Cloning and expression of pigeon IFN-γ gene

This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498 bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.     

Expression of VEGFR and PDGFR‐α/‐β in 187 canine nasal carcinomas

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR)‐α and PDGFR‐β in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic‐membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic‐membranous expression pattern (70.9%). PDGFR‐α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR‐β was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co‐expression of rTKs was common. These results confirm expression of VEGFR, PDGFR‐α and PDGFR‐β in canine intranasal carcinomas and support the utility of TKIs.     

Paired box 7 inhibits differentiation in 3T3‐L1 preadipocytes

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3‐L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets.     

Bactrian camel (Camelus bactrianus) integrins αvβ3 and αvβ6 as FMDV receptors: Molecular cloning, sequence analysis and comparison with other species

Integrins are heterodimeric adhesion receptors that participate in a variety of cell–cell and cell–extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the αv integrin family of cellular receptors, αvβ3, αvβ6, αvβ1 and αvβ8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the αv integrins from a susceptible species as viral receptors, we have cloned Bactrian camel αv, β3 and β6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin αv, β3 and β6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel αv, β3 and β6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.     

Olive leaves (Olea europea L.) and α‐tocopheryl acetate as feed antioxidants for improving the oxidative stability of α‐linolenic acid‐enriched eggs

Ninety‐six brown Lohmann laying hens were equally assigned into four groups with six replicates. Hens within the control group were fed a corn–soybean‐based diet supplemented with 4% linseed oil. Two other groups were given the same diet further supplemented with 5 or 10 g ground olive leaves/kg feed, while the diet of the fourth group was further supplemented with 200 mg α‐tocopheryl acetate/kg. Supplementing diets with olive leaves had no effect on egg production, feed intake and egg traits. Eggs collected 28 days after feeding the experimental diets were analysed for lipid hydroperoxides and malondialdehyde (MDA) content, fatty acid profile, α‐tocopherol concentrations and susceptibility to iron‐induced lipid oxidation. Olive leaves were also analysed for total and individual phenolics, and total flavonoids, whereas their antioxidant capacity was determined using both the DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) and ABTS (2,2‐azinobis3‐ethylbenzothiazoline‐6‐sulphonic acid) radical scavenging activity assays. Results showed that neither α‐tocopheryl acetate nor olive leaves supplementation exerted (p > 0.05) any effect on the fatty acid composition of n‐3 eggs. Supplementing the diet with 5 g olive leaves/kg had no (p > 0.05) effect on the hydroperoxide levels of n‐3 eggs, while supplementing with 10 g olive leaves/kg or 200 mg α‐tocopheryl acetate/kg, the lipid hydroperoxide levels were reduced (p ≤ 0.05) compared to control. However, although hydroperoxides were reduced, MDA, a secondary lipid oxidation product, was not affected (p > 0.05). Iron‐induced lipid oxidation increased MDA values in eggs from all groups, the increase being higher (p ≤ 0.05) in the control group and the group supplemented with 5 g olive leaves/kg. The group supplemented with 10 g olive leaves/kg presented MDA values lower (p ≤ 0.05) than the control but higher (p ≤ 0.05) than the α‐tocopheryl acetate group, which presented MDA concentrations lower (p ≤ 0.05) than all other experimental diets at all incubation time points.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.     

Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells

Changes in the proportion of peripheral blood T cell subsets after subcutaneous inoculation of cattle with Mycobacterium bovis Bacille Calmette-Guerin (BCG) were studied. Calves were injected with approximately 8 × 10⁶ BCG bacillus and blood samples collected at weekly intervals for flow-cytometric analyses to determine the proportion of CD4⁺, CD8⁺ and γδ T cells. In addition, whole blood samples were stimulated in vitro with M. bovis purified protein derivative (PPD) and the secreted IFN-γ quantified by ELISA. Results showed cellular and cytokine changes which could be categorized into three phases. The first phase occurred within the first 2 weeks after vaccination involving an increase in proportion of WC1⁺ γδ T cells and a concomitant increase in the secretion of IFN-γ. These two responses peaked at 2 weeks and waned thereafter. The second phase involved an increase in the CD4/CD8 ratio as a result of an increase in the proportion of CD4⁺ T cells between 4 and 6 weeks. The third phase involved a decrease in the CD4/CD8 ratio due to an increase in the proportion of CD8⁺ T cells between 8 and 10 weeks. Surprisingly, the IFN-γ response was associated with changes in the γδ rather than the CD4⁺ or CD8⁺ T cells, suggesting that this cytokine was secreted by γδ-T cells. These results are consistent with the reported ability of γδ T cells to act rapidly and bridging the innate and classically adaptive immune responses.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

Duration of feeding linseed diet influences peroxisome proliferator-activated receptor γ and tumor necrosis factor gene expression, and muscle mass of growing–finishing barrows

The aim of the study was to investigate the effect of duration of feeding linseed (rich in n-3 PUFA) on peroxisome proliferator-activated receptor γ (PPARγ) and tumor necrosis factor (TNF-α) gene expression, and muscle mass of growing–finishing barrows. Two isoenergetic and isonitrogenous diets were formulated, and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%. Twenty-four Landrace × Yorkshire barrows weighing 35 ± 3.7 kg were randomly assigned to four treatments with six individuals per treatment. Pigs in treatment 1 (T1) fed the control diet throughout the experimental period, while pigs in T2, T3 and T4 fed the control diet except for 30, 60, and 90 d prior to slaughter when the linseed diet were fed. The experiment was conducted for 90 days. The longissimus muscle mass and each muscle mass in the hind leg were weighted. PPARγ and TNF-α mRNA expression levels in muscle, spleen and adipose tissue, and plasma concentrations of TNF-α data were measured and analyzed. The results showed that the longissimus muscle mass, quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly (P < 0.01) as prolonged the time of feeding linseed diet. The expression of PPARγ in longissimus muscle and spleen increased (P < 0.01) linearly as prolonged the time of feeding linseed diet, while the expression of PPARγ in adipose tissue were not affected (P = 0.095). Duration of linseed addition linearly decreased (P < 0.01) TNF-α gene expression levels in the longissimus dorsi muscle, adipose and spleen, and serum concentration of TNF-α as well. The expression levels of PPARγ negatively correlated with the expression of TNF-α in muscle (R² = 0.70, P < 0.001) and spleen (R²R² = 0.77, P < 0.001) respectively. Likewise, PPARγ expression level in spleen (R²R² = 0.59, P < 0.01) or muscle (R²R² = 0.52, P < 0.05) negative correlated with serum TNF-α concentration, while there were significant quadratic relation between muscular PPARγ (R²R² = 0.80, P < 0.01) or muscular TNF-α (R²R² = 0.87, P < 0.01) expression and the longissimus dorsi muscle mass. These data suggested that duration of feeding linseed diet lead to a linear decrease of TNF-α gene expression, which may increase the muscle mass in growing–finishing barrows, at least in part, through a PPARγ-dependent mechanism.     

Therapeutic effect of anti-TNF-α antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-α antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-α antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-α antibody and LVFX. Anti-TNF-α antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

Analysis of monoclonal antibodies that recognize γδ T/null cells

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γδ TcR established the majority of null cells are γδ T cells. Use of this mAb in further comparisons demonstrated the γδ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2⁻ γδ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.     

Effects of diet and weight gain on circulating tumour necrosis factor‐α concentrations in Thoroughbred geldings

Low‐grade inflammation precedes the development of obesity‐related metabolic disorders in humans, but whether the same is true in the horse is not known. The objective of this study was to examine the effects of weight gain and diet on the inflammatory state of horses as determined by serum concentrations of tumour necrosis factor‐α (TNF), an inflammatory cytokine. Fifteen mature Thoroughbred geldings with an initial body weight (BW) of 519 ± 12 kg and body condition score (BCS) of 4.3 ± 0.1 were fed a diet of hay plus a concentrate that was either high in non‐structural carbohydrates (NSC) (i.e. starch and sugar), similar to those commercially available (CON) or one that had the energy source replaced with fat and fibre (FAT) for 32 weeks. Weight gain was achieved by feeding an additional 20 Mcal/day in excess of digestible energy maintenance requirements and resulted in a final BW of 608 ± 12 kg and BCS of 6.9 ± 0.1. Horses were exercised twice daily at a walk during the weight gain period. Horses were assessed bi‐weekly for BW and BCS. Serum TNF was analysed from blood samples collected at 4‐week intervals. Although treatment groups began the study with similar mean serum TNF concentrations, 12 weeks of FAT feeding promoted a decrease in circulating TNF that was maintained throughout the study with the exception of weeks 20 and 32. For either diet, there were no linear correlations between serum TNF concentration and BCS when horses increased in BCS from four to seven. The higher level of TNF observed in horses fed the CON diet indicates an increase in some level of systemic inflammation that was independent of their weight gain from a moderately thin to fleshy condition. The influence of diet on serum TNF concentrations should be investigated in horses fed to maintain body condition.     

The effects of concentrations of lysine in media on differentiation of 3T3‐L1 preadipocytes

Intramuscular fat content is increased by feeding of low lysine diets in pigs. Reduction in dietary lysine intake results in low plasma lysine concentration and low cytosolic lysine concentration in skeletal muscles. From these observations, we hypothesized that low plasma lysine concentration in pigs fed on low lysine diets reduced supply of lysine from blood circulation to preadipocytes, and this limited supply of lysine might promote adipocyte differentiation in porcine muscles. In order to verify the hypothesis, we investigated the effects of low concentrations of lysine in culture medium on differentiation of 3T3‐L1 preadipocytes. Low concentration of lysine suppressed lipid accumulation and messenger RNA (mRNA) expression and enzyme activity of fatty acid synthase. mRNA expressions of peroxisome proliferator‐activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were lower in cells cultured in low lysine medium. On the other hand, mRNA and protein expressions of C/EBPβ and C/EBPδ were not inhibited by low concentrations of lysine in culture medium. These results indicate that low lysine concentrations in culture medium inhibit differentiation of 3T3‐L1 preadipocytes through inhibiting the mRNA expressions of PPARγ and C/EBPα.