Inhibition of tumor growth by the peptidoglycan from Bacillus megaterium.

When injected in admixture with tumor cells, the peptidoglycan extracted from Bacillus megaterium inhibited the growth of a chemically induced fibrosarcoma in syngeneic rats. In some instances, the tumor growth was totally suppressed. Animals rejecting mixed inocula exhibited a tumor-specific immunity. Peptidoglycan was not cytotoxic for tumor cells; it rendered macrophages nonspecifically cytotoxic.

Release of a cytotoxic factor by macrophages stimulated with adjuvant-active peptidoglycans.

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.

Cytotoxic responses induced by peptidoglycans with or without in vivo antitumor activity.

Cytotoxic responses mediated by effector cells stimulated in vivo were studied after ip injection in mice of peptidoglycans extracted from gram-positive bacteria. A comparison was done between Staphylococcus aureus peptidoglycan (PGS), which possessed an antitumor effect, and Micrococcus lysodeikticus peptidoglycan, which was ineffective against tumors. Both peptidoglycans induced similar effects on the modulation of T-cell cytotoxic response. Both were able to stimulate splenic and peritoneal nonspecific cytotoxicity against YAC-1 lymphoid tumor cells, but only PGS could induce cytotoxicity and cytostasis against solid tumor target cells.

Peptidoglycans extracted from gram-positive bacteria: expression of antitumor activity according to peptide structure and route of injection.

The antitumor effect of peptidoglycans of various structures extracted from different gram-positive bacteria was studied, on chemically induced fibrosarcomas, in C3H/He, C57BL/6, and (C57BL/6 X C3H/He)F1 mice. When given sc admixed with tumor cells, only some peptidoglycans (those extracted from Bacillus megaterium and Staphylococcus aureus) enhanced tumor resistance in syngeneic and semiallogeneic hosts, whereas other peptidoglycans (those extracted from Micrococcus lysodeikticus and Corynebacterium poinsettiae) possessed no antitumor effect. When tumor cells were given ip, administration of peptidoglycans by the same route was either without effect on tumor growth or it induced tumor enhancement. Enhancement could be observed with all of the peptidoglycans tested. The antitumor effect when given sc and the ability to stimulate the proliferation of B-lymphocytes were shared by the same two peptidoglycans, while the other two peptidoglycans were devoid of both activities. It appears that these biological activities depend on the structure of the peptide moiety of peptidoglycans and that mitogenic and antitumor responses are stimulated by similar structures.

Mycobacterium genavense infection in normal and immunodeficient mice.

Mycobacterium genavense is a recently described microorganism causing disseminated infections in AIDS patients. In this study, we investigate its pathogenicity in mice and some mechanisms of the host response to this bacterium. Following an intravenous challenge of 10(6) organisms, M. genavense grew progressively in the spleens and livers of BALB/c and CBA mice over at least an 8-month period. Granulomas were present in the spleens, livers and lungs of the animals. The numbers of bacteria recovered from the spleens and livers were higher in BALB/c (Bcg(s)) than in CBA (Bcg(r)) mice from day 30. The role of the Bcg gene, in the early phase of infection, was supported by the fact that the bacterial load, on day 15, was higher in BALB/c than in the congenic C.D2 (Bcg(r)) mice. The role of T cells in the host response was suggested by the high susceptibility of nude mice to M. genavense infection. In vivo depletion experiments in CBA mice indicated that gamma interferon and both CD4(+) and CD8(+) T cells participate in the containment of the bacterial load.

Immunogenicity of foreign peptide epitopes expressed in bacterial envelope proteins.

We have used two bacterial proteins from Escherichia coli to express heterologous peptides. Both proteins are situated in the E. coli cell envelope but have different properties: LamB is an integral outer membrane protein, and MalE a soluble periplasmic protein. The peptides were expressed as genetic inserts within "permissive sites" of these recipient proteins, i.e. sites which allow the insertion of foreign peptides without affecting the biological properties of the host protein. In this paper, we summarize preliminary rules governing the immunogenicity of resulting LamB and MalE hybrid proteins when expressed in E. coli. We focus on two model epitopes: either peptide 132-145 from the preS(2) region of hepatitis B virus or peptide 93-103 from poliovirus VP1 capsid protein. We also present first results obtained when the same hybrid proteins were expressed in attenuated Salmonella typhimurium. Plasmids encoding the hybrid proteins were transferred to aroA S.typhimurium by electroporation. In vitro, the hybrid proteins could be expressed at high levels by S. typhimurium. Mice were immunized by parenteral and oral routes. The effect of the carrier protein and the level of its expression on the in vivo behaviour of the immunizing bacteria and on the immune response induced will be discussed.

JC virus detection in the cerebrospinal fluid of AIDS patients with progressive multifocal leucoencephalopathy and monitoring of the antiviral treatment by a PCR method.

Twenty-four cerebrospinal fluid (CSF) samples from 19 AIDS patients with neurological signs were analysed by the polymerase chain reaction (PCR) for the presence of JC virus (JCV). Eleven of the 19 patients tested presented with progressive multifocal leucoencephalopathy (PML). Two specific JCV target sequences were used for the PCR analysis: a sequence specific for the T antigen genes from both BK virus (BKV) and JCV (PCR1) and a sequence specific for the large T antigen gene from JCV (PCR2). The JCV genome was detected in 10 of 11 patients with PML by the PCR1 method and in all 11 patients by the PCR2 method. With samples from the eight patients without PML, one positive result was obtained with the PCR1 method and this sample and another gave positive results with PCR2. Multiple CSF samples were collected from three patients with PML at different times, including after intrathecal cytarabine treatment, and were tested by the PCR2 method for the presence of the JCV genome. The PCR result became negative for two of the three patients during the cytarabine treatment. However, the absence of a PCR signal was not associated with clinical improvement in these patients. The PCR method is useful for the detection of JCV in CSF samples and in the diagnosis of PML. However, the application of PCR for monitoring the effect of treatment remains to be established.

Molecular epidemiology of Legionella pneumophila serogroup 1 by ribotyping with a non-radioactive probe and PCR fingerprinting.

Hybridization with acetylaminofluorene-labelled 16 + 23 S rRNA from Escherichia coli was used to detect DNA polymorphism among Legionella pneumophila serogroup 1 isolates. Isolates from unrelated patients showed at least four different rRNA restriction patterns, whereas those from related patients showed a single pattern. Amplification of genomic regions with an arbitrary primer by polymerase chain reaction was used to further analyze the isolates. Related isolates showed closely related patterns while unrelated isolates displayed six distinct patterns. We could differentiate the majority of unrelated isolates with the combination of the patterns obtained with the ribotyping and the PCR fingerprinting, while strains from the same outbreak remained highly related. The ribotyping and the PCR fingerprinting are proposed as useful and easy to perform epidemiological markers of L. pneumophila serogroup 1 infection.

Interleukin 1, a mediator of immunoadjuvant peptidoglycans.

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.

[Molecular markers in the epidemiologic study of Legionella pneumophila infections]. / Les marqueurs moléculaires pour l'étude épidémiologique des infections à Legionella pneumophila.

PURPOSE: Legionnaires' disease is due to the inhalation of contaminated aerosols. The identification of the source of contamination in the aquatic environment is necessary to prevent the occurrence of new cases. A comparative study of clinical and environmental isolates is the basis of epidemiological investigations. CURRENT KNOWLEDGE AND KEY POINTS: Genotypic methods are now mainly used to compare bacterial strains. Some of these methods are based on the electrophoretic separation of DNA restriction fragments. When electrophoretic profile are complex, some fragments can be visualized after hybridization (ribotyping). Large-sized fragments can be separated by pulsed-field gel electrophoresis. Other techniques are based on gene amplification, such as AP-PCR. This technique is easy to perform but its discriminatory power and reproducibility are lower. Some procedures are combining enzymatic cleavage and gene amplification. Finally methods based on the nucleotide sequence analysis of some genes are being evaluated. FUTURE PROSPECTS AND PROJECTS: Techniques enabling the rapid comparison of various Legionella isolates will permit a quick detection of outbreaks and contribute to the identification of the source of contamination.

[Defense mechanisms against bacteria of intracellular development]. / Mécanismes de défense contre les bactéries à développement intracellulaire.

Intracellular bacteria are not inhibited by antibodies. Therefore the main mechanisms of resistance against these pathogens are the influx and activation of mononuclear phagocytes by the synergistic interaction of gamma-interferon and tumour necrosis factor. During the early phase of infection, gamma-interferon is produced by IL-12-activated natural killer cells (IL-12 being mainly produced by macrophages). In the later phase of infection acquired immunity is T-cell-mediated and involves the Th1 CD4+ subpopulation. These cells produce gamma-interferon when triggered by antigen. There is a complex network of interactions among cytokines. Some cytokines act synergistically to enhance resistance to infection, yet antagonistic interactions can sometimes occur.

[Legionnaires' disease in the Paris area: epidemiology and mortality. Apropos of a series of 81 culture-positive cases]. / Légionellose en région parisienne: épidémiologie et mortalité. A propos d'une série de 81 cas à culture positive.

OBJECTIVES: Evaluate etiological circumstances and prognosis in Legionnaires' disease. METHODS: A series of 81 culture-proven cases of Legionnaires' disease was collected in the Paris area between 1989 and 1994. RESULTS: Direct immunofluorescence assay was positive for Legionella pneumophilia in 48% of the cases. Serogroup 1 was isolated in 88% of the cases. The median age of the patients was 51 years and 74% were males. Infection was nosocomial in 28% of the cases. Immunosuppression was present in 45% of the patients (transplantation, cancer, leukemia). Among the immunosuppressed patients, 7 were HIV-infected. Mortality due to legionellosis reached 27%. This high mortality was probably related to patient selection criteria. CONCLUSION: Mortality from Legionnaires' disease remains high as confirmed in this series.

[Quality of blood salvaged in orthopedic surgery by washing swabs]. / Qualité du sang récupéré en chirurgie orthopédique par le lavage des compresses.

OBJECTIVE: To establish the feasibility and safety of recuperating blood absorbed by swabs used during orthopaedic surgery. STUDY DESIGN: Open, prospective study. PATIENTS: Included were children undergoing potentially haemorrhagic orthopaedic surgery for whom intraoperative blood salvage seemed possible. Excluded were those with contraindications for this procedure such as septic surgery and cancer surgery. METHOD: Intraoperative swabs used within the surgical field were collected by a surgical assistant, also in charge of weighing and washing them. The liquid was collected by the aspiration system of a recuperation-washing machine (RWM). The salvaged red blood cells were collected and retransfused at the end of surgery. Several samples of the washing liquid of the swabs and salvaged blood were taken during the procedure. The correlation between the quantity of blood shed and salvaged was calculated. The biological and clinical tolerance of the transfusion was assessed. RESULTS: Twelve patients undergoing surgery for scoliosis have been included. An average of 278 mL of blood were salvaged. In the washed cell concentrates the haematocrit was 54% and the free haemoglobin concentration was 3.84 g.L-1. All the bacteriological tests were negative over the first 24 hours. CONCLUSION: Provided that a strict operatory protocol is followed, this study demonstrates the possibility of recuperating blood from swabs used during major orthopaedic surgery.

[Enterobacter cloacae and E. aerogenes septicemia: emergence of resistant variants (derepressed cephalosporinase) during treatment with third-generation cephalosporins]. / Septicémies à Enterobacter cloacae et E. aerogenes: émergence de variants résistants (céphalosporinase déréprimée) en cours de traitement par des céphalosporines de troisième génération.

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.

Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection.

Two aspects of acquired resistance to Salmonella typhimurium infection in BALB/c mice, i.e., the ability to clear the primary inoculum from the spleen and resistance to a secondary challenge, were studied with the use of mAb against T cell subsets. The ability to clear a temperature-sensitive mutant of S. typhimurium from the spleen (assessed at day 21) was abrogated by in vivo treatment with anti-CD4 mAb. Accelerated bacterial clearance could be adoptively transferred into naive mice. In vitro depletion experiments also showed the role of CD4+ T cells in this phenomenon. Depletion of CD8+ T cells had only a marginal effect. Resistance to reinfection in the late phase of the primary infection (day 50) was markedly depressed by in vivo treatment with anti-CD4 mAb, whereas this was not the case during the early phase (day 14). Furthermore, during the early phase of infection athymic nude mice showed increased nonspecific resistance to reinfection. Taken together these results suggest that T-independent mechanisms play a major role in acquired resistance during the early phase of infection.

Influence of mouse genotype and bacterial virulence in the generation of interferon-gamma-producing cells during the early phase of Salmonella typhimurium infection.

Interferon-gamma (IFN-gamma) is known to play a major role in resistance to Salmonella typhimurium infection. In this study, the IFN-gamma production in spleens of mice infected with S. typhimurium was analysed at the single cell level using an ELISPOT method. The in vivo IFN-gamma production during the early phase of infection with virulent and avirulent S. typhimurium strains was examined in four mouse strains. Data show that infection with a virulent strain of S. typhimurium caused a much greater enhancement in the frequency of IFN-gamma-producing cells in innately resistant (ltyr) mice (CBA and DBA/2) than in susceptible (ltys) mice (C57BL/6 and BALB/c). In contrast, infection with an avirulent strain of S. typhimurium induced a clear increase in the number of IFN-gamma-producing cells in susceptible mice which was even greater than in resistant ones. These results indicate that both the host genetic background and bacterial virulence play a critical role in the regulation of IFN-gamma production during the early phase of S. typhimurium infection.

[Comparative efficacy of ampicillin, chloramphenicol, cefotaxime, ceftriaxone and pefloxacin in experimental Salmonella typhimurium infection]. / Efficacité comparée de l'ampicilline, du chloramphénicol, du céfotaxime, de la ceftriaxone et de la péfloxacine dans l'infection expérimentale a Salmonella typhimurium.

The efficacies of various antibiotics were compared in Salmonella typhimurium infection in mice. Treatment began 24 h after challenge. Antibiotics were given subcutaneously every 12 or 24 h in the following daily doses: 200 mg/kg for ampicillin, 100 mg/kg for chloramphenicol, cefotaxime and ceftriaxone, 50 mg/kg for pefloxacin. The number of bacteria in the spleens was determined after 3 and 7 days of treatment. In animals treated with ceftriaxone or pefloxacin the mean number of bacteria per spleen was from one to two log10 lower than in animals treated with other antibiotics. Similar results were obtained in genetically susceptible or resistant mice. The MIC being similar for cefotaxime, ceftriaxone and pefloxacin, the better in vivo activity of the two latter drugs appears to be related to their pharmacokinetic properties.

Adjuvant activity of bacterial peptidoglycans.

Purified peptidoglycans isolated from various Gram negative and Gram positive bacteria can substitute for Mycobacteria in Freund's adjuvant. Monomeric subunits possess the same adjuvant activity. This activity seems to be related to the peptidic fraction.

Production of interleukin-12 by murine macrophages in response to bacterial peptidoglycan.

Peptidoglycan (PG), a component of the bacterial cell wall, has various immunomodulating activities, including the capacity to induce delayed-type hypersensitivity reactions to antigens administered in Freund's adjuvant. We report that PG induces interleukin-12 (IL-12) mRNA production and IL-12 secretion by mouse macrophages. The capacity of PG to induce IL-12 production, like its previously reported immunomodulating activities, was dependent on the structure of its peptide subunit. PG from Bacillus megaterium and Staphylococcus aureus induced IL-12 production, whereas PG from Micrococcus luteus and Corynebacterium poinsettiae did not. The ability of most bacterial PGs to induce IL-12 production suggests that they play an important role in triggering host defense mechanisms against bacterial infections.

Suppression of primary antibody response in genetically susceptible mice infected with Salmonella typhimurium: restoration by catalase.

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.