Disparate effects of non-steroidal anti-inflammatory drugs on apoptosis in guinea-pig gastric mucous cells: inhibition of basal apoptosis by diclofenac

Non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo-oxygenase-2 selective inhibitor, NS-398, on a primary culture of guinea-pig gastric mucous epithelial cells. Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase-3-like activity. Necrosis was evaluated from release of cellular enzymes. Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N-Hexanoyl-D-sphingosine (C6-ceramide). Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase-3-like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6-ceramide. The inhibitory effect of diclofenac on basal caspase-3-like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid. Inhibition of superoxide production by the cells increased caspase-3-like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production. Diclofenac inhibited caspase-3-like activity in cell homogenates and also inhibited human recombinant caspase-3. In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea-pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity

Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (β)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Molecular Mechanisms of Airway Epithelial Progenitor Cell Maintenance and Repair.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Reduced epithelial suppressor of cytokine signalling 1 in severe eosinophilic asthma

Severe asthma represents a major unmet clinical need. Eosinophilic inflammation persists in the airways of many patients with uncontrolled asthma, despite high-dose inhaled corticosteroid therapy. Suppressors of cytokine signalling (SOCS) are a family of molecules involved in the regulation of cytokine signalling via inhibition of the Janus kinase-signal transducers and activators of transcription pathway. We examined SOCS expression in the airways of asthma patients and investigated whether this is associated with persistent eosinophilia.

Healthy controls, mild/moderate asthmatics and severe asthmatics were studied. Whole genome expression profiling, quantitative PCR and immunohistochemical analysis were used to examine expression of SOCS1, SOCS2 and SOCS3 in bronchial biopsies. Bronchial epithelial cells were utilised to examine the role of SOCS1 in regulating interleukin (IL)-13 signalling in vitro.

SOCS1 gene expression was significantly lower in the airways of severe asthmatics compared with mild/moderate asthmatics, and was inversely associated with airway eosinophilia and other measures of T-helper type 2 (Th2) inflammation. Immunohistochemistry demonstrated SOCS1 was predominantly localised to the bronchial epithelium. SOCS1 overexpression inhibited IL-13-mediated chemokine ligand (CCL) 26 (eotaxin-3) mRNA expression in bronchial epithelial cells.

Severe asthma patients with persistent airway eosinophilia and Th2 inflammation have reduced airway epithelial SOCS1 expression. SOCS1 inhibits epithelial IL-13 signalling, supporting its key role in regulating Th2-driven eosinophilia in severe asthma.

Sub lethal mercuric chloride toxicity induced stress related alterations in the epithelial lining of foot of the freshwater mussel Lamellidens marginalis (Lamarck)

Sub lethal (0.2 ppm) mercuric chloride induced stress related histopathological alterations in the epithelial linings of foot (podium) of the edible freshwater mussel Lamellidens marginalis (Lamarck) were studied using histochemical techniques up to 60 days of exposure. The histomorphological changes were manifested only slowly and its intensity was somewhat proportional to the duration of exposure. The immediate response of the exposed mussels was the altered mucous secretion. There was a progressive incorporation of sulphated glycoproteins into the secretory contents of the mucous cells especially in the first half of the experiment. Marked histopathological changes including necrosis, appearance of pyknotic nuclei, sloughing of epithelial cells and appearance of non-tissue spaces, etc., started appearing during the later half of the experiment. The fag end of the experiment, which witnessed prominent histomorphological changes, was accompanied by highly decreased mucous secretion. KEYWORDS: heavy metal toxicity, mercuric chloride, Lamellidens marginalis, freshwater mussel, histopathology.

Dual Roles of Diadenosine Polyphosphates in Corneal Epithelial Cell Migration

Purpose. To investigate the influence of diadenosine polyphosphates on the rate of corneal epithelial cell migration. Methods. Primary corneal epithelial cell cultures were obtained from New Zealand White rabbits. Immunocytochemical experiments were performed by fixing the cells with 4% paraformaldehyde (PFA) and incubated with cytokeratin 3 primary antibody, which was subsequently incubated with a secondary IgG mouse labeled with FITC, and the cells were observed under confocal microscopy. Migration studies were performed by taking confluent monolayers that were wounded with a pipette tip and challenged with different di- and mononucleotides with or without P2 antagonist (n = 8 each treatment). For concentration–response analysis, compounds were tested in doses ranging from 10−8 to 10−3 M (n = 8). The stability of the dinucleotides was assayed by HPLC, with an isocratic method (n = 4). Results. Cells under study were verified as corneal epithelial cells via the immunocytochemical analysis. Cell migration experiments showed that Ap4A, UTP, and ATP accelerated the rate of healing (5, 2.75, and 3 hours, respectively; P < 0.05; P < 0.001), whereas Ap3A, Ap5A, and UDP delayed it (6.5, 10, and 2 hours, respectively; P < 0.05). ADP did not modify the rate of migration. Antagonists demonstrated that Ap4A and Ap3A did activate different P2Y receptors mediating corneal wound-healing acceleration and delay. Concerning the possible degradation of the dinucleotides, it was almost impossible to detect any products resulting from their cleavage. Conclusions. Based on the pharmacological profile of all the compounds tested, the two main P2Y receptors that exist in these corneal cells are a P2Y2 receptor accelerating the rate of healing and a P2Y6 receptor that delays this process.

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

Indole-3-carbinol Attenuates The Deleterious Gestational Effects Of Bisphenol A Exposure On The Prostate Gland Of Male F1 Rats.

Bisphenol A (BPA) is a chemical that has been investigated for it potential to cause prostate diseases. In this study, pregnant Sprague-Dawley rats were treated with 25 or 250 μg/kg BPA from gestational day (GD) 10 to GD21 with or without concurrent indole-3-carbinol (I3C) feeding. I3C is a phytochemical, and it affords chemoprotection against many types of neoplasia. Male F1 rats from different litters were euthanized on post-natal day (PND) 21 and PND180. BPA-treated groups showed a significant increase in histopathological lesions, but I3C feeding reversed many of these changes, mainly at PND180. Maternal I3C feeding increased prostate epithelial apoptosis in the BPA-treated groups and across age groups. Furthermore, I3C induced partial normalization of the prostate histoarchitecture. The results pointed to a protective effect of maternal I3C feeding during pregnancy in the BPA-exposed male offspring, thereby indicating reduction in the harmful effects of gestational BPA imprinting on the prostate.

Pituitary Macroadenoma Presenting As A Nasal Tumor: Case Report.

Pituitary macroadenomas are rare intracranial tumors. In a few cases, they may present aggressive behavior and invade the sphenoid sinus and nasal cavity, causing unusual symptoms. In this paper, we report an atypical case of pituitary adenoma presenting as a nasal mass. The patient was a 44-year-old woman who had had amenorrhea and galactorrhea for ten months, with associated nasal obstruction, macroglossia and acromegaly. Both growth hormone and prolactin levels were increased. Magnetic resonance imaging showed a large mass originating from the lower surface of the pituitary gland, associated with sella turcica erosion and tumor extension through the sphenoid sinus and nasal cavity. Histopathological analysis demonstrated a chromophobe pituitary adenoma with densely packed rounded epithelial cells, with some atypias and rare mitotic figures. There was no evidence of metastases. Macroadenoma invading the nasal cavity is a rare condition and few similar cases have been reported in the literature. This study contributes towards showing that tumor extension to the sphenoid sinus and nasopharynx needs to be considered and investigated in order to make an early diagnosis when atypical symptoms like nasal obstruction are present.

Gut Bacteria Products Prevent Aki Induced By Ischemia-reperfusion.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.

Aspectos ultra-estruturais dos canais secretores em órgãos vegetativos e reprodutivos de Anacardiaceae

The study assessed phloem canal development and ultra-structure in shoot apices of Spondias dulcis G. Forst., phloematic canal ultra-structure in shoot apices of Tapirira guianensis Aubl., and floral canal ultra-structure and development and fruit canal ultra-structure of the latter specie. The flower and fruit canals of Anacardium humile St.Hil. were also studied ultra-structurally. The canals in shoot apices of S. dulcis show schizo-lysigenous formation and the floral canals of T. guianensis show schizogenous development. Epithelial cells of S. dulcis and T. guianensis canals have rough endoplasmic reticulum, free ribosomes, elongated plastids of several shapes with osmiophilic inclusions and dictyosomes with production of vesicles. Such organelles participate in the secretion of a heterogeneous exudate, which is comprised of hydrophilic and lipophilic substances. The epithelial cells of the fruit of A. humile present elongated plastids with circular membrane system, which are involved in the synthesis of lipophilic substances. The results of the ultra-structural analyses of the epithelial cells corroborate the results previously obtained in a histochemical study. In the histochemical study, lipophilic and hydrophilic substances were identified in the canals of T. guinanensis and S. dulcis and only lipophilic substances were identified in the canals of A. humile. Based on the ultrastructural aspects of the secretory canals of T. guianensis and S. dulcis we concluded that the plastids of the epithelial cells of the two species are different although they produce secretion of similar composition. A new record for the family is the presence of a great number of circular plastids in epithelial cells of the fruit of Anacardium humile. The pattern found in the secretory canals of the studied species is the ecrine type of secretion release.

Vimentin expression and the influence of Matrigel in cell lines of head and neck squamous cell carcinoma

Vimentin is a cytoeskeletal intermediate filament protein commonly observed in mesenchymal cells; however, it can also be found in malignant epithelial cells. It is demonstrated in several carcinomas, such as those of the cervix, breast and bladder, in which it is widely used as a marker of the epithelial to mesenchymal transition that takes place during embryogenesis and metastasis. Vimentin is associated with tumors that show a high degree of invasiveness, being detected in invasion front cells. Its expression seems to be influenced by the tumor microenvironment. The aim of this study was to evaluate vimentin expression in head and neck squamous cell carcinoma (HNSCC) cell lines, and to investigate the contribution of the microenvironment to its expression. HNSCC cell lines (HN6, HN30 and HN31) and an immortalized nontumorigenic cell line (HaCaT) were submitted to a three-dimensional assay with Matrigel. Cytoplasmatic staining of the HN6 cell line cultured without Matrigel and of the HN30 and HN31 cell lines cultured with Matrigel was demonstrated through immunohistochemistry. Western Blotting revealed a significant decrease in vimentin expression for the HN6 cell line and a significant increase for the HN30 and HN31 cell lines cultured with Matrigel. The results suggest that vimentin can be expressed in HNSCC cells and its presence is influenced by the microenvironment of a tumor.

Nerve endings of filliform, fungiform and vallate papillae of dorsal tongue mucosa of White-lipped peccary (Tayassu pecari): Neurohistological observations

The neurohistologic observations were performed using the specimens prepared by Winkelmann and Schmitt silver impregnation method. The tissues were fixed in 10% formalin solution and sections of 40µm thickness were obtained by Leica Cryostat at -30ºC. The sections of dorsal mucosa of White-lipped peccary tongue showed numerous filliform and fungiform papillae, and two vallate papillae on the caudal part. The epithelial layer revealed queratinized epithelial cells and the connective tissue papillae of different sizes and shapes. Thick nerve fiber bundles are noted into the subepithelial connective tissue of the papillae. The connective tissue of fungiform and vallate papillae contained numerous sensitive nerves fibers bundles forming a complex nerve plexus.