Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Accuracy of a rapid enzyme-linked immunosorbent assay to measure progesterone in mares

The aim of this study was to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of progesterone (P4) in mares. Specifically, the objectives were as follows: (1) to determine the specificity and sensitivity of the ELISA test for determination of P4, (2) to measure the potential agreement between the 2 people performing the test, and 3) to evaluate the effect of time on the outcome. Ten mares were sampled on the day before ovulation (D-1), and on days 1 (D1), 3 (D3), and 5 (D5) following ovulation, during the reproductive season. While mares were cycling regularly, estrus was induced by the injection of 5 mg of prostaglandin (PGF2) and monitored starting on the 4th day by daily transrectal palpation and ultrasonography to determine the time of ovulation. Blood was collected and all samples (n=96) were assayed for P4 by a semiquantitative ELISA, by chemiluminescent immunoassay, and by radioimmunoassay (RIA). Based on the RIA, values of P4 on D-1, D1, D3, and D5 were significantly different (P < 0.0001) with mean and standard deviation(s) of 0.004, s = 0.52; 2.05, s = 2.58; 8.37, s = 4.17; and 12.76, s = 4.00 ng/mL respectively. The sensitivity and specificity of the semiquantitative assay were 94% and 95%, respectively for the lowest values of P4 (< 1.0 ng/mL). The value of kappa was 0.90 between 2 individuals performing the test. In conclusion, these results suggest that the semiquantitative test may be used reliably and economically to evaluate P4 levels in equine plasma in the clinical     

Increased abundance of patatins,lipoxygenase and miraculins in a thaxtomin A-habituated potato Russet Burbank somaclone with enhanced resistance to common scab

Potato common scab caused by the actinobacterium Streptomyces scabiei is characterized by the formation of corky lesions on tubers that reduce their marketability. Management of common scab is very complex and often ineffective under various environmental conditions. Using potato varieties that are more resistant to common scab remains one of the most efficient strategies to control this disease. However, very little is known about the factors associated with resistance to common scab. Somaclone RB9 regenerated from thaxtomin A-habituated potato Russet Burbank calli produced tubers more resistant to common scab than the original variety. Comparison of the RB9 tuber proteome with that of Russet Burbank using label-free quantitative proteomic analysis revealed changes in the accumulation of defence-related proteins from the patatin and lipoxygenase (LOX) families, which are involved in the metabolism of lipids, and of two miraculins of the Kunitz-type protease inhibitors family. The implication of LOX during common scab infection was studied using synchronized minitubers developed from leaf-bud cuttings. S. scabiei infection stimulated the accumulation of LOX in both Russet Burbank and RB9 minitubers, but this accumulation was intensified in RB9 minitubers. Infection also increased LOX activity in Russet Burbank and RB9 minitubers. However, LOX activity measured in noninfected RB9 minitubers was similar to that of infected Russet Burbank minitubers, indicating endogenous activation of LOX activity in RB9 minitubers. We discuss how increased LOX abundance and activity in the somaclone RB9 may contribute to improving tuber defence against common scab.     

The Exploitation of Nematode-Responsive Plant Genes in Novel Nematode Control Methods

The molecular interactions between plants and sedentary nematodes are undergoing intense study, not only for reasons of fundamental research but also for the potential benefits to agriculture. The present technology allows the transformation of an increasing number of crop plants, providing new ways to introduce resistance against plant-parasitic nematodes. The ability of sedentary nematodes to induce specialized feeding sites in plant roots is one of the most fascinating aspects of this host–parasite interaction. Molecular approaches have been initiated to identify and characterize plant genes altered in expression after infection by sedentary nematodes. The results obtained indicate that many genes indeed become up-regulated upon nematode infection. Surprisingly, several so-called constitutive promoters that are normally used to achieve high expression in plant cells are completely ‘silenced’ in the feeding sites within days after nematode infection. Generally, there are two options available for the genetic engineering of nematode resistance: the synthesis of anti-nematode proteins or the localized production of a cytotoxic protein that interferes with the development of feeding cells. Nematode-induced promoters are very useful for the production by plants of sufficiently high levels of anti-nematode proteins at feeding sites. Alternatively, interfering with feeding-cell development is somewhat similar to the hypersensitive response evoked by nematodes in a naturally resistant plant. Here, destruction of specific plant cells can be achieved by the localized expression of a cytotoxin such as barnase, a potent ribonuclease. This approach, however, calls for a highly specific ‘non-leaky’ promoter, which is active only in the feeding cells. Another possibility is to use a two-component system, where the leakiness of the promoter in other tissues is counterbalanced by the constitutive expression of a neutralizing gene.     

Fragment size is associated with post-operative complications following elective arthroscopy of the tibiotarsal joint of horses

The objective of this retrospective study was to determine the occurrence of joint-related complications after elective arthroscopy of the tibiotarsal joint (TTJ) in 329 horses, and the association with specific clinical parameters. Data were collected from medical records of horses undergoing elective tibiotarsal joint arthroscopy for fragment removal. Exact conditional univariate regression was used to determine significant risk factors for joint-related post-operative complications.Of 485 joints, 2 (0.4%) developed surgical site infection, 4 (0.8%) developed septic arthritis, 1 (0.2%) developed synovial fistula. There was a significantly increased odds of having septic arthritis as height and length of the distal intermediate ridge of the tibia (DIRT) lesion increased. The median height and length of the DIRT fragments in affected cases was 13.5 mm and 18.0 mm, respectively. For each unit (1 mm) increase in height, there was a 42% increase in the risk of septic arthritis occurrence (P = 0.0042), and a 15% increase for each unit increase in length (P = 0.035). Horses were significantly less likely to develop septic arthritis when suture smaller than USP 0 was used.Horses with larger osteochondritis dissecans lesions of the DIRT region have an increased risk of developing septic arthritis following fragment removal.     

Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis.

Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 (hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin-Starry-stained tissue sections. Results of PCR analysis and histologic examination suggested a leptospiral infection in the newborn foal. At the moment of death, the infection coexisted with a streptococcal-associated aspiration bronchopneumonia and postpartum septicemia. These findings indicate that the PCR assay based on the amplification of the hap1 gene represents a useful tool for specific detection of pathogenic leptospira in field samples taken from horses.     

Abnormal Sysmex XT‐2000iV DIFF scattergram in a cat with a prominent mastocytemia

A 2‐year‐old neutered male domestic shorthair cat was presented to the emergency service of the National Veterinary School of Toulouse (France) for acute vomiting and diarrhea with lethargy, inappetence, and adypsia for the past 48 hours. Complete blood counts were performed with the ProCyte DX at the emergency department and with the Sysmex XT‐2000iV at the laboratory 2 weeks later. The scattergrams from the two analyzers revealed similar unusual and abnormal dot plots. The Sysmex XT‐2000iV DIFF scattergram also showed no clear separation between different leukocyte populations. The eosinophil cluster was in an abnormal location compared with that of the “typical” location in a normal cat. A blood smear evaluation revealed the presence of numerous mast cells. Thus, we hypothesized that the Sysmex XT‐2000iV had detected the mast cell population, and this led to errors in the differential counts. To explore this hypothesis, we manually gated on the DIFF scattergram and performed a manual differential on the blood smear. With this new gating strategy, the Sysmex XT‐2000iV and manual differentials were similar. Thus, in the case of systemic mastocytosis, mast cells can be located between the lymphocyte, monocyte, and eosinophil clusters on scattergrams.     

Immunohistochemical characterization of a hepatic neuroendocrine carcinoma in a cat.

A hepatic mass was identified in a 5-year-old, female mixed-breed cat that died spontaneously after a clinical history of progressive emaciation, ptyalism, and persistent coryza. At necropsy, a 7-cm-diameter, yellow-brown, firm, multilobulated tumor was identified in the liver. Microscopically, the mass consisted of neoplastic cells arranged in small, closely packed nests within a thin fibrovascular stroma. These cells were of medium sized and polygonal, with fine argyrophilic cytoplasmic granules. Nuclei were predominantly round with finely stippled chromatin and indistinct nucleoli. Mitotic figures were numerous. Immunohistochemically, most of the neoplastic cells were immunoreactive for chromogranin A, neuron-specific enolase (NSE), and cytokeratin AE1/AE3 and weakly labeled for synaptophysin. The tumor was negative for glial fibrillary acidic protein (GFAP), vimentin, and cytokeratins 5, 6, 8, and 17. Vascular emboli and intrahepatic micrometastasis were also identified with chromogranin A. All these features were consistent with a hepatic neuroendocrine carcinoma and emphasized the importance of using a panel of antibodies to diagnose such rare tumors.     

Early hepatic immune response in rats infected with Fasciola hepatica

We investigated the phenotype of the T cells (CD4+ and CD8+) that produced Th1 (IFN-gamma) and Th2 cytokines (IL-4 and IL-10) during the firsttwo weeks of experimental fasciolosis in rats. We also followed the kinetics of the cytokine and proliferative responses of hepatic mononuclear cells (HMNC) over the same period. We found that HMNC were more numerous in the infected animals than in the controls. The percentage of CD4+ cells increased significantly after infection, whereas the percentage of CD8+ cells did not change. Moreover, the frequency of the cells producing (CP) cytokine changed after infection. The frequency of CP IFN-gamma on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-gamma was clearly lower on day 14 pi, whereas the frequency of CP IL-4 and CP IL-10 had increased. The CP IL-10-were mostly CD4+. Mitogenic stimulation (phorbol myristate acetate/ionomycin) of HMNC led to an increase in the amounts of the Th2 cytokines in the supernatant on days 7 and 14 pi, with the increase more pronounced on day 14. In contrast, IFN-gamma levels also increased by day 7 pi but then decreased to below control levels by day 14. In addition, HMNC proliferation in response to mitogen followed a similar pattern to IFN-gamma production. These findings suggested that, during the first 2 weeks of infection, F hepatica induced a transient ThO cytokine profile followed by downregulation of the cellular response and the induction of a Th2 cytokine profile.