Intestinal Patches Research Articles

Effect of probiotic strains upon intestinal immune system under cold stress

Gut microbiome changes can be considered as a causative factor of stress-associated intestinal diseases. The aim of the investigation was to study the effects of the oral probiotic strains Lactobacillus casei LB 148 and E. coli EB 387 onto gut microbiota, as well as the intestinal immune system of rats Sprague Dawley in a healthy state and under cold stress. There are no strict official recommendations here in the Russian Federation for preclinical studies of new probiotic strains. Here we describe a method used to estimate the safety and effectiveness of the oral use of a probiotic. It is cheap and simple, so we recommend using it at least for the screening search of effective and safe probiotics. The number of Peyer’s patches in the small intestine of rats in different compartments was counted and then compared between different groups of rats. After cold stress there was a change in the number of Peyer’s patches together with microbiota shifts. Lactobacillus casei LB 148 as well as E. coli EB 387 protected the immune system against the stress effects.

THE STRUCTURE OF LYMPHOID-ASSOCIATED EPITHELIUM OF PEYERS’ PATCHES OF THE ALBINO RATS’ SMALL INTESTINE

Over the past two decades, there have been many publications dealing with the further development of an urgent issue on the immune system of the mucous membranes of the digestive tract, called mucoseassociated lymphoid tissue (MALT), which includes spheres of innate (non-specific) and adaptive (specific) immunity. Most structured formations and indicators of adaptive immunity in the intestinal mucosa are lymphoepithelial formation (Peyer's patches). The data on the formation of the peripheral part of the immune system are carried through the epithelium, mechanisms of interaction between pathogenic intestinal microflora and immunocompetent cells, thereby initiating the development of immune responses in the mucous membranes. A concept has long been established in the literature, according to which a leading role in mediating these reactions belongs to a special type of enterocytes, called M-cells. Before the concept of the initial role of these cells in the development of immune responses in the mucous membranes of the intestinal tract they were known as caveolated cells. The purpose of this study was to determine the shape and topological relations of M-cells with other types of enterocytes, and also with lymphoid elements Peyer's patches of the small intestine. 30 mature albino male rats weighted 200,0±20,0 g were involved into the study. After vivisection, which was carried out by an overdose of thiopental anesthesia (75 mg / kg of animal body weight intramuscularly in the upper third of the thigh of the hind paw) [1] in compliance with the requirements for dissection of the abdominal cavity, the entire complex of the gastrointestinal tract was removed, which was preserved in 10 % formalin solution. Subsequently, short sections of the small intestine, containing Peyer’s patches, were selectively excised. Finding the latter was not difficult due to their clear visualization on the external (nonmesenteric) surface of the small intestine in the form of whitish spots. The specimens, after washing from formalin and dehydration in alcohol of increasing concentration, were embedded into paraffin blocks, from which serial sections of 4 µm thick (Microm HM 325) were obtained with subsequent staining with hematoxylin-eosin and Van Gieson. Their study and documentation was carried out using the “Konus” light microscope equipped with the Sigeta DCM-900 9.0MP digital microphoto attachment and the Biorex 3 program (serial number 5604) adapted for these studies. In the study of many series of paraffin sections stained with hematoxylin-eosin, it was found that while maintaining the general shape of the structure, lymphoid nodules are susceptible to plastic variability, which depends on situationally changing factors of antigenic effect, i.e., functional polymorphism is characteristic of them. This is especially true of their lymphoidassociated epithelium, which appears in a rather diverse form, which depends not only on the section angle, but also, probably, on its reactive state. Thus, in some cases it is a relatively even monolayer of intestinal epithelium, consisting mainly of absorbing enterocytes, among which the most distinct are goblet cells. At the same time, it draws attention to itself that in the apical sections of some of them there are clear signs of rupture of the plasmolemma and the presence in the cytoplasm of basophilic granular fibrous material of an unknown nature. Along with this picture, other histological sections of large lymphoid nodules of Peyer's patches of the small intestine demonstrate a different configurational character of lymphoid-associated epithelium, in which the cluster principle of cell distribution in the form of limited portioned sets is clearly noted. While maintaining the general structural shape, Peyer's patches were found to be subjected to plastic variability, which depends on situationally changing factors of antigenic exposure, i.e., functional polymorphism is characteristic of them. This is especially true of their lymphoid-associated epithelium. Identification of M-cells using only traditional histological methods in practice is complicated. And yet, in the process of a focused study of serial paraffin sections, it was possible to detect some morphological signs indicating their location.

The Roles of Peyer's Patches and Microfold Cells in the Gut Immune System: Relevance to Autoimmune Diseases

Microfold (M) cells are located in the epithelium covering mucosa-associated lymphoid tissues, such as the Peyer's patches (PPs) of the small intestine. M cells actively transport luminal antigens to the underlying lymphoid follicles to initiate an immune response. The molecular machinery of M-cell differentiation and function has been vigorously investigated over the last decade. Studies have shed light on the role of M cells in the mucosal immune system and have revealed that antigen uptake by M cells contributes to not only mucosal but also systemic immune responses. However, M-cell studies usually focus on infectious diseases; the contribution of M cells to autoimmune diseases has remained largely unexplored. Accumulating evidence suggests that dysbiosis of the intestinal microbiota is implicated in multiple systemic diseases, including autoimmune diseases. This implies that the uptake of microorganisms by M cells in PPs may play a role in the pathogenesis of autoimmune diseases. We provide an outline of the current understanding of M-cell biology and subsequently discuss the potential contribution of M cells and PPs to the induction of systemic autoimmunity, beyond the mucosal immune response.

Enhanced paracellular delivery of vaccine by hydrogel microparticles-mediated reversible tight junction opening for effective oral immunization

The current conventional injectable vaccines face several drawbacks such as inconvenience and ineffectiveness in mucosal immunization. Therefore, the current development of effective oral vaccines is vital to enable the generation of dual systemic and mucosal immunity. In the present study, we examine the potential of pH-responsive bacterial nanocellulose/polyacrylic acid (BNC/PAA) hydrogel microparticles (MPs) as an oral vaccine carrier. In-vitro entrapment efficiency and release study of Ovalbumin (Ova) demonstrated that as high as 72% of Ova were entrapped in the hydrogel, and the release of loaded Ova was pH-dependent. The released Ova remained structurally conserved as evident by Western blot and circular dichroism. Hydrogel MPs reduced the TEER measurement of HT29MTX/Caco2/Raji B triple co-culture monolayer by reversibly opening the tight junctions (TJs) as shown in the TEM images. The ligated ileal loop assay revealed that hydrogel MPs could facilitate the penetration of FITC-Ova into the Peyer's patches in small intestine. Ova and cholera toxin B (CTB) were utilized in in-vivo oral immunization as model antigen and mucosal adjuvant. The in-vivo immunization revealed mice orally administered with Ova and CTB-loaded hydrogel MPs generated significantly higher level of serum anti-Ova IgG and mucosal anti-Ova IgA in the intestinal washes, compared to intramuscular administrated Ova. These results conclude that BNC/PAA hydrogel MPs is a potential oral vaccine carrier for effective oral immunization.

Nonsecretory intestinocystoplasty: postoperative outcomes of 25 years.

ABSTRACTObjective The objective of bladder augmentation (BA) is to create a low-pressure reservoir with adequate capacity. Despite its benefits, the use of intestinal patches in bladder enlargement provides a high risk of developing complications and BA with demucosalised bowel represents a potential alternative. Therefore, this study evaluated urological parameters and long-term clinical follow-up of patients submitted to non-secretory BA in a single center with 25 years of experience.Materials and Methods Patients treated with BA underwent urological evaluation, which included history, physical examination and urodynamic study. The main urodynamic parameters (bladder capacity and bladder compliance) were assessed in the pre and postoperative moments, and compared by the Wilcoxon Signed Rank test. The main long-term complications were described.Results 269 patients (mean age 14±13 years, 47% male) underwent BA with the use of demucolised intestinal segments. Among the patients in the sample, 187 (69.52%) had neurogenic bladder, 68 (25.28%) had bladder exstrophy, nine had tuberculosis (3.34%), four had a posterior urethral valve (1.49%) and one with hypospadia (0.37%). After the surgical procedure, a significant increment in both urodynamic parameters was found, with a 222% increase in bladder capacity and 604% in bladder compliance (p <0.001 in both analyzes). Mean follow-up time ranged from 2 to 358 months, with a median of 72 months (IQR 74-247). Among all patients, 5 presented spontaneous perforation.Conclusion The study showed statistically significant increase in both compliance and bladder capacity after non-secretory BA, with a low rate of severe complications.

Effect of Chronic Commercial Sweeteners Consumption in Lymphocytes of Peyer’s Patches

Aims: To know the effect of chronic commercial sweeteners consumption in lymphocytes of Peyer’s patches.&#x0D; Study Design: A prospective, longitudinal, comparative and experimental study.&#x0D; Place and Duration of Study: The study was conducted in the Nutrition Research Laboratory of the Faculty of Medicine of Universidad Autónoma del Estado de México (UAEMéx) between August 2018 and May 2019 and was approved by the Bioethics Committee.&#x0D; Materials and Methods: Two groups of male mice of different strains were used: 1) Balb/c and 2) CD1, both at 8 weeks-old age. The groups were divided into 4 subgroups: 1) Control (without sweetener), 2) Sucrose (table sugar, 41.66 mg/mL), and two groups of commercial sweeteners 3) Splenda® (sucralose 1.2%, with a concentration of 4.16 mg/mL), and 4) Svetia® (Steviol glycoside 0.025 g with a concentration of 4.16 mg/mL). The mice consumed the supplementation for 6 weeks. Also, were quantified plasma glucose, percentage of lymphocytes from Peyer’s patches, water and food consumption weekly.&#x0D; Results: Mice increased their body weight after 6 weeks of treatment. The animals of Control and Sucrose subgroups showed a significant body weight gain of 5 g compared with the Splenda® and Svetia® subgroups, which increased only 4 g. In the subgroup treated with Splenda®, the blood glucose was reduced significantly. Svetia® and Control groups consumed more water without sweetener. The differences in food consumption were between the subgroups, not between the strains. By the end, the percentage of lymphocytes from Peyer´s patches increased in the Sucrose subgroup but decreased significantly in other subgroups.&#x0D; Conclusion: The consumption of sweeteners may modify the lymphocyte population of Peyer's patches in the small intestine and this variation depends on the frequency of consumption the strain of the rodents and the type of sweetener.

Acetylcholine-producing T cells augment innate immune-driven colitis but are redundant in T cell-driven colitis.

Clinical trials suggest that vagus nerve stimulation presents an alternative approach to classical immune suppression in Crohn's disease. T cells capable of producing acetylcholine (ChAT+ T cells) in the spleen are essential mediators of the anti-inflammatory effect of vagus nerve stimulation. Besides the spleen, ChAT+ T cells are found abundantly in Peyer's patches of the small intestine. However, the role of ChAT+ T cells in colitis pathogenesis is unknown. Here, we made use of CD4creChATfl/fl mice (CD4ChAT-/- mice) lacking ChAT expression specifically in CD4+ T cells. Littermates (ChATfl/fl mice) served as controls. In acute dextran sulfate sodium (DSS)-induced colitis (7 days of 2% DSS in drinking water), CD4ChAT-/- mice showed attenuated colitis and lower intestinal inflammatory cytokine levels compared with ChATfl/fl mice. In contrast, in a resolution model of DSS-induced colitis (5 days of 2% DSS followed by 7 days without DSS), CD4ChAT-/- mice demonstrated a worsened colitis recovery and augmented colonic histological inflammation scores and inflammatory cytokine levels as compared with ChATfl/fl mice. In a transfer colitis model using CD4+CD45RBhigh T cells, T cells from CD4ChAT-/- mice induced a similar level of colitis compared with ChATfl/fl T cells. Together, our results indicate that ChAT+ T cells aggravate the acute innate immune response upon mucosal barrier disruption in an acute DSS-induced colitis model, whereas they are supporting the later resolution process of this innate immune-driven colitis. Surprisingly, ChAT expression in T cells seems redundant in the context of T cell-driven colitis.NEW & NOTEWORTHY By using different mouse models of experimental colitis, we provide evidence that in dextran sulfate sodium-induced colitis, ChAT+ T cells capable of producing acetylcholine worsen the acute immune response, whereas they support the later healing phase of this innate immune-driven colitis.

Enrichment of intestinal Lactobacillus by enhanced secretory IgA coating alters glucose homeostasis in P2rx7\u2212/\u2212 mice

The secretory immunoglobulin A (SIgA) in mammalian gut protects the organism from infections and contributes to host physiology by shaping microbiota composition. The mechanisms regulating the adaptive SIgA response towards gut microbes are poorly defined. Deletion of P2rx7, encoding for the ATP-gated ionotropic P2X7 receptor, leads to T follicular helper (Tfh) cells expansion in the Peyer’s patches (PPs) of the small intestine, enhanced germinal centre (GC) reaction and IgA secretion; the resulting alterations of the gut microbiota in turn affects host metabolism. Here, we define gut microbiota modifications that correlate with deregulated SIgA secretion and metabolic alterations in P2rx7−/− mice. In particular, Lactobacillus shows enhanced SIgA coating in P2rx7−/− with respect to wild-type (WT) mice. The abundance of SIgA-coated lactobacilli positively correlates with Tfh cells number and body weight, suggesting Lactobacillus-specific SIgA response conditions host metabolism. Accordingly, oral administration of intestinal Lactobacillus isolates from P2rx7−/− mice to WT animals results in altered glucose homeostasis and fat deposition. Thus, enhanced SIgA production by P2X7 insufficiency promotes Lactobacillus colonization that interferes with systemic metabolic homeostasis. These data indicate that P2X7 receptor-mediated regulation of commensals coating by SIgA is important in tuning the selection of bacterial taxa, which condition host metabolism.

Candida albicans Elicits Pro-Inflammatory Differential Gene Expression in Intestinal Peyer's Patches.

The details of how gut-associated lymphoid tissues such as Peyer's patches (PPs) in the small intestine play a role in immune surveillance, microbial differentiation and the mucosal barrier protection in response to fungal organisms such as Candida albicans are still unclear. We particularly focus on PPs as they are the immune sensors and inductive sites of the gut that influence inflammation and tolerance. We have previously demonstrated that CD11c+ phagocytes that include dendritic cells and macrophages are located in the sub-epithelial dome within PPs sample C. albicans. To gain insight on how specific cells within PPs sense and respond to the sampling of fungi, we gavaged naïve mice with C. albicans strains ATCC 18804 and SC5314 as well as Saccharomyces cerevisiae. We measured the differential gene expression of sorted CD45+ B220+ B-cells, CD3+ T-cells and CD11c+ DCs within the first 24h post-gavage using nanostring nCounter® technology. The results reveal that at 24h, PP phagocytes were the cell type that displayed differential gene expression. These phagocytes were able to sample C. albicans and discriminate between strains. In particular, strain ATCC 18804 upregulated fungal-specific pro-inflammatory genes pertaining to innate and adaptive immune responses. Interestingly, PP CD11c+ phagocytes also differentially expressed genes in response to C. albicans that were important in the protection of the mucosal barrier. These results highlight that the mucosal barrier not only responds to C. albicans, but also aids in the protection of the host.

ATP released by intestinal bacteria limits the generation of protective IgA against enteropathogens

T cell dependent secretory IgA (SIgA) generated in the Peyer’s patches (PPs) of the small intestine shapes a broadly diverse microbiota that is crucial for host physiology. The mutualistic co-evolution of host and microbes led to the relative tolerance of host’s immune system towards commensal microorganisms. The ATP-gated ionotropic P2X7 receptor limits T follicular helper (Tfh) cells expansion and germinal center (GC) reaction in the PPs. Here we show that transient depletion of intestinal ATP can dramatically improve high-affinity IgA response against both live and inactivated oral vaccines. Ectopic expression of Shigella flexneri periplasmic ATP-diphosphohydrolase (apyrase) abolishes ATP release by bacteria and improves the specific IgA response against live oral vaccines. Antibody responses primed in the absence of intestinal extracellular ATP (eATP) also provide superior protection from enteropathogenic infection. Thus, modulation of eATP in the small intestine can affect high-affinity IgA response against gut colonizing bacteria.

АНГИОАРХИТЕКТОНИКА ПЕЙЕРОВЫХ БЛЯШЕК ТОНКОЙ КИШКИ БЕЛЫХ КРЫС

Background. In the general bloodstream of the small intestine, it is common to distinguish superficially located main blood vessels that distribute blood throughout the intestinal tube, and intramural networks, represented mainly by microvascular communications of the muscular and mucous membranes. The aim. To study the features of blood supply to the groups of lymphoid nodules (Peyer's patches) of the small intestine of albino rats. Material and Methods. The study was performed on the basis of the injecting of a 5% solution of gelatin, colored with filtered black ink, into the bloodstream of abdominal organs of 20 white male rats. The material for the study was the specimens of the small intestine with Peyer's patches. The specimens were subjected to dehydration in alcohols with transition to pure acetone; after that they were enclosed in epoxy. Photographing of the obtained specimens was carried out using a digital camera, as well as in a binocular magnifier MBS-9, equipped with a digital photo attachment Sigeta DCM-900 9.0MP. Results. The trophism of an individual Peyer's patch of the small intestine is provided by several passing to it from different directions nutritive arteries, which are branches of the corresponding arterial vessels segmented around the perimeter of the intestinal tube. Conclusions. Based on the major principles of microangiology, there is reason to believe that perinodal arterial vessels are the sources of formation of radially oriented metabolic microcirculatory vessels, capillary links of which are located between the subunits of lymphoid nodules.

Oral Prion Neuroinvasion Occurs Independently of PrPC Expression in the Gut Epithelium.

The early replication of certain prion strains within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally acquired prions utilize specialized gut epithelial cells known as M cells to enter Peyer's patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer's patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer's patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in Peyer's patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility.IMPORTANCE The accumulation of orally acquired prions within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within Peyer's patches. Some gastrointestinal pathogens utilize molecules, such as the cellular prion protein PrPC, expressed on gut epithelial cells to enter Peyer's patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer's patches was dependent upon PrPC expression in the gut epithelium. We show that orally acquired prions can establish infection in Peyer's patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.

Особливості локалізації лімфоїдної тканини в імунних утвореннях стінки кишечнику, дивертикулі меккеля і сліпокишкових дивертикулах качок

Mucous associated lymphoid tissue has a special place in the immune system, forming the first protective barrier against antigens that enter the body with food and air. In birds 70% of lymphoid tissue that forms the parenchyma of peripheral immune organs localized in the mucosa of tubular digestive organs. Recently, in the literature there have been reports that lymphoid tissue in tubular digestive organs of waterfowl (geese, musk ducks) can not be localized only in the mucosa, but also in muscularis. In this context, the aim of the study was to determine the features of lymphoid tissue localization in immune formations (Peyer’s patches) of the intestine wall of ducks, Meckel diverticulum and apical diverticula in postnatal period ontogenesis. Established that lymphoid tissue in Peyer’s patches of duck intestine, Meckel’s diverticulum and apical diverticula located in the mucosa and muscularis of the wall. From the one-day age of duck, the lymphoid tissue is found in the mucosa of all investigated structures and in the muscularis of the apical diverticula, and in the muscularis of Peyer’s patches of the intestine and the Meckel’s diverticulum – from 10–20-days of age.The complete morphofunctional maturity of the lymphoid tissue of mucosa Peyer's patches of intestine is reached in the 15 days-age of duck, the lymphoid tissue of the mucosa of the Meckel diverticulum – in 20-day age, and in the apical diverticula – in 10-day age. The complete morphofunctional maturity of the lymphoid tissue of muscularis Peyer's patches of intestine is reached in the 15–25 days-age of duck, the lymphoid tissue of the muscularis of the Meckel diverticulum – in 25-day age, and in the apical diverticula – in 10-day age.Area of lymphoid tissue in the mucosa of duodenal and ileum Peyer’s patches increased to 210-day-old ducks and jejunum – to 150-day-old age. Lymphoid tissue occupies the largest area in 150-day ducks in the muscularis of these structures. Area of lymphoid tissue in the mucosa of cecal Peyer’s patches increased to 90-day-old ducks, and in the muscularis – to 240-day-old age.Lymphoid tissue in the mucosa of Meckel’s diverticulum takes up the largest area in 20-day-old ducks, in apical diverticula – in 180-day-old. In the muscularis of these structures, lymphoid tissue occupies the largest area in 150-day ducks.

Oral Immunization of FMDV Vaccine Using pH-Sensitive and Mucoadhesive Thiolated Cellulose Acetate Phthalate Microparticles.

Several barriers such as gastric pH, enzymatic degradation and rapid transit should be overcome to orally deliver antigens for taking up by epithelial microfold cells in Peyer's patches of small intestine. To solve the above mentioned problems, we designed pH-sensitive and mucoadhesive polymeric microparticles (MPs) prepared by double emulsion technique using cellulose acetate phthalate (CAP) to enhance immune response of foot-and-mouth disease (FMD) virus (FMDV) subunit vaccine. Thiolation of CAP improved mucoadhesive property of CAP to prolong the MPs transit time through the gastrointestinal tract. Thiolated CAP (T-CAP) also slowed down antigen release in acidic pH of stomach but released more antigens in neutral pH of small intestine due to the pH-sensitivity of the T-CAP. Oral immunization of a chimerical multi-epitope recombinant protein as the FMD subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer's patches eliciting mucosal IgA response. It will make a step forward into a promising oral subunit vaccine development in livestock industry.

Oral Prion Disease Pathogenesis Is Impeded in the Specific Absence of CXCR5-Expressing Dendritic Cells.

ABSTRACTAfter oral exposure, the early replication of certain prion strains upon stromal cell-derived follicular dendritic cells (FDC) in the Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. However, little is known of how prions are initially conveyed from the gut lumen to establish infection on FDC. Our previous data suggest that mononuclear phagocytes such as CD11c+ conventional dendritic cells play an important role in the initial propagation of prions from the gut lumen into Peyer's patches. However, whether these cells conveyed orally acquired prions toward FDC within Peyer's patches was not known. The chemokine CXCL13 is expressed by FDC and follicular stromal cells and modulates the homing of CXCR5-expressing cells toward the FDC-containing B cell follicles. Here, novel compound transgenic mice were created in which a CXCR5 deficiency was specifically restricted to CD11c+ cells. These mice were used to determine whether CXCR5-expressing conventional dendritic cells propagate prions toward FDC after oral exposure. Our data show that in the specific absence of CXCR5-expressing conventional dendritic cells the early accumulation of prions upon FDC in Peyer's patches and the spleen was impaired, and disease susceptibility significantly reduced. These data suggest that CXCR5-expressing conventional dendritic cells play an important role in the efficient propagation of orally administered prions toward FDC within Peyer's patches in order to establish host infection.IMPORTANCE Many natural prion diseases are acquired by oral consumption of contaminated food or pasture. Once the prions reach the brain they cause extensive neurodegeneration, which ultimately leads to death. In order for the prions to efficiently spread from the gut to the brain, they first replicate upon follicular dendritic cells within intestinal Peyer's patches. How the prions are first delivered to follicular dendritic cells to establish infection was unknown. Understanding this process is important since treatments which prevent prions from infecting follicular dendritic cells can block their spread to the brain. We created mice in which mobile conventional dendritic cells were unable to migrate toward follicular dendritic cells. In these mice the early accumulation of prions on follicular dendritic cells was impaired and oral prion disease susceptibility was reduced. This suggests that prions exploit conventional dendritic cells to facilitate their initial delivery toward follicular dendritic cells to establish host infection.

Intestinal micropatches for oral insulin delivery

Diabetes mellitus has become a major public health issue that has almost reached epidemic proportions worldwide. Injectable insulin has been typically utilized for the management of this chronic disease. However, lack of patient compliance with injectable formulations has spurred the development of oral insulin formulations, which although appealing, face several delivery challenges. We have developed novel mucoadhesive intestinal patches, several hundred micrometers in dimension (micropatches) that address the challenges of oral insulin delivery. The micropatches adhere to the intestinal mucosa, release their drug load rapidly within 30 min and are effective in lowering blood glucose levels in vivo. When insulin-loaded micropatches were administered with a permeation enhancer and protease inhibitor, a peak efficacy of 34% drop in blood glucose levels was observed within 3 h. Efficacy further improved to 41% when micropatches were administered in multiple doses. Here, we describe the design of micropatches as an oral insulin formulation and report their in vivo efficacy.

T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP

SummaryThe ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh) cell abundance in the Peyer’s patches (PPs) of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help.

Protective effects of lactic acid bacteria on influenza A virus infection

Gut microbiota is essential to regulate the whole body metabolism and the immune system of the host animals. Lactic acid bacteria have as a major characteristic to produce lactic acid from glucose. As probiotics, lactic acid bacteria provide many beneficial effects on human health by regulating the gut microbiota or the cell function. Recently, we have reported that oral administration of a probiotic strain,Lactobacillus gasseri SBT2055 (LG2055) induced IgA production by increasing the population of IgA+ B cell in Peyer’s patch and propria mucosae of small intestine in mice. Furthermore, we demonstrated that oral administration of LG2055 increased the survival rate of mice infected with influenza A virus and decreased the ratio of body weight losses. The virus titer was significantly decreased and the amount of inflammatory cytokine, IL-6 in the lung tissue was reduced by LG2055 administration. The expression of antiviral genes in the lung tissues was increased by LG2055. Therefore, LG2055 administration is shown to be effective for the protection against influenza A virus infection by inhibition of viral replication through the induction of antiviral genes expression. In the future, the constituents of LG2055 to have the effects on the induction of antiviral genes should be clarified for the development of novel influenza drugs.

Development and Function of Secondary and Tertiary Lymphoid Organs in the Small Intestine and the Colon.

The immune system of the gut has evolved a number of specific lymphoid structures that contribute to homeostasis in the face of microbial colonization and food-derived antigenic challenge. These lymphoid organs encompass Peyer’s patches (PP) in the small intestine and their colonic counterparts that develop in a programed fashion before birth. In addition, the gut harbors a network of lymphoid tissues that is commonly designated as solitary intestinal lymphoid tissues (SILT). In contrast to PP, SILT develop strictly after birth and consist of a dynamic continuum of structures ranging from small cryptopatches (CP) to large, mature isolated lymphoid follicles (ILF). Although the development of PP and SILT follow similar principles, such as an early clustering of lymphoid tissue inducer (LTi) cells and the requirement for lymphotoxin beta (LTβ) receptor-mediated signaling, the formation of CP and their further maturation into ILF is associated with additional intrinsic and environmental signals. Moreover, recent data also indicate that specific differences exist in the regulation of ILF formation between the small intestine and the colon. Importantly, intestinal inflammation in both mice and humans is associated with a strong expansion of the lymphoid network in the gut. Recent experiments in mice suggest that these structures, although they resemble large, mature ILF in appearance, may represent de novo-induced tertiary lymphoid organs (TLO). While, so far, it is not clear whether intestinal TLO contribute to the exacerbation of inflammatory pathology, it has been shown that ILF provide the critical microenvironment necessary for the induction of an effective host response upon infection with enteric bacterial pathogens. Regarding the importance of ILF for intestinal immunity, interfering with the development and maturation of these lymphoid tissues may offer novel means for manipulating the immune response during intestinal infection or inflammation.