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.     

Kinetics of IL-6 and TNF-α changes in a canine model of sepsis induced by endotoxin

Sepsis is a major cause of death in veterinary medicine, although a better prognosis can result from an early diagnosis. To speed the diagnosis, the biomarkers TNF-α and IL-6 can provide valuable information regarding systemic inflammatory response. The purpose of this study was to investigate the changes in cytokine levels in an experimental model of sepsis using ELISA and real-time PCR. Ten adult Beagles were studied; seven received an IV bolus of high dose lipopolysaccharide solution (1mg/kg) to induce sepsis. The remaining three beagles were the control group. Blood samples were collected before and 1, 3, 6, 12, 24 and 48 h after administering LPS. Serum IL-6 level peaked at 3h (1.89 ± 0.10 ng/ml) and serum TNF-α peaked at 1h (1.11 ± 0.01 ng/ml). The expression of IL-6 mRNA in peripheral blood mononuclear cells (PBMC) increased 62-fold compared to the control group at 1h; TNF-α mRNA increased by 4.5-fold at 1h. The expressions of IL-6 and TNF-α mRNA in PBMCs changed more rapidly than serum IL-6 and TNF-α concentrations. In addition, TNF-α mRNA levels in PBMCs remained elevated longer than serum TNF-α. Our study establishes the basis for future work aimed at a better understanding of the systemic inflammatory response to infection and sepsis in canine patients.     

The production and distribution of IL-6 and TNF-α in subcutaneous adipose tissue and their correlation with serum concentrations in Welsh ponies with equine metabolic syndrome

A main symptom of equine metabolic syndrome (EMS) in ponies is pathological obesity characterized by abnormal accumulation of fat deposits and inflammation. In this study, we analyzed the expression of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in subcutaneous adipose tissue and the correlation with serum concentrations in peripheral blood of Welsh ponies. Based on clinical examination findings, the animals were divided into two groups: ponies affected with EMS (n = 8) and obese ponies (n = 8). The adipose tissue was examined using immunohistochemical analysis while concentrations IL-6 and TNF-α were measured using enzyme-linked immunosorbent assays (ELISAs). Additionally, histological characterization of the adipose tissue was performed. The results obtained showed that IL-6 expression in adipose tissue biopsies derived from animals with EMS was enhanced while TNF-α levels of both groups were comparable. Compared to the obese ponies, EMS animals also had significantly elevated levels of serum IL-6 and TNF-α. Histological analysis revealed macrophage infiltration and fibrosis in adipose tissue preparations from the EMS group. These data suggest that IL-6 may play a key role in the course of EMS in Welsh ponies. Our findings also demonstrated that analysis of pro-inflammatory cytokines levels in serum may serve as an additional tool for diagnosing EMS.     

The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the β2-toxin in enteric disease of domestic animals,wild animals and humans

The virulence of Clostridium perfringens, a bacterium causing enteritis and enterotoxaemia in domestic and wild animals and humans, results largely from its ability to produce toxins. In 1997, an unknown toxin of C. perfringens, the β2-toxin, and its encoding gene cpb2 were described. Since that time numerous studies have been published dealing with a possible association of cpb2-harbouring strains of C. perfringens and the occurrence of enteric disease in domestic and wild animals and humans. This article offers an overview of the current literature on the spread and pathological significance of cpb2-harbouring C. perfringens. Unambiguous conclusions on the prevalence of cpb2 and the contribution of β2-toxin to the disease cannot be drawn from existing studies but in some animal species a strong correlation between the presence of cpb2-harbouring C. perfringens, the β2-toxin and enteric disease has been reported.     

Classical swine fever virus infection modulates serum levels of INF-α, IL-8 and TNF-α in 6-month-old pigs

Several studies have highlighted the important role of cytokines in disease development of classical swine fever virus (CSFV) infection. In the present study, we examined the kinetics of 7 porcine cytokines in serum from pigs infected with 3 different CSFV strains. Based on the clinical picture in 6-month-old Danish pigs, the strains used for inoculation were classified as being of low (Bergen), low to moderate (Eystrup) and moderate to high (Lithuania) virulence. The cytokines interferon-alpha (INF-α), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) showed increased levels after CSFV infection with more or less comparable course in the 3 groups. However, the cytokine level peaked with a 2–3 days delay in pigs infected with the low virulent strain compared to those infected with a moderately or highly virulent strain. These findings may indicate that INF-α, IL-8 and TNF-α are involved in the immune response during CSFV infection with strains of different virulence.     

Inflammatory cytokines IL-6 and TNF-α regulate lymphocyte trafficking through the local lymph node

Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a 'steady-state' to a bimodal flow. Initially, a 'shutdown' phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a 'recruitment' phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6.     

硒对雏鸡空肠和回肠IL-1β、IL-6和TNF-α mRNA表达的影响

通过复制缺硒雏鸡模型,对雏鸡空肠和回肠中主要细胞因子的变化进行不同时间点的检测,为硒调控肠黏膜免疫的机制研究奠定基础。选取120只1日龄的SPF雏鸡,随机分为对照组和试验组2组。对照组雏鸡饲喂全价饲料,试验组雏鸡饲喂缺硒饲料。分别在试验期7,14,21,28d对雏鸡进行剖杀并取其空肠与回肠,利用实时荧光定量PCR法检测IL-1β、IL-6和TNF-α的mRNA表达水平。结果显示,试验组与对照组相比,缺硒雏鸡空肠在试验期14,21d时,IL-1β、IL-6和TNF-α细胞因子的表达量均升高,且差异极显著(P0.01);28d时,除IL-1β的表达量降低(P0.05),其他细胞因子的相对表达量升高(P0.01)。试验组与对照组相比,缺硒雏鸡回肠在试验期14,21d时,IL-1β、IL-6和TNF-α细胞因子表达量均升高(P0.01);在28d时,IL-6和TNF-α的相对表达量升高(P0.05)。结果表明,缺硒能够上调肠黏膜炎性细胞因子IL-1β、IL-6和TNF-αmRNA的相对表达量,这可能与硒缺乏导致雏鸡肠黏膜中氧自由基含量升高有关。     

Association of TNF-α, IL-10 and IL-6 promoter polymorphisms in pulmonary tuberculosis patients and their household contacts of younger age group

Single nucleotide polymorphisms (SNPs) of cytokine genes have been found to be involved in the clinical outcome of Tuberculosis. The present study was aimed to identify the high risk genotypes in Tuberculosis patients and their household contacts. A total of 490 subjects were studied which includes 150 active pulmonary tuberculosis patients (APTB), 190 household contacts (HHC) and 150 healthy controls (HC). The SNPs of TNF-α (-308A/G), IL-10(-1082G/A) and IL-6(-174G/C) were performed by ARMs PCR. The IL-10 GA genotype showed significant association in APTB and HHC and was 2.3 times higher risk in APTB and 3.7 times in HHC compared to HCs. The A allele was found to be significantly associated with the risk of disease. The CC genotype of IL-6 was found to be significantly associated in APTB and an insignificant positive association in HHCs. The multifactor dimensionality reduction (MDR) analysis indicated that the genotypes of IL-6 were showing high risk with GA genotype of IL-10. In conclusion the gene interaction may be useful for identification of genotypes as biomarkers to distinguish high risk individuals.     

The role of histamine and fish meal in the incidence of gizzard erosion and pro‐ventricular abnormalities in the fowl

1. The role of certain fish meals in the production of localised gizzard erosion is confirmed.

2. Lesions typical of gizzard erosion could be produced by the addition of histamine to the diet.

3. The amount of histamine occurring naturally in fish meal depends on the species of fish and the extent and nature of bacterial spoilage.

4. These variables may explain why histamine has not been implicated previously and also why there have not been consistent associations between the condition and geographical source or common factors in bulk consignments of the meal.     

黄芩苷黄芩素小檗碱对LLC-PK1细胞炎症模型中TNF-α IL-1β及IL-6表达的影响

研究中药黄芩苷、黄芩素、小檗碱对细菌脂多糖(LPS)诱导的猪肾小管上皮细胞(LLC-PK1)中肿瘤坏死因子-α(TNG-α)、白细胞介素-1β(IL-1β)及白细胞介素-6(IL-6)表达的影响。用噻唑蓝比色法(MTT法)筛选LPS、黄芩苷、黄芩素和小檗碱对细胞作用的最佳浓度,用RT-PCR检测TNF-α、IL-1βmRNA表达,以确定细胞炎症模型是否建立成功;炎症模型建立后,用最佳浓度的黄芩苷、黄芩素和小檗碱分别作用细胞24h,用qPCR检测TNF-α、IL-1β及IL-6mRNA的表达量。结果LPS刺激组细胞TNF-α、IL-1βmRNA表达量极显著高于正常对照组(P0.01),黄芩苷、黄芩素和小檗碱作用组细胞TNF-α、IL-1β及IL-6mRNA表达量比LPS刺激组显著降低(P0.05),与对照组无显著差异(P0.05)。上述结果表明,黄芩苷、黄芩素和小檗碱通过抑制炎症细胞TNF-α、IL-1β及IL-6 mRNA的表达,从而发挥抗炎作用。     

Evaluation of HSP70, 27 and TNF-α espression in different canine cutaneous and gland diseases

Veterinary Research Communications -     

The role of nuclear technologies in the diagnosis and control of livestock diseases—a review

Nuclear and nuclear-related technologies have played an important role in animal health, particularly in relation to disease diagnosis and characterization of pathogenic organisms. This review focuses primarily on how and where nuclear technologies, both non-isotopic and isotopic methods, have made their impact in the past and where it might be expected they could have an impact in the future. The review outlines the extensive use of radiation attenuation in attempts to create vaccines for a multiplicity of pathogenic organisms and how the technology is being re-examined in the light of recent advances in irradiation techniques and cryopreservation/lyophilization that might obviate some of the problems of maintenance of viable, attenuate vaccines and their transport and use in the field. This approach could be used for a number of parasitic diseases where vaccination has been problematic and where investigations into the development of molecular vaccines have still failed to deliver satisfactory candidates for generating protective immune responses. Irradiation of antigens or serum samples also has its uses in diagnosis, especially when the samples need to be transported across international boundaries, or when handling the pathogens in question when carrying out a test presents serious health hazards to laboratory personnel. The present-day extensive use of enzyme immunoassays and molecular methods (e.g., polymerase chain reaction) for diagnosis and characterization of animal pathogens has its origins in the use of isotope-labeled antigens and antibodies. These isotopic techniques that included the use of ⁷⁵Se, ³²P, ¹²⁵I, and ³⁵S isotopes enabled a level of sensitivity and specificity that was hitherto unrealized, and it is prescient to remind ourselves of just how successful these technologies were, in spite of their infrequent use nowadays. Finally, the review looks at the potential for stable isotope analysis for a variety of applications??in the tracking of animal migrations, where the migrant are potential carriers of transboundary animal diseases, and where it would be useful to determine the origins of the carrier, e.g., Highly Pathogenic Avian Influenza and its dissemination by wild water fowl. Other applications could be in monitoring sequestered microbial culture (e.g., rinderpest virus) where in the case of accidental or deliberate release of infective culture it would be possible to identify the laboratory from which the isolate originated.     

The effect of tonicity of storage media for fowl semen on the occurrence of neck‐bending spermatozoa,fertility and hatchability 1

1. Semen of White Leghorn and Rhode Island Red males was stored for 24 h in diluents hypertonic (460 mOsm/kg H₂O) and isotonic (340 mOsm/kg H₂O) to cock seminal plasma.

2. Compared with the fertility results with semen that had been stored in the hypertonic diluent or was fresh, the fertility of the White Leghorns was not affected after storage in the isotonic diluent; a decrease (P < 0.lb05) was observed, however, using Rhode Island Red semen and isotonic diluent.

3. Fresh RIR semen contained 2.lb31% “neck‐bent spermatozoa” (NBS) which was increased to 4.lb23% and 5.lb76% after dilution in hypertonic and isotonic diluents respectively and stored for 24 h. It is doubtful whether this increase (P < 0.lb05) is the sole reason for the lowered fertility obtained with this breed after storage in the isotonic diluent.     

The composition of the soil seedbank and its role in ecosystem dynamics and rehabilitation potential in the arid Tankwa Karoo Region,South Africa§

Very few soil seedbank studies have been conducted in South Africa, especially in arid rangelands. Insight into the soil seedbank could therefore improve assessment of rangeland dynamics and enhance rehabilitation efforts. This study aimed to characterise the soil seedbanks in various vegetation types of the Tankwa Karoo National Park, an arid environment in South Africa. At 43 sites soil was sampled twice at the end of spring and the end of autumn, and the composition and structure of aboveground vegetation were described at the peak growth period in spring. Seeds were isolated from the soil samples by means of flotation in a salt solution, seedbank species composition was obtained by seedling germination, and the vegetation was investigated using a line-point survey method. Overall seed density was 8 034 seeds m^?2 of which 55% was viable, and the common life-forms were therophytes and chamaephytes. Species richness and diversity were lower in the seedbank compared with those of the vegetation, and the two vegetation spheres were 25% similar. While seedbank composition suggested poor veld condition, there was enough seed density and viability for future regeneration and rehabilitation initiatives. However, this may be impeded by the absence of many perennial species in the soil seedbank.     

The role of Bcl-xL and nuclear factor-��B in the effect of taxol on the viability of dendritic cells

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-κB (NF-κB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-_L-phenylalanine chloromethyl ketone confirmed that NF-κB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.     

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.