Tumor Necrosis Factor‐α and Interleukin‐6 Concentrations in Cerebrospinal Fluid of Dogs After Seizures

Background

Idiopathic and acquired epilepsy are common in dogs. Up to 30% of these dogs are refractory to pharmacological treatment. Accumulating experimental evidence indicates that brain immune response and presence of inflammatory mediators decrease the threshold for individual seizures and contribute to epileptogenesis.

Hypothesis

Dogs with seizures have higher cerebrospinal interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) concentrations compared to dogs with no seizures.

Methods

A prospective double blinded study; cerebrospinal fluid (CSF) and serum IL‐6, TNF‐α and total protein (TP) concentrations were measured by a blinded investigator for the study group and CSF IL‐6 and TNF‐α levels and TP concentrations were measured in the control group (CG).

Animals

Dogs presented with seizures that had enough CSF collected to allow analysis were included in the study group. Twelve apparently healthy, quarantined, stray dogs served as control (CG).

Results

Cerebrospinal fluid TNF‐α and IL‐6 concentrations were significantly higher (P = .011, P = .039) in dogs with seizures (0 ± 70.66, 0.65 ± 10.93 pg/mL) compared to the CG (0 ± 19, 0.73 ± 0.55 pg/mL). When assessing cytokine concentrations of specifically the idiopathic epilepsy (IE) dogs compared to the CG, only TNF‐α concentrations (8.66 ± 62, 0 ± 19 pg/mL) were significantly higher (P = .01). CSF TP concentrations were not significantly higher in the study dogs compared to the CG.

Conclusions and Clinical Importance

Higher TNF‐α and IL‐6 concentration in the CSF of dogs with naturally occurring seizures. The higher supports the hypothesis that inflammatory processes through certain mediators play a role in the pathogenesis of seizures in dogs.     

Development of a Real-time PCR Assay for Detection of Chicken IL-1β, IL-18 and Tumor Necrosis Factor α Genes

In order to develop a SYBR GreenⅠ Real-time PCR assay for detection of chicken IL-1β,IL-18 and tumor necrosis factor-α (TNF-α) genes, four specific primer pairs were designed according to the chicken's IL-1β,IL-18, TNF-α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences in GenBank. The four fragments were amplified by RT-PCR from chicken embryo fibroblasts, cloned and sequenced. The recombinant plasmids containing the target gene were constructed and used as the Real-time PCR standard templates. Real-time PCR assays based on SYBR GreenⅠfor detection of chicken IL-1β,IL-18, TNF-α and GAPDH were established. The results showed that each gene's melting curve also had a single peak,each gene's amplification efficiency was 101.2%, 95.6%, 100.1% and 98.2%, R² was 0.9996,0.9998,0.9957 and 0.9989. Moreover,the assays were highly sensitive,the detection limit of 100 copies in 35 Ct and each gene's coefficient of variation less than 1.4 percent for intra-assay. This reliable Real-time PCR assay might be used for decting chicken's IL-1β,IL-18 and TNF-α mRNA expressing and provided the basis for quantitative analysis of cytokine expression in host cell after virus infect which cause immunosuppressive diseases.     

A Comparison of the Concentrations of C-reactive Protein and α1-Acid Glycoprotein in the Serum of Young and Adult Dogs with Acute Inflammation

The concentrations of C-reactive protein (CRP) and ₁-acid glycoprotein (AAG) were evaluated in 1-, 3- and 18-month-old dogs (four of each age) that had been inoculated with turpentine oil. The CRP and AAG in 3-month-old and younger dogs subjected to surgery or inoculated with either Staphylococcus aureus or a viral vaccine were also evaluated. The average CRP concentration in the sera peaked 2 days after inoculation of turpentine oil. The peak CRP concentrations in 3- and 18-month-old dogs were significantly (p<0.05) greater than those in 1-month-old dogs. The average AAG concentration in the sera peaked 4 days after inoculation of turpentine oil. No significant difference was found in AAG concentrations between any of the age groups. When experimentally inoculated with S. aureus or subjected to oophorohysterectomy, the CRP and AAG concentrations increased in 3-month-old dogs, but they increased little in 1-month-old dogs. The CRP and AAG in dogs inoculated with the viral vaccine did not increase. In dogs with fractures or subjected to percutaneous gastrostomy, the CRP and AAG concentrations correlated with the condition of dogs.     

Effects of EDTA-Induced Hypocalcaemia and Stress on Plasma TNF-α, IL-1-ra,G-CSF,GM-CSF and S-100 in Dairy Cows

The pathophysiology of postparturient paresis is still not completely understood. Knowledge recently acquired in immunology, endocrinology and cell physiology has still to be integrated in order to elucidate the aetiopathogenesis of the disease. For that purpose, the effect of the EDTA infusion model on the plasma concentrations of selected cytokines and growth factors, and of a calcium binding protein was examined in dairy cows. Six 6- to 11-year-old Brown Swiss cows in mid lactation were infused with a 5% solution of Na²EDTA in one jugular vein over a period of 5 h. Blood samples were collected from the contralateral side daily two days before, and then hourly for five hours during the infusion, hourly for five hours after the end of the infusion, and once daily for 10 days thereafter. The plasma concentrations of cortisol, tumour necrosis factor-, interleukin-1 receptor antagonist, granulocyte colony-stimulating factor, granulocyte and macrophage colony-stimulating factor, and the calcium binding protein S-100 were determined. Before the EDTA infusion, during the infusion and for two days thereafter, the mean plasma concentrations of cortisol were significantly higher than those from days 4 to 10 after the infusion. The plasma concentrations of tumour necrosis factor- and interleukin-1 receptor antagonist followed a similar profile. At the end of EDTA infusion, low concentrations of granulocyte colony-stimulating factor were detected in one cow only. On days 3 and 4, the mean plasma concentrations of granulocyte colony-stimulating factor were significantly higher than the pre-infusion values, but this was followed by a significant decrease on post-infusion day 5. From day 4 to 7, the plasma concentrations of S-100 were significantly lower than the pre-infusion values. The importance of these findings in the pathophysiology of postparturient paresis remains to be established.     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

Effects of Nandrolone and TGF-β1 in growing rabbits with osteopenia induced by over-supplementation of calcium and vitamin D3

The study was undertaken to find out the effects of over supplementation of dietary calcium and vitamin D3 on the mineralization of growing skeleton, taking rabbit as an animal model; further to study the effects of Nandrolone deconoate and TGF-β1 on the mineralization of osteopenic bones. Twenty four New Zealand White rabbits of either sex, 60 day old, were randomly divided in 4 equal groups, A, B, C and D. The animals of groups B, C and D were administered with oral supplementation of calcium (2000 mg/kg of standard rabbit feed) and vit-D3 (1000 IU/kg of standard feed) for 60 days. The animals of group A were given standard ration without any supplementation. After 60 days, the Ca-vit.D3 supplementation was discontinued; and the animals of group C were administered with TGF-β1 (10 ng, i.m.) once in every three days and animals of group D were given Nandrolone deconoate (10 mg, i.m.) once every week for 30 days, whereas in animals of group B, no treatment was given. All the animals were evaluated based on different observations like body weight, radiographic observations, circulating biochemical and hormone profile (plasma Ca, IP, AP, OC and iPTH) every 15 days up to 60 days after initiation of treatment. The results indicated that the body weight of rabbits in different groups increased gradually and steadily at different intervals till the end of observation period, however, the increase was non-significantly more in group D. The CI in group A increased gradually at different intervals; whereas in groups B, C and D, there was no appreciable increase in the CI during the period of Ca-vit.D3 supplementation, suggesting development of osteopenia. Treatment with TGF-β1 did not increase the CI significantly, whereas Nandrolone treatment resulted in significant increase in the CI on days 45 and 60. The plasma Ca levels showed slight but gradual increase from day 0 to 60 in almost all groups. Subsequently also, there was no marked change at different intervals in groups A and B; however, significant reduction in plasma Ca was noticed in group C on 15^th day and in group D on 60^th day after initiation of treatment. Plasma IP levels in groups B and C showed a decreasing trend up to day 60. After discontinuation of Ca-vit.D3 supplementation, in group B, it further decreased to remain significantly lower on 15^th day, and in groups C and D, it increased significantly on 60^th post-treatment day. There was no significant change in the AP activity during the entire period of study in group A; whereas significant reduction in AP activity was measured on 30^th and 60^th days of treatment in groups B and C, and on 15^th day of treatment in group D. Plasma iPTH values did not show any significant change at any interval during the first 60 days in all groups. On 30^th and 60^th days of treatment, the mean iPTH level remained significantly lesser in group B. In all groups treated with over supplementation of Ca and vit.D3, there was a non-significant increase in the plasma OC levels up to day 60; however, there was no significant difference between the groups. It can be concluded that additional supplementation of Ca and vit.D3 results in osteopenia in growing rabbits. Administration of Nandrolone helps to increase the mineral density in osteopenic bones, whereas TGF-β1 does not seem to have positive effect on the skeletal mineralization.     

Plasma alpha 1‐acid glycoprotein concentration in broilers: Influence of age,sex and injection of escherichia coli lipopolysaccharide

1. The influence of age, sex and injection of Escherichia coli lipopolysaccharide (LPS) on the plasma concentration of alpha‐1 acid glycoprotein (AGP) was determined in broilers using the single radial immunodiffusion method.

2. Plasma AGP concentration increased in the 3 d after hatching, and then stabilised at 240 ± 33 μg/ml up to 14 d of age.

3. No sex‐related differences in plasma AGP concentration were observed up to 6 weeks of age.

4. A single injection of 900 μg LPS per chick resulted in a 5‐fold increase in AGP concentration compared with that in saline‐injected chicks. Multiple injections of LPS (200 μg/chick every 2d for 14d) caused only a 50% increase in AGP concentration.     

The role of chicken IL-1β, IL-6 and TNF-α in the occurrence of amyloid arthropathy

In this study, the roles of IL-1β, IL-6 and TNF-α in amyloid arthropathic chickens with variable amounts (severe, moderate and mild) of amyloid accumulation were investigated. The presence and the levels of cytokines were evaluated in serum and in joint tissues by using ELISA and immunohistochemistry, respectively. One hundred brown layer chicks were allocated into four groups and intra-articular injections of Freund’s adjuvant were used to induce amyloid arthropathy in Groups II, III and IV. Vitamin A in group II, and methylprednisolone in Group IV were added to enhance and to reduce the severity of amyloidosis, respectively. At the end of the study, a positive correlation was observed among the incidence and severity of amyloidosis, the serum amyloid A levels and the IL-1β values both in the serum and tissues. Elevation in the tissue TNF-α levels in parallel with the severity of amyloidosis has also been noted. As a conclusion, IL-1β appears to play an important role in avian AA amyloidosis either alone or in combination with TNF-α. Further investigation is needed for understanding the role of the pro-inflammatory cytokines in avian AA amyloidosis.     

The Analysis for the Single Nucleotide Polymorphism of TGF-β1 and Its Association with Reproduction in Different Sheep Breeds

A single nucleotide polymorphism(SNP)of 805 bp region in the intron 6 of transforming growth factor β1(TGF-β1)gene was identified by polymerase chain reaction-single-strand composition polymorphism(PCR-SSCP)in 196 sheep among Small-tailed Han sheep,Tong sheep,Tan sheep and Oula sheep.Comparative sequence analysis of cloned products revealed an AGAC deletion at 294 bases upstream of exon 7 of the TGF-β1 gene(site 14201 in gi76871756).Statistical results of the genotype and allele frequencies in different breeds showed that genotype AB was dominant in the Small-tailed Han sheep.Genotype BB,however,was in majority in low-fecundity sheep.The results of a Chi-square test indicated that all the populations were in Hardy-Weinberg equilibrium.     

Effect of Conceptus on Transforming Growth Factor (TGF) β1 mRNA Expression and Protein Concentration in the Porcine Endometrium—In Vivo and In Vitro Studies

Transforming growth factor (TGF) β and its receptors are expressed at the conceptus-maternal interface during early pregnancy in the pig. The present studies were conducted to examine: (1) the effect of conceptus products on TGFβ1 mRNA expression and protein concentration in the porcine endometrium using in vivo and in vitro models, and (2) the effect of TGFβ1 on proliferation of porcine trophoblast cells in vitro. During in vivo experiments, gilts with one surgically detached uterine horn were slaughtered on days 11 or 14 of the estrous cycle and pregnancy. For in vitro studies, endometrial explants and luminal epithelial (LE) cells co-cultured with stromal (ST) cells were treated with conceptus-exposed medium (CEM). Moreover, porcine trophoblast cells were treated with TGFβ1, and the number of viable cells was measured. On day 11, the presence of conceptuses had no effect on TGFβ1 mRNA expression, but decreased the TGFβ1 protein concentration in the connected uterine horn compared with the detached uterine horn. In contrast to day 11, on day 14 after estrus, TGFβ1 mRNA expression and protein content in the endometrium collected from the gravid uterine horn were greater when compared with the contralateral uterine horn. The treatment of endometrial slices with CEM resulted in greater TGFβ1 mRNA expression and protein secretion. LE cells responded to CEM with an increased TGFβ1 mRNA level. Moreover, TGFβ1 stimulated the proliferation of day 14 trophoblast cells. In summary, porcine conceptuses may regulate TGFβ1 synthesis in the endometrium at the time of implantation. TGFβ1, in turn, may promote conceptus development by increasing the proliferation of trophoblast cells.     

Development of prednisone resistance in naïve canine lymphoma: Longitudinal evaluation of NR3C1α, ABCB1, and 11β-HSD mRNA expression

Prednisone resistance develops rapidly and essentially universally when dogs with lymphoma are treated with corticosteroids. We investigated naturally occurring mechanisms of prednisone resistance in seven dogs with naïve multicentric lymphoma, treated with oral prednisone; four dogs were administered concurrent cytotoxic chemotherapy. Expression of NR3C1α, ABCB1 (formerly MDR1), 11β-HSD1, and 11β-HSD2 mRNA was evaluated in neoplastic lymph nodes by real-time RT-PCR. Changes of expression levels at diagnosis and at time of clinical resistance to prednisone were compared longitudinally using a Wilcoxon signed-rank test. Clinical resistance to prednisone was observed after a median of 68 days (range: 7–348 days) after initiation of treatment. Relative to pretreatment samples, prednisone resistance was associated with decreased NR3C1α expression in biopsies of all dogs with high-grade lymphoma (six dogs, p=.031); one dog with indolent T-zone lymphoma had increased expression of NR3C1α. Resistance was not consistently associated with changes in ABCB1, 11β-HSD1, or 11β-HSD2 expression. Decreased expression of the glucocorticoid receptor (NR3C1α) may play a role in conferring resistance to prednisone in dogs with lymphoma. Results do not indicate a broad role for changes in expression of ABCB1, 11β-HSD1, and 11β-HSD2 in the emergence of prednisone resistance in lymphoma-bearing dogs.     

Expression of APOB,ADFP and FATP1 and their correlation with fat deposition in Yunnan’s top six famous chicken breeds

ABSTRACT

1. Adipose differentiation related protein (ADFP), fatty acid transport protein 1 (FATP1) and apolipoprotein B (APOB) are suspected to play an important role in determining intramuscular fat and in overall meat quality.

2. Yunnan’s top six famous chicken breeds (the Daweishan Mini, Yanjin Black-bone, Chahua, Wuding, Wuliangshan Black-bone and Piao chicken) are known for the high quality of their meat, but little is known about their expression of these three genes.

3. The present study aimed to examine the ADFP, FATP1 and APOB genes in different tissues of these six breeds at different development stages. The subcutaneous fat from the back midline and front, abdominal fat, liver and muscle tissue was sampled at 28, 49, 70, 91 and 112 days. The expression of ADFP, FATP1 and APOB was measured by real-time PCR.

4. The results showed that the expression of the three genes differed depending on age, tissue types and breeds. However, the expression of the three genes correlated with fat traits. In conclusion, the expression of the ADFP, FATP1 and APOB genes is associated with the fat traits of Yunnan’s top six chicken breeds. These results could help with molecular marker screening and marker-assisted breeding to improve the quality of poultry for meat production.     

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor,and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) β1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72 h later. Gilts in the control group (n = 23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P < 0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P < 0.05) and 15 (P < 0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFβ, LIF, and PGHS-2 on day 12 of pregnancy (P < 0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.     

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1β, IL-8 and TNF-α in porcine alveolar macrophages

ABSTRACT: Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs.