Francisco Javier Hernandez-Blazquez

Progranulin is a stress-response factor in fibroblasts subjected to hypoxia and acidosis.

The growth factor progranulin (granulin–epithelin precursor, PC-derived growth factor or acrogranin) regulates proliferation and migration and is implicated in cancer, development, wound repair and neurodegenerative diseases. Under most conditions fibroblasts do not express progranulin in vivo, however its expression is activated following wounding. We hypothesised that progranulin is part of a fibroblast stress response. Fibroblasts in culture were exposed to two physiologically and clinically relevant microenvironmental stresses; hypoxia (1% oxygen) and acidosis, both of which increase progranulin expression. The greatest increases occurred when hypoxia and acidosis were combined. Increased progranulin expression is not a direct response to apoptosis since it occurred under conditions of pH and hypoxia under which cell viability remained high. Low concentrations of progranulin (2 nM) protected fibroblasts from apoptosis induced by extreme acidosis (pH 5.0 and 4.0). We propose that progranulin is part of a fibroblast stress response and is cytoprotective to acidotic stress.

Identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma

Hepatic progenitor cells (HPCs) are bipotential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic or cholangiocytic lineages. HPCs are present in both hepatocellular (HCC) and cholangiocellular carcinoma (CC) in humans; and a small percentage of HCC can originate from cancer stem cells. However, its distribution in canine liver tumour has not been studied. Herein, we searched for stem/progenitor cells in 13 HCC and 7 CC archived samples by immunohistochemical analysis. We found that both liver tumours presented a higher amount of K19-positive HPCs. Besides, 61.6% of HCC cases presented immature CD44-positive hepatocytes. Nevertheless, only two cases presented CD133-positive cells. As observed in humans, hepatic canine tumours presented activated HPCs, with important differentiation onto hepatocytes-like cells and minimal role of cancer stem cells on HCC. These findings reiterate the applicability of canine model in the search for new therapies before application in humans.

Morphological and molecular pathology of CCL4-induced hepatic fibrosis in connexin43-deficient mice.

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl4)‐induced hepatic fibrosis in mice. We administered CCl4 to both Cx43‐deficient (Cx43+/−) and wild‐type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real‐time PCR. We demonstrated that Cx43+/− mice developed excessive liver fibrosis compared with wild‐type mice after CCl4‐induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43+/− mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43+/− mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl4‐treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech., 2011.

Roles of Gap Junctions and Connexins in Non-Neoplastic Pathological Processes in which Cell Proliferation Is Involved

Cell proliferation is an important process for reproduction, growth and renewal of living cells and occurs in several situations during life. Cell proliferation is present in all the steps of carcinogenesis, initiation, promotion and progression. Gap junctions are the only specialization of cell membranes that allows communication between adjacent cells. They are known to contribute to tissue homeostasis and are composed of transmembrane proteins called “connexins.” These junctions are also known to be involved in cell proliferation control. The roles of gap junctions and connexins in cell proliferation are complex and still under investigation. Since pioneer studies by Loewenstein, it is known that neoplastic cells lack communicating junctions. They do not communicate with their neighbors or with non-neoplastic cells from the surrounding area. There are many studies and review articles dedicated to neoplastic tissues. The aim of this review is to present evidence on the roles of gap junctions and connexins in non-neoplastic processes in which cell proliferation is involved.

Immunohistochemical Characterization of Canine Prostatic Intraepithelial Neoplasia

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR.

Intestinal histology of a detritivorous (iliophagous) fish Prochilodus scrofa (characiformes, prochilodontidae)

Abstract The intestinal epithelium of Prochilodus scrofa, a freshwater teleost fish, was studied by means of light microscopic techniques. The samples were obtained from the initial, middle and terminal portions of each intestinal loop. The pyloric caeca are lined by a simple columnar epithelium with a small striated border and a very thick basal membrane. Goblet cells, lymphocytes and eosinophil granular cells (ECGs) were observed in the epithelium of all the intestinal loops. The lamina propria of the third intestinal loop is characterized by a high glandular density. At the fourth loop and up to the rectal segment, thick flaps of mucous membrane project into the lumen, resembling the valves found in mammalian veins, apparently preventing the reflux of intestinal contents. At the fourth and fifth intestinal loops an intense intraepithelial invasion of phagocytic cells occurs (macrophage, ECG and lymphocyte). Goblet cell concentration is higher at the sixth intestinal loop and rectal segment.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat.

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL.kg-1.day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-alpha1 (-53%), TIMP-1 (-31.7%), TGF-beta1 (-57.7%), and MMP-2 (-41.6%), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1%), ALT (-17.6%), and AST (-12.2%) serum levels; c) increased liver weight (11.3%), and reduced liver collagen (-37.1%), regenerative nodules size (-22.1%), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Deletion of a single allele of Cx43 is associated with a reduction in the gap junctional intercellular communication and increased cell proliferation of mouse lung pneumocytes type II

Abstract.  Objectives: Connexins (Cx) are proteins that form the gap junctional channels at neighbouring plasma membranes between adjacent cells. Cxs are involved in cell communication, which is reportedly correlated with cell proliferation and differentiation. Alterations in connexin expression and/or gap junctional intercellular communication (GJIC) capacity have long been postulated to be important in a number of pathological conditions including cancer. This study was performed to determine the consequences of the deletion of a single allele of Gja1 (Cx43 gene) in Alveolar Type II cells (APTIIs), and its impact on GJIC and cell proliferation. Material and methods: In order to do so, APTIIs from wild type (Cx43+/+) and heterozygous (Cx43+/–) mice were harvested and cultured for 4 days. The GJIC capacity was evaluated by scrape‐loading method, with the transfer of lucifer yellow dye. The expression of Cx43 was evaluated by immunofluorescence method and Western blotting. Cell proliferation was evaluated by 3‐(4,5‐dimethylthazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Results: It was observed that GJIC capacity was significantly reduced and cell proliferation index was significantly higher in Cx43+/– cells compared to Cx43+/+ cells. Conclusions: These results show that knocking out one allele of Cx43 leads to a lower cell to cell communication capacity, and consequently induces a higher cell proliferation. Because chemically induced lung adenomas in mice are known to originate from APTIIs, these alterations may play a critical role in their susceptibility to lung carcinogenesis.

IgA production, coliforms analysis and intestinal mucosa morphology of piglets that received probiotics with viable or inactivated cells

Two types of probiotics were used in piglets. One product is a mixed culture of viable Lactobacillus acidophilus, Enterococcus faecium e Bifidobacterium bifidum. The second product is composed of inactivated Lactobacillus acidophilus cells. The piglets received two weekly oral doses for 30 days while a control group did not receive probiotics. All piglets were euthanized at the 30th day of life and the mesenteric lymph nodes, the small intestine, and blood samples were collected. The tissue samples were studied by light microscopy and the blood serum was analyzed by ELISA method. The treatment with the probiotic with viable cells produced higher serum levels of IgA (P<0.05) and more IgA expressing cells were found in the mesenteric lymph nodes than observed in the inactivated cells treatment or control groups (P<0.05). Also, intestinal villi were longer, crypts were deeper (P<0.05) and fecal coliform count was lower than found in the inactivated product (P<0.05). These results suggest that viable probiotics are more efficient than inactivated probiotics to induce immunostimulation and intestinal modifications in piglets, thus improving their health and development.

Differential effect of chronic aflatoxin B1 intoxication on the growth performance and incidence of hepatic lesions in triploid and diploid rainbow trout (Oncorhynchus mykiss)

El proposito del presente estudio fue comparar el crecimiento y la incidencia de lesiones hepaticas en truchas triploides y diploides tratadas con aflatoxina B1(AFB1). 240 truchas fueron divididas en 4 grupos: DC: truchas diploides alimentadas con racion sin AFB1; TC: truchas triploides alimentadas con racion sin AFB1; DT: truchas tratadas con racion con 80 ppb de AFB1 y TT: truchas triploides alimentadas con racion con 80 ppb de AFB1. Durante doce meses, mensualmente, cinco ejemplares de cada grupo fueron anestesiados y sacrificados. Con posteiroridad a la obtencion del peso y medicion del tamano de los pesces, muestras hepaticas fueron fijadas en solucion de formalina salina 10% y procesadas para analisis histopatologico. El analisis comparativo del rendimiento en crecimiento indicaron diferencias significativas entre truchas diploides del grupo control y del tratado, sugiriendo que AFB1 afecta el crecimiento de las truchas diploides. En truchas triploides no se observaron diferencias entre pesces del grupo control y los pesces tratados. El analisis histopatologico senalo que truchas triploides son mas resistentes a AFB1, ya que ambos grupos tratados presentaron lesiones preneoplasicas, sin embargo, el grupo TT demostro menor incidencia de lesiones e igualmente un desarrollo mas lento de las mismas. En cuanto a la ocurrencia de neoplasia, en el grupo DT 4 pesces desarrolaron carcinoma hepatocelular en el ultimo trimestre del experimento, mientras que ningun animal triploide desarrollo lesion neoplasica