolar space and macrophages. To test the direct effect of Zn deficiency on macrophage dysfunction, we treated rat alveolar macrophage cell line with a Zn chelator, N,N,N',N'-tetrakis-(2-pyridyl-methyl) ethylenediamine, and this decreased GM-CSFRbeta expression and phagocytosis. In parallel, treatment with Zn acetate in vitro for 48 hours restored intracellular Zn levels and phagocytic function in alveolar macrophages from HIV-1-transgenic rats. Taken together, these data suggest that pulmonary Zn deficiency could be one of the mechanisms by which chronic HIV-1 infection impairs alveolar macrophage immune function and renders these individuals susceptible to serious lung infections.