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The functional and structural performance of a 5cm synthetic small diameter vascular graft (SDVG) produced by the copolymerization of polyvinyl alcohol hydrogel with low molecular weight dextran (PVA/Dx graft) associated to mesenchymal stem cells (MSCs)-based therapies and anticoagulant treatment with heparin, clopidogrel and warfarin was tested using the ovine model during the healing period of 24 weeks. The results were compared to the ones obtained with standard expanded polyetetrafluoroethylene grafts (ePTFE graft). Blood flow, vessel and graft diameter measurements, graft appearance and patency rate (PR), thrombus, stenosis and collateral vessel formation were evaluated by B-mode ultrasound, audio and color flow Doppler. Graft and regenerated vessels morphologic evaluation was performed by scanning electronic microscopy (SEM), histopathological and immunohistochemical analysis. All PVA/Dx grafts could maintain a similar or higher PR and systolic/diastolic laminar blood flow velocities were similar to ePTFE grafts. CD14 (macrophages) and α-actin (smooth muscle) staining presented similar results in PVA/Dx/MSCs and ePTFE graft groups. Fibrosis layer was lower and endothelial cells were only detected at graft-artery transitions where it was added the MSCs. In conclusion, PVA/Dx graft can be an excellent scaffold candidate for vascular reconstruction, including clinic mechanically challenging applications, such as SDVGs, especially when associated to MSCs-based therapies to promote higher endothelialization and lower fibrosis of the vascular prosthesis, but also higher PR values., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The therapeutic effect of three polyvinyl alcohol (PVA) membranes loaded with electrically conductive materials - carbon nanotubes (PVA-CNTs) and polypyrrole (PVA-PPy) - were tested in vivo for neuro-muscular regeneration after an axonotmesis injury in the rat sciatic nerve. The membranes electrical conductivity measured was 1.5 ± 0.5 × 10 -6 S/m, 579 ± 0.6 × 10 -6 S/m, and 1837.5 ± 0.7 × 10 -6 S/m, respectively. At week-12, a residual motor and nociceptive deficit were present in all treated groups, but at week-12, a better recovery to normal gait pattern of the PVA-CNTs and PVA-PPy treated groups was observed. Morphometrical analysis demonstrated that PVA-CNTs group presented higher myelin thickness and lower g-ratio. The tibialis anterior muscle, in the PVA-PPy and PVA-CNTs groups showed a 9% and 19% increase of average fiber size area and a 5% and 10% increase of the "minimal Feret's diameter," respectively. No inflammation, degeneration, fibrosis or necrosis were detected in lung, liver, kidneys, spleen, and regional lymph nodes and absence of carbon deposits was confirmed with Von Kossa and Masson-Fontana stains. In conclusion, the membranes of PVA-CNTs and PVA-PPy are biocompatible and have electrical conductivity. The higher electrical conductivity measured in PVA-CNTs membrane might be responsible for the positive results on maturation of myelinated fibers. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1267-1280, 2017., (© 2017 Wiley Periodicals, Inc.)

This report describes an ocular mast cell tumor in a 13-year-old female sport horse. Clinical examination revealed a solitary firm mass located in the ocular mucosa, protruding from behind the left lower eyelid. The lesion was surgically removed and submitted to histopathology. Microscopically, the mass was composed of sheets of well-differentiated neoplastic round cells circumscribed by delicate connective tissue. Positive Giemsa and Toluidine Blue staining confirmed the presence of cytoplasmic granules. Neoplastic cells showed strong membranous and mild diffuse cytoplasmic immunoreactivity for c-KIT and a low KI-67 proliferative index. Based on these findings, a diagnosis of ocular mast cell tumor was made. Six months after surgical removal, no evidence of ocular lesion recurrence was detected.

The functional and structural performance of a 5cm synthetic small diameter vascular graft (SDVG) produced by the copolymerization of polyvinyl alcohol hydrogel with low molecular weight dextran (PVA/Dx graft) associated to mesenchymal stem cells (MSCs)-based therapies and anticoagulant treatment with heparin, clopidogrel and warfarin was tested using the ovine model during the healing period of 24 weeks. The results were compared to the ones obtained with standard expanded polyetetrafluoroethylene grafts (ePTFE graft). Blood flow, vessel and graft diameter measurements, graft appearance and patency rate (PR), thrombus, stenosis and collateral vessel formation were evaluated by B-mode ultrasound, audio and color flow Doppler. Graft and regenerated vessels morphologic evaluation was performed by scanning electronic microscopy (SEM), histopathological and immunohistochemical analysis. All PVA/Dx grafts could maintain a similar or higher PR and systolic/diastolic laminar blood flow velocities were similar to ePTFE grafts. CD14 (macrophages) and α-actin (smooth muscle) staining presented similar results in PVA/Dx/MSCs and ePTFE graft groups. Fibrosis layer was lower and endothelial cells were only detected at graft-artery transitions where it was added the MSCs. In conclusion, PVA/Dx graft can be an excellent scaffold candidate for vascular reconstruction, including clinic mechanically challenging applications, such as SDVGs, especially when associated to MSCs-based therapies to promote higher endothelialization and lower fibrosis of the vascular prosthesis, but also higher PR values., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Increasing relevance has been attributed to hydrogels due to their ability to provide an extracellular matrix (ECM)-like environment for cellular adhesion and proliferation, acting as mechanical scaffolds for tissue remodeling or as delivery matrices. In vivo biocompatibility of a hybrid dextrin hydrogel produced from oxidized dextrin and adipic acid dihydrazide and its combinations with human mesenchymal stem cells (hMSCs), ECM from a porcine bladder (urinary bladder matrix) and ceramic granules (Bonelike ® ), was evaluated following ISO 10993 after subcutaneous implantation in a rat model. Histological analysis after 3 and 15 d showed typical acute and chronic inflammatory responses, respectively, with a more severe reaction exhibited whenever the ceramic granules were present. However, the dextrin hydrogel was able to stabilize granules in the implant site. Dextrin hydrogel was scored as slight irritant after 3 d, similar to its combination with UBM, and as non-irritant after 15 d. The presence of viable hMSCs in the subcutaneous tissue could be confirmed by the presence of anti-human nuclei antibody (HuNu + ) cells. The production of growth factors and inflammatory and immunomodulatory cytokines by these cells was also quantified in peripheral blood confirming the successful encapsulation of hMSCs into the hydrogel matrix for cell survival promotion. The presence of hMSCs seemed to modulate the inflammatory response by accelerating its progression when compared to the acellular experimental groups. Dextrin hydrogel has proven to be a biocompatible multifunctional matrix for minimally invasive biomedical procedures, including orthopedic surgeries when associated with bone substitutes and also as a possible encapsulation matrix for cell-based therapies.

Halicephalobus gingivalis (H. gingivalis) causes a rare and fatal infection in horses and humans. Despite the zoonotic potential and severity of the disease, the epidemiology and pathogenesis of halicephalobiasis are still poorly understood. Several European cases of equine halicephalobiasis have been documented; however, in South-Eastern European countries, including Romania, equine neurohelminthiasis caused by H. gingivalis has not been previously described. Two Lipizzaner horses with a clinical history of progressive neurological signs were referred to the Pathology Department of the Cluj-Napoca (Romania) for necropsy. Both horses died with severe neurological signs. Gross examination and cytological, histological, and molecular analyses were performed. The stallions came from two different breeding farms. No history of traveling outside Romania was recorded. At necropsy, granulomatous and necrotizing lesions were observed in the kidneys, lymph nodes, brain, retroperitoneal adipose tissue, and lungs, indicating a systemic infection. Parasitological and histopathological analyses evidenced larval and adult forms of rhabditiform nematodes consistent with Halicephalobus species. Parasites were observed in both lymph and blood vessels of different organs and were also identified in urine samples. A subunit of the large-subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified from lesions in both horses.To the authors' knowledge, this is the first report of equine systemic H. gingivalis infection in Romania and in South-Eastern Europe. Our findings provide new insights into the geographic distribution of specific genetic lineages of H. gingivalis, while also raising public health awareness, as the parasite is zoonotic.

Background/aim: Peripheral nerve sheath tumors (PNSTs) belong to a very heterogeneous group of neoplasms occurring both in dogs and humans. The aim of the present study was to evaluate the histological and immunohistochemical features of canine cutaneous PNSTs contributing to further refine their diagnosis., Materials and Methods: The histopathological phenotype and biological behavior of 40 canine cutaneous PNSTs were evaluated and vimentin, S-100, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), desmin, alpha-smooth muscle actin (α-SMA) and Ki-67 immunoreactivity were assessed., Results: Respectively, 17 and 23 lesions were classified as benign and malignant PNSTs. The malignant lesions were more often positive for S-100 and presented a proliferation index significantly higher when compared to the benign ones (p<0.05)., Conclusion: The differential diagnosis of PNST on routine stained samples is difficult and the immunohistochemical examination may contribute to the final diagnosis. However, these lesions present a complex histogenesis and show very variable individual features; thus, an unequivocally immunohistochemical panel that could have supported the PNST diagnostic was not achieved. Nevertheless, we concluded that Ki-67 can be a useful marker helping to discriminate the biological behavior of canine PNST., (Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: To study the therapeutic effect of three tube-guides with electrical conductivity associated to mesenchymal stem cells (MSCs) on neuro-muscular regeneration after neurotmesis., Methods: Rats with 10-mm gap nerve injury were tested using polyvinyl alcohol (PVA), PVA-carbon nanotubes (CNTs) and MSCs, and PVA-polypyrrole (PPy). The regenerated nerves and tibialis anterior muscles were processed for stereological studies after 20 wk. The functional recovery was assessed serially for gait biomechanical analysis, by extensor postural thrust, sciatic functional index and static sciatic functional index (SSI), and by withdrawal reflex latency (WRL). In vitro studies included cytocompatibility, flow cytometry, reverse transcriptase polymerase chain reaction and karyotype analysis of the MSCs. Histopathology of lung, liver, kidneys, and regional lymph nodes ensured the biomaterials biocompatibility., Results: SSI remained negative throughout and independently from treatment. Differences between treted groups in the severity of changes in WRL existed, showing a faster regeneration for PVA-CNTs-MSCs (P < 0.05). At toe-off, less acute ankle joint angles were seen for PVA-CNTs-MSCs group (P = 0.051) suggesting improved ankle muscles function during the push off phase of the gait cycle. In PVA-PPy and PVA-CNTs groups, there was a 25% and 42% increase of average fiber area and a 13% and 21% increase of the "minimal Feret's diameter" respectively. Stereological analysis disclosed a significantly (P < 0.05) increased myelin thickness (M), ratio myelin thickness/axon diameter (M/d) and ratio axon diameter/fiber diameter (d/D; g-ratio) in PVA-CNT-MSCs group (P < 0.05)., Conclusion: Results revealed that treatment with MSCs and PVA-CNTs tube-guides induced better nerve fiber regeneration. Functional and kinematics analysis revealed positive synergistic effects brought by MSCs and PVA-CNTs. The PVA-CNTs and PVA-PPy are promising scaffolds with electric conductive properties, bio- and cytocompatible that might prevent the secondary neurogenic muscular atrophy by improving the reestablishment of the neuro-muscular junction.

Background: Non-Helicobacter pylori Helicobacters (NHPH) are also able to cause disease in humans. Dogs are a natural reservoir for many of these species. Close and intense human contact with animals has been identified as a risk factor and therefore, an important zoonotic significance has been attributed to NHPH., Methods: To determine the prevalence of Helicobacter species and the gastric histopathological changes associated, gastric mucosa samples of 69 dogs were evaluated., Results: Only one dog presented a normal histopathological mucosa with absence of spiral-shaped organisms. A normal gastric mucosa and the presence of spiral-shaped bacteria was observed in two dogs. All remaining animals presented histopathological changes representative of gastritis. Helicobacter species were detected in 60 dogs (87.0%) by at least one detection method. Histological, histochemical and immunohistochemical evaluations revealed that Helicobacter spp. were present in 45 (65.2%), 52 (75.4%) and 57 (82.6%) dogs, respectively. Spiral-shaped bacteria were detected by qPCR analysis in 33 (47.8%) dogs. H. heilmannii-like organisms were identified in 22 animals (66.7%) and predominantly in the antral gastric region. H. salomonis was the second most prevalent species (51.5%) although it was mainly found in association with other Helicobacter spp. and in the body gastric region. H. bizzozeronii and H. felis were less frequently detected., Conclusions: It was concluded that, despite the high incidence and worldwide distribution of gastric NHPH in dogs, the presence of specific Helicobacter species may vary between geographic regions. NHPH infections were significantly accompanied by mild to moderate intraepithelial lymphocyte infiltration and mild to moderate gastric epithelial injury, but a clear relationship between gastritis and Helicobacter infection could not be established.

Background/aim: Claudins (CLDNs) are crucial structural and functional components of tight junctions playing an important role in maintaining cell polarity, controlling paracellular diffusion and regulating cell growth and differentiation of epithelial cells. CLDNs are differentially expressed in neoplastic lesions compared to the corresponding healthy tissues; therefore, they are thought to play a role in tumorigenesis and tumor progression. Reduced expression of CLDN-1 has been observed in several types of human cancers, including thyroid tumors. There are no reports of CLDN-1 immunoexpression in normal and neoplastic canine thyroid tissues., Material and Methods: CLDN-1 immunoexpression was investigated in normal canine thyroid gland (n=2), benign (n=1) and malignant (n=10) tumors, as well as neoplastic emboli (n=6)., Results: CLDN-1 was constitutively expressed in normal canine follicular epithelium. Ninety percent of the malignant canine thyroid lesions showed absence or reduced CLDN-1 expression compared to that of normal thyroid gland. Additionally, a cytoplasmic subcellular location of CLDN-1 was recorded in the malignant epithelial cells and neoplastic emboli., Conclusion: These findings link altered expression of CLDN-1 to neoplastic transformation and suggest that CLDN-1 expression is associated with malignant canine thyroid tumors and their vascular invasion., (Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Polyvinyl alcohol hydrogel (PVA) is a synthetic polymer with an increasing application in the biomedical field that can potentially be used for vascular grafting. However, the tissue and blood-material interactions of such gels and membranes are unknown in detail. The objectives of this study were to: (a) assess the biocompatibility and (b) hemocompatibility of PVA-based membranes in order to get some insight into its potential use as a vascular graft. PVA was evaluated isolated or in copolymerization with dextran (DX), a biopolymer with known effects in blood coagulation homeostasis. The effects of the mesenchymal stem cells (MSCs) isolated from the umbilical cord Wharton's jelly in the improvement of PVA biocompatibility and in the vascular regeneration were also assessed. The biocompatibility of PVA was evaluated by the implantation of membranes in subcutaneous tissue using an animal model (sheep). Histological samples were assessed and the biological response parameters such as polymorphonuclear neutrophilic leucocytes and macrophage scoring evaluated in the implant/tissue interface by International Standards Office (ISO) Standard 10993-6 (annex E). According to the scoring system based on those parameters, a total value was obtained for each animal and for each experimental group. The in vitro hemocompatibility studies included the classic hemolysis assay and both human and sheep bloods were used. Relatively to biocompatibility results, PVA was slightly irritant to the surrounding tissues; PVA-DX or PVA plus MSCs groups presented the lowest score according to ISO Standard 10993-6. Also, PVA was considered a nonhemolytic biomaterial, presenting the lowest values for hemolysis when associated to DX., (© 2014 Wiley Periodicals, Inc.)

Background: Gastric polyps (GP) are characterised by luminal overgrowths projecting above the plane of the mucosal surface that can be classified as non-neoplastic and neoplastic lesions. In humans, recent studies have drawn attention to the malignant potential of some of these lesions. However, gastric polyps are uncommon lesions in dogs., Findings: In this study, the presence of Helicobacter spp., the cellular proliferative activity, potential phenotypic alterations, COX-2 and p53 expression in canine spontaneous gastric polyps were investigated. The expression of these molecules was also studied in normal canine gastric mucosa in order to gain further knowledge of the significance of their loss or overexpression in gastric lesions., Conclusions: The normal expression of almost all the factors evaluated, along with the reduced proliferative activity is strongly suggestive that, in dogs, spontaneous gastric polyps are not only a rare finding but also of benign nature., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000&#95;2014&#95;166.

Background: The gastric mucosa of dogs is often colonized by non-Helicobacter pylori helicobacters (NHPH), while H. pylori is the predominant gastric Helicobacter species in humans. The colonization of the human gastric mucosa by H. pylori is highly dependent on the recognition of host glycan receptors. Our goal was to define the canine gastric mucosa glycophenotype and to evaluate the capacity of different gastric Helicobacter species to adhere to the canine gastric mucosa., Materials and Methods: The glycosylation profile in body and antral compartments of the canine gastric mucosa, with focus on the expression of histo-blood group antigens was evaluated. The in vitro binding capacity of FITC-labeled H. pylori and NHPH to the canine gastric mucosa was assessed in cases representative of the canine glycosylation pattern., Results: The canine gastric mucosa lacks expression of type 1 Lewis antigens and presents a broad expression of type 2 structures and A antigen, both in the surface and glandular epithelium. Regarding the canine antral mucosa, H. heilmannii s.s. presented the highest adhesion score whereas in the body region the SabA-positive H. pylori strain was the strain that adhered more., Conclusions: The canine gastric mucosa showed a glycosylation profile different from the human gastric mucosa suggesting that alternative glycan receptors may be involved in Helicobacter spp. binding. Helicobacter pylori and NHPH strains differ in their ability to adhere to canine gastric mucosa. Among the NHPH, H. heilmannii s.s. presented the highest adhesion capacity in agreement with its reported colonization of the canine stomach., (© 2014 John Wiley & Sons Ltd.)

The healing process of the skin is a dynamic procedure mediated through a complex feedback of growth factors secreted by a variety of cells types. Despite the most recent advances in wound healing management and surgical procedures, these techniques still fail up to 50%, so cellular therapies involving mesenchymal stem cells (MSCs) are nowadays a promising treatment of skin ulcers which are a cause of high morbidity. The MSCs modulate the inflammatory local response and induce cell replacing, by a paracrine mode of action, being an important cell therapy for the impaired wound healing. The local application of human MSCs (hMSCs) isolated from the umbilical cord Wharton's jelly together with a poly(vinyl alcohol) hydrogel (PVA) membrane, was tested to promote wound healing in two dogs that were referred for clinical examination at UPVET Hospital, showing non-healing large skin lesions by the standard treatments. The wounds were infiltrated with 1000 cells/µl hMSCs in a total volume of 100 µl per cm(2) of lesion area. A PVA membrane was applied to completely cover the wound to prevent its dehydration. Both animals after the treatment demonstrated a significant progress in skin regeneration with decreased extent of ulcerated areas confirmed by histological analysis. The use of Wharton's jelly MSCs associated with a PVA membrane showed promising clinical results for future application in the treatment of chronic wounds in companion animals and humans.

Fifteen cases of canine gastric polyps, collected over a 4-year period, were investigated using gross inspection, histological procedures and immunohistochemical techniques for Helicobacter infection. No breed or sex predisposition was found for gastric polyps, although they occurred mainly in elderly animals. There were 9 pedunculated and 6 sessile polypoid growths, between 5 to 30 mm in diameter developed mainly in the pyloric region of the stomach. The most common type of gastric polyps was the hyperplastic one. The inflammatory type was identified in three cases. Foci of AB/PAS Goblet positive cells resembling intestinal metaplasia, mild dysplasia of gastric epithelium, well delimited calcified areas, islands of osteoid matrix and nematodes were present in some of these lesions. Histological examination of the adjacent gastric polyp (surrounding gastric mucosa) revealed a severe chronic inflammation in 13 cases and a high grade of Helicobacter species colonization in all cases, but Kendall test analysis showed no correlation between Helicobacter spp. colonization degree and gastritis scores (τ = 0289; p = 0.204). A significant correlation was found between Helicobacter spp. location and gastritis scores (τ = 0.497; p = 0.035). Immunohistochemistry performed with a polyclonal antibody confirmed Helicobacter spp. infection in all cases. Based on their morphology, Helicobacter pylori - like organisms were described in 3 of 15 cases. No high degree of dysplasia nor neoplasia were identified in these lesions. The etiology and pathogenesis of gastric polyps in dogs are still unknown, although a severe chronic antral gastritis may be a predisposing condition for development of gastric polyps in dogs.

Human ERBB2 is a proto-oncogene that codes for the erbB-2 epithelial growth factor receptor. In human breast cancer (HBC), erbB-2 protein overexpression has been repeatedly correlated with poor prognosis. In more recent works, underexpression of this gene has been described in HBC. Moreover, it is also recognised that oncogenes that are commonly amplified or deleted encompass point mutations, and some of these are associated with HBC. In cat mammary lesions (CMLs), the overexpression of ERBB2 (27%-59.6%) has also been described, mostly at the protein level and although cat mammary neoplasias are considered to be a natural model of HBC, molecular information is still scarce. In the present work, a cat ERBB2 fragment, comprising exons 10 to 15 (ERBB2&#95;10-15) was achieved for the first time. Allelic variants and genomic haplotype analyses were also performed, and differences between normal and CML populations were observed. Three amino acid changes, corresponding to 3 non-synonymous genomic sequence variants that were only detected in CMLs, were proposed to damage the 3D structure of the protein. We analysed the cat ERBB2 gene at the DNA (copy number determination), mRNA (expression levels assessment) and protein levels (in extra- and intra protein domains) in CML samples and correlated the last two evaluations with clinicopathological features. We found a positive correlation between the expression levels of the ERBB2 RNA and erbB-2 protein, corresponding to the intracellular region. Additionally, we detected a positive correlation between higher mRNA expression and better clinical outcome. Our results suggest that the ERBB2 gene is post-transcriptionally regulated and that proteins with truncations and single point mutations are present in cat mammary neoplastic lesions. We would like to emphasise that the recurrent occurrence of low erbB-2 expression levels in cat mammary tumours, suggests the cat mammary neoplasias as a valuable model for erbB-2 negative HBC.

Behçet disease is a chronic relapsing systemic disease with possible life-threatening presentations. Management of this disease can be challenging and reports of the off-label use of anti-TNFα agents for the treatment of severe manifestations are increasing, with good results. The authors report a case of Behçet disease with a sudden and severe multi-systemic onset successfully treated with infliximab.

Purpose: Evaluation of the effectiveness of highly focalised thermotherapy (HFT) in a melanoma mouse model, using a ferrimagnetic cement (FC) and repeated low hyperthermia treatments., Materials and Methods: A melanoma mouse model was induced with B16F10 cells in C57BL6 mice. The FC, injected into the tumour, was used as the magnetic vehicle for HFT. FC location within the tumour was assessed by radiography and its capability to generate heat, when exposed to an external high frequency magnetic field (HFMF), monitored by thermal camera. The HFT treatment consisted of three HFMF exposures, with 48-h intervals, each one lasting 30 min, with a 5-6°C tumour temperature increase. At the end of the experiment, FC samples were characterised by scanning electron microscopy (SEM) and energy dispersion spectroscopy (EDS). The presence of iron contents was analysed in the tumour, lungs, liver and spleen. Histological evaluation and immunohistochemical staining for caspase-3 were performed. Tumour growth was monitored during the experiment., Results: Surface analysis showed FC stabilisation within the tumour, and iron was absent. The thermal camera confirmed the localised temperature increase in the tumour. HFT treatments inhibited the tumour growth by ∼70% compared to controls. This was due to cell destruction by necrosis and apoptosis., Conclusions: The HFT, using the FC, proved to be a minimally invasive technique that statistically inhibited tumour growth. Results suggested that this methodology seems to be a promising technique for the treatment of solid tumours, allowing repeated low hyperthermia treatments, which can be easier and less traumatic than other hyperthermia techniques.

Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival. MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective(s): Autologous-labeled leukocytes combined with sulfur colloid bone marrow scan is the current nuclear medicine gold standard for the diagnosis of prosthetic joint infection. The goal of this study is to assess the diagnostic accuracy of a new nuclear medicine modality for detecting infection in this context., Methods: Twenty-seven patients with suspicious hip and knee arthroplasties were enrolled prospectively and underwent nuclear medicine testing using 99mTc-sulesomab and 99mTc-nanocolloids sequentially. These results were then crossed with the final diagnosis to determine the test(s) diagnostic accuracy., Results: An isolated 99mTc-sulesomab scan shows 100% sensitivity (0.40-1) and negative predictive value (0.31-1) but only 20% specificity (0.05-0.48). Combining it with a 99mTc-nanocolloid bone marrow scan increases the specificity (0.75-1) and the positive predictive value (0.40-1) to 100%. Furthermore, the combined test has fewer equivocal readings and higher interreader agreement: κ test value 0.59 vs. 0.44., Conclusion: The results support the hypothesis that these technically simpler and ready-to-use products may be an alternative to autologous-labeled leukocytes/sulfur colloid marrow scan.

Urokinase plasminogen activator (uPA) has been associated with aggressive behaviour and poor prognosis in human breast cancer, but there is no information on its expression in canine mammary tumours (CMT). uPA immunohistochemical expression was studied in 119 CMT (24 benign, 95 malignant) to investigate its relationship with clinical and histopathological parameters. Dogs with malignant mammary tumours (MMT) underwent a 2-year follow-up evaluation. MMT expressed significantly more uPA than benign tumours. In MMT, high uPA stromal expression was significantly associated with larger tumour size, high Ki-67 expression, invasive growth, high histological grade, regional lymph node metastases, development of distant metastases, and lower overall survival (OS) and disease-free survival (DFS). On the basis of these results, uPA could be considered a useful prognostic factor in dogs with MMT., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Diffuse plane normolipemic xanthomatosis (DPNX) is a rare, non-inherited disease that is often associated with systemic diseases, mainly malignant hematological (especially multiple myeloma) or lymph proliferative disorders. The DPNX can precede the appearance of such conditions by several years, so careful follow-up and periodic laboratory examinations are recommended even for patients that seemed to have no underlying disease. We describe a case associated with monoclonal gammopathy. This case shows that dermatological lesions can be the first manifestation of important hematological diseases and so physicians should be familiarized with this entity.

The histopathological and clinical aspects of canine mammary tumours (CMTs) have been widely studied, but the variation in the biological behaviour of these neoplasms hampers the identification of prognostic factors. Sustained angiogenesis has been suggested to be one of the most important factors underlying tumour growth and invasion. This process involves the action of several growth factors including vascular endothelial growth factor (VEGF). The present study characterizes the relationship between immunohistochemical expression of VEGF and gross (e.g. size and tissue fixation) and microscopical (e.g. type, growth, necrosis, lymphoid infiltration, lymph node metastasis, histological grade and proliferation index) features of CMTs. Forty-eight benign and 64 malignant CMTs were evaluated. Statistical analysis failed to show a significant relationship between VEGF expression and the pathological features, suggesting that VEGF expression occurs in both benign and malignant tumours and is independent of histological type, proliferation, tissue invasion or local metastatic capacity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Dogs represent the major reservoir of Leishmaniao chagasi and vaccination against the canine disease is a potential control strategy. However, seroconversion occurs post-vaccine and hence, there is need to discriminate between the former group and naturally infected dogs. The present study represents a comparison of the humoral immunological profiles of both groups using Leishmania soluble antigen (LSA) and fucose-mannose ligand (FML). For both categories, ear skin samples were evaluated immunohistochemically and through PCR, that was also performed in blood specimens, as well as their ability to infect Lutzomyia (Lutzomyia) longipalpis. All these tests showed negative results for the vaccinated dogs. Differences between groups were observed regarding IgG, IgG2 and IgE absorbances as determined by FML-ELISA, and for IgG1 and IgE absorbances as measured by LSA-ELISA, showing that Leishmune-immunised animals and VL naturally infected dogs present different immunological profiles, even though these differences cannot be used to distinguish between these two groups of dogs., (Copyright 2010 Elsevier B.V. All rights reserved.)

Caveolins are the major structural proteins of caveolae and play a role in tumour surveillance. The expression of caveolin-1 (Cav-1) was investigated immunohistochemically in samples of normal canine mammary tissue (n=5), benign (n=23) and malignant (n=45) mammary tumours, tumour emboli (n=10) and metastatic lesions (n=10). Cav-1 was not expressed by normal luminal epithelium, but was consistently expressed by normal myoepithelial cells. There was no epithelial expression of Cav-1 by the benign mammary lesions, but the molecule was detected in 35.6% of the malignant lesions, 80% of the tumour emboli and in 40% of the metastatic lesions. These findings link increased expression of Cav-1 to neoplastic transformation and suggest that Cav-1 expression is associated with more malignant canine mammary tumours and their vascular invasion and regional lymph node metastasis., ((c) 2010 Elsevier Ltd. All rights reserved.)

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.

Feline endometrial adenocarcinomas are uncommon malignant neoplasms that have to date been poorly characterized. The present immunohistochemical study describes the expression of the pancytokeratins AE1 and AE3, cytokeratin-14, vimentin, alpha-actin, cyclo-oxygenase-2, E-cadherin, beta-catenin, the progesterone receptor, the oestrogen receptor and caveolin-1 within normal feline uterine tissue and tissue from six cats with endometrial adenocarcinoma. Synthesis of cyclo-oxygenase-2 and reduced expression of progesterone receptors may be involved in the neoplastic transformation of feline endometrium. The loss of cellular adhesion that occurs within these tumours does not require down-regulation of E-cadherin expression and nuclear translocation of beta-catenin is not a feature of these neoplasms.

Introduction and Objective: The clinical relevance of myocardial bridging (MB) is a matter of debate. The objective of this work was to examine its prevalence, clinical presentation, prognosis, and functional features on scintigraphy in a group of patients with no significant atherosclerotic coronary disease., Methods: We retrospectively examined 7039 consecutive cardiac angiograms performed between January 1994 and December 2004, selecting cases in which MB was not accompanied by significant coronary disease (defined as luminal stenosis > 50%), valvular heart disease or cardiomyopathy. Patient characteristics such as age, gender, vascular risk factors and clinical presentation were recorded. We then contacted these patients by telephone to determine cardiovascular events since diagnosis (hospitalization, infarction, death) or symptoms of angina, and to invite them for myocardial perfusion scintigraphy (MPS) with stress testing., Results: The prevalence of MB was 0.97% (68 patients). The study population without significant coronary disease, valvular heart disease or cardiomyopathy was composed of 31 subjects, mean age 51.45 +/- 13.77 years, 94% (29 patients) male. Clinical presentations were: non-ST elevation acute coronary syndromes with or without biomarkers of necrosis 34% (14 patients), stable angina 23% (7 patients), atypical chest pain 16% (5 patients), ST-elevation myocardial infarction 13% (4 patients), other 3% (1 patient). Left ventricular function was normal in all patients, and angiography showed MB of the left anterior descending artery, 63% (19 patients) in the mid portion, and 33% (12 patients) in the distal portion. All subjects were treated medically. We were able to contact 24 (77%) of the 31 patients, all of whom underwent MPS; mean time since angiography was 61.25 months, with no cardiovascular events (hospitalization, infarction, death) or symptomatic angina. MPS only identified three patients with findings consistent with anterior wall ischemia., Conclusions: In this series, 58% of the patients with MB and no significant atherosclerotic coronary disease presented with acute coronary syndromes. In all the subjects contacted, the prognosis had been good during the time since diagnosis. In the majority of them, no signs of ischemia were detected on MPS.

Leukemia cutis represents a skin infiltration by leukemic cells. Clinically it can mimic a wide variety of dermatoses. We describe the case of a 64-year-old man with psoriasis who presented with a 4-day history of erythematous, slightly scaly, asymptomatic plaques distributed on the trunk and upper-extremities, and associated asthenia, myalgias, and anorexia. A skin biopsy revealed a leukemic infiltrate. Studies of peripheral blood and bone marrow provided a diagnosis of acute monocytic leukemia. This case report shows the importance of the clinical suspicion for the diagnosis of leukemia.

A procedure for the determination of cadmium, chromium, and lead in marine sediment slurries by electrothermal atomic absorption spectrometry is proposed. Slurry was prepared by mixing 10mg of ground sample with particle size smaller than 50 microm completed to the weight of 1.0 g with a 3% nitric acid and 10% hydrogen peroxide solution. The slurry was maintained homogeneous with an aquarium air pump. For cadmium, the best results were obtained using iridium permanent with optimum pyrolysis and atomization temperatures of 400 and 1300 degrees C, respectively, a characteristic mass, m(o) (1% absorption), of 2.3 pg (recommended 1 pg). Without modifier use, zirconium, ruthenium, and rhodium m(o) were 3.4, 4.1, 4.6, and 4.8 pg, respectively. For chromium, the most sensitive condition was obtained with zirconium permanent with optimum pyrolysis and atomization temperatures of 1500 and 2500 degrees C, m(o) of 6.6 pg (recommended 5.5 pg); and without modifier use, rhodium, iridium, and ruthenium m(o) were 5.3, 8.8, 8.8, and 8.9 pg, respectively. For lead, the best modifier was also zirconium, m(o) of 8.3 pg for the optimum pyrolysis and atomization temperatures of 600 and 1400 degrees C, respectively, (recommended m(o) of 9.0 pg). For iridium, ruthenium, without modifier, and rhodium, m(o) were 14.7, 15.5, 16.5, and 16.5 pg, respectively. For all the modifiers selected in each case, the peaks were symmetrical with r(2) higher than 0.99. Being analyzed (n=10), two marine sediment reference materials (PACS-2 and MESS-2 from NRCC), the determined values, microg l(-1), and certified values in brackets, were 2.17+/-0.05 (2.11+/-0.15) and 0.25+/-0.03 (0.24+/-0.01) for cadmium in PACS-2 and MESS-2, respectively. For chromium in PACS-2 and MESS-2 the values were 94.7+/-5.6 (90.7+/-4.6) and 102.3+/-10.7 (106+/-8), respectively. Finally, for lead in PACS-2 and MESS-2, the results obtained were 184+/-7 (183+/-8) and of 25.2+/-0.40 (21.9+/-1.2), respectively. For cadmium and lead in both samples and chromium in PACS-2, calibration was accomplished with aqueous calibration curves. For chromium in MESS-2, only with the standard addition technique results were in agreement with the certified ones. The limits of detection (k=3, n=10) obtained with the diluents were 0.1, 3.4, and 3.6 microg l(-1) for cadmium, chromium, and lead, respectively.

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

The authors examined the relationship between coping style and perceived quality of life in patients with non-insulin-dependent diabetes mellitus. Diabetic patients (N=123) and nondiabetic comparison patients (N=124) treated at a Portuguese health center completed the Nottingham Health Profile, a quality-of-life measure. The diabetic patients also completed the Coping Responses Inventory, a measure of coping styles. In both groups, female subjects had poorer quality-of-life ratings than male subjects. Diabetic patients were more likely to regard diabetes and the consequent changes in lifestyle as a threat than as a challenge. A greater proportion of diabetic patients used avoidance coping styles, which overall were related to worse quality of life, than used active confrontation coping styles. Coping style was significantly correlated with several dimensions of quality of life in diabetic patients.

The gene polo encodes a highly conserved serine/threonine protein kinase that has been implicated in several functions during cell division. Polo-like kinases are important positive regulators of cell cycle progression and have also been implicated in the exit from mitosis through the activation of the anaphase-promoting complex. Several data indicate that Plks are required for centrosome function, bipolar spindle organisation and cytokinesis. The intracellular localisation of Plks reflects their multiple roles in cell division, however, in vivo studies that describe the distribution of this protein during different stages of mitosis have never been performed. In the present work, we report the in vivo distribution of a GFP-POLO fusion protein expressed in stable transformants and analysed during the early embryonic development of Drosophila melanogaster. The GFP-POLO protein can be detected in unfertilised oocytes associated with the centromeric region of chromosomes of the polar body and followed until the formation of mitotic domains in later development. Detailed analysis of the dynamic localisation of GFP-POLO during syncytial mitotic cycles shows the timing of localisation to the centrosomes, centromeres and midbody. The results also indicate that GFP-POLO is present in astral microtubules early in mitosis, accumulates around the nuclear envelope until nuclear envelop breakdown and at metaphase associates to spindle microtubules. These in vivo studies show a highly dynamic association of POLO with multiple compartments of the mitotic apparatus. Furthermore, the wide distribution of the GFP-POLO protein to all compartments of the mitotic apparatus provides a valuable tool for future studies on cell cycle during development.

The polo gene of Drosophila melanogaster is the founding member of the polo-like kinase family which is conserved among eukaryotes. POLO has been implicated in the organisation and function of the mitotic apparatus. Furthermore, POLO has been shown to be required for normal spermatogenesis. To characterize further the role of POLO in spermatogenesis, polo mutants were analysed by immunostaining with specific antibodies and phase contrast microscopy. Immunofluorescence shows that POLO localises to the centrosomes, the centromere/kinetochore and the spindle midzone. The meiotic phenotype of various mutant allelic combinations was also studied in detail. Observation of mutant live testes indicates cytological abnormalities in all meiotic cell types, including variable DNA content and multipolar spindles. Primary spermatocytes in polo mutant testes contain an abnormal DNA content, suggesting failure of chromosome segregation during gonial division. Immunostaining of polo mutant cells with alpha-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis.

In Portugal, piroxicam is one of the most prescribed non-steroidal anti-inflammatory agents (NSAIDs) and is frequently associated to photosensitivity skin reactions, well known by all dermatologists. The authors review the physiopathology and clinical patterns of this adverse drug reaction. Importance is given to the high prevalence of thimerosal sensitisation in our country, a marker of piroxicam photosensitivity. The authors propose that piroxicam prescription should be avoided in Portugal, as this drug can be substituted by any other NSAID, without marked photosensitivity.

Mitochondrial DNA control region sequences were analyzed from 162 wolves at 27 localities worldwide and from 140 domestic dogs representing 67 breeds. Sequences from both dogs and wolves showed considerable diversity and supported the hypothesis that wolves were the ancestors of dogs. Most dog sequences belonged to a divergent monophyletic clade sharing no sequences with wolves. The sequence divergence within this clade suggested that dogs originated more than 100,000 years before the present. Associations of dog haplotypes with other wolf lineages indicated episodes of admixture between wolves and dogs. Repeated genetic exchange between dog and wolf populations may have been an important source of variation for artificial selection.

Two ultraviolet A (UVA) regimens for oral 8-methoxypsoralen (8-MOP) photochemotherapy (PUVA) for moderate/severe chronic plaque psoriasis using a half body study technique were compared. Each patient received both regimens. A higher-dose regimen based on minimal phototoxic dose (MPD) with percentage incremental increases was given to one-half of the body. The other half received a lower dose regimen based on skin type with fixed incremental UVA increases. Patients were treated twice weekly. Symmetrical plaques were scored to determine the rate of resolution with each regimen. In addition, the number of treatments, cumulative UVA dose and number of days in treatment to achieve overall clearance were recorded. Patients were reviewed monthly for 1 year to record remission data. Thirty-three patients completed the study. Both regimens were effective and well tolerated. With the MPD-based approach, the number of exposures was significantly less for patients with skin types I and II but not III. Although the cumulative UVA dose was higher with the MPD regimen for all skin types studied, the reduced number of exposures required for clearance for skin types I and II but not III, combined with the security of individualized MPD testing, has practical attractions. MPD testing also identified five patients who required an increased psoralen dose and six patients who required a reduction of the initial UVA dose with the skin type regimen. Forty-two percent were still clear 1 year after treatment and there was no significant difference in the number of days in remission between the regimens for those whose psoriasis had recurred. The reduction in the number of exposures required for clearance with the MPD-based regimen may be safer and more cost effective in the long term.

The effect of irradiation from Sylvania PUVA lamps (emitting predominantly in the ultraviolet (UVA) region) and broadband Philips TL-12 lamps (peaking in the UVB region) on two inflammatory mediators, 12(R)- and 12(S)-hydroxy-eicosatetraenoic acid (HETE) was studied. A high-performance liquid chromatography study showed significant photodegradation of both enantiomers at a concentration of 5 micrograms/ml in phosphate-buffered saline following irradiation with 10 J.cm-2 UVA or 0.375 J.cm-2 UVB. The in vitro chemokinetic microdroplet migration response of human peripheral polymorphonuclear leukocytes from normal and psoriatic subjects was significantly reduced following irradiation of 12(R)-HETE at a concentration of 1 micrograms/ml in medium with 40 J.cm-2 UVA and 1.5 J.cm-2 UVB respectively. No such effect was seen with 12(S)-HETE. The effect of ultraviolet on skin physiology, and in particular in the successful phototherapy of a range of inflammatory skin disorders, is not fully understood. The photodegradation of inflammatory mediators such as 12-HETE, as shown in this study, provides another factor of possible therapeutic significance.