Superficial Epithelium Research Articles

Examinations on the prevalence of footpad lesions and breast skin lesions in British United Turkeys Big 6 fattening turkeys in Germany. Part I: Prevalence of footpad lesions

The present study demonstrates the prevalence of footpad lesions in turkeys and their level of expression in both live birds and in carcasses. In clinical investigations of 11,860 turkeys (5,740 males, 6,120 females) of the strain British United Turkeys Big 6, individuals of all the observed flocks showed alterations to the plantar skin. In general, the degree and severity of skin alterations were higher in the 16th week of life than in the 6th or 11th week of life. Epithelial necrosis, however, was already apparent in about 45% of individuals in the 6th week of life. At this point, deep lesions on the footpad surface were still rare findings. In the 11th week of life, such skin changes were apparent in 14.7% of males and 25.7% of hens. In general, the feet of male turkeys were less affected by footpad damage than those of females in the subsequent fattening period. In hens, the prevalence of lesions of the footpads at wk 16 of age was 60.0%, almost twice as high as in males of the same age group (33.8%). A comparison of the findings from the right and left feet showed that the state of the 2 extremities was correlated (Spearman correlation: r = 0.830). During the visual inspection of 16,200 slaughtered turkeys (7,800 males, 8,400 hens), almost all carcasses studied showed alterations to the footpad skin. Only 2.1% of males and 0.6% of females were without lesions. The most frequently observed lesions measured up to 2cm in diameter (males: 59.2%; hens: 57.7%). Pronounced skin lesions of the footpad (males: 21.1%; females: 29.5%) and necrosis of the superficial epithelium (males: 17.5%; females: 12.1%) were less common. Foot abscesses were diagnosed only sporadically (males: 0.1%; females: 0.1%).

Bimodal Electric Tissue Ablation (BETA): A Study on Ablation Size When the Anode is Placed on the Peritoneum and the Liver

In bimodal electric tissue ablation (BETA), the cathode of the DC circuit is attached to the radiofrequency (RF) electrode to increase the surrounding tissue hydration. This will delay tissue desiccation and allowing the ablation process to continue for a longer period of time before "roll-off" occurs, resulting in larger ablations compared with standard radiofrequency ablation (RFA). Previous research showed that attaching the anode to the skin using electrosurgical grounding pads would reduce the efficacy of BETA because of the high electrical resistivity of the skin. This study investigated the ablation size produced when the anode was attached to the peritoneum (BETA-peritoneum) and the liver (BETA-liver) respectively. The anode of the DC circuit in BETA was attached to the peritoneum and the liver in a pig model using ECG dots. In BETA, 9 V of DC was provided for 10 min, after which the radiofrequency generator were switched on and both electrical circuits allowed to run concurrently until "roll-off." The size of ablations produced was compared to when the anode attached to the skin (BETA-skin) and standard RFA, respectively. The sites of anode placement were examined for local tissue injury. The transverse diameters in BETA-peritoneum and BETA-liver were significantly larger compared with BETA-skin and standard RFA, respectively (P < 0.001). The axial diameter in the BETA-peritoneum and BETA-liver groups were also larger compared with the BETA-skin and RFA groups, although the differences did not reach statistical significance (P = 0.09). Hematoxylin and eosin (H and E) examination of the peritoneum and the liver where the anode was attached showed coagulation necrosis involving the superficial epithelium and the liver capsule, respectively. BETA can be used to treat larger liver tumors more effectively and may reduce the tumor recurrence rates compared with standard RFA. The efficacy of BETA depends on ensuring good electrical conductivity between the cathode and the anode of the DC circuit. Research so far has shown that BETA works best when the anode is placed deep to the skin as the stratum corneum consisted of a layer of a-nucleated cells, which have high electrical resistivity. The liver could be the ideal location to place the anode as it has excellent electrical conductivity, therefore ensuring maximum tissue hydration around the cathode to produce the largest ablations possible.

Epithelial–Mesenchymal Interactions as a Working Concept for Oral Mucosa Regeneration

Oral mucosa consists of two tissue layers, the superficial epithelium and the underlying lamina propria. Together, oral mucosa functions as a barrier against exogenous substances and pathogens. In development, interactions of stem/progenitor cells of the epithelium and mesenchyme are crucial to the morphogenesis of oral mucosa. Previous work in oral mucosa regeneration has yielded important clues for several meritorious proof-of-concept approaches. Tissue engineering offers a broad array of novel tools for oral mucosa regeneration with reduced donor site trauma and accelerated clinical translation. However, the developmental concept of epithelial-mesenchymal interactions (EMIs) is rarely considered in oral mucosa regeneration. EMIs in postnatal oral mucosa regeneration likely will not be a simple recapitulation of prenatal oral mucosa development. Biomaterial scaffolds play an indispensible role for oral mucosa regeneration and should provide a conducive environment for pivotal EMIs. Autocrine and paracrine factors, either exogenously delivered or innately produced, have rarely been and should be harnessed to promote oral mucosa regeneration. This review focuses on a working concept of epithelial and mesenchymal interactions in oral mucosa regeneration.

Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.

Uterine Progesterone Receptor Expression, Conceptus Development, and Ovarian Function in Pigs Treated with RU 486 During Early Pregnancy1

Establishment of pregnancy in the pig depends on down-regulation of progesterone receptor (PGR) in the uterine luminal and glandular epithelium during the first week after breeding. The present study evaluated the regulation of endometrial PGR by progesterone and the possible role of endometrial tumor necrosis factor (ligand) superfamily member 11 (TNFSF11) and nuclear factor-kappa B (NFKB) activation in PGR expression. Mature, cycling gilts were inseminated (Day 0) and assigned to either untreated control (n = 9) or one of two treatments that employed RU 486 to block progesterone action either before (treatment 1 [T1]) or after (treatment 2 [T2]) the initiation of PGR down-regulation. The T1 gilts were treated with RU 486 (400 mg/day) on Days 3-5 of pregnancy (n = 9), and T2 gilts were treated with RU 486 on Days 6 and 7 of pregnancy (n = 9). Uteri and ovaries were collected on Day 8 or 12 of gestation. The diameter of the conceptuses in T1 gilts was approximately half that in controls by Day 8, and normal conceptuses were not collected from any T1 gilts on Day 12. Endometrial PGR mRNA was more abundant in T1 and T2 gilts compared with control gilts. The PGR-B protein decreased from Day 8 to Day 12 in the luminal epithelium and, to some extent, in superficial glandular epithelium in control and T2 gilts. In T1 gilts, the PGR-B protein remained elevated (i.e., failed to undergo down-regulation) on Day 12. Blocking PGR action early in the cycle (i.e., on or before Day 5), therefore, prevented normal conceptus development, caused elevated PGR mRNA, and prevented the decrease in PGR protein that typically occurs in pigs. We could not confirm a role for NFKB activation in PGR down-regulation, because pigs with extreme differences in PGR and TNFSF11 expression (T1 and controls) had similar NFKB activation on Day 8. Activated NFKB within the luminal epithelium and glandular epithelium (both superficial and deep) was observed in T2 and control pigs on Day 12 when elongating conceptuses (presumably releasing interleukin 1 beta to activate NFKB) were recovered. Gilts treated with RU 486 had greater ovarian follicular growth and greater plasma estradiol concentrations. We conclude that the mechanisms controlling PGR down-regulation are progesterone-dependent and occur between Day 3 and Day 6 of pregnancy. NFKB activation did not appear to have a role in PGR down-regulation within the period that we studied. Blocking progesterone action after Day 6 did not reverse the process of PGR down-regulation, nor did it appear to affect the development of conceptuses collected on Day 12.

Influence of different calcium supplies and a single vitamin D injection on vitamin D receptor and calbindin D9k immunoreactivities in the gastrointestinal tract of goat kids

The purpose of this study was to investigate whether diets differing in Ca concentration would have an influence on vitamin D (VitD) receptor (VDR) and calbindin D9k (Calb9k) immunoreactivities in the gastrointestinal tract of growing goats. In addition, the effect of a single VitD injection was studied, to clarify whether exogenous VitD would further increase the active Ca absorption mechanisms. The hypothesis of the study was that reduced Ca intake would lead to greater active Ca absorption, and with that, to greater amounts of VDR and Calb9k immunoreactivities. The normal Ca kid group (according to age requirements) received 2.5 to 6 g of Ca/d, whereas the lesser Ca kid group (less than requirements) received 1.5 to 4 g of Ca/d from wk 6 (weaning) to 15 (slaughter). In addition, 5 and 6 goat kids, respectively, of each group (normal Ca kid group, lesser Ca kid group), were injected with VitD (0.05 mg of cholecalciferol/kg of BW) in wk 14 of life. Blood samples were taken in wk 14 and 15. Calcium and VitD (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) concentrations were determined in serum. Immediately after slaughter, the duodenum (DD) and rumen (RU) were mounted in conventional Ussing chambers. Unidirectional flux rates of Ca across gastrointestinal tissues were measured. Additionally, tissue specimens of the gastrointestinal tract were collected, and formaldehyde-fixed paraffin sections were used for VDR and Calb9k immunohistochemistry. In all kid groups, a net absorption in the RU and a net secretion of Ca in the DD were observed. Immunoreactions of VDR were greatest in the duodenal mucosa, whereas Calb9k immunoreactions were observed in the forestomach and intestinal tissues. The greatest expression was observed in the duodenal surface epithelium. Additionally, in the VitD-injected groups, an immunoreaction occurred in the jejunal superficial and basal glands and the ileal superficial epithelium. In contrast, the other groups showed no Calb9k immunoreactions at these sites. In conclusion, there is clear evidence for the RU as a main site for Ca absorption. The results of this study also indicate that VDR and Calb9k are highly expressed in the duodenal mucosa. The active absorption may not have such an important role in the DD because active transport was also evident in the RU. However, Calb9k expression seems to be stimulated by VitD administration.

PAR-1 promotes primary neurogenesis and asymmetric cell divisions via control of spindle orientation

In both invertebrate and vertebrate embryonic central nervous systems, deep cells differentiate while superficial (ventricular) epithelial cells remain in a proliferative, stem cell state. The conserved polarity protein PAR-1, which is basolaterally localised in epithelia, promotes and is required for differentiating deep layer cell types, including ciliated cells and neurons. It has recently been shown that atypical protein kinase C (aPKC), which is apically enriched, inhibits neurogenesis and acts as a nuclear determinant, raising the question of how PAR-1 antagonises aPKC activity to promote neurogenesis. Here we show that PAR-1 stimulates the generation of deep cell progeny from the superficial epithelium of the neural plate and that these deep cells have a corresponding (i.e. deep cell) neuronal phenotype. We further show that gain- and loss-of-function of PAR-1 increase and decrease, respectively, the proportion of epithelial mitotic spindles with a vertical orientation, thereby respectively increasing and decreasing the number of cleavages that generate deep daughter cells. PAR-1 is therefore a crucial regulator of the balance between symmetric (two superficial daughters) and asymmetric (one superficial and one deep daughter) cell divisions. Vertebrate PAR-1 thus antagonises the anti-neurogenic influence of apical aPKC by physically partitioning cells away from it in vivo.

Changes in the histomorphology of the canine cervix through the oestrous cycle

The importance of canine reproduction is steadily increasing and little is known about the canine cervix so far. The aim of this study was to describe the histomorphology of the canine cervix and to determine its correlation to the stage of oestrous cycle and to circulating concentrations of progesterone (P4) and estradiol-17beta (E2), respectively. Ovariohysterectomy (OHE) was performed in 33 non-pregnant, healthy, intact bitches in defined stages of oestrous cycle (proestrus N = 5, estrus N = 7, early diestrus N = 5, late diestrus N = 11, anestrus N = 5). The entire cervix was collected for histological evaluation of the epithelial layer (number of layers, thickness), the stroma (number of layers, thickness, density, structure, and distribution of elastic fibres) and the average area and density of cervical glands as well as blood vessels. These parameters were evaluated in all the three parts of the cervix (cranial, middle, and caudal or vaginal part). The cervix showed the typical structure with a superficial epithelium, a lamina propria with cervical glands and vascular structures and a tunica muscularis below. Folds in the superficial epithelium were only observed in 54% females (N = 18). Epithelial thickness (P < 0.0001), number of glands (P < 0.05), mean area of glands (P < 0.0001), mean area of venous vessels (P < 0.0001), number of arterial vessels (P = 0.02), number of mast cells and number of eosinophilic granulocytes per mm 2 (P < 0.01) were significantly influenced by the stage of cycle. The following factors were significantly influenced by the localisation: number of epithelial layers (P < 0.0001), thickness of stroma (P < 0.0001) and mean number of glands (P < 0.01). Only mean area of venous vessels was significantly influenced by the stage of cycle and the localisation (P < 0.01). Besides, P4 was significantly correlated to number of glands per mm 2 (P < 0.0001), mean area of venous vessels (P < 000.1) and number of mast cells (P < 0.01). This study provided detailed information about the histomorphological structure of the cervix in non-pregnant bitches and showed that the cervix undergoes cyclic changes during the canine oestrous cycle, in particular in association with circulating progesterone concentration.

PAR-1 promotes primary neurogenesis and asymmetric cell divisions via control of spindle orientation

In both invertebrate and vertebrate embryonic central nervous systems, deep cells differentiate while superficial (ventricular) epithelial cells remain in a proliferative, stem cell state. The conserved polarity protein PAR-1, which is basolaterally localised in epithelia, promotes and is required for differentiating deep layer cell types, including ciliated cells and neurons. It has recently been shown that atypical protein kinase C (aPKC), which is apically enriched, inhibits neurogenesis and acts as a nuclear determinant, raising the question of how PAR-1 antagonises aPKC activity to promote neurogenesis. Here we show that PAR-1 stimulates the generation of deep cell progeny from the superficial epithelium of the neural plate and that these deep cells have a corresponding (i.e. deep cell) neuronal phenotype. We further show that gain- and loss-of-function of PAR-1 increase and decrease, respectively, the proportion of epithelial mitotic spindles with a vertical orientation, thereby respectively increasing and decreasing the number of cleavages that generate deep daughter cells. PAR-1 is therefore a crucial regulator of the balance between symmetric (two superficial daughters) and asymmetric (one superficial and one deep daughter) cell divisions. Vertebrate PAR-1 thus antagonises the anti-neurogenic influence of apical aPKC by physically partitioning cells away from it in vivo.

P16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Oncogenic human papillomavirus (HPV) type 16 has been strongly associated with tonsillar squamous cell carcinoma (TSCC) and appears to be of prognostic significance. Because HPV+ TSCC also accumulates p16(INK4A), this cyclin-dependent kinase inhibitor has been proposed as a potential biomarker for HPV in clinical diagnosis. The aim of this study was to determine the prevalence of HPV in tumour-free tonsillar tissue and the value of p16(INK4A) overexpression in predicting its presence. p16(INK4A) overexpression was detected by immunohistochemistry in tissue sections of tumour-free tonsils of 262 patients. They were treated for non-oncological reasons (snoring or chronic/recurrent tonsillitis) consisting of tonsillectomy. Genomic DNA isolated from these tissues was subjected to HPV-specific polymerase chain reaction (PCR) analysis. p16(INK4A) immunoreactivity was detected in 28% of samples in both crypt epithelium (49/177) and lymphoid germinal centres (52/187), which correlated with each other (P < 0.0001). No reactivity was observed in superficial squamous cell epithelium. HPV16 and 18 were detected by PCR analysis in 2/195 cases (1%), which, however, were negative on fluorescence in situ hybridization analysis and discrepant on p16(INK4A) immunostaining. No proof was found for the presence of HPV in tumour-free tonsil tissue, despite increased p16(INK4A) expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16(INK4A) up-regulation.

Up-regulated EMMPRIN/CD147 protein expression might play a role in colorectal carcinogenesis and its subsequent progression without an alteration of its glycosylation and mRNA level

Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of colorectal carcinomas (CRC). EMMPRIN expression was examined on tissue microarray containing colorectal carcinomas, adenoma and non-neoplastic mucosa (NNM) by immunohistochemistry and in situ hybridization (ISH). Colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) and tissues were studied for EMMPRIN expression by Western blot or RT-PCR, followed by sequencing. All carcinoma cell lines showed EMMPRIN expression at both mRNA and protein levels. Two synonymous mutations were found in carcinoma cell lines at codon109 (GCT → GCC: Ala) or 179 (GAT → GAC: Asp). Frozen CRC tissues displayed higher EMMPRIN expression than paired NNM (P < 0.05). EMMPRIN expression was immunohistochemically stronger in colorectal high-grade adenoma, adenocarcinoma and metastatic carcinoma than non-neoplastic superficial epithelium and low-grade adenoma (P < 0.05). In contrast, its mRNA level was similar from colorectal NNM, adenoma to adenocarcinoma by ISH, in line with the findings of RT-PCR (P > 0.05). Immunohistochemically, EMMPRIN expression was positively correlated with tumor size, depth of invasion, vascular or lymphatic invasion, grade of infiltration (INF), ki-67 and VEGF expression of CRCs (P < 0.05). Among them, depth of invasion was an independent associated factor for EMMPRIN expression in CRCs (P < 0.05). Up-regulated EMMPRIN protein expression might contribute to colorectal carcinogenesis without the alteration of its glycosylation and mRNA level. Aberrant EMMPRIN protein expression might promote growth or invasion of CRCs possibly through increased ki-67 expression and inducible angiogenesis via up-regulating VEGF expression.

Subsite-specific differences of estrogen receptor beta expression in the normal colonic epithelium: implications for carcinogenesis and colorectal cancer epidemiology

This study aimed at investigating whether a differential estrogen receptor beta (ER-beta) expression between the colonic subsites could correspond to a modification in proliferation, apoptosis, and adhesion of the normal colonocytes. ER-beta, Ki-67, Bcl-2, and E-cadherin expressions were investigated immunohistochemically, in normal epithelium biopsies from the ascending and the descending colon of 53 individuals, who underwent colonoscopy for the investigation of anemia and in whom no local pathology was identified. ER-beta immunoreactivity has been shown to be stronger at the superficial epithelium than the crypts' base, the difference being important only for the ascending colon. In addition, ER-beta expression was higher in the superficial epithelium of the ascending colon than that of the descending colon. The variations of ER-beta expression did not correspond to the alterations in Ki-67, Bcl-2, and E-cadherin expression. A subsite-specific variation of ER-beta expression has been shown in the normal colonic epithelium. This modulation of ER-beta might account for some well established specificities of colorectal cancer epidemiology like the right-sided predominance of the neoplasm in women and its gradual shift to more proximal sites over time.

T1770 Enteric Nematodes Regulates Host Proteolytic Pathway on Immune Cells: Influence on Host Immune Response

Background and aims: Mucosal immunity and epithelial integrity are interconnected factors involved in intestinal homeostasis. The hedgehog (Hh) family of morphogens is essential in embryogenesis, but it has been also associated with inflammation and tissue repair. We hypothesized that the Hh-pathway could affect the inflammatory response and cell survival, crucial factors in IBD pathogenesis. Methods: Endoscopic biopsies were obtained from the inflamed colon of 14 patients with Crohn's disease (CD), 12 ulcerative colitis (UC), and 15 controls. Frozen sections of mucosal samples were analyzed by immunohistochemistry using antibodies against Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Gli-1. Double immunofluorescence was used for co-localization studies. RT-PCR was used for analyzing the expression of Gli-1 mRNA. Tissue cultures of colon samples were performed during 24h in the presence of Shh peptide, anti-Shh antibody, cyclopamine, a specific Hh inhibitor, or diluent. Supernatants were analyzed for cytokine production, by ELISA, and total protein was extracted from tissue for caspase-3 activity assay. Results: CD colonic mucosa showed a lower expression of Gli-1 mRNA compared to UC and controls (p=0.03). Hh-proteins were characteristically expressed at the superficial epithelium, and a marked reduction was observed in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria, low densities of Hh-family members basically do not co-localize with infiltrating T-cells or macrophages. Baseline levels of cytokines in culture supernatants were higher in IBD compared to controls. In CD colon explants, treatment with Shh peptide resulted in a significant decrease in the levels of TNF-alpha (p=0.04), IL-17 (p=0.01), and TGF-beta (p=0.03). Caspase-3 activity was higher in CD and significantly decreased after blockade of Hh, either with anti-Shh antibody(p=0.04) or with cyclopamine (p=0.03), in contrast to UC and controls. Levels of Gli-1 negatively correlated to caspase-3 activity (CC -0.75, p=0.01). Conclusion: Normal colonic mucosa constitutively expresses Hh-proteins, which appear to constitute a key pathway for intestinal homeostasis. Hh-signaling affects cytokine production and the control of apoptosis within the colonic mucosa. Disruption of the intestinal Hhpathway might be implicated in the pathogenesis of CD.

T1769 Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A Novel Role in Intestinal Inflammation

Background and aims: Mucosal immunity and epithelial integrity are interconnected factors involved in intestinal homeostasis. The hedgehog (Hh) family of morphogens is essential in embryogenesis, but it has been also associated with inflammation and tissue repair. We hypothesized that the Hh-pathway could affect the inflammatory response and cell survival, crucial factors in IBD pathogenesis. Methods: Endoscopic biopsies were obtained from the inflamed colon of 14 patients with Crohn's disease (CD), 12 ulcerative colitis (UC), and 15 controls. Frozen sections of mucosal samples were analyzed by immunohistochemistry using antibodies against Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Gli-1. Double immunofluorescence was used for co-localization studies. RT-PCR was used for analyzing the expression of Gli-1 mRNA. Tissue cultures of colon samples were performed during 24h in the presence of Shh peptide, anti-Shh antibody, cyclopamine, a specific Hh inhibitor, or diluent. Supernatants were analyzed for cytokine production, by ELISA, and total protein was extracted from tissue for caspase-3 activity assay. Results: CD colonic mucosa showed a lower expression of Gli-1 mRNA compared to UC and controls (p=0.03). Hh-proteins were characteristically expressed at the superficial epithelium, and a marked reduction was observed in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria, low densities of Hh-family members basically do not co-localize with infiltrating T-cells or macrophages. Baseline levels of cytokines in culture supernatants were higher in IBD compared to controls. In CD colon explants, treatment with Shh peptide resulted in a significant decrease in the levels of TNF-alpha (p=0.04), IL-17 (p=0.01), and TGF-beta (p=0.03). Caspase-3 activity was higher in CD and significantly decreased after blockade of Hh, either with anti-Shh antibody(p=0.04) or with cyclopamine (p=0.03), in contrast to UC and controls. Levels of Gli-1 negatively correlated to caspase-3 activity (CC -0.75, p=0.01). Conclusion: Normal colonic mucosa constitutively expresses Hh-proteins, which appear to constitute a key pathway for intestinal homeostasis. Hh-signaling affects cytokine production and the control of apoptosis within the colonic mucosa. Disruption of the intestinal Hhpathway might be implicated in the pathogenesis of CD.

Pre-gastrula expression of zebrafish extraembryonic genes

BackgroundMany species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).ResultsWe hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent.ConclusionWe have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.

Different Thymosin Beta 4 Immunoreactivity in Foetal and Adult Gastrointestinal Tract

BackgroundThymosin beta 4 (Tβ4) is a member of beta-thymosins, a family of peptides that play essential roles in many cellular functions. A recent study from our group suggested a role for Tβ4 in the development of human salivary glands. The aim of this study was to analyze the expression of Tβ4 in the human gut during development, and in the adult.Methodology/Principal FindingsImmunolocalization of Tβ4 was studied in autoptic samples of tongue, oesophagus, stomach, ileum, colon, liver and pancreas obtained from two human foetuses and two adults. Tβ4 appeared unevenly distributed, with marked differences between foetuses and adults. In the stomach, superficial epithelium was positive in foetuses and negative in adults. Ileal enterocytes were strongly positive in the adult and weakly positive in the foetuses. An increase in reactivity for Tβ4 was observed in superficial colon epithelium of adults as compared with the foetuses. Striking differences were found between foetal and adult liver: the former showed a very low reactivity for Tβ4 while in the adult we observed a strong reactivity in the vast majority of the hepatocytes. A peculiar pattern was found in the pancreas, with the strongest reactivity observed in foetal and adult islet cells.SignificanceOur data show a strong expression of Tβ4 in the human gut and in endocrine pancreas during development. The observed differential expression of Tβ4 suggests specific roles of the peptide in the gut of foetuses and adults. The observed heterogeneity of Tβ4 expression in the foetal life, ranging from a very rare detection in liver cells up to a diffuse reactivity in endocrine pancreas, should be taken into account when the role of Tβ4 in the development of human embryo is assessed. Future studies are needed to shed light on the link between Tβ4 and organogenesis.

Eimeria tenella: Effects of diclazuril treatment on microneme genes expression in second-generation merozoites and pathological changes of caeca in parasitized chickens

The effects of diclazuril on mRNA expression levels of invasion-related microneme genes were examined in second-generation merozoites of Eimeria tenella (E. tenella) by quantitative real-time (QRT) PCR. Diclazruil treatment of infected chickens significantly decreased the number of second-generation merozoites by 65.13%, and resulted in downregulation of EtMIC genes: EtMIC1 by 65.63%, EtMIC2 by 64.12%, EtMIC3 by 56.82%, EtMIC4 by 73.48%, and EtMIC5 by 78.17%. SEM images of caecum tissue from uninfected chickens showed regular intestinal villus structure. In infected chickens, a distinct loss of the superficial epithelium, with a flattened mucosa and large-area necrosis and anabrosis, was evident. In diclazruil-treated chickens, a decrease in merozoite number and a visibly improved appearance of the caeca were noted. These improvements appeared to be mediated in part by downregulation of the expression of invasion-related EtMIC genes in response to diclazuril.

Esophagitis Dissecans Superficialis (“Sloughing esophagitis”)

Esophagitis dissecans superficialis (EDS) is a term applied to a rare endoscopic finding characterized by sloughing of large fragments of the esophageal squamous mucosa that may be coughed up or vomited. Although EDS has been reported in association with certain medications and esophageal strictures, most cases remain unexplained and the histopathologic features of EDS are inadequately described. We undertook this study to define useful diagnostic criteria based on the examination of a series of well-characterized cases of EDS. To identify patients with EDS, we searched our endoscopy and pathology databases, reviewed the esophageal biopsy specimens from candidate cases, and correlated them with pertinent clinical information. Twelve patients (11 men and 1 woman) had endoscopic and histologic findings of EDS and 9 had the histologic features without the endoscopic correlates. Biopsies from confirmed EDS patients showed sloughing and flaking of superficial squamous epithelium with occasional bullous separation of the layers, parakeratosis, and varying degrees of acute or chronic inflammation. Fungal elements were identified in 3 patients, but were not associated with acute inflammation. None of the EDS patients were on bisphosphonate therapy or had bullous skin disorders. Follow-up endoscopy in 5 patients showed complete resolution of the esophageal abnormalities in 4 and mild esophagitis in one. In spite of its sometimes, dramatic presentation, EDS is a benign condition that resolves without lasting esophageal pathology. Although an association with medications, skin conditions, heavy smoking, and physical trauma has been reported, the pathogenesis of EDS remains unexplained.

Novel pathways for implantation and establishment and maintenance of pregnancy in mammals

Uterine receptivity to implantation varies among species, and involves changes in expression of genes that are coordinate with attachment of trophectoderm to uterine lumenal and superficial glandular epithelia, modification of phenotype of uterine stromal cells, silencing of receptors for progesterone and estrogen, suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, angiogenesis and vasculogenesis, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine epithelial and stromal cells in response to progesterone, glucocorticoids, prostaglandins and interferons may influence uterine receptivity to implantation in mammals. Uterine receptivity to implantation is progesterone-dependent; however, implantation is preceded by loss of expression of receptors for progesterone (PGR) so that progesterone most likely acts via PGR-positive stromal cells throughout pregnancy. Endogenous retroviruses expressed by the uterus and/or blastocyst also affect implantation and placentation in various species. Understanding the roles of the variety of hormones, growth factors and endogenous retroviral proteins in uterine receptivity for implantation is essential to enhancing reproductive health and fertility in humans and domestic animals.

Tissue transglutaminase expression in celiac mucosa: an immunohistochemical study

Tissue transglutaminase (tTG) constitutes the main autoantigen in celiac disease (CD). The aim of the study was to clarify weather celiac disease is associated with changes in tTG expression in duodenal mucosa. Tissue transglutaminase was assessed immunohistochemically (clone CUB 7402) in duodenal biopsy specimens from 22 untreated CD patients, ten normal controls (NC) with unremarkable duodenal mucosa, and nine disease nonceliac controls (DC). In 15 CD patients duodenal biopsy specimens were repeatedly assessed after these patients had been prescribed gluten-free diet. Positive pixel count algorithm of ImageScope was used for quantitative evaluation of immunohistochemistry. Tissue transglutaminase expression in superficial epithelium differed significantly between the three groups (p < 0.001). It was increased in DC in relation to NC (p < 0.001) and in CD--in relation to NC (p < 0.001) and DC (p = 0.003). In CD and DC, cryptal epithelium was stained more intensively than in NC (p < 0.001), but there was no difference between CD and DC (p = 0.507). The same pattern was seen in lamina propria. A significant decrease in tTG expression in all the compartments was seen in repeatedly assessed samples. Untreated CD is associated with tTG overexpression, which is reversible. Tissue transglutaminase up-regulation does not seem to be specific for CD and can appear in other pathological conditions.