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Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-α release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-α by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials. © 2001 Kluwer Academic Publishers

Resume Le paradoxe de l'infection retrovirale reside dans le fait qu'elle induit une hyperactivation du systeme immunitaire conduisant a une reponse immunitaire inefficace. Dans ce contexte, le reseau des cytokines, qui jouent un role fondamental dans la regulation du systeme immunitaire, est fortement sollicite. Une modification du profil des cytokines, resultant de la deregulation de ce systeme par l'infection virale, peut etre associee a la progression de la maladie; et les cytokines peuvent etre impliquees dans le processus de replication virale. Des cytokines, des inhibiteurs de cytokines ainsi que des mediateurs cellulaires anti-VIH sont a la base de strategies d'immunotherapie. Cette revue analyse l'implication des cytokines dans l'hyperactivation du systeme immunitaire induite par VIH-1, les consequences sur le desequilibre du profil cytakinique et le deficit des cellules T, ainsi que l'implication des cytokines dans les troubles hematologiques et oncogenes lies au VIH-1. Le potentiel diagnostique et pronostique lie a la detection des cytokines ainsi que leur importance dans les strategies d'immuno-intervention sont discutes.

Resume Une complication possible consecutive a l'implantation de protheses a base d'hydroxyapatite (HA) est la liberation de particules d'usure etant susceptibles d'induire une reaction inflammatoire sur le site d'implantation apres phagocytose de ces particules par des monocytes. Il a ete precedemment montre qu'en utilisant un rapport surfacique de 1 pour 1 (surface particules–surface cellules) certaines particules d'HA induisaient l'expression et la production de cytokines impliquees lors d'une reaction inflammatoire. Dans cette presente etude, nous avons etudie l'effet d'un rapport surfacique de 10 pour 1 sur la synthese de cytokines (TNF-α, IL-6, IL-8 et IL-10) produites par des monocytes humains en reponse a des particules d'HA de differentes formes. Nous avons mis en evidence que l'expression des cytokines augmentait avec le rapport surfacique alors que la production etait regulee et ce, semble-t-il, de facon post-transcriptionnelle.

The aims of this study were (a) to determine if rat alveolar type II (ATII) cells and human pulmonary epithelial-derived cells (A549 cell line) could generate IL-6 in vitro, (b) to characterize the cytokine regulation of IL-6 gene and protein expression in these cells, and (c) to detect the in vivo expression of immunoreactive IL-6 by human ATII cells. Rat ATII cells in primary culture secreted bioactive IL-6 and immunostained with an anti-IL-6 antiserum. Spontaneous IL-6 secretion by rat ATII cells amounted to 5,690 +/- 770 pg/ml/10(6) cells (n = 12) and was fivefold higher than spontaneous rat alveolar macrophages IL-6 secretion (1,052 +/- 286 pg/ml/10(6) cells, n = 8, P = 0.001). Rat alveolar macrophage conditioned media (CM) increased IL-6 secretion by rat ATII cells through the effect of IL-1 and TNF. IL-6 gene expression and IL-6 secretion by A549 cells was induced by IL-1 beta, TNF alpha, and by human alveolar macrophages and THP1 cells CM. Induction was abolished when CM were preincubated with anti-IL-1 beta and anti-TNF alpha antibody. The combination of IFN gamma and LPS induced the expression of IL-6 mRNA by A549 cells whereas LPS alone had no effect. Immunohistochemical staining evidenced the expression of immunoreactive IL-6 by hyperplastic ATII cells in fibrotic human lung, a condition in which alveolar macrophages are known to be activated. ATII cells in normal human lung did not express immunoreactive IL-6. Our findings demonstrate that ATII cells may be an important source of IL-6 in the alveolar space thereby participating to the regulation of the intra-alveolar immune response.

Cytokine synthesis and secretion by blood mononuclear cells is a well-documented phenomenon in hemodialyzed patients. The present study was conducted in 17 chronically hemodialyzed patients to test the relative effect of uremic toxicity, membrane biocompatibility, dialysate composition, and the risk of endotoxinemia on the serum level of tumor necrosis factor-alpha (TNF-alpha). The only significant parameter that influenced circulating TNF-alpha was the chemical characteristics of the dialyzer membrane. Tumor necrosis factor-alpha levels significantly increased during the session with cuprophane, whereas they decreased with AN69. The TNF-alpha increase was documented whatever the dialysate buffer and the presence or absence (negative Limulus amoebocyte lysate test) of endotoxin in the dialysate. In the subgroup of patients treated with a contaminated dialysate and AN69, none had clinical symptoms and the central body temperature remained constant throughout the session. In these patients, serum TNF-alpha levels did not change after priming the dialyzer with sterile saline. In conclusion, the serum TNF-alpha level during hemodialysis appears to be modulated by biocompatibility, permeability, and binding properties of dialysis membrane rather than dialysate composition. Endotoxin in the dialysate did not result in positive TNF-alpha balance no matter what its possible priming effect on mononucleated blood cells.

Murine monoclonal antibodies to lipopolysaccharides (LPS) from rough J5 mutant of Escherichia coli O111:B4 protected D-galactosamine-treated mice against lethal effects of LPS and provided protection in an experimental infection model with live E. coli. Three previously prepared anti-LPS antibodies were evaluated for ability to inhibit LPS-induced tumor necrosis factor (TNF) secretion. In vivo, TNF production was induced in mice treated with D-galactosamine and challenged with LPS. Two antibodies (D6B3 and D6B4) decreased serum TNF levels and prevented lethal effects of LPS. Nonprotective anti-LPS antibody (D9A2) and an unrelated antibody to neomycin did not reduce circulating TNF levels after LPS challenge. Pretreatment of mice with D6B3 and D6B4 protected mice from infection with E. coli and prevented serum TNF increases induced by E. coli infection. In nonprotected animals, high levels of TNF were detected in serum 3-6 h after infection; animals died within 24 h. In vitro, addition of protective antibodies to macrophage cultures at initiation of LPS stimulation inhibited TNF production. Nonprotective antibody D9A2 failed to block LPS-induced TNF production.

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)(3) peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI(3)-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI(3)-kinase p110gamma sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI(3)-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.

TNF-alpha is considered as one of the potential determinants of insulin resistance. However several data suggest that TNF-alpha expression itself, could be modulated by the degree of adiposity and/or plasma insulin levels. To clarify the determinants of plasma TNF-alpha levels in type 2 diabetes mellitus, we studied the impact of intensive insulin treatment on plasma TNF-alpha levels in 16 type 2 diabetic subjects with failure to oral antidiabetic medication (HbA1c: 10.8 +/- 1.2 %). Furthermore, we analyzed the relationship between plasma TNF-alpha levels and total or regional body fat measurements using anthropometry, bienergetic absorptiometry and computed tomography in a cohort of 33 caucasian obese type 2 diabetic subjects (BMI: 32.2 +/- 4.4 kg/m(2) ). The plasma TNF-alpha level was neither affected by plasma glucose level variations nor intensive insulin treatment despite a 37 % decrease in daily insulin needs at the end of insulin therapy (total duration: 11.5 +/- 2.0 days). The plasma TNF-alpha level was similar in men and women and unrelated to age, fasting glycemia or HbA1c. A relationship was highlighted with BMI (r =0.39, p0.02), but not with total fat mass. This relationship was only dependent on the intra-abdominal fat mass amount as assessed by the waist-to-hip circumference ratio (r =0.52, p0.01) and the visceral adipose tissue area (r =0.56, p0. 01). These results show that plasma TNF-alpha levels are essentially dependent on visceral fat amount, thus suggesting that TNF-alpha could be one of the factors mediating insulin resistance and cardiovascular risk in obese type 2 diabetic patients.

Objectives: Sixteen Mycobacterium avium strains were isolated from the blood of eight AIDS patients over a period of months. All the patients were on combination therapies including clarithromycin, and all had treatment failure and relapses of M. avium bacteremia. Paired clarithromycin-sensitive and resistant M. avium strains isolated at the beginning of treatment and at the first relapse of bacteremia were compared. Methods: The M. avium isolates were identified after hybridization with DNA probes specific for M. avium rRNA and typed epidemiologically with random amplified polymorphic DNA analyses using three arbitrary primers. The rate of intracellular cell entry or the tumour necrosis factor alpha induction by the M. avium isolates were studied in human monocytes and J774 cells. Results: When the M. avium isolates were hybridized with the rRNA probes, we obtained lower hybridization values with clarithromycin-resistant isolates than with clarithromycin-sensitive isolates. This appeared to be due to smaller amounts of rRNA available for hybridization than to mutation of the 23S rRNA sequences in clarithromycin-resistant strains. The RAPD analyses showed that the clarithromycin-resistant isolates were clonally related to the clarithromycin-sensitive strains in six of the eight patients. The other two patients had a RAPD profile, suggesting a re-infection and/or polyclonal infection. The M. avium isolates obtained on day 0 and after the emergence of resistance to clarithromycin did not differ in terms of their intracellular entry rate, or in terms of tumour necrosis factor alpha induction. Conclusions: We infer that M. avium strains isolated during bacteraemic relapses on combination therapies including clarithromycin are epidemiologically related to the initial strain and do not show changes in the rate of intracellular cell entry and in terms of tumour necrosis factor α induction. Re-infections and/or polyclonal infections however, although less frequent, can also occur.

The purpose of this study was to determine the clinical and immunological outcome of 78 children with congenital toxoplasmosis treated with the pyrimethamine-sulfadoxine combination between 1980 and 1997.Children were divided into 3 groups according to the initial duration of treatment (always including folinic acid, 5 mg/week by mouth), as follows: pyrimethamine (1.25 mg/kg every 15 d) + sulfadoxine (25 mg/kg every 15 d) for 12 months (Group 1, 47 children), or for 24 months, with or without prenatal therapy (respectively, Group 2, 19 children, and Group 3, 12 children).Chorioretinitis occurred in 23% of these 78 children. Four children had unilateral blindness, 1 had mild epileptic fits and 1 had psychomotor retardation. The lowest rate of sequelae were in Groups 2 and 3. Immunological rebounds, generally without clinical repercussions, occurred frequently (90% of cases on average) during, or more often after therapy, regardless of the treatment duration. Treatment was always well tolerated.Our current treatment strategy for congenital toxoplasmosis consists of a 24-month course of pyrimethamine-sulfadoxine (Fansidar) combined with folinic acid (Lederfoline). If the prenatal diagnosis is positive, we also prescribe this treatment to the mother until delivery. This combination offers satisfactory compliance, adequate serum concentrations, and good preventive efficacy.

Allergic reactions are under the control of several events that occur sequentially following allergen exposure, recognition by the immune system, IgE production and their interaction with effector cells bearing Fc epsilon receptors. The lymphocyte activation in response to allergens determines the intensity and the nature of the immune response. Cytokines produced by T (and non-T) cells are involved in the polarized development of the specific immune response. In particular, type 1 and type 2 cytokines are responsible for the control of the different steps during allergic reactions. Th2 cytokines and particularly IL4 are responsible for switching the immunoglobulin synthesis by B cells to IgE production. They also play a key role in the activation of effector cells that occurs following allergen interaction with fixed specific IgE and participate to the local inflammatory reaction. Cytokine profile determination appears to represent a helpful laboratory parameter in the understanding of the mechanisms underlying allergic diseases. The development of new technological tools may allow the use of cell activation parameters, and cytokine profiles determination in clinical biology. This review aims to analyze the involvement of the cytokine network in the mechanisms leading to IgE production and the involvement of cytokines in effector mechanisms of allergic reactions. It also analyses the potential use of cytokine profile determination for diagnosis purpose and survey of immune desensitization of allergic diseases.

International audience; Three hybrids derived from CD5+ B cell chronic lymphocytic leukemia (B-CLL) and their parental B cells were studied for phenotypic evolution, immunoglobulin (Ig), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) secretion. When phenotypic evolution was examined, hybrids showed the loss of classical B cell markers, indicating that they follow the same pattern of phenotypic differentiation as normal B cells. Hybrids displayed spontaneous high Ig secretion, which did not appear to be modified through stimulation by phorbol 12-myristate 13-acetate (PMA), recombinant interferon-gamma (rIFN-gamma) and Staphylococcus aureus Cowan I (SAC). Parental cells secreted minimal amounts of Ig spontaneously or through IFN-gamma and SAC stimulation, whereas PMA succeeded in increasing this secretion. An opposite pattern was observed when TNF-alpha and IL-6 secretion an expression at the mRNA level were assessed in hybrids and parental cells. TNF-alpha and IL-6 were spontaneously secreted by parental cells and this secretion was increased after PMA and SAC stimulation, both cytokine secretion and expression at the mRNA level were negative in hybrid cells. The absence of expression of these cytokines could be explained either by chromosomal loss or by down regulation. These results indicate that when parental CLL cells are induced to differentiate in the heterohybrid model, they acquire high spontaneous secretion of Ig, lose the classical B cell phenotypic markers and down regulate the expression of the cytokines studied.

Many clinical and experimental data are in favour of a participation of leukocytes in vascular disease. Diabetes, a risk factor, is associated with a dysfunction of neutrophils. If chemotaxis and phagocytosis are deficient, it is not clearly established whether superoxide generation is conserved in these patients. We have measured this function in 35 noninsulin dependent diabetic patients, compared with a control population. We have assessed, in parallel, a profile of the cytokines involved in vascular phenomenons including TNF alpha, IL-1 beta et IL-6. Our results indicate that the generation of free radicals is normal in diabetics, with a significant elevation of TNF alpha. These results suggest a possible participation of this cytokine in the modulation of granulocyte reactivity.

The cytokine network is involved in normal immune reaction and in the progression of several pathologies. Antisense (AS) oligonucleotides, which allow specific inhibition of expression of proteins, offer a new methodology to investigate this complex network. This review focuses on the use of AS to modulate cytokine expression. AS may act in different ways such as blocking fixation or progression of the ribosome along the mRNA, mRNA cleavage by RNase H, or preventing normal RNA maturation. In order to improve AS efficiency, chemical modifications have been developed, and improvement of oligonucleotide uptake has been achieved with different systems of vectorization including liposomes (neutral, cationic, immunoliposome), nanoparticles, or covalent attachment of a carrier. In oncogenesis, intracellular or extracellular autocrine loops have been demonstrated by the use of cytokine AS. Involvement of cytokines in immunological reactions (TH1 and TH2 subset, IgE response, lymphokine activated killer, cytotoxic T lymphocyte...) and in hematopoiesis have also been studied with this approach. Therapeutic application of AS has been suggested by inhibition of inflammatory cytokines in vivo. Clinical trials using AS are under investigation in virological and in oncological diseases. At present, cytokine antisenses primarily represent a tool for dissecting the function of a cytokine in vitro, but they may offer in the future a new way for immunomodulation intervention.

International audience; We investigated the role of tumor necrosis factor alpha (TNF-alpha) in human peripheral monocytes infected with Legionella pneumophila in vitro. Exogenous TNF-alpha significantly inhibited the intracellular multiplication of the bacterium. This effect was concentration and time dependent and was abrogated by anti-TNF antibodies. TNF-alpha levels in the culture supernatants were low but were enhanced by the addition of gamma interferon. When monocytes were cultured and infected in the presence of pentoxyphilline, a potent inhibitor of TNF-alpha synthesis, the intracellular bacterial growth was enhanced. The effect of pentoxyphilline was concentration and time dependent and was due to the inhibition of TNF-alpha production, as shown by Northern (RNA) blot hybridization of total RNA. In addition, the pentoxyphilline partially abolished the inhibitory effect of gamma interferon on bacterial intracellular multiplication. These results suggest that gamma interferon inhibits, at least partially, the intracellular multiplication of L. pneumophila by enhancing TNF-alpha synthesis.

Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in AIDS patients. In vitro, splenic cells of noninfected euthymic mice produced tumor necrosis factor-alpha (TNF alpha) in greater amounts when incubated with live R. equi rather than with killed bacteria. In vivo, interferon-gamma (IFN-gamma) and TNF alpha serum levels of infected euthymic mice remained below the level of detectability. Treatment of infected nude mice, which developed chronic infection, with discontinuous injections of IFN-gamma, TNF alpha, or both did not decrease bacterial colony-forming units in liver, spleen, or lungs. However, treatment of infected euthymic mice, which cured a R. equi inoculum within 3 weeks, with anti-IFN-gamma or anti-TNF alpha antibodies (or both) significantly increased tissue colony counts. These data argue that, in this murine model, endogenous IFN-gamma and TNF alpha are involved in the cell-mediated immunologic response against R. equi infection.

The Enterobacteriaceae producing extended broad-spectrum beta-lactamases (ESB) must be detected even when their MIC to oxyiminocephalosporins are low. We tested 164 isolates (133 ESB, 31 non ESB) on two experimental ATB MIC plates containing cefotaxime or ceftazidime (0.06-128 mg/l) alone or associated with sulbactam (4 mg/l). Fifty four per cent of the ESB isolates are sensitive to cefotaxime, and 30% to ceftazidime. Respectively 95% and 94% of these bacteria have a synergism index (MIC CIII alone/MIC CIIII + sulbactam) greater than or equal to 4 with cefotaxime and ceftazidime. The isolates that do not produce ESB have all a rate less than 4. The ATB system can detect the extended broad spectrum beta-lactamases when low concentrations of cephalosporins are used, alone and in association with an inhibitor of beta-lactamases.

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.

Minimal inhibitory concentrations (MIC) of pefloxacin (PEF), norfloxacin (NOR), ofloxacin (OFL) and ciprofloxacin (CIP) are evaluated by agar dilution against 100 Klebsiella pneumoniae strains isolated in hospital. MIC 50 and 90%, micrograms/ml, are respectively: CIP (0.25/8), OFL (1/16), NOR (1/32), PEF (2/64). We determined the phenotypes PEF/NOR/OFL/CIP by taking into account the critic concentrations of the French Committee for Antibiogram. The results are: SSSS = 45%, RI/RI/RI/RI = 20%, RI/SSS = 19%, RI/RI/RI/S = 8%, RI/RI/SS = 7%. When a strain is resistant to pefloxacin alone, the MICs of the other fluoroquinolones are higher than those of the sensitive strains. The resistance to fluoroquinolones is most frequent in strains that have acquired resistance to aminoglycosides or betalactams, but exists also in strains that have no acquired resistance to these antibiotics.

Summary RU-41740, a glycoprotein extract from Klebsiella pneumoniae K2O1 strain, is an immunomodulating compound which has been shown to reduce infectious episodes in immunodeficient patients. Data from preliminary experimental designs suggested that RU-41740 could affect several target cells, such as T cells, B cells and macrophages. In the present report, we show that RU-41740 is a selective B-lymphocyte activator. It induces blast transformation in Nude mouse spleen cell cultures and in B-cell-enriched fractions obtained from normal mice. It does not activate T lymphocytes to proliferate. Activation of mouse B lymphocytes by RU-41740 is not affected by removal of adherent cells. RU-41740 also activates immunoglobulin secrection by murine B lymphocytes. Incubating spleen cells from C3H/HeJ mice with RU-41740 results in cell proliferation and activation of antibody-forming cells. This suggests that B-cell activation is not due to LPS contamination. Other experiments show that RU-41740 can also trigger mouse macrophages to produce interleukin-1 activity. Indeed, supernatants from peritoneal adherent cells incubated in the presence of RU-41740 can stimulate blastogenesis in thymocytes from C3H/HeJ mice. Thus, B-cell activation and IL-1 production by macrophages could constitute two additive mechanisms involved in immunomodulation induced by RU-41740.

Two preparations designated as PWE and NCE were obtained from H, influenzae type a by treatment with either phenol water or hypertonic NaCl citrate respectively. The protein content of NCE was three fold higher than for PWE. Electroimmunodiffusion against H. influenzae antiserum disclosed an immunological relationship between NCE and PWE. The toxicity for mice was lower for both antigenic extracts (LD50 PWE: 47 +/- 7 mg/kg; LD50 NCE: 943 +/- 5 mg/kg) than for E. coli LPS. Limulus assay showed that both H. influenzae extracts had a weaker potency (100 to 1000 times less) than E. coli LPS. Sanarelli and Shwartzman reactions failed for PWE and NCE. The blast transformation of DBA/2 and nude mouse spleen cells were strikingly different for the three preparations: PWE, NCE, and E. coli LPS. Neither extract from H. influenzae cells showed the biological properties of a classical endotoxin. We suggest that they are endotoxin-like substances.

RU-41740, a glycoprotein extract from Klebsiella pneumoniae, is an immunomodulating agent with a broad spectrum of activities. It enhances several macrophage functions, including interleukin-1 (IL-1) secretion. Present data show that RU-41740 was able to promote murine macrophage cytotoxicity against tumour cells. A soluble cytotoxic factor (CF) was found in supernatants from macrophage cultures stimulated with RU-41740. These supernatants were shown to contain IL-1. CF was detected on L-929 cells sensitized by actinomycin D. CF was analysed on the basis of MW and pHi: after gel filtration on "Ultrogel Aca54", a single peak of CF was found in the range of 50-60 Kd and was then distinct from IL-1, which was eluted at 15 Kd. After chromatofocusing, CF was found in a narrow peak of pH 4.8. CF was detected in supernatants 2 h after macrophage stimulation by RU-41740, and its release was abolished by pretreatment of macrophages with cycloheximide (2 micrograms/ml). CF described here shares several properties with tumour necrosis factor (TNF).

Resume Les auteurs rapportent le cas d'une septicemie a Yersinia enterocolitica survenue chez un diabetique dont l'hospitalisation a permis de decouvrir un deficit en glucose 6 phosphate deshydrogenase (G 6 PD) et une double hemoglobinopathie S-C. Malgre un terrain deficient, la guerison a ete obtenue grâce a l'antibiotherapie. Le germe, isole du sang du malade, n'a pas ete retrouve dans les selles. La souche a ete inoculee a des souris normales et a des souris athymiques (Nude). Elle n'a ete retrouvee qu'au niveau des organes des souris athymiques, hormis le contenu intestinal.

Mild hydrolysis of Haemophilus influenzae type a lipopolysaccharide by ion exchangers in the presence of chloroform, to remove the lipid moiety, yielded a nontoxic and immunogenic polysaccharide fraction. This polysaccharide selectively triggered murine B lymphocytes in vitro: (i) it induced enhancement of thymidine incorporation and stimulated antibody secretion in cultures of normal and nude mouse spleen cells; (ii) it did not stimulate splenic T lymphocytes; (iii) the activation of B lymphocytes was not absolutely dependent on the presence of macrophages. Sepharose 4B gel filtration showed that this polysaccharide consisted at least of two fractions: PS I (molecular weight [MW] 10(6)) and PS II (MW 10(4)). Only PS I was found to act as a polyclonal B cell activator. EDTA treatment dissociated the polysaccharide into PS III (MW 10(6)) and PS IV (MW 10(4)), which was not reassembled after the addition of 0.02 M CaCl2. Both fractions PS III and PS IV were unable to stimulate B lymphocytes. The immunological active fraction of H. influenzae polysaccharide is PS I. This fraction consists of a high-molecular-weight group (10(6)) and an association of 10(4)-MW aggregated units.

andoleic acids andtwoother unidentified long-chain fatty acids werefound. Thefree lipid A wasnottoxic formiceatdoses ofupto50mg/kganddidnotprovoke a Shwartzman reaction. TheLimulus testactivity waspositive upto10-12 g/ml, butthepyrogenicity inrabbits waslower thanwiththeoriginal lipopolysaccharide. However, thelipid Adidinduced amitogenic response andpolyclonal B-cell activation inmousespleen cell cultures. Complexing lipid A withbovine serum albumin gaveanontoxic preparation whichlost these immunological activities. Immunochemical studies showedthatthemajorreactive determinants ofthis lipid-protein complex werealtered after suchalinkage. Consequently, thenontoxic andmitogenic lipid Aisolated fromH.influenzae typeadidnotexbibit all oftheclassical activities oflipid Apreparations. Lipid A isacommonconstituent ofthelipopolysaccharides (LPS) ofall gram-negative bacteria, anditisbelieved tobethepartofthe molecule responsible forendotoxicity. However, ourknowledge ofitschemistry andbiological activity israther fragmentary. Previous studies showthatthelipid A components derived fromvarious bacterial groups, including Salmonella spp.andother genera of theEnterobacteriaceae family, seemtobealike

The concanavalin A-unreactive variants of alpha 1-acid glycoprotein introduced in culture medium of monocytes/macrophages induces the inhibition of thymocyte proliferative activity of interleukin 1. LPS or LPS receptors were not involved in the activity of alpha 1-acid glycoprotein on macrophages. alpha 1-acid glycoprotein did not show any direct effect on thymocytes whereas the monocyte/macrophage-supernatant inhibited the interleukin 1 proliferative effect. The activity of tumor necrosis factor and interleukin 2 was not altered by the alpha 1-acid glycoprotein-macrophage supernatant.

Resume Cette revue generale concerne la phagocytose ; notamment les mecanismes bactericides et les methodes permettant leurs explorations. Les differentes etapes impliquees dans la phagocytose et la bactericidie sont les suivantes : chimiotactisme, opsonisation, contact cellulaire, ingestion et digestion. L'adhesion fait intervenir les recepteurs specifiques de membrane (fragment Fc des IgG et la fraction C 3 du complement) ainsi que des facteurs extra-cellulaires. Apres la description de l'internalisation, les aspects morphologiques et physiologiques de la bactericidie sont decrits. Des modifications metabolique interviennent durant l'ingestion et la digestion. Ces activites bactericides peuvent etre separees en deux classes qui sont : -- soit oxygene independantes, -- soit oxygene dependantes. Dans la 1 re classe, on distingue les mecanismes enzymatiques impliquant le lysozyme et ceux, non enzymatiques, utilisant la lactoferrine et les proteines cationiques. Differents facteurs participent au second systeme. Des complexes letaux peuvent etre generes par oxydation des halogenes. De plus, d'autres composants sont aussi impliques comme l'eau oxygenee (H 2 O 2 ), les anions superoxyde (O 2 ), les radicaux hydroxyls et le singlet oxygene ( 1 0 2 ). Ils necessitent l'intervention de la NADPH, H + oxydase et de la superoxyde dismutase. L'etude de l'activite bactericide intra-cellulaire s'effectue : -- soit par l'evaluation des bacteries ingerees, -- soit par la mesure des modifications metaboliques : consommation d'oxygene, production de H 2 O 2 , generation de (O − 2 ) et de O 2 et augmentation de diverses activites enzymatiques (NADPH, H + oxydase, superoxide dismutase, myeloperoxydase). Cependant, notons que chacune de ces methodes n'explore seulement qu'un des nombreux mecanismes bactericides.

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.

RU-41740, a glycoprotein complex extracted from Klebsiella pneumoniae, is an immunomodulating agent which acts on B cells and macrophages. It has been shown that RU-41740 is composed mainly of two macromolecular fractions, F1, having an LPS-related structure, and P1, with a proteoglycan structure. In the present paper, the effects of these molecules on B cells and on IL-1 and tumour necrosis factor (TNF), production by macrophages were compared. Data reveal that both fractions were mitogenic for murine B cells and induced IL-1 and TNF production by macrophages. The LPS-like fraction (F1) was sensitive to polymyxin B and was unable to activate macrophages and spleen cells from LPS non-responder mice. The P1 fraction was mitogenic for B cells and induced the production of IL-1 and TNF activities by macrophages from LPS non-responder C3H/HeJ mice. The cytotoxic activity was due to TNF alpha, since treatment with anti-TNF alpha antiserum abrogated the lytic activity of supernatants from stimulated macrophages. The differences observed between P1 and F1 fractions in terms of sensitivity to polymyxin B and activity towards C3H/HeJ spleen cells and macrophages suggest that the two structurally distinct molecules isolated from RU-41740 could act at different sites on immunocompetent cells.

Sodium periodate treatment of human peripheral blood lymphocytes at submitogenic concentrations (10 −4 M ) completely inhibits their ability to mount a primary in vitro antibody response to trinitrophenyl-polyacrylamide beads. Transfer experiments show that (i) mitogenic concentrations (2 × 10 −3 M ) of sodium periodate induce suppressor cells to the in vitro antibody response: (ii) the generation of these suppressor cells is decreased but not abolished after mitomycin treatment, whereas that of Con A-induced suppressors is prevented; (iii) the periodate-induced suppression is present in the T-enriched fraction; and (iv). upon periodate treatment, T-depleted cells have a moderate but significant suppressive effect.

Antisense oligonucleotides (ODN), complementary to mRNA of human tumor necrosis factor alpha (TNF alpha) and lymphotoxin (LT) were tested for their ability to inhibit TNFs. TNFs production was studied in cell-free systems including wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL). All ODN were effective in WGE at low concentration (0.2 microM), except those targeted to the 3' region of TNF alpha mRNA. A short ODN complementary to a common region between TNF alpha and LT inhibited both TNFs. In contrast, high ODN concentration (50 microM) was needed to inhibit LT mRNA translation in RRL, whereas no clear inhibition of TNF alpha was observed unless RNase H was added to the translation mixture. ODN effects on TNFs production by stimulated cell line in culture were also investigated. Three ODN-one located in the 5'-untranslated region, one spanning the AUG initiation codon and one downstream of this AUG-were the most effective sequences to decrease TNF alpha production. Two ODN targeted to the AUG initiation codon of LT were also able to inhibit its production. In conclusion we confirm the role of RNase H in cell free systems, and we found that there is no correlation between ODN efficiency in a cell-free system nor in cell culture. Efficient ODN could be used for in vitro investigation of the role of TNF alpha and LT in mechanism in which they are involved.

Resume Une observation d'epidurite a Haemophilus aphrophilus est rapportee. Le diagnostic bacteriologique repose sur la mise en evidence de cette bacterie, a 3 reprises, a l'hemoculture. La guerison complete a ete obtenue par immobilisation et administration d'ampicilline et gentamicine.

RU-41740, a glycoprotein extract from Klebsiella pneumoniae, is an immunomodulating agent with a broad spectrum of activities. It enhances several macrophage functions, including interleukin-1 (IL-1) secretion. Present data show that RU-41740 was able to promote murine macrophage cytotoxicity against tumour cells. A soluble cytotoxic factor (CF) was found in supernatants from macrophage cultures stimulated with RU-41740. These supernatants were shown to contain IL-1. CF was detected on L-929 cells sensitized by actinomycin D. CF was analysed on the basis of MW and pHi: after gel filtration on "Ultrogel Aca54", a single peak of CF was found in the range of 50-60 Kd and was then distinct from IL-1, which was eluted at 15 Kd. After chromatofocusing, CF was found in a narrow peak of pH 4.8. CF was detected in supernatants 2 h after macrophage stimulation by RU-41740, and its release was abolished by pretreatment of macrophages with cycloheximide (2 micrograms/ml). CF described here shares several properties with tumour necrosis factor (TNF).

The ability of differently structured, purified peptidoglycans (PG) to induce interleukin 1 (IL1) secretion was compared. PG from Bacillus megaterium and Staphylococcus aureus stimulated the production of IL1 by mouse peritoneal macrophages and human adherent mononuclear cells, whereas PG from Micrococcus lysodeikticus and Corynebacterium poinsettiae were inactive. There was a correlation between the ability of PG to induce IL1 secretion and previously demonstrated immunoenhancing activities (adjuvant effect, increase of resistance to tumor growth) of PG. PG solubilization by lysozyme decreased but did not abolish the PG effect on IL1 secretion. Active PG induced IL1 production in nude mice and in the C3H/HeJ strain (which is unresponsive to lipopolysaccharides).

RU 41740, a glycoprotein extract from K. pneumoniae K2O1 strain, is an immunomodulating compound which has been shown to reduce infectious episodes in patients prone to recurrent infections. Data from preliminary experiments suggest that RU 41740 may affect several target cells, including T cells, B cells or macrophages. In the present report we show that RU 41740 can trigger mouse macrophages and human adherent mononuclear cells to produce interleukin 1 activity. Indeed, supernatants from mouse peritoneal adherent cells and human monocytes incubated in presence of RU 41740, can stimulate blastogenesis in thymocytes from C3H/HeJ mice. The data suggest that the immunomodulating effect of RU 41740 could be related to its ability to induce interleukin 1 production.

The adjuvant and mitogenic properties of bacterial peptidoglycans (PG) of different structures have been compared. The PG from Bacillus megaterium and Staphylococcus aureus were adjuvant-active in the guinea-pig, and mitogenic for guinea-pig and mouse lymphocytes. On the other hand the PG from Micrococcus lysodeikticus and Corynebacterium poinsettiae exerted neither adjuvant nor mitogenic activities. Mitogenic PG were selective B mitogens. They were mitogenic for splenocytes from Nude mice and inactive towards T-cell-enriched populations. They were mitogenic in both germ-free mice and in the C3H/HeJ strain which is unresponsive to lipopolysaccharide.

RU-41740, a glycoprotein complex extracted from Klebsiella pneumoniae, is an immunomodulating agent which acts on B cells and macrophages. It has been shown that RU-41740 is composed mainly of two macromolecular fractions, F1, having an LPS-related structure, and P1, with a proteoglycan structure. In the present paper, the effects of these molecules on B cells and on IL-1 and tumour necrosis factor (TNF), production by macrophages were compared. Data reveal that both fractions were mitogenic for murine B cells and induced IL-1 and TNF production by macrophages. The LPS-like fraction (F1) was sensitive to polymyxin B and was unable to activate macrophages and spleen cells from LPS non-responder mice. The P1 fraction was mitogenic for B cells and induced the production of IL-1 and TNF activities by macrophages from LPS non-responder C3H/HeJ mice. The cytotoxic activity was due to TNF alpha, since treatment with anti-TNF alpha antiserum abrogated the lytic activity of supernatants from stimulated macrophages. The differences observed between P1 and F1 fractions in terms of sensitivity to polymyxin B and activity towards C3H/HeJ spleen cells and macrophages suggest that the two structurally distinct molecules isolated from RU-41740 could act at different sites on immunocompetent cells.

Oxidation of human peripheral mononuclear cells with sodium periodate results in lymphocyte activation. Period-date, at optimal mitogenic concentrations, oxidizes membrane sialyl residues (NeuNAc) essentially into the 7 carbon analogue (C7-NeuNAc). Fucosyl and galactosyl residues are also oxidized by periodate, since propane 1,2-diol and glycerol are isolated in acid hydrolysates of lymphocytes oxidized by periodate and reduced by tritiated borohydride. The neuraminidase pretreatment of lymphocytes induces a 40-50% decrease of their response to periodate. Neuraminidase treatment of 108 human peripheral lymphocytes liberated 9.6 microgram NeuNAc (31 nmol), representing 68.5% of the total content. The neuraminidase treatment dramatically enhances the recovery of glycerol in hydrolysates of lymphocytes treated successively with periodate and tritiated borohydride.

From three patients hospitalised in intensive care units with Enterobacter septicaemia (two cases with E. cloacae, and one with E. aerogenes), cefotaxime therapy, alone or in combination with an aminoglycoside, selected variants (R) with increased resistance to beta-lactam antibiotics. The cross-resistance extended to all the beta-lactam antibiotics tested, penicillins and cephalosporins, including third-generation cephalosporins. The crude extracts of uninduced cultures of R variants showed high beta-lactamase activity and of the cephalosporinase type. These variants were selected in vitro with a frequency of 10(-6) to 10(-7) and may result from a mutation involving the regulation of Enterobacter cephalosporinases, usually inducible. Data from the literature indicated that this new type of resistance is actually emerging and observed not only in Enterobacter sp. The problem of emergence of R variants exhibiting cross-resistance to beta-lactam antibiotics should be considered when third-generation cephalosporins are used.

Mild periodate treatment is mitogenic for T lymphocytes. With murine spleen cells periodate oxidation is effective between C8 and C9 on sialyl acid residues. With human blood lymphocytes this oxidation occurs between C7 and C8 of these residues. In vitro immune response is inhibited by periodate treatment. Activation of an immunosuppressive T lymphocyte population is obtained. Similar results are performed with human blood lymphocytes.

C57BL/6 (susceptible) and A/J (resistant) mice were infected intravenously with a temperature-sensitive mutant of Salmonella typhimurium. In C57BL/6 mice a marked depression of the proliferative response of spleen cells to B and T mitogens occurred and was maximal at 2-3 weeks post-infection, whereas only minor changes were found in A/J mice. This immunodepression was mediated, at least in part, by adherent cells. Moreover, spleen cells from infected C57BL/6 mice did not produce interleukin-2 (IL-2) after concanavalin A stimulation. The impairment of IL-2 production was not related to a defect in IL-1 release. The addition of IL-2 to spleen cells did not restore their ability to respond to mitogens. The depression of mitogenic responses in infected C57BL/6 mice occurred at a time when increased resistance was present.

Bacterial peptidoglycans and the synthetic analog muramyl dipeptide possess various immunomodulating properties (adjuvant effect, increase of resistance to infectious agents and to tumor growth). They are able to induce B cell activation and to stimulate macrophages to produce monokines such as Interleukin 1 (IL 1). IL 1 plays an essential role in immune response. It promotes thymocytes maturation and Interleukin 2 secretion by antigen sensitive T cells, which in turn triggers regulatory T cells. Moreover, it is involved in the proliferation and differentiation of B cells. There is a correlation between the immunoenhancing effect of PG of a definite structure and their ability to induce IL 1 secretion. Non-adjuvant PG were inactive. This suggests that one of the major mechanisms of action of adjuvant PG could be the stimulation of IL 1 synthesis.

Sodium periodate treatment of Mouse spleen cells activated suppressor cells which act on the in vitro antibody response towards sheep erythrocytes or trinitrophenyl-polyacrylamide beads. Present study shows that suppression can be mediated by soluble factor (s) released in the cultures of periodate treated cells. Production of such suppressor factor (s) was associated to the presence of free aldehyde groups generated by periodate on the lymphocyte membrane. Control or cells successively treated by periodate and then reduced by borohydride were unable to produce suppressor factor (s). Suppressor factor (s) were stable at 56 degrees C (30 mn) and inactivated at 80 degrees C (10 mn).

RU-41740, a glycoprotein extract from Klebsiella pneumoniae K2O1 strain, is an immunomodulating compound which has been shown to reduce infectious episodes in immunodeficient patients. Data from preliminary experimental designs suggested that RU-41740 could affect several target cells, such as T cells, B cells and macrophages. In the present report, we show that RU-41740 is a selective B-lymphocyte activator. It induces blast transformation in Nude mouse spleen cell cultures and in B-cell-enriched fractions obtained from normal mice. It does not activate T lymphocytes to proliferate. Activation of mouse B lymphocytes by RU-41740 is not affected by removal of adherent cells. RU-41740 also activates immunoglobulin secretion by murine B lymphocytes. Incubating spleen cells from C3H/HeJ mice with RU-41740 results in cell proliferation and activation of antibody-forming cells. This suggests that B-cell activation is not due to LPS contamination. Other experiments show that RU-41740 can also trigger mouse macrophages to produce interleukin-1 activity. Indeed, supernatants from peritoneal adherent cells incubated in the presence of RU-41740 can stimulate blastogenesis in thymocytes from C3H/HeJ mice. Thus, B-cell activation and IL-1 production by macrophages could constitute two additive mechanisms involved in immunomodulation induced by RU-41740.

Similar batches of five-week-old C57 Bl/6 mice were given either a magnesium-deficient diet (4mg Mg/100 g), or a control diet (40 mg/100 g). Control diet intake was either ad libitum or reduced. After immunization with SRBC (sheep red blood cells), the immune response was studied by estimating the number of spleen AFC (antibody-forming cells) capable of lysing SRBC, and by a cytoadhesion test to determine the number of RFC (rosette forming cells). Limitation of the control diet slowed the growth rate in mice. Whenever food intake was reduced from 4g/day to 2.9 or 2.6g/day, the AFC response intensified but the RFC response remained similar. Food limitation might therefore mainly affect immature IgM producing cells with a high dividing rate. Magnesium deficiency produced a drastic fall in the primary and secondary immune responses, as measured by the number of spleen AFC. The number RFC was also much lower in the spleen of deficient animals. Consequently, the spleen immune system is deeply affected by this deficiency.

Bacterial peptidoglycans (PG) possess various immunomodulating activities, including the ability to inhibit the growth of some experimental tumors. We report here that PG induce the release by mouse (DBA/2 and C3H/HeJ) peritoneal macrophages of a cytotoxic factor (CF) that is active on L-929 cells and a 3-methylcholanthrene-induced tumor cell line but inactive on normal fibroblasts. Only the adjuvant-active PG (extracted from Bacillus megaterium and Staphylococcus aureus) were good inducers of the CF, whereas the adjuvant inactive PG (extracted from Micrococcus lysodeikticus and Corynebacterium poinsettiae) exhibited only weak activity. The CF was released within 2 hours of contact with PG either in serum-free or serum-containing media. The CF was inhibited neither by serum nor by the protease inhibitors that have been tested. It was stable for 30 minutes at 56 degrees C but inactivated after being heated for 10 minutes at 80 degrees C. After gel filtration, a single peak of activity at 80-90 kilodaltons was found. Chromatofocusing showed that the isoelectric point of the CF was 4.8.

Susceptible C57BL/6 mice infected with a temperature-sensitive mutant of Salmonella typhimurium exhibited a marked depression of in vivo and in vitro primary antibody response to sheep erythrocytes which persisted for several weeks. The antibody response to a T-independent antigen (TNP-polyacrylamide) was also depressed. Cell-mixing experiments indicated that spleen cells from infected animals contained adherent suppressor cells and that the functional activity of T and B cells was unaffected. The antibody response of spleen cells from infected mice was restored by the addition of catalase, suggesting that hydrogen peroxide is involved in the mechanism of suppression.