IGF-I transcript levels in whole-liver tissue, in freshly isolated hepatocytes, and in cultured hepatocytes from lean and obese Zucker rats.

BACKGROUND: The mechanisms underlying the maintenance of normal to high rates of linear growth and plasma insulin-like growth factor I (IGF-I) levels in spite of a low growth hormone secretion in obese children remain unknown. Among the animal models of early-onset obesity, obese Zucker (FA/FA) rats (which are homozygous for an inactivating missense mutation in the leptin receptor) are particularly appropriate, because their linear growth shows this growth hormone independence. METHODS: To study the regulation of IGF-I synthesis in this model, we have established primary cultures of hepatocytes derived from 12-week-old Zucker male obese and lean rats. The rat IGF-I gene contains six exons, and alternative splicing generates different mRNAs, one of which (called IGF-1B) has been shown to be decreased by fasting. We report steady state mRNA levels for IGF-I (all transcripts) and for IGF-IB in hepatocytes after 3 days in culture, in freshly isolated hepatocytes, and in whole-liver tissue. RT-PCRs using primers specific for IGF-I or IGF-IB were performed with two different internal competitors for quantification. RESULTS: In primary cultures of hepatocytes, the IGF-IB mRNA was increased by >50-fold (p = 0.01) in cells derived from obese animals as compared with cells from lean animals. However, these transcript levels were not significantly different when measured in freshly isolated hepatocytes or in whole-liver tissue. CONCLUSIONS: Increased IGF-IB transcription could be an intrinsic characteristic of cultured hepatocytes harbouring leptin receptors that bear the FA mutation. However, the modulation of this characteristic by cell-cell interactions and by in vivo hormone and metabolic status remains to be studied.

Increased priming for interleukin-12 and tumour necrosis factor alpha in CD64 monocytes in HIV infection: modulation by cytokines and therapy.

BACKGROUND: A key factor leading to impaired immunity in HIV infection is an alteration of the pattern of cytokine response, although its precise nature remains controversial, particularly the in vivo influence of HIV on interleukin (IL)-12 synthesis. DESIGN: A cross-sectional study in 73 HIV-infected persons (28 of them receiving highly active antiretroviral therapy) and 18 HIV-seronegative healthy donors. METHODS: The frequency of monocytes/macrophages (M/M) synthesizing IL-12, IL-10 and tumour necrosis factor alpha (TNF-alpha) was determined in peripheral blood mononuclear cells. The cells were cultured in medium or were stimulated with lipopolysaccharide; proportions of CD64 M/M producing IL-12, TNF-alpha or IL-10 was determined by cytofluorometric analysis. The influence of exogenous interferon gamma (IFN-gamma), IL-10 or IL-15 on IL-12 synthesis was tested. RESULTS: Chronic HIV disease is associated with increased priming of M/M for IL-12 (involving both p40 and p70 molecules) and TNF-alpha synthesis; this was associated with cosynthesis of both cytokines by a fraction of M/M. Priming for IL-12 was physiologically enhanced by IFN-gamma and decreased by IL-10; IL-15 had no effect. The proportion of IL-10-producing CD64 M/M was not altered in patients compared with controls but there was an inverse correlation between IL-10-producing M/M and viral load. IL-12 production was not correlated with viral load but was increased following antiretroviral therapy. Following LPS stimulation, IL-12 and TNF-alpha responses were not altered in HIV-positive patients; however, the IL-10 response was decreased but restored by antiretroviral therapy. CONCLUSION: These observations argue for a preserved intrinsic CD64 M/M of IL-12 production in HIV pathogenesis.

Alteration in CD29(high) CD4(+) lymphocyte subset is a common feature of early HIV disease and of active tuberculosis.

Peripheral CD4 T-cell depletion has been observed in human immunodeficiency virus (HIV)-negative patients with pulmonary tuberculosis (TB). To investigate more accurately this alteration, we studied peripheral blood CD45RA(+) and CD29(high) CD4 subsets in 79 TB patients with (HIV(+)TB(+)) or without (HIV(-)TB(+)) HIV infection, 85 HIV-infected patients without TB (HIV(+)TB(-)), and 43 healthy controls, all living in West Africa. The high proportion of CD4(+)CD29(high) T cells observed in controls was dramatically decreased in CDC-A stage HIV(+)TB(-) patients. CD45RA(+) CD4(+) T cells were depleted during the CDC-B stage. Both the percentage and the absolute count of CD29(high)CD4(+) T cells were decreased in HIV(-)TB(+) and HIV(+)TB(+) patients versus controls, but CD45RA(+)CD4(+) T cells were not decreased in TB patients without HIV-infection. Although distinct alterations in the CD4(+) T-cell homeostasis are involved in TB(-) versus HIV-infected subjects, our data suggest that the CD29(+)CD4(+) T-cell depletion observed during the early HIV disease contributes to the risk of active TB, by reducing the pool of T cells able to relocalize to the sites of the M. tuberculosis multiplication.

H19 sense and antisense transgenes modify insulin-like growth factor-II mRNA levels.

The oppositely-imprinted genes insulin-like growth factor-II (IGF2) and H19, a putative tumor suppressor, often show coordinate, reciprocal regulation and are believed to play a role in carcinogenesis. To explore the possible interactions between these genes, we stably transfected diHepG2 cells with a plasmid containing either the sense or the antisense H19 cDNA sequences and verified their expression by Northern analysis and by RNase protection analysis. Levels of H19, IGF2 and gamma-actin mRNA were quantified by competitive RT-PCR analysis. Although H19 sense transgene overexpression (n = 24 clones) did not decrease the low, basal levels of IGF2 mRNA compared to control cells, levels of IGF2 mRNA were positively correlated with the levels of H19 antisense mRNA (P < 0.0001, n = 40 clones). Furthermore, the increase in IGF2 mRNA level was accompanied by an elevation of IGF-II peptide in conditioned media. To see if H19 mRNA had a specific effect on transcription, we also performed transient transfections with reporter gene constructs containing IGF2 promoter 3 in the presence of sense or antisense H19 cDNA sequences under control of a cytomegalovirus promoter. We show a lower reporter gene activity from reporter gene constructs in the presence of sense H19 cDNA than from those with antisense or neomycin. Our results suggest that H19 participates in the repression of IGF2, at least in part through effects on IGF2 transcription, an effect which may contribute to its action as a tumor suppressor.

Alteration of tumor necrosis factor-alpha T-cell homeostasis following potent antiretroviral therapy: contribution to the development of human immunodeficiency virus-associated lipodystrophy syndrome.

Highly-active antiretroviral therapy (HAART) has lead to a dramatic decrease in the morbidity of patients infected with the human immunodeficiency virus (HIV). However, metabolic side effects, including lipodystrophy-associated (LD-associated) dyslipidemia, have been reported in patients treated with antiretroviral therapy. This study was designed to determine whether successful HAART was responsible for a dysregulation in the homeostasis of tumor necrosis factor-alpha (TNF-alpha), a cytokine involved in lipid metabolism. Cytokine production was assessed at the single cell level by flow cytometry after a short-term stimulation of peripheral blood T cells from HIV-infected (HIV(+)) patients who were followed during 18 months of HAART. A dramatic polarization to TNF-alpha synthesis of both CD4 and CD8 T cells was observed in all patients. Because it was previously shown that TNF-alpha synthesis by T cells was highly controlled by apoptosis, concomitant synthesis of TNF-alpha and priming for apoptosis were also analyzed. The accumulation of T cells primed for TNF-alpha synthesis is related to their escape from activation-induced apoptosis, partly due to the cosynthesis of interleukin-2 (IL-2) and TNF-alpha. Interestingly, we observed that LD is associated with a more dramatic TNF-alpha dysregulation, and positive correlations were found between the absolute number of TNF-alpha CD8 T-cell precursors and lipid parameters usually altered in LD including cholesterol, triglycerides, and the atherogenic ratio apolipoprotein B (apoB)/apoA1. Observations from the study indicate that HAART dysregulates homeostasis of TNF-alpha synthesis and suggest that this proinflammatory response induced by efficient antiretroviral therapy is a risk factor of LD development in HIV(+) patients.

HIV, cytokines and programmed cell death. A subtle interplay.

HIV infection is marked by the progressive destruction of the CD4 T lymphocyte subset, an essential component of the immune system and a vital source of cytokines required for differentiation of natural killer (NK) and gamma delta T cells, for maturation of B lymphocytes into plasmocytes, and for differentiation of CD8+ T cells into virus-specific cytotoxic T lymphocytes. CD4 T lymphocytes are also a source of chemokines which control migration of lymphocytes to the site of infection and which also inhibit HIV entry into CD4-expressing targets. Continuous production of viral proteins leads to an unbalanced immune activation and to the triggering of apoptotic programs, turning mononuclear cells, including CD4 T cells, CD8 T cells and APC, into effectors of apoptosis, leading to fratricidal destruction of healthy uninfected cells expressing the death receptors. Inappropriate PCD is also responsible for the disappearance of T helper cells primed for type-1 cytokine synthesis, thus contributing to the lack of survival factors which could prevent spontaneous lymphocyte apoptosis. Under potent anti-retroviral therapies, a significant decrease in spontaneous, TCR- and CD95-induced lymphocyte apoptosis is observed, concomitant with a partial quantitative and qualitative restoration of the immune system in treated patients. However, owing to the suppressive effect of anti-retroviral drugs on physiological apoptosis, these therapies are associated with alteration of TNF-alpha-regulated T cell homeostasis, leading to an accumulation in the blood of T cells primed for TNF-alpha synthesis, and contributing to the development of a new syndrome associated with these treatments, the lipodystrophy syndrome.

Evaluation of a quantitative determination of CD4 and CD8 molecules as an alternative to CD4+ and CD8+ T lymphocyte counts in Africans.

In the developed word, monitoring HIV-infected patients is routinely determined by CD4+ T lymphocyte absolute counts. The reference procedure, flow cytometry, is expensive, requires sophisticated instrumentation and operators with specific training. Due to these limitations, CD4 counting is often unavailable in developing countries. The Capcellia assay is an enzyme-linked immunoassay for quantitative determination of CD4 and CD8 molecules. We evaluated this method in West Africa on blood samples collected from 39 HIV-uninfected and 44 HIV-infected adult subjects. CD4 concentration ranges were determined according to the clinical stages of the disease. We then studied the relationship between the two methods in the HIV-infected patients. The Spearman's rank correlation was 0.61 (95% confidence interval: 0.38-0.76, P < 0.0001). Nevertheless, determination of limits of agreement revealed discrepancies between the two methods, especially for CD4 counts > 0.4 x 10(9)/l, which are discussed. We conclude that the Capcellia assay is a convenient means to determine the immunodepression level where flow cytometric instrumentation is unavailable, and can be complementary to CD4 T lymphocyte enumeration.

Relationship between interleukin-5 production and variations in eosinophil counts during HIV infection in West Africa: influence of Mycobacterium tuberculosis infection.

Eosinophils are important effectors of the non-specific immune response and we studied whether perturbations in the production of the type 2 cytokine, interleukin-5 (IL-5), could account for the variations in eosinophil counts observed in human immunodeficiency virus (HIV) infection. HIV-infected patients without helminthiasis were investigated in a cross-sectional study in West Africa. Eosinophil counts were significantly higher in CDC-B patients than in controls, but were dramatically decreased at the CDC-C stage. Phorbol 12-myristate 13-acetate (PMA)+ ionomycin-induced IL-5 production by peripheral blood mononuclear cells (PBMC) was decreased from the A stage of the disease, and significant correlations were observed between IL-5 production and eosinophil counts in tuberculosis (TB)-negative HIV-1-positive, TB-positive HIV-1-positive and TB-positive HIV-negative patient groups. Nevertheless, the production of IL-5 was not decreased in HIV-positive patients with TB, in contrast to HIV-positive patients without TB presenting with the same ranges of CD4+ counts. Our data suggest that, during HIV infection, the impairment in IL-5 production is one of the factors associated with the 'paradoxal' eosinopenia observed in tropical areas, but that IL-5 production during active TB is compensated by cellular subsets, yet to be identified.

PIP: Eosinophils are important effectors of nonspecific immune response, with eosinophilia being a classic sign of helminthic infection, allergies, and some inflammatory processes. The authors explored whether perturbations in the production of interleukin-5 (IL-5) could account for the variations in eosinophil counts seen in HIV infection. The 491 study subjects were recruited between 1993 and 1995 in Bobo-Dioulasso, Burkina Faso. Eosinophil counts were significantly higher in CDC-B AIDS patients than in controls, but were dramatically lower among CDC-C stage subjects. Phorbol 12-myristate 13-acetate (PMA)+ionomycin-induced IL-5 production by peripheral blood mononuclear cells (PBMC) was decreased from the A stage of the disease, and significant correlations were observed between IL-5 production and eosinophil counts in tuberculosis (TB)-negative HIV-1-positive, TB-positive HIV-1-positive, and TB-positive HIV-negative patient groups. The production of IL-5 was not decreased among HIV-positive patients with TB, in contrast to HIV-positive patients without TB presenting with the same ranges of CD4+ counts. These data suggest that during HIV infection, impairment in IL-5 production is one factor associated with the paradoxal eosinopenia observed in tropical areas, but that IL-5 production during active TB is compensated by as yet unidentified cellular subsets.

A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.

The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFNgamma, TNFalpha, IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFNgamma, TNFalpha, IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.

A proposal for basic management of HIV disease in west Africa: use of clinical staging and haemogram data.

Our objective was to propose a strategy to screen HIV-infected African people for biological immunodeficiency easily. In a cross-sectional study, we analysed the patterns of diseases and of CD4 counts among 266 HIV-infected adults. Peripheral facial paralysis and chronic cutaneo-mucous diseases were the earlier B-stage diseases. Pulmonary tuberculosis was close to B-stage diseases, and chronic diarrhoea was borderline between B and C stages. Cachexia was the most frequent C-stage symptom (47.8%). Ninety per cent of CDC-C stage people had CD4 counts below 350/microliter, whereas only 75% had CD4 counts below 200/microliter. Regression analysis identified the lymphocyte count, clinical stage and platelet count as predictors of CD4 count below 350/microliter. A simple score (lymphocyte count < or = 2500/microliter and clinical stage > or = B) is proposed to determine this CD4 threshold (positive predictive value: 83%) and to determine those patients needing treatment to prevent wasting and opportunistic infections.

PIP: Findings are presented from a cross-sectional study conducted in 1995 in Bobo-Dioulasso, Burkina Faso, in which the patterns of diseases and CD4 counts among 266 HIV-infected adults of mean age 33 years were analyzed. The bioclinical spectrum of subjects' HIV disease is described and a simple alternative proposed to CD4 enumeration for screening and monitoring HIV-infected Africans. Dermatological symptoms and diarrhea were the most frequent signs associated with B-stage disease, while cachexia and digestive candidosis were the most frequent AIDS-defining diseases (ADD). Peripheral facial paralysis and cutaneo-mucous diseases were associated with weak immune deficiency. Pulmonary tuberculosis (TB) was close to B-stage diseases, and chronic diarrhea was borderline between B and C stages. Cachexia was the most frequent C-stage symptom (47.8%). 90% of CDC C-stage subjects had CD4 counts of less than 350 per mcl, while only 75% had CD4 counts under 200/mcl. Regression analysis identified the lymphocyte count, clinical stage, and platelet count as predictors of CD4 count below 350/mcl. A lymphocyte count of less than or equal to 2500/mcl and clinical stage of B or higher is proposed to determine the CD4 threshold and to determine those patients in need of treatment to prevent wasting and opportunistic infections.

Lack of direct correlation between CD4 T-lymphocyte counts and induration sizes of the tuberculin skin test in human immunodeficiency virus type 1 seropositive patients.

SETTING: The study was conducted in Bobo-Dioulasso, Burkina Faso, where Mycobacterium tuberculosis infection and human immunodeficiency virus type 1 (HIV-1) infection are prevalent. OBJECTIVE: To identify proportions of representative (test) populations who are reactive to the tuberculin skin test, and to study the relationship between CD4 T-lymphocyte counts and the induration size of the tuberculin skin test in these groups. DESIGN: A group of 435 healthy students was tuberculin skin tested in order to evaluate the intensity of skin testing in a 'normal' population. The study group consisted of 195 subjects with or without tuberculosis, and with or without HIV-1 infection, who received a tuberculin skin test and a CD4 T lymphocyte count on the same day. RESULTS: In total, 90% of the control (nontuberculous, HIV negative) subjects, 32% of the HIV-1 seropositive subjects, 76.5% of the tuberculous patients and 57% of the tuberculous HIV-1 seropositive patients were tuberculin positive. There was no direct correlation between the induration size of reactions to the tuberculin skin test and CD4 T-lymphocyte count in these study groups using linear regression analysis. CONCLUSION: In vivo skin testing using tuberculin yields clinically significant information on the degree of immunodeficiency which is different from that of CD4 T-lymphocyte counts. The tuberculin skin test should therefore be used as an independent marker of the weakened immunological status of HIV-1 seropositive subjects.

Differential susceptibility to activation-induced apoptosis among peripheral Th1 subsets: correlation with Bcl-2 expression and consequences for AIDS pathogenesis.

It has been proposed that HIV infection is associated with an imbalance in Th1 and Th2 subsets. Recent reports indicate that Th1 and Th2 effectors differ in their susceptibility to activation-induced apoptosis. To determine whether increased T cell apoptosis in HIV-infected patients contributes to alterations in cytokine synthesis, we performed single-cell analysis of type 1 and type 2 cytokine production by CD4 and CD8 T cells, simultaneously with detection of apoptosis. We demonstrate that a differential alteration in representation of Th1 subsets, rather than commitment of T cells to secrete Th2 cytokines, occurs throughout HIV infection. A significant decrease in the number of IL-2- or TNF-alpha-producing T cells was observed, whereas those producing IFN-gamma remained preserved. Furthermore, there is a gradient of susceptibility to activation-induced apoptosis (IL-2 < IFN-gamma < TNF-alpha) among the different Th1 subsets. This gradient was detected in both CD4 and CD8 subsets, as well as in control donors and HIV-infected patients, in whom the susceptibility to apoptosis of IL-2 and IFN-gamma producers was increased compared with controls. This differential intrinsic apoptosis susceptibility of Th1 effectors was found to be tightly regulated by Bcl-2 expression. In HIV-infected persons, disappearance of IL-2-producing T cells was a good indicator of disease progression and was correlated with the progressive shrinkage of the CD4+ CD45RA+ T cell compartment and a gradual increased susceptibility to activation-induced apoptosis of the IL-2-producing subset. This close relationship between the CD45RA/CD45R0 ratio, the level of type 1 cytokine production, and susceptibility to apoptosis should be considered in HIV-infected patients under antiviral or immune-based therapies.

CD4+ T-lymphocyte counts in HIV infection: are European standards applicable to African patients?

CD4+ lymphocyte count (CD4+ LC) is a widely used marker of Human Immunodeficiency Virus (HIV) immune impairment. Physiological lymphocytosis is frequently encountered in Africans. Therefore, we tried to determine if given CD4+ LC levels are of similar significance in European versus African HIV-infected individuals. Lymphocyte phenotyping of 750 HIV-infected adults was retrospectively analyzed. Three hundred and seventy patients were consecutively selected in Paris, France; 185 in Abidjan, Côte d'Ivoire; and 195 in Bobo-Dioulasso, Burkina Faso. In the three settings, lymphocyte phenotyping was performed by flow cytometry using similar protocols. Data from Abidjan and Bobo-Dioulasso were combined on the basis of geographic proximity and contrasted with those from Paris. Geometric mean levels of Total Lymphocyte Count (TLC), CD4+ LC, CD8+ lymphocyte count (CD8+ LC), and CD4:CD8 ratio, adjusted for percentage of CD4+ T-cells (%CD4+), were compared between Africans and Europeans. For a given %CD4+, TLC and CD4+ LC but not CD8+ LC tended to be about one third higher in West African than in French adults (p < 0.0001). Approximate equivalencies of absolute CD4+ counts in French and West African HIV-infected adults suggest that where thresholds of 200 and 500 CD4+ cells/microliter are applied in Europe, it might be appropriate to apply a threshold of approximately 250 and 700 CD4+ cells/microliter in West Africa, respectively. Establishing indicators of progression of HIV infection with locally appropriate thresholds may represent important steps toward improvement of HIV disease management in Africa.

PIP: CD4+ T-lymphocyte count (CD4+ LC) is a widely used marker of HIV immune impairment. The authors explored whether given CD4+ LC levels have the same significance in European HIV-infected individuals as they do in similarly infected Africans. 370 HIV-infected adults were consecutively selected in Paris, France, 185 in Abidjan, Cote d'Ivoire, and 195 in Bobo-Dioulasso, Burkina Faso, to undergo retrospective lymphocyte phenotyping using flow cytometry. Data from Abidjan and Bobo-Dioulasso were combined on the basis of geographic proximity and contrasted with those from Paris. Geometric mean levels of total lymphocyte count (TLC), CD4+ LC, CD8+ LC, and CD4:CD8 ratio, adjusted for the percentage of CD4+ T-cells, were compared between Africans and Europeans. For a given percent CD4+, TLC and CD4+ LC, but not CD8+ LC, tended to be about one-third higher in West African than in French adults. Approximate equivalencies of absolute CD4+ counts in French and West African HIV-infected adults suggest that where thresholds of 200 and 500 CD4+ cells/mcl are applied in Europe, thresholds of approximately 250 and 700 CD4+ cells/mcl may be more suitable in West Africa. Establishing indicators of the progression of HIV infection with locally appropriate thresholds may lead to the improved management of HIV disease in Africa.

Lack of chronic immune activation in HIV-infected chimpanzees correlates with the resistance of T cells to Fas/Apo-1 (CD95)-induced apoptosis and preservation of a T helper 1 phenotype.

Chimpanzees are one of the few species, along with humans, susceptible to persistent HIV-1 infection. However, HIV-infected chimpanzees do not exhibit the marked immune system alterations seen in humans and remain relatively resistant to AIDS. In humans, HIV infection leads to unresponsiveness of T cells in response to TCR stimulation, associated with increased T cell death by apoptosis. In an effort to understand some of the mechanisms used to limit lentivirus infection in African nonhuman primates, we compared apoptosis in infected humans vs chimpanzees in CD4 and CD8 T cells in relation with the expression of Bcl-2 and Fas molecules. The intensity of apoptosis in CD4 and CD8 T cells from infected chimpanzees was very low, was not inducible by several TCR-dependent activators, and was comparable to that detected in noninfected chimpanzees. Moreover, CD45RO+ and HLA-DR+ subsets, which were shown to exhibit ex vivo a high propensity to undergo apoptosis in infected humans, were not modified in infected chimpanzees. Interestingly, in contrast to the situation found in infected humans, Fas ligation by agonistic Abs or recombinant human Fas ligand on CD4 and CD8 T cells from infected chimpanzees did not induce apoptosis in these subsets even when Bcl-2 was down-regulated. Finally, this resistance to apoptosis was associated with the predominance of CD3 T cells with a Th1 phenotype. Together these observations argue for a strong relationship among the absence of chronic immune stimulation in HIV-1-infected chimpanzees, the normal control of lymphocyte survival, and the resistance to disease progression.

Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.

Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods.

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD(low) with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.