The impact of weight loss on circulating cytokines in Beagle dogs

Chronic low-grade inflammation in obesity is characterized by an increased production of pro-inflammatory and chemotactic cytokines that are contributing to insulin resistance and related co-morbidities. Cytokines act in networks and exhibit pleiotropic effects so we investigated the circulating levels of a wide array of cytokines (pro and anti-inflammatory, chemotactic and growth factors) in a canine model of weight loss. The dogs served as their own control in order to study the impact of weight loss independent of potential confounding factors, such as history of excess weight or gender. While low-grade inflammation had been previously investigated in obese dogs by measuring changes in adipokines, acute phase proteins and key pro-inflammatory cytokines, to the best of our knowledge this is the first study to evaluate how weight loss impacts a wide array of circulating cytokines.Eighteen overweight Beagle dogs were recruited (six spayed females and 12 neutered males), and none of them were grossly obese according to the body condition score (BCS). All the dogs reached an ideal weight by the end of the program. Parameters were assessed before (baseline), at mid-point (month 3) and at end-point (month 6). Plasma GM-CSF, IL-2, Il-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IFNγ, IP-10, TNFα, monocyte chemotactic protein 1 (MCP-1), keratinocyte chemokine (KC) were measured with canine multiplex immunoassays. Fat mass was assessed by dual energy X-ray absorption (DEXA).Several cytokines decreased throughout the weight loss program (p < 0.01) and were correlated with the percentage of fat measured by DEXA (p < 0.05): chemotactic (MCP-1), growth factors (GM-CSF, IL-7 and IL-2), and pro-inflammatory (KC and IL-18). We could not show trends for several cytokines, possibly because their level may be lower than the assay sensitivity: anti-inflammatory (IL-4 and IL-10), and pro-inflammatory (IL-6 and TNFα).In conclusion, while our findings for several pro-inflammatory and chemotactic cytokines are in accordance with human and rodent studies, we may have identified additional cytokines, such as growth factors, related to obesity-induced low-grade inflammation. Considering the weight loss was enabled by an adjusted diet, the role of this association of cytokines in insulin resistance and related co-morbidities needs to be clarified. Our results could help better understand the cytokine biology in dogs, and as such are relevant for further elucidating the relationship between immune function and metabolism/nutrition.     

The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.     

Evidence of LAT1 expression in canine caput epididymis

L-type amino acid transporter 1 (LAT1), the first isotype of amino acid transport system L, transports aromatic and branched amino acids pivotal for fundamental cellular activities such cellular growth and proliferation. LAT1 expression was high only in the brain in contrast to its limited distribution and low level of expression in normal tissues. We found potent LAT1 expression in canine caput epididymis by quantitative RT-PCR and Western blotting analysis. Immnuno-histochemical examination revealed observable LAT1 in microvillous epithelial cells.     

Relationship between three novel SNPs of BRCA1 and canine mammary tumors

The BRCA1 gene plays an important role in the development of human breast cancer, and recent research indicated that genetic variations of BRCA1 are also related to canine mammary tumors (CMTs). Here, using rapid amplification of cDNA ends (RACE), we cloned the 5′- and 3′-UTRs of BRCA1. By direct sequencing of the flanking sequences of the 5′- and 3′-UTRs of BRCA1, three previously unreported single-nucleotide polymorphisms (SNPs) were identified, two (−1228T >C, −1173C >T) in the putative promoter regions and one non-synonymous SNP (63449G >A) in exon 23. Compared with 16 normal samples, the sequences from 34 CMTs suggested that SNP (−1173C >T) was associated with the development of CMTs (odds ratio (OR)=2.57, 95% confidence interval (CI): 1.07–6.15).     

Mdivi-1, mitochondrial fission inhibitor,impairs developmental competence and mitochondrial function of embryos and cells in pigs

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.     

Pathogenicity of emerging Japanese type 1 porcine reproductive and respiratory syndrome virus in experimentally infected pigs

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.     

Downregulation of the expression of inhibin α subunit and betaglycan in porcine cystic follicles

Inhibins, as members of the transforming growth factor beta (TGF-β) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.     

Effect of dietary Schizochytrium microalga oil and fish oil on plasma cholesterol level in rats

The purpose of the study was to test the hypothesis that the dietary oils with different content of n‐3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) affect plasma lipid level in rats in a different degree. The diets with 6% of fish oil (FO) and Schizochytrium microalga oil (SchO; EPA+DHA content in the diets 9.5 + 12.3 and 2.6 + 29.5% of the sum of total fatty acids, respectively) were used; the diet with 6% of safflower oil (high content of n‐6 PUFA linoleic acid, 65.5%; EPA+DHA content 0.7 + 0.9%) was used as a control. The difference between FO and SchO was established only in the case of plasma triacylglycerol (TAG) level: plasma TAG of the FO‐fed rats did not differ from the control rats (p > 0.05), while SchO decreased (p < 0.05) plasma TAG to 46% of the control. On the other hand, FO and SchO decreased (p < 0.05) total plasma cholesterol (TC) in rats in the same extent, to 73% of the control. Regarding the underlying mechanisms for the TC decrease, both SchO and FO up‐regulated hepatic Insig‐1 gene (181 and 133% of the control; p < 0.05), which tended (p = 0.15 and p = 0.19 respectively) to decrease the amount of hepatic nSREBP‐2 protein (61 and 66% of the control). However, neither SchO nor FO influenced hepatic 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase gene expression (p > 0.05); SchO (but not FO) increased (p < 0.05) low‐density lipoprotein receptor mRNA in the liver. It was concluded that the decrease of total plasma cholesterol might be caused by an increased cholesterol uptake from plasma into the cells (in the case of SchO), but also by other (in the present study not tested) mechanisms.