Suppression of the primary IgM response by environmental teichoic acid.

It has been shown previously that ingestion of glycerol teichoic acid (GTA) in the conventional laboratory diet (8 mg/kg) is the stimulus for natural background responses to GTA in rats. Since injected GTA suppresses responses to sheep red blood cells (SRBC), it was suspected that dietary GTA also might be acting suppressively. A comparison of rats fed the conventional diet with rats fed a GTA-free diet showed that ingested GTA markedly suppressed immune and background direct plaque-forming cell (PFC) responses to SRBC. It appeared that a direct causal relationship existed between the degree of suppression and the amount of GTA exposure. When GTA-deprived rats were force-fed varying doses of GTA or when conventional animals were injected (i.p.) with GTA, increasing the total GTA dose resulted in decreased direct PFC responses to SRBC. Suppression was also observed when GTA-deprived rats were force-fed GTA-containing Bacillus sp. ATCC 29726. The phenomenon of suppression by dietary GTA was not restricted to responses to SRBC, as similar results were obtained with chicken erythrocytes. When IgG PFC were measured, no difference between conventional and GTA-deprived groups was observed. Thus, an IgM-IgG shift does not seem to play a role in the mechanism of suppression by GTA.

Modulation of the immune response to sheep erythrocytes by lipid-free glycerol teichoic acid.

The 4-day response of C3H/HeJ mice to sheep erythrocytes was suppressed by a lipid-free teichoic acid with an average molecular weight of 2,900 when it was administered by the intraperitoneal route. Enhancement was not observed at that time, and neither suppression nor enhancement could be demonstrated by the intravenous route. Either suppression or enhancement of background plaques could be induced, depending upon the timing. Dosage influenced the degree of suppression from 8 to 100 micrograms, whereas suppression of background plaques required only 1 microgram of lipid-free teichoic acid. The kinetics of the sheep erythrocyte response was altered by treatment of the mice with lipid-free teichoic acid, delaying the peak until day 5 and producing enhancement at that time. Although lipid-free teichoic acid was shown to be toxic for mouse splenocytes (50% lethal dose, ca. 200 micrograms) in vitro, no effect at the levels employed was observed in vivo. The data presented indicate that modulatory activity is influenced by route, timing, dosage, and apparently the number of antibody-secreting cells.

Immunogenicity of soluble versus cellular glycerol teichoic acid.

Guinea pigs which were injected with either whole bacilli or purified soluble glycerol teichoic acid (GTA) usually exhibited a rise in hemolysin titer to GTA-coated erythrocytes. The only exceptions were those animals having high baseline titers of natural anti-GTA antibodies. Rats yielded better responses than guinea pigs and produced significantly higher responses to the soluble antigen than to the cellular GTA. Rats reared on a GTA-free diet were predominantly free of natural antibodies to GTA and furnished a more clear-cut model for assaying immune responses. Using this model, it was shown that adsorption of GTA to homologous erythrocytes before injection resulted in poor responses, suggesting that such spontaneous adsorption does not account for the good responses to soluble antigen. In GTA-deprived rats, positive skin tests were induced only with bacilli, whereas migration inhibitory factor was induced with both bacilli and soluble antigen. Hemolytic plaques in immunized rats were increased over controls with both kinds of immunogen, but the GTA-deprived rats responded better than conventional ones, and hemolytic plaque responses to bacilli were better than those to soluble antigen. This reversal of the serum hemolysin results may be due to delayed suppression by soluble GTA or to antibody cycling. The guinea pig data, combined with results from GTA-deprived rats, suggest that high antibody levels resulted in depressed antibody synthesis, perhaps because antibody cycling was initiated. No evidence was found to explain the superior responses to soluble antigen, but it did not seem related to formation of immune complexes or adsorption to erythrocyte membranes.

Chemical carcinogenesis and immunity: immunologic status of rats treated with methylnitrosourea.

During the treatment of outbred Sprague-Dawley rats with methylnitrosourea (MNU) or the noncarcinogenic analog diphenylnitrosamine, antibody levels to teichoic acid as well as several parameters of lymphocyte and macrophage function were assessed in animals not overtly stimulated with antigen. Treatment with MNU did not appear to alter most immunologic parameters studied. Some alterations occurred in natural antibody levels, in spleen weight, and in peripheral blood differentials of rats that had received the highest carcinogen dose (4.5 mg/kg). These alterations, however, appeared to coincide with the development of tumors and were not observed in animals with premalignant lesions. Levels of chemical carcinogens that were capable of inducing tumors did not necessarily suppress the immune response.

Lipid-free glycerol teichoic acids with potent membrane-binding activity.

Lipid analysis of several glycerol teichoic acid preparations strongly indicated that covalently bound lipid is not required for spontaneous adsorption of glycerol teichoic acid to erythrocyte membranes. Although fatty acids were detected in each of four batches, none were covalently bound. Chloroform-ether-extracted antigens retained potent erythrocyte membrane-binding activity as measured by passive hemagglutination, even though they were shown to contain less than one fatty acid residue per 4,869 teichoic acid chains. Mild ammonolysis abolished erythrocyte-sensitizing activity in passive hemagglutination, but further studies indicated the loss of activity was due to partial destruction of the polyglycerophosphate backbone and not to the removal of esterified lipid. The amount of hydrolyzed antigen required to produce 100% passive hemagglutination inhibition was between 170 and 330 times the amount required to produce the same result using unhydrolyzed glycerol teichoic acid. The average chain length was reduced from 19.1 to 9.7, 7.4, and 5.1 glycerophosphate residues for antigen samples hydrolyzed for 1, 5, and 16 h, respectively.

Environmental origin of natural antibodies to teichoic acid.

In an effort to determine the origin of natural antibodies to teichoic acid, rats were fed a sterile liquid diet free of detectable teichoic acid and virtually free of gram-positive bacteria. Both germ-free and conventional Sprague-Dawley rats raised on this diet failed to produce antibodies to polyglycerophosphate, whereas 100% of their counterparts fed the usual teichoic acid-containing diet did produce these antibodies. The intestinal flora was similar in both groups of animals. When the test animals were immunized intraperitoneally or orally with gram-positive bacteria, 100% displayed immunocompetency by producing significant levels of antibody. These results demonstrate the environmental nature of the antigenic stimulus for these antibodies and suggest the importance of food as the major source of stimulation. The experimental model described here furnishes a valuable tool for studies of immunologic responses where a single known specificity and a controlled system would be advantageous.

Cyclic antibody formation to polyglycerophosphate in normal and injected rats.

Normal adult Sprague-Dawley rats were bled serially over a 30-week period and their sera were examined for antibodies to polyglycerophosphate (PGP) by a standardized passive hemolysis test. Levels of "natural" antibodies to PGP fluctuated during this period with a majority of animals exhibiting pronounced cycling of serum antibody levels, however, the individual cycles were not synchronized with each other. Feeding of radiolabeled Gram-positive bacilli to these animals and examination of lymphoid tissue by liquid scintillation counting revealed that the antigen persisted mainly in the mesenteric lymph nodes. A second group of rats was injected i.v. with radiolabeled Gram-positive bacilli and tissues were examined for plaque-forming cells (PFC) of PGP specificity, and the sera were examined by passive hemolysis. Cycling of both anti-PGP antibodies and PFC became synchronized in the injected animals with peaks of serum antibody evident at 16 and 28 days post-injection and splenic PFC peaks at 4 and 16 days post-injection. Cycling was also observed in the mesenteric lymph nodes and bone marrow Examination of lymphoid tissue from the rats injected i.v. revealed that antigen introduced by this route also perisisted mainly in the mesenteric lymph nodes, This report demonstrates individual cycling of natural responses to environmental antigen and to the same determinant in secondary responses, indicating its importance as a regulatory mechanism.

Cross-reactions of Streptococcus mutans due to cell wall teichoic acid.

Antisera to the whole cells of Streptococcus mutans cross-reacted with antigen extracts from four other gram-positive species, as well as with those of three other oral streptococci. Similarly, antisera to these bacteria cross-reacted with extracts from S. mutans and with those from each other. Using a purified phenol extract of the walls of S. mutans, which was identified by chemical, immunochemical, and enzymatic analyses as glycerol teichoic acid, the cross-reactions were shown to be specific for a determinant of the teichoic acid backbone. Results were confirmed in immunodiffusion tests where clear bands of identify were shown. These observations point out the need for caution in sereological research empolying extracts of gram-positive bacteria and may be of interest in investigations of periodontal disease.

An experimentally pathogenic Bacillus species. II. The pathogenicity of the organism for mice.

The pathogenic effects produced in mice by intraperitoneal (i.p.) injection of a Bacillus species (OSU 372) are presented. This organism belongs to group 3 of the genus, and members of this group have not heretofore been shown to be pathogenic for mice even under experimental conditions. However, this organism is capable of producing a fatal involvement in doses which are not considered to be overwhelming. The mean lethal dose (LD50) of the organism for 20-25 g mice by the i.p. route is about 1 times 10-8 bacteria/mouse. A rapid drop in body temperature along with severe dehydration were noted in infected animals, and hematologic studies indicated that leukopenia and hemoconcentration also occurred. Although a transient septicemia developed, the bacteria could not be recovered from the tissues of fatally infected mice after a certain point in time. Results prevented indicate that the animals died of hypovolemic shock. A possible parallel with human bacillary infection is drawn.

An experimentally pathogenic Bacillus species. I. Description and characterization of the organism.

A previously underscribed Bacillus species has been characterized. The organism was isolated from a blood transfusion bottle implicated in a fatal bacteriogenic transfusion reaction and has been examined from a number of different aspects to characterize it as completely as necessary to provide identification. It was placed into group 3 of the genus Bacillus on the basis of its cellular morphology, the morphology of the sporangium, and the location of the spore within the cell. There are only three other species described in this group. Since the organism differed markedly from each of these, it appears to represent a new species.

Preparation and chemical composition of the cell walls of Streptococcus mutans.

Purified cell walls from Streptococcus mutans strain BHT were prepared without the use of proteolytic enzymes in order to retain all cell wall constituents for chemical analysis. Of four methods employed, the Ribi cell fractionator produced disrupted cell suspensions which could be most thoroughly purified on sucrose gradients. Results of chemical analyses on purified cell walls prepared in this 8.9% glycerol teichoic acid, 33.6% non-peptidoglycan polysaccharide, and 49.9% peptidoglycan.