Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses

The lower intestine of adult mammals is densely colonized with nonpathogenic (commensal) microbes. Gut bacteria induce protective immune responses, which ensure host-microbial mutualism. The continuous presence of commensal intestinal bacteria has made it difficult to study mucosal immune dynamics. Here, we report a reversible germ-free colonization system in mice that is independent of diet or antibiotic manipulation. A slow (more than 14 days) onset of a long-lived (half-life over 16 weeks), highly specific anticommensal immunoglobulin A (IgA) response in germ-free mice was observed. Ongoing commensal exposure in colonized mice rapidly abrogated this response. Sequential doses lacked a classical prime-boost effect seen in systemic vaccination, but specific IgA induction occurred as a stepwise response to current bacterial exposure, such that the antibody repertoire matched the existing commensal content.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Intravenous immunoglobulin contains a broad repertoire of anticarbohydrate antibodies that is not restricted to the IgG2 subclass

BACKGROUND: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. OBJECTIVE: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. METHODS: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. RESULTS: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. CONCLUSIONS: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non-pathogen-associated prokaryotic glycans.

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.

BACKGROUND Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses.

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.