Gastrointestinal tract mucosal histomorphometry and epithelial cell proliferation and apoptosis in neonatal and adult dogs

In this study, the hypothesis was tested that the size of gastrointestinal tract (GIT) mucosal components and rates of epithelial cell proliferation and apoptosis change with increasing age. The aims were to quantitatively examine GIT histomorphology and to determine mucosal epithelial cell proliferation and apoptosis rates in neonatal (<48 h old) and adult (8 to 11.5 yr old) dogs. Morphometrical analyses were performed by light microscopy with a video-based, computer-linked system. Cell proliferation and apoptosis of the GIT epithelium were evaluated by counting the number of Ki-67 and caspase-3-positive cells, respectively, using immunohistochemical methods. Thickness of mucosal, glandular, subglandular, submucosal and muscular layers, crypt depths, villus heights, and villus widths were consistently greater (P < 0.05 to P < 0.001), whereas villus height/crypt depth ratios were smaller (P < 0.001) in adult than in neonatal dogs. The number of Ki-67-positive cells in stomach, small intestine, and colon crypts, but not in villi, was consistently greater (P < 0.01) in neonatal than in adult dogs. In contrast, the number of caspase-3-positive cells in crypts of the stomach, small intestine, and colon and in villi was not significantly influenced by age. In conclusion, canine GIT mucosal morphology and epithelial cell proliferation rates, but not apoptosis rates, change markedly from birth until adulthood is reached.

Host cell responses of Salmonella typhimurium infected human dendritic cells

Live attenuated Salmonella are attractive vaccine candidates for mucosal application because they induce both mucosal immune responses and systematic immune responses. After breaking the epithelium barrier, Salmonella typhimurium is found within dendritic cells (DC) in the Peyer's patches. Although there are abundant data on the interaction of S. typhimurium with murine epithelial cells, macrophages and DC, little is known about its interaction with human DC. Live attenuated S. typhimurium have recently been shown to efficiently infect human DC in vitro and induce production of cytokines. In this study, we have analysed the morphological consequences of infection of human DC by the attenuated S. typhimurium mutant strains designated PhoPc, AroA and SipB and the wild-type strains of the American Type Culture Collection (Manassas, VA, USA), ATCC 14028 and ATCC C53, by electron microscopy at 30 min, 3 h and 24 h after exposure. Our results show that genetic background of the strains profoundly influence DC morphology following infection. The changes included (i) membrane ruffling; (ii) formation of tight or spacious phagosomes; (iii) apoptosis; and (iv) spherical, pedunculated membrane-bound microvesicles that project from the plasma membrane. Despite the fact that membrane ruffling was much more pronounced with the two virulent strains, all mutants were taken up by the DC. The microvesicles were induced by all the attenuated strains, including SipB, which did not induce apoptosis in the host cell. These results suggest that Salmonella is internalized by human DC, inducing morphological changes in the DC that could explain immunogenicity of the attenuated strains.

Spindle cell oncocytoma of the adenohypophysis: report of a case with a 16-year follow-up

Spindle cell oncocytoma (SCO) is a recently described, rare neoplasm of the anterior pituitary. Clinically and radiologically simulating a non-functioning macroadenoma, its eponymous fusiform cells display a non-epithelial phenotype with conspicuous cytoplasmic accumulation of mitochondria. We report a case of SCO retrospectively identified in a biopsy specimen 16 years after transsphenoidal operation of a 48-year-old woman. Presenting symptoms were adynamia and transient decrease of visual acuity. Neuroimaging showed an isointense, enhancing, sellar-centered mass 1.8 cm in diameter without evidence of invasive growth. No postoperative adjuvant therapy was administered. The patient was left with panhypopituitarism, yet no recurrence was seen during follow-up. Initially diagnosed as a null cell adenoma of oncocytic type, repeat immunohistochemistry showed the characteristic coexpression of S100 protein, vimentin, and epithelial membrane antigen. Oncocytic granula stained intensely with antimitochondrial antibody 113-1, and were negative with the lysosomal marker CD68. Anterior pituitary hormones tested negative, and there was no evidence of neuroendocrine differentiation using antibodies to synaptophysin and chromogranin. Few cells stained for glial fibrillary acidic protein (GFAP). SCO has been proposed to represent a neoplasm of folliculo-stellate cells (FSCs). While the dynamic properties of the latter are incompletely characterized, and indeed no specific marker allows for their identification, overlapping features of SCO with look alikes, in particular pituicytoma, point to FSCs being a potential adult stem cell. The favorable outcome of the present case further argues for SCO to be considered a low-grade neoplasm. Moderate tumor size, lack of invasiveness, and low proliferation rate are likely predictors of benign behavior.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis

Diarrhea remains a significant cause of worldwide morbidity and mortality. Over 4 million children die of diarrhea annually. Although antibiotics can be used as prophylaxis or for treatment of diarrhea, concern remains over antibiotic resistance. Rifaximin is a semi-synthetic rifamycin derivative that can be used to treat symptoms of infectious diarrhea, inflammatory bowel syndrome, bacterial overgrowth of the small bowel, pouchitis, and fulminant ulcerative colitis. Rifaximin is of particular interest because it is poorly adsorbed in the intestines, shows no indication of inducing bacterial resistance, and has minimal effect on intestinal flora. In order to better understand how rifaximin functions, we sought to compare the protein expression profile of cells pretreated with rifaximin, as compared to cells treated with acetone, rifamycin (control antibiotic), or media (untreated). 2-D gel electrophoresis identified 38 protein spots that were up- or down-regulated by over 2-fold in rifaximin treated cells compared to controls. 16 of these spots were down-regulated, including keratin, annexin A5, intestinal-type alkaline phosphatase, histone h4, and histone-binding protein RbbP4. 22 spots were up-regulated, including heat shock protein HSP 90 alpha, alkaline phosphatase, and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. A better understanding of the functionality of rifaximin will identify additional potential uses for rifaximin and determine for whom the drug is best suited. ^

Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2 domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation

Sik, the mouse homologue of the breast tumor kinase Brk, is expressed in differentiating cells of the gastrointestinal tract and skin. We examined expression and activity of Sik in primary mouse keratinocytes and a mouse embryonic keratinocyte cell line (EMK). Calcium-induced differentiation of these cells has been shown to be accompanied by the activation of tyrosine kinases and rapid phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65). We demonstrate that Sik is activated within 2 min after calcium addition in primary keratinocytes and EMK cells. In EMK cells, Sik binds GAP-A.p65, and this interaction is mediated by the Sik Src homology 2 domain. Although Sik directly complexes with GAP-A.p65, overexpression of wild-type or kinase defective Sik in EMK cells does not lead to detectable changes in GAP-A.p65 phosphorylation. These data suggest that Sik is not responsible for phosphorylation of GAP-A.p65. GAP-A.p65 may act as an adapter protein, bringing Sik into proximity of an unidentified substrate. Overexpression of Sik in EMK cells results in increased expression of filaggrin during differentiation, supporting a role for Sik in differentiation.

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.

Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation

Chemically induced skin carcinomas in mice are a paradigm for epithelial neoplasia, where oncogenic ras mutations precede p53 and INK4a/ARF mutations during the progression toward malignancy. To explore the biological basis for these genetic interactions, we studied cellular responses to oncogenic ras in primary murine keratinocytes. In wild-type keratinocytes, ras induced a cell-cycle arrest that displayed some features of terminal differentiation and was accompanied by increased expression of the p19ARF, p16INK4a, and p53 tumor suppressors. In ARF-null keratinocytes, ras was unable to promote cell-cycle arrest, induce differentiation markers, or properly activate p53. Although oncogenic ras produced a substantial increase in both nucleolar and nucleoplasmic p19ARF, Mdm2 did not relocalize to the nucleolus or to nuclear bodies but remained distributed throughout the nucleoplasm. This result suggests that p19ARF can activate p53 without overtly affecting Mdm2 subcellular localization. Nevertheless, like p53-null keratinocytes, ARF-null keratinocytes were transformed by oncogenic ras and rapidly formed carcinomas in vivo. Thus, oncogenic ras can activate the ARF-p53 program to suppress epithelial cell transformation. Disruption of this program may be important during skin carcinogenesis and the development of other carcinomas.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Contactus adherens, a special type of plaque-bearing adhering junction containing M-cadherin, in the granule cell layer of the cerebellar glomerulus.

In the glomeruli of the granule cell layer of mammalian cerebellum, neuronal extensions are interconnected by numerous small, nearly isodiametric (diameters up to 0.1 micron), junctions previously classified as puncta adherentia related to the vinculin-containing, actin microfilament-anchoring junctions of the zonula adherens of epithelial and certain other cells. Using immunofluorescence and immunoelectron microscopy, we have found, however, that these junctions are negative for E- and VE-cadherin, for desmosomal cadherins, and also for vinculin, alpha-actinin, and desmoplakin, but they do contain, in addition to the protein plakoglobin common to all forms of adhering junctions, the plaque proteins alpha- and beta-catenin and the transmembrane glycoprotein M-cadherin previously found as a spread--i.e., not junction bound--plasma membrane protein in certain fetal and regenerating muscle cells and in satellite cells of adult skeletal muscle. We conclude that these M-cadherin-containing junctions of the granule cell layer represent a special type of adhering junction, for which we propose the term contactus adherens (from the Latin contactus, for touch, site of bordering upon, also influence), and we discuss the differences between the various adhering junctions on the basis of their molecular constituents.

Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Retromer controls epithelial cell polarity by trafficking the apical determinant crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.