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Previous work has shown that Secretory-IgA (SIgA) binding to the intestinal microbiota is variable and may regulate host inflammatory bowel responses. Nevertheless, the impact of the SIgA functional binding to the microbiota remains largely unknown in preterm infants whose immature epithelial barriers make them particularly susceptible to inflammation. Here, we investigated SIgA binding to intestinal microbiota isolated from stools of preterm infants

Objectives Francisella tularensis, the causative agent of tularaemia, is an exceptionally infectious bacterium, potentially fatal for humans if left untreated and with the potential to be developed as a bioweapon. Both natural infection and live‐attenuated vaccine strain (LVS) confer good protection against tularaemia. LVS vaccination is traditionally administered by scarification, and the formation of a cutaneous reaction or take at the vaccination site is recognised as a clinical correlate of protection. Although previous studies have suggested that high antibody titres following vaccination might serve as a useful surrogate marker, the immunological correlates of protection remain unknown. Methods We investigated the host T‐cell‐mediated immune (T‐CMI) responses elicited following immunisation with LVS vaccine formulated by the DynPort Vaccine Company (DVC‐LVS) or the United States Army Medical Research Institute of Infectious Diseases (USAMRIID‐LVS). We compared T‐CMI responses prompted by these vaccines and correlated them with take size. Results We found that both LVS vaccines elicited similar T‐CMI responses. Interestingly, take size associated with the T cells’ ability to proliferate, secrete IFN‐γ and mobilise degranulation, suggesting that these responses play an essential role in tularaemia protection. Conclusions These results renew the appreciation for vaccination through the scarification as a prime route of inoculation to target pathogens driving specific T‐CMI responses and provide further evidence that T‐CMI plays a role in protection from tularaemia., The actual immunological correlate(s) of protection from tularaemia in humans remain(s) unknown. As with smallpox, take is widely accepted as a clinical correlate of protection against tularaemia. We report, for the first time, a correlation between take‐lesion size and cellular‐mediated immune responses in humans after tularaemia immunization.

Background & Aims: Clostridium difficile toxin A (TcdA) and C difficile toxin toxin B (TcdB), the major virulence factors of the bacterium, cause intestinal tissue damage and inflammation. Although the 2 toxins are homologous and share a similar domain structure, TcdA is generally more inflammatory whereas TcdB is more cytotoxic. The functional domain of the toxins that govern the proinflammatory activities of the 2 toxins is unknown. Methods: Here, we investigated toxin domain functions that regulate the proinflammatory activity of C difficile toxins. By using a mouse ilea loop model, human tissues, and immune cells, we examined the inflammatory responses to a series of chimeric toxins or toxin mutants deficient in specific domain functions. Results: Blocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells. Conclusions: In this study, we identified a novel mechanism of regulating the biological activities of C difficile toxins in that cysteine protease-mediated autoprocessing regulates toxins’ proinflammatory activities. Our findings provide new insight into the pathogenesis of C difficile infection and the design of therapeutics against the disease. Keywords: C difficile, Toxins, Cysteine Protease, Autoprocessing, Inflammation

Enteric fevers, caused by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB), are life-threatening illnesses exhibiting very similar clinical symptoms but with distinct epidemiologies, geographical distributions and susceptibilities to antimicrobial treatment. Nevertheless, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using a three-dimensional organotypic model of the human intestinal mucosa and PA, PB, and ST, we observed significant differences in the secretion patterns of pro-inflammatory cytokines and chemokines elicited by these serovars. These cytokines/chemokines were likely to be co-regulated and influenced the function of epithelial cells, such as the production of IL-8. We also found differing levels of polymorphonuclear leukocyte (PMN) migration among various infection conditions that either included or excluded lymphocytes and macrophages (Mϕ), strongly suggesting feedback mechanisms among these cells. Blocking experiments showed that IL-1β, IL-6, IL-8, TNF-α and CCL3 cytokines were involved in the differential regulation of migration patterns. We conclude that the crosstalk among the lymphocytes, Mϕ, PMN and epithelial cells is cytokine/chemokine-dependent and bacterial-serotype specific, and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes., Author summary Enteric fevers are acute illnesses caused mainly by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB). The incidence of enteric fevers is very low in industrialized countries, with most infections occurring in military personnel and individuals traveling to endemic areas. Nevertheless, worldwide, more than 20.6 million cases of enteric fever occur annually in low- and middle-income countries and are associated with approximately 129,000–223,000 deaths. ST, PA, and PB exhibit very similar clinical symptoms but distinct epidemiologies, geographical distributions, and susceptibilities to antimicrobial treatment. However, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using an in vitro model of the human intestinal mucosa, we found that the crosstalk among leukocytes—lymphocytes, Mϕ, and PMN- and epithelial cells is cytokine/chemokine-dependent, and bacterial-serotype specific and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes.

Summary Secretory immunoglobulin A (SIgA) antibodies play an important role in protecting the mucosal surfaces against pathogens and maintaining homeostasis with the commensal microbiota. Because a substantial portion of the gut microbiota is coated with SIgA, we hypothesized that microbiota–SIgA complexes are important for the maintenance of gut homeostasis. Here we investigated the relationship between microbiota–SIgA complexes and inflammatory epithelial cell responses. We used a multi-cellular three-dimensional (3D) organotypical model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes/monocytes, endothelial cells and fibroblasts. We also used human SIgA from human colostrum, and a prominent bacterial member of the first colonizers, Escherichia coli, as a surrogate commensal. We found that free and microbiota-complexed SIgA triggered different epithelial responses. While free SIgA up-regulated mucus production, expression of polymeric immunoglobulin receptor (pIgR) and secretion of interleukin-8 and tumoir necrosis factor-α, microbiota-complexed SIgA mitigated these responses. These results suggest that free and complexed SIgA have different functions as immunoregulatory agents in the gut and that an imbalance between the two may affect gut homeostasis.

A notable proportion of Salmonella-associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S. Typhimurium vaccine candidate CVD 1921 (I77 ΔguaBA ΔclpP) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA. We determined that S. Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S. Typhimurium ΔpipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (10(9) CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD(50) (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S. Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model.

Here, we investigated toxin domain functions that regulate the proinflammatory activity ofBlocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells.In this study, we identified a novel mechanism of regulating the biological activities of

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA's Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

We investigated the ability of live attenuatedSalmonella entericaserovar Typhi strains delivered to mice intranasally to induce specific cytotoxic T-lymphocyte (CTL) responses at regional and systemic levels. Mice immunized with two doses (28 days apart) ofSalmonellaserovar Typhi strain Ty21a, the licensed oral typhoid vaccine, and genetically attenuated mutants CVD 908 (ΔaroCΔaroD), CVD 915 (ΔguaBA), and CVD 908-htrA(ΔaroCΔaroDΔhtrA) induced CTL specific forSalmonellaserovar Typhi-infected cells in spleens and cervical lymph nodes. CTL were detected in effector T cells that had been expanded in vitro for 7 days in the presence ofSalmonella-infected syngeneic splenocytes. A second round of stimulation further enhanced the levels of specific cytotoxicity. CTL activity was observed in sorted αβ+CD8+T cells, which were remarkably increased after expansion, but not in CD4+T cells. CTL from both cervical lymph nodes and spleens failed to recognizeSalmonella-infected major histocompatibility complex (MHC)-mismatched cells, indicating that the responses were MHC restricted. Studies in which MHC blocking antibodies were used showed that H-2Ldwas the restriction element. This is the first demonstration thatSalmonellaserovar Typhi vaccines delivered intranasally elicit CD8+MHC class I-restricted CTL. The results further support the usefulness of the murine intranasal model for evaluating the immunogenicity of typhoid vaccine candidates at the preclinical level.

The engineering of multicellular organotypic models of the human intestinal mucosa has wide-ranging potential as a tool for discovery in both health and disease, including interaction with pathogens, antigen trafficking, inflammatory and physiological processes. However, models reported to date remain relatively simple composed of few cell types. Here, we report the development of an organotypic model with close structural and functional resemblance to the human intestinal mucosa; it is comprised of fibroblasts, lymphocytes, epithelial cells, and endothelial cells. Moreover, epithelial cells in our 3-D system can differentiate into multiple lineages (e.g., goblet cells, M cells and differentiated enterocytes). We hypothesized that under both microgravity and proper culture conditions, aggregates of randomly distributed cells will self-organize into a spatial configuration resembling those in native tissues. Our hypothesis was based on recent developments showing that: (1) the culture of epithelial cells as well as endothelial cells under microgravity can be accompanied by a commensurate increase in the cell differentiation1–3 and (2) gelled collagen-I can constitute a flexible extracellular matrix (ECM) to be used under microgravity.4 It is known that collagen-I possesses most of the properties of an ideal scaffold, such as specific gravity similar to the culture medium and the capability to incorporate other relevant ECM proteins.4 In our model, fibroblasts and endothelial cells were embedded in a collagen-I matrix enriched with additional gut basement membrane proteins (i.e., laminin, collagen IV, fibronectin and heparin sulfate proteoglycan) and added to Rotating Wall Vessel (RWV) bioreactors containing epithelial cells (Supplementary Methods). These epithelial cells consisted of a human enterocyte cell line (i.e., HCT-8) that was originally derived from the junction of the small and large bowel.5 The choice of this cell line was based on its ability to successfully differentiate in a low shear microgravity environment1 provided by the RWV bioreactor.6 Activated lymphocytes were added twice to the culture at days 4 (± 1 day) and 9 (± 1 day) (Supplementary Methods). After 10 to 15 days, histological staining and electron microscopy demonstrated the presence of villus-like structures in the constructs. About 60–80% of these epithelial cells were organized as a monolayer of polarized cells with nuclei located in a basal position near the ECM, a major feature of well-differentiated cells (Figure 1A). H&E staining also demonstrated the presence of a brush border, or microvilli, seen collectively as a fuzzy fringe in the apical surface of the epithelium (Figure 1B). The presence of microvilli was further evidenced by immunochemical staining using an anti-villin mouse monoclonal antibody (mAb) or by electron microscopy (Figure 1C and D, respectively). Assessment by immunostaining for zonula occludens (ZO)-1, a protein involved in the formation of tight junctions, and electron microscopy demonstrated that our 3-D organotypic model recapitulates the formation of tight junctions and desmosomes (Figure 1E and F). In addition, because immortalized epithelial cells in 3-D systems may lose the growth pattern found in the normal epithelium, we assessed the expression of E-cadherin and the Ki-67 proliferation marker in our 3-D model. As expected, HCT-8 cell lines expressed E-cadherin but only negligible amount of Ki-67 antigen, a pattern consistent with normal highly-differentiated epithelium (Figures 1G–I). Finally, these HCT-8 cells also expressed numerous markers for multi-lineage differentiation to globet (mucin 2, MUC 2) and M (Sialyl Lewis Antigen) cell lineages (Figures 1J and K). Goblet and M cells were distributed throughout the epithelial layer (Figures 1J and K, respectively). We also investigated the presence of the machinery for the specific digestion and transfer of glucose. We observed apical expression of dissacharidases (i.e., sucrase-isomaltase, SI) and sugar transporters (i.e., energy-dependent sodium-glucose co-transporter 1, SGLT1) which strongly suggests that epithelial cells in our 3-D model are able to absorb and transport glucose (data not shown). Taken together, these results demonstrate a remarkable degree of differentiation of HCT-8 epithelial cells in our 3-D model. Figure 1 Epithelial cell differentiation. Hematoxylin and eosin staining of 14 day-cells cultured in the 3-D microgravity model: tissues were stained purple and scaffold stained pink. Lower (A) and higher magnification (B) shows the presence of polarized epithelial ... We also examined the presence and persistence of other cell types by monitoring the presence of defined cellular markers for each individual cell type (i.e., CD45 for hematopoietic-derived cells, including lymphocytes; CD31 for endothelial cells and Vimentin for fibroblasts). These studies were performed by immunochemistry. We observed that the lymphocytes migrated through the epithelial cell layer and localized to the ECM. We were able to detect lymphocytes for at least 7 days (last day examined) (Figure 1L). As in the native gut tissue, in our 3-D model fibroblasts and endothelial cells remained dispersed throughout the ECM. Interestingly, we observed that individual endothelial cells exhibited cytoplasmic extensions that form bridges between them, suggesting that these endothelial cells were activated and underwent a remodeling phase (Figures 1M and N). However, the vast majority of these endothelial cells do not appear to differentiate towards the formation of vessel-like conduits. Additional studies are needed to determine the proper conditions that will further enable vessel-like conduits to form. Interestingly, fibroblasts showed different shapes, ranging from classical spindle shaped to rounded-fat cell-like shaped with the nucleus pushed to one side (Figures 1O and P). These results suggest that cells were activated and remain able to both receive and respond to differentiation signals. Finally, several studies have demonstrated that good viability for primary cells lasting more than 10 days in culture systems is difficult to achieve.7 Thus, we assessed cell viability by immunochemical staining using antibodies to cleaved Caspase-3. Up to 20 days after the initiation of the cultures, few cleaved Caspase-3-positive cells were observed in the ECM, the major location of primary cells including lymphocytes, fibroblasts and endothelial cells (data not shown). Although the combination of the features described above give some insights into the similarities of our 3-D organotypic model to in vivo tissues, we further assessed its functionality by observing its response to exposure to a human enteric bacterial pathogen. Specifically, we examined the changes in the epithelial cell morphology, as well as cytokine production, following exposure to wild-type Salmonella enterica serovar Typhi (S. Typhi) strain ISP1820, to evaluate whether it mimicked the reported stages of the infection. The identification and characterization of S. Typhi-infected cells was accomplished using a polyclonal antibody that specifically detects common Salmonella antigens (CSA-1). A visible feature of the Salmonella entry process in vivo, i.e., membrane rearrangements or ruffles, were observed in our system as early as 1 hour after infection (Supplementary Figure 1B) and increased over time (Supplementary Figures 1C and D). Of note, these ruffles were absent in the control (not-infected cells, Supplementary Figure 1A). One day after infection, the ruffles fade away and bacteria, when present, was mostly observed on the cell surface/brush border of healthy looking epithelial cells or intracellular in epithelial cells sloughed from the monolayer which likely died as a result of the infection (Supplementary Figures 1E and F). We also observed that the exposure to Salmonella resulted in production of cytokines, including IL-1β, IL-6, IL-8, IL-11, IL-12p70, IL-17a, IL-21 and TNF-α (Supplementary Figures 1G, H and I). These results are in agreement, and extend previous studies showing the importance of these cytokines in the control of Salmonella infection8, 9 and further confirming the functional capacity of this 3-D organotypic model to properly react to the presence of an enteric pathogen. To summarize, although similar approaches have been attempted by other investigators with moderate success,1, 10 our system provides major innovations and advances such as a matrix enriched with gut basement membrane proteins. Also, to our knowledge, except for our system described above, attempts to integrate multiple cell types in 3-D constructs grown under microgravity have been unsuccessful. Finally, we provided evidence that our 3-D model might be a helpful tool to investigate the early events of the host interaction with gastrointestinal pathogens, including invasion, pathogenesis and immune responses.

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, and S. Paratyphi A and B, causative agents of paratyphoid fever, are major public health threats throughout the world. Although two licensed typhoid vaccines are currently available, they are only moderately protective and immunogenic necessitating the development of novel vaccines. A major obstacle in the development of improved typhoid, as well as paratyphoid vaccines is the lack of known immunological correlates of protection in humans. Considerable progress has been made in recent years in understanding the complex adaptive host responses against S. Typhi. Although the induction of S. Typhi-specific antibodies (including their functional properties) and memory B cells, as well as their cross-reactivity with S. Paratyphi A and S. Paratyphi B has been shown, the role of humoral immunity in protection remains undefined. Cell mediated immunity (CMI) is likely to play a dominant role in protection against enteric fever pathogens. Detailed measurements of CMI performed in volunteers immunized with attenuated strains of S. Typhi have shown, among others, the induction of lymphoproliferation, multifunctional type 1 cytokine production, and CD8(+) cytotoxic T-cell responses. In addition to systemic responses, the local microenvironment of the gut is likely to be of paramount importance in protection from these infections. In this review, we will critically assess current knowledge regarding the role of CMI and humoral immunity following natural S. Typhi and S. Paratyphi infections, experimental challenge, and immunization in humans. We will also address recent advances regarding cross-talk between the host's gut microbiota and immunization with attenuated S. Typhi, mechanisms of systemic immune responses, and the homing potential of S. Typhi-specific B- and T-cells to the gut and other tissues.

A common finding when measuring T cell immunity to enteric bacterial vaccines in humans is the presence of background responses among individuals before immunization. Yet the nature of these background responses remains largely unknown. Recent findings show the presence in uninfected individuals of mucosal associated invariant T (MAIT) cells that mount broad spectrum immune responses against a variety of microorganisms including Mycobacterium tuberculosis and enteric bacteria such as Escherichia coli and Salmonella. Therefore, we investigated whether MAIT immune responses to intestinal bacteria might account for the background responses observed before immunization. Here we measured MAIT immune responses to commensal and enteric pathogenic bacteria in healthy individuals with no history of oral immunization with enteric bacteria. We found that MAIT cells were activated by B cells infected with various bacteria strains (commensals and pathogens from the Enterobacteriaceae family), but not by uninfected cells. These responses were restricted by the non-classical MHC-related molecule 1 (MR1) and involved the endocytic pathway. The quality of these responses (i.e., cytokine profile) was dependent on bacterial load but not on the level expression of MR1 or bacterial antigen on B cell surface, suggesting that a threshold level of MR1 expression is required to trigger MAIT activation. These results provide important insights into the role of B cells as a source of antigen-presenting cells to MAIT cells and the gut immune surveillance of commensal microbiota.

Objective : During HIV-1 infection, CD4+ T lymphocytes migrate to immune-reactive lymphoid organs where they are infected by the virus and/or killed by apoptosis on immunoregulatory stimuli - a potential mechanism underlying fatal CD4+ T-cell depletion observed in AIDS. This study seeks to determine the effects of glucocorticoids (GCC) on the activation-induced T-cell apoptosis triggered by HIV-1. Methods : CD4+ and CD8+ T cells were purified from HIV-negative donor peripheral blood mononuclear cells (PBMC) by positive selection and exposed to HIV-1 (primary isolates). HIV-1-exposed CD4+ and CD8+ T cells as well as PBMC derived from HIV-1-infected patients were cultured with medium alone or anti-CD3 monoclonal antibodies (MAb)/mitogens in the presence or absence of hydrocortisone or prednisolone. Viral infection kinetics were assessed by polymerase chain reaction and viral replication was measured by p24 enzyme-linked immunosorbent assay. Cell survival, apoptosis, T-cell proliferation, blast cell transformation, and interleukin (IL)-2 receptor (CD25) expression were monitored in parallel for each cell population. Results : Fractionated CD4+ T cells acutely infected by HIV-1 underwent apoptotic death on anti-CD3 MAb/mitogen stimulation. This activation-induced apoptotic cell killing was antagonized by pharmacological doses of prednisolone or hydrocortisone added up to 6h after stimulation. GCC were also found to be capable of inhibiting the accelerated apoptosis in PBMC (including both CD4+ and CD8+ T-cell fractions) from HIV-1-infected patients. This anti-apoptotic action of GCC overbalanced their downregulatory effect on T-cell proliferation, resulting in an overall improvement of CD4+ T-cell survival in patient PBMC. These effects of GCC were abrogated by the anti-GCC RU 486 and were not associated with significant suppression of CD25 expression and IL-2-dependent T-cell blast transformation ; moreover, GCC had no impact on viral infection and replication. Conclusion : GCC exert a receptor-mediated anti-apoptotic activity in mature T cells through both activation-induced and HIV-1-triggered pathways, and could be potent inhibitors of T-cell apoptosis in HIV-1-infected patients.