Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes

Comparison between open and closed methods of herniorrhaphy in calves affected with umbilical hernia

Umbilical hernias in calves commonly present to veterinary clinics, which are normally secondary to failure of the normal closure of the umbilical ring, and which result in the protrusion of abdominal contents into the overlying subcutis. The aim of this study was to compare the suitability of commonly-used herniorrhaphies for the treatment of reducible umbilical hernia in calves. Thirty-four clinical cases presenting to the Veterinary Teaching Hospital, Chittagong Veterinary and Animal Sciences University, Chittagong, Bangladesh from July 2004 to July 2007 were subjected to comprehensive study including history, classification of hernias, size of the hernial rings, presence of adhesion with the hernial sacs, postoperative care and follow-up. They were reducible, non-painful and had no evidence of infection present on palpation. The results revealed a gender influence, with the incidence of umbilical hernia being higher in female calves than in males. Out of the 34 clinical cases, 14 were treated by open method of herniorrhaphy and 20 were treated by closed method. Complications of hernia were higher (21%) in open method-treated cases than in closed method-treated cases (5%). Hernia recurred in three calves treated with open herniorrhaphy within 2 weeks of the procedure, with swelling in situ and muscular weakness at the site of operation. Shorter operation time and excellent healing rate (80%) were found in calves treated with closed herniorrhaphy. These findings suggest that the closed herniorrhaphy is better than the commonly-used open method for the correction of reducible umbilical hernia in calves

Congenital portosystemic shunt concurrent with an atrial septal defect in a Maltese dog

Background: Portosystemic shunt and atrial septal defect (ASD) are generally congenital diseases in dogs. Rarely, dogs with congenital vascular anomalies could be related to other vascular anomalies.Case Description: A 1-year-old male Maltese dog, neutered and weighing 1.7 kg, was brought in for an additional assessment of a congenital portosystemic shunt (CPSS). CPSS was diagnosed as portocaval shunt by computed tomography. Surgical attenuation was performed. Although prognosis after CPSS attenuation was good, the dog was presented with exercise intolerance 1 year after the operation. Thoracic radiographs observed generalized cardiomegaly. Echocardiography revealed pulmonary hypertension and right-to-left shunting ASD.Conclusion: The present study reports a rare case of CPSS concurrent with ASD in a dog. As dogs with CPSS might have been associated with other vascular anomalies; therefore, echocardiography is recommended for early diagnosis of other cardiovascular anomalies

In vitro antiproliferative and apoptosis inducing effect of a methanolic extract of Azadirachta indica oil on selected cancerous and noncancerous cell lines

Objective: To find the cytotoxic and apoptotic effects of neem oil extract on the selected cancerous (A-549, PC-3 and DU-145) and noncancerous (NIH3T3 and CCD-18Co) cell lines. Methods: Viability and cytotoxic effect induced by the extract was measured by using MTT assay and apoptotic effect of the extract was evaluated by using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and activity of caspases 3, 8 and 9 through colorimetric assay kits. Results: The results showed that neem oil extract significantly reduced the viability in all selected cancer cells treated with varying concentrations of extract as compared with untreated cells and had less effect on noncancerous cell lines. It significantly increased the percentage of necrotic and apoptotic cells, and caspases 3, 8 and 9 activities in all cancer cells treated with extract as compared with untreated cells whereas no effect on noncancerous cell lines. It suggested that neem oil extract exerted a higher cytotoxic effect on cancer cells than normal cells and lower concentration induced apoptosis only in cancer cells. One of the apoptosis-inducing mechanism was through the activation of caspases signaling pathways. Conclusion: Conclusively, it implies that neem oil extract may contain one or more potential agents that can be used as a safe and effective anticancer therapy

Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system

Bovine bone marrow mesenchymal stem cells（MSCs）cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative“Real Time”reverse transcriptase polymerase chain reaction（qRT-PCR）．Results showed that bovine MSCs underwent complete chondrogenesis ; the initial stage was characterized by expression of sox９ messenger ribonucleic acid（mRNA），followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day ７to day１４of differentiation increased mRNA expression of the transforming growth factors β１and β２，basic fibroblast growth factor（FGF 2），bone morphogenic protein６（BMP 6），insulin-like growth factors１，parathyroid hormone related peptide and indian hedgehog（Ihh）were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day２１of the culture, FGF 2，BMP 6 and Ihh were highly expressed, compared to cells culextertured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: Influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation

Basosquamous carcinoma with systemic metastasis in a miniature Pinscher

Basosquamous carcinoma (BSCC) is a rare malignancy, primarily composed of basal cells with foci of squamous differentiation. It is considered to be histologically an intermediate type between basal cell carcinoma and squamous cell carcinoma, and is known to have aggressive behaviors. BSCC occurred in a 17-year-old female minipin with a history of surgical excision for a mammary tumor. The right upper hindlimb was severely enlarged to 8 × 5 cm. Cross-section showed a homogenous white to yellow-white mass compressing the surrounding muscular tissues. The tumor metastasized also to the lungs, heart, abdominal cavity, liver and salivary gland. Microscopically, basaloid cells were crowded into solid nests or lobules separated by well-developed fibrous tissues with occasional keratinizations. Since there was no skin lesions, the tumor is assumed to be originated from the formerly present tumor in mammary gland. To our literature review, this case is the first BSCC with systemic metastasis in a dog

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system

OBJECTIVE: Pluripotent mesenchymal stem cells (MSC) have been isolated and well characterized from several tissue sources, including bone marrow stroma. MSC from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated MSC from bovine bone marrow and induced chondrogenesis in order to establish a new experimental model of stem cell research. METHODS: Bone marrow was harvested from 8 calves. For inducing chondrogenesis, MSC were cultured in pellet culture system in a chemically defined medium supplemented with 0 and 10 ng/mL of transforming growth factor beta1 (TGF-beta1). Chondrogenic differentiation was evaluated by histological, immunohistochemical, and in situ hybridization techniques. The degrees of genes expression were measured by quantitative RT-PCR. RESULTS: Metachromatic alcian blue staining and immunoreactivity for type II collagen were detected in both pellet groups (0 and 10 ng/mL TGF-beta1) after 7 days of culturing. In situ hybridization demonstrated strong expression of type II collagen and aggrecan mRNAs in the round cells located at the center region of pellets and at densely organized areas. On the other hand, type I collagen mRNA was strongly expressed in the superficial layer of the pellets. After 20 days of pellet culture, expression of type II collagen mRNA in the cells which were not treated by TGF-beta1 was 1.7-fold higher compared with that treated by TGF-beta1. CONCLUSION: Independent, spontaneous chondrogenesis of bovine MSC in pellet culture occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary

Effects of ascorbic acid on proliferation and biological properties of bovine chondrocytes in alginate beads

Bovine chondrocytes were cultured in monolayers and alginate beads with or without ascorbic acid (Asc) for 16 days. Cell proliferation was examined every 4 days by staining with Hoechst 33258 dye. The gene expression of aggre can, and collagen type I and II was analyzed at 16 days by reverse transcription and polymerase chain reaction. Cell morphology and the production of extracellular matrix (ECM) were evaluated by cytochemical, immunocytochemical and electron microscopical methods. Cells were continuously cultured in alginate beads with Asc for 2 months, and the cell morphology and ECM were examined. The proliferation of chondrocytes was significantly stimulated with Asc in both monolayers and alginate beads at 16 days. Expression of the collagen type I gene in both cultures was increased, and that of the collagen type I] gene in alginate beads was decreased, by Asc. There were no significant cytochemical and immunocytochemical differences between the cultures in alginate beads with or without Asc at 16 days. In alginate beads cultured with Asc for 2 months, proliferating cells were observed mainly at the periphery of the beads, and glycosaminoglycan and collagen type II were found around the cells. These results suggest that Asc stimulated the proliferation of chondrocytes and maintained the chondrogenic properties of the cells in an alginatebeads culture