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The complex homeostasis of tissues is coordinated by the cytokine network and imbalances in this network may result in chronic immune disorders. Key specific cytokines, such as TNF-α, IFN-α, IL-4 or VEGF have been demonstrated to be overproduced or abnormally released in the microenvironment of pathologic tissues. These findings have opened up the way to passive immunotherapy with anticytokine monoclonal antibodies. Even though passive immunotherapy has proved to be efficient, it is hampered by specific limitations. The discovery of a family of immunogens, the kinoids, consisting of inactivated cytokine derivatives, has led some to propose them for active immunotherapy as an alternative to passive immunotherapy. This review focuses on kinoids – on their validation in experimental mouse models and ongoing clinical trials. The advantages offered by this active immune therapy in terms of efficacy, safety and patient compliance will be stressed.

Current therapeutic vaccine trials in major chronic diseases including AIDS, cancer, allergy and autoimmunity, target antigenic pathogens but not the pathogenic stromal cytokines which can be major sources of histopathologic processes. Considering that the limited efficacy of these vaccines has been ascribed to local pathogen-induced cytokine dysfunction, we propose to antagonize pathogenic cytokine(s) by high affinity neutralizing auto-Abs triggered by specific anti-cytokine vaccines. As anticipated by theoretical considerations, animal experiments and initial clinical trials showed that anti-cytokine immunization was safe, well tolerated and triggered transient high titers Abs neutralizing pathogenic cytokines but, in contrast to conventional vaccines, no relevant cellular response was observed. Advantages of active versus passive anti-cytokine Ab therapy, particularly for long-term treatments, as those required in AIDS, cancer, allergy and autoimmunity include greater ease of maintaining high Ab titers, lack of anti-antibody responses and low cost.

Summary The need to modify tumor cells in order to render them more “immunogenic” was based on the assumption that normal, nonmodified tumor cells are non or weakly immunogenic and as such are unable to raise an efficient protective immune response. Various methods for “xenogenization” (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens. Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-[3,3-dimethyl-1-triazeno]-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4 [3H]-one 8-carbamoyl-3-[2-chloroethyl]imidazole [5,1 -d]-1,2,3,5-tetrazin-4[3H]-one) and antiemetic drugs, were tested in experimental models of murine leukemia. Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma. Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde. Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells. In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization. The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.

HPV-16 E6 and E7 oncoproteins impair the cell cycle in human uterine cervix carcinoma cells (HUCC) by acting on p53 and retinoblastoma proteins, respectively. We recently reported that E7 related into the extracellular compartment by HUCC SiHa cells could inhibit immune T-cell response to recall and alloantigens by a mechanism involving an overproduction of the immunosuppressive IFN alpha by antigen presenting cells (APCs). In this study, we found that besides E7, E6 protein and the vascular endothelium growth factor (VEGF) were released into the SiHa cell supernatants, and we further showed that extracellular E7 but not E6 oncoprotein 1) inhibits the immune cell response to recall and alloantigens, and 2) enhances the release of angiogenic cytokines, including TNF alpha, IL-1 beta and IL-6 by macrophages and/or dendritic cells. VEGF unexpectedly released by cancer cells could also contribute to angiogenesis. Thus in HUCC the same E7 oncoprotein which contributes to controlling the cancer cell cycle has the means in its extracellular configuration to contribute to microenvironmental immunosuppressive and angiogenic processes. Neutralizing anti-E7 antibodies either passively administered or induced by active immunization could represent a new immunotherapeutic endeavour to combat the immunosuppression and/or neoangiogenesis effects of extracellular E7 protein.

This randomized, double-blind, placebo-controlled, phase II/III study was designed to evaluate safety, immunogenicity, and efficacy of an active anti-interferon-alpha (anti-IFN-alpha) vaccine in asymptomatic HIV-1-infected patients. The active immunization was aimed at inducing anti-IFN-alpha antibodies to counteract IFN-alpha overproduction. In all, 242 patients, recruited between December 1995 and July 1996 in eight centers in Europe and Israel, with CD4+ counts from 100 to 634 cells/mm3 who were receiving or not receiving antiretroviral therapy (including protease inhibitors) were randomized to receive either anti-IFN-alpha vaccine or placebo. The anti-IFN-alpha immunization regimen consisted of three priming injections delivered intramuscularly at 1-month intervals in a water-in-oil emulsion of inactivated recombinant IFN-alpha-2b (i-IFN-alpha) followed by intramuscular booster injections of i-IFN-alpha adsorbed onto calcium phosphate every 3 months. Immunogenicity to vaccine was defined as an increase of anti-IFN-alpha antibody level of more than twofold the preimmunization value. Clinical progression, changes in antiretroviral treatment, and decrease of CD4+ counts to200 cells/mm3 were considered endpoints for efficacy evaluation. Contrary to our previous experience, in which six to seven oil priming injections induced a90% response rate, the three oil-adjuvanted injections in this trial were suboptimal because only 40 of 122 vaccinees (33%) had raised anti-IFN-alpha antibody following immunization. In vaccinees, both antibody responders (AbRV) and nonresponders (AbNRV), the tolerance to the vaccine was good and was without evidence of significant safety concerns. During the course of the trial, 62% of vaccine responders, 64% of nonresponders, and 63% of placebo patients elected to add protease inhibitor-containing regimens as new treatment guidelines were established, resulting in a marked decrease in clinical and laboratory progression such that the expected endpoints of the study could not be achieved and further follow-up was halted. Despite the unexpectedly low immunogenicity and fewer than expected endpoints, anti-IFN-alpha vaccine recipients, in comparison with placebo recipients, showed a lower rate of disease progression, nonelective treatment changes, and/or CD4+ count decrease to200 cells/mm3, but the difference was not statistically significant. Nevertheless, the subgroup of patients immunized to IFN-alpha who experienced a rise in anti-IFN-alpha antibodies had a significantly lower rate of occurrence of HIV-1-related events and of any combination of the endpoints compared with those of either placebo patients or vaccinees who failed to develop anti-IFN-alpha antibodies, the latter two groups behaving similarly. Further studies of this approach are warranted because these data suggest a beneficial effect of this adjuvant approach.

The Duffy Antigen Receptor for Chemokines (DARC) belongs to a family of erythrocyte chemokine receptors that bind C-X-C and C-C chemokines such as interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1) and regulated-on-activation, normal T cell-expressed and -secreted (RANTES), but not macrophage inflammatory protein 1 alpha (MIP-1 alpha) or MIP-1 beta. DARC has also been identified to a receptor for malaria parasites Plasmodium vivax and Plasmodium knowlesi. In the present study, we show that HIV-1 binds to RBCs from Caucasian individuals via DARC making RBCs able to transmit HIV to peripheral blood mononuclear cells (PBMCs). Furthermore, binding of HIV-1 particles to RBCs is inhibited by treating these cells with recombinant RANTES, but not with recombinant MIP-1 alpha prior to their incubation with HIV-1. This finding suggests that RBCs may function as a reservoir for HIV-1 or as a receptor for the entry of HIV-1 into CD4-cell subsets as well as neurons or endothelial cells.

Summary Extracellular Tat can act as a viral toxin on uninfected cells of different tissues, including the CNS and the immune system, thus in order to immunize humans against Tat we have prepared a biologically inactivated but immunogenic Tat (Tat Toxoid). Tat Toxoid is not toxic in mice even at high doses. It triggers high levels of specific Tat Abs in the mouse and rabbit. Furthermore, in humans Tat Toxoid immunization was safe and induced in seronegatives persistent high levels of Tat Abs and in immunodeficient patients a significant rise of these specific Abs. Facing acute HIV-1 infection, the presence of high level of circulating Tat Abs promoted by Tat Toxoid vaccine should prevent Tatinduced immunosuppression and allow anti-HIV-1 cellular response to develop. As a consequence, early release of β-chemokines could enhance host resistance towards HIV-1, and, in infected people, inhibit viral replication and evolution towards AIDS.

Exposure to HIV type 1 (HIV-1) does not usually lead to infection. Although this could be because of insufficient virus titer, there is now abundant evidence that some individuals resist infection even when directly exposed to a high titer of HIV. This protection recently has been correlated with homozygous mutations of an HIV-1 coreceptor, namely CCR5, the receptor for the β-chemokines. Moreover, earlier results already had shown that the same chemokines markedly suppress the nonsyncitial inducing variants of HIV-1, the chief virus type transmitted from person to person. CCR5 mutation, as a unique mechanism of protection, is, however, suspect because HIV-1 variants can use other chemokine receptors as their coreceptor. Moreover, recent results have established that infection can indeed sometimes occur with such mutations. Here, we report on transient natural resistance over time of most of 128 hemophiliacs who were inoculated repeatedly with HIV-1-contaminated Factor VIII concentrate from plasma during 1980–1985 before the development of the HIV blood test. Furthermore, and remarkably, 14 subjects remain uninfected to this date, and in these subjects we found homozygous CCR5 mutations in none but in most of them overproduction of β chemokines. In vitro experiments confirmed the potent anti-HIV suppressive effect of these chemokines.

Circulating interferon (IFN) was investigated in HIV-1 seropositive patients by measuring the IFN alpha antiviral effect in the serum. While serum of healthy seronegative individuals exhibits an antiviral effect, not due to IFNs, considered as background, serum of seropositive patients showed an additional antiviral effect due to the abnormal presence of IFN alpha. Increased titers of IFN alpha were found in the course of the HIV infection and seemed to correlate with the evolution of AIDS disease. Furthermore, patients immunized against IFN alpha had both stabilized CD4 cell count and decreased IFN alpha in their serum. HIV-1-infected patients also exhibited higher titers of natural anti-IFN antibodies than seronegative controls and the level of specific antibodies (Abs) markedly increased in immunized patients. Finally, serum from immunized patients, when compared to seronegative controls, exhibits an interferon neutralizing capacity.

Probiotics are living microorganisms (e.g., bacteria) that are either the same as or similar to organisms found naturally in the human body and may be beneficial to health. Current researches have shown that the balance between beneficial and pathogenic bacteria is essential in order to maintain the oral health. Therefore, oral cavity has recently been suggested as a relevant target for probiotic applications. Dental caries can be seen as a microbial imbalance where the oral microbiota shift towards community dominance which produces acidogenic and acid-tolerant gram positive bacteria. Similarly, the accumulation of bacteria within the biofilm, facilitated by poor oral hygiene, predisposes to allogenic shifts in the microbial community, leading to the onset of periodontal inflammation. Probiotic bacteria belonging to the genus of Lactobacillus, Bifidobacterium and Streptococcus have been proven effective for preventing caries by reducing the number of cariogenic bacteria in saliva after a short period of consuming the probiotic. In contrast, the effect of probiotics on improving gingivitis and periodontitis has been less investigated. The currently available studies on the effect of probiotics on periodontal pathogens and clinical periodontal parameters showed differing results depending on the strains used and the endpoints analyzed. Many of the clinical studies are pilot in nature and with low quality, therefore, properly conducted clinical trials, using probiotic strains with in vitro proven periodontal probiotic effects, are needed. The putative beneficial effects of probiotics on oral malodour have also been evaluated, but further evidence is needed to fully explore the potential of probiotics for preventing malodour.

Since the immune system is impaired in the course of HIV-infection, the purpose of any AIDS vaccine therapy should be the restoration in the patient of an adequate immunocompetence to enable him to respond to the antigenic stimulus represented by the virus. In the present investigation we have shown the antiproliferative action on activated T-cells in culture of: sera taken from HIV-infected, but not seronegative individuals; T lymphocytes taken from seronegative subjects and infected in vitro with HIV but not non infected cells; native alpha-IFN and the time-dependent inactivation of this activity by formaldehyde treatment of alpha-IFN. Thus is confirmed the major contribution provided by alpha-IFN to the immunosuppression occurring in the course of HIV-infection. These results also strongly support the new AIDS vaccine therapy strategy based on the administration to HIV-infected patients of inactivated, but still immunogenic alpha-IFN. To the alpha-IFN treatment could also be combined the administration of fixed autologous suppressive cells. The induction of gamma-IFN in addition to alpha-IFN production by stimulation of cells from healthy donors with gp120 should encourage the use of a vaccine combining both inactivated alpha-IFN and gamma-IFN. On the other hand, the IL-12 cytokine with its potential to restore compromised cell-mediated functions associated with HIV infection should also be a valuable adjuvant treatment.

The purification and analysis of IgGs from sera of HIV-1-infected and non infected individuals are reported. The effect of antibodies purified from sera of infected individuals on antigen-induced T cell proliferation was investigated in relation to their possible involvement in an autoimmune reaction in AIDS, in view of the previously unravelled striking peptide similarities between HIV-1 gp120 and the immunoregulatory CD4 and Fas molecules. However, our data do not allow definite conclusions to be drawn. The necessity of purifying antibodies against specific peptides to show their direct effect on T-cell activation is further stressed.

Four cardinal immune disorders interacting with each other may promote the progressive T cell depletion and immunosuppression characterizing AIDS. Immune activation of HIV-1 infected T4 cells leads to virus release and premature cell death. Both virus release with its resulting viral load and dead cells are the source of gp120 stimulus. Anergy of non-infected CD4 cells, resulting in cytokine dysregulation may be promoted by impairing the CD4-MHC interaction during CD4 cell activation either directly through the SLWDQ pentapeptide identity with the CD4 molecule and the CD4 binding region or through a gp120-induced autoimmune reaction to CD4. Overproduction of IFNα, the known antiproliferative and cytolytic cytokine may promote in a paracrine manner to neighbouring cells the immunosuppression generated by the lack of IL2 secretion following CD4 cell anergy. Apoptosis of activated non infected T cells could be induced by effector components of the sutoimmune reaction (CTL, Lymphotoxins or Abs?) directed towards the 2 consensus gp120 sequence identity/similarity (INCTR and FYCNST) shared with the APO/Fas molecule. These two sequences are known as immunodominant sites of the gp120. Furthermore, IFNa overproduction may also render circulating memory T cells competent to apoptosis by upregulating the cascade of metabolic events leading to programmed cell death.

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10–secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti–IFN-α Ab immunotherapy in lupus patients.

Adjuvants have been used to augment the immune response in experimental immunology as well as in practical vaccination for more than 60 years. The chemical nature of adjuvants, their mode of action and the profile of their side effects are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions. The most common adjuvants for human use today are still aluminium hydroxide, aluminium phosphate and calcium phosphate although oil emulsions, products from bacteria and their synthetic derivatives as well as liposomes have also been tested or used in humans. In recent years monophosphoryl lipid A, ISCOMs with Quil-A and Syntex adjuvant formulation (SAF) containing the threonyl derivative of muramyl dipeptide have been under consideration for use as adjuvants in humans. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side effects.

Antiviral therapy, including antiprotease treatment, suppresses viral replication, but it does not restore the HIV-1 induced immunopathogenesis which includes IFN alpha overproduction and cellular immunosuppression. To combat HIV-1 induced immunopathogenesis, anti-IFN alpha kinoid immunization in combination with tritherapy may be beneficial to HIV-1 infected immunodeficient patients.

HAART (highly active antiretroviral therapy) suppresses but does not eradicate HIV-1 infection. However, since the antiretroviral agents used in HAART may also be toxic in the long-term, immunotherapies which correct HIV-1 immunosuppression or the cytokine dysregulation associated with it may be beneficial. In this respect, a double blind multicentric placebo-controlled phase II/III anti-IFN alpha vaccine trial has been carried out on 242 HIV-1 patients, the majority of whom were undergoing HAART treatment. In vaccinated patients (vaccinees) who responded to immunization by increased levels of IFN alpha Abs (whether under HAART or not) when compared to placebo or non-responder vaccinees, a strong correlation was found between an increased IFN alpha neutralizing capacity and the reduction of clinical manifestations.

The abnormal cytokine release in the stromal microenvironment of pathologic tissues, contributes to the pathogenesis of viral infections such as AIDS, cancer and auto-immune diseases. Neovacs developed therapeutic vaccines, named Kinoids, which induce anti-cytokine Antibodies. Kinoids are non toxic but immunogenic cytokine derivatives. Kinoid immunizations induce high titre of neutralizing Abs to the corresponding cytokine, is well tolerated and experimentally effective. In transgenic mice expressing huTNFalpha, the TNFalpha kinoid decreases clinical signs of Rheumatoid Arthritis and in mice challenged with syngenic CT26 tumor cell line huVEGF kinoid inhibits lung metastases. After validation by clinical trials, kinoid vaccines could represent a second generation of specific immune therapy to be used to combat ectopic cytokines.

A procedure for the routine and simultaneous laboratory detection of IgG antibodies produced in humans in the course of various infectious diseases is described. The procedure, based on dot-enzyme-linked immunosorbent assay (ELISA), used single nitrocellulose strips onto which several antigens were dotted in close proximity. Optimal conditions were specified that allowed the unequivocal and simultaneous detection of IgG antibodies specifically directed against Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus type 1 and type 2 antigens. This technique has proved to be simultaneously specific, sensitive, and reliable, and it has been applied to prenatal screening of sera from pregnant women. It is suggested that this technique should also be used for the screening of large numbers of sera under field trial conditions.

International audience; Tumor growth depends on blood supply, requiring the development of new vessels, and vascular endothelial growth factor (VEGF) plays a central role in neoangiogenic processes. For this reason, VEGF represents a target for the development of new therapeutic antiangiogenic molecules. Clinical trials using anti-VEGF mAbs such as bevacizumab have validated the efficacy of this therapeutic approach but have also revealed adverse effects. Here we report that a VEGF-derived immunogen, consisting of a heterocomplex of a murine (m)VEGF and keyhole limpet hemocyanin, called "mVEGF kinoid," triggered a strong Ab immune response in mice. The anti-VEGF Abs inhibited both the proliferation of human umbilical vein endothelial cells cultured in the presence of mVEGF and the binding of mVEGF to its receptor-2 Flk-1. In mVEGF kinoid-immunized BALB/c mice challenged with syngeneic CT26 colorectal tumor cells, the number and size of lung metastases were significantly decreased. In human (h)VEGF kinoid-immunized BALB/c mice, high levels of serum Abs to hVEGF were present, and purified IgG from these mice decreased by > or =50% the tumor growth of human A673 rhabdomyosarcoma cells and HT29 colon carcinoma xenografted in Swiss nude and NOD/SCID mice, respectively. Tumor cell growth inhibition was similar to that observed in mice receiving therapeutic doses of bevacizumab. These experiments suggest that a therapeutic vaccine containing VEGF kinoid may represent a strategy for safely combating VEGF-dependent neovascularization and metastases occurring in malignant tumors.

Cytotoxic T cells are the main antigen-specific effector cells of the cellular immune system and MHC class I restricted cytotoxic T-lymphocyte (CTL) responses in mice, acting against the HIV-1 envelope protein, are known to be predominantly directed against an amino acid sequence in the third hypervariable domain. We have investigated the epitope specificity of anti-HIV-1 CTL in healthy human volunteers inoculated with a recombinant vaccinia expressing the HIV-1 gp160 envelope gene. Their isolated lymphocytes were stimulated in vitro with autologous HIV-1 infected cells. Our results show that immunization with recombinant virus is able to generate virus-specific CTLs to the HIV-1 gp160 envelope protein and to a 15-residue synthetic peptide corresponding to a highly variable region of the envelope p18(IIIB). The CTL response was restricted by class I MHC molecules HLA-A2 and A3 that commonly occur in the human population.

Summary Extracellular Tat can exercise its deleterious effects on cells surrounding HIV-1-infected cells and allow spreading of virus. Extracellular Tat should be neutralized by anti-Tat vaccination using as an immunogen a functionally inactivated but immunogenic Tat preparation (Tat toxoid). In the present paper we show that native Tat can be inactivated without impairment of its immunogenicity by subjecting it to various chemical treatments. Since the carboxyamidation reaction can be easily monitored and the carboxyamidated Tat retained the whole immunogenicity of the native molecule, it should be the toxoid of choice for mass immunization.

Pneumococcal infections are still a cause of high morbidity and mortality in elderly populations. Since antibiotics are not wholly effective, vaccination should be performed. The response to pneumococcal vaccination of a limited group of elderly persons aged 60 to 90 years was studied. The sera of most individuals were found to contain antipneumococcal antibodies prior to vaccination. Vaccination resulted in antibody level increases in sera of all subjects except two. On the basis of antibody levels, 19 out of 20 vaccinees proved to be protected after vaccination. Combination of vaccination with a non-specific immunostimulation enhanced the antibody response.

The human papillomavirus type 16 (HPV-16) E7 oncogenic protein is found in the culture supernatant of SiHa cells, a cervical carcinoma cell line. Extracellular E7 protein, acting as a viral toxin in human immune cells, induces the overproduction of the immune suppressive IFN alpha cytokine by APCs, and inhibits the T-cell response to recall and allogenic antigens. These effects should be taken into account for the design of anti-human cervical carcinoma vaccines.

Summary Cultured human peripheral blood monocytes (PBMC) and the cell line H9 release a lectin. This lectin is not the previously described sarcolectin, since it does not specifically recognize the sugars lactose and sialic acid. The lectinic T-cell factor reduces the release by APCs of IFNα — a key cytokine known to inhibit the proliferation of activated T-lymphocytes.

Because administration of Tat protein, the HIV-1 toxin that induces immunosuppression and apoptosis, may be deleterious to the host immune system, a chemically inactivated but nonetheless immunogenic Tat preparation, Tat toxoid, was used to immunize seronegative individuals against Tat. In an open, controlled, phase I clinical trial, Tat toxoid turned out to be safe, well tolerated, and able to trigger a specific immune reaction. In particular, a threefold to more than 10-fold increase of circulating antibodies directed against the native Tat was observed after immunization in all of 5 immunized study subjects, together with a positive reaction to delayed-type hypersensitivity (DTH) skin test with Tat toxoid in vivo and increased lymphoproliferative response to native Tat in vitro. Persistent (> or =1 year) high levels of circulating anti-Tat antibodies could prevent the Tat-induced immune suppression and, following HIV-1 exposure, allow the anti-HIV-1 cellular immune response, with its early release of protective beta-chemokines, to occur leading to an increase of host resistance, that is, protection.

HIV type 1 (HIV-1) not only directly kills infected CD4+T cells but also induces immunosuppression of uninfected T cells. Two immunosuppressive proteins, interferon α (IFNα) and extracellular Tat, mediate this process because specific antibodies against these proteins prevent generation of suppressor cells in HIV-1-infected peripheral blood mononuclear cell cultures. Furthermore, the production of C-C chemokines in response to immune cell activation, initially enhanced by IFNα and Tat, ultimately is inhibited by these proteins in parallel with their induction of immunosuppression. The clinical corollary is the immunosuppression of uninfected T cells and the decline in C-C chemokine release found at advanced stages of HIV-1 infection paralleling rising levels of IFNα and extracellular Tat. We, therefore, suggest that IFNα and Tat may be critical targets for anti-AIDS strategies.

Summary Peripheral blood mononuclear cells (PBMCs) are physiologically activated by interleukin (IL)-2. We found that oyster extract (JCOE) currently used as a functional nutrient enhanced in vitro the IL-2 dependent activation as measured by cell count, 3 H-thymidine uptake and up-regulation of a IL-2 receptor. In human immunodeficiency virus (HIV) seropositive individuals, this oyster extract-induced effect was marked in asymptomatic individuals with quasi-normal CD4 cell counts, but was weakly reflected in acquired immunodeficiency syndrome (AIDS) patients.

Twenty-seven HIV-1-infected patients, 16 at early stage of disease and without concomitant antiretroviral therapy and 11 at more advanced stage of disease receiving antiretroviral therapy, have been followed since their enrollment, November 1992 and July 1993, respectively, in phase I/II studies to evaluate safety and immunogenicity of an anti-interferon-alpha (IFN-alpha) vaccine, aimed at modulating the impaired cytokine network in AIDS patients by counteracting IFN-alpha overproduction. We compared clinical, virological, and immunological markers of disease progression, including circulating IFN-alpha levels in a 24- to 30-month follow-up period with those of 62 patients fulfilling the same enrollment criteria and comparable for sex, risk factor, and age, regularly followed at our center. Anti-IFN-alpha immunization consisted of four-six intramuscular injections 1 month apart of a water-in-oil emulsion of 500 micrograms formalin-inactivated recombinant IFN-alpha-2b (iIFN-alpha) followed by intramuscular injections of 250 micrograms iIFN-alpha adsorbed onto calcium phosphate every 3 months. Neither clinical deterioration nor a CD4+ cell count decrease from pretreatment values was observed in IFN-alpha-immunized patients in the follow-up period, whereas clinical and immunological disease progressions were observed among open-comparison patients. Furthermore, statistical analysis showed a strong association between occurrence of clinical manifestations and high circulating IFN-alpha titers, while nonprogression of IFN-alpha-immunized patients was associated with decreased levels of circulating IFN-alpha.

Summary The acute human immunodeficiency virus type 1 (HIV-1) infection of activated peripheral blood mononuclear cells (PBMCs) from normal donors results in inhibition of cell proliferation and generation of functional suppressive T cells. Cultured HIV-1 infected PBMCs but not uninfected PBMCs, following irradiation, can inhibit the proliferation of antigen-activated autologous T cells in a dose-dependent way. CD8+ cell subpopulation is responsible for this inhibition. The presence of anti-alpha interferon (IFNα) and anti-Tat antibodies in the culture medium counteracts the HIV-1-induced immunosuppression and prevents the generation of suppressive T cells by these PBMCs. The reported data should have major implications for strategies of AIDS treatment which, in association with antiviral drugs, aim at targetting immune disorders.

Automat is a novel program which finds exhaustively all the oligopeptide segments shared by a given protein of any size with the proteins of a whole databank. It allows the user to collect statistics on the composition of the sequence studied in reference to the databank used. We present here the rationale and the algorithm underlying this powerful software. Besides its immediate interest for identifying efficiently similarities between proteins (or DNAs), biological applications of this software have already been described in the case of HIV-1 viral proteins, and Automat should prove useful also for studying more generally autoimmune diseases.

A decreased number of macrophages has previously been demonstrated by means of skin-window assays in operable breast cancer patients. However, in this type of cellular inflammatory response, a varying intensity of cellular activation, cellular recruitment and macrophage chemotaxis exists. In this study, we performed skin windows after stimulation by a chemo-attractant (FMLP) and a recall antigen (Candidin-latex). Cellular responses were classified in 19 breast cancer patients according to the presence (A+) or absence (A0) of macrophage activation by comparison to healthy controls. In 13 A0 patients, the admixture of a cytokine (IFN alpha) and an immunodulator (P40) to one agent or the other or both resulted in the restoration of macrophage functions. Our study shows that uniform cellular responses to chemo-attractants and antigens are observed in healthy and not immunocompromised individuals as assessed by skin-window tests. However, the cellular response recorded in immunodeficient cancer patients is altered. It also shows that the admixture to chemoattractants and antigens of particular cytokines or better still of an immunomodulator displaying a wide spectrum of activity offers a way of restoring macrophage functions. Individual responses to immunotherapy in clinical oncology may be related to such results.

An ELISA for assessing pneumococcal anticapsular antibodies in human sera is described. Sensitization was carried out by adding to avidin-coated polystyrene plates biotinylated capsular polyosides. This ELISA was used for the determination of anticapsular antibody levels in pre- and post-immunization sera from 20 elderly individuals. The anticapsular antibody titres differed significantly in sera taken prior to and after vaccination. Eighteen of the 20 sera tested showed mean fold increases of the antibody level greater than 2 (2.1 to 21.0) as a result of vaccination. Since morbidity and mortality rates from S. pneumoniae infections are high in the elderly, immunization should be systematically performed. However, the elderly subject is frequently immunosuppressed, and it is therefore advisable to control the result of the vaccination, using a simple procedure, such as the one described here.

An enzyme-linked immunosorbent assay using nitrocellulose strips (dot-ELISA) for the routine laboratory detection of IgG antibodies to mumps and varicella viruses is described. The virus antigens are dotted onto nitrocellulose strips, and the dotted strips are incubated with the sera to be tested. The bound antibodies are revealed using enzyme-labeled antihuman IgG antibodies. Reliable results are obtained when the assay is carried out at 37 degrees C. The reported data indicate that the dot-ELISA can reliably be used for the detection of IgG antibodies to mumps and varicella viruses in human sera.

Immunity to tetanus was investigated in 157 individuals aged between 1 and 77 years using, for the evaluation of both tetanus antitoxin and antibody to fragment BIIb (anti-BIIb), an easily performed ELISA that gave reproducible results. Among these people 72% were protected against tetanus (tetanus antitoxin titres greater than 0.06 IU ml-1). Anti-BIIb titres greater than 0.15 U ml-1 were found in 75% of the males vs 57% of the females (P less than 0.02) with marked variations according to age. Furthermore, 13 out of 41 individuals with antitoxin titres less than 0.06 IU ml-1 (not protected against tetanus) were found to have anti-BIIb titres greater than 0.15 U ml-1. These data raise questions as to the significance and protective effect of anti-BIIb against tetanus.

T cell blasts isolated from six-day cultures of peripheral blood mononuclear cells stimulated with tetanus toxoid (TT) were cloned in a two-layer agar system in the presence of autologous irradiated PBM (iPBM) and TT. Colonies were individually isolated and expanded in interleukin 2-containing medium. The antigen specificity of three T cell clones was attested by their capacity to proliferate under restimulation by TT and not by an unrelated antigen. The clones were specific for either the alpha or the beta chain of the toxin. T cells from these clones expressed Ia determinants and antigens of the helper/inducer T cell subset as defined by anti-T monoclonal antibodies. In the case of the alpha chain-specific clone MA 11 from the donor MA, allogeneic iPBM from HLA-compatible unrelated donors, including seven donors sharing one HLA DR specificity with MA, were found inefficient as antigen-presenting cells. A familial study, however, demonstrated that antigen presentation could be obtained using nonautologous cells. The presenting capability of cells from relatives of the donor MA segregated in association with the HLA-DRw6-bearing maternal haplotype present in MA. Results suggest that MA 11 cells recognized the antigen in the context of a surface determinant closely linked to HLA-DRw6.

O tratamento de infecções crônicas recidivantes (I.C.R.) encontra muitas dificuldades. No presente estudo, o uso do imunoadjuvante P40, quer isolado, quer associado a vacinoterapia no tratamento de I.C.R., demonstrou ser muito eficaz, ao passo que a vacinoterapia isoladamente não o foi. Foi aventada a hipótese de que a imunidade mediada por células pode desempenhar papel importante no contrôle das I.C.R., uma vez que a melhora clínica dos pacientes foi acompanhada de positivação de testes cutâneos anteriormente negativos, testes estes realizados quer com o agente infeccioso específico, quer com antígeno inespecífico de refôrço. The treatment of relapsing chronic infections (RCI) encounters many difficulties. In the present study, the use of the immuno adjuvant P40 either alone or in association with vaccinotherapy for the treatment of RCI turned out to be very effective, whereas vaccinotherapy alone was not. It is hypothesized that cell-mediated immunity may play a major role in controlling RCI, since the clinical improvement of the patients kept up with the positivation of previously negative skin tests carried out with either the specific infecting agent or with recall-anti gens.

We describe a Dot-ELISA system for the rapid, specific and reliable assessment of a subject's antitetanus immune status. The conditions for Dot-ELISA were selected to give positive reactions only for sera with antitetanus antibody titers equal to or higher than 0.06 I.U./ml, considered to represent protection when using "in vitro" methods. This Dot-ELISA method could advantageously be applied to the monitoring of vaccination campaigns, as well as for assessing the antitetanus immune status of the wounded.

In the course of our studies on oxidative stress as a component of pathological processes in humans, we showed that microintrusion into cells with microcapillary and ultramicroelectrochemical detection could mimic many types of mechanical intrusion leading to an instant (0.1 s) and high (some femtomoles) burst release of H 2 O 2 . Specific inhibitors of NADPH enzymes seem to support the assumption that this enzyme is one of the main targets of our experiments. Also, human immunodeficiency virus type 1 (HIV-1) gp160 inhibits the cooperative response of uninfected T cells as well as Tat protein release by infected cells does. In this study, we analyzed in real time, lymphocyte per lymphocyte, the T-cell response following activation in relation to the redox state. We showed that the immunosuppressive effects of HIV-1 Tat and gp160 proteins and oxidative stress are correlated, since the native but not the inactivated Tat and gp160 proteins inhibit the cellular immune response and enhance oxidative stress. These results are consistent with a role of the membrane NADPH oxidase in the cellular response to immune activation.