Molecular characterization of newly identified Klebsiella PKBSG14 and analysis of its effect on immune response and cell cycle progression using common catfish (Channa punctatus) as a model.

Microbial studies on Catfish revealed that Klebsiella is the most common pathogen causing prevalence of ulcers, fin erosion, and other lesions. During this study, a new strain of bacteria was isolated from Channa punctatus, and molecular identification by 16srRNA revealed the strain was Klebsiella PKBSG14 (Accession no KJ162158). The strain was also PCR positive for two virulent gene wcaG (Accession no LN606595) and rmpA (Accession no LN606594) responsible for inflammatory reactions and induction of innate immune response in the host cell. To study innate immune response induced by pathogenic infection the phagocytic interactive process between the spleen macrophages and KlebsiellaPKBSG14 was investigated using optical microscopy. FACS of splenic macrophages revealed that the phagocytic interaction leads to the process of macrophage cell cycle progression. A detailed study on the macrophage DNA content by performing DNA fragmentation and comet allowed us to study simultaneously host cell division as a function of phagocytosis and the findings unveiled the fact that Phagocytosis of KlebsiellaPKBSG14 aided in macrophage cell cycle progression but was less likely to complete mitosis. Here we also report the cytotoxic effect linked to the infection with KlebsiellaPKBSG14 by performing Cell viability assay, intracellular production of ROS, and mitochondrial transmembrane potential where it manifested itself in impaired cellular function. So, in summary, we simultaneously discovered a new strain of bacteria ie. Klebsiella PKBSG14 as well as deliberately attempted to study the immunomodulatory effect of isolated new stain on Channa punctatus by performing host-pathogen phagocytic interactive experiments, the cell cycle state of the host cell and pathogen-mediated cytotoxicity along with genotoxicity, and our results evidence a new immunomodulatory effect of KlebsiellaPKBSG14 infection on fish splenic macrophages.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-ß Synthesis Induced by Lipopolysaccharide.

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-ß. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-ß production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-ß synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-ß production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-ß production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-ß is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-ß during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors.

Efficacy of vaccination with a Klebsiella pneumoniae siderophore receptor protein vaccine for reduction of Klebsiella mastitis in lactating cattle.

Clinical mastitis caused by Klebsiella spp. is an emerging problem in the US dairy industry and results in a high degree of financial losses to dairy workers. This study was conducted as a randomized, blinded, and placebo-controlled efficacy study of a Klebsiella pneumoniae siderophore receptor protein (SRP) vaccine (Kleb-SRP), with a total of 569 cows and heifers enrolled. The study was designed to look at vaccine effect on Klebsiella mastitis; however, the SRP in Klebsiella are highly conserved across coliform bacteria, which means that the vaccine has potential for cross-protection against all coliforms. Cows were paired based on parity, days in milk at enrollment, and somatic cell count. Within pairs, individuals were randomized to receive either Kleb-SRP or a placebo formulation. Following vaccination, the incidence of Klebsiella spp. and total coliform mastitis from natural exposure were compared to determine the efficacy of the vaccine. When analyzing all cows, the reduction of mastitis risk was not significant, though milk production increased 0.31 kg/d and somatic cell counts were reduced by 20.1%. When administered before calving, the vaccine reduced the risk of Klebsiella and total coliform mastitis by 76.9 and 47.5% respectively; however, we observed no significant effect when administered after calving. The vaccine, when administered before calving, also increased milk production by an average of 1.74 kg/d and reduced somatic cell counts by 64.8%. When administered after calving, we noted a slight decrease in daily milk production (0.39 kg) but no significant effect on somatic cell counts. All cows in the study (including vaccinates and placebo) received multiple doses of a commercially available licensed Escherichia coli bacterin. It should be noted that this herd was chosen because of the high number of clinical Klebsiella clinical mastitis cases this herd experienced before the trial and the extreme environmental challenge that was present from bedding with dried manure solids. The data from this study demonstrate efficacy of the Kleb-SRP vaccine against Klebsiella mastitis alone and coliform mastitis in general (including all coliforms) when administered before the initiation of a lactation cycle.

Attenuating immune pathology using a microbial-based intervention in a mouse model of cigarette smoke-induced lung inflammation.

BACKGROUND: Cigarette smoke exposure is the major risk factor for developing COPD. Presently, available COPD treatments focus on suppressing inflammation and providing bronchodilation. However, these options have varying efficacy in controlling symptoms and do not reverse or limit the progression of COPD. Treatments strategies using bacterial-derived products have shown promise in diseases characterized by inflammation and immune dysfunction. This study investigated for the first time whether a novel immunotherapy produced from inactivated Klebsiella (hereafter referred to as KB) containing all the major Klebsiella macromolecules, could attenuate cigarette smoke exposure-induced immune responses. We hypothesized that KB, by re-directing damaging immune responses, would attenuate cigarette smoke-induced lung inflammation and bronchoalveolar (BAL) cytokine and chemokine production. METHODS: KB was administered via a subcutaneous injection prophylactically before initiating a 3-week acute nose-only cigarette smoke exposure protocol. Control mice received placebo injection and room air. Total BAL and differential cell numbers were enumerated. BAL and serum were analysed for 31 cytokines, chemokines, and growth factors. Lung tissue and blood were analysed for Ly6CHI monocytes/macrophages and neutrophils. Body weight and clinical scores were recorded throughout the experiment. RESULTS: We demonstrate that KB treatment attenuated cigarette smoke-induced lung inflammation as shown by reductions in levels of BAL IFNγ, CXCL9, CXCL10, CCL5, IL-6, G-CSF, and IL-17. KB additionally attenuated the quantity of BAL lymphocytes and macrophages. In parallel to the attenuation of lung inflammation, KB induced a systemic immune activation with increases in Ly6CHI monocytes/macrophages and neutrophils. CONCLUSIONS: This is the first demonstration that subcutaneous administration of a microbial-based immunotherapy can attenuate cigarette smoke-induced lung inflammation, and modulate BAL lymphocyte and macrophage levels, while inducing a systemic immune activation and mobilization. These data provide a foundation for future studies exploring how KB may be used to either reverse or prevent progression of established emphysema and small airways disease associated with chronic cigarette smoke exposure. The data suggest the intriguing possibility that KB, which stimulates rather than suppresses systemic immune responses, might be a novel means by which the course of COPD pathogenesis may be altered.

Comparative analysis of CRISPR-Cas systems in Klebsiella genomes.

Prokaryotic CRISPR-Cas system provides adaptive immunity against invasive genetic elements. Bacteria of the genus Klebsiella are important nosocomial opportunistic pathogens. However, information of CRISPR-Cas system in Klebsiella remains largely unknown. Here, we analyzed the CRISPR-Cas systems of 68 complete genomes of Klebsiella representing four species. All the elements for CRISPR-Cas system (cas genes, repeats, leader sequences, and PAMs) were characterized. Besides the typical Type I-E and I-F CRISPR-Cas systems, a new Subtype I system located in the ABC transport system-glyoxalase region was found. The conservation of the new subtype CRISPR system between different species showed new evidence for CRISPR horizontal transfer. CRISPR polymorphism was strongly correlated both with species and multilocus sequence types. Some results indicated the function of adaptive immunity: most spacers (112 of 124) matched to prophages and plasmids and no matching housekeeping genes; new spacer acquisition was observed within the same sequence type (ST) and same clonal complex; the identical spacers were observed only in the ancient position (far from the leader) between different STs and clonal complexes. Interestingly, a high ratio of self-targeting spacers (7.5%, 31 of 416) was found in CRISPR-bearing Klebsiella pneumoniae (61%, 11 of 18). In some strains, there even were multiple full matching self-targeting spacers. Some self-targeting spacers were conserved even between different STs. These results indicated that some unknown mechanisms existed to compromise the function of self-targets of CRISPR-Cas systems in K. pneumoniae.

M2b macrophage elimination and improved resistance of mice with chronic alcohol consumption to opportunistic infections.

Alcohol abuse was found to predispose persons to opportunistic infections. In this study, we tried to improve the host antibacterial resistance of chronic alcohol-consuming (CAC) mice to opportunistic infections. Bactericidal macrophages with functions to produce IL-12 and to express mRNAs for CXCL9 and inducible nitric oxide synthase (M1 macrophages) were characterized as the main effector cells in host antibacterial innate immunities against infections with opportunistic pathogens. However, CAC mice were found to be carriers of M2b macrophages [macrophages with functions to produce IL-10 and to express mRNAs for CD163, chemokine ligand (CCL)1, and LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for high-voltage electron microscopy on T cells)], which were inhibitory on macrophage conversion from resident macrophages to M1 macrophages. Under treatment with CCL1 antisense oligodeoxynucleotides, a specific inhibitor of M2b macrophages, CAC mouse macrophages reverted to resident macrophages, and M1 macrophages were induced by a bacterial antigen from macrophages of CAC mice that were previously treated with the oligodeoxynucleotides. Opportunistic infections (enterococcal translocation and Klebsiella pneumonia) in CAC mice were completely controlled by CCL1 antisense oligodeoxynucleotides. These results indicate that certain opportunistic infections in alcoholics are controllable through the modulation of M2b macrophages.

The airway microbiome in patients with severe asthma: Associations with disease features and severity.

BACKGROUND: Asthma is heterogeneous, and airway dysbiosis is associated with clinical features in patients with mild-to-moderate asthma. Whether similar relationships exist among patients with severe asthma is unknown. OBJECTIVE: We sought to evaluate relationships between the bronchial microbiome and features of severe asthma. METHODS: Bronchial brushings from 40 participants in the Bronchoscopic Exploratory Research Study of Biomarkers in Corticosteroid-refractory Asthma (BOBCAT) study were evaluated by using 16S ribosomal RNA-based methods. Relationships to clinical and inflammatory features were analyzed among microbiome-profiled subjects. Secondarily, bacterial compositional profiles were compared between patients with severe asthma and previously studied healthy control subjects (n = 7) and patients with mild-to-moderate asthma (n = 41). RESULTS: In patients with severe asthma, bronchial bacterial composition was associated with several disease-related features, including body mass index (P < .05, Bray-Curtis distance-based permutational multivariate analysis of variance; PERMANOVA), changes in Asthma Control Questionnaire (ACQ) scores (P < .01), sputum total leukocyte values (P = .06), and bronchial biopsy eosinophil values (per square millimeter, P = .07). Bacterial communities associated with worsening ACQ scores and sputum total leukocyte values (predominantly Proteobacteria) differed markedly from those associated with body mass index (Bacteroidetes/Firmicutes). In contrast, improving/stable ACQ scores and bronchial epithelial gene expression of FK506 binding protein (FKBP5), an indicator of steroid responsiveness, correlated with Actinobacteria. Mostly negative correlations were observed between biopsy eosinophil values and Proteobacteria. No taxa were associated with a TH2-related epithelial gene expression signature, but expression of TH17-related genes was associated with Proteobacteria. Patients with severe asthma compared with healthy control subjects or patients with mild-to-moderate asthma were significantly enriched in Actinobacteria, although the largest differences observed involved a Klebsiella genus member (7.8-fold increase in patients with severe asthma, adjusted P < .001). CONCLUSIONS: Specific microbiota are associated with and may modulate inflammatory processes in patients with severe asthma and related phenotypes. Airway dysbiosis in patients with severe asthma appears to differ from that observed in those with milder asthma in the setting of inhaled corticosteroid use.

Placental transfer of IgG antibodies specific to Klebsiella and Pseudomonas LPS and to group B Streptococcus in twin pregnancies.

Group B Streptococcus (GBS), Klebsiella spp. and Pseudomonas spp. are important aetiological agents of neonatal infections in Brazil. There is a lack of data in the literature regarding the specific transport of immunoglobulin G (IgG) against these pathogens in multiple pregnancies. Maternal (n = 55) and umbilical cord (n = 110) blood samples were prospectively collected at birth from 55 twin pregnancies. The factors associated with cord levels and transfer ratios of IgG against GBS, Klebsiella and Pseudomonas were examined. The IgG umbilical cord serum levels specific to GBS, Klebsiella LPS and Pseudomonas LPS were significantly associated with maternal-specific IgG concentrations and the presence of diabetes. The anti-Klebsiella IgG cord serum concentrations were also related to birthweight and the presence of hypertension. The transfer ratios against GBS and Pseudomonas LPS were associated with maternal-specific IgG concentrations. The transfer ratios for GBS and Pseudomonas LPS were associated with gestational age at delivery and the presence of diabetes, respectively. None of the examined parameters were related to Klebsiella LPS transfer ratios. We conclude that in twin pregnancies, specific maternal IgG serum concentrations and diabetes were the parameters associated with umbilical cord serum IgG concentrations reactive with the three pathogens investigated. All the other parameters investigated showed different associations with neonatal-specific IgG levels according to the antigen studied. There was no uniformity of the investigated parameters regarding association with placental IgG transfer ratios against the GBS, Pseudomonas LPS and Klebsiella LPS.

Raised incidence of ankylosing spondylitis among Inuit populations could be due to high HLA-B27 association and starch consumption.

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis mainly affecting the spinal joints. It would appear that the most likely causative agent in the development of AS is an environmental factor in the genetically susceptible, HLA-B27 positive, individuals. Extensive data from several countries support the notion that Klebsiella pneumonia bacteria are the most likely culprit in the causation of AS. These microbes possess antigens which resemble HLA-B27 and spinal collagens. Increased intake of high-starch diet is directly proportional to the gut-associated bacterial load, especially in the large intestine, and among these microbial agents, Klebsiella is considered as one of the main constituting components. Therefore, a low-starch diet intake alongside the currently used medical therapeutic modalities could be beneficial in the management of patients with early AS. It is suggested that a change in the dietary habits from high protein, low-starch marine components to the Westernized high-starch diet among the Inuit peoples of Alaska and Canada could be considered as one of the main contributing factors in the increased prevalence of AS during the last few decades within this genetically unmixed native population.

TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells.

Neonates heavily depend on the innate immune system for defence against invading pathogens. Toll-like receptors (TLRs) represent a primary line of host defence and play an important role in orchestrating the inflammatory response to invading pathogens. The most commonly infecting pathogens in neonates are E. coli, Klebsiella pneumoniae and Staphylococcus aureus. Also, co-infection with more than one organism is common in neonatal sepsis. Therefore, we aimed to study the TLR2 and TLR4 mediated neonatal inflammatory response to these pathogens. For this, we stimulated mononuclear cells from cord blood with LPS, PGN, E. coli, K.pneumoniae andS.aureus and analyzed the surface expression of TLR2 and TLR4 on CD14(+)CD16(+) and CD14(dim)CD16(+) and its inflammatory response in comparison with peripheral blood. We found that the TLR2 and TLR4 were differentially expressed on both monocyte subpopulations. Cytokines such as IL-6, IL-1ß, IL-23, IL-10, IL-13, MCP-1 and IL-8 were measured using ELISA and we observed that although, neonatal cells were able to produce similar levels of the classical pro-inflammatory (IL-6, IL-1ß) and anti-inflammatory (IL-10, IL-13) cytokines as that of adult cells, the amounts of IL-23 and MCP-1 were lower in CBMCs while the chemokine IL-8 was higher in CBMCs when compared with PBMCs. In addition, using Human Inflammation Antibody array technique we found that multiple cytokine production was impaired in cord blood when cells were co-infected with LPS and PGN. In conclusion, the TLR-mediated inflammatory response to neonatal pathogens is differentially regulated by different pathogens.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

Characterization of a factor(s) present in Klebsiella culture filtrates that specifically modifies an HLA-B27-associated cell-surface component.

It has been shown that HLA-B27 lymphocytes from healthy individuals (B27+ ankylosing spondylitis [AS]-), which are not lysed by an antiserum against Klebsiella K43, can be rendered susceptible to lysis after incubation in the culture filtrate of Klebsiella K43. This finding is compatible with a specific modification by a Klebsiella K43-derived soluble factor of a B27-associated lymphoid cell component. Preliminary characterization of the factor has indicated that it is nondialyzable, but it is heat labile at 56 degrees C for 30 min and has a 35,000-50,000 mol wt. The modifying factor activity of the filtrate is destroyed by neuraminidase but not by trypsin and alpha-chymotrypsin. Furthermore, the ability of the factor to convert B27+AS- lymphocytes can be specifically absorbed by B27+AS- lymphocytes, but not by B27+AS+, B27-AS+, or by B27-AS- lymphocytes, which suggests that B27+AS- cells carry a hypothetical receptor which can specifically bind a Klebsiella K43 antigenic determinant. These results imply that the modification by environmental agents of specific major histocompatibility complex-associated gene products may be an important element in the pathogenesis of the HLA-B27-linked seronegative arthropathies.

Human monoclonal macroglobulins with specificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6-pyruvylated-D-galactose.

Two human IgM myeloma proteins, IgMWEA and IgMMAY, were found to react with agar and Klebsiella polysaccharides that contain pyruvylated D-galactose (DGal). Quantitative precipitin data and precipitin inhibition studies with methyl alpha- and beta-glycosides of 4,6-pyruvylated-D-galactose showed their combining sites to be different, although each was directed against the pyruvylated-D-Gal, one reacting most specifically with Klebsiella polysaccharides with terminal nonreducing beta-linked 2,4 pyruvylated-D-Gal, whereas the other reacted equally well with Klebsiella polysaccharides that contain 3,4 beta-linked and 4,6 alpha-linked terminal nonreducing pyruvylated-DGal. Inhibition studies showed that both sites are directed toward one of the two space isomers of 3,4- or 4,6-pyruvylated DGal, the form in which the methyl group of the pyruvate is equatorial, or endo, and its carboxyl group axial, or exo, to the plane of the acetal ring. Coprecipitation studies showed the combining site of IgMWEA to be located on an (Fab')2 fragment and not on the (Fc)5mu fragment. The monoclonal peak in the serum of IgMMAY was specifically precipitated by Klebsiella polysaccharide. Myeloma proteins with specificities of this type may occur with reasonable frequency in humans and may be a consequence of clonal expansion from inapparent infection, carrier states, or disease produced by various Klebsiella organisms.

Targeting the Sugary Armor of Klebsiella Species.

The emergence of multidrug-resistant strains of Gram-negative Klebsiella species is an urgent global threat. The World Health Organization has listed Klebsiella pneumoniae as one of the global priority pathogens in critical need of next-generation antibiotics. Compared to other Gram-negative pathogens, K. pneumoniae accumulates a greater diversity of antimicrobial-resistant genes at a higher frequency. The evolution of a hypervirulent phenotype of K. pneumoniae is yet another concern. It has a broad ecological distribution affecting humans, agricultural animals, plants, and aquatic animals. Extracellular polysaccharides of Klebsiella, such as lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, play crucial roles in conferring resistance against the host immune response, as well as in colonization, surface adhesion, and for protection against antibiotics and bacteriophages. These extracellular polysaccharides are major virulent determinants and are highly divergent with respect to their antigenic properties. Wzx/Wzy-, ABC-, and synthase-dependent proteinaceous nano-machineries are involved in the biosynthesis, transport, and cell surface expression of these sugar molecules. Although the proteins involved in the biosynthesis and surface expression of these sugar molecules represent potential drug targets, variation in the amino acid sequences of some of these proteins, in combination with diversity in their sugar composition, poses a major challenge to the design of a universal drug for Klebsiella infections. This review discusses the challenges in universal Klebsiella vaccine and drug development from the perspective of antigen sugar compositions and the proteins involved in extracellular antigen transport.

The pathogenesis of ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease that can cause significant functional complications by affecting the sacroiliac joints and axial skeleton. Despite a longstanding knowledge about the familial associations of this disease, particularly among patients positive for human leukocyte antigen (HLA)-B27, the fundamental pathogenetic mechanism by which this disease arises in genetically susceptible individuals remains ill defined. Furthermore, the molecular predilection for characteristic articular site involvement remains under ongoing investigation. Current theories about the HLA-B27 association range from the presentation of novel arthritogenic peptides, to abnormal autoimmune stimulation, to anomalous microbial tolerance. The immune effectors of this damage include CD4+, CD8+, and natural killer cells, with marked heterogeneity at different sites. Biomechanical stresses may trigger this disease by exposing the body to previously immune-sequestered autoantigens or by providing a route for bacterial seeding. Environmental triggers such as infection have not been definitively established but may represent a primary pathogenic step in a molecular-mimicry process. In this article, the authors review the current literature on the origin and pathophysiology of AS, focusing on genetic and molecular associations, consequent pathomechanisms, and associated triggers. An improved understanding of the sequence of molecular events that predispose and initiate the onset of this disease will allow for more specific and targeted therapy and better avoidance of the significant side effects of systemic immunomodulation.

Iron and innate antimicrobial immunity-Depriving the pathogen, defending the host.

The acute-phase response is triggered by the presence of infectious agents and danger signals which indicate hazards for the integrity of the mammalian body. One central feature of this response is the sequestration of iron into storage compartments including macrophages. This limits the availability of this essential nutrient for circulating pathogens, a host defence strategy known as 'nutritional immunity'. Iron metabolism and the immune response are intimately linked. In infections, the availability of iron affects both the efficacy of antimicrobial immune pathways and pathogen proliferation. However, host strategies to withhold iron from microbes vary according to the localization of pathogens: Infections with extracellular bacteria such as Staphylococcus aureus, Streptococcus, Klebsiella or Yersinia stimulate the expression of the iron-regulatory hormone hepcidin which targets the cellular iron-exporter ferroportin-1 causing its internalization and blockade of iron egress from absorptive enterocytes in the duodenum and iron-recycling macrophages. This mechanism disrupts both routes of iron delivery to the circulation, contributes to iron sequestration in the mononuclear phagocyte system and mediates the hypoferraemia of the acute phase response subsequently resulting in the development of anaemia of inflammation. When intracellular microbes are present, other strategies of microbial iron withdrawal are needed. For instance, in macrophages harbouring intracellular pathogens such as Chlamydia, Mycobacterium tuberculosis, Listeria monocytogenes or Salmonella Typhimurium, ferroportin-1-mediated iron export is turned on for the removal of iron from infected cells. This also leads to reduced iron availability for intra-macrophage pathogens which inhibits their growth and in parallel strengthens anti-microbial effector pathways of macrophages including the formation of inducible nitric oxide synthase and tumour necrosis factor. Iron plays a key role in infectious diseases both as modulator of the innate immune response and as nutrient for microbes. We need to gain a more comprehensive understanding of how the body can differentially respond to infection by extra- or intracellular pathogens. This knowledge may allow us to modulate mammalian iron homeostasis pharmaceutically and to target iron-acquisition systems of pathogens, thus enabling us to treat infections with novel strategies that act independent of established antimicrobials.

Spontaneous in vitro differentiation of antigen-specific lymphocytes from a patient with immunoglobulin M gammopathy.

Recently we have identified two monoclonal immunoglobulin M (IgM) proteins that bind Klebsiella polysaccharides. The lymphocytes of one of these patients (M.A.Y.) were available for study. A substantial proportion of the B lymphocytes isolated from this patient's peripheral blood also bound Klebsiella polysaccharides with a pattern of specificity identical to that of the monoclonal IgM, and reacted with an anti-idiotypic antiserum directed against this IgM. Stripping the surface immunoglobulin from these lymphocytes eliminated this reactivity. Although no plasma cells were detected in the freshly isolated peripheral blood lymphocytes of this patient, plasma cells binding Klebsiella polysaccharide appeared after 7 d of in vitro culture. This occurred regardless of whether the cultures were supplemented with autologous plasma, normal human plasma, or fetal calf serum. Pokeweed mitogen neither stimulated nor inhibited the in vitro differentiation of the monoclonal B lymphocytes into plasma cells. This differentiation was, however, abrogated by F(ab')2 fragments of anti-human IgM and by anti-idiotypic antibodies, as well as by the Klebsiella polysaccharide with which the monoclonal IgM reacted.

Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation.

Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (TH1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease.

Use of animal models for space flight physiology studies, with special focus on the immune system.

Animal models have been used to study the effects of space flight on physiological systems. The animal models have been used because of the limited availability of human subjects for studies to be carried out in space as well as because of the need to carry out experiments requiring samples and experimental conditions that cannot be performed using humans. Experiments have been carried out in space using a variety of species, and included developmental biology studies. These species included rats, mice, non-human primates, fish, invertebrates, amphibians and insects. The species were chosen because they best fit the experimental conditions required for the experiments. Experiments with animals have also been carried out utilizing ground-based models that simulate some of the effects of exposure to space flight conditions. Most of the animal studies have generated results that parallel the effects of space flight on human physiological systems. Systems studied have included the neurovestibular system, the musculoskeletal system, the immune system, the neurological system, the hematological system, and the cardiovascular system. Hindlimb unloading, a ground-based model of some of the effects of space flight on the immune system, has been used to study the effects of space flight conditions on physiological parameters. For the immune system, exposure to hindlimb unloading has been shown to results in alterations of the immune system similar to those observed after space flight. This has permitted the development of experiments that demonstrated compromised resistance to infection in rodents maintained in the hindlimb unloading model as well as the beginning of studies to develop countermeasures to ameliorate or prevent such occurrences. Although there are limitations to the use of animal models for the effects of space flight on physiological systems, the animal models should prove very valuable in designing countermeasures for exploration class missions of the future.

Is a Klebsiella plasmid involved in the aetiology of Ankylosing Spondylitis in HLA-B27-positive individuals?

The possibility that plasmid genes, carried by enteric organisms previously indirectly implicated as disease agents, play a role in the pathogenesis of Ankylosing Spondylitis (AS) was explored. A particular Klebsiella isolate (K21) previously found to cross-react with cells from HLA-B27 positive (B27+) patients with AS, but not with cells from normal individuals, was found to contain a plasmid(s). This coded for the organism's ability to produce a factor which could modify B27+ normal cells (AS-) rendering them lysable by the anti-Klebsiella serum. Curing of this isolate resulted in the loss of the plasmid concerned and a loss of ability of its culture filtrate to modify B27+ lymphocytes of clinically healthy subjects. When plasmids from K21 were transferred to a plasmid free laboratory strain, E. coli JP995, the recipient strain acquired the ability to elaborate modifying factor. These data suggest that plasmids, harboured by some enteric bacteria, and their products, may be implicated in modifying cells bearing certain Major Histocompatibility Complex genes, and that such modification may be an important factor in the pathogenesis of a number of diseases including the seronegative arthropathies.