Lipodystrophy in 685 HIV-1-treated patients: influence of antiretroviral treatment and immunovirological response.

PURPOSE: To describe the clinical features in HIV-1-infected patients treated with protease inhibitors (PIs) or not, and to determine factors related to occurrence of lipodystrophy (LD). METHOD: We performed a cross-sectional analysis of 685 treated HIV-1-infected patients that were routinely followed in 6 Paris hospital centers between January and May 1999. Demographic data, familial and personal vascular risk factors, history of antiretroviral treatment, HIV plasma viral load, CD4 cell count, and metabolic data were collected. Clinical examination was based on an assessment of changes in abdominal, dorso-cervical, and breast girth and wasting of the limbs, face, and skin as quoted by the clinician. RESULTS: The mean age at inclusion in the study was 38 years; 29.5% were women. At study assessment, 77.5% of patients were PI-treated and 22.5% had never received a PI. LD was observed in 403 (58.8%) patients, of whom 340 were currently receiving a PI and 63 had never received a PI. More than half of the lipodystrophic patients had a mixed form (53.9%), while 25.3% were classified as exclusive lipoatrophic and 20.8% as exclusive hypertrophic. In multivariate analysis, older age, duration of antiretroviral treatment (ART), antiretroviral combinations including stavudine, antiretroviral combinations including a PI, AIDS status, and a low HIV RNA were independently associated with occurrence of LD. CONCLUSION: LD is frequently observed in PI-treated patients, but it is also observed in patients receiving an ART regimen without PIs. Our study suggests different underlying mechanisms, because immunovirological response to treatment as well as certain therapies were linked to the occurrence of LD. This hypothesis would be best clarified in a large prospective cohort of naive patients.

Acute upregulation of CCR-5 expression by CD4+ T lymphocytes in HIV-infected patients treated with interleukin-2. ANRS 048 IL-2 Study Group.

BACKGROUND: The treatment of HIV-infected patients with interleukin (IL)-2 causes a sustained increase in CD4+ T-lymphocyte counts, involving both naive and memory cells. However, the short-term immunological effects of IL-2, which may shed light on the mechanism of immune reconstitution by this cytokine, are unknown. OBJECTIVE: To evaluate the acute effect of IL-2 on circulating T-lymphocyte subpopulations and their expression of chemokine receptors. DESIGN AND METHODS: Flow cytometry, reverse transcriptase polymerase chain reaction and chemokine receptor function experiments were performed before and after 5 days of IL-2 administration in 30 HIV-infected patients. RESULTS: IL-2 induced an acute lymphopenia of both naive and memory T-helper (TH) lymphocytes. This was associated with a large increase in CC-chemokine receptor (CCR)-5 and CCR-2b expression by TH cells. Before IL-2 treatment, CCR-5 was mostly produced by CD62L- memory TH lymphocytes. After 5 days of IL-2 administration, the level of CCR-5 mRNA in circulating cells was 18.6 times higher than before treatment (P < 0.002). CCR-5 expression was upregulated in CD62L- memory TH lymphocytes, but also in CD62L+ memory and in naive (CD62L+ CD45RO-) TH lymphocytes. IL-2 treatment also increased the function of CCR-5 in TH cells. CONCLUSIONS: Chemokine receptors are involved in trafficking of lymphocytes. The IL-2-induced upregulation of chemokine receptors in TH cells may thus play a role in the acute effects of this cytokine in TH lymphocyte redistribution.

Constrained evolution of human immunodeficiency virus type 1 protease during sequential therapy with two distinct protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) variants that have developed protease (PR) inhibitor resistance most often display cross-resistance to several molecules within this class of antiretroviral agents. The clinical benefit of the switch to a second PR inhibitor in the presence of such resistant viruses may be questionable. We have examined the evolution of HIV-1 PR genotypes and phenotypes in individuals having failed sequential treatment with two distinct PR inhibitors: saquinavir (SQV) followed by indinavir (IDV). In viruses where typical SQV resistance mutations were detected before the change to IDV, the corresponding mutations were maintained under IDV, while few additional mutations emerged. In viruses where no SQV resistance mutations were detected before the switch to IDV, typical SQV resistance profiles emerged following the introduction of IDV. We conclude that following suboptimal exposure to a first PR inhibitor, the introduction of a second molecule of this class can lead to rapid selection of cross-resistant virus variants that may not be detectable by current genotyping methods at the time of the inhibitor switch. Viruses committed to resistance to the first inhibitor appear to bear the "imprint" of this initial selection and can further adapt to the selective pressure exerted by the second inhibitor following a pathway that preserves most of the initially selected mutations.

Immunosenescence in HIV pathogenesis.

Telomeres are complex protein-DNA structures located at the ends of eukaryotic chromosomes. In a normal cell, telomere DNA shortens with cell divisions. Such a telomere loss may act as a mitotic clock to eventually signal cell cycling exit and cellular senescence. In a transversal study, we found a marked decrease in telomere length of peripheral blood mononuclear cells in HIV-infected patients with advanced immunodeficiency. This telomere reduction concerns T4, T8, and B lymphocytes, providing evidence of high turnover of these cells in the course of HIV infection. These data suggest that replicative senescence could be involved in the final immunosuppression and may have important therapeutical implications.

Programmed cell death in peripheral lymphocytes from HIV-infected persons: increased susceptibility to apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with disease progression.

We analyzed the potential causes of the increased susceptibility to apoptosis of peripheral lymphocytes from a large cohort of HIV-infected persons that we followed during a 3-yr period. By using quantitative cytofluorometric methods, we demonstrate that all lymphocyte populations were contributing proportionally to the apoptotic population in both groups of donors, but the percentage of T and B cells involved in this cell death process was significantly increased in HIV-infected persons. To study the relationship between the increased apoptosis in HIV infection and the activation state of the immune system, we analyzed cell surface expression of activation markers on apoptotic and nonapoptotic cells. We found that in the chronic phase of HIV infection, 50 to 60% of the apoptotic cells exhibited an activated phenotype (they were HLA-DR+, CD38+, CD45RO+, and Fas+), and interestingly, the CD45RO+ subset appeared to be more prone to apoptosis in HIV-positive persons. This study also indicates that the activated phenotype found on apoptotic cells was not a distinctive feature of patients' lymphocytes since it was in similar proportion in apoptotic cells from control lymphocytes. However, a significant correlation was found between the intensity of anti-CD3-induced apoptosis in both CD4 and CD8 T cells from HIV-infected persons and their in vivo expression of CD45RO and HLA-DR molecules. Finally, a significant correlation was found between the intensity of spontaneous or anti-CD3-induced apoptosis in total lymphocytes and disease progression; this was confirmed when the CD4 and CD8 T cell subsets were analyzed separately. Altogether these observations indicate that the increased susceptibility to apoptosis of peripheral T cells from HIV-infected persons correlates with disease progression and support the hypothesis that the chronic activation of the immune system occurring throughout HIV infection is the primary mechanism responsible for this cell deletion process.

In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals.

Mycoplasma penetrans is a mycoplasma species newly isolated from the urine of human immunodeficiency virus (HIV)-infected individuals and presents the only case in which an association has been found between antibodies against a mycoplasma and HIV infection. To further explore the effects of M. penetrans on the immune system, we studied the influence of this mycoplasma on peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-infected individuals. M. penetrans induced, in addition to blastogenesis of PBMCs, a significant proliferative response associated with the expression of some activation markers such as CD69, HLA-DR, and CD25. This M. penetrans-dependent lymphocyte activation was observed not only in healthy donors but also in HIV-infected persons at different stages of the disease. In addition, our study revealed that both CD4+ and CD8+ T lymphocytes were responsive to M. penetrans. Interestingly, the mitogenic activity of M. penetrans was associated with mycoplasma cells but not with the supernatants of mycoplasma culture. The potent stimulating activity of M. penetrans on T lymphocytes from HIV-infected individuals is of particular interest in view of the supposed contribution of immune activation to HIV replication and disease progression.

Production of PAF-acether by synovial fluid neutrophils in rheumatoid arthritis.

PAF-acether (PAF) is a pro-inflammatory phospholipid molecule potentially involved in the pathogenesis of arthritis. PAF and related metabolites have been isolated in the synovial fluid from patients with arthritis. The aim of this study was to determine fluid and blood in patients with rheumatoid arthritis. Blood neutrophils from normal donors were also studied for their capacity to form PAF. Neutrophils were stimulated with the calcium ionophore A23187 (2 microM) for 1 to 60 min. PAF released in the medium and PAF associated to cells were measured. In synovial fluid neutrophils. PAF production began as soon as 1 min of stimulation (16.1 +/- 6.3 pmol per 1 x 10(6) cells) and reached a maximum at 20 min: 29.2 +/- 2.8 pmol per 1 x 10(6) cells (mean +/- SEM, n = 5). The amount of PAF released in the supernatant increased with the length of stimulation, similar amounts of PAF were produced by blood neutrophils isolated from the joint had a lower capacity to produce PAF than blood neutrophils from the same patients. The present results demonstrate the synthesis and release of PAF by synovial fluid neutrophils. They suggest that neutrophils may be source of PAF locally present in the joint. Newly synthesized PAF could participate in the amplification of the local inflammatory reaction.

Quantification of cytokine gene expression by competitive PCR using a colorimetric assay.

Competitive reverse transcription-polymerase chain reaction (RT-PCR) is a new technique allowing quantification of cytokine gene expression from either experimental or clinical samples. In this assay, a time-consuming step is the quantification of amplified products. To improve this step, we set up a colorimetric assay in which the amplified product from either the cDNA or the competitor can be reliably quantified. Using this approach, which can be completely automatized, up to 320 PCR products can be quantified each day. In this report, we describe the quantification of IL-10 mRNA molecules as compared to that of beta-actin mRNA molecules. The sensitivity of the quantification was 7.7 x 10(7) molecules for the amplified beta-actin cDNA and the amplified IL-10 cDNA, corresponding to approximately 9.6 pg amplified beta-actin cDNA and 11 pg amplified IL-10 cDNA, respectively. The intra-assay variation coefficient was < 12%. This technique can be readily extended to all cytokines, and it thus allows routine monitoring of cytokine gene expression, either from experimental samples or from clinical trials.

Paf-acether acetylhydrolase activity is increased in patients with rheumatic diseases.

Paf-acether (platelet-activating factor) is a phospholipid described as a potent mediator of inflammatory response. We have recently shown that the level of paf bound to lipoproteins was significantly higher in the serum from patients with rheumatic diseases, compared to that of control subjects. In serum, paf is inactivated in part by a paf acetylhydrolase that catalyses the hydrolysis of the acetate residue. Acetylhydrolase activity was measured in the serum and synovial fluid of patients with rheumatoid arthritis and other arthritides, i.e. osteoarthritis and chondrocalcinosis. In serum, the activity of acetylhydrolase was significantly increased in patients with rheumatic diseases when compared with that in the control group. However, it was enhanced to a lesser degree in rheumatoid arthritis than in non inflammatory rheumatic diseases. These results suggest a role for acetylhydrolase in controlling paf levels in rheumatic diseases.

Immunoregulatory functions of paf-acether. III. Down-regulation of CD4+ T cells high-affinity IL-2 receptor expression.

In the present report, we further explored the mechanisms by which 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), a phospholipid mediator of inflammation inhibited PHA-induced CD4+ cell proliferation. Evidence was obtained that CD4+ cells stimulated with either PHA or immobilized OKT3 in the presence of paf at concentrations that block CD4+ cell proliferation, exhibited a marked decrease in high affinity IL-2R expression. Importantly, paf did not prevent the binding of IL-2 to its receptor. Scatchard analysis of the binding data indicated that paf caused more than 50% decrease in the number of IL-2 high affinity sites per cell, whereas the receptor ligand affinity remained essentially constant. Moreover, the down-regulation of high affinity IL-2R was also accompanied by a loss of IL-2-dependent proliferative capacity. Together these data suggest that decreased expression of high affinity IL-2R may contribute to the diminished proliferative activity observed in CD4+ cells stimulated with PHA or immobilized OKT3 in the presence of paf. They further emphasize the potential role of lipid proinflammatory mediators such as paf in the regulation of T cell activation.

Immunoregulatory functions of paf-acether. IV. Enhancement of IL-1 production by muramyl dipeptide-stimulated monocytes.

paf-Acether (paf) is a phospholipid mediator of inflammation released from monocytes along with IL-1. In this study, we have examined the role of paf on IL-1 production by human monocytes. When paf from 1 nM to 5 microM, but not its precursor lyso paf, was added to monocytes in the presence of muramyl dipeptide (MDP) or LPS, a marked increase in IL-1 activity over the value with MDP alone was observed. In contrast, paf alone had minimal activity over the same dose range. Antibodies against rHu IL-1 alpha and rHu IL-1 beta neutralized the increased IL-1 activity. Interestingly, MDP that prompts monocytes to synthesize IL-1, induced the synthesis of paf, as well. Most of the paf produced remained cell-associated and always preceded IL-1 synthesis. When the paf receptor antagonist, L-652,731 was added to monocytes, it prevented the enhancement of IL-1 activity induced by exogenous paf. In contrast, L-652,731 had little effect on MDP-induced IL-1 synthesis in the absence of exogenous paf. This may indicate that there are alternative mechanisms involved in the sequences of events leading to IL-1 production. It is also conceivable that the paf receptor antagonist is not able to compete or inhibit endogenous paf as well as it does for exogenous paf. Nevertheless, exogenous paf in association with a second signal, modulates IL-1 production from human monocytes in a positive manner. This may constitute another means through which paf can modulate inflammatory and immune reactions.

Presence of paf-acether in human thymus.

Paf-acether (paf) is a phospholipid mediator of inflammation endowed with major immunoregulatory properties. The present study demonstrates that human thymus contains large amounts of paf, as well as paf precursors. In addition, isolated thymic cells produced paf under ionophore stimulation. Paf from thymus exhibited the same biological and physiochemical properties as synthetic paf. The purity and molecular structure of paf from thymus were further characterized by reverse-phase HPLC and gas chromatography with electron-capture detection. These findings may have important implications since thymus microenvironment is essential in the proper development of bone marrow progenitors committed to the T cell lineage into thymocytes capable of emigrating to the periphery as functional T lymphocytes.

Biosynthesis of paf-acether. Paf-acether but not leukotriene C4 production is impaired in cultured macrophages.

After adherence for 24 or 48 h mouse peritoneal macrophages, upon a zymosan challenge, synthesized 114 +/- 55 and 82 +/- 31 pmol of paf-acether (paf)/mg of protein respectively, as compared with 513 +/- 195 pmol of paf/mg of protein in 2 h-adherent macrophages (means +/- S.D., n = 10). By contrast, 24 h- and 48 h-adherent macrophages exposed to zymosan produced more leukotriene C4 (2.7 +/- 1.1 and 1.4 +/- 0.2 nmol/mg of protein respectively, n = 5) than did 2 h-adherent macrophages (0.5 +/- 0.2 nmol/mg of protein, n = 5). Paf production was not altered when 2 h- and 24 h-adherent cells were cultured and/or stimulated in the presence of 5 microM-indomethacin, 10 microM-nordihydroguaiaretic acid or 100 microM-BW755C as compared with untreated cells. These results indirectly exclude the regulation of paf production by arachidonic acid metabolites. We investigated the efficiency of the enzymic steps which govern paf synthesis. We showed that the anabolic process was not impaired since (1) the amounts of alkylacylglycerophosphocholine and lyso-paf were similar in 2 h-, 24 h- and 48 h-adherent macrophages; (2) adding synthetic lyso-paf or acetyl-CoA to intact cells did not increase paf production in zymosan-stimulated 24 h- and 48 h-adherent macrophages; (3) the basal level of acetyltransferase was comparable in 2 h-, 24 h- and 48 h-adherent macrophages and in all cases was increased by 2-3 times upon zymosan challenge. We also showed that impaired paf production in 24 h- and 48 h-cultured macrophages was not due to the nature of the stimulus used to induce its synthesis.

Biosynthesis of paf-acether. XIV. Paf-acether output in murine peritoneal macrophages is regulated by the level of acetylhydrolase.

Paf-acether (paf) synthesis was previously shown to be impaired in 24 hr-adherent and Bacillus Calmette-Guérin-activated murine peritoneal macrophages as compared to resident macrophages. We report here that the induction of acetylhydrolase was responsible for the decreased paf output in 24 hr-adherent macrophages. The kinetic analysis of the enzymes derived from 2 hr-, 24 hr- and BCG-activated adherent macrophages and from plasma revealed that the Km for paf was similar whatever the source of the acetylhydrolase whereas the Vmax was five-fold increased in 24 hr-cultured macrophages. The acetylhydrolase activity was Ca2+-independent and was not inhibited by addition of alkyl-acyl (long chain)-glycero-phosphocholine suggesting that the enzyme was not a phospholipase A2.

Immunoregulatory functions of paf-acether. II. Decrease of CD2 and CD3 antigen expression.

Paf-acether (platelet-activating factor) is a phospholipid initially described as a potent platelet-aggregating compound. It is produced by numerous cell types and is now considered as an important mediator of cell-cell interactions. The effect of paf-acether on the expression of CD2 and CD3, two human T cell surface glycoproteins, was investigated by indirect immunofluorescence and flow cytometry. Paf-acether partially down-regulated, in a time- and dose-dependent manner, CD2 and CD3 but not HLA class I antigen expression on peripheral human T cells and Jurkat cells. Lysophosphatidylcholine, a phospholipid closely related to paf-acether, had no detectable modulatory effect on CD2 and CD3 expression. In addition to CD2/CD3 modulation, paf-acether markedly inhibited T cell proliferative response not only to phytohemagglutinin or concanavalin A but also to anti-CD3 or a stimulatory combination of anti-CD2 monoclonal antibodies. These data demonstrate for the first time that lipid mediators such as paf-acether might be involved in the regulation of the expression of cell surface glycoproteins that are essential in the execution of T cell function.

Immunoregulatory functions of paf-acether. I. Effect of paf-acether on CD4+ cell proliferation.

Paf-acether or platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation initially described as a potent platelet-aggregating compound. It is newly formed by a variety of cells including monocytes and is now recognized as a major mediator of cell-cell interactions. The present studies were undertaken to determine whether paf-acether could modulate T cell function. We found that addition of paf-acether to CD4+ cells cultured with phytohemagglutinin markedly inhibited the proliferative response in a dose-dependent manner. Maximal inhibition occurred when paf-acether was present during the first 24 hr of cell culture and the presence of paf-acether did not alter the kinetics of CD4+ cell proliferation. Importantly, the mechanism by which paf-acether inhibited the proliferative response was not related to inhibition of interleukin 2 (IL-2) secretion since the amount of IL-2 in cultures was not altered and addition of exogenous IL-2 failed to restore the CD4+ cell proliferative response. Further, as judged by indirect immunofluorescence, paf-acether did not inhibit IL-2 receptor expression. Taken together, these data indicate that paf-acether interferes with some processes leading to CD4+ cell proliferation. This new role for the chemically defined monokine paf-acether emphasizes the potential role of inflammatory lipid mediators in the regulation of T cell response.