Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and ß-catenin expression as well as nuclear ß-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and ß-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/ß-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

Epithelial-mesenchymal transition in an EcPV2-positive vulvar squamous cell carcinoma of a mare.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. OBJECTIVE: To describe a case of naturally occurring EcPV2-induced VSCC, by investigating tumour ability in undergoing the epithelial-to-mesenchymal transition (EMT). STUDY DESIGN: Case report. METHODS: A 13-year-old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was consistent with a VSCC. Real-time qPCR, real-time reverse transcriptase (RT)-qPCR and RNAscope were carried out to detect EcPV2 infection and to evaluate E6/E7 oncogenes expression. To highlight the EMT, immunohistochemistry (IHC) was performed. Expression of EMT-related and innate immunity-related genes was investigated through RT-qPCR. RESULTS: Real-time qPCR, RT-qPCR and RNAscope confirmed EcPV2 DNA presence and expression of EcPV2 oncoproteins (E6 and E7) within the neoplastic vulvar lesion. IHC highlighted a cadherin switch together with the expression of the EMT-related transcription factor HIF1α. With RT-qPCR, significantly increased gene expression of EBI3 (45.0 ± 1.62, p < 0.01), CDH2 (2445.3 ± 0.39, p < 0.001), CXCL8 (288.7 ± 0.40, p < 0.001) and decreased gene expression of CDH1 (0.3 ± 0.57, p < 0.05), IL12A (0.04 ± 1.06, p < 0.01) and IL17 (0.2 ± 0.64, p < 0.05) were detected. MAIN LIMITATIONS: Lack of ability to generalise and danger of over-interpretation. CONCLUSION: The results obtained were suggestive of an EMT event occurring within the neoplastic lesion.

Metaplastic Ossification of a Giant Paraprostatic Cyst in a Dog: A Case Report.

A 7-year-old male Caucasian shepherd presented with a 3 month history of intermittent hematuria, penile discharge, and abdominal pain and distension. The dog had a history of prostatic hyperplasia with multiple cysts, diagnosed by the referring clinician two years prior to the case presentation. Two oral courses of antibiotics and antiandrogens were administered by the treating veterinarian without resolution. At the case presentation visit, massive swelling was present in the mid- and caudal parts of the abdomen. Abdominal ultrasound and exploratory laparotomy revealed a paraprostatic cyst (size: 25 × 20 × 18 cm) in the caudal part of the abdomen. The cyst had a bony ridge along the wall with multiple cauliflower-like lesions extending inside. Histopathologic examination revealed an intermittently epithelial-lined inner wall of the cyst, which was partially degenerated and flattened due to the pressure of the intraluminal fluid. The luminal surface of the cyst appeared markedly irregular with multiple structures made of dense, fibrous connective tissue protruding inside with metaplastic ossification foci, consistent with severe osseous metaplasia. The epithelium showed focal secretory activity. Numerous subepithelial multifocal neutrophil and mononuclear cellular infiltrates were found. The lumen of the cyst contained a reddish-brown (blood and protein-rich) fluid. Complete surgical excision of the cyst and omentalization of the capsular remnant resulted in successful resolution of the clinical signs.

Investigation of the Epithelial to Mesenchymal Transition (EMT) Process in Equine Papillomavirus-2 (EcPV-2)-Positive Penile Squamous Cell Carcinomas.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, ß-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL, together with BCATN1, LEF1, and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas.

Peripheral Nerve Sheath Tumors Resembling Human Atypical Neurofibroma in Goldfish (Carassius auratus, Linnaeus, 1758).

Skin spindle cell tumors (SSTs) frequently occur in fishes, with peripheral nerve sheath tumors (PNSTs) being the most commonly reported neoplasms in goldfish. However, distinguishing PNSTs from other SCTs is not always possible when relying exclusively on routine cytological and histopathological findings. Therefore, the aim of this study is to characterize six skin nodules, resembling atypical neurofibromas in humans, found in six cohabiting goldfish (Carassius auratus), and to determine a minimal subset of special stains required to correctly identify PNSTs in this species. Routine cytology and histopathology were indicative of an SCT with nuclear atypia in all cases, with randomly distributed areas of hypercellularity and loss of neurofibroma architecture. Muscular and fibroblastic tumors were excluded using Azan trichrome staining. Alcian blue and Gomori's reticulin stains revealed the presence of intratumoral areas of glycosaminoglycans or mucins and basement membrane fragments, respectively. PAS and PAS-diastase stains confirmed the latter finding and revealed intra- and extracellular glycogen granules. Immunohistochemistry displayed multifocal, randomly distributed aggregates of neoplastic cells positive for S100 protein and CNPase, intermingled with phosphorylated and non-phosphorylated neurofilament-positive axons. Collectively, these findings are consistent with a PNST resembling atypical neurofibroma in humans, an entity not previously reported in goldfish, and suggest that Azan trichrome staining, reticulin staining, and immunohistochemistry for S100 protein and CNPase represent a useful set of special stains to identify and characterize PNSTs in this species.

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas.

In horses, penile squamous cell carcinomas (epSCCs) are among the most common cutaneous neoplastic lesions. These tumors usually arise in benign lesions such as viral plaques and papillomas frequently induced by Equus caballus papillomavirus type 2 (EcPV2) infection. In the last decade, the introduction of immune checkpoint inhibitors (ICI) for the treatment of human cancers has demonstrated promising results. Among the most commonly targeted pathways, there is PD-1/PD-L1 and CTLA-4. The aim of this study is to investigate the expression of the PD-1/PD-L1 pathway and CTLA-4 in the tumor microenvironment of epSCCs to assess the feasibility of an immunotherapeutic approach. Twenty equine epithelial tumors were retrospectively selected and submitted to RT-qPCR for PD-1 and PD-L1 genes. After testing antibodies cross-reactivity by western blotting, immunohistochemistry for PD-L1 and CTLA-4 was performed. Results from RT-qPCR demonstrated that 3/20 cases expressed the PD-L1 gene, whereas the PD-1 gene was not detected. Immunohistochemical positivity for PD-L1 was found only in one case. CTLA-4-positive cells were observe in all cases but were few (Mdn = 4.8; IQR = 2.3-7.1 cells/HPF). In this study group, PD-1/PD-L1 and CTLA-4 do not appear to be highly expressed and therefore the use of ICI in epSCCs may not have promising rates of response.

Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate.

Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.

Equine Genital Squamous Cell Carcinoma Associated with EcPV2 Infection: RANKL Pathway Correlated to Inflammation and Wnt Signaling Activation.

Equine genital squamous cell carcinomas (egSCCs) are among the most common equine tumors after sarcoids, severely impairing animal health and welfare. Equus caballus papillomavirus type 2 (EcPV2) infection is often related to these tumors. The aim of this study was to clarify the molecular mechanisms behind egSCCs associated with EcPV2 infection, investigating receptor activator of nuclear factor-kappa B ligand (RANKL) signaling in NF-kB pathway, together with the Wnt and IL17 signaling pathways. We analyzed the innate immune response through gene expression evaluation of key cytokines and transcription factors. Moreover, Ki67 index was assessed with immunohistochemistry. EcPV2-E6 DNA was checked, and viral presence was confirmed in 21 positive out to 23 cases (91%). Oncogene expression was confirmed in 14 cases (60.8%) for E6 and in 8 (34.7%) for E2. RANKL, nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB)-p50, NFKBp65, interleukin (IL)-6, IL17, IL23p19, IL8, IL12p35, IL12p40, ß-catenin (BCATN1), FOS like 1 (FOSL1), and lymphoid enhancer binding factor 1 (LEF1) showed a significant upregulation in tumor samples compared to healthy tissues. Our results describe an inflammatory environment characterized by the activation of RANKL/RANK and IL17 with the relative downstream pathways, and a positive modulation of inflammatory cytokines genes such as IL6 and IL8. Moreover, the increase of BCATN1, FOSL1, and LEF1 gene expression suggests an activation of both canonical and non-canonical Wnt signaling pathway that could be critical for carcinogenesis and tumor progression.

First report of Echinococcus granulosus sensu lato genotype G6/G7 in domestic pigs from mainland Italy.

Cystic echinococcosis (CE; Echinococcus spp.) is widespread in many domestic animal species in Italy, with the G1-G3 genotype predominating. The G7 genotype ("pig strain"), which is much less common, has only been reported in pigs and wild boar from the island of Sardinia and in wild boars from southern mainland Italy. Ten pig livers with hydatid cysts were identified in a slaughterhouse in northwestern Italy. Multiplex PCR for Echinococcus granulosus gave positive results for two of these and subsequent sequencing confirmed the species as Echinococcus granulosusu s.l. G6/G7. Affected pigs came from an intensive farm in northeastern Italy. This is, to the authors' knowledge, the first report of CE by Echinococcus granulosusu s.l. G6/G7 in the domestic pig in mainland Italy. E. granulosus s.l. G6/G7 is zoonotic and its circulation in Italy should be of concern for public health.

Epithelial to Mesenchymal Transition (EMT) in a Laryngeal Squamous Cell Carcinoma of a Horse: Future Perspectives.

Squamous cell carcinoma (SCC) is one of the most frequent tumors of skin and muco-cutaneous junctions in the horse. Equine papillomavirus type 2 (EcPV2) has been detected in equine SCC of the oral tract and genitals, and recently also in the larynx. As human squamous cell carcinoma of the larynx (SCCL), it is strongly etiologically associated with high-risk papillomavirus (h-HPV) infection. This study focuses on tumor cells behavior in a naturally occurring tumor that can undergo the so-called epithelial to mesenchymal transition (EMT). A SCCL in a horse was investigated by immunohistochemistry using antibodies against E-cadherin, pan-cytokeratin AE3/AE1, ß-catenin, N-cadherin, vimentin, ZEB-1, TWIST, and HIF-1α. EcPV2 DNA detection and expression of oncogenes in SCC were investigated. A cadherin switch and an intermediate filaments rearrangement within primary site tumor cells together with the expression of the EMT-related transcription factors TWIST-1, ZEB-1, and HIF-1α were observed. DNA obtained from the tumor showed EcPV2 positivity, with E2 gene disruption and E6 gene dysregulation. The results suggest that equine SCCL might be a valuable model for studying EMT and the potential interactions between EcPV2 oncoproteins and the EMT process in SCCL.

Equine Penile Squamous Cell Carcinomas as a Model for Human Disease: A Preliminary Investigation on Tumor Immune Microenvironment.

Penile squamous cell carcinomas (SCCs) are common tumors in older horses, with poor prognosis mostly due to local invasion and recurrence. These tumors are thought to be mainly caused by Equus caballus papillomavirus type 2 (EcPV-2). The aim of this study is to characterize the tumor immune environment (TIME) in equine penile tumors. Equine penile epithelial tumors (17 epSCCs; 2 carcinomas in situ, CIS; 1 papilloma, P) were retrospectively selected; immune infiltrate was assessed by histology and immunohistochemistry; RT-qPCR tested the expression of selected chemokines and EcPV-2 DNA and RNA. The results confirmed EcPV-2-L1 DNA in 18/20 (90%) samples. L1 expression was instead retrieved in 13/20 cases (65%). The samples showed an increased infiltration of CD3+lymphocytes, macrophages (MAC387; IBA1), plasma cells (MUM1), and FoxP3+lymphocytes in the intra/peritumoral stroma when compared to extratumoral tissues (p < 0.05). Only MAC387+neutrophils were increased in EcPV-2high viral load samples (p < 0.05). IL12/p35 was differentially expressed in EcPVhigh and EcPVlow groups (p = 0.007). A significant decrease of IFNG and IL2 expression was highlighted in TGFB1-positive samples (p < 0.05). IBA1 and CD20 were intratumorally increased in cases where IL-10 was expressed (p < 0.005). EpSCCs may represent a good spontaneous model for the human counterpart. Further prospective studies are needed in order to confirm these preliminary results.

Platelet lysate reduces the chondrocyte dedifferentiation during in vitro expansion: Implications for cartilage tissue engineering.

In vitro studies have demonstrated that platelet lysate (PL) can serve as an alternative to platelet-rich plasma (PRP) to sustain chondrocyte proliferation and production of extracellular matrix components in chondrocytes. The present study aimed to evaluate the direct effects of PL on equine articular chondrocytes in vitro in order to provide a rationale for in vivo use of PL. An in vitro cell proliferation and de-differentiation model was used: primary articular chondrocytes isolated from horse articular cartilage were cultured at low density under adherent conditions to promote cell proliferation. Chondrocytes were cultured in serum-free medium, 10% foetal bovine serum (FBS) supplemented medium, or in the presence of alginate beads containing 5%, 10% and 20% PL. Cell proliferation and gene expression of relevant chondrocyte differentiation markers were investigated. The proliferative capacity of cultured chondrocytes, was sustained more effectively at certain concentrations of PL as compared to that with FBS. In addition, as opposed to FBS, PL, particularly at percentages of 5% and 10%, could maintain the gene expression pattern of relevant chondrocyte differentiation markers. In particular, 5% PL supplementation showed the best compromise between chondrocyte proliferation capacity and maintenance of differentiation. The results of the present study provide a rationale for using PL as an alternative to FBS for in vitro expansion of chondrocytes for matrix-assisted chondrocyte implantation, construction of 3D scaffolds for tissue engineering, and treatment of damaged articular cartilage.

An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes, CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8ß+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion.

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT. KP induced an early dose-dependent shift to pro-inflammatory CD172α+CD14+high monocytes and an increase of CD3+CD16+ natural killer (NK) T cells. KP triggered CD8α and CD8ß expression on classical CD4-CD8αß+ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4+CD8α+ Th memory cells (CD4+CD8α+low CD8ß+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4+CD8α+high CD8ß+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.

Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line.

BACKGROUND: Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 µM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment. RESULTS: Cell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 µM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 µM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 µM (P < 0.01). CONCLUSIONS: The adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.

Phenotypic characterization of a highly pathogenic Italian porcine reproductive and respiratory syndrome virus (PRRSV) type 1 subtype 1 isolate in experimentally infected pigs.

Highly pathogenic (HP) isolates of the PRRS virus started emerging in North America and Asia in the late 1990s. More recently, they have emerged in Europe. These isolates are characterized by high viral loads, severe general clinical signs and high mortality, in sows, weaners and growers. Their genome shows a discontinuous aminoacids deletion in the non-structural protein 2 (NSP2). The present study was aimed at characterizing the clinical, pathological and immunological features of a highly pathogenetic, Italian PRRSV-1 subtype 1 isolate (PRRSV1_PR40/2014), following experimental infection in conventional 4-weeks-old pigs. The PRRSV1_PR40/2014 infected group showed severe clinical signs (high fever and dispnoea). Pathological lesions, including severe lymphocytopenia in bronchial lymph-nodes and thymus were also recorded. Higher serum PRRSV genome copies and lower virus neutralizing antibody titer were observed in the PR40 group, when compared to the group infected with a conventional PRRSV strain. The genetic analysis of the strain, and the phenotypic features observed in the field and reproduced in the experimental study, confirmed the high pathogenicity of the Italian PRRSV-1 subtype 1 PR40 isolate.

The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes.

The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan. In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes.

Effects of histamine H4 receptor ligands in a mouse model of gastric ulceration.

AIM: In the present study we examined whether histamine H(4) receptors (H(4)Rs) have a role in gastric ulcerogenesis using a mouse model of gastric damage. METHODS: The H(4)R antagonist JNJ7777120 and the H(4)R agonists VUF8430 and VUF10460 were investigated in fasted CD-1 mice against the ulcerogenic effect induced by co-administration of indomethacin(IND, 30 mg/kg s.c.) and bethanechol (BET, 5 mg/kg i.p.). Both macroscopic and histologic lesions were examined. Strain-related differences were investigated by testing JNJ7777120 also in NMRI, BALB/c and C57BL/6J mice. RESULTS: Neither JNJ7777120 nor the H(4)R agonists displayed effects in the normal stomach at any dose tested (10 and 30 mg/kg s.c.). As expected, IND+BET provoked several lesions in the fundic mucosa, which were significantly reduced by JNJ7777120 (10 and 30 mg/kg s.c.). The gastroprotective effect of JNJ7777120 (10 and 30 mg/kg s.c.) was observed in CD-1, NMRI and BALB/c, but not in C57BL/6J, mice. In CD-1 mice, the H(4)R agonists VUF8430 and VUF10460 (both at 10 and 30 mg/kg s.c.) did not modify the damage induced by IND+BET, however VUF8430 (10 mg/kg s.c.) prevented the gastroprotection induced by JNJ7777120 (10 mg/kg s.c.). CONCLUSIONS: Data obtained with selective ligands suggest that the H(4)R may have a role in mouse gastric ulcerogenesis. If confirmed in humans, these data would emphasize the potential advantage of H(4)R blockers as gastrosparing anti-inflammatory drugs. The lack of effects of JNJ7777120 in C57BL/6J mice has to be carefully considered in the pharmacological characterization of H(4)R functions and/or new selective ligands.

Clinical, genetic, and pathological features of male pseudohermaphroditism in dog.

Male pseudohermaphroditism is a sex differentiation disorder in which the gonads are testes and the genital ducts are incompletely masculinized. An 8 years old dog with normal male karyotype was referred for examination of external genitalia abnormalities. Adjacent to the vulva subcutaneous undescended testes were observed. The histology of the gonads revealed a Leydig and Sertoli cell neoplasia. The contemporaneous presence of testicular tissue, vulva, male karyotype were compatible with a male pseudohermaphrodite (MPH) condition.

Diazoxide attenuates indomethacin-induced small intestinal damage in the rat.

ATP-sensitive potassium (K(ATP)) channel openers have been shown to protect against cellular damage in neurons, cardiac muscle, and kidney and to effectively reduce nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage in rats. We investigated the effects of K(ATP) channel opener diazoxide on small intestinal injury induced in rats by indomethacin administration. The effect of glibenclamide, a K(ATP) channel blocker, was also evaluated. Diazoxide (15, 45 and 135mg/kg) or glibenclamide (18mg/kg), were given by oral gavage 1h before and 6h after indomethacin treatment (20mg/kg p.o.). After 24h, macroscopic and histologic lesions, myeloperoxidase (MPO) activity and lipid peroxidation levels were evaluated. Diazoxide at 15mg/kg was ineffective, while at doses of 45mg/kg and 135mg/kg was able to significantly improve all damage parameters. Glibenclamide administration enhanced intestinal injury. These results show for the first time a beneficial effect of diazoxide in indomethacin-induced enteritis in the rat. Several mechanisms, such as oxidative phosphorylation uncoupling and hypermotility seem particularly important in NSAID-induced intestinal injury. Such events lead to increased mucosal permeability and to penetration of noxious lumen components, which ignite the inflammatory response. Since K(ATP) channel openers were shown to protect against mitochondrial damage, to reduce intercellular permeability and to relax smooth muscle, we suggest that diazoxide could exert its beneficial effects by one or more of these actions.