Shigella Antigens Research Articles

Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other gram-negative bacteria.

Incubation of intact Salmonella typhi Ty21a, Salmonella enterica serovar Typhimurium (Salmonella typhimurium) aroA or Escherichia coli DH5 alpha with membrane vesicles (MVs) derived from either Shigella flexneri M90T or Pseudomonas aeruginosa dsp89 resulted in a significant incorporation of vesicle antigens into the outer membrane of the bacteria; each recipient strain possessed a surface mosaic of new Shigella and Pseudomonas antigens intermixed with the native antigens of the Salmonella or Escherichia strains. Electron microscopy of preparations during the integration of vesicle antigens revealed that the MVs rapidly fused with the outer membrane of the host strains. Western blot analysis of host bacteria confirmed the integration of foreign antigens. Quantitative analysis for binding and fusion of antigens using an ELISA showed that approximately 78.7 +/- 12.8 ng of the Pseudomonas and 67.5 +/- 13.8 ng of the Shigella LPSs (microgram host protein)-1 were integrated into the Sal. typhimurium strain. Similar integrations of the Shigella or Pseudomonas vesicles were found with the E. coli or Sal. typhi strains. There was no loss of viability in the recipient bacteria after incorporation of the MVs, although vesicle antigens became diluted during continued growth as daughter cells shared the vesicle antigens. The new antigens were highly stable after being incorporated into recipient strains, being able to withstand storage of several months at 4 degrees C as well as several cycles of freezing and thawing. Since the recipient bacteria are common vaccine strains, the procedure described here offers a simple efficient means of introducing exogenous surface antigens, in their native form, into the outer membranes of Gram-negative bacteria for possible vaccine use.

Dissociation between cytokine mRNA expression and protein production in shigellosis.

In our study, infection with Shigella dysenteriae type 1 (n = 16) or Shigella flexneri in adults (n = 5) was associated with a gradual accumulation of mRNA for interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, transforming growth factor-beta, IL-10, IL-4, TNF-beta, interferon (IFN)-gamma and perforin in the rectal biopsy samples during the convalescent stage of the disease demonstrated by in situ hybridization. In contrast, immunohistochemical staining in rectal tissues of cytokine protein-producing cells at the single-cell level exhibited a steady-state expression during 2-36 days after the onset of the disease. The frequency of cytokine mRNA-expressing cells varied in the range of 3-100-fold higher than that of the corresponding protein-synthesizing cells. The accumulation of cytokine mRNA in vivo during shigellosis represented a long-lasting phenomenon throughout the disease course, and may be linked to its immunopathogenesis. The results also indicate that assessment of both protein and mRNA in vivo may provide complementary information. Stimulation in vitro of peripheral blood mononuclear cells from normal healthy donors with Shigella-derived lipopolysaccharide or shiga toxin was carried out to elucidate the role of Shigella antigens in the regulation of translation of cytokine-specific mRNA. The incidence of cytokine (IFN-gamma, IL-6 and TNF-alpha) mRNA- and cytokine protein-expressing cells was very similar and congruent after both these Shigella-derived stimuli. We could, thus, not find evidence for shiga toxin-induced down-regulation of cytokine mRNA translation as the explanation for the observed discrepancy between cytokine mRNA and protein levels in the tissue biopsies.

Changes in the peripheral blood T-Cell receptor V beta repertoire in vivo and in vitro during shigellosis.

A sequential activation of T cells in peripheral blood during shigello sis has been observer (D. Islam, P.K. Bradham, A. A. Lindberg, and B. Christensson, Infect. Immun 63:2941-2949, 1995). To further investigate the cellular response during the course of Shigella infection, changes in the T-cell receptor (TCR) repertoire in the subsets in blood in patients during shigellosis was that Shigella antigens may modulate the function of T cells carrying TCRs capable of recognizing Shigella-specific epitopes or superantigens. Such a selective preference for T cells expressing certain TCR Vbeta types could lead to the expansion or deletion of these T cells. In the present study of 27 adult male Bangladeshi patients with dysentery (14 cases caused by Shigella Dysenteriae 1 and 13 cases caused by Shigella flexneri), the changes in the TCR Vbeta repertoire of peripheral blood CD4+ and CD8+ T-cell subsets have been analyzed with a panel of nine anti-Vbeta monoclonal antibodies by flow cytometry. Twenty healthy males from Bangladesh and 20 healthy males from Sweden served as controls. Compared with the Bangladeshi controls, the patients had an increased frequency of CD4+T cells expression Vbeta2, Vbeta3, and Vbeta17, with a maximum at day 7 after the onset of disease. The frequency of CD4+T cells expressing Vbeta5.1 was increased only in patients with S. flexneri infection. Peripheral blood T cells from Shigella-infected patients also responded to in vitro stimulation in a TCR Vbeta-specific manner. Stimulation with heat-killed S. dysenteriae 1 and Shiga toxin enhanced the frequency of cells expressing Vbeta2, Vbeta3, Vbeta5.1, Vbeta13.6, and Vbeta17, especially in samples obtained at day 7. The enhanced frequency of cells expressing Vbeta2, Vbeta3, Vbeta5.1, and Vbeta17 found both in in vivo and in vitro could suggest that in shigellosis antigens or superantigens are presented to the immune system and preferentially activate certain TCR Vbeta types in T-cell subsets. The kinetics of the change in the TCR Vbeta repertoire in blood during shigellosis may indicate that following local activation, the antigen activated T cells can be retrieved in the blood and restimulated in vitro. If confirmed by parallel analysis of T cells in the gut and blood by TCR sequence analysis, the possibility suggested by our findings would facilitate further analysis of the role of cell-mediated immune responses in the pathogenesis of and protection against Shigella infection.

Modulation and recovery of immune response of BALB/c mice to Shigella dysenteriae antigens after cyclophosphamide treatment.

We have analysed the suppressive effect of cyclophosphamide, when given in vivo, on the antibody response of BALB/c mice against Shigella dysenteriae antigens using ELISA and immunoblot. Out of various protocols tried, it was found that co-injection of cyclophosphamide at 150 mg/kg bodyweight, i.p., at the time of antigen administration and then after a lapse of 24 h during both primary and secondary immunizations, was the most effective in suppressing antibody response of mice. Analysis of sera by ELISA demonstrated the presence of some antibodies to S. dysenteriae antigens after secondary immunization, but immunoblot analysis using the same sera revealed complete suppression of antibody response. Animals whose antibody response was almost completely suppressed after two immunizations with co-injection of cyclophosphamide, when immunized again after the lapse of 14 days from the date of secondary immunization with Shigella antigens but without administration of cyclophosphamide, partially recovered their ability to respond to the same antigens. This protocol can now be used in mice to analyse the hierarchy of immunogenic epitopes present in a complex mixture of antigens.

Antibody–oligosaccharide interactions: the synthesis of 2-deoxy-α-L-rhamnose containing oligosaccharide haptens related to Shigella flexneri variant Y antigen

A series of di- and trisaccharide glycosides based on the α-L-Rha(1 → 3)β-D-GlcNAc and α-L-Rha(1 → 3)α-L-Rha(1 → 3)β-D-GlcNAc elements have been synthesized to locate the minimal oligosaccharide determinant of the Shigella flexneri O-polysaccharide, which is built from a tetrasaccharide repeating unit, [ → 2) α-L-Rhap(1 → 2)α-L-Rhap(1 → 3)α-L-Rhap(1 → 3)β-D-GlcNAcp(1-]n. These compounds also serve to identify the carbohydrate surface of the Shigella antigen that interacts with a monoclonal antibody, currently the subject of crystallographic studies. Two strategies utilizing suitably protected glycals 1 and 19 were employed to obtain analogs bearing either terminal or glycosylated 2,6-dideoxy-α-L-arabino-hexopyranosyl (2-deoxy-α-L-rhamnopyranosyl) residues. N-Iodosuccinimide activation of the glycals in the presence of selectively protected mono- and disaccharide alcohols afforded 2-deoxy-2-iodo-α-L-rhamnopyranosides and these were ultimately reduced during deprotection stages to afford the desired functionality. Di-O-acetyl L-rhamnal 1 reacted with monosaccharides 2 and 7, and with disaccharide 11, to yield disaccharides 4 and 8, and trisaccharide 12, each bearing a terminal 2-deoxy-α-L-rhamnopyranosyl residue. The selectively protected 3-O-benzoyl-4-O-benzyl-L-rhamnal 19 was synthesized from L-rhamnal and used to prepared trisaccharide 22, which contained an internal 2-deoxy-2-iodo-α-L-rhamnopyranosyl unit. Removal of protecting groups gave the oligosaccharides 6, 10, 14, and 23. Oligosaccharides that contained a 2-deoxy-α-L-rhamnopyranosyl residue showed enhanced inhibitory power: in the case of trisaccharide 23 a 1.8 kcal mol−1 relative increase in free energy of binding compared to a larger pentasaccharide epitope, α-L-Rhap(1 → 2)α-L-Rhap(1 → 3)α-L-Rhap(1 → 3)β-D-GlcNAcp(1 → 2)α-L-Rhap-1 → OMe. These data suggest that the rhamnose O–2 hydroxyl of residue C points toward and has important interactions with binding site amino acids.

Shigella-specific IgA in saliva of children with bacillary dysentery.

To study the secretory immune response after Shigella infection, the anti-lipopolysaccharide and anti-Shiga-toxin response in saliva, obtained from children with confirmed shigellosis and healthy children, were determined by enzyme-linked immunosorbent assay and by Western blot. Children with infection showed high titers compared to healthy controls. After Shigella dysenteriae type 1 infection a significant change in titer could be observed in a large number of cases, in contrast to Shigella flexneri infection. It appeared that, in children living in endemic areas, infection with one serotype can give a rise in antibody titer to another serotype. This could be ascribed to polyclonal B cell activation since children in endemic areas routinely show relatively high titers to Shigella antigens. We conclude that the dynamics of salivary anti-Shigella LPS and anti-Shiga-toxin in children with dysentery indicate that it can be applied to studies of immune response in shigellosis for epidemiological and vaccination purposes.

Shigella-specific IgA in saliva of children with bacillary dysentery

To study the secretory immune response after Shigella infection, the anti-lipopolysaccharide and anti-Shiga-toxin response in saliva, obtained from children with confirmed shigellosis and healthy children, were determined by enzyme-linked immunosorbent assay and by Western blot. Children with infection showed high titers compared to healthy controls. After Shigella dysenteriae type 1 infection a significant change in titer could be observed in a large number of cases, in contrast to Shigella flexneri infection. It appeared that, in children living in endemic areas, infection with one serotype can give a rise in antibody titer to another serotype. This could be ascribed to polyclonal B cell activation since children in endemic areas routinely show relatively high titers to Shigella antigens. We conclude that the dynamics of salivary anti- Shigella LPS and anti-Shiga-toxin in children with dysentery indicate that it can be applied to studies of immune response in shigelosis for epidemiological and vaccination purposes.

Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides

Shigellosis is a major cause of infant morbidity and mortality in developing countries. To find immunological correlates of specific protection against shigellosis, we examined chronological samples of sera, stool extracts, duodenal aspirates, and saliva samples from 39 adults and 22 children with shigellosis from Peru for the presence of specific antibody to invasion plasmid antigens (Ipa) common to all virulent Shigella strains, by using both a whole-organism enzyme-linked immunosorbent assay (ELISA) and a Western blot (immunoblot) assay. Antibody responses to lipopolysaccharide (LPS) from Shigella serotypes both homologous and heterologous to the infecting strain were also determined by ELISA. ELISAs showed that the highest serum immunoglobulin G (IgG) antibody titers to Shigella whole organisms both with and without surface Ipa were found in adults and malnourished children, the two groups with the shortest and longest durations of disease, respectively. Mucosal IgA antibody titers to Shigella strains decreased over time to a much greater extent than serum IgG titers, and IgA to Ipa in mucosal secretions was found in adults and well-nourished children but not in malnourished children. The presence of mucosal antibody to Ipa may limit the spread and severity of the infection, as indicated by the prolonged illness observed in malnourished children who have no significant mucosal antibody to Shigella Ipa. Serum antibody titers to the Ipa antigens were high relative to anti-Shigella LPS antibody titers, especially in pediatric patients. In contrast to the anti-Ipa responses observed, no differences in antibody responses to LPS in children compared by nutritional status were found. High levels of serum and mucosal cross-reacting antibody to heterologous serotype LPS were found between Shigella flexneri serotypes 1a and 2a. Different patterns of immune response to Ipa proteins and LPS that may aid in the definition of Shigella antigens important in host protection were observed in adults, well-nourished children, and malnourished children.

Introduction of Shigella flexneri 2a type and group antigen genes into oral typhoid vaccine strain Salmonella typhi Ty21a.

For protection against dysentery caused by Shigella flexneri 2a, an in vivo-constructed recombinant plasmid with genes specifying the S. flexneri type and group antigens located near the pro (min 6) and his (min 44) chromosomal markers, respectively, was made and transferred to the galE Salmonella typhi strain Ty21a. Strain Ty21a carrying this recombinant plasmid was shown by immunological and biochemical analyses to express the S. flexneri 2a type and group antigens. Mice immunized with this vaccine strain were found to be protected against challenge with virulent S. flexneri 2a, but not significantly against S. typhi challenge, presumably because synthesis of the Shigella antigens interfered with expression of the typhoid antigens. Elimination of the recombinant plasmid from Ty21a allowed this strain to again express typical S. typhi O antigens. Mouse protection against both S. typhi and S. flexneri 2a challenges was achieved with a whole-cell vaccine mixture composed of equal parts of Ty21a and the Ty21a-S. flexneri 2a hybrid strain.

Oral vaccination against enteric bacterial infections: an overview.

The present situation and the future prospects for the use of oral vaccines against the major enteric diseases typhoid fever, shigellosis and cholera are discussed in this paper. No significant protection could be demonstrated for oral inactivated whole-cell vaccines. In contrast, an oral live vaccine based on the attenuated Salmonella typhi strain Ty 21a was highly efficacious in volunteer challenge studies and in a controlled field trial. Two attenuated strains are presently being tested in volunteer studies as candidate vaccines against shigellosis; one uses S. typhi Ty 21a and the other Escherichia coli K-12 as the carrier for shigella antigens. Experimental challenge studies in volunteers showed that recovery from clinical cholera confers solid and long-lasting protection. The goal of present research is to develop a vaccine that mimics the events of clinical cholera without causing disease.

Immunologic observations in macaques with Shigella-associated periodontal disease.

The purpose of this study was to determine if macaques with Shigella‐associated periodontal disease (SAPD) were mounting humoral and cellular immunologic responses to Shigella flexneri. Responses to Bacteroides melaninogenicus ss. intermedius, a recognized periodontal pathogen, were also examined. Of the 12 monkeys studied, 4 were uninfected controls, 6 had SAPD, and 2 had been successfully treated for SAPD. At or near the time of immunologic testing, all animals were free of intestinal shigellosis. Antibody titers to the test organisms were determined by direct bacterial agglutination; cellular responses were determined by a serum‐free blast transformation assay. The highest antibody titers and blastogenic responses to both Shigella and Bacteroides antigens were observed in animals from the SAPD group. Monkeys with SAPD gave significantly higher antibody titers to both S. flexneri (p < 0.05) and B. melaninogenicus ss. intermedius (p < 0.01) than did the control monkeys. They also gave significantly higher blastogenic responses to PHA (p < 0.05) and S. flexneri (p < 0.05) than did control monkeys, but did not differ significantly in their blastogenic responses to Bacteroides. These results support the suggestion that Shigella is of etiologic importance in SAPD. They also suggest that a highly virulent microorganism which is a numerically minor component (< 0.1%) of the cultivable flora can elicit significant host responses.

Role of antigen form in development of mucosal immunoglobin A response to Shigella flexneri Antigens.

One major stumbling block in the development of an effective means to immunize against shigellosis and other enteric diseases has been the lack of a means to assess sequential mucosal immune responses to different potential immunogens. In the present study, we compared the abilities of live invasive organisms, noninvasive organisms, and nonviable antigen preparations of shigella to elicit mucosal immune responses. Whereas previous studies have found that effective immunity was produced best by vaccination with live invasive strains of shigella, in the present study, live noninvasive strains that did not produce any histopathological damage were consistently able to produce local (immunoglobulin A) immune responses as vigorous as those of the invasive strains. Further, acetone-killed shigella antigen was also an effective mucosal immunogen, whereas hot phenol-water-extracted shigella lipopolysaccharide was ineffective, possibly due to the method of preparation. A single oral or parenteral priming was ineffective in enhancing the mucosal immune response when restimulated 1 month later with the same antigen. However, a mucosal memory response was found to be present several months after a triple mucosal stimulation with a locally invasive vaccine strain.

Macrophage functions in high and low cancer incidence strains of mice. A comparative study

Various functions of macrophage derived from different age groups of SJL/J mice, a high-cancer-incidence inbred strain, and BALB/c or C57B1/6 mice, two low-cancer-incidence strains, were studied and compared. SJL/J macrophages derived from 2–4 month-old mice were functionally hyperactive. The cells demonstrated elevated acid phosphatase levels and enhanced phagocytic, bacteriostatic, and cytotoxic activities when compared to BALB/c or C57B1/6 macrophages derived from mice of the same age. Bacteriostasis and two lysosomal enzyme activities were still potentiated in SJL/J macrophages derived from 12–17 month-old mice, compared to BALB/c mice, whereas macrophages from 12–17 month-old SJL/J mice, with or without tumors, demonstrated depressed macrophage-mediated cytotoxicity as well as a functional defect in their ability to handle Shigella antigen.

Cellular and molecular aspects of the immune response to a bacterial somatic antigen.

The immune response to the lipopolysaccharide (LPS) somatic antigen of Shigella paradysenteriae was studied at the levels of humoral antibody and individual antibody-forming cells. Mice immunized with various doses of either LPS or wholecell vaccine rapidly developed serum agglutinins to the bacteria. Antibody activity was due to IgM globulins throughout the primary and secondary responses. LPSsensitized ovine erythrocytes permitted assessment of the serum antibody response and were useful for detection and enumeration of specific antibody plaque-forming cells in spleens of immunized animals. Antibody-forming cells rapidly appeared after immunization and peaked on days 5 and 6. Only 19S IgM antibodyforming cells were stimulated by the shigella antigen. Immunologic tolerance to shigella LPS was induced by injection of a relatively large inoculum into neonatal mice and persisted for at least six to eight weeks, with markedly depressed cellular and humoral antibody responses after challenge. Extracts rich in ribonucleic acid (RNA) from spleens of normal immune and tolerant donor mice transferred antibody-forming activity to normal or shigella-primed mice. Immunogenic activity of these RNA extracts was abolished by in-vitro treatment with RNAase or specific antibody to Shigella; thus both RNA and a persisting antigen fragment appear to contribute to biologic activity. The significance of immunogenic RNA extracts during immunogenesis and tolerogenesis was studied.

EPIDEMIC IN SOUTHERN MEXICO OF DISEASE RESEMBLING VIRUS HÆMORRHAGIC FEVERS

In 1967 an epidemic of a previously unrecognised disease occurred in man in Chiapas State, Mexico. It was characterised by fever, vomiting, diarrhoea, hæmorrhagic manifestations, shock, and some deaths. The outbreak was in an isolated, tropical area of southern Mexico and coincided with a striking increase in the rodent population in and around homes. Although no ætiological diagnosis has been established, the clinical and epidemiological findings were similar to the virus hæmorrhagic fever (H.F.) diseases and lead to the hypothesis that the outbreak was caused by a virus, perhaps one in the H.F. group. Sera obtained from patients 2 years after recovery from the disease were tested against arbovirus, Leptospira, Shigella, and rickettsia antigens, as well as antigens of the Tacaribe complex of viruses. Complement-fixation antibodies of low titre (1/2 to 1/16) against Tacaribe complex viruses, Latino and Paraná, found in sera from eight of eleven persons tested were compatible with, but not confirmatory of, the hypothesis.

Biologic Activities of Lymph Node Cells Fractionated on Bovine Plasma Albumin

Abstract For the study of some characteristics of antibody-forming cells optimal conditions were determined for enrichment of plaque-forming cells (PFC) in suspensions of rabbit lymph node cells (LNC) by density gradient centrifugation in bovine plasma albumin. The most enriched fractions contained 5 to 15 times their concentration in the original unfractionated cell suspension. There was a correlation between the size distribution of the LNC and their density, the smallest cells appearing in the high density region of the gradient, and the medium and large cells in the low density region. PFC obtained from rabbits 3 to 4 days after secondary injection of Shigella paradysenteriae were all in low density fractions. On days 4 to 5 some PFC appeared among the highest density fractions, with a gradual shift in that direction until, by day 10, all PFC were in high density fractions. The plaques produced by cells of the low density region of the gradient were prevented by treatment of the plates with 2-mercaptoethanol (2-ME), whereas the antibody produced by the high density cells was 2-ME resistant. Mouse spleen cells and LNC did not show such high density PFC at these times. The antigen-binding capacity of LNC from normal and immunized rabbits was studied with 131I-labeled soluble Shigella antigen. It was found that the antigenbinding profile of LNC from immunized rabbits followed the profile of the PFC, but among cells obtained from normal rabbits, or rabbits immunized with an unrelated antigen, high density cells were found to bind the antigen. The biosynthetic activity of mouse spleen cells was measured by the incorporation of radioactive amino acids, uridine and thymidine into macromolecules. The density gradient fractions containing the PFC showed greater incorporation of the precursors than the rest of the population.

Decreased 19S antibody response to bacterial antigens in systemic lupus erythematosus.

The antibody response to immunization with Brucella and the levels of natural antibody to Escherichia coli and Shigella were compared in patients with systemic lupus erythematosus and control groups. After Brucella immunization, SLE patients showed a significantly lower antibody response in whole serum and in the macroglobulin antibody fraction separated by sucrose density gradient centrifugation. Sucrose gradient fractionation of natural antibodies to E. coli and a polyvalent Shigella antigen showed a significant decrease in macroglobulin antibody against four of the five E. coli antigens tested and the Shigella polyvalent antigen in SLE patients when compared with a group of normal individuals and a matched control group with pulmonary tuberculosis. Whole serum natural antibody titers against 5 of 13 Shigella antigens were significantly lower in the SLE patients when compared with the normal group, and against 7 of 13 when compared with the matched tuberculosis controls. Whole serum titers against 8 of 13 E. coli antigens were significantly lower in the SLE patients when compared with normal subjects. The observed decreased antibody response to bacterial antigens in SLE patients, occurring mainly in the macroglobulin fraction, is discussed in relation to the increased incidence of infection commonly observed in these patients.

Plesiomonas shigelloides in South Australia.

Plesiomonas shigelloides, a motile, oxidase-positive, Gram-negative rod that may possess shigella antigens, was isolated from the faeces of 36 children and two adults. In 13 children the organism was thought to be possibly the cause of enteritis and in eight children there was no evidence of intestinal disease. In nine children with gastroenteritis other enteropathogenic bacteria were found in addition, and six children had intestinal disease due, or possibly due, to other factors. Only four of the 38 strains had shigella antigens.

Depressed Cellular Formation of Antibody toShigellaAntigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.

Immunological tolerance to microbial antigens. II. Suppressed antibody plaque formation to Shigella antigen by spleen cells from tolerant mice.

Friedman, Herman (Albert Einstein Medical Center, Philadelphia, Pa.). Immunological tolerance to microbial antigens. II. Suppressed antibody plaque formation to Shigella antigen by spleen cells from tolerant mice. J. Bacteriol. 92:820-827. 1966.-An indirect, localized, antibody plaque procedure has been used to demonstrate a marked difference in the number of antibody plaques formed with spleen cell suspensions from normal and Shigella-tolerant mice. Whereas challenge with soluble Shigella antigen (SSA) into normal mice, ranging in age from 4 to 40 weeks, resulted in a rapid rise in antibody plaque formation to Shigella-treated sheep erythrocytes, there was only a slight increase in plaque formation with spleen cell suspensions from similarly challenged mice which had been made tolerant to Shigella antigen during neonatal life. Apparently, the suppression of plaque formation to Shigella in tolerant animals was specific, since both Shigella-tolerant and normal mice responded equally well to untreated sheep red blood cells after challenge with sheep erythrocytes only. Spleen cells from nonchallenged Shigella-tolerant mice did not form significant numbers of antibody plaques to SSA-treated red blood cells during an observation period of 4 to 30 weeks after neonatal administration of antigen. "Nonspecific" increases in plaque formation to untreated sheep red cells occurred with spleen cell suspensions from both normal and SSA-tolerant mice after challenge injection with Shigella antigen, with or without sheep erythrocytes. Such a response suggested an adjuvant effect for the endotoxin-containing Shigella antigen even in mice tolerant to the agglutinogenic moiety of SSA. The results of these experiments support the view that specific antibody-forming cells are either absent or in low number in lymphoid tissue from mice specifically tolerant to Shigella antigens. It seems unlikely that the low postchallenge agglutinin titers of tolerant mice are due to suppressed antibody formation by normal numbers of individual antibody-producing cells, or due to "masking" of normal antibody production by persisting circulating antigen.