Lung (1990) Suppl:716-719 @

Springer-Verlag New York Inc. 1990

Influence of a Bacterial Extract on Antigen Presentation and Protection against Experimental Infections R. Fontanges, C. Bottex, B, Cristau, and M. F. Burckhart French Army Medical Research Center, La Tronche, France

Abstract. Although substances known as immunodulators are usually used to stimulate nonspecific immunity, their mode of action is not well understood. In an effort to clarify this mechanism, we investigated the effect of a lyophilized bacterial extract (Broncho-Vaxom) on experimental infections, on normal or irradiated mice, and on antigen processing. Key words: Immunomodulators--Broncho-Vaxom. Broncho-Vaxom's Protective Effect on an Experimental Klebsiella Pneumoniae Infection

Two batches of 20 six-week-old Balb/c mice received 600 mg/kg/day of Broncho-Vaxom on five consecutive days by oesophageal intubation. These mice were infected intraperitoneally with 103/0.5 rill Klebsiella pneumonia, 15 days and 30 days after the last intubation. We observed that Broncho-Vaxom provided significant protection compared with the control group and the protection depended on the time after the intubation. We investigated if Broncho-Vaxom had a preventive effect on the treatment of an infection with Bactrim (trimethoprim-sulfamethoxazole). Mice had orally received 600 mg/kg/day of Broncho-Vaxom on five consecutive days (the optimal antiinfection dose of Bactrim was previously determined as 0.5 mg/day for five days, after the infection). Ten days after Broncho-Vaxom intubation, mice were infected intraperitoneaUy with 103/0.5 ml Klebsiella pneumoniae and treated with Bactrim.

Offprint requests to: Dr. R. Fontanges, Maitre de Recherches du Service de Sant6, C.R.S.S.A., 24 Av. des Maquis du Gr6sivaudan, 38702 La Tronche, Cedex, France.

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We observed that the combined administration of Broncho-Vaxom and Bactrim increased the level of protection compared to Bactrim alone. In vivo Study of Broncho-Vaxom's Protective Efficiency against

Pseudomonas aeruginosa Infection in Irradiated Mice Radiation may modify interleukin production [1], cause variations in the levels of prostaglandins and cyclic nucleotides [2] and reduce antibody production [3]. These effects show that an irradiated animal is in a condition of immunosuppression which increases its vulnerability to the infection with an opportunistic germ. During our study, we investigated if Broncho-Vaxom had a protective effect on irradiation which was followed by experimental infection with Pseudomonas aeruginosa. We also compared the effect of Broncho-Vaxom intubation alone with that of the combined administration of Broncho-Vaxom and indomethacin, used as a prostaglandin-synthesis inhibitor. In fact, in recent studies, we showed that indomethacin was able to increase Broncho-Vaxom's immunostimulatory effects. We used 80 eight-week-old male Balb/c mice divided into four batches of 20 mice each. On day 0, each mouse received, by oesophageal intubation, either 0.1 ml of Broncho-Vaxom (80 mg/ml) or 0.1 ml of the BronchoVaxom-indomethacin (BVI) [mixture containing Broncho-Vaxom (80 mg/ml) and indomethacin (500/xg/ml in 1% alcohol)] or 0.1 ml of isotonic aqueous solution for control batches. On day 1, the mice were irradiated with a dose of 8 gy with the aid of a Saturne-type particle accelerator (C.G.R. MeV) using a photon radiation of 18 MeV. On day 2, the mice received 106/0.5 ml Pseudomonas aeruginosa suspension intraperitoneally. The control batch received 0.5 ml of distilled water. The survival rates were calculated up to the 30th day following the irradiation. A X2 distribution test supplemented by a Fischer's test showed that in the Broncho-Vaxom group at day 30 the survival was significantly higher than in the control group. Study of Broncho-Vaxom's Effect on the Processing of a Radiolabeled Viral Antigen T cells respond to an antigen only after it is captured by another cell called antigen-presenting cell (APC). This antigen is then partially degraded by still badly defined mechanisms leading to short immunogenic peptides which can stimulate an immune response. These determinants may take the form of native proteins if the epitope is located directly on an accessible flexible portion [4] of the unfolded polypeptidic chain or peptidic fragments [5]. This intermediate step of antigen processing is situated in an intracellular acid compartment to avoid the inhibition of protease inhibitors. This hypothesis

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was made by Germain [6] who showed that an antigen is processed by proteolysis in the endosome. After this level of degradation, the antigen combines with a molecule of type Ia, which apparently protects the determinant from total lysis, as it was shown by Allen [7]. During our study, we investigated pretreatment of macrophages with Broncho-Vaxom on the capture and degradation of a viral antigen. J774-1 macrophages were cultivated in 96-well plates at the rate of 105 cells per well in 100/zl of fetal calf RPMI serum medium at 37°C with a 5% CO2 atmosphere. Cells were left adhering for two hours. The supernatants were then eliminated and replaced by 100/zl of medium containing either 1.6 mg/ml of Broncho-Vaxom or I0/~g/ml of lipopolysaccharide (Escherichia coli 0111: B4 Sigma). After a two hour incubation at 37°C, the supernatants were decanted and the cells were washed with RPMI. Measles virus radiolabeled with 35S methionine was then added at the rate of 4.104 cpm/well for 30 min or 19 h. The J774-1 cells were then washed and destroyed in a lytic buffer Tri-HCI pH 7.8 containing 2% of Triton X-100 and were analyzed on 12.5% polyacrylamide gel with the presence of beta-mercaptoethanol from the Laemmli method [8] modified by Sheshberadaran [9]. We found that after a 30 min macrophage-antigen incubation, radiolabelled polypeptides appeared in the zone 24, 25 KD, only with the ceils treated by Broncho-Vaxom. After a 19 h incubation, we found the radiolabelled viral material, i.e., HA (78 KD), NP (60 KD), F1 (42 KD), M (39 KD) and additional polypeptides which molecular weight was less than 40 KD (24, 25 KD, 18 KD and 16 KD). On the opposite, LPS did not modify these processes in our experimental conditions when compared to the control test. We are presently studying, with differential ultracentrifugation, different cell compartments, particularly plasma membranes and lysosomes to locate these polypeptides depending on the time. The quality of purification is controlled determining activities of 5' nucleotidase and acid phosphatase. We also have to define the nature of these polypeptides and their function as a signal which activates an immune response. It may be concluded that pretreatment of macrophages with BronchoVaxom accelerates the capture and the degradation of a viral antigen by macrophages. This mechanism, among others, could explain the immunopotentiation of Broncho-Vaxom and its protection against infections.

References 1. Manori I, Kushelevsky A, Segal S, Weinstein Y (1985) Effect of radiation on the production of interleukins and T lymphocytes activities. J Natl Cancer Inst 74:1215-1221 2. E1-Ghazaly M, Salem S, Kenawy S, Roushdy HM, Khayyal MT (1986) The protective value of piroxicam on the enhanced inflammatory response after whole body irradiation. Pharmacol Res Comm 18:563-580

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3. Behling UH, Nowotny A (1978) Long-term adjuvant effect of bacterial endotoxin in prevention and restoration of radiation-caused immunosuppression. Proc Soc Exp Biol Med 157:348-353 4. Lee P, Matsueda GR, Allen PM (1988) T cell recognition of fibrinogen. A determinant on the A chain does not require processing. J Immunol 140:1063 5. Allen PM (1987) Antigen processing at the molecular level. Immunology Today 8:270 6. Germain RN (1986) The ins and outs of antigen processing and presentation. Nature 322:687 7. Donermeyer DL, Allen PM (1989) Binding to I a protects an immunogenic peptide from proteolytic degradation. J Immunol 142:1063 8. Laemmli UK (1970) Cleavage of structural proteins during Assembly of the Head of Bacteriophage T4. Nature 227:680 9. Scheshberadaran H, Chert SN, Norby E (1983) Monoclonal antibody against five structural components of measles virus: characterization of antigenic determinants on nine strains of measles virus. Virology 128:341-353

Influence of a bacterial extract on antigen presentation and protection against experimental infections.

Although substances known as immunodulators are usually used to stimulate nonspecific immunity, their mode of action is not well understood. In an eff...
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