Neural Wiskott-Aldrich syndrome protein modulates Wnt signaling and is required for hair follicle cycling in mice.

The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Pseudomonas aeruginosa adhesion to normal and injured respiratory mucosa

Human nasal polyps outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibrocnectin-containing fibrillar material The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

Altered expression of CD44 and DKK1 in the progression of Barrett's esophagus to esophageal adenocarcinoma.

Barrett's esophagus (BE) is an acquired condition in which the normal lining of the esophagus is replaced by intestinal metaplastic epithelium. BE can evolve to esophageal adenocarcinoma (EAC) through low-grade dysplasia (LGD) and high-grade dysplasia (HGD). The only generally accepted marker for increased risk of EAC is the presence of HGD, diagnosed on endoscopic biopsies. More specific markers for the prediction of EAC risk are needed. A tissue microarray was constructed comprising tissue samples from BE, LGD, HGD, and EAC. Marker expression was studied by immunohistochemistry using antibodies against CD44, DKK1, CDX2, COX2, SOX9, OCT1, E-cadherin, and beta-catenin. Immunostaining was evaluated semi-quantitatively. CD44 expression decreased in HGD and EAC relative to BE and LGD. DKK1 expression increased in HGD and EAC relative to BE and LDG. CDX2 expression increased in HGD but decreased in EAC. COX2 expression decreased in EAC, and SOX9 expression increased only in the upper crypt epithelial cells in HGD. E-cadherin expression decreased in EAC. Nuclear beta-catenin was not significantly different between BE, LGD, and HGD. Loss of CD44 and gain of DKK1 expression characterizes progression from BE and LGD to HGD and EAC, and their altered expression might indicate an increased risk for developing an EAC. This observation warrants inclusion of these immunohistochemically detectable markers in a study with a long patient follow-up.

Tyzzeria boae n. sp., (Apicomplexa: Eimeriidae), a New Coccidium from the Kidney of the snake Boa constrictor constrictor (Serpentes: Boidae)

A new species of Tyzzeria is described from the kidney of the snake Boa constrictor constrictor Linnaeus, from the State of Pará, north Brazil. Oocysts from the coacal contents matured in eight days, at approximately 24°C. They measured 19.0 x 18.0 (15.0 x 15.0 - 22.5 x 21.5) µm, shape-index (length/width) 1.0 (1.0 - 1.1). The oocyst wall is of an extremely delicate single, colourless layer, with no micropyle. Division of the oocyst contents into the 8 naked sporozoites leaves a bulky, spherical oocyst residuum averaging 15.5 x 14.8 (13.5 x 13.5 - 18.5 x 17.5) µm; the sporozoites measure an average of 11.0 x 1.8 (8.5 x 1.25 - 12.5 x 2.0) µm, and possess both anterior and posterior refractile bodies. Tyzzeria boae n.sp. is unique among the recorded species of the genus by virtue of its development in the epithelial cells of the distal convoluted tubules and collecting tubules of the kidney: stages in the merogony and gametogony of the parasite are described and figured.

Occurrence of Sciadicleithrum mexicanum Kritsky, Vidal-Martinez et Rodríguez-Canul, 1994 (Monogenea: Dactylogyridae) in the Cichlid Cichlasoma urophthalmus from a flooded quarry in Yucatan, Mexico

Cichlids, Cichlasoma urophthalmus, collected in a flooded quarry in the Yucatan Peninsula, Mexico, from January through June 1992, had high levels of infection with the ancyrocephaline Sciadicleithrum mexicanum (Monogena: Dactylogyridade) in all montlhly samples. Neither occurrence nor maturation of the worms eshibited any pronounced monthly fluctuation. The infection rate was found to be sizedependent, greater in longer fish. The worms occurred on primary lamellae of gill filaments of all arches, with lower numbers of parasites attached to the fourth gill arch. Otherwise, there was no significant site preference of worms. Only minor histopathological changes were found at the sites of attachment, and these were restricted to the epithelial cells of the primary lamellae of thegill filaments. The lack of seasonal periodicity in this tropical monogenean is compared to seasonal cycles typical of temperate species.

Schellackia (Apicomplexa: Eimeriidae) of the brazilian tree-frog, Phrynohyas venulosa (Amphibia: Anura) from Amazonian Brazil

Endogenous stages of a Schellackia species are described in histological sections of the intestine of the tree-frog, Phrynohyas venulosa, from North Brazil. Most oocysts sporulate within the epithelial cells of the gut, but a few were detected in the lamina propria.

Lipoxin A4 is a novel estrogen receptor modulator.

Inflammation is intimately linked with naturally occurring remodeling events in the endometrium. Lipoxins comprise a group of short-lived, nonclassic eicosanoids possessing potent anti-inflammatory and proresolution properties. In the present study, we investigated the role of lipoxin A(4) (LXA(4)) in the endometrium and demonstrated that 15-LOX-2, an enzyme necessary for LX biosynthesis, is expressed in this tissue. Our results establish that LXA(4) possesses robust estrogenic activity through its capacity to alter ERE transcriptional activity, as well as expression of estrogen-regulated genes, alkaline phosphatase activity, and proliferation in human endometrial epithelial cells. Interestingly, LXA(4) also demonstrated antiestrogenic potential, significantly attenuating E2-induced activity. This estrogenic activity was directly mediated through estrogen receptors (ERs). Subsequent investigations determined that the actions of LXA(4) are exclusively mediated through ERα and closely mimic those of the potent estrogen 17β-estradiol (E2). In binding assays, LXA(4) competed with E2 for ER binding, with an IC(50) of 46 nM. Furthermore, LXA(4) exhibited estrogenic activity in vivo, increasing uterine wet weight and modulating E2-regulated gene expression. These findings reveal a previously unappreciated facet of LXA(4) bioactions, implicating this lipid mediator in novel immunoendocrine crosstalk mechanisms.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Ultrastructure of Endogenous Stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in Experimentally Infected Goat

The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 105 oocysts/kg. The secondary meronts developed above the nucleus of the host cell. The nucleus first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nucleolus and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleolus. The macrogametes contain a nucleus, wall-forming bodies of type I and type II. The young oocysts present a wall containing two layers and a sporont

Expression and function of beta1 integrins on human eosinophils

Eosinophils preferentially accumulate at sites of chronic allergic diseases such as bronchial asthma. The mechanisms by which selective eosinophil migration occurs are not fully understood. However, interactions of cell-surface adhesion molecules on the eosinophil with molecular counterligands on endothelial and epithelial cells, and on extracellular matrix proteins, are likely to be critical during the recruitment process. One possible mechanism for selective eosinophil recruitment involves the alpha4beta 1 (VLA-4) integrin which is not expressed on neutrophils. Correlations have been found between infiltration of eosinophils and endothelial expression of VCAM-1, the ligand for VLA-4, in the lungs of asthmatic individuals as well as in late phase reactions in the lungs, nose and skin. Epithelial and endothelial cells respond to the Th2-type cytokines IL-4 and IL-13 with selective de novo expression of VCAM-1, consistent with the possible role of VCAM-1/VLA-4 interactions in eosinophil influx during allergic inflammation. Both beta 1 and beta 2 integrins on eosinophils exist in a state of partial activation. For example, eosinophils can be maximally activated for adhesion to VCAM-1 or fibronectin after exposure to beta 1 integrin-activating antibodies or divalent cations, conditions that do not necessarily affect the total cell surface expression of beta 1 integrins. In contrast, cytokines like IL-5 prevent beta 1 integrin activation while promoting beta 2 integrin function. Furthermore, ligation of integrins can regulate the effector functions of the cell. For example, eosinophil adhesion via beta 1 and/or beta 2 integrins has been shown to alter a variety of functional responses including degranulation and apoptosis. Thus, integrins appear to be important in mediating eosinophil migration and activation in allergic inflammation. Strategies that interfere with these processes may prove to be useful for treatment of allergic diseases.

Eimeria peltocephali n. sp., (Apicomplexa:Eimeriidae) from the Freshwater Turtle Peltocephalus dumerilianus (Chelonia:Pelomusidae) and Eimeria molossi n. sp., from the Bat, Molossus ater (Mammalia:Chiroptera)

The oocyst is described of Eimeria peltocephali n.sp. from faeces of the freshwater turtle Peltocephalus dumerilianus from Barcelos, State of Amazonas, Brazil. Sporulation is exogenous and fully developed oocysts are elongate, ellipsoidal or cylindrical, frequently curved to a banana-shape, 54.4 x19.1 (37.5 - 68.7 x 18.7-20.0 µm), shape-index 2.8 (1.8 -3.9). The oocyst wall is a single thin, colourless layer about 1 µm thick, with no micropyle. There is a bulky oocyst residuum, at first spherical to ellipsoidal, 19 x 16 (16. 2 -26.2 x 16 - 21.5µm) , but becoming dispersed on maturation. There are no polar bodies. The sporocysts, 19.1 x 6.8 ( 17.5 -21.2 x 6.2 -7.5 µm), shape- index 2.8 (2.3 -3.2), are usually disposed in pairs at each end of the oocyst, and bear an inconspicuous Stieda body in the form of a flat cap. The sporozoites are elongate and slightly curved around the residuum. No refractile bodies were seen. Eimeria molossi n.sp., is described from the molossid bat Molossus ater. Sporulation is exogenous and the mature oocysts are predominantly broadly ellipsoidal, 23.4 x 17.5 (18-30 x 15-22.5 µm), shape-index 1.3 (1-1.6). The oocyst wall is about 2 µm thick, and of three layers: an inner thin, colourless one and two outer layers which are thicker, yellowish-brown, prominently striated and in close apposition. There is no micropyle or oocyst residuum, but one and occasionally two polar bodies are usually present. Sporocysts are ellipsoidal, 10.2 x 7.5 (10-12.5 x 7.5 µm), shape-index 1.4 (1.3-1.7) with an inconspicuous Stieda body. Endogenous stages are described in the epithelial cells of the small intestine

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Progesterone/RANKL is a major regulatory axis in the human breast.

Estrogens and progesterones are major drivers of breast development but also promote carcinogenesis in this organ. Yet, their respective roles and the mechanisms underlying their action in the human breast are unclear. Receptor activator of nuclear factor κB ligand (RANKL) has been identified as a pivotal paracrine mediator of progesterone function in mouse mammary gland development and mammary carcinogenesis. Whether the factor has the same role in humans is of clinical interest because an inhibitor for RANKL, denosumab, is already used for the treatment of bone disease and might benefit breast cancer patients. We show that progesterone receptor (PR) signaling failed to induce RANKL in PR(+) breast cancer cell lines and in dissociated, cultured breast epithelial cells. In clinical specimens from healthy donors and intact breast tissue microstructures, hormone response was maintained and RANKL expression was under progesterone control, which increased RNA stability. RANKL was sufficient to trigger cell proliferation and was required for progesterone-induced proliferation. The findings were validated in vivo where RANKL protein expression in the breast epithelium correlated with serum progesterone levels and the protein was expressed in a subset of luminal cells that express PR. Thus, important hormonal control mechanisms are conserved across species, making RANKL a potential target in breast cancer treatment and prevention.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2.

Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic beta-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice. In beta-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low or almost undetectable levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and beta-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, beta-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the alpha- to beta-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.