MHC-I-restricted presentation of HIV-1 virion antigens without viral replication.

Dendritic cells and macrophages can process extracellular antigens for presentation by MHC-I molecules. This exogenous pathway may have a crucial role in the activation of CD8+ cytotoxic T lymphocytes during human viral infections. We show here that HIV-1 epitopes derived from incoming virions are presented through the exogenous MHC-I pathway in primary human dendritic cells, and to a lower extent in macrophages, leading to cytotoxic T-lymphocyte activation in the absence of viral protein synthesis. Exogenous antigen presentation required adequate virus-receptor interactions and fusion of viral and cellular membranes. These results provide new insights into how anti-HIV cytotoxic T lymphocytes can be activated and have implications for anti-HIV vaccine design.

Loss of CD4 membrane expression and CD4 mRNA during acute human immunodeficiency virus replication.

Using mAbs and genomic probe to the CD4 molecule, the HIV receptor, we demonstrated that HIV replication induces the disappearance of its functional receptor from the cell surface by two distinct mechanisms. First, after being expressed onto the cell surface, HIV envelope gp110 will complex CD4, efficiently masking the CD4 epitope used by the virus to bind its receptor. This phenomenon occurs on the surface of each infected cell and is not due to the release of soluble gp110; infection with recombinant HIV/vaccinia viruses expressing a mutated HIV env gene designed to prevent gp110 release from the cell surface induces a similar gp/CD4 complexes formation. Second, virus replication induces a dramatic and rapid loss of CD4 mRNA transcripts, preventing new CD4 molecules from being synthesized. These two mechanisms of receptor modulation could have been developed to avoid reinfection of cells replicating the virus as well as to produce more infectious particles. These results suggest that the classical virus interference documented for other retroviruses might not only be due to receptor/envelope interaction, but might also depend on receptor gene expression.

Severity of lymphocytic choriomeningitis virus disease in different strains of suckling mice correlates with increasing amounts of endogenous interferon.

A marked difference was observed in the severity of disease in lymphocytic choriomeningitis (LCM) virus-infected suckling BALB/c, Swiss, and C3H mice. BALB/c mice had minimal liver lesions and none died, whereas C3H mice had extensive liver lesions and all mice died. An intermediate pattern was oberved for Swiss mice (36% mortality). Although there was no difference in the titers of LCM virus in the plasma or liver between these three strains of mice, there was a marked difference in the amount of interferon produced and the duration of interferonemia. C3H mice produced more interferon than Swiss mice which produced more interferon than BALB/c mice, indicating a direct correlation between the amount of interferon induced by LCM virus and the extent of disease. Inoculation of a potent anti-mouse interferon globulin markedly reduced the incidence of mortality in virus-infected C3H mice. BALB/c mice were as sensitive to the effects of interferon as C3H mice because daily administration of potent interferon preparations did induce disease in this strain. This ensemble of results supports our contention that endogenous interferon is in large part responsible for the manifestaions of acute LCM virus disease in suckling mice.

Direct action of interferon and inducers of interferon on tumor cells in athymic nude mice.

Two interferon-mediated enzyme activities, the protein kinase and pppA (2'p5'A)n synthetase (2-5A synthetase) were used to assess the presence and action of interferon on HeLa tumor cells in athymic nude mice. The protein kinase is manifested by the phosphorylation of endogenous proteins with a molecular weight of 67,000 and 72,000 in mouse and human cells, respectively. Treatment of HeLa tumor-bearing mice with mouse interferon (alpha and beta) resulted in enhanced levels of 2-5A synthetase and protein kinase (Mr 67,000) activities in the spleen and lung while there were no apparent effects on HeLa cells. In these HeLa tumor cells of human origin, the 2-5A synthetase and protein kinase (Mr 72,000) activities were enhanced considerably only after treatment of mice with human fibroblastic (beta) interferon. When HeLa tumor-bearing mice were given injections of polyadenylate-polyuridylate or with polyinosinylate-polycytidylate, then the 2-5A synthetase and the protein kinase activities were enhanced in tumor cells [protein kinase] as well as in the different tissues [protein (Mr 67,000) kinase] of mice since both mouse and human interferons were produced under these conditions. These results indicate a direct action of interferon on homologous tumor cells, and furthermore they indicate that tumor cells in an organism may themselves produce interferon and respond to their own interferon.

Wastewater stabilisation ponds: Sludge accumulation, technical and financial study on desludging and sludge disposal case studies in France.

Waste stabilisation pond treatment was widely developed during the 1980s in France, where there are now over 3,000 plants. Desludging the ponds has now become essential. In 19 primary facultative ponds, in operation for 12-24 years, the net average sludge accumulation rate was 19 mm/yr. The average per capita accumulation rates ranged from 0.04-0.148 m3/person.year (mean of 0.08 m3/person.year). In primary facultative ponds the volume of sludge represented 15-39% of the total volume of the basin. A filling rate above 30% necessitates desludging. In France, a desludging interval of 15 years is recommended for primary facultative ponds. The cost evaluation of desludging and landspreading showed differences according to the desludging technique used. Desludging after emptying the water had an average cost of 38 Euro/m3 of sludge with 10% dry solid (range from 20 to 83 Euro/m3). Under-water desludging was 50% more expensive. Although desludging is carried out only after several years of operation, its cost must be allowed for in the annual operation and maintenance costs of the process. It can be estimated to be 3 l/person.year. Even with this additional cost, waste stabilisation pond treatment remains less expensive than other treatment processes.

HIV-specific CD8+ T-cell immune responses and viral replication.

AIM: To review current knowledge of CD8+ T cells in relation to their effect on the replication of HIV and on disease progression. PRESENT KNOWLEDGE: Both CD8+ cytotoxic T lymphocytes capable of killing cells expressing HIV antigens and CD8+ lymphocytes that suppress HIV replication in vitro are detectable in response to HIV infection. CONCLUSION: These CD8+ T cells may help to maintain a low viral load in vivo, thus allowing a long asymptomatic period of infection.

Autoantibodies typical of non-organ-specific autoimmune diseases in HIV-seropositive patients.

OBJECTIVE: To analyse serological aspects of systemic autoimmunity in HIV-1-seropositive patients and in individuals at risk for AIDS. DESIGN AND METHODS: The reactivity of antibodies in the serum of 100 HIV-1-seropositive patients was investigated by enzyme-linked immunosorbent assay (ELISA) using a series of antigens known to be recognized by antibodies from patients with multisystemic autoimmune diseases, such as systemic lupus erythematosus, mixed-connective tissue disease and Sjögren's syndrome. RESULTS: High levels of immunoglobulin G (IgG) antibodies reacting with double-stranded DNA (dsDNA), synthetic peptides of ubiquitinated histone H2A, Sm-D antigen, U1-A RNP antigen and 60 kD SSA/Ro antigen were found in 44-95% of HIV-infected patients. Among histone antibodies, the most frequent reactions were towards the carboxy-terminal region of histone H1 and to histone H2B and its amino-terminal domain 1-25. Eight HIV-1-seropositive patients at different stages of disease according to the Centers for Disease Control classification were also studied. In most cases, no obvious fluctuations were observed over several years. Antibodies were found early, and their specificity and apparent level of activity remained relatively constant. There was no evidence of such an autoimmune response in the serum of high-risk homosexual seronegative men. CONCLUSIONS: Although the aetiology of AIDS is known, in general the aetiology of multisystemic autoimmune diseases remains to be determined, and the sequence of events taking place remains obscure in both cases. It is possible that the large spectrum of antibodies found in HIV-infected patients reflects a specific stimulation of B-cells by nuclear antigens released by apoptosis during an early stage of disease.

Plasma protein kinase activity enhanced by interferon is found in platelets.

A protein kinase activity analogous to that found in interferon-treated HeLa cells is detectable in human plasma rich in platelets. This kinase activity is manifested by the phosphorylation of an endogenous Mr 72000 protein which could be conveniently assayed after partial purification on poly(G)-Sepharose. Here, we show that the protein kinase system in the plasma consists of at least 2 components. The protein kinase is found to be localised in the platelets whereas most of the substrate (the Mr 72000 protein) is found free in the plasma and a fraction of it associated with the surface of platelets.

A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.

Characterization of an HIV-1 p24gag epitope recognized by a CD8+ cytotoxic T-cell clone.

A CD8+ cytotoxic T-cell clone that recognized HIV p24gag was isolated from an infected individual. The minimal epitope was localized to amino acids 308-316 (QASQEVKNW). Using allogeneic target cells, we found that lysis was restricted by the HLA-Cw0401 molecule. We observed that C1R cells, that express the HLA-Cw0401 allele are able to present the peptide to the cytotoxic clone, but with reduced efficiency. Other B-cell lines, that have been genotyped as HLA-Cw0401+ were unable to present the peptide to the clone, suggesting the existence of other variants of HLA-Cw0401 or a loss of cell surface expression of this molecule.

Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

Induction of interferon by streptococcus pyogenes extracellular products.

Lymphocyte-activating streptococcal exoproteins, which were previously characterized, have been tested for their capacity to induce interferon in vitro. Two out of the 3 different streptococcal fractions, studied on mice splenocytes, were shown to elicit the production of a significant amount of interferon. A large proportion of the interferon detected in the supernatants from mice activated spleen cells was acid-labile interferon. The highest level of interferon titer was obtained with the streptococcal fraction identified as the erythrogenic toxin.

Efficient antigen presentation to cytotoxic T lymphocytes by cells transduced with a retroviral vector expressing the HIV-1 Nef protein.

In the classic model of antigen processing and presentation, viral antigens must be synthesized within the cytoplasm of infected cells to be processed and presented to CD8+, MHC class I-restricted cytotoxic T lymphocytes (CTLs). We have examined the utility of a retroviral vector (pNeoNef) expressing the human immunodeficiency virus type (HIV-1)Lai Nef protein for the development of target cells to study HIV-specific CTLs. Autologous Epstein-Barr-transformed B cell lines (EBV-B cells) transduced with pNeoNef were efficiently lysed by CTL lines from donors capable of lysing EBV-B cells infected with a recombinant vaccinia virus (rVV) expressing Nef. Also, the transduced cells were efficient stimulator cells for the generation of Nef-specific CTL lines. The CTL lines thus generated recognized the same epitopes as CTL lines from the same donor generated by nonspecific stimulation. The use of similar cell lines transduced with retroviral vectors expressing HIV proteins may be useful in the study of CTLs in HIV-infected donors and in the study of the ability of candidate vaccines, including rVV, to induce HIV-specific CTLs. As antigen-presenting cells, the cell lines may be useful in the generation of antigen-specific CTL lines.

A long-term follow-up of an HIV type 1-infected patient reveals a coincidence of Nef-directed cytotoxic T lymphocyte effectors and high incidence of epitope-deleted variants.

Cytotoxic T lymphocytes (CTL) play a critical role in controlling human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, in spite of developing a strong CTL response most HIV-1-infected patients eventually progress to AIDS. Amino acid changes in CTL epitope have been previously described and may permit HIV to escape from CTL immune responses. The importance of CTL selection pressure in controlling the course of viral evolution in HIV-infected patient remains debatable. For over a 10-year period, we longitudinally followed a patient for bulk unstimulated effector (eCTL) and stimulated memory CTL responses (mCTL) against the viral proteins Gag, Pol, and Nef. The patient showed a strong CTL response against Nef in unstimulated peripheral blood mononuclear cells with a peak during Month 40 of the follow-up. The mCTL response was also higher against Nef than Gag and Pol. PCR amplification and nucleotide sequencing of the plasma viral variants showed a viral variant with the epitope deletion that was detected early during the follow-up and essentially replaced the wild-type virus during the peak eCTL response. These studies support the importance of Nef epitope deletion as a mechanism for HIV-1 escape from CTL immune pressure.

Membrane expression of HIV envelope glycoproteins triggers apoptosis in CD4 cells.

The cytopathic effect of HIV-1 and HIV-2 in CD4+ lymphocytes has been shown to be associated with apoptosis or programmed cell death. Using different experimental conditions, we demonstrate here that apoptosis is triggered by cell membrane expression of the mature HIV envelope glycoproteins, gp120-gp41 complex, and their interaction with CD4 receptor molecules. Viral entry alone did not induce apoptosis but virus replication was required in order to produce the gp120-gp41 complex. Indeed, expression of the HIV env gene alone in the CD4+ T cell line (CEM) was sufficient for the induction of apoptosis. In general, syncytium formation and apoptosis induction were closely associated as both events require functional envelope glycoproteins and CD4 molecules. Nevertheless, apoptosis but not syncytium formation was suppressed by a monoclonal antibody against CD4 that does not affect gp120 binding. Furthermore, single-cell killing by apoptosis was observed in infected cell cultures treated with a monoclonal antibody against gp41, which completely abolishes the formation of syncytia. These results indicate that apoptosis is not the consequence of toxic effects induced by the formation of syncytia but is triggered by the HIV envelope glycoproteins. Therefore, cell death during HIV infection in CD4+ lymphocyte cultures is due to a specific event triggered by the gp120-gp41 heterodimer complex programming death in metabolically active cells.

A prime-boost approach to HIV preventive vaccine using a recombinant canarypox virus expressing glycoprotein 160 (MN) followed by a recombinant glycoprotein 160 (MN/LAI). The AGIS Group, and l'Agence Nationale de Recherche sur le SIDA.

The safety and the immunogenicity of a recombinant canarypox live vector expressing the human immunodeficiency virus type 1 (HIV-1) gp160 gene from the MN isolate, ALVAC-HIV (vCP125), followed by booster injections of a soluble recombinant hybrid envelope glycoprotein MN/LAI (rgp160), were evaluated in vaccinia-immune, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 20) received vCP125 (10(6) TCID50) at 0 and 1 month, followed randomly by rgp160 formulated in alum or in Freund's incomplete adjuvant (FIA) at 3 and 6 months. Local and systemic reactions were mild or moderate and resolved within the first 72 hr after immunization. No significant biological changes in routine tests were observed in any volunteer. Two injections of vCP125 did not elicit antibodies. Neutralizing antibodies (NA) against the HIV-1 MN isolate were detected in 65 and 90% of the subjects after the first and the second rgp 160 booster injections, respectively. Six months after the last boost, only 55% were still positive. Seven of 14 sera with the highest NA titers against MN weakly cross-neutralized the HIV-1 SF2 isolate; none had NA against the HIV-1 LAI or against a North American primary isolate. Specific lymphocyte T cell proliferation to rgp 160 was detected in 25% of the subjects after vCP125 and in all subjects after the first booster injection and 12 months after the first injection. An envelope-specific cytotoxic lymphocyte activity was found in 39% of the volunteers and characterized for some of them as CD3+, CD8+, MHC class I restricted. The adjuvant formulation did not influence significantly the immune responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Early HIV-specific cytotoxic T lymphocytes and disease progression in children born to HIV-infected mothers.

The activities of HIV-specific cytotoxic T lymphocytes (CTLs) were evaluated in 10 HIV-infected children, born to infected mothers who did not receive AZT during pregnancy. CTL activities were present as early as 4 months of age. The five children that progressed to AIDS before 1 year of age had reduced in vivo and in vitro CTL activities, when compared with children who remained AIDS free after 1 year of age. The latter children had weak in vivo activated CTL responses but strong memory CTLs. No relation was found between viral load, lymphocyte populations, and CTL responses between birth and 6 months of age. Between 7 and 12 months old, children with broader in vitro activated CTLs had higher absolute numbers of CD4+ and CD8+ T lymphocytes and lower plasma viral load. These data support a beneficial role of CTLs in pediatric HIV infection.

Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS.

The duration of human immunodeficiency virus (HIV-1) infection prior to the development of AIDS is variable, and for most patients the exact time of infection is not known. A group of 38 HIV-1-infected subjects was tested while asymptomatic for comparative cytotoxic lymphocyte responses to the Gag and envelope antigens of HIV-1. Twenty of the 38 patients had no detectable primary cytotoxic T lymphocyte (CTL) response to Gag, and this was associated with a relative risk of 1.89 for progression to ARC or AIDS during the subsequent 3 to 40 months of observation when compared with patients who had Gag-specific CTL activity at the beginning of the observation period. In contrast, no significant association was observed between envelope-specific cytotoxic activity and disease progression. Other patient characteristics, including CD4+ T lymphocyte counts and antibody levels to the p24gag protein, measured at the start of observation, did not correlate with disease progression during the observation period. This suggests that the anti-Gag CTL response may be protective during HIV-1 infection.

Cell-mediated immune proliferative responses to HIV-1 of chimpanzees vaccinated with different vaccinia recombinant viruses.

The only animal that can be reproducibly infected with HIV, and that thus provides an experimental system for testing the effectiveness of prototype vaccines, is the chimpanzee. We compared proliferative responses to HIV and to vaccinia virus (VV) antigens of lymphocytes taken at various times from chimpanzees vaccinated with recombinant VV expressing different HIV genes. Animals were immunized with the original VV strain, as control, or with constructs expressing gp160 (VV160) given exclusively or in combination with one or two other constructs producing p25 (VV25), F/3'-orf (VVF), or the human interleukin-2 (IL-2) gene, which was included in an attempt to amplify immune responses. Irrespective of the HIV gene utilized, lymphocyte proliferation to HIV was usually weak and rapidly decreased after each inoculation, contrasting with strong and sustained responses to VV. Lack of adequate recall reactivity after challenge with fixed autologous lymphocytes expressing VV-produced HIV antigens indicated that vaccination resulted only in low levels of HIV-specific memory cell priming. The use of IL-2-producing VV did not lead to increased responsiveness. Reactivity to soluble purified gp160, but not to p25, could be detected in PBL from animals that had received both VV160 and VV25, while immunization with VVF resulted in a significant response to this protein in one of two animals. The transient nature of T cell reactivity to HIV might explain why, in similar studies, chimpanzees were not protected from infection with live HIV.

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation.

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50-25000 µg g-1 ) and cadmium (1-170 µg g-1 ). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.