Immunohistochemical, pharmacovigilance, and omics analyses reveal the involvement of ATP-sensitive K+ channel subunits in cancers: role in drug-disease interactions.

Background: ATP-sensitive-K+ channels (KATP) are involved in diseases, but their role in cancer is poorly described. Pituitary macroadenoma has been observed in Cantu' syndrome (C.S.), which is associated with the gain-of-function mutations of the ABCC9 and KCNJ8 genes. We tested the role of the ABCC8/Sur1, ABCC9/Sur2A/B, KCNJ11/Kir6.2, and KCNJ8/Kir6.1 genes experimentally in a minoxidil-induced renal tumor in male rats and in the female canine breast cancer, a spontaneous animal model of disease, and in the pharmacovigilance and omics databases. Methods: We performed biopsies from renal tissues of male rats (N = 5) following a sub-chronic high dosing topical administration of minoxidil (0.777-77.7 mg/kg/day) and from breast tissues of female dogs for diagnosis (N = 23) that were analyzed by immunohistochemistry. Pharmacovigilance and omics data were extracted from EudraVigilance and omics databases, respectively. Results: An elevated immunohistochemical reactivity to Sur2A-mAb was detected in the cytosol of the Ki67+/G3 cells other than in the surface membrane in the minoxidil-induced renal tumor and the breast tumor samples. KCNJ11, KCNJ8, and ABCC9 genes are upregulated in cancers but ABCC8 is downregulated. The Kir6.2-Sur2A/B-channel opener minoxidil showed 23 case reports of breast cancer and one case of ovarian cancer in line with omics data reporting, respectively, and the negative and positive prognostic roles of the ABCC9 gene in these cancers. Sulfonylureas and glinides blocking the pancreatic Kir6.2-Sur1 subunits showed a higher risk for pancreatic cancer in line with the positive prognostic role of the ABCC8 gene but low risks for common cancers. Glibenclamide, repaglinide, and glimepiride show a lower cancer risk within the KATP channel blockers. The Kir6.2-Sur1 opener diazoxide shows no cancer reactions. Conclusion: An elevated expression of the Sur2A subunit was found in proliferating cells in two animal models of cancer. Immunohistochemistry/omics/pharmacovigilance data reveal the role of the Kir6.1/2-Sur2A/B subunits as a drug target in breast/renal cancers and in C.S.

Thoracic and Abdominal Mesothelioma in an Older Horse in Lazio Region.

A Quarter Horse, a gelding aged 22, was subjected to a clinical examination for colic syndrome. During admission to the clinic, blood counts and ultrasound examination were performed. Ultrasound revealed abdominal masses and abundant accumulation of pleural (50 L) and abdominal fluid (100 L). Cytology was performed on the aspirated fluid. The patient was euthanized. The autopsy examination revealed abundant effusion and nodular masses on the peritoneum, omentum, lungs, heart, and mediastinum. A diagnosis of epithelioid mesothelioma was made via histopathology and confirmed with immunohistochemistry; it showed positive antibodies against cytokeratin (CK) and vimentin. Mesothelioma is a rare cancer in older horses. It is important to employ the correct differential diagnostics using the available methods, providing valid ante-mortem support to the clinical veterinarian and monitoring the territory using this species as a valid biological indicator.

Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome.

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10-5 M in SUR2wt/AV and 8.6 ± 0.4 × 10-6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] "knock-in" mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

Dirofilarioses in two cats in southern Italy.

Two cats infected by Dirofilaria immitis and Dirofilaria repens, respectively, were taken to two different private practitioners for a clinical examination. The analyses conducted on the first cat revealed a microfilaraemia due to D. repens of 66 mfs/mL by a modified Knott's test. No clinical signs of D. repens infection were observed in the cat. The animal was euthanised because of a lymphoma condition, and two adult females of D. repens were found in the subcutaneous tissue of the lumbar and left scapular regions at the post-mortem examination. The second cat showed severe abnormalities in the white blood cells, including eosinophil count. Microfilariae of D. immitis were detected in the blood smear, with an average length (n = 2) of 296.2 µm. These clinical cases represent the first reports of feline dirofilarioses in southern Italy and are indicative of a common occurrence of dirofilarial infection in the local canine population.

Absorbable fixation straps for laparoscopic gastropexy in dogs.

OBJECTIVE: To evaluate the feasibility and efficacy of laparoscopic absorbable fixation straps (AFS) for laparoscopic gastropexy in dogs. STUDY DESIGN: Cadaveric and prospective clinical study. ANIMALS: Five dog cadavers for the cadaveric study; 12 dogs for the clinical study. METHODS: The pyloric antrum was affixed to the abdominal wall laparoscopically by applying a series of straps. The cadaveric study assessed potential challenges during the procedure and stomach mucosal penetration. For the clinical study, the total duration of surgery, time to complete the gastropexy, and the number of straps used were recorded. Ultrasound evidence of adhesion, complications, and weight were monitored at 7, 30, and 90 days after surgery. Owner satisfaction was evaluated at the 6-month follow-up. RESULTS: The total duration of surgery was 25.8 minutes (range, 19-39; SD, 6.7), and the time to complete the gastropexy was 10.1 minutes (range, 7-19; SD, 3.9). The linear regression analysis revealed an inverse correlation between the time to complete the gastropexy and the order of the surgeries (r2  = 0.75, P < .05). No complications were recorded. Ultrasound examination was used to confirm gastropexy at all follow-ups. CONCLUSION: Laparoscopic gastropexy with AFS was performed in both cadavers and clinical animals with minimal complications. Persistent adhesion was demonstrated during ultrasound evaluations and in one postmortem evaluation. CLINICAL SIGNIFICANCE: This novel laparoscopic technique can be employed safely, effectively, and reasonably quickly, and the learning curve is expected to be relatively short.

Microvascular Density, Endothelial Area, and Ki-67 Proliferative Index Correlate Each Other in Cat Post-Injection Fibrosarcoma.

Soft tissue sarcomas are a large group of different tumor types both in humans and in animals. Among them, fibrosarcoma is the most frequent malignant mesenchymal tumoral form in cats, representing up to 28% of all cat skin tumors, while human fibrosarcoma, fortunately, only represents 5% of all sarcomas and 0.025% of the world-wide burden of tumors. This low incidence in humans leads to consideration of this group of tumoral diseases as rare, so therapeutic options are few due to the difficulty of starting clinical trials. In this context, the identification of research models for fibrosarcomas could be of great interest to deepen knowledge in this field and recognize new or possible biological pathways involved in tumor progression and metastasis. Angiogenesis is considered a fundamental scattering cause of tumor aggressiveness and progression in all forms of cancer, but only a few research parameters were developed and reported to express them quantitatively and qualitatively. The role in angiogenesis of microenvironmental stromal cells, such as fibroblasts, lymphocytes, mast cells, and macrophages, was largely demonstrated since this topic was first approached, while quantification of new vessels and their blood capacity in tumoral area is a relatively recent approach that could be well developed thanks to expertise in immunohistochemistry and image analysis. In this paper, a crossing study evaluating microvascular density (MVD), endothelial area (EA), and Ki-67 proliferative index was reported for a series of formalin-fixed and paraffin-embedded tissue samples from 99 cat patients, affected by cat post-injection fibrosarcoma, by using a till ×400 magnification light microscopy. We aim to demonstrate that cat pets may be considered a useful animal model for better studying the correspondent human diseases and we report, for the first time to our knowledge, experimental data in terms of correlation among MVD, EA, and Ki-67 strictly involved in aggressiveness and tumoral progression.

The hydroxypropyl-ß-cyclodextrin-minoxidil inclusion complex improves the cardiovascular and proliferative adverse effects of minoxidil in male rats: Implications in the treatment of alopecia.

The efficacy of minoxidil (MXD) ethanolic solutions (1%-5% w/v) in the treatment of androgenetic alopecia is limited by adverse reactions. The toxicological effects of repeated topical applications of escalating dose (0.035%-3.5% w/v) and of single and twice daily doses (3.5% w/v) of a novel hydroxypropyl-ß-cyclodextrin MXD GEL formulation (MXD/HP-ß-CD) and a MXD solution were investigated in male rats. The cardiovascular effects were evaluated by telemetric monitoring of ECG and arterial pressure in free-moving rats. Ultrasonographic evaluation of cardiac morphology and function, and histopathological and biochemical analysis of the tissues, were performed. A pharmacovigilance investigation was undertaken using the EudraVigilance database for the evaluation of the potential cancer-related effects of topical MXD. Following the application of repeated escalating doses of MXD solution, cardiac hypertrophy, hypotension, enhanced serum natriuretic peptides and K+ -ion levels, serum liver biomarkers, and histological lesions including renal cancer were observed. In addition, the administration of a twice daily dose of MXD solution, at SF rat vs human = 311, caused reductions in the systolic, diastolic, and mean blood pressure of the rats (-30.76 ± 3%, -28.84 ± 4%, and -30.66 ± 5%, respectively, vs the baseline; t test P < .05). These effects were not reversible following washout of the MXD solution. Retrospective investigation showed 32 cases of cancer associated with the use of topical MXD in humans. The rats treated with MXD HP-ß-CD were less severely affected. MXD causes proliferative adverse effects. The MXD HP-ß-CD inclusion complex reduces these adverse effects.

Effect of cat litters on feline coronavirus infection of cell culture and cats.

OBJECTIVES: Feline infectious peritonitis (FIP) is caused by infection with feline coronavirus (FCoV). FCoV is incredibly contagious and transmission is via the faecal-oral route. FCoV infection, and therefore FIP, is most common in breeder and rescue catteries, where many cats are kept indoors, using litter trays. Whether it is possible to break the cycle of FCoV infection and reinfection using cat litters has never been investigated. The aim of the study was to examine the effect of cat litters on FCoV infectivity and virus load in multi-cat households, and transmission frequency. METHODS: Fifteen cat litters were mixed and incubated with FCoV, centrifuged and the supernatants tested in vitro for the ability to prevent virus infection of cell culture. To test applicability of in vitro results to real life, virus load was measured in two households in a double crossover study of four Fuller's earth-based cat litters by testing rectal swabs using FCoV reverse transcriptase quantitative PCR. RESULTS: Four litters abrogated FCoV infection of cell culture, nine reduced it to a greater or lesser extent and two had no effect. One brand had different virus inhibitory properties depending on where it was manufactured. Fuller's earth-based litters performed best, presumably by adsorbing virus. In the field study, there appeared to be less virus shedding on one Fuller's earth-based cat litter. CONCLUSIONS AND RELEVANCE: The in vitro study successfully identified cat litters that inactivate FCoV; such litters exist so do not need to be developed. Fuller's earth-based litters best prevented infection of cell culture, but did not completely abrogate FCoV transmission in two multi-cat households. A dust-free clumping Fuller's earth litter appeared to fare best, but virus shedding also varied on the control litters, complicating interpretation. Sawdust-based cat litters are not useful in FCoV-endemic households because they track badly and have a poor effect on virus infection.

Histological features of Rickettsia-like organisms in the European flat oyster (Ostrea edulis L.).

The European flat oyster (Ostrea edulis L.) represents an economically important oyster production in Southern Italy, widespread in natural beds along the coast. The practice to be eaten raw is an everlasting concern for possible health risk with a need to stringently monitor the health of aquatic environment. A screening survey using histopathological examination was undertaken by harvesting O. edulis from different sites along the Apulian coast of Italy. Tissue samples of the digestive gland, kidney, gonad, and gill were provided for morphologic study in light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) analysis. The LM observations revealed spherical cytoplasmic inclusions as basophilic prokaryote colonies in 13/250 oysters. The TEM and SEM confirmed the presence of intracytoplasmic inclusions of Rickettsia-like organisms (RLOs), merely in the epithelial cells of the digestive gland tubule tissues in the 13 oysters. Within intracytoplasmic vacuoles, RLOs exhibited a prokaryotic characteristic ultrastructure with transverse binary fission, a DNA zone full of chromatin fibers and a granular periplasmic ribosome zone. O. edulis were found positive for RLOs in wild oysters from Manfredonia, while the other sites were found free of pathological inclusions. Thus, we present the first report of a Rickettsia-like infection in the Apulian wild oyster (O. edulis) from Italy, including an ultrastructural description and pathological characterization.

Thymidine Phosphorylase Expression and Microvascular Density Correlation Analysis in Canine Mammary Tumor: Possible Prognostic Factor in Breast Cancer.

Purpose: The thymidine phosphorylase (TP) is a key enzyme involved in the metabolism of pyrimidines. Inhibition or downregulation of this enzyme causes accumulation of metabolites with consequences in DNA replication. TP regulates angiogenesis and chemotactic activity of endothelial cells. Different studies showed the presence of TP upregulation in human cancer but the correlation between TP expression and the microvascular density (MVD) in canine mammary tumors is unknown. The aim of this study was to investigate a possible correlation between the MVD and TP expression in tumor cells of canine mammary tumors of different degree of severity (G1-G3) by immunohistochemical analysis. Methods: Sixty-eight samples of spontaneous mammary neoplasia of 5-12 cm in diameter were collected from purebred and mixed-breed dogs (mean aged = 9.5 ± 7), not subject to chemotherapy treatments in veterinary clinics. Histopathological analysis and immunostaining were performed. Results: Carcinoma simple samples have been classified as 72.06% of tubule-papillary, 20.59% cysto-papillary, and 7.35% tubular carcinomas. Immunostainings revealed a marked cytoplasmic expression of TP in 30.88% of samples, mild in 32.35%, weaker in 22.07%, and negative in 14.70%. The correlation analysis and two-way ANOVA showed a linear correlation between MVD and TP with a coefficient of correlation (r) > 0.5 (p < 0.05) in G2 and G3. No correlation between variables was found in G1. Conclusions: These findings suggest that cytoplasmic TP overexpression is correlated with microvascular density in canine mammary tumors, in severe grade, and it can be a potential prognostic factor in breast cancer.

Sarcocystis bertrami in skeletal muscles of donkeys (Equus africanus asinus) from Southern Italy.

Among the protozoa of the genus Sarcocystis (Apicomplexa; Sarcocystidae), Sarcocystis bertrami (syn. Sarcocystis fayeri) is an obligate intracellular parasite of donkeys and horses with worldwide distribution. Here, we report the detection of S. bertrami in naturally infected donkeys from southern Italy and describe their structure by light microscopy (LM) and transmission electron microscopy (TEM). Protozoal cysts were detected both morphologically and molecularly in skeletal muscles of 28.57% (40/140) donkeys. Mature cysts of S. bertrami were found in skeletal muscle measuring 31-102 µm long and 19-83 µm wide with radially striated thick cyst wall. The high prevalence of infected donkeys suggests that dogs, the definitive hosts of S. bertrami, are contaminating environment with environmentally resistant sporocysts. Considering the increased consumption of raw donkey meat results also suggest a potential risk for human health.

Nectin-4 and p63 immunohistochemical expression in canine prostate tumourigenesis.

Nectin-4 is an E-cadherin-based adherens junction protein of normal epithelial cells, as well as a potent mediator of anchorage-independent cancer colony formation. It is considered a tumour-associated histological and serological marker in various human cancers. The transcription factor p63 is a basal cell marker in the normal prostate, involved in cell adhesion, as well as in the formation and survival of circulating tumour cell clusters. The aim of this study was to evaluate Nectin-4 and p63 immunohistochemical expression in 42 canine prostate tissues including 2 normal prostates, 10 benign prostatic hyperplasias (BPHs), 30 prostatic carcinomas (PCs), 1 pulmonary and 1 lymph node metastasis. From normal to neoplastic tissues, Nectin-4 showed a progressive switching from membranous (m-Nectin-4) to cytoplasmic (c-Nectin-4), regardless of the histological subtypes, except for lack of expression in solid PCs. Metastatic cells exhibited both strong membranous and cytoplasmic positivity. c-Nectin-4 expression was significantly (P < 0.0001) increased in PCs/metastasis compared to BPHs cases and a decrease (P < 0.05) of nuclear p63 immunostaining was also detected in the two groups. Furthermore, data showed a significant association (P < 0.05) between p63 and m-Nectin-4 distribution, although their colocalization was detected only in scattered cells by double immunofluorescence. Our results suggest the involvement of m-Nectin-4 in canine prostate tumourigenesis and metastatic potential, while the exact role of c-Nectin-4 expression detectable in primary PCs requires further investigations.

Cell Cycle Regulation by Ca2+-Activated K⁺ (BK) Channels Modulators in SH-SY5Y Neuroblastoma Cells.

The effects of Ca2+-activated K⁺ (BK) channel modulation by Paxilline (PAX) (10-7⁻10-4 M), Iberiotoxin (IbTX) (0.1⁻1 × 10-6 M) and Resveratrol (RESV) (1⁻2 × 10-4 M) on cell cycle and proliferation, AKT1pSer473 phosphorylation, cell diameter, and BK currents were investigated in SH-SY5Y cells using Operetta-high-content-Imaging-System, ELISA-assay, impedentiometric counting method and patch-clamp technique, respectively. IbTX (4 × 10-7 M), PAX (5 × 10-5 M) and RESV (10-4 M) caused a maximal decrease of the outward K⁺ current at +30 mV (Vm) of -38.3 ± 10%, -31.9 ± 9% and -43 ± 8%, respectively, which was not reversible following washout and cell depolarization. After 6h of incubation, the drugs concentration dependently reduced proliferation. A maximal reduction of cell proliferation, respectively of -60 ± 8% for RESV (2 × 10-4 M) (IC50 = 1.50 × 10-4 M), -65 ± 6% for IbTX (10-6 M) (IC50 = 5 × 10-7 M), -97 ± 6% for PAX (1 × 10-4 M) (IC50 = 1.06 × 10-5 M) and AKT1pser473 dephosphorylation was observed. PAX induced a G1/G2 accumulation and contraction of the S-phase, reducing the nuclear area and cell diameter. IbTX induced G1 contraction and G2 accumulation reducing diameter. RESV induced G2 accumulation and S contraction reducing diameter. These drugs share common actions leading to a block of the surface membrane BK channels with cell depolarization and calcium influx, AKT1pser473 dephosphorylation by calcium-dependent phosphatase, accumulation in the G2 phase, and a reduction of diameter and proliferation. In addition, the PAX action against nuclear membrane BK channels potentiates its antiproliferative effects with early apoptosis.

C-Kit receptor and tryptase expressing mast cells correlate with angiogenesis in breast cancer patients.

C-Kit protein is a transmembrane tyrosine kinase (TK) receptor (c-KitR-TK), which is predominantly expressed on mast cells (MCs) playing a role in tumor angiogenesis. It could be also expressed on epithelial breast cancer cells (EBCCs), but no data have been published regarding the correlation between mast cells positive to c-KitR (MCs-c-KitR), EBCCs positive to c-KitR (EBCCs-c-KitR), BC angiogenesis in terms of microvessel density (MVD) and the main clinic-pathological features. This study aims to evaluate the above parameters and their correlations in a series of selected 121 female early BC patients. It has been found a strong correlation between MVD and MCDPT, and MCs-c-KitR, MVD and MCs density positive to tryptase (MCDPT), and MCs-c-KitR and MCDPT by Pearson correlation. These data suggest an involvement of both MCDPT and MCs-c-KitR in BC tumor angiogenesis. Furthermore, BC tissue expressing c-KitR could be a putative predictive factor to c-KitR-TK inhibitors. In this way, selected patients with higher MCs-c-KitR could be candidate to receive c-KitR-TK inhibitors (e.g. masitinib, sunitinib) or tryptase inhibitors (e.g. nafamostat mesilate, gabexate mesilate).

First report of Angiostrongylus vasorum in a wild red fox (Vulpes vulpes) from Apulia (Italy).

Severe lung strongylosis was detected in a wild red fox (Vulpes vulpes) (1/12) from Apulia (Italy). We performed routine diagnostics on 12 foxes found dead in Apulia. Eleven of them showed lesions consistent with a vehicle collision. However, the remaining fox appeared to have died from other causes. At necropsy we observed, catarrhal enteritis, fatty liver, lung congestion with some areas rm in consistence and brain haemorrhages and malacia. Histopathology revealed lung brosis with mononucleate cells in ltration, thrombosis a several larval nematodes spread in the parenchyma, interstitial nephritis, interstitial myocarditis, encephalitis, encephalomalacia, and a brain granuloma. The larvae recovered from the lung parenchyma were identi ed as the rst stage larvae of Angiostrongylus vasorum. This is the rst documented report of angiostrongylosis in a fox in Southern Italy.

Expression of proto-oncogene C-kit and correlation with morphological evaluations in canine cutaneous mast cell tumors.

Canine cutaneous mast cell tumor (MCT) is very common disease in dogs, this is more aggressive than in other species. The biologic behavior of MCT is highly variable and a more accurate prognosis for these tumors needs to performed. The proto-oncogene c-kit is known to play a critical role in development and function of mast cells (MC). The aim of this study was to evaluate the expression of immunohistochemical pattern of c-kit in MCTs and to correlate these results with MC density (MCD) and intratumoral microvessel density (MVD). Our results confirm that a more aggressive biologic behavior of canine MCT is associated with the increased c-kit expression, further suggesting a new role for c-kit, as a useful marker, in diagnostic pathology and in tumor progression.

The dog mast cell tumour as a model to study the relationship between angiogenesis, mast cell density and tumour malignancy.

Substantial experimental data suggest that tumour progression is associated with angiogenesis and that increase in microvessel density (MVD) is associated with increase in mast cells density (MCD). Dog mast cell tumour (MCT) is common in dog with an incidence much higher than that found in human and in both species several common biological and clinical characteristics have been demonstrated. To evaluate the role of angiogenesis in progression of this tumour and to correlate MVD and MCD, in this study a series of 78 MCT was investigated. Serial sections obtained from biopsy specimens were processed with toluidine blue staining, specific for MC identification, and by immunohistochemistry using a polyclonal antibody anti factor VIII-related antigen (FVIII-RA), used as an endothelial marker, and MVD and MCD were determined. Results showed that MVD was significantly higher in poorly differentiated (G3) MCTs than in intermediate (G2) and well differentiated (G1) MCTs and that MCD and MVD were significantly correlated in G3, but not in G1 and G2 subgroups. These data indicate that angiogenesis and MCD are significantly correlated in MCTs progression.