Characteristics and outcome of suspected cerebrovascular disease in dogs: 66 cases (2009-2016).

OBJECTIVES: To characterise the clinical signs of suspected cerebrovascular disease in dogs. MATERIALS AND METHODS: Medical records of one hospital were searched from November 2009 to December 2016 for dogs that suffered of cerebrovascular disease. We diagnosed cerebrovascular disease based on acute onset, clinical signs and magnetic resonance imaging findings. The medical history, clinical signs, concurrent disease, area of infarction, cerebrospinal fluid results, month at onset and outcome were investigated in the cerebrovascular disease group and in a control group (dogs with brain disorders other than cerebrovascular disease). RESULTS: A total of 122 CVD cases were extracted from the 5312 patients that visited during the study period. Of these 122 cases, 66 (1.2%) matched the subject selection criteria of our study and were included in the analysis. Forebrain infarction was observed in 51 of 66 cases, of which 24 (47.1%) suffered from seizures. The number of dogs diagnosed with cerebrovascular disease was disproportionately high in August (nine of 59 cases) and December (13 of 59 cases). In the outcome survey, deterioration was observed in 11 of 55 cases. CLINICAL SIGNIFICANCE: Seizure is an important clinical sign of cerebrovascular disease in dogs. There was a significant seasonal variation in the number of dogs diagnosed with cerebrovascular disease in Japan. Clinical features observed in this report differ from those of previous reports and highlight the need for additional research in this area.

Shift-and-add image processing incorporated with the unsharp masking method.

Shift-and-add (SAA) is a simple image processing procedure. SAA was devised to reconstruct a diffraction-limited image from atmospherically degraded stellar images. Recently SAA has been applied to biological imaging. There are several variants of SAA. Here proposed is an SAA procedure incorporated with unsharp masking (USM). The SAA procedure proposed here encompasses an extended version of USM. The proposed SAA method retains the simplicity and easiness, and the basic features of SAA. The effectiveness of the proposed method is examined by restoring atmospherically degraded solar images. It is shown that the USM SAA reconstructed image exhibits high contrast and reveals fine structures blurred by atmospheric turbulence. It is also shown that the USM SAA performs better with a data frame selection scheme.

Case Report of Living Kidney Transplantation Using Ectopic Pelvic Kidney Harvested by Reduced Port Laparoscopic Donor Nephrectomy.

A 28-year-old woman was diagnosed as having an ectopic kidney in adolescence. She desired to donate her ectopic kidney to her mother, who was diagnosed as having renal failure. The ectopic kidney was located behind the sigmoid colon with 3 renal arteries and 3 renal veins. Laparoscopic donor nephrectomy was performed by reduced port surgery using the GelPOINT access platforms at a midline incision below the umbilicus with 1 accessory port. A thin artery of the donated kidney was ligated. An artery of the upper pole was anastomosed to the internal iliac artery, and a second artery was anastomosed directly to the inferior epigastric artery. Three veins were anastomosed to the external iliac vein: 1 anastomosed directly, 1 interposed by saphenous vein graft, and 1 interposed by harvested ovarian vein. To our knowledge, this is the first successful case of transplantation using an ectopic pelvic kidney by reduced port laparoscopic donor nephrectomy.

Investigation of methicillin-resistant Staphylococcus aureus among clinical isolates from humans and animals by culture methods and multiplex PCR.

BACKGROUND: Staphylococcus aureus is responsible for large numbers of hospital-related and community-acquired infections. In this study, we investigated the presence of S. aureus and methicillin-resistant S. aureus (MRSA) in 100 samples from animals (55 cattle, 36 dogs, and 9 cats) and 150 samples from hospitalized human patients. The samples were collected from healthy and diseased animals and from diseased humans and included milk, wound swab, pus, exudates, nasal swab and diabetic ulcer. Initially, S. aureus was isolated and identified by colony morphology, Gram staining, and biochemical tests (catalase and coagulase tests). The S. aureus-positive samples were examined by polymerase chain reaction (PCR) to determine their MRSA status. RESULTS: Of the 100 animal samples, 29 were positive for S. aureus. Four samples (13.8%) from dogs were MRSA-positive, but samples from cattle and cats were MRSA-negative. Of the 150 human samples we collected, 64 were S. aureus-positive and, of these, 34 (53.1%) were MRSA-positive. Most (28%) of the MRSA samples were isolated from surgical wound swabs, followed by the pus from skin infections (11%), exudates from diabetic ulcers (6%), exudates from burns (4%), and aural swabs (3%). By contrast, a low MRSA detection rate (n = 4) was seen in the non-human isolates, where all MRSA bacteria were isolated from nasal swabs from dogs. The antimicrobials susceptibility testing results showed that S. aureus isolates with mecA genes showed resistance to penicillin (100%), oxacillin (100%), erythromycin (73.5%), ciprofloxacin (70.6%), and gentamicin (67.7%). The lowest resistance was found against ceftazidime, and no vancomycin-resistant isolates were obtained. CONCLUSIONS: We detected S. aureus and MRSA in both human and canine specimens. Isolates were found to be resistant to some of the antimicrobials available locally. MRSA carriage in humans and animals appears to be a great threat to effective antimicrobials treatment. The prudent use of antimicrobials will reduce the antimicrobial resistance. Our findings will help to find the most appropriate treatment and to reduce antimicrobial resistance in the future by implementing prudent use of antimicrobials. Further studies are required to better understand the epidemiology of MRSA human-animal inter-species transmission in Bangladesh.

Aberrant expression of microRNAs and the miR-1/MET pathway in canine hepatocellular carcinoma.

Canine hepatocellular carcinoma (HCC) is the most common primary hepatic tumour in dogs. MicroRNA (miRNA) dysregulation has been reported in human HCC and shown to have diagnostic and prognostic value; however, there are no data on miRNA expression in canine HCC. The aim of the present study was to investigate differentially expressed miRNAs in canine HCC. Analysis of miRNA expression in canine HCC tissues and cell lines by quantitative reverse transcription PCR showed that miR-1, miR-122, let-7a, and let-7g were downregulated, whereas miR-10b and miR-21 were upregulated in canine HCC. MET is one of the target genes of miR-1. MET was upregulated in canine HCC at the gene and protein levels, and a significant correlation between the concomitant downregulation of miR-1 and upregulation of MET was observed. Fast/intermediate-proliferating canine HCC cell lines had higher MET gene and protein expression levels than the slow-proliferating cell line. These findings suggest that miRNAs are differentially expressed in canine HCC, and that the miR-1/MET pathway may be associated with canine HCC cell proliferation.

Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism.

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Rapid Communication: MiR-92a as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Prognostic significance of gene amplification of ACTN4 in stage I and II oral tongue cancer.

Despite complete resection of the early stage of oral tongue cancer by partial glossectomy, late cervical lymph node metastasis is frequently observed. Gene amplification of ACTN4 (protein name: actinin-4) is closely associated with the metastatic potential of various cancers. This retrospective study was performed to demonstrate the potential usefulness of ACTN4 gene amplification as a prognostic biomarker in patients with stage I/II oral tongue cancer. Fifty-four patients with stage I/II oral tongue cancer were enrolled retrospectively, in accordance with the reporting recommendations for tumour marker prognostic studies (REMARK) guidelines. The copy number of ACTN4 and the protein expression of actinin-4 were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. The overall survival time of patients with gene amplification of ACTN4 was significantly shorter than that of patients without gene amplification (P=0.0010, log-rank test). Gene amplification of ACTN4 was a significant independent risk factor for death in patients with stage I/II oral tongue cancer (hazard ratio 6.08, 95% confidence interval 1.66-22.27). Gene amplification of ACTN4 is a potential prognostic biomarker for overall survival in oral tongue cancer.

Phosphorylation of serine 367 of FOXC2 by p38 regulates ZEB1 and breast cancer metastasis, without impacting primary tumor growth.

Metastatic competence is contingent upon the aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), which bestows stem cell properties as well as migratory and invasive capabilities upon differentiated tumor cells. We recently identified the transcription factor FOXC2 as a downstream effector of multiple EMT programs, independent of the EMT-inducing stimulus, and as a key player linking EMT, stem cell traits and metastatic competence in breast cancer. As such, FOXC2 could serve as a potential therapeutic target to attenuate metastasis. However, as FOXC2 is a transcription factor, it is difficult to target by conventional means such as small-molecule inhibitors. Herein, we identify the serine/threonine-specific kinase p38 as a druggable upstream regulator of FOXC2 stability and function that elicits phosphorylation of FOXC2 at serine 367 (S367). Using an orthotopic syngeneic mouse tumor model, we make the striking observation that inhibition of p38-FOXC2 signaling selectively attenuates metastasis without impacting primary tumor growth. In this model, circulating tumor cell numbers are significantly reduced in mice treated with the p38 inhibitor SB203580, relative to vehicle-treated counterparts. Accordingly, genetic or pharmacological inhibition of p38 decreases FOXC2 protein levels, reverts the EMT phenotype and compromises stem cell attributes in vitro. We also identify the EMT-regulator ZEB1-known to directly repress E-cadherin/CDH1-as a downstream target of FOXC2, critically dependent on its activation by p38. Consistent with the notion that activation of the p38-FOXC2 signaling axis represents a critical juncture in the acquisition of metastatic competence, the phosphomimetic FOXC2(S367E) mutant is refractory to p38 inhibition both in vitro and in vivo, whereas the non-phosphorylatable FOXC2(S367A) mutant fails to elicit EMT and upregulate ZEB1. Collectively, our data demonstrate that FOXC2 regulates EMT, stem cell traits, ZEB1 expression and metastasis in a p38-dependent manner, and attest to the potential utility of p38 inhibitors as antimetastatic agents.

Inhibition of FOXC2 restores epithelial phenotype and drug sensitivity in prostate cancer cells with stem-cell properties.

Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties.

ACTN4 copy number increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer.

BACKGROUND: Several clinical trials have compared chemotherapy alone and chemoradiotherapy (CRT) for locally advanced pancreatic cancer (LAPC) treatment. However, predictive biomarkers for optimal therapy of LAPC remain to be identified.We retrospectively estimated amplification of the ACTN4 gene to determine its usefulness as a predictive biomarker for LAPC. METHODS: The copy number of ACTN4 in 91 biopsy specimens of LAPC before treatment was evaluated using fluorescence in situ hybridisation (FISH). RESULTS: There were no statistically significant differences in overall survival (OS) or progression-free survival (PFS) of LAPC between patients treated with chemotherapy alone or with CRT. In a subgroup analysis of patients treated with CRT, patients with a copy number increase (CNI) of ACTN4 had a worse prognosis of OS than those with a normal copy number (NCN) of ACTN4 (P=0.0005, log-rank test). However, OS in the subgroup treated with chemotherapy alone was not significantly different between patients with a CNI and a NCN of ACTN4. In the patients with a NCN of ACTN4, the median survival time of PFS in CRT-treated patients was longer than that of patients treated with chemotherapy alone (P=0.049). CONCLUSIONS: The copy number of ACTN4 is a predictive biomarker for CRT of LAPC.

GD3 synthase regulates epithelial-mesenchymal transition and metastasis in breast cancer.

The epithelial-mesenchymal transition (EMT) bestows cancer cells with increased stem cell properties and metastatic potential. To date, multiple extracellular stimuli and transcription factors have been shown to regulate EMT. Many of them are not druggable and therefore it is necessary to identify targets, which can be inhibited using small molecules to prevent metastasis. Recently, we identified the ganglioside GD2 as a novel breast cancer stem cell marker. Moreover, we found that GD3 synthase (GD3S)--an enzyme involved in GD2 biosynthesis--is critical for GD2 production and could serve as a potential druggable target for inhibiting tumor initiation and metastasis. Indeed, there is a small molecule known as triptolide that has been shown to inhibit GD3S function. Accordingly, in this manuscript, we demonstrate that the inhibition of GD3S using small hairpin RNA or triptolide compromises the initiation and maintenance of EMT instigated by various signaling pathways, including Snail, Twist and transforming growth factor-ß1 as well as the mesenchymal characteristics of claudin-low breast cancer cell lines (SUM159 and MDA-MB-231). Moreover, GD3S is necessary for wound healing, migration, invasion and stem cell properties in vitro. Most importantly, inhibition of GD3S in vivo prevents metastasis in experimental as well as in spontaneous syngeneic wild-type mouse models. We also demonstrate that the transcription factor FOXC2, a central downstream effector of several EMT pathways, directly regulates GD3S expression by binding to its promoter. In clinical specimens, the expression of GD3S correlates with poor prognosis in triple-negative human breast tumors. Moreover, GD3S expression correlates with activation of the c-Met signaling pathway leading to increased stem cell properties and metastatic competence. Collectively, these findings suggest that the GD3S-c-Met axis could serve as an effective target for the treatment of metastatic breast cancers.

Heart transplantation in pediatric population and in adults with congenital heart disease: long-term follow-up, critical clinical analysis, and perspective for the future.

BACKGROUND: Heart transplantation is a treatment option for children as well as for adults with congenital heart disease. OBJECTIVE: To report the experience of a tertiary center with heart transplant program in pediatric population and in adults with congenital heart disease. PATIENTS AND METHODS: The study consisted of the evaluation of pediatric as well as adult patients undergoing heart transplantation for congenital heart disease. We evaluated the following indication and complications such as renal dialysis, graft vascular disease, tumors and survival. RESULTS: From October 1992 to November 2013, 134 patients had transplantation, and there were 139 transplantations and 5 retransplantations. The immunosuppression regimen is based on calcineurin inhibitors and cytostatic drugs. The type of heart disease indicated for transplantation was cardiomyopathies in 70% and congenital heart disease in 30%. Of these 134 patients, 85 patients were alive. Actuarial survival is 77.4%, 69.6%, 59.3% at 1, 5, and 10 years after transplantation. Three patients underwent renal transplantation, 1 patient is in renal dialysis, and 8.2% of patients had post-transplant lymphoproliferative disease. Two patients had retransplantation for graft vascular disease; 1 of them required a simultaneous kidney transplant and died 30 days after the procedure and 1 patient is clinically well 2 years after retransplantation. CONCLUSION: Heart transplantation in children and in adults with congenital heart disease is a promising therapeutic option and enables long-term survival for these patients.

Multipotency of equine mesenchymal stem cells derived from synovial fluid.

Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellular matrices after induction of differentiation. The total number of SF-derived mesenchymal stromal cells reached >1 × 10(7) by the fourth passage. SF-derived cells were strongly positive (>90% cells positive) for CD44, CD90 and major histocompatibility complex (MHC) class I, and moderately positive (60-80% cells positive) for CD11a/CD18, CD105 and MHC class II by flow cytometry. SF-derived cells were negative for CD34 and CD45. Under specific nutrient conditions, SF-derived cells differentiated into osteogenic, chondrogenic, adipogenic and tenogenic lineages, as indicated by the expression of specific marker genes and by the production of specific extracellular matrices. Chondrogenic induction in culture resulted in a change in cell shape to a 'stone-wall' appearance and formation of a gelatinous sheet that was intensely stained with Alcian blue. SF may be a novel source of multipotent mesenchymal stem cells with the ability to regenerate chondrocytes.

Diagnostic potential of antibody titres against Candida cell wall ß-glucan in Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis syndrome of unknown aetiology in children. The administration of Candida cell wall antigens induced KD-like coronary vasculitis in mice. However, the responses of KD patients to Candida cell wall antigen are unknown. In this study, we examined the response of KD patients to ß-glucan (BG), one of the major fungal cell wall antigens, by measuring the anti-BG titre. In KD patients, the anti-C. albicans cell wall BG titre was higher than that in normal children. The anti-BG titre was also higher in KD patients compared to children who served as control subjects. The efficacy of intravenous immunoglobulin (IVIG) therapy in KD is well established. We categorized the KD patients into three groups according to the therapeutic efficacy of intravenous immunoglobulin (IVIG) and compared the anti-BG titre among these groups. Anti-BG titres were similar in the control group and the non-responsive group. In the fully responsive group, the anti-BG titre showed higher values than those in the normal children. This study demonstrated clinically that KD patients have high antibody titres to Candida cell wall BG, and suggested the involvement of Candida cell wall BG in the pathogenesis of KD. The relationship between IVIG therapy and anti-BG titre was also shown. These results provide valuable insights into the therapy and diagnosis of KD.

Distinct outcome of stage I lung adenocarcinoma with ACTN4 cell motility gene amplification.

BACKGROUND: Even if detected at an early stage, a substantial number of lung cancers relapse after curative surgery. However, no method for distinguishing such tumors has yet been established. PATIENTS AND METHODS: The copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridization on tissue microarrays comprising 543 surgically resected adenocarcinomas of the lung. RESULTS: Amplification (an increase in the copy number by ≥ 2.0 fold) of the ACTN4 gene was detected in two of seven lung adenocarcinoma cell lines and 79 (15%) of 543 cases of pathological stage I-IV lung adenocarcinoma. Multivariate analysis revealed that ACTN4 gene amplification was the most significant independent factor associated with an extremely high risk of death (hazard ratio, 6.78; P = 9.48 × 10(-5), Cox regression analysis) among 290 patients with stage I lung adenocarcinoma. The prognostic significance of ACTN gene amplification was further validated in three independent cohorts totaling 1033 patients. CONCLUSIONS: Amplification of the ACTN4 gene defines a small but substantial subset of patients with stage I lung adenocarcinoma showing a distinct outcome. Such patients require intensive medical attention and might benefit from postoperative adjuvant chemotherapy.

Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis.

Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt4 (wingless-type MMTV integration site family member-4) promoter region. The resulting elevated expression of bone morphogenetic protein-4 (Bmp4) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt4 and Bmp4 signaling pathways.