Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues.

We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT ((125)I-CT) or CT-B subunit ((125)I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with (125)I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas (125)I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.

Mice deficient in Th1- and Th2-type cytokines develop distinct forms of hapten-induced colitis.

BACKGROUND & AIMS: Most experimental models for inflammatory bowel disease in mice are associated with production of interferon (IFN)-gamma and other proinflammatory cytokines. We hypothesized that T-helper 2 (Th2)-type cells could also contribute to the colitis and cause inflammation different than that mediated by Th1-type cells. METHODS: Trinitrobenzene sulfonic acid (TNBS)-induced colitis in C57BL/6 background mice genetically deficient in interleukin (IL)-12 p40 (IL-12(-/-)), IFN-gamma (IFN-gamma(-/-)), or IL-4 (IL-4(-/-)) was examined in comparison with control mice (C57BL/6(+/+)). RESULTS: C57BL/6(+/+), IFN-gamma(-/-), and IL-12(-/-) mice developed patterns of colitis characterized by distortion of crypts, loss of goblet cells, and mononuclear cell infiltration with fibrosis of the mucosal layer. IL-4(-/-) mice had greater mortality than other groups because of penetrating ulcers; however, survivors developed milder lesions that were limited to focal acute ulceration. Colonic CD4(+) T cells from normal, IFN-gamma(-/-), or IL-12(-/- )mice produced both IL-4 and IL-5. CONCLUSIONS: In TNBS colitis, Th1-like cytokine responses induce fatal, acute, transmural, and focal types of lesions, whereas Th2-like cytokine responses play a significant role in the diffuse atrophic changes in crypts and the mucosal layer that occur in the late stages of this disease.

Direct effects on antigen-presenting cells and T lymphocytes explain the adjuvanticity of a nontoxic cholera toxin mutant.

The present study has elucidated two distinct mechanisms that may explain how a mutant of cholera toxin (mCT), E112K, retains adjuvant effects though it lacks ADP-ribosyltransferase activity and associated toxicity. In the first mechanism, we show that mCT E112K, like native cholera toxin (nCT), enhances B7-2 expression, but, to some extent, also enhances B7-1 on Peyer's patch B cells and macrophages. Cocultivation of CD4+ T cells with E112K- or nCT-treated B cells and macrophages in the presence of anti-CD3 stimulation resulted in the induction of T cell-proliferative responses. Further, the responses were blocked by mAbs to B7-1 and/or B7-2; however, the effect of anti-B7-1 was minimal. In the second mechanism, addition of mCT E112K or nCT to anti-CD3 mAb-stimulated Peyer's patch CD4+ T cells inhibited proliferative responses, while recombinant CT-B subunit (rCT-B) did not. Analysis of cytokine responses showed that both mCT E112K and nCT preferentially inhibited IFN-gamma production. Interestingly, however, nCT, but not mCT E112K, induced apoptosis in CD4+ T cells activated via the TCR-CD3 complex. These results indicate that CT uses at least two pathways for inhibition of Th1 responses and that, while nCT induces cAMP accumulation that in turn leads to apoptosis in Th1-type cells, mCT E112K, which lacks ADP-ribosyltransferase activity, inhibits IFN-gamma synthesis by a separate mechanism. Thus, mCT E112K, like nCT, induces adjuvant responses via up-regulation of mainly B7-2 on APCs and through preferential inhibition of Th1-type CD4+ T cell responses in the absence of ADP-ribosyltransferase activity.

Chemical properties and immunobiological activities of streptococcal lipoteichoic acids.

Lipoteichoic acids (LTAs) were chromatographically purified from crude phenol-water extract of whole cells of some streptococcal species, which included Streptococcus pyogenes Sv, Streptococcus mutans 6715, and Streptococcus sanguis ATCC 10556. Among these, special attention was paid to S. pyogenes LTA for analyses of chemical composition and biological activities. All LTA preparations contained equimolar amounts of glycerol and phosphorus. Chemical analyses showed that S. pyogenes LTA contained glycerophosphate, alanine, glucose, and fatty acids (as palmitic acid) at molar ratio of 1 : 0.1 : 0.1 : 0.25. The crude phenol-water extract and isolated LTA from S. pyogenes Sv were found to be mitogenic for spleen cells of BALB/c and BALB/c (nu/nu) mice, but not for thymus cells of BALB/c mice. The mitogenicity of deacylated LTA (dLTA) was significantly lower than that of LTA. It was also found that various LTA preparations possessed polyclonal B cell activation ability and adjuvant activity both in vivo and in vitro, as demonstrated by using hemolytic plaque assay. LTA, but not dLTA, induced macrophage activation which resulted in tumor cytotoxicity in mice. Limulus lysate activity of S. pyogenes LTA was approximately 1,000 fold lower than that of Escherichia coli lipopolysaccharide. These results indicate that streptococcal LTA possesses various immunobiological activities that modulate lymphoreticular system in vivo and in vitro.

Human colostral cells. I. Separation and characterization.

Analyses of the cells present in human colostrum obtained from fifty-four healthy donors during the first four days of lactation revealed that there were 3.3 x 10(6) (range 1.1 x 10(5)--1.2 x 10(7)) cells per ml of colostrum. Based on histochemical examinations, it was found that this population consisted of 30--47% macrophages, 40--60% polymorphonuclear leucocytes, 5.2--8.9% lymphocytes, and 1.3--2.8% colostral corpuscles; epithelial cells were rarely encountered. The identity of various cell types was confirmed by Wright's stain and by a series of histochemical techniques which disclosed the presence of non-specific esterase, peroxidase, and lipids. For further characterization, the different types of cells were separated by various methods, such as Ficoll-Hypaque density centrifugation, isokinetic centrifugation on a linear Ficoll gradient, adherence to glass or plastic, and phagocytosis of carbonyl iron. Immunohistochemical staining with FITC- and/or TRITC-labelled reagents to IgA, IgM, IgG, K- and lambda-chains, secretory component, lactoferrin, and alpha-lactalbumin were applied to unseparated as well as separated colostral cells. Polymorphonuclear leucocytes (staining for peroxidase) as well as macrophages and colostral corpuscles (staining for non-specific esterase) exhibited numerous intracellular vesicles that contained lipids as well as various combinations of milk proteins. Lymphoid cells did not stain with any of these reagents and plasma cells were not detected among the colostral cells. Individual phagocytic cells contained immunoglobulins of the IgA and IgM classes, both K and lambda light chains, secretory component, lactoferrin, and alpha-lactalbumin. The coincidental appearance of these proteins in single, phagocytic cells but not in lymphoid cells indicate that the cells acquired these proteins by ingestion from the environment. Markers commonly used for the identification of B lymphocytes (surface immunoglobulins) and T lymphocytes (receptors for sheep red blood cells) were unreliable for the analysis of colostral cells (unless accompanied by subsequent morphological characterization) because strong fluorescence was observed on the surface of many non-lymphoid cells and because numerous macrophages and colostral corpuscles formed rosettes with sheep red blood cells (SRBC). Lymphocytes, often found in association with colostral macrophages or corpuscles, were classified as T cells.

Effective immunity to dental caries: dose-dependent studies of secretory immunity by oral administration of Streptococcus mutans to rats.

Rats (COBS/CD) provided Formalin-killed Streptococcus mutans 6715, C211 in their drinking water (10(8) to 10(9) equivalent colony-forming units [CFU] per ml) had high levels of specific antibodies in saliva, colostrum, and milk. Rats provided a lower concentration of S. mutans antigen (10(7) CFU per ml) in water had agglutinin titers in secretions that were similar to those in controls. Gnotobiotic rats provided S. mutans antigen in food (10(7) to 10(8) equivalent CFU per g of diet) manifested a secretory immune response as evidenced by the presence of specific immunoglobulin A antibodies in saliva, colostrum, and milk. Gnotobiotic rats provided a higher concentration of antigen (10(9) CFU per g) in food had levels of specific antibodies in their secretions that were similar to those in controls. No significant antibody activity to S. mutans was observed in sera of any group of animals. Furthermore, the presence of specific salivary immunoglobulin A antibodies in gnotobiotic rats correlated with a reduction in the level of plaque, numbers of viable S. mutans in plaque, and levels of S. mutans-induced dental caries. This paper discusses the importance of antigen dosage for induction of a secretory immune response that is protective against S. mutans-induced dental caries.

Effective immunity to dental caries: selective induction of secretory immunity by oral administration of Streptococcus mutans in rodents.

1) The selective induction of secretory antibody is antigen dose dependent. A dose of 10(7) or 10(8) S. mutans CFU (per gram of diet) elicited a significant response, while a higher dose (10(9) CFU/gram) resulted in an apparent state of unresponsiveness. 2) A lowly virulent mutant of S. mutans 6715 (C4) induced protective s-IgA antibodies against challenge with a highly virulent species (C211). Cross protection among serotypes of S. mutans has been achieved. 3) Bovine milk with antibodies to S. mutans significantly reduced the level of caries lesions in rats fed this lyophilized milk and challenged with the homologous, virulent S. mutans strains.

Virulence of Streptococcus mutans: an antibiotic-suppressed rat model for studies of pathogenesis.

This investigation describes a simple and effective method of antibiotic suppression of the total indigenous microbiol flora of rats. Specifically, the results of this study demonstrate that: (1) antibiotics which inhibit microbial growth of oral and fecal cultures from individual rats are effective in suppressing the indigenous flora of 95% of these rats when provided as dietary supplements; (2) S mutans 6715 colonized 95% of the antibiotic-suppressed animals while only 50% of the untreated rat pups yielded S mutans following challenge with a single inoculum of this micro-organism; and (3) significant smooth surface caries developed in S mutans-infected rats within 20 days when provided caries-promoting Diet 305, while noninfected, antibiotic-suppppressed offspring remained essentially caries-free.

Naturally occurring secretory immunoglobulin A antibodies to Streptococcus mutans in human colostrum and saliva.

Human colostrum, parotid saliva, and serum were assayed for the presence of naturally occurring antibodies to five serotypes of Streptococcus mutans. Appreciable levels of agglutinins to strains AHT, BHT, 10449, 6715, and LM-7 (groups a leads to e, respectively) were detected in normal colostrum and saliva, whereas relatively low levels were found in serum. No agglutinins could be detected in the colostrum or saliva of immunodeficient patients. Molecular sieve chromatography of the colostrum on Sephadex G-200 revealed agglutinin activity in the secretory immunoglobulin A (s-IgA)-rich fraction only. Titration of purified colostral s-IgA confirmed the IgA nature of this agglutinating activity. Indirect immunofluorescence tests with anti-s-IgA, -IgG, and -IgM revealed S. mutans specificity only in the s-IgA class. The presence of s-IgA antibodies to indigenous oral microorganisms in colostrum, as well as in saliva, suggests that antigenic stimulation occurs at a site remote from the oral mucosa.

Rat immunoglobulins in serum and secretions: comparison of IgM, IgA and IgG in serum, colostrum, milk and saliva of protein malnourished and normal rats.

Immunoglobulin levels in colostrum and milk of normal and protein malnourished rat dams and in serum and saliva of their offspring were quantitated by radial immunodiffusion employing purified anti-rat alpha, gamma and mu. In general, no significant difference was observed in the level of colostral IgA in malnourished as compared to normal rat dams or in the level of serum IgA and IgM and salivary IgA and IgG2a of their offspring. On the other hand, malnourished mothers demonstrated approximately twofold lower levels of colostral IgG2a than normal mothers throughout the entire period of lactation. In addition, at every interval tested, the level of IgG was approximately 1.5- to 2-fold less in malnourished as compared to normal offspring.