Intestinal Patches Research Articles

Changes in the Tissue Barrier after Exposure to Lipopolysaccharide on the Apical Side of Enterocytes and the Follicle-Associated Epithelium in Peyer's Patches of the Rat Intestine.

To study the para- and transcellular permeability of columnar epithelium and follicle-associated epithelium of Peyer's patches in the rat intestine, LPS was applied from the mucosal side to simulate the action of endotoxins from gram-negative bacteria of gut microbiota. LPS did not affect transepithelial resistance or sodium fluorescein permeability, but increased the levels of claudin-3 and claudin-4 in enterocytes, suggesting strengthening of the paracellular intestinal barrier. Transcellular permeability was evaluated by electron microscopy based on the number of vesicular structures in the cytoplasm of different cell types. LPS increased the number of small vesicles in follicle-associated epithelium of Peyers' patches. In columnar epithelial cells, LPS reduced the number of smaller vesicles and increased the number of larger ones. LPS did not damage the tissue barrier, but enhanced transcytosis, which could potentiate the effects of endotoxin on its receptors in the intestinal mucosa.

Exercise improves intestinal IgA production by T-dependent cell pathway in adults but not in aged mice.

Hypermutated high-affinity immunoglobulin A (IgA), neutralizes toxins and drives the diversification of bacteria communities to maintain intestinal homeostasis although the mechanism underlies the impact of moderate aerobic exercise (MAE) on the IgA-generation via T-dependent (TD) is not fully know. Therefore, the aim of this study was to determine the effect of long-time MAE on the production of IgA through the TD pathway in Peyer´s patches of the small intestine from aged mice. MAE protocol consisted of twenty 3-month-old (young) BALB/c mice running in an endless band at 0° inclination and a speed of 10 m/h for 5 days a week and resting 2 days on the weekend until reaching 6-month-old (adulthood, n=10) or 24-month-old (aging, n=10). Groups of young, adult, or elderly mice were included as sedentary controls (n=10/per group). At 6 or 24 months old, all were sacrificed, and small intestine samples were dissected to prepare intestinal lavages for IgA quantitation by ELISA and to obtain suspensions from Peyer´s patches (PP) and lamina propria (LP) cells for analysis of T, B, and plasma cell subpopulations by flow cytometry and mRNA analysis expression by RT-qPCR of molecular factors related to differentiation of B cells to IgA+ plasma cells, class switch recombination, and IgA-synthesis. Statistical analysis was computed with two-way ANOVA (factor A=age, factor B=group) and p<0.05 was considered for statistically significant differences. Compared to age-matched sedentary control, in exercised elderly mice, parameters were either increased (IgA concentration, IL-21, IL-10 and RDH mRNA expression), decreased (α-chain mRNA, B cells, mIgA+ B cells, mIgM+ B cells and IL-4 mRNA) or unchanged (PP mIgA+ plasmablasts and LP cyt-IgA+ plasma cells). Regarding the exercised adult mice, they showed an up-modulation of IgA-concentration, mRNA expression IL-21, IL-10, and RDH and cells (PP B and T cells, mIgM+ plasmablasts and LP cyt-IgA+plasma cells). Our findings suggest that MAE restored the IgA production in adult mice via the TD cell pathway but does not in aged mice. Other studies are necessary to know in more detail the impact of long-time MAE on the TD pathway to produce IgA in aging.

Alterations of the gut microbial community structure modulates the Th17 cells response in a rat model of asphyxial cardiac arrest

Th17 cells triggered inflammation is a critical element in cerebral ischemic injury, and the gut microbiota intricately impacts T lymphocytes. Nevertheless, it remains unclear whether the gut microbiota involves in cardiac arrest/cardiopulmonary resuscitation (CA/CPR) induced-brain injury through Th17 cells. The present study investigated the interaction between gut microbiota and Th17 cells in a rat model. We observed that CA/CPR induced the alterations of the gut microbial community structure, and elevated the level of IL-17 in the serum, and a slight infiltration of Th17 cells into the brain. The Th17 cells were increased significantly in the peripheral blood, 28.33 ± 6.18% of these Th17 cells were derived from the Peyer’s patches of small intestine. Furthermore, fecal microbiota transplantation (FMT) from rats with CA/CPR induced Th17 cell response, promoting hippocampal cell apoptosis and declining learning ability and memory in recipient rats. Taken together, CA/CPR-induced alterations of the gut microbial community structure stimulated Th17 cell response which aggravated brain injury.

Pharmaconutraceutical Chondroguard®TRIO – chondroprotector with immunomodulatory activity

Understanding the major pathological pathways and the key molecules involved in the pathogenesis of inflammatory processes in joints, particularly in osteoarthritis (OA), is crucial for drug and pharmaconutraceuticals development. OA is a degenerative joint disease that predominantly affects articular cartilage. Destruction of hyaline cartilage and restructuring of subchondral bone are accompanied by synovial inflammation in the joint, including the facet joint of the spine, manifested by pain in the joint, low back pain (LBP), and limitation of functional activity. The article discusses the relationship between immune and inflammatory mechanisms in OA of any location, including the joints of the spine. One of the mechanisms for the formation of a “vicious circle of inflammation” during the activation of discoidin receptors by endogenous type II collagen is discussed, leading to the induction of the synthesis of pro-inflammatory mediators: tumor necrosis factor α(TNFα), metalloproteinases (MMPs) 1 and 13, interleukins (IL) 1 and 6. Inflammation, in turn, leads to a decrease in the synthesis and destruction of endogenous type II collagen and, subsequently, to cartilage destruction. Cartilage fragments entering the joint space of the intercellular matrix enhance the synthesis of TNFα, IL, and MMP and exacerbate the inflammatory process. Oral ingestion of exogenous undenatured type II collagen(NK-II) helps, first, to inactivate the binding of fragments of destroyed endogenous type II collagen to discoidin receptors and to break the "vicious circle of inflammation"; secondly, through the mechanism of oral/intestinal tolerance via the lymphoid system in Peyer's patches of the small intestine, leads to the activation of immune cells (T-lymphocytes) and initiation of the immune response – the synthesis of anti-inflammatory mediators (transforming growth factor β, IL4 and IL10). The new pharmaconutraceutical Chondroguard®TRIO, which contains chondroprotectors (chondroitin sulfate and glucosamine sulfate) as well as NK-II, will make it possible to influence the key sites of the pathological process in OA.

S-57-6: OBSTRUCTIVE SLEEP APNEA INDUCED GUT DYSBIOSIS STIMULATES A PROINFLAMMATORY RESPONSE THAT PROMOTES NEUROINFLAMMATION AND HYPERTENSION

Objective: Obstructive sleep apnea (OSA) is an independent risk factor for hypertension (HT). We have demonstrated that OSA induces gut dysbiosis, and a dysbiotic microbiota is causal in the development of HT. The mechanisms linking gut dysbiosis to blood pressure (BP) regulation are unclear. Recent studies demonstrate gut dysbiosis induces a pro-inflammatory host response resulting in peripheral- and neuro-inflammation, key factors in the development of HT. We hypothesized that OSA induced gut dysbiosis elicits a pro-inflammatory response that promotes neuroinflammation and HT. Methods: OSA was induced in 8-week-old male rats by repeatedly inflating a surgically implanted tracheal balloon. Sham rats underwent balloon implantation without inflations. After two weeks of apneas, lymphocytes were isolated from aorta, brain, cecum, ileum, mesenteric lymph node, and spleen for assessment by flow cytometry. In studies examining IL-17, a monoclonal antibody to neutralize IL-17 was injected every 48 hours during the 2 weeks of OSA. To track lymphocytes originating from the gut, cells in the Peyers patches of the small intestine were labelled by injection of carboxyfluorescein succinimidyl ester (CFSE) dye. Results: Two weeks of OSA significantly decreased anti-inflammatory regulatory T (Treg) cells and increased TH17 (IL-17+) in the brain, cecum, and ileum (n = 7, p &lt; 0.05). A significant increase in the Th17/Treg ratio was observed in brain, cecum and ileum of OSA, versus sham, rats (n = 7, p &lt; 0.05). To examine the role of TH17 cells, we injected an IL-17 neutralizing antibody or control IgG antibody during the 2 weeks of OSA. Compared to OSA rats receiving IgG control, neutralization of IL-17 significantly reduced BP of OSA rats (n = 6, p &lt; 0.05). Additionally, neutralization of IL-17 resulted in a significant increase of Tregs and decreased Th1 cells in brain, cecum and ileum of OSA rats (n = 6, p &lt; 0.01). To examine the distribution of lymphocytes originating from the gut, cells in the Peyers patches of sham and OSA rats were labelled with CFSE. We observed significant increases in CFSE+ Th1, Th2, and Th17 cells in the brain, MLN and spleen of OSA as compared to sham rats (n = 6, p &lt; 0.05). Conclusion: Overall, we found that OSA induced gut dysbiosis is associated with a pro-inflammatory response in the gut and brain that involves IL-17 signaling. Our findings suggest that gut dysbiosis may serve as the trigger for widespread inflammation, and treatment strategies to prevent or reverse gut dysbiosis may prove useful in reducing neuroinflammation and HT.

Abstract 095: Obstructive Sleep Apnea Induced Gut Dysbiosis Stimulates A Proinflammatory Response In Gut And Brain That Contribute To Hypertension

Objective: Obstructive sleep apnea (OSA) is an independent risk factor for the development of hypertension (HT). OSA induces gut dysbiosis, and a dysbiotic microbiota is causal in the development of HT. The mechanisms linking gut dysbiosis to blood pressure (BP) regulation remain unclear. Gut dysbiosis can induce a pro-inflammatory response of the host resulting in peripheral- and neuro-inflammation, key factors in the development of HT. We hypothesized that OSA induced gut dysbiosis elicits a pro-inflammatory response that promotes neuroinflammation and HT. Methods: OSA was induced in 8-week-old male rats (60 apneas/hr for 8 hrs during sleep) by inflating a tracheal balloon. Sham rats underwent balloon implantation without inflations. After 2 weeks of apneas, lymphocytes were isolated from brain, cecum, ileum, mesenteric lymph node (MLN), and spleen for flow cytometry. The role of IL-17 was tested by treating rats with a monoclonal antibody to IL-17 to neutralize circulating IL-17. To track the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches of the small intestine were labelled by injection of carboxyfluorescein succinimidyl ester (CFSE) dye. Results: Following 2 weeks of OSA we found a significant decrease in anti-inflammatory regulatory T (Treg) cells along with an increase in T H 17 (IL-17+) in the brain, cecum, and ileum (n=7, p&lt;0.05 for each). To examine the role of T H 17, we injected an IL-17 neutralizing antibody or control IgG during the 2 weeks of OSA. Compared to OSA rats receiving IgG control, neutralization of IL-17 significantly reduced BP of OSA rats (n=6, p&lt;0.05). IL-17 neutralization also resulted in a significantly increased Tregs and decreased Th1 cells in brain, cecum and ileum of OSA rats (n=6, p&lt;0.01 for each). To examine the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches were labelled with CFSE. We observed significant increases in CFSE+ Th1, Th2, and Th17 cells in the brain, MLN and spleen of OSA as compared to sham rats (n=6, p&lt;0.05). Conclusion: OSA induced gut dysbiosis is associated with a pro-inflammatory response in the gut and brain that involves IL-17 signaling. Treatment strategies to prevent gut dysbiosis may prove useful in reducing neuroinflammation and HT.

Obstructive Sleep Apnea Induced Gut Dysbiosis Stimulates a Proinflammatory Response that Promotes Neuroinflammation and Hypertension Sriram Ayyaswamy, Bojun Zhang, Huanan Shi, Sharon Phillips, Robert Bryan and David Durgan Baylor College of Medicine, Department of Anesthesiology, Houston, Texas, United States

Background and HypothesisObstructive sleep apnea (OSA) is an independent risk factor for the development of hypertension (HT). We have previously demonstrated that OSA induces gut dysbiosis, and a dysbiotic microbiota is causal in the development of HT. However, the mechanisms linking gut dysbiosis to blood pressure (BP) regulation are unclear. Recent studies have demonstrated that gut dysbiosis can induce a pro‐inflammatory response of the host resulting in peripheral‐ and neuro‐inflammation, key factors in the development of HT. We therefore hypothesized that OSA induced gut dysbiosis elicits a pro‐inflammatory response that promotes neuroinflammation and hypertension.MethodsOSA was induced in 8‐week‐old male rats (60 apneas/hour for 8 hours during the sleep phase) by repeatedly inflating a surgically implanted tracheal balloon. Sham rats underwent balloon implantation without inflations. After two weeks of apneas, lymphocytes were isolated from aorta, brain, cecum, ileum, mesenteric lymph node, and spleen for assessment by flow cytometry. In studies examining the role of IL‐17, a monoclonal antibody to IL‐17 was injected every 48 hours during the 2 weeks of apneas to neutralize circulating IL‐17. To track the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches of the small intestine were labelled by microinjection of carboxyfluorescein succinimidyl ester (CFSE) dye.Summary of resultsFollowing 2 weeks of OSA we found a significant decrease in anti‐inflammatory regulatory T (Treg) cells along with an increase in TH17 (IL‐17+) in the brain, cecum, and ileum and TH1 (IFN‐γ+) in cecum and ileum (n=7, p&lt;0.05 for each). A significant increase in the Th17/Treg ratio, a sign of inflammation associated with hypertension, was observed in brain, cecum and ileum of OSA, versus sham, rats (n=7, p&lt;0.05 for each). To examine the role of TH17 cells, we injected an IL‐17 neutralizing antibody or control IgG antibody during the 2 weeks of OSA. Compared to OSA rats receiving IgG control, neutralization of IL‐17 significantly reduced BP of OSA rats after 1 and 2 weeks (n=6, p&lt;0.05 for each). Additionally, neutralization of IL‐17 resulted in a significant increase of Tregs and decreased Th1 cells and TNFα expressing cells in brain, cecum and ileum of OSA rats (n=6, p&lt;0.01 for each). To examine the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches of sham and OSA rats were labelled with CFSE. We observed significant increases in CFSE+ Th1, Th2, and Th17 cells in the brain, MLN and spleen of OSA as compared to sham rats (n=6, p&lt;0.05).ConclusionOverall, we found that OSA induced gut dysbiosis is associated with a pro‐inflammatory response in the gut and brain that involves IL‐17 signaling. Our findings suggest that gut dysbiosis may serve as the trigger for widespread inflammation, and treatment strategies to prevent or reverse gut dysbiosis may prove useful in reducing neuroinflammation and HT.

Intestinal Immune System and Amplification of Mouse Mammary Tumor Virus.

Mouse mammary tumor virus (MMTV) is a virus that induces breast cancer in mice. During lactation, MMTV can transmit from mother to offspring through milk, and Peyer’s patches (PPs) in mouse intestine are the first and specific target organ. MMTV can be transported into PPs by microfold cells and then activate antigen-presenting cells (APCs) by directly binding with Toll-like receptors (TLRs) whereas infect them through mouse transferrin receptor 1 (mTfR1). After being endocytosed, MMTV is reversely transcribed and the cDNA inserts into the host genome. Superantigen (SAg) expressed by provirus is presented by APCs to cognate CD4+ T cells via MHCII molecules to induce SAg response, which leads to substantial proliferation and recruitment of related immune cells. Both APCs and T cells can be infected by MMTV and these extensively proliferated lymphocytes and recruited dendritic cells act as hotbeds for viral replication and amplification. In this case, intestinal lymphatic tissues can actually become the source of infection for the transmission of MMTV in vivo, which results in mammary gland infection by MMTV and eventually lead to the occurrence of breast cancer.

THE ISSUE OF HISTOLOGICAL IDENTIFICATION OF М-CELLS IN THE PEYER'S PATCHES OF ALBINO RAT SMALL INTESTINE.

The aim: Based on the above cytological signs of M-cells, we set the goal of more detailed clarification of some of their topological relationships with other enterocytes in the follicle-associated epithelium of Peyer's patches of albino rat small intestine. Materials and methods: 10 mature albino male rats weighted 200,0±20,0 g were involved into the study. Anatomical dissection with the sampling of the sections of the small intestine containing Peyer's patches was carried out with subsequent embedment of the latter into paraffin blocks and making of serial histological sections of 4 μm thick in the cross-section of the small intestine, followed with hematoxylin-eosin staining. The specimens were studied and documented on the "Konus" light microscope equipped. Morphometric characteristics of the specimen tissue structures were studied using the Sigeta X 1 mm/100 Div.x0.01mm stage micrometer. Results: The findings of the study revealed enterocytes with phagocytic properties found in the lymphoid-associated epithelium of Peyer's patches of the small intestine of albino rats. Moreover, if they are clearly visualized at the light-optical level, then M-cells are poorly recognizable, which is consistent with a similar assessment made by other authors. Conclusions: Given this, the issue on the topology and functional purpose of M-cells remains uncertain to date and, thereby, the prospect of further research is being outlined, which, in our opinion, can be successful using the method of stereomorphological analysis. For this purpose, multilayer plastic reconstruction methods can be used for serial semi-thin sections of Peyer's patches embedded in epoxy resin, according to the requirements of transmission electron microscopy.

Using crystallography tools to improve vaccine formulations.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

IL-4-Producing Vγ1+/Vδ6+ γδ T Cells Sustain Germinal Center Reactions in Peyer's Patches of Mice.

The mucosal immune system is the first line of defense against pathogens. Germinal centers (GCs) in the Peyer’s patches (PPs) of the small intestine are constantly generated through stimulation of the microbiota. In this study, we investigated the role of γδ T cells in the GC reactions in PPs. Most γδ T cells in PPs localized in the GCs and expressed a TCR composed of Vγ1 and Vδ6 chains. By using mice with partial and total γδ T cell deficiencies, we found that Vγ1+/Vδ6+ T cells can produce high amounts of IL-4, which drives the proliferation of GC B cells as well as the switch of GC B cells towards IgA. Therefore, we conclude that γδ T cells play a role in sustaining gut homeostasis and symbiosis via supporting the GC reactions in PPs.

Renal revascularization by a pedicled intestinal segment wrapping the kidney: a new method for kidney revascularization.

Renal artery stenosis is caused by a heterogeneous group of diseases, including atherosclerosis and fibromuscular dysplasia, which can be treated medically, via endovascular techniques, or by open revascularization; however, satisfactory and effective results are not always obtained. We aimed to assess the possibility of renal revascularization by a pedicled intestinal segment wrapping the kidney. Five dogs were operated on at three steps. At the first step, laparotomy was performed, and the right kidney was released. Subsequently,an 8-10cm segment of jejunum was separated longitudinally, and mucosectomy was done. This intestinal patch wrapped up the kidney. After eightweeks, the kidney and the intestinal patch were analyzed, and the renal artery was ligated. After fourweeks, the kidney and the intestinal patch were sent for pathological evaluation. At the 12th week of evaluation, no evidence of abscess formation or collection was seen. All kidneys had a normal color, consistency, and size. All renal cells were alive, and neither atrophy nor necrosis was seen. Glomerulus and tubules were intact, and no inflammatory change was visible. Furthermore, thick wall vasculature was inspected in a fibromuscular tissue, rising from the intestinal flap toward the kidney. One of the dogs expired due to peritonitis and sepsis in the fifth week. In our study, indirect perfusion of the kidney by an intestinal patch was achieved successfully. This represents new hope in patients suffering from chronic renal failure who underwent former medical and surgical interventions with undesirable results.

Physical properties of lactic acid bacteria influence the level of protection against influenza infection in mice.

We evaluated whether the water dispersibility of lactic acid bacteria (Enterococcus faecalis KH2) affects their efficacy. When cultured lactic acid bacteria are washed, heat-killed, and powdered, adhesion occurs between results in aggregation (non-treated lactic acid bacteria, n-LAB). However, dispersed lactic acid bacteria (d-LAB) with a lower number of aggregates can be prepared by treating them with a high-pressure homogenizer and adding an excipient during powdering. Mice were administered n-LAB or d-LAB Peyer's patches in the small intestine were observed. Following n-LAB administration, a high amount of aggregated bacteria drifting in the intestinal mucosa was observed; meanwhile, d-LAB reached the Peyer's patches and was absorbed into them. Evaluation in a mouse influenza virus infection model showed that d-LAB was more effective than n-LAB in the influenza yield of bronchoalveolar lavage fluids on day 3 post-infection and neutralizing antibody titers of sera and influenza virus-specific immunoglobulin A in the feces on day 14 post-infection. Therefore, the physical properties of lactic acid bacteria affect their efficacy; controlling their water dispersibility can improve their effectiveness.

Identification, isolation and analysis of human gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) comprise key intestinal immune inductive sites, including the Peyer's patches of the small intestine and different types of isolated lymphoid follicle (ILF) found along the length of the gut. Our understanding of human GALT is limited due to a lack of protocols for their isolation. Here we describe a technique that, uniquely among intestinal cell isolation protocols, allows identification and isolation of all human GALT, as well as GALT-free intestinal lamina propria (LP). The technique involves the mechanical separation of intestinal mucosa from the submucosa, allowing the identification and isolation of submucosal ILF (SM-ILF), LP-embedded mucosal ILF (M-ILF) and LP free of contaminating lymphoid tissue. Individual SM-ILF, M-ILF and Peyer's patch follicles can be subsequently digested for downstream cellular and molecular characterization. The technique, which takes 4-10 h, will be useful for researchers interested in intestinal immune development and function in health and disease.

Dietary Indole-3-Carbinol Activates AhR in the Gut, Alters Th17-Microbe Interactions, and Exacerbates Insulitis in NOD Mice.

The diet represents one environmental risk factor controlling the progression of type 1 diabetes (T1D) in genetically susceptible individuals. Consequently, understanding which specific nutritional components promote or prevent the development of disease could be used to make dietary recommendations in prediabetic individuals. In the current study, we hypothesized that the immunoregulatory phytochemcial, indole-3-carbinol (I3C) which is found in cruciferous vegetables, will regulate the progression of T1D in nonobese diabetic (NOD) mice. During digestion, I3C is metabolized into ligands for the aryl hydrocarbon receptor (AhR), a transcription factor that when systemically activated prevents T1D. In NOD mice, an I3C-supplemented diet led to strong AhR activation in the small intestine but minimal systemic AhR activity. In the absence of this systemic response, the dietary intervention led to exacerbated insulitis. Consistent with the compartmentalization of AhR activation, dietary I3C did not alter T helper cell differentiation in the spleen or pancreatic draining lymph nodes. Instead, dietary I3C increased the percentage of CD4+RORγt+Foxp3- (Th17 cells) in the lamina propria, intraepithelial layer, and Peyer’s patches of the small intestine. The immune modulation in the gut was accompanied by alterations to the intestinal microbiome, with changes in bacterial communities observed within one week of I3C supplementation. A transkingdom network was generated to predict host-microbe interactions that were influenced by dietary I3C. Within the phylum Firmicutes, several genera (Intestinimonas, Ruminiclostridium 9, and unclassified Lachnospiraceae) were negatively regulated by I3C. Using AhR knockout mice, we validated that Intestinimonas is negatively regulated by AhR. I3C-mediated microbial dysbiosis was linked to increases in CD25high Th17 cells. Collectively, these data demonstrate that site of AhR activation and subsequent interactions with the host microbiome are important considerations in developing AhR-targeted interventions for T1D.

Berberine augments hypertrophy of colonic patches in mice with intraperitoneal bacterial infection

Colonic patches, the counterparts of Peyer’s patches in the small intestine, are dynamically regulated lymphoid tissues in the colon that have an important role in defensing against microbial infections. Berberine is an isoquinoline alkaloid extracted from medicinal herbs including Rhizoma coptidis and has long been used for the treatment of infectious gastroenteritis, but its impact on the colonic lymphoid tissues (such as colonic patches) is unknown. In this study, we aimed to investigate whether berberine had any influences on the colonic patches in mice with bacterial infection. The results showed that oral berberine administration in bacterial infected mice substantially enhanced the hypertrophy of colonic patches, which usually possessed the features of two large B-cell follicles with a separate T-cell area. Moreover, the colonic patches displayed follicular dendritic cell networks within the B-cell follicles, indicative of mature colonic patches containing germinal centers. Concomitant with enlarged colonic patches, the cultured colon of infected mice treated with berberine secreted significantly higher levels of interleukin-1β (IL-1β), IL-6, TNF-α, and CCL-2, while NLRP3 inhibitor MMC950 or knockout of NLRP3 gene abrogated berberine-induced hypertrophy of colonic patches, suggesting the involvement of the NLRP3 signaling pathway in this process. Functionally, oral administration of berberine ameliorated liver inflammation and improved formed feces in the colon. Altogether, these results indicated that berberine was able to augment the hypertrophy of colonic patches in mice with bacterial infection probably through enhancing local inflammatory responses in the colon.

Coptis chinensis Franch polysaccharides provide a dynamically regulation on intestinal microenvironment, based on the intestinal flora and mucosal immunity

Ethnopharmacological relevanceCoptis chinensis Franch is one of the most widely used traditional Chinese herbs in China and was firstly recorded in "Shennong's Classic of Materia Medica” in the Han Dynasty. The medical records in past thousands years have fully confirmed the clinical efficacies of Coptis chinensis Franch against intestinal diseases. The polysaccharides in herbal medicines can be digested by the flora and uptaken by the Peyer's patches (PPs) in intestine. It can be reasonably presumed that the polysaccharides in Coptis chinensis Franch (CCP) should be one of the critical element in the regulation of intestinal microenvironment. Aim of the studyThis study intended to explore the dynamic regulation of CCP on intestinal microenvironment from the perspective of the intestinal mucosal immunity and the intestinal flora, in order to provide a new research perspective for the pharmacological mechanism of Coptis chinensis Franch. Materials and methodsThe absorption and distribution of CCP in intestinal tissues were observed after the perfusion of FITC labeled CCP. The influences of CCP on intestinal flora were evaluated by the 16sRNA gene illumina-miseq sequencing after gavage. The regulations of CCP on intestinal mucosal immunity were evaluated by the immunohistochemical analysis of the interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) secretion in PPs and intestinal epithelial tissue. ResultsWith the self-aggregation into particles morphology, CCP can be up-taken by PPs and promote the IFN-γ, IL-4, IL-17 and TGF-β secretion in PPs in a dose-dependent manner. The CCP can also be utilized by the intestinal flora and dynamically regulate the diversity, composition and distribution of the intestinal flora. The temporal regulations of CCP on IFN-γ, IL-4, IL-17 and TGF-β secretions in intestinal epithelial tissues are consistent with the variation tendency of intestinal flora. ConclusionCCP can provide effective, dynamical and dose-dependent regulations on intestinal microenvironment, not only the intestinal flora but also the PPs and intestinal epithelium related immune response. These may be involved in the multiple biological activities of Coptis chinensis Franch.

GENERAL HISTOLOGICAL CHARACTERISTICS OF LYMPHOID NODULES OF PEYER’S PATCHES OF THE SMALL INTESTINE IN ALBINO RATS AFTER ADMINISTRATION OF A BROAD-SPECTRUM ANTIBIOTIC

Administration of an antibiotic by any route can cause dysbacteriosis, but its risk is the highest when taken orally, since the drug gets directly into the intestine, affecting microflora. After administration of a broad-spectrum antibiotic, Peyer’s patches of the small intestine of albino rats remained unchanged both topographically and in their total amount. But at the same time, their total area is more than doubled, which, according to our data, becomes possible due to the appearance of a new generation of lymphoid nodules in them. The aim of the research was to study the histological characteristics of lymphoid nodules of Peyer’s patches of the small intestine in albino rats after administration of clarithromycin. 30 mature albino male rats weighing 200.0±20.0 g were involved in the experiment. The antibiotic was administered to the rodents as a supplement to food during their two-meals-a-day feeding. Areas of the small intestine with Peyer’s patches have been studied. Serial paraffin sections have been analyzed using the “Konus” light microscope. Morphometric characteristics of the tissue structures were obtained using the Sigeta X 1 mm / 100 Div.x0.01mm stage micrometer. It has been established that after the course of administration of a broad-spectrum antibiotic (clarithromycin) in the small intestine of albino rats, the topography and total number of Peyer’s patches remain unchanged, while their total area increased by more than twice. This hyperplasia of structured lymphoid tissue in the mucous membrane of the small intestine of animals under the influence of the antibacterial drug occurs due to the appearance of new generations of lymphoid nodules in the Peyer’s patches, among which small, medium and large forms are distinguished, similar to the stages of their development. Each of them is distinguished by its morphological features, primarily, the conversion of the lymphoid-associated epithelium. Therefore, the genetically programmed total number of Peyer’s patches in the small intestine of mature albino rats is constant, whereas the number of lymphoid nodules in them is a variable that depends on the state of the intestinal microbiocenosis.

Special Issue No. – 10, June, 2020 Journal &gt; Special Issue &gt; Special Issue No. – 10, June, 2020 &gt; Page 5 “Quantative Methods in Modern Science” organized by Academic Paper Ltd, Russia MORPHOLOGICAL AND ANATOMICAL FEATURES OF THE GENUS GAGEA SALISB., GROWING IN THE EAST KAZAKHSTAN REGION Authors: Zhamal T. Igissinova,Almash A. Kitapbayeva,Anargul S. Sharipkhanova,Alexander L. Vorobyev,Svetlana F. Kolosova,Zhanat

Special Issue No. – 10, June, 2020 Journal &gt; Special Issue &gt; Special Issue No. – 10, June, 2020 &gt; Page 5 “Quantative Methods in Modern Science” organized by Academic Paper Ltd, Russia MORPHOLOGICAL AND ANATOMICAL FEATURES OF THE GENUS GAGEA SALISB., GROWING IN THE EAST KAZAKHSTAN REGION Authors: Zhamal T. Igissinova,Almash A. Kitapbayeva,Anargul S. Sharipkhanova,Alexander L. Vorobyev,Svetlana F. Kolosova,Zhanat

High-resolution imaging of living gut mucosa: lymphocyte clusters beneath intestinal M cells are highly dynamic structures

In the Peyer's patches of the small intestine, specialized epithelial cells, the membranous (M) cells, sample antigenic matter from the gut lumen and bring it into contact with cells of the immune system, which are then capable of initiating specific immune reactions. Using autofluorescence 2-photon (A2P) microscopy, we imaged living intestinal mucosa at a 0.5-μm resolution. We identified individual M cells without the aid of a marker and in vivo analyzed their sampling function over hours. Time-lapse recordings revealed that lymphocytes associated with M cells display a remarkable degree of motility with average speed rates of 8.2μm/min, to form new M cell-associated lymphocyte clusters within less than 15min. The lymphocytes drastically deform the M cells' cytoplasm and laterally move from one lymphocyte cluster to the next. This implies that the micro-compartment beneath M cells is a highly efficient container to bring potentially harmful antigens into contact with large numbers of immunocompetent cells. Our setup opens a new window for high-resolution 3D imaging of functional processes occurring in lymphoid and mucosal tissues.