Panx1 knockout promotes preneoplastic aberrant crypt foci development in a chemically induced model of mouse colon carcinogenesis.

Colorectal cancer, which is the third leading cause of cancer-related deaths worldwide, is a multistep disease, featuring preneoplastic aberrant crypt foci (ACF) as the early morphological manifestation. The roles of hemichannel-forming transmembrane Pannexin 1 (Panx1) protein have not been investigated in the context of colon carcinogenesis yet, although it has contrasting roles in other cancer types. Thus, this study was conducted to examine the effects of Panx1 knockout (Panx1-/- ) on the early events of chemically induced colon carcinogenesis in mouse. Wild type (WT) and Panx1-/- female C57BL6J mice were submitted to a chemically induced model of colon carcinogenesis by receiving six intraperitoneal administrations of 1,2-dimethylhydrazine (DMH) carcinogen. Animals were euthanized 8 h (week 7) or 30 weeks (week 37) after the last DMH administration in order to evaluate sub-acute colon toxicity outcomes or the burden of ACF, respectively. At week 7, Panx1 genetic ablation increased DMH-induced genotoxicity in peripheral blood cells, malondialdehyde levels in the colon, and apoptosis (cleaved caspase-3) in colonic crypts. Of note, at week 37, Panx1-/- animals showed an increase in aberrant crypts (AC), ACF mean number, and ACF multiplicity (AC per ACF) by 56%, 57% and 20%, respectively. In essence, our findings indicate that Panx1 genetic ablation promotes preneoplastic ACF development during chemically induced mouse colon carcinogenesis, and a protective role of Panx1 is postulated.

Evaluation of functional candidate biomarkers of non-genotoxic hepatocarcinogenicity in human liver spheroid co-cultures.

Validated in vitro assays for testing non-genotoxic carcinogenic potential of chemicals are currently not available. Consequently, the two-year rodent bioassay remains the gold standard method for the identification of these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of the non-genotoxic carcinogenic processes, however, functional changes induced by effects at transcriptional and translational levels have not been addressed. The present study was set up to test a number of proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity at the functional level using a translational 3-dimensional model. Spheroid cultures of human hepatocytes and stellate cells were exposed to 5 genotoxic carcinogenic, 5 non-genotoxic carcinogenic, and 5 non-carcinogenic chemical compounds and assessed for oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, and inflammation. The spheroid model could capture many of these events triggered by the genotoxic carcinogenic chemicals, particularly aflatoxin B1 and hydroquinone. Nonetheless, no clear distinction could be made between genotoxic and non-genotoxic hepatocarcinogenicity. Therefore, spheroid cultures of human liver cells may be appropriate in vitro tools for mechanistic investigation of chemical-induced hepatocarcinogenicity, however, these mechanisms and their read-outs do not seem to be eligible biomarkers for detecting non-genotoxic carcinogenic chemicals.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates ß-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

BACKGROUND: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. ß-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. RESULTS: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. ß-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. CONCLUSIONS: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces ß-catenin expression in ob/ob mice.

Sorafenib reduces steatosis-induced fibrogenesis in a human 3D co-culture model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) affects around 25% of the worldwide population. Non-alcoholic steatohepatitis (NASH), the more progressive variant of NAFLD, is characterized by steatosis, cellular ballooning, lobular inflammation, and may culminate on hepatic stellate cell (HSC) activation, thus increasing the risk for fibrosis, cirrhosis, and HCC development. Conversely, the antifibrotic effects of sorafenib, an FDA-approved drug for HCC treatment, have been demonstrated in 2D cell cultures and animal models, but its mechanisms in a NAFLD-related microenvironment in vitro requires further investigation. Thus, a human 3D co-culture model of fatty hepatocytes and HSC was established by culturing hepatoma C3A cells, pre-treated with 1.32 mM oleic acid, with HSC LX-2 cells. The fatty C3A/LX-2 spheroids showed morphological and molecular hallmarks of altered lipid metabolism and steatosis-induced fibrogenesis, similarly to the human disease. Sorafenib (15 µM) for 72 hours reduced fatty spheroid viability, and upregulated the expression of lipid oxidation- and hydrolysis-related genes, CPT1 and LIPC, respectively. Sorafenib also inhibited steatosis-induced fibrogenesis by downregulating COL1A1, TGFB1, PDGF, and TIMP1 and by decreasing protein levels of IL-6, TGF-ß1, and TNF-α in fatty spheroids. The demonstration of the antifibrotic properties of sorafenib on steatosis-induced fibrogenesis in a 3D in vitro model of NAFLD supports its clinical use as a therapeutic agent for the treatment of NAFLD/NASH patients.

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals.

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines.

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

Connexin and Pannexin (Hemi)Channels: Emerging Targets in the Treatment of Liver Disease.

Connexin proteins are the building blocks of hemichannels, which dock further between adjacent cells to form gap junctions. Gap junctions control the intercellular exchange of critical homeostasis regulators. By doing so, gap junctions control virtually all aspects of the hepatic life cycle. In the last decade, it has become clear that connexin hemichannels also provide a pathway for cellular communication on their own independent of their role as structural precursors of gap junctions, namely between the cytosol of an individual cell and its extracellular environment. In contrast to gap junctions, connexin hemichannels become particularly active in liver disease by facilitating inflammation and cell death. This equally holds true for cellular channels composed of pannexins, being connexin-like proteins recently identified in the liver that gather in structures reminiscent of hemichannels. This paper gives an overview of the involvement of connexin-based and pannexin-based channels in noncancerous liver disease.

Targeting gap junctional intercellular communication by hepatocarcinogenic compounds.

Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.

Burdock (Arctium lappa L.) root attenuates preneoplastic lesion development in a diet and thioacetamide-induced model of steatohepatitis-associated hepatocarcinogenesis.

Nonalcoholic steatohepatitis (NASH) is considered growing risk factor for hepatocellular carcinoma development in high-income countries. Diet- and chemically induced rodent models have been applied for the translational study of NASH-associated hepatocarcinogenesis due to their morphological and molecular similarities to the corresponding human disease. Arctium lappa L. (burdock) root tea has been extensively consumed in Traditional Chinese Medicine due to its potential therapeutic properties. Indeed, the bioactive compounds of A. lappa root, as the polyphenols, have already showed antioxidant and anti-inflammatory properties in different in vivo and in vitro bioassays. In this study, we investigated whether burdock root ethanolic extract (BRE) administration attenuates NASH-associated hepatocarcinogenesis. Eight-week-old male Wistar rats received choline-deficient high-fat diet for 8 weeks and multiple thioacetamide doses for 4 weeks in order to induce NASH and preneoplastic glutathione-S-transferase pi (GST-P)+ preneoplastic foci. Subsequently, rats were treated with BRE (100 or 200 mg/kg body weight) or vehicle by oral gavage for 2 weeks. BRE displayed high levels of chlorogenic and caffeic acids and BRE administration reduced total fatty acid and lipid hydroperoxide levels, while increasing the activities of antioxidant superoxide dismutase and catalase enzymes in the liver. Furthermore, burdock intervention diminished the size of GST-P+ remodeling preneoplastic lesions (PNLs) and displayed a trend on reducing hepatocyte proliferation (Ki-67) inside them. These findings suggest that short-term exposure to BRE alleviated remodeling PNL development in NASH-associated hepatocarcinogenesis.

Cholestasis Differentially Affects Liver Connexins.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function.

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

Pannexin1 as mediator of inflammation and cell death.

Pannexins form channels at the plasma membrane surface that establish a pathway for communication between the cytosol of individual cells and their extracellular environment. By doing so, pannexin signaling dictates several physiological functions, but equally underlies a number of pathological processes. Indeed, pannexin channels drive inflammation by assisting in the activation of inflammasomes, the release of pro-inflammatory cytokines, and the activation and migration of leukocytes. Furthermore, these cellular pores facilitate cell death, including apoptosis, pyroptosis and autophagy. The present paper reviews the roles of pannexin channels in inflammation and cell death. In a first part, a state-of-the-art overview of pannexin channel structure, regulation and function is provided. In a second part, the mechanisms behind their involvement in inflammation and cell death are discussed.

Connexins and their channels in inflammation.

Inflammation may be caused by a variety of factors and is a hallmark of a plethora of acute and chronic diseases. The purpose of inflammation is to eliminate the initial cell injury trigger, to clear out dead cells from damaged tissue and to initiate tissue regeneration. Despite the wealth of knowledge regarding the involvement of cellular communication in inflammation, studies on the role of connexin-based channels in this process have only begun to emerge in the last few years. In this paper, a state-of-the-art overview of the effects of inflammation on connexin signaling is provided. Vice versa, the involvement of connexins and their channels in inflammation will be discussed by relying on studies that use a variety of experimental tools, such as genetically modified animals, small interfering RNA and connexin-based channel blockers. A better understanding of the importance of connexin signaling in inflammation may open up towards clinical perspectives.

Protective effect of genetic deletion of pannexin1 in experimental mouse models of acute and chronic liver disease.

Pannexins are transmembrane proteins that form communication channels connecting the cytosol of an individual cell with its extracellular environment. A number of studies have documented the presence of pannexin1 in liver as well as its involvement in inflammatory responses. In this study, it was investigated whether pannexin1 plays a role in acute liver failure and non-alcoholic steatohepatitis, being prototypical acute and chronic liver pathologies, respectively, both featured by liver damage, oxidative stress and inflammation. To this end, wild-type and pannexin1-/- mice were overdosed with acetaminophen for 1, 6, 24 or 48h or were fed a choline-deficient high-fat diet for 8weeks. Evaluation of the effects of genetic pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, lipid accumulation, protein adduct formation, oxidative stress and inflammation. In parallel, in order to elucidate molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. The results of this study show that pannexin1-/- diseased mice present less liver damage and oxidative stress, while inflammation was only decreased in pannexin1-/- mice in which non-alcoholic steatohepatitis was induced. A multitude of genes related to inflammation, oxidative stress and xenobiotic metabolism were differentially modulated in both liver disease models in wild-type and in pannexin1-/- mice. Overall, the results of this study suggest that pannexin1 may play a role in the pathogenesis of liver disease.

Sex steroids drive the remodeling of oviductal extracellular matrix in cattle.

The extracellular matrix (ECM) is a group of molecules that offer structural and biochemical support to cells and interact with them to regulate their function. Also, growth factors (GFs) stored in the ECM can be locally released during ECM remodeling. Here, we hypothesize that the balance between ECM components and remodelers is regulated according to the ovarian steroid milieu to which the oviduct is exposed during the periovulatory period. Follicular growth was manipulated to generate cows that ovulated small follicles (SF-small corpus luteum [SCL]; n = 20) or large follicles (LF-large corpus luteum [LCL]; n = 21) and possess corresponding Estradiol (E2) and Progesterone (P4) plasmatic concentrations. Ampulla and isthmus samples were collected on day 4 (day 0 = ovulation induction) and immediately frozen or fixed. The transcriptional profile (n = 3/group) was evaluated by RNA sequencing. MMP Antibody Array was used to quantify ECM remodelers' protein abundance and immunohistochemistry to quantify type I collagen. Transcriptome analysis revealed the over-representation of ECM organization and remodeling pathways in the LF-LCL group. Transcription of ECM components (collagens), remodelers (ADAMs and MMPs), and related GFs were upregulated in LF-LCL. Protein intensities for MMP3, MMP8, MMP9, MMP13, and TIMP4 were greater for the LF-LCL group. Type I collagen content in the mucosa was greater in SF-SCL group. In conclusion, that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates ECM remodeling. We speculate that differential ECM regulation may contribute to oviductal receptivity to the embryo.

Genetic ablation of pannexin1 counteracts liver fibrosis in a chemical, but not in a surgical mouse model.

Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis.

TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice.

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

Involvement of connexin43 in acetaminophen-induced liver injury.

BACKGROUND AND AIMS: Being goalkeepers of liver homeostasis, gap junctions are also involved in hepatotoxicity. However, their role in this process is ambiguous, as gap junctions can act as both targets and effectors of liver toxicity. This particularly holds true for drug-induced liver insults. In the present study, the involvement of connexin26, connexin32 and connexin43, the building blocks of liver gap junctions, was investigated in acetaminophen-induced hepatotoxicity. METHODS: C57BL/6 mice were overdosed with 300mg/kg body weight acetaminophen followed by analysis of the expression and localization of connexins as well as monitoring of hepatic gap junction functionality. Furthermore, acetaminophen-induced liver injury was compared between mice genetically deficient in connexin43 and wild type littermates. Evaluation of the toxicological response was based on a set of clinically relevant parameters, including protein adduct formation, measurement of alanine aminotransferase activity, cytokines and glutathione. RESULTS: It was found that gap junction communication deteriorates upon acetaminophen intoxication in wild type mice, which is associated with a switch in mRNA and protein production from connexin32 and connexin26 to connexin43. The upregulation of connexin43 expression is due, at least in part, to de novo production by hepatocytes. Connexin43-deficient animals tended to show increased liver cell death, inflammation and oxidative stress in comparison with wild type counterparts. CONCLUSION: These results suggest that hepatic connexin43-based signaling may protect against acetaminophen-induced liver toxicity.

Cystic endometrial hyperplasia-pyometra syndrome in bitches: identification of hemodynamic, inflammatory, and cell proliferation changes.

Cystic endometrial hyperplasia (CEH)-pyometra syndrome is one of the most common diseases of noncastrated female dogs. However, determination of etiological mechanisms and differential diagnosis of CEH-pyometra syndrome are undefined. The aim of this study is to compare immunohistochemically the expression of cyclooxygenase-2 (COX-2) inflammatory mediator, Ki-67 antigen proliferation marker, vascular endothelial growth factor (VEGF-A) angiogenesis mediator and its FLT-1 and KDR receptors, and correlate with Doppler velocimetry of uterine artery and endometrial vascularization in bitches with CEH-pyometra syndrome. Bitches were allocated into CEH-mucometra Group (n = 13), Pyometra Group (n = 11), and Control Group (n = 8). Pyometra Group presented cytoplasmatic staining intensity for COX-2, VEGF-A, and FLT-1 and KDR receptors in luminal epithelium cells significantly higher compared to CEH-mucometra and Control groups. For the glandular epithelium, Pyometra Group had higher immunostaining score for VEGF-A and its receptors (FLT-1 and KDR). Hemodynamic indexes showed negative correlation with VEGF-A and its receptors as well as with COX-2. On the other hand, uterine vascularization score showed positive correlation in relation to immunostaining of COX-2, VEGF-A, and receptors in the endometrium luminal epithelium. In conclusion, uterus of bitches with CEH-pyometra syndrome show inflammatory process characterized by COX-2 expression, resulting in greater expression of proliferative Ki-67 marker as tissue response against the infectious agent. Furthermore, the increased VEGF-A expression and its receptors in CEH-pyometra reflect the increased blood flow and lower vascular resistance. Therefore, canine pyometra is characterized by an inflammatory, proliferative, and vascular disorder.

Cryptococcus gattii molecular type VGII infection associated with lung disease in a goat.

BACKGROUND: Cryptococcus gattii-induced cryptococcosis is an emerging infectious disease of humans and animals with worldwide distribution and public health importance due to its significant morbidity and mortality rate. The present study aimed to report a case of pulmonary infection by C. gattii molecular type VGII in State of São Paulo, Brazil. CASE PRESENTATION: A 5-year-old goat showing intermittent dry cough, ruminal tympany, anorexia, fever, tachycardia and tachypnea was presented for necropsy at the Veterinary Hospital of the School of Veterinary Medicine and Animal Sciences, São Paulo University, São Paulo, Brazil. Postmortem examination revealed numerous 2.0-6.0 cm diameter yellow gelatinous pulmonary masses. Tissues were evaluated by a combination of pathological, mycological, and molecular diagnostic techniques. Microscopically, pneumonia granulomatous, multifocal to coalescing, moderate, with many intralesional carminophilic yeasts was observed. The immunohistochemistry and mycological culture confirmed Cryptococcus spp. Internal transcribed spacers and orotidine monophosphate pyrophosphorylase nucleotide differentiation demonstrated that the isolate corresponds to the C. gattii VGII molecular subtype. CONCLUSIONS: To our knowledge, this is the first report of a pulmonary infection in a goat linked to C. gattii molecular type VGII in Southeastern Brazil. Our findings emphasize the need for an active surveillance program for human and animal new infections to improve the current public health policies due to expansion of the epidemiological niche of this important microorganism.