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A major involvement of IFNα in the etiopathogenesis of systemic lupus erythematosus has been suggested by clinical observations, including the increase of serum levels of this cytokine in patients with active disease. Supporting this hypothesis, we have shown that expression of IFNα from a recombinant adenovirus (IFNα Adv) precipitates lupus manifestations in genetically susceptible New Zealand Black (NZB) × New Zealand White (NZW)F1mice (NZB/W) but not in BALB/c mice. In the present investigation, we have prepared an IFNα immunogen, termed IFNα kinoid, which, appropriately adjuvanted, induces transient neutralizing antibodies (Abs) but no cellular immune response to the cytokine and without apparent side effects. Using this preparation, we also showed that, in kinoid-vaccinated NZB/W mice, lupus manifestations, including proteinuria, histological renal lesions, and death triggered by IFNα Adv challenge were delayed/prevented as long as an effective threshold of anti-IFNα inhibitory capacity was present in the serum.

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.

Objective: To evaluate the effect in mice with arthritis of active anti-TNFα immunotherapy based on a keyhole limpet hemocyanin-human TNFα heterocomplex (hTNFα kinoid or TNFK) adjuvanted in incomplete Freund adjuvant. Immunotherapy was evaluated also with methotrexate. Methods: Human TNFα-transgenic mice received hTNFα kinoid with or without methotrexate. Follow-up ranged from 6 weeks (short-term) to 17 weeks (long-term). Arthritis was evaluated clinically and histologically. Monitoring included titration of anti-hTNFα antibodies by ELISA and neutralization assay. Results: Vaccination with TNFK was associated with rapid-onset, long-lasting protection. Long-term results showed significantly milder arthritis in vaccinated animals than in control animals at the peak of the disease. Vaccination was followed by resolution of the clinical evidence of arthritis, contrasting with severe progressive arthritis in the control group. Histological improvements with decreased inflammation and destruction were noted in all immunized groups, even after the shortest follow-up (6 weeks). High titers of neutralizing anti-hTNFα antibodies were detected as early as the fifth post-immunization week and persisted over time. Methotrexate given concomitantly with the vaccine did not influence either the effect on arthritis or the anti-hTNFα antibody titers. Conclusion: Anti-cytokine induction of autoimmune protection against chronic hTNFα overproduction is an efficient alternative to TNFα blockade in experimental arthritis and can be achieved using a TNFK vaccine.

Pathogenesis of allergic inflammatory disorders is characterized by allergen-induced IgE stimulated by Th2 cytokines including mainly IL-4 overproduction. To counteract IL-4 effects in sensitized-BALB/c mice, we prepared an IL-4 derivative immunogen, made of KLH and murine IL-4 heterocomplex, termed mIL-4 kinoid. Murine IL-4 kinoid immunized mice produced high titer of anti-IL-4 neutralizing Abs. In contrast to KLH control immunization kinoid immunization reversed the allergic IgE:IgG ratio hallmark in rBet v 1a sensitized mice and reduced pulmonary eosinophil recruitment and bronchial hyperreactivity in Ova-sensitized mice. These data pave the way to alternative therapies to combat allergic conditions.

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.

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.

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.

We have designed a computer strategy in order to detect systematically peptidic sites with the potential of interfering with the immune regulatory processes. Applying this software to HIV-1 proteins has led us to unravel a few peptidic sites which could either act directly or be the targets of an auto-immune reaction during HIV-1 infection. We previously reported that the SLWDQ pentapeptide identity with a critical site of CD4 could trigger in HIV-1 infected individuals both an humoral and a cellular autoimmune reaction. In this study, we focused on surprising similitudes unravelled by our software Automat, between HIV-1/2 and another immunoregulatory molecule, the Fas protein which is also called the apoptosis-mediating cell-surface antigen.

Cultures of spinal cord neurons and cocultures of rat embryo neurons and muscle cells have been studied in the presence of tetanus toxin (TT) at a concentration of 40 μg/ml of medium. TT strongly stimulated neurite outgrowth, notably branching from the cell bodies. In addition it induced a marked, overall increase in acetylcholine receptor (AChR), but inhibited focalisation of AChR and acetylcholinesterase (AChE) at the synaptic sites. TT seems to act on neurite emergence, on the neuronal factor(s) controlling AChE and AChR concentrations, and on the factor(s) modulating degradation and/or synthesis of AChR.

Since influenza morbidity and inortalily arc high in the elderly, the usefulness of vaccination has been evaluated. One hundred and twenty-six older persons aged 60–90 yr were given a vaccine consisting of the 3 antigens Singapore, Shanghai and Yamagata, the latter 2 being new antigens. Antibody levels for each of the antigens were determined by ELISA calibrated in HA units prior to and I month after vaccination. Before vaccination, antibodies to Shanghai and Singapore antigens were found in practically all sera tested, and antibodies to Yamagala antigen in only 66% of sera. After vaccination, significant levels of antibodies to all 3 antigens were found in all sera tested. Thus, it was observed that influenza vaccination proved to be effective in the elderly. The elderly should be immunized against influenza, since this is a safe and efficacious preventive measure. In addition, vaccination should limit the spreading of influenza in nursing homes.

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.

While 4 micrograms of Fragment A-B of tetanus toxin (which lacks the binding site for nervous tissue) causes flaccid paralysis and death in mice, 26 micrograms has no toxic effect in goldfish. Antibodies to either A-B or to fragment C (which contains the binding site) block the paralytic effect of whole toxin in goldfish. It is concluded that binding is necessary for the neuromuscular blocking action of the toxin in goldfish.

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.

Anti-Tat vaccination experiments were carried out in mice with a view to inducing systemic in addition to mucosal immunity. For this. three types of immunizing preparations were tested, which consisted of Tat toroid embedded in either an adjuvant oily structure (IMS), or nanoparticles of chitosan, or microparticles of polylactide-co-glycolide (PLG). Administered by either the intranasal or oral route all preparations triggered anti-Tat IgG and IgA antibodies. Sera from mice immunized with either of these preparations could also inhibit significantly the Tat transactivating activity. These results open up a new avenue to the development of an effective anti-AIDS protective vaccine. (C) 2001 Editions scientifiques et medicates Elsevier SAS.

The Tat protein is essential for HIV type 1 (HIV-1) replication and may be an important virulence factor in vivo . We studied the role of Tat in viral pathogenesis by immunizing rhesus macaques with chemically inactivated Tat toxoid and challenging these animals by intrarectal inoculation with the simian/human immunodeficiency virus 89.6PD. Immune animals had significantly attenuated disease with lowered viral RNA, interferon-α, and chemokine receptor expression (CXCR4 and CCR5) on CD4 + T cells; these features of infection have been linked to in vitro effects of Tat and respond similarly to extracellular Tat protein produced during infection. Immunization with Tat toxoid inhibits key steps in viral pathogenesis and should be included in therapeutic or preventive HIV-1 vaccines.

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.

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.

Tetanus toxin decreased the frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocytosis and peptidase activity on synaptobrevin, is discussed.

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.

The capacity of the Corynebacterium granulosum-derived P40 immunomodulator to induce in mice the formation of various cytokines IFN, IL-1, IL-2, alpha-TNF as well as to activate the complement system in rats was investigated. The results showed that P40 injected by the intravenous route was capable of inducing the formation of all four cytokines. High levels of IFN were measured 2 h after P40 stimulation and were still present at 24 h. The kinetic study of IL-1, IL-2 and alpha-TNF induction showed that it was a biphasic phenomenon. The patterns of IL-1 and alpha-TNF induction were quite comparable, whereas the release of IL-2 was delayed with respect to that of IL-1 and alpha-TNF. Oral administration of P40 to rats strongly activated the alternative pathway of the complement system. It was concluded that most of the non-specific effects of P40 on the immune system are likely to be mediated by its capacity to induce cytokine formation and to activate the complement system.

It is known that C granulosum -derived P40 immunomodulator displays strong anti-microbial effects in mice by the intravenous route. Since microbial contamination of humans occurs in many instances via the airways, the effect of P40 on infections was investigated when it was given intranasally or by aerosolization. In order to augment its bioavailability, P40 was derivalized by coupling with polylysine chains (P40-PL). The results showed that P40-PL exercised a significant protective effect, both by the intranasal route and by aerosolization on both influenza and K pneumonlae infections produced by aerosolization or intranasal instillation. Stimulation of the phagocytic capacity of alveolar macrophages by these types of treatment is likely to account for the increased resistance of mice toward microbial infections influenza / K pneumoniae infection / immunomodulator

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.

We have studied the action of tetanus toxin on the release of acetylcholine from a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. We have also studied the morphological changes induced by chemical stimulation on the presynaptic plasma membrane of poisoned synaptosomes. These changes were studied by means of freeze-fracture techniques. We found that tetanus toxin blocks the release of acetylcholine from isolated nerve terminals in a dose-dependent manner. The maximal inhibition is achieved at a concentration of 12.5 nM in 10 min. This effect is prevented by tetanus toxin antiserum. Tetanus toxin also blocks the rearrangement of intramembrane particles at plasma membrane of poisoned synaptosomes, specifically the decrease of small (less than or equal to 9.5 nm diameter) intramembranous particles at the protoplasmic hemimembrane leaflet and the increase of large (greater than 9.5 nm diameter) intramembrane particles at the external hemimembrane leaflet induced by potassium stimulation. These results suggest that intramembrane particle rearrangement could be related to acetylcholine secretion.

Anti-Tat therapeutic vaccination has been clinically investigated by different groups [1-4], given that 1) extracellular Tat protein induces T cell apoptosis and cellular immune suppression, 2) epidemiological data showed that LTNP exhibit high level of serum anti-Tat Ab, negatively correlated with p24 antigenemia, 3) in Tat immunized macaques, viremia decreased following SHIV challenge. Anti-Tat therapeutic vaccination using Tat Toxoid adjuvanted either with Seppic [1,2] or with alum or DcChol (Aventis Pasteur) proved to be safe. A prospective structured treatment interruption study (STI) monitored according to EU guidelines was conducted at Hospital St-Pierre, Brussels (Pr. N. Clumeck) on 31 vaccinees who received a DcChol adjuvanted Tat Toxoid (n = 12), a DcChol placebo (n = 8) or non adjuvanted Tat toxoid (n = 11). The 2 year study follow-up showed that vaccinees developing high titer of Abs neutralizing Tat bioactivity prolonged HAART-interruption., info:eu-repo/semantics/published, Oral presentation. From 2006 International Meeting of The Institute of Human VirologyBaltimore, USA. 17–21 November, 2006

Resume Dans cette experimentation, nous suivons les effets d'une injection IV de Corynebacterium granulosum sur les activites complementaire et phagocytaire chez les rats normaux ou brules. Une etude prealable a montre que des doses variables de Corynebacterium granulosum entre 1 et 10 mg par animal sont bien tolerees : l'allure generale de la courbe ponderale est analogue a celle des animaux normaux, non stimules. Avec une dose minimale de 4mg de Corynebacterium granulosum, le titre du complement augmente de maniere significative. Parallelement, l'activite phagocytaire prealablement deprimee par une brulure devient semblable a celle des rats normaux. Cette immunostimulation est suivie d'une augmentation importante du poids de la rate et est sans effet sur le poids du foie. Par leur action sur au moins deux des facteurs de defense antimicrobienne non specifiques, complement et phagocytes, les corynebacteries anaerobies pourraient etre utilisees comme traitement d'appoint des complications septiques survenant chez l'homme a la suite de brulures severes.

B. Bizzini , P. Toth and A. A. Fedince . Defining a region on tetanus toxin responsible for neuromuscular blockade. Toxicon 26, 309 – 318, 1988. — Administering high doses of tetanus toxin to animals produces neuromuscular blockade. Previous studies, in which specific F(ab) antibody fragments were used to mask the 50,000 MW COOH-terminal portion of the heavy chain (fragment ♠ c ) on the toxin molecule, have shown that the paralyzing effect of the toxin was most probably located in an area comprising the light chain and the 50,000 MW NH 2 -terminal portion of the heavy chain (Fragment I bc ). In our study, the toxin was also complexed with F(ab) fragments directed to the light chain (α), heavy chain (β), β minus II c , and with monoclonal antibodies to epitopes on II c and β minus II c . Investigating the effect of the resulting complexes both in mice and on the sphincter pupillae muscle in rabbits permitted us to circumscribe further the tetanus toxin neuromuscular blocking activity in a region of the NH 2 -terminal fragment ( M t = 50,000) of the heavy chain (fragment β minus II c ). Our results are consistent with the assumption that the β minus II c fragment is critical for the neuromuscular blockade activity of tetanus toxin. However, it cannot be ruled out that both the peripheral and central effects of the toxin result from the same portion of the toxin molecule, the nature of the action depending on where the toxin is carried after its introduction into the organism.

Resume Le fractionnement des corps bacteriens delipides de Corynebacterium granulosum par le procede de Migliore-Samour et Jolles a permis l'isolement d'une fraction complexe insoluble appelee fraction P40 douee d'activite immunostimulante. L'analyse chimique de la fraction P40 a revele la presence de tous les acides amines usuels, d'oses neutres et d'hexosamines, ainsi que d'acide diaminopimelique et d'acide muramique. Les essais de degradation de la fraction P40 par voie chimique ou enzymatique ont echoue. Sa composition chimique suggere qu'il s'agit d'un morceau de la paroi bacterienne a laquelle est associee une glycoproteine. La fraction P40 augmente la capacite granulopexique du S.R.E. et la resistance a l'egard des infections bacteriennes. Elle accroit tant la reponse primaire que secondaire a l'injection d'un antigene particulaire. Elle permet dans certaines conditions de supprimer l'effet immunosuppresseur de la cyclophosphamide. Enfin, elle exerce une action inhibitrice sur le developpement de Tumeurs greffees de la souris, lorsqu'elle est injectee apres la greffe tumorale.

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Dev. Brain Res. 5, 139–147) . Using a combined approach of immunocytochemistry and [3H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 ± 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.

The ontogenesis of cells expressing surface membrane binding sites for tetanus toxin (Tt) was studied in the mouse nervous system. Cells were labeled shortly after the tissue dissociation and the toxin bound was revealed by immunofluorescence. In the brain, spinal cord and dorsal root ganglia the toxin binding cells (TBC) are found as of very early stages of nervous system organogenesis, i.e. at 10 days of gestation. There is a close temporal correlation between the pattern of emergence and accumulation of TBC and the known pattern of appearance of post-mitotic neurons in mouse cerebral cortex, cerebellum and spinal cord. The curves of TBC abundance as a function of fetal age in various nervous system areas are different. They show regional fluctuations in the proportion of TBC that reflect the cumulative changes in the dynamics of neuronal subpopulations. The results indicate that Tt can be used as an ontogenetically early marker of neuronal differentiation and that the acquisition of Tt receptors may represent one of the earliest detectable characteristics of the developing neurons.

In the present investigation, mouse infection models with either intracellular or extracellular bacteria were designed in order to assess the effect of nonspecific immunostimulation with the C. granulosum -derived P40 immunomodulator on infection treatment, performed with doses of antibiotics achieving a low percentage of cures. The results obtained showed that nonspecific immunostimulation was able to significantly enhance antibiotic therapy efficacy. In addition, combined treatments of bacterial infections should allow for a reduction in dosages of antibiotics while at the same time, achieving increased efficacy. Possible mechanisms for the potentiating effect of nonspecific immunostimulation on antibiotic therapy effectiveness are discussed. antibiotic therapy/nonspecific immunostimulation/bacterial infections

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.

Tetanus toxin, like botulinum toxin type A, blocks cholinergic synaptic transmission at the central and peripheral nervous systems. Nevertheless, the diseases induced by the two toxins are different since tetanus toxin induces a spastic paralysis and botulinum toxin elicits a flaccid paralysis. Thus, we have investigated the sensitivity of a central and a peripheral cholinergic synapse to these two toxins. We have studied the action of both poison on the release of acetylcholine from slices of the rat striatum and from the isolated electric organ of Torpedo, which is homologous to the neuromuscular junction. Acetylcholine release from the rat striatum was continuously monitored by a chemiluminescent method. The secretion of acetylcholine from the electric organ was estimated both by measuring the amplitude of the evoked electrical discharge from stacks of electroplaques, and by continuously monitoring the neurotransmitter release from isolated nerve terminals. Tetanus toxin blocks the electrical discharge of electric organ prisms, and also impairs the release of acetylcholine from the Torpedo electric organ nerve endings. Our results on acetylcholine release show that tetanus toxin is more potent than botulinum toxin type A at the central cholinergic synapse (tetanus/botulinum toxins potency ratio about 100-200) whereas botulinum toxin is the most potent at the peripheral cholinergic synapse (botulinum/tetanus toxins potency ratio about 100).

The tyrosyl residues of tetanus toxin can be specifically nitrated by tetranitromethane. The introduction of NO2 groups into the toxin molecule is followed by a rapid decrease in toxicity. Although tryptophyl residues are differently involved and some aggregation occurs, these two phenomena do not take any significant part in the toxicity loss. The immunological reactivity in vitro is preserved. However, it is modified when a certain amount of NO2 groups have been introduced. The thus-formed derivative no longer gives a reaction of identity with the native toxin. Furthermore, when a sufficiently large number of NO2 groups have been introduced, two lines of precipitation are seen. Antisera raised in rabbits with a few nitro-derivatives of the toxin exhibit a low avidity. Precipitating power with pure toxin of these antisera is lower than with a normal antitetanus serum. The capability of tetanus toxin to give rise to neutralizing antibodies is not impaired until 33 NO2 groups have been introduced, then it gradually decreases until it finally disappears. Modification of the toxin by nitration allowed us to discriminate three groups of determinants. In particular, the results presented are further substantiated by those given by 3-aminotyrosyl derivatives of tetanus toxin.