The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock.

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

Moderate Alcohol Consumption Uniquely Regulates Sodium-Dependent Glucose Co-Transport in Rat Intestinal Epithelial Cells In Vitro and In Vivo.

BACKGROUND: Chronic alcohol use often leads to malnutrition. However, how the intestinal absorption of nutrients such as glucose may be affected during moderate ethanol use has not been investigated. Glucose is absorbed via sodium (Na)-dependent glucose co-transport (SGLT1; SLC5A1) along the brush border membrane (BBM) of intestinal absorptive villus cells. OBJECTIVE: The aim of this study was to investigate how moderate alcohol consumption affects the absorption of glucose via SGLT1. METHODS: Intestinal epithelial cells (IEC-18; rat) were exposed to 8.64 mM ethanol over 1, 3, 6, and 12 h. Rats (16-wk-old, male, Sprague-Dawley) were administered 2 g/kg ethanol over 1, 3, and 6 h. Na-dependent 3H-O-methyl-d-glucose uptake was measured to assess SGLT1 activity. Na-K-ATPase activity was measured as a function of inorganic phosphate release. Protein expression was analyzed by Western blot analysis and immunohistochemical staining. RESULTS: Ethanol significantly decreased Na-dependent glucose absorption in enterocytes in vitro (ethanol treatment: 48.4% of controls at 1 h; P < 0.01) and in vivo (ethanol treatment: 60.0% of controls at 1 h; P < 0.01). Na-K-ATPase activity was significantly inhibited in vitro (ethanol treatment: 36.9% of controls at 1 h; P < 0.01) and in vivo (ethanol treatment: 42.1% of controls at 1 h; P < 0.01). Kinetic studies showed that the mechanism of inhibition of Na-glucose co-transport was secondary to a decrease in the affinity (1/Km) of the co-transporter for glucose both in vitro and in vivo. Western blots and immunohistochemistry further demonstrated unaltered amounts of SGLT1 after ethanol treatment. CONCLUSIONS: Moderate ethanol significantly decreases glucose absorption in IEC-18 cells and in villus cells of Sprague-Dawley rats. The inhibition of SGLT1 is secondary to an altered Na gradient at the cellular level and secondary to diminished affinity of the co-transporter for glucose at the protein level in the BBM. These observations may, at least in part, explain 1 possible mechanism of the onset of malnutrition associated with alcohol consumption.

Prophylactic Potential of Synbiotic (Lactobacillus casei and Inulin) in Malnourished Murine Giardiasis: an Immunological and Ultrastructural Study.

Giardiasis is a re-emerging infectious disease with outbreaks reported globally specially in children and malnourished individuals leading to malabsorption, growth retardation, and severe diarrhea. Thus, in the present study, prophylactic administration of synbiotic as the functional food was used to assess its antigiardial potential in malnourished murine giardiasis. Interestingly, prior administration of synbiotic (Lactobacillus casei + inulin) even to malnourished-Giardia-infected mice led to increased body mass, small intestine mass, lactobacilli counts, and reduced severity of giardiasis as evident by decreased cyst and trophozoite counts. Synbiotic therapy further boosted the innate and acquired immune response resulting into increase in nitric oxide, antigiardial secretory IgA and IgG antibody levels along with IL-6 and IL-10 cytokines, and decreased levels of inflammatory TNF-α cytokine in both serum and intestinal fluid in malnourished-synbiotic-Giardia-infected mice compared with malnourished-Giardia-infected mice. More specifically, histopathological and scanning electron microscopy analysis of the small intestine also confirmed the modulatory potentials of synbiotic in malnourished-synbiotic-Giardia mice which had less cellular and mucosal damage compared with severely damaged, mummified, and blunted villi in malnourished-Giardia-infected mice. Taken together, this is the first experimental study to report that prior supplementation of synbiotic restored the gut morphology and improved the immune status of the malnourished-Giardia-infected mice, and could be considered as the prophylactic adjunct therapy for malnourished individuals.

Influences of Short-Term Fasting and Carbohydrate Supplementation on Gut Immunity and Mucosal Morphology in Mice.

BACKGROUND: Preoperative carbohydrate (CHO) supplementation has been recommended in enhanced recovery after surgery protocols. However, the effects of CHO supplementation on gut and systemic immunity are not well understood. METHODS: Mice (n = 60) were randomized to 1 of the following 5 groups: control (ad lib feeding), 12-hour fasting without CHO administration (fasting), and 12 hours of fasting with CHO administration at 2, 4, and 8 hours before sacrifice. Then, lymphocytes were isolated from gut-associated lymphoid tissue, that is, Peyer's patches, the intraepithelial space, and the lamina propria of the small intestine. These lymphocyte numbers and phenotypes were evaluated. IgA levels in respiratory and small-intestinal washings were determined by ELISA. Morphology, proliferation, and apoptosis of the intestinal epithelium were also evaluated histologically. RESULTS: Although there were no significant differences in IgA levels among the 5 groups, fasting decreased intraepithelial and lamina propria, but not Peyer's patches lymphocyte numbers. CHO at 2 hours prevented lymphocyte loss in intraepithelial, whereas CHO at 4 hours reversed lamina propria lymphocytes numbers. Percentages of lymphocyte phenotypes were similar in each site among the 5 groups. Fasting caused villous atrophy; however, CHO at 2 hours restored villous structure along with maintenance of epithelial cell proliferation rate. CONCLUSIONS: Only 12 hours of fasting causes marked gut-associated lymphoid tissue cell loss along with gut atrophy. However, CHO at 2 hours preserves gut immunity and morphology not completely but moderately.

Microscopic features of small bowel mucosa of patients with Crohn's disease.

BACKGROUND: Double-balloon enteroscopy enables performing numerous small bowel biopsies for pathologic analysis. However, most histopathological characteristics of Crohn's disease are non-specific characteristics. We aimed to explore the small bowel mucosal histopathologic characters of Crohn's disease and identify some disease-specific changes. METHODS: We included 253 patients without tumors and grouped them into Crohn's disease, suspected Crohn's disease, and non-Crohn's disease groups. These patients underwent double-balloon endoscopy examination and small bowel biopsy at Renji Hospital, Shanghai. All histopathological sections were reviewed, and > 20 histopathological parameters were assessed. Immunohistochemistry was conducted when necessary. RESULTS: There were different forms of granulomatous lymphangitis on the small bowel mucosa in Crohn's disease. They showed as various macrophages or epithelioid cells in the lumina of lymphatics or in the center of the villi with or without evident obstruction. These features were only observed in Crohn's disease patients. Furthermore, they were correlated with granuloma and lymphangiectasia. Additionally, 15 other features showed significant differences among the three groups, and Crohn's disease patients showed an average of almost seven histopathological characteristics. CONCLUSIONS: We described the detailed morphologies of granulomatous lymphangitis on the small bowel mucosa and recommend it as a useful histopathological feature for the diagnosis of Crohn's disease. In terms of specificity and sensitivity, it was superior to non-caseating epithelioid granuloma.

An intracellular diamine oxidase triggered hyperpolarized 129Xe magnetic resonance biosensor.

Here, a novel method was developed for suppressing 129Xe signals in cucurbit[6]uril (CB6) until the trigger is activated by a specific enzyme. Due to its noncovalent interactions with amino-groups and CB6, putrescine dihydrochloride (Put) was chosen for blocking interactions between 129Xe and CB6. Upon adding diamine oxidase (DAO), Put was released from CB6 and a 129Xe@CB6 Hyper-CEST signal emerged. This proposed 129Xe biosensor was then tested in small intestinal villus epithelial cells.

Micro-patterned endogenous stroma equivalent induces polarized crypt-villus architecture of human small intestinal epithelium.

The small intestine is the major site for digestion, drug and nutrient absorption, as well as a primary site for many diseases. Current in vitro gut models fail in reproducing the complex intestinal extracellular matrix (ECM) network of the lamina propria and the peculiar architecture of the crypt-villus axis. Here we proposed a novel in vitro human intestine model that mimics the intestinal stromal topography and composition and strictly reproduces the tissue polarity with the crypt-villus architecture. First we developed a 3D human intestinal stromal equivalent (3D-ISE) composed of human intestinal subepithelial myofibroblasts (ISEMFs) embedded in their own extracellular matrix. Then, we seeded human colon carcinoma-derived cells (Caco-2) onto flat or patterned cell-synthetized stromal equivalent structure and cultured them until the formation of a well-oriented epithelium. We demonstrated that the patterned stroma increases the absorbing surface area, the epithelial proliferation rate, and the density of microvilli. In addition it induces changes in the biological functions of the epithelial cells such as enzymes and mucus production, polarization and tightness showing a physiological cell-lineage compartmentalization along the crypt/villi axes with the undifferentiated phenotypes at the base. At last, we reproduced an inflamed intestinal tissue model in which we identified the contribution of the stromal microenvironment by molecular (cytokines release and MMPs production) and immunofluorescence analyses and the effects of the epithelial-stromal cross-talk in the intestinal innate immunity by multiphoton investigation that revealed differences in the collagen network architecture. STATEMENT OF SIGNIFICANCE: The intestinal stroma morphology and composition has a fundamental role in crypt-villus development and appropriate epithelial cell-lineage compartmentalization. On this base, here we develop an engineered organotypic model of human intestine equivalent in which a functional epithelial/ECM crosstalk is recapitulated. Due to its accessible luminal surface it provides a new platform for preclinical studies of mucosal immunology and bowel inflammation as well as the assessment of pharmaco-toxicity studies.

Natural Sources of Extracellular Matrix for Cardiac Repair.

Tissue engineering and regenerative medicine have adopted the use of extracellular matrix (ECM) as a cell delivery device and bioactive regenerative agent. To this end, many ECMs have been investigated for cardiac tissue engineering and regenerative medicine applications with variable success. Many sources of natural ECMs have been tested for cardiac applications. Typically, natural ECMs have been made from decellularized organs or tissues and processed into either sheets or injectable hydrogels. This chapter will review natural sources of ECM materials that have been tested as therapeutic agents in models of heart failure.

Protective Effect of Aplysin Supplementation on Intestinal Permeability and Microbiota in Rats Treated with Ethanol and Iron.

Aplysin, a kind of phytochemicals or phytonutrients, is purified from red alga Laurencia tristicha. The present study aims to investigate the influence of aplysin on changes of intestinal permeability and microbiota induced by excessive ethanol and iron. Thirty male rats were randomly divided into three groups (10/group): control group (normal saline); ethanol + iron group as EI treated with ethanol (8⁻12 mL/kg/day) and iron (1000 mg/kg) in diet; EI supplemented with aplysin (150 mg/kg/day) group as AEI; the trial lasts for 12 weeks. The result showed that levels of plasma endotoxin, fatty acid-binding protein 2, D-lactic acid, diamine oxidase were increased in rats in the EI group; and significantly decreased by 14%, 17%, 26%, 16%, respectively (p < 0.05) in the AEI group after the 12-week aplysin treatment. Moreover, in the AEI group the amount of Escherichia coli and Bacteroides fragilis were higher, while the amount of Lactobacillus, Bifidobacterium and Clostridium were lower than those in the EI group. The expressions of iron transporters divalent-metal transporter 1(DMT1) and ferroportin 1(FPN1) were significantly upregulated in the EI group compared to those in the control group. In conclusion, aplysin could effectively improve intestinal permeability and intestinal flora disorder induced with excessive ethanol and iron.

Logarithmic expansion of LGR5+ cells in human colorectal cancer.

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.

Effect of Piper betle on Giardia intestinalis infection in vivo.

Piper betle has been used as a medicinal plant in traditional medical systems throughout South and South East Asia. Experimental studies have revealed its wide and diverse biological and pharmacological effects. In this study, antigiardial activity of Piper betle was tested using experimental infections of Giardia intestinalis, the most common cause of protozoal diarrhoea worldwide, in Mongolian gerbils. Plants were extracted in water, methanol and methanol:tetrahydrofuran. Gerbils were treated for ten days intragastrically twice a day, with the dose of 40 mg of the extract per 100 g of body weight. Drug metronidazole was used as a negative control. Gerbils' faeces were taken every day and examined by flotation method, the number of shed cysts were counted using a haemocytometer. After gerbils' sacrifice and dissection, their duodena were then processed for examination using histological sectioning and scanning electron microscopy. The antigiardial activity was evaluated by the course of cyst shedding throughout the entire experiment. A significant decline in cyst shedding, evaluated by linear regression was found in gerbils treated with the aqueous extract. Our results indicate that the aqueous extract of P. betle shows giardicidal effects.

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

Buffered Romanowsky-Giemsa method for formalin fixed, paraffin embedded sections: taming a traditional stain.

Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96-100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H & E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H & E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H & E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.

Implication of artemisinin nematocidal activity on experimental trichinellosis: In vitro and in vivo studies.

Benzimidazole drugs are used for treatment of trichinellosis, but they have a limited effect against encapsulated larval stages of Trichinella spiralis. Hence, there is a considerable interest in developing new anthelmintic drugs. Our aim is to investigate the possible effect of artemisinin on T. spiralis in in vitro and in vivo studies. T. spiralis worms were isolated from infected mice and transferred to 3 culture media; group I: with no drugs, group II: contained artemisinin and group III: contained mebendazole, then they were subjected to electron microscopic study. An in vivo study was done where mice were divided into three groups; group I: infected and untreated, group II: received artemisinin and group III: received mebendazole. The efficacy of treatment was assessed by adult and total larval counts, histopathological study of the small intestinal and muscle tissues and immunohistochemical staining of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in muscles. Adult worm teguments showed significant degeneration and destruction with both drugs. Also, significant reduction of total adult and larval counts occurred in treated groups in comparison to the control group. Histopathological examination of the small intestine and muscles showed marked improvement with reduction in the inflammatory infiltrates with both drugs. COX-2 and VEGF expressions were reduced in both treated groups with more reduction in the artemisinin-treated group. This study revealed that artemisinin has the potential to be an alternative drug against trichinellosis.

TM6SF2 rs58542926 impacts lipid processing in liver and small intestine.

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).

Progressive Depletion of Rough Endoplasmic Reticulum in Epithelial Cells of the Small Intestine in Monosodium Glutamate Mice Model of Obesity.

Chronic obesity is a known risk factor for metabolic syndrome. However, little is known about pathological changes in the small intestine associated with chronic obesity. This study investigated cellular and subcellular level changes in the small intestine of obese mice. In this study, a mouse model of obesity was established by early postnatal administration of monosodium glutamate. Changes in body weight were monitored, and pathological changes in the small intestine were evaluated using hematoxylin-eosin and Nissl staining and light and electron microscopy. Consequently, obese mice were significantly heavier compared with controls from 9 weeks of age. Villi in the small intestine of obese mice were elongated and thinned. There was reduced hematoxylin staining in the epithelium of the small intestine of obese mice. Electron microscopy revealed a significant decrease in and shortening of rough endoplasmic reticulum in epithelial cells of the small intestine of obese mice compared with normal mice. The decrease in rough endoplasmic reticulum in the small intestine epithelial cells of obese mice indicates that obesity starting in childhood influences various functions of the small intestine, such as protein synthesis, and could impair both the defense mechanism against invasion of pathogenic microbes and nutritional absorption.

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Pathway underlying small intestine apoptosis by dietary nickel chloride in broiler chickens.

The aims of this study were to investigate the pathways which dietary nickel chloride (NiCl2) affects small intestine apoptosis in broiler chickens by observing the ultrastructure, and bcl-2, bax, and caspase-3 protein expression and mRNA expression, and cytochrome C, bak and caspase-9 mRNA expression of the small intestine. A total of 240 one-day-old avian broilers were divided into four groups and fed a corn-soybean basal diet as the control diet or three experimental diets supplemented with 300, 600, and 900 mg/kg of NiCl2 for 42 days. Ultrastructurally, the microvilli were apparently exfoliated, and the mitochondria were swollen and the number of lysosomes increased in the intestinal cells of three experimental groups. As measured by TUNEL and flow cytometry (FCM), the percentage of apoptotic cells in the small intestine and the lymphocytes in the ileum were significantly increased in three experimental groups when compared with those of the control group. Meanwhile, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immuno-sorbent assay (ELISA) tests showed that the protein expression, mRNA expression levels were decreased in the bcl-2, whereas those of bax and caspase-3, and the cytochrome C, bak and caspase-9 mRNA expression levels were increased in three experimental groups. The abovementioned results show that pathway of dietary NiCl2-induced small intestine apoptosis is related to the mitochondrial damage and promotion of the cytochrome C release from mitochondria, which activates the mitochondrion-mediated apoptosis pathway.

High-frequency ultrasound of Peyer's patches in the small intestine of young cats.

OBJECTIVES: A previously unreported, asymmetrically positioned hypoechoic extra layer (APHEL) in the submucosa of the feline distal jejunum and ileum has been recognised using high-frequency ultrasound. The objectives of this study were to characterise the APHEL histologically, and to describe the prevalence and ultrasonographic features of the APHEL in a population of clinically healthy young cats. METHODS: In an anatomical study, two cats were autopsied and histopathology of the small intestine was performed. An APHEL was detected with ultrasound in the distal jejunum and ileum ante-mortem in the first cat and post mortem in the second cat. Samples for histopathology were obtained from these areas. In the second, prospective part of the study, to document the presence or absence of an APHEL, high-frequency (18 MHz) ultrasound was performed of the intestinal tract in 20 other cats. These cats were client-owned cats aged 6-18 months presented for neutering. The cats were included in the study based on a normal clinical examination, lack of previous or concurrent signs of disease, and having no abnormalities detected at abdominal ultrasound. RESULTS: Histopathology from the distal jejunum and ileum in the two cats in the anatomical part of the study showed that the APHEL represented asymmetrically positioned normal lymphatic tissue (Peyer's patches) in the lamina propria and submucosa. In the second part of the study, an APHEL was identified in the submucosa of the distal part of the jejunum and ileum in all 20 cats. Additionally, a similar layer could also be seen further proximally in the jejunum in 10 (50%) of the cats. The thickness of the APHEL was 1.0 mm in both jejunum and ileum. CONCLUSIONS AND RELEVANCE: Presumed normal lymphatic tissue in the small intestinal submucosa can be seen with high-frequency ultrasound and is a common finding in young cats.

Morphological and Immunohistochemical Identification of Villous M Cells in the Small Intestine of Newborn Piglets / Identificación Morfológica e Inmunohistoquímica de los Epiteliocitos Microplegados de las Vellosidades en el Intestino Delgado de Lechones Recién Nacidos

Microfold (M) cells act as antigen-sampling sites for initiating antigen specific mucosal immune responses, but they may also provide a gateway for enteropathogen entry. In this study we demonstrated villous M cells by morphological and immunohistochemical methods to be present in the three regions of the small intestine from newborn piglets. Immunohistochemical analysis, using anti- cytokeratin 18 (CK18) primary antibodies, showed a gradually decreased density of M cells from the lower crypt epithelium to the upper villous epithelium. The proportion of villous M cells was greater in the ileum than in the duodenum or the mid-jejunum. Ultrastructural observation revealed that villous M cells were mainly columnar in shape in the duodenum and the mid-jejunum, and appeared as more pocket-like structure in the ileum. These villous M cells exhibited short and irregular microvilli, rich vesicles and reduced glycocalyx, but lacked the lymphocyte-containing basolateral invagination. Our results support evidence that M cells can develop in the small intestinal villous epithelium of newborn piglets, implying that villous M cells may begin developing in the pig's small intestine during fetal stages, which depends neither on the influence of the mucosal lymphoid tissue nor the antigen from the intestinal lumen stimulation. In addition, the variable morphology and heterogeneity distribution of villous M cells in the three regions of the small intestine may be indicative of its different functional properties. This information extent our understanding of the diversity of M cells and provides important basic knowledge for further research on the actual role of villous M cells in neonate.

Los epiteliocitos microplegados (células M) actúan como receptores de antígeno para iniciar la respuesta inmune específica de las mucosas, pero también pueden proporcionar una puerta de entrada para enteropatógenos. En este estudio, se demostró por métodos morfológicos e inmunohistoquímicos que los epiteliocitos microplegados de las vellosidades están presentes en las tres regiones del intestino delgado de lechones recién nacidos. Se utilizaron anticuerpos primarios de citoqueratina 18 (CK18) para el análisis inmunohistoquímico, el cual mostró una disminución gradual de la densidad de los epiteliocitos microplegados desde el epitelio de las criptas inferiores hasta el epitelio de las vellosidades superiores. La proporción de los epiteliocitos microplegados, fue mayor en el íleon que el duodeno o yeyuno medio. La observación ultraestructural reveló que los epiteliocitos microplegados fueron principalmente de forma columnar en el duodeno y el yeyuno medio. Además, mostraron microvellosidades cortas e irregulares, muchas vesículas y glucocáliz reducidos, pero carecían de invaginaciones basolaterales contenedoras de linfocitos. Nuestros resultados apoyan la evidencia de que los epiteliocitos microplegados pueden desarrollarse en el epitelio de las vellosidades intestinales de los lechones recién nacidos, lo que implica que estas células pueden comenzar a desarrollarse en el intestino delgado del cerdo durante las etapas fetales, y no dependen ni de la influencia del tejido linfoide de las mucosas ni del antígeno para la estimulación del lumen intestinal. Además, la morfología y heterogeneidad de distribución de los epiteliocitos microplegados en las tres regiones del intestino delgado pueden ser indicativas de sus diferentes propiedades funcionales. Esta información mejora nuestra comprensión de la diversidad de los epiteliocitos microplegados y proporciona conocimientos básicos importantes para la investigación sobre el papel de los epiteliocitos microplegados en las vellosidades del neonato.