Localization of cytokines in tendinocytes of the superficial digital flexor tendon in the horse

Although inflammatory activation of cytokines have been analyzed in various tissues, there have only been a few and as-yet-inconclusive studies on cytokines in equine tendons. In this study, the localizations of 4 cytokines (IL-1alpha, IL-1beta, TNFalpha and IFNgamma) in tendinocytes of the equine superficial digital flexor tendon (SDFT) were analyzed by the use of an immunohistochemical method. In inflamed tendons positive staining for all 4 cytokines antibodies were detected in endotedinieum cells and vascular epithelial cells. In contrast, negative or trace immunoreactions were obtained in many tendinocytes in the normal tendon. The variation in cellular immune responses depending on the kind of cytokine may reflect the physiological/pathological condition of the SDFT.     

Production of aromatic amino acids and related compounds from p-hydroxyphenylacetic acid by rumen microorganisms in vitro

Suspensions of mixed rumen bacteria (B), protozoa (P), and mixed rumen microorganisms (BP) prepared from rumen contents of fistulated goats were anaerobically incubated with 1 mM p‐hydroxyphenylacetic acid (HPA) at 39°C for 24 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of microbial cells in all incubations were analyzed by HPLC. Large amounts of Tyr (32.1, 42.7 and 36.1% of disappeared HPA in B, P and BP, respectively) were produced from HPA during a 12 h incubation period. The formation of Tyr in P (178.6 µmol/g MN) was 1.5 and 2 times higher than in B and BP, respectively. Phe (7–11% of the disappeared HPA) and Trp (3–6% of the disappeared HPA) were also synthesized from HPA in B, P, and BP. Phe synthesis in P (46.3 µmol/g MN) was 1.7 times higher than in B but, in contrast, Trp synthesis in B, was 1.6 times higher than in P. The metabolites p‐hydroxyphenylpyruvic acid (in the range of 5–14% of disappeared HPA), phenylacetic acid (1–11%), p‐hydroxybenzoic acid (3–7%) and benzoic acid (1–6%) were produced from HPA in B, P and BP. Phenylpropionic acid (6% of the disappeared HPA) was produced only in B and BP.     

Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations

BACKGROUND: The occurrence of carboxylic acid amide (CAA)‐fungicide‐resistant Plasmopara viticola populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a method, which utilises PCR‐RFLP, for the rapid detection of resistance to the CAA fungicide mandipropamid in P. viticola populations. With this method, a glycine‐to‐serine substitution at codon 1105 of the cellulose synthase gene PvCesA3 of CAA‐fungicide‐resistant P. viticola was easily detected, although no resistant P. viticola was detected from 398 isolates in Japan. CONCLUSION: It is proposed that the PCR‐RFLP method is a reliable tool for the rapid detection of CAA‐fungicide‐resistant P. viticola isolates. Only 4 h was required from the sampling of symptoms to the phenotyping of fungicide resistance. Copyright © 2011 Society of Chemical Industry     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

Distribution of TNF receptors and TNF receptor-associated intracellular signaling factors on equine tendinocytes in vitro

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were treated with 10 ng/ml equine TNFalpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF -R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFalpha-treated cells (inflamed condition). Intense TRAF2 and NF-kappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in vitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.     

Comparative studies on the distribution and population of immunocompetent cells in bovine hemal node, lymph node and spleen

The distribution and population of immunocompetent cells in bovine hemal node, mesenteric lymph node and spleen were analyzed comparatively by immunohistochemistry and flow cytometry. Many CD8(+) cells, CD172a(+) cells and γδ T cells were found in the lymphatic cord along the sinus of the hemal node and the splenic red pulp. A few CD8(+) cells and γδ T cells were distributed diffusely in the paracortex and medullary cord of the mesenteric lymph node. Many germinal centers were recognized in the lymphatic regions such as the cortex and white pulp of these lymphoid organs. The populations of CD8(+) cells and γδ T cells in the hemal node and the spleen were higher than those of the mesenteric lymph node. In addition, the populations of CD21(+) cells and MHC class II(+) cells in the hemal node and the mesenteric lymph node were higher than those of the spleen. The results suggest that the hemal node has an important role in both cellular and humoral immunity as well as the lymph node and the spleen in cattle.     

Inhibition of transient receptor potential vanilloid type 1 through α2 adrenergic receptors at peripheral nerve terminals relieves pain

The activation of α₂ adrenergic receptors contributes to analgesia not only in the central nervous system but also in the peripheral nervous system. We reported that noradrenaline inhibits the activity of transient receptor potential vanilloid 1 (TRPV1) evoked by capsaicin through α₂ receptors in cultured rat dorsal root ganglion (DRG) neurons. However, it is unclear whether activation of TRPV1 expressed in peripheral nerve terminals is inhibited by α₂ receptors and whether this phenomenon contributes to analgesia. Therefore, we examined effects of clonidine, an α₂ receptor agonist, on several types of nociceptive behaviors, which may be caused by TRPV1 activity, and subtypes of α₂ receptors expressed with TRPV1 in primary sensory neurons in rats. Capsaicin injected into hind paws evoked nociceptive behaviors and clonidine preinjected into the same site inhibited capsaicin-evoked responses. This inhibition was not observed when clonidine was injected into the contralateral hind paws. Preinjection of clonidine into the plantar surface of ipsilateral, but not contralateral, hind paws reduced the sensitivity to heat stimuli. Clonidine partially reduced formalin-evoked responses when it was preinjected into ipsilateral hind paws. The expression level of α_2C receptor mRNA quantified by real-time PCR was highest followed by those of α_2A and α_2B receptors in DRGs. α_2A and α_2C receptor-like immunoreactivities were detected with TRPV1-like immunoreactivities in the same neurons. These results suggest that TRPV1 and α₂ receptors are coexpressed in peripheral nerve terminals and that the functional association between these two molecules causes analgesia.