Follicle-associated Epithelium Research Articles

Discrimination of distinct chicken M cell subsets based on CSF1R expression.

In mammals, a subset of follicle-associated epithelial (FAE) cells, known as M cells, conduct the transcytosis of antigens across the epithelium into the underlying lymphoid tissues. We previously revealed that M cells in the FAE of the chicken lung, bursa of Fabricius (bursa), and caecum based on the expression of CSF1R. Here, we applied RNA-seq analysis on highly enriched CSF1R-expressing bursal M cells to investigate their transcriptome and identify novel chicken M cell-associated genes. Our data show that, like mammalian M cells, those in the FAE of the chicken bursa also express SOX8, MARCKSL1, TNFAIP2 and PRNP. Immunohistochemical analysis also confirmed the expression of SOX8 in CSF1R-expressing cells in the lung, bursa, and caecum. However, we found that many other mammalian M cell-associated genes such as SPIB and GP2 were not expressed by chicken M cells or represented in the chicken genome. Instead, we show bursal M cells express high levels of related genes such as SPI1. Whereas our data show that bursal M cells expressed CSF1R-highly, the M cells in the small intestine lacked CSF1R and both expressed SOX8. This study offers insights into the transcriptome of chicken M cells, revealing the expression of CSF1R in M cells is tissue-specific.

Quercetin Improves Barrier Properties in Porcine Small Intestine but Not in Peyer's Patches.

Peyer's patches (PPs) are part of the gut-associated lymphatic tissue (GALT) and represent the first line of the intestinal immunological defense. They consist of follicles with lymphocytes and an overlying subepithelial dome with dendritic cells and macrophages, and they are covered by the follicle-associated epithelium (FAE). A sealed paracellular pathway in the FAE is crucial for the controlled uptake of luminal antigens. Quercetin is the most abundant plant flavonoid and has a barrier-strengthening effect on tight junctions (TJs), a protein complex that regulates the paracellular pathway. In this study, we aimed to analyze the effect of quercetin on porcine PPs and the surrounding villus epithelium (VE). We incubated both tissue types for 4 h in Ussing chambers, recorded the transepithelial electrical resistance (TEER), and measured the unidirectional tracer flux of [3H]-mannitol. Subsequently, we analyzed the expression, protein amount, and localization of three TJ proteins, claudin 1, claudin 2, and claudin 4. In the PPs, we could not detect an effect of quercetin after 4 h, neither on TEER nor on the [3H]-mannitol flux. In the VE, quercetin led to a higher TEER value, while the [3H]-mannitol flux was unchanged. The pore-forming claudin 2 was decreased while the barrier-forming claudin 4 was increased and the expression was upregulated. Claudin 1 was unchanged and all claudins could be located in the paracellular membrane by immunofluorescence microscopy. Our study shows the barrier-strengthening effect of quercetin in porcine VE by claudin 4 upregulation and a claudin 2 decrease. Moreover, it underlines the different barrier properties of PPs compared to the VE.

RGD-modified solid lipid nanoparticles improve oral doxorubicin absorption: In vitro and in vivo study

ObjectiveDoxorubicin (DOX), an effective antitumor drug, is widely used in cancer treatment. However, DOX has low oral bioavailability. Owing to its ability to specifically bind the β1 receptor on the surface of M cells, promote the phagocytosis of drugs by M cells, and improve the oral efficacy of drugs, arginine-glycine-aspartic peptide (RGD) is used to modify the oral nanodrug delivery system. MethodsAn emulsion solvent evaporation method was used to prepare DOX-loaded solid lipid nanoparticles (DOX-SLNs), which were further modified with RGD (RGD-DOX-SLNs) by noncovalent interactions. The physicochemical properties, dissolution rate, and cytotoxicity of the SLNs were investigated. An in vitro transparency experiment was performed using the follicle-associated epithelium (FAE) model. In addition, oral bioavailability and gastrointestinal (GI) mucosal irritation were evaluated in rats. ResultsThe cumulative release percentage of DOX at pH 1.2 decreased significantly when DOX was incorporated into the SLNs, which may be dependent on drug diffusion and matrix erosion mechanisms. The cell toxicity was low in Caco-2 cells. No GI mucosal irritation was found in rats. The cellular uptake of RGD-DOX-SLNs in the FAE model was higher than in Caco-2 model. Compared with the DOX group, the area under the plasma concentration-time curve (AUC) of DOX in the RGD-DOX-SLNs group increased 2.25-fold after oral administration to rats. ConclusionsRGD-DOX-SLNs improved DOX transport in the FAE model and enhanced the oral bioavailability of DOX in rats.

Comparison of the Development and Involution Periods of Bursa of Fabricius with Histological and Histochemical Methods

The aim of this study is to histologically and histochemically determine the developmental and involutional stages of bursa of Fabricius of henna partridge (Alectoris chukar). In the study, bursa of Fabricius of 12 3-month-old (6 males, 6 females), 12 6-month-old (6 males, 6 females) henna partridges purchased from a private farm were used. It was observed that bursa of Fabricius was surrounded by a connective tissue capsule and consisted of tunica serosa, tunica muscularis, and tunica mucosa layers from the outside to the inside. It was seen that the tunica muscularis consisted of outer longitudinal and inner circular smooth muscle fibers. It was observed that the tunica mucosa made plicae towards the lumen of the organ and consisted of 10-15 plicae. It was seen that lamina epithelialis and lymph follicles were present in each plica. It was determined that the lamina epithelialis consisted of two parts called FAE (Follicle-Associated Epithelium) and IFE (İnter Follicular Epithelium). It was noted that the lymph follicles contained cortex and medulla sections and were separated locally by capillaries together with cortical medullary boundry cells. In the Methyl Green-pyronin staining method, plasma cells were found in the bursa of Fabricius of the henna partridge, in the connective tissue surrounding the organ, around the blood vessels and inside the follicles. In AB pH=2.5 staining, AB-positive reaction was seen only in the apical part of the epithelial cells forming FAE and IFE in the pre- and post-involution period. In PAS staining, PAS-positive reaction was observed only in the apical part of the epithelial cells forming FAE and IFE in the pre- and post-involution period. In PAS/AB pH=2.5 combined staining method, AB-positive reaction was observed only in the apical part of epithelial cells in the pre- and post-involution period. As a result of this study, it was found that bursa of Fabricius of the henna partridge did not undergo any histochemical changes despite its histologically large differences after involution.

Diurnal changes in bacterial settlement on the Peyer's patch and surrounding mucosa in the rat ileum and its effect against the intestinal immune system.

Our previous study revealed the diurnal change in the indigenous bacteria settling on the terminal region of the rat ileum. In the present study, we investigated the diurnal change in indigenous bacteria on the most distal ileal Peyer's patch (PP) and surrounding ileal mucosa and explored how stimulation from indigenous bacteria for a day affects the intestinal immune system at the beginning of the light phase. Histological measurement revealed that bacteria adjacent to the follicle-associated epithelium of PP and to the villous epithelium of the surrounding ileal mucosa are more abundant at zeitgeber time (ZT)0 and ZT18 than at ZT12. On the other hand, tissue-section 16S rRNA amplicon sequencing revealed no significant difference between ZT0 and ZT12 in the bacterial composition on the ileal tissue including the PP. One-day treatment with an antibiotic (Abx) successfully impaired the settlement of bacteria around the ileal PP. In transcriptome analysis, 1-day Abx treatment led to the downregulation of several chemokines in both PP and ordinary ileal mucosa at ZT0. Histological analysis of the 1-day Abx group revealed decreases in both CD68+ macrophages in PP and naphthol AS-D chloroacetate esterase stain-positive mast cells in the ileal villi. Together, these findings suggest that the colonies of indigenous bacteria on the distal ileal PP and surrounding mucosa expand during the dark phase, which might lead to the expression of genes to regulate the intestinal immune system and contribute to the homeostasis of at least macrophages in PP and mast cells in the ileal mucosa.

M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer’s patch

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ Tcell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

In vivo tracing of immunostimulatory raw starch microparticles after mucosal administration

Raw starch microparticles (SMPs) proved efficient antigen carriers with adjuvant properties when administered via the mucosal route; however, the underlying mechanisms associated with this bioactivity are unknown. In the present study, we explored the mucoadhesion properties, fate, and toxicity of starch microparticles after mucosal administration. Nasally administered microparticles were mainly retained in nasal turbinates, reaching the nasal-associated lymphoid tissue; this step is facilitated by the ability of the microparticles to penetrate through the mucous epithelium. Likewise, we found intraduodenally administered SMPs on the small intestinal villi, follicle-associated epithelium, and Peyer's patches. Furthermore, under simulated gastric and intestinal pH conditions, we detected mucoadhesion between the SMPs and mucins, regardless of microparticle swelling. SMPs' mucoadhesion and translocation to mucosal immune responses induction sites explain the previously reported role of these microparticles as vaccine adjuvants and immunostimulants.

Intestinal Peyer's Patches: Structure, Function, and In Vitro Modeling.

Considering the important role of the Peyer's patches (PPs) in gut immune balance, understanding of the detailed mechanisms that control and regulate the antigens in PPs can facilitate the development of immune therapeutic strategies against the gut inflammatory diseases. In this review, we summarize the unique structure and function of intestinal PPs and current technologies to establish in vitro intestinal PP system focusing on M cell within the follicle-associated epithelium and IgA+ B cell models for studying mucosal immune networks. Furthermore, multidisciplinary approaches to establish more physiologically relevant PP model were proposed. PPs are surrounded by follicle-associated epithelium containing microfold (M) cells, which serve as special gateways for luminal antigen transport across the gut epithelium. The transported antigens are processed by immune cells within PPs and then, antigen-specific mucosal immune response or mucosal tolerance is initiated, depending on the response of underlying mucosal immune cells. So far, there is no high fidelity (patho)physiological model of PPs; however, there have been several efforts to recapitulate the key steps of mucosal immunity in PPs such as antigen transport through M cells and mucosal IgA responses. Current in vitro PP models are not sufficient to recapitulate how mucosal immune system works in PPs. Advanced three-dimensional cell culture technologies would enable to recapitulate the function of PPs, and bridge the gap between animal models and human.

Cellular Uptake and Transport Mechanism of 6-Mercaptopurine Nanomedicines for Enhanced Oral Bioavailability.

Nanomedicines have significant advantages in enhancing the oral bioavailability of drugs, but a deeper understanding of the underlying mechanisms remains to be interpreted. Hence, the present study aims to explain the uptake and trafficking mechanism for 6-MP nanomedicines we previously constructed. 6-MP loaded poly(lactide-co-glycolide) (PLGA) nanomedicines (6-MPNs) were prepared by the multiple emulsion method. The transcytosis mechanism of 6-MPNs was investigated in Caco-2 cells, Caco-2 monolayers, follicle associated epithelium (FAE) monolayers and rats, including transmembrane pathway, intracellular trafficking, paracellular transport and the involvement of transporter. Pharmacokinetics in rats showed that the area under the curve (AUC) of 6-MP in the 6-MPNs group (147.3 ± 42.89 μg/L·h) was significantly higher than that in the 6-MP suspensions (6-MPCs) group (70.31 ± 18.24 μg/L·h). The uptake of 6-MPNs in Caco-2 cells was time-, concentration- and energy-dependent. The endocytosis of intact 6-MPNs was mediated mainly through caveolae/lipid raft, caveolin and micropinocytosis. The intracellular trafficking of 6-MPNs was affected by endoplasmic reticulum (ER)-Golgi complexes, late endosome-lysosome and microtubules. The multidrug resistance associated protein 4 (MRP4) transporter-mediated transport of free 6-MP played a vital role on the transmembrane of 6-MPNs. The trafficking of 6-MPNs from the apical (AP) side to the basolateral (BL) side in Caco-2 monolayers was obviously improved. Besides, 6-MPNs affected the distribution and expression of zona occludens-1 (ZO-1). The transport of 6-MPNs in FAE monolayers was concentration- and energy-dependent, while reaching saturation over time. 6-MPNs improved the absorption of the intestinal Peyer's patches (PPs) in rats. 6-MPNs improve the oral bioavailability through multiple pathways, including active transport, paracellular transport, lymphatic delivery and MRP4 transporter. The findings of current study may shed light on the cellular uptake and transcellular trafficking mechanism of oral nanomedicines.

Morphological and Phenotypic Characteristics of the Bovine Nasopharyngeal Mucosa and Associated Lymphoid Tissue

The mammalian nasopharynx is an anatomically complex region of the upper respiratory tract that directly communicates with the nasal cavity, laryngopharynx, oesophagus and trachea. The nasopharyngeal mucosa contains moderate quantities of mucosa-associated lymphoid tissue (MALT) that is appropriately located for immunological sampling but also creates vulnerability to pathogens. In recent years, the nasopharynx has been inculpated in the pathogenesis of important diseases of cattle (foot-and-mouth disease) and humans (COVID-19), yet the tissue has never been described in detail in any species. In order to characterize the morphology and cellular composition of the bovine nasopharynx, samples of mucosa were collected from the nasopharynx of five 8-13-month-old steers and examined using light microscopy, immunohistochemistry and multichannel immunofluorescence. Morphologically, the nasopharyngeal epithelium was highly heterogeneous, with a continuum ranging from stratified squamous epithelium to highly attenuated, follicle-associated epithelium (FAE). Distribution of MALT was similarly regionally variable ranging from absent to clusters of multiple lymphoid follicles. Phenotypic characterization demonstrated dense distributions of dendritic cells and T lymphocytes surrounding lymphoid follicles, which comprised mostly B lymphocytes. The FAE overlaying the lymphoid follicles also contained higher numbers of dendritic cells and lymphocytes compared with the adjacent non-lymphoid epithelium, although cytotoxic T cells were notably scarce in the FAE. The bovine nasopharyngeal lymphoid tissue had comparable elements to other MALTs with specific differences that may help to elucidate the pathogenesis of infectious agents that have specific tropism for this tissue.

M cells of mouse and human Peyer's patches mediate the lymphatic absorption of an Astragalus hyperbranched heteroglycan

The gut cell wall is considered an impenetrable barrier to orally administrated polysaccharides. We recently reported a selective lymphatic route for Radix Astragali polysaccharide RAP to enter Peyer's patches (PPs) to trigger immune responses. However, how RAP enters PPs is unclear. Herein, we screened the intestinal epithelial cells of mice and found that the follicle-associated epithelium cells were specifically bound with FITC-RAP. Further studies in vitro and in vivo revealed that RAP was efficiently transported by microfold (M) cells. We also confirmed that M cell-transported RAP directly contacted dendritic cells. More importantly, for the first time, we verified this interesting M cell-mediated transcytosis of RAP in the human distal ileum. Mechanistically, we identified M cells to be the transporter cells that independently deliver RAP into the lymphatic system to trigger immune responses. This interesting transcytosis mechanism might apply to many other immunomodulatory polysaccharides orally dosed to human body.

Barrier Perturbation in Porcine Peyer's Patches by Tumor Necrosis Factor is Associated With a Dysregulation of Claudins.

The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer’s patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer’s patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer’s patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer’s patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer’s patches, in agreement with the major role of this receptor in Peyer’s patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer’s patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer’s patches as a primary mucosal immune defense.

Intestinal Absorption Study of a Granular Form of Ferric Pyrophosphate.

Iron deficiency is one of the most prevalent nutritional disorders worldwide. The standard treatment involves iron supplementation, but this task is challenging because of poor solubility and organoleptic issues. Moreover, the need to increase iron bioavailability represents a challenge for treating iron-related disorders. In this study, gastroresistance and iron intestinal absorption of an innovative granular formulation composed of ferric pyrophosphate, modified starch and phospholipids branded as Ferro Fosfosoma® was investigated. Gastroresistant properties were studied using standard protocols, and a bioaccessible fraction was obtained by exposing a food supplement to in vitro digestion. This fraction was used for investigating iron absorption in Caco-2 and human follicle-associated intestinal epithelium (FAE) models. Ferro Fosfosoma® showed an improved resistance to gastric digestion and higher intestinal absorption than ferric pyrophosphate salt used as a control in both models. In the FAE model, Ferro Fosfosoma® induces larger iron absorption than in the Caco-2 monolayer, most likely due to the transcytosis ability of M cells. The larger iron absorption in the Ferro Fosfosoma®-treated FAE model corresponds to higher ferritin level, proving physiological iron handling that was once delivered by granular formulation. Finally, the formulation did not induce any alterations in viability and barrier integrity. To conclude, Ferro Fosfosoma® favors iron absorption and ferritin expression, while preserving any adverse effects.

Coronin 1A is Uniquely Expressed in the Human Follicle Associated Epithelium and is Required For Human M Cell Maturation and Function

IntroductionMicrofold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut luminal environment. They functionally act as gatekeepers within the follicle‐associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut‐associated lymphoid tissue. Studies have associated damage in the FAE with chronic inflammatory conditions, such as IBD; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis under pathogenic conditions remain incomplete. OBJECTIVE: Since M cells are differentiated from Lgr5+ intestinal stem cells, we proposed to utilize human enteroids to determine the factors that regulate human M cell differentiation and function. Quantitative proteomics analysis of human enteroids differentiated to express M cells revealed enriched expression of coronin 1A, an actin‐remodeling protein involved in phagocytosis and expressed immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that coronin 1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.MethodsHuman ileal enteroids derived from biopsies obtained from healthy donors were grown as monolayers and differentiated (no Wnt3a, R‐spondin‐1) for at least 5 days. To express M cells, cultures were exposed to RANKL and TNF during differentiation. Human expression of coronin 1A was validated by immunoblot and confocal microscopy of ileal biopsies obtained from healthy donors. Stable knockdown (KD) of coronin 1A in human enteroids was achieved by shRNA lentivirus and puromycin selection. M cell monolayers, expressing or lacking coronin 1A, were exposed apically to commensal (HS) and pathogenic strains of E. coli (EPEC, AIEC) to determine the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent polystyrene latex beads and immunoglobulins.ResultsCoronin 1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similar results were obtained in biopsy sections of the human ileum with coronin 1A detected in the FAE, but not in crypt or villus epithelia. This staining pattern was unique for human, as coronin 1A expression in the mouse ileum was confined to lamina propria and lymphoid follicle immune cells. Coronin 1A KD resulted in decreased expression of glycoprotein 2, a marker of mature M cells, while other markers of the FAE and M cells, including CD14, ICAM1, Spi‐B, SOX8, and FcRN remained unchanged. Coronin 1A KD also resulted in impaired uptake/transcytosis of IgG (via FcRN) as well as pathogen‐stimulated uptake/transcytosis of polystyrene beads by human M cells.ConclusionsThe studies show: (1) human enteroids can be differentiated to model coronin 1A expression in the intact human FAE; (2) coronin 1A expression is necessary for terminal differentiation and function of human M cells; and (3) coronin 1A is required for pathogen‐stimulated uptake and transcytosis of various types of luminal gut cargo. We conclude that M cell‐expressing enteroid monolayers can be used as a relevant human model to further interrogate tropism for host‐pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.

Histoarchitecture of Large Intestine and Lympho Glandular Complex of Pig

Background: To have a better understanding of the lympho glandular complexes (LGC) along with the histoarchitecture of large intestine and associated M cells, the proposed study have been undertaken. Methods: Present study was conducted on 12 (twelve) numbers of apparently healthy weaned crossbred piglets of 3 to 4 months irrespective of sex. After humane sacrifice of the animals with the approval of institutional animal ethics committee, the representative tissue samples were collected preserved, processed and stained as per standard methods of Luna (1968). Result: Columnar cells predominated the follicle associated epithelium (FAE) and the crypts. M cells were recorded in the follicle associated epithelium as well as in lower crypt epithelium in toluidine blue stained ultra thin sections. M cells were observed with intraepithelial lymphocyte and basally placed euchromatic nucleus. In the colon and rectum of crossbred pigs the lympho glandular complex (LGC) typically consisted of a pore like depression on surface epithelium from which extensions of mucosal glands passed through the circular gap into lamina muscularis mucosae. The lymphatic aggregation of LGC in the lamina propria and tunica submucosa showed three distinct zone viz. follicle, interfollicular area and dome area and distinct FAE could be appreciated. The epithelium lining the solitary lymphatic nodule (SLN) showed (Periodic acid-Schiff) PAS positive reaction. The mucous substance showed strong reaction in the gland in LGC and the FAE epithelium the number of goblet was less. The LGC was formed by aggregation of lymphoid follicles with invagination of crypt gland inside the follicle in the tunica sub mucosa. The follicle associated epithelium devoid of goblet cells were observed on the LGC with interspersed of diffuse migrating lymphocytes in the lamina propria. In the LGC, secondary follicle with interfollicular area was appreciated. In mesenteric lymph node the secondary follicles with inner germinal centre and outer corona could be seen in cortico medullary area. Meshwork of reticular fibers was abundantly present around the crypts in the lamina propria and in the pore like depression of LGC and around the secondary follicle. Collagen fibers could be seen surrounding the LGC and glands. Scanty collagen fibres were observed in the inter-follicular glands. Innervations of nerve fibre was observed around the lymphoid follicle and penetrating the LGC. The mucosal glands were PAS positive.

Engineered Nanoparticles inside a Microparticle Oral System for Enhanced Mucosal and Systemic Immunity.

Antigen delivery through an oral route requires overcoming multiple challenges, including gastrointestinal enzymes, mucus, and epithelial tight junctions. Although each barrier has a crucial role in determining the final efficiency of the oral vaccination, transcytosis of antigens through follicle-associated epithelium (FAE) represents a major challenge. Most of the research is focused on delivering an antigen to the M-cell for FAE transcytosis because M-cells can easily transport the antigen from the luminal site. However, the fact is that the M-cell population is less than 1% of the total gastrointestinal cells, and most of the oral vaccines have failed to show any effect in clinical trials. To challenge the current dogma of M-cell targeting, in this study, we designed a novel tandem peptide with a FAE-targeting peptide at the front position and a cell-penetrating peptide at the back position. The tandem peptide was attached to a smart delivery system, which overcomes the enzymatic barrier and the mucosal barrier. The result showed that the engineered system could target the FAE (enterocytes and M-cells) and successfully penetrate the enterocytes to reach the dendritic cells located at the subepithelium dome. There was successful maturation and activation of dendritic cells in vitro confirmed by a significant increase in maturation markers such as CD40, CD86, presentation marker MHC I, and proinflammatory cytokines (TNF-α, IL-6, and IL-10). The in vivo results showed a high production of CD4+ T-lymphocytes (helper T-cell) and a significantly higher production of CD8+ T-lymphocytes (killer T-cell). Finally, the production of mucosal immunity (IgA) in the trachea, intestine, and fecal extracts and systemic immunity (IgG, IgG1, and IgG2a) was successfully confirmed. To the best of our knowledge, this is the first study that designed a novel tandem peptide to target the FAE, which includes M-cells and enterocytes rather than M-cell targeting and showed that a significant induction of both the mucosal and systemic immune response was achieved compared to M-cell targeting.

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.

Gross, Histomorphological and Scanning Electronic Microscopic Studies on Gut Associated Lymphoid Tissue of Intestine of Pati Duck (Anas platyrhynchos domesticus) of Assam

Background: The study on Gut Associated Lymphoid Tissue (GALT) of Pati duck of Assam is of great value in regard to normal academic and bio-medical research aspects. The aim of the study was to evaluate the gross, histomorphological and scanning electronic microscopic examination of gut-associated lymphoid tissue of the intestine of Pati duck at different age group. Methods: For this study, forty five Pati ducks were divided into five groups depending on its age viz., 1st week, 4th week, 16th week, 24th week and 42nd weeks old. The pieces of gut having lymphoid tissue or Peyer’s patches were collected immediately after slaughter. These samples were fixed in 10% neutral buffered formalin solution and were processed as per the standard technique of procedure (Luna, 1968). The paraffin blocks were sectioned in Shandon Finesse microtome at 5 µm thickness and the sections were stained with Mayer’s Haematoxylin and Eosin staining technique for Cellular details, Van Gieson’s method for collagen fibres, Gomori’s method for reticular fibres, Hart’s method for elastic fibres and Bielchowsky’s method for axis cylinder and dendrites as per the method of Luna (1968). Result: Gut-associated lymphoid tissue was found in the duodenum, jejunum, ileum, caecum and the terminal part of the rectum in all the age group of Pati duck. The lymphoid compartment of the gut-associated lymphoid tissue in duck included a follicular structure, dome, follicle associated epithelia and interfollicular area. Lamina propria of jejunum was heavily infiltrated with diffuse lymphatic tissue in the 16th, 24th and 42nd week of age of Pati duck. The scattered and diffuse lymphatic infiltration occurred in all age groups. The lamina propria of the colorectum revealed an extensive network of reticular fibre with diffused lymphatic tissue in all the age group of duck. In Scanning Electron Microscope, the lumen of the jejunum was covered by finger-like villi with numerous opening of goblet cells. The lymphoid follicle of Lamina propria contains numerous lymphocytes along with connective tissue fibres.

Light and transmission electron microscopic evaluation of the lamina epithelialis mucosae in the ileum of pre- and post-hatch broiler chicken.

Morphological development of the lamina epithelialis mucosae (LEM) of the ileum of broiler chicken was evaluated using light and transmission electron microscopic techniques. Ileal sections were collected on embryonic days (ED) 14, 17 and 19, as well as post-hatch days (PD) 1, 3, 5, 7, 14, 21, 28, 35, 42 and 56. The result showed that the ileal LEM, which were poorly defined at embryonic days 14 and 17, consisted of enterocytes and some atypically shaped goblet cells. Apico-lateral tight junctions and irregularly shaped microvilli were associated with the enterocytes at ED 14 and 17. The enterocyte microvilli were more uniform in shape and distribution at ED 19. The embryonic goblet cells were varied in shape and possessed basally displaced, star-shaped nuclei and small apical cytoplasmic vacuoles. During the post-hatch ages, the LEM was a typical epithelium with a single layer of columnar-shaped enterocytes that became highly elongated at post-hatch day (PD) 7. The goblet cells were characteristically 'wine-glass' shaped. Follicle-associated epithelium (FAE) showing numerous lymphocytes among the enterocytes occurred in the post-hatch LEM. The intra-epithelial lymphocytes (IEL) were first encountered at PD 1, but increased several folds within the first two weeks post-hatch. Entero-endocrine cells were observed in the epithelium from PD 21. Finally, from this study, it is obvious that enterocytes and small atypically shaped goblet cells occur in the ileal LEM during the pre-hatch period, but these cells assume adult morphological characteristics after hatch. Thus, the cells of the ileal LEM play strategic absorptive, secretory and protective roles in the gut.

Aging-Related Impairments to M Cells in Peyer's Patches Coincide With Disturbances to Paneth Cells.

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer’s patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer’s patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer’s patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.