Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

Expression, purification and characterisation of recombinant Escherichia coli derived chicken interleukin-12

Zoonotic viruses, such as H5N1 Avian Influenza, pose major threats to both animals and humans, and with this in mind there is a need for the development of new anti-viral strategies. The cytokine interleukin-12 (IL-12) is known to play a pivotal regulatory role in the anti-viral response due to its role in the induction of the key anti-viral cytokine IFN-gamma. Therefore, strategies which provide a means for the production of therapeutic quantities of IL-12 may be of major benefit. Here we describe the development of biologically active Escherichia coli (E. coli) derived chicken IL-12 (ChIL-12). The single chain ChIL-12 gene was cloned into the pET32b expression vector, transformed into the BL-21 E. coli strain and expression induced with IPTG. Over expressed protein was solubilised with zwittergent detergent and isolated utilising Nickel ion affinity chromatography. Biological activity was determined as ChIL-12 stimulated proliferation of pre-treated T-cells in vitro. This study is the first example of a biologically active E. coli derived IL-12 from a non-mammalian vertebrate subsequently providing a means for testing the anti-viral therapeutic potential of ChIL-12 in an in vivo model.     

Myopathy and alterations in serum 3-methylhistidine in dogs with liver disease

Liver disease can influence the metabolism of various other organs. Regarding the influence of liver diseases on muscles, only a few studies done on people exist. The goal of our study was to investigate the influence of liver diseases on muscles in dogs. Twenty-eight dogs with different liver diseases were investigated in this study. The diagnosis of muscle alteration was based on electromyography (EMG), creatine kinase serum activity, 3-methylhistidine serum concentration and a muscle biopsy in some cases. Our results suggest that liver diseases in dogs can be accompanied with muscle alteration. 3-Methylhistidine serum concentration as a new parameter for muscle destruction in dogs was significantly increased compared to clinical healthy dogs and was comparable to those concentrations in dogs with histologically confirmed myopathy of different types. The differentiation of the liver diseases into severe hepatitis, moderate hepatitis and liver tumours showed a significant elevation of 3-methylhistidine serum concentration in cases of liver tumours (P=0.03) and a tendency in cases of severe hepatitis (P=0.07). Based on our study we can conclude that liver diseases have an influence on muscles in dogs and 3-methylhistidine could be a useful parameter for muscle destruction.     

What is your diagnosis? Muculent pleural effusion from a dog

Pleural effusion was examined from a 5-year-old, female Brittany Spaniel with a 7-day history of dyspnea, anorexia, and diarrhea. The fluid was yellow, cloudy, and slightly gelatinous, and had a total protein concentration of 2.8 g/dL, a total nucleated cell concentration of 1.1 x 10(3)/muL, and a triglyceride concentration of 177 mg/dL. A cytocentrifuged preparation contained a mixed inflammatory cell population with a predominance of small lymphocytes and abundant mucinous material in the background. The dog died 3 days later and a mass was found within the lumen and wall of the right auricle of the heart at necropsy. Histopathologic sections of the mass contained a population of anaplastic spindle cells diffusely suspended in a pale basophilic matrix, consistent with myxosarcoma. The cells were positive for vimentin and negative for cytokeratin, desmin, and von Willebrand factor VIII-related antigen. A myxoid matrix was confirmed by positive staining with Alcian blue. Myxosarcoma is a rare cardiac tumor in dogs that should be considered, along with mucus-producing carcinomas and bile, as a cause of muculent effusion.     

Evidence for vertical transmission of Mycoplasma haemocanis,but not Ehrlichia ewingii,in a dog

A 2‐year‐old female intact pregnant Beagle was evaluated after the owner surrendered her to a shelter. Prepartum and 2 months postpartum at the time of routine spay, the dam was whole‐blood polymerase chain reaction (PCR) positive for Ehrlichia ewingii. She was also whole‐blood PCR positive for Mycoplasma haemocanis prepartum and continuously for 5 months thereafter. The dam delivered 5 healthy puppies, 1 of which was whole‐blood PCR positive for M. haemocanis. All 5 puppies had antibodies against Ehrlichia spp. at 1 month of age but not thereafter, and all puppies were Ehrlichia spp. PCR negative for 5 months of follow‐up. Therefore, this study supports a potential role for vertical transmission in the maintenance of M. haemocanis in dogs as reservoir hosts. In contrast, in this case there was no evidence that E. ewingii was transmitted transplacentally or during the perinatal period.     

Unilateral polydactyly in two foals

This case report describes unilateral supernumerary digits of the forelimb in two foals. A diagnosis of polydactyly of the right forelimb in one case and of the left forelimb in the other was made by clinical examination and radiographic interpretation. Surgical excision was performed under general anaesthesia with a good cosmetic and functional outcome in both cases.