Negative regulation of interleukin 1β expression in response to DnaK from Pseudomonas aeruginosa via the PI3K/PDK1/FoxO1 pathways

Interleukin (IL)-1β is crucial for a wide range of inflammatory responses. Previously, we reported that IL-1β is produced in response to Pseudomonas aeruginosa-derived DnaK via NF-κB and JNK pathways; however, the signaling pathways that counter the process to maintain IL-1β homeostasis are unknown. Here, we show that DnaK-mediated expression of IL1β is increased markedly in macrophages upon blockade of PI3K/PDK1. This was verified by measuring released IL-1β protein. The negative effect of PI3K on IL-1β production was dependent on suppression of both NF-κB and JNK activation. Intriguingly, PDK1 (an underlying mediator of PI3K) acted as an upstream regulator for the activation of NF-κB, but downregulated JNK activation. Furthermore, production of IL-1β and activation of JNK were triggered by inhibition of phosphorylated FoxO1; phosphorylation of FoxO1 was controlled by PDK1 signaling in response to DnaK. Thus, IL-1β production is modulated by P. aeruginosa-derived DnaK via cross-talk between JNK and PI3K/PDK1/FoxO1 pathways.     

Elevated expression of CAPON and neuronal nitric oxide synthase in the sciatic nerve of rats following constriction injury

Neuronal nitric oxide synthase (nNOS) has been implicated in peripheral nerve lesions and regeneration. The CAPON adaptor protein interacts with the PDZ domain of nNOS, helping to regulate nNOS activity at post-synaptic sites in neurones, but it is not known whether its expression is altered in sciatic nerves after chronic nerve constriction injury. In the present study, the spatiotemporal expression of CAPON was determined in chronically constricted rat sciatic nerves. Similar to the level of protein expression, CAPON mRNA was significantly up-regulated for almost 5 weeks following sciatic nerve injury. Immunohistochemistry demonstrated that increased CAPON was found mainly in S-100-positive Schwann cells. In addition, co-immunoprecipitation demonstrated an interaction between CAPON and nNOS in Schwann cells and the interaction was enhanced in injured sciatic nerves. CAPON may be involved in peripheral nerve regeneration through regulation of nNOS activity.     

Increased phosphorylation of caveolin-1 in the spinal cord of irradiated rats

Phosphorylation of caveolin-1 occurs during cell activation by various stimuli. In this study, the involvement of caveolin-1 in an irradiation injured spinal cord was examined by analyzing the phosphorylation of caveolin-1 in the spinal cord of rats after irradiation with a single dose of 15 Gray from a ⁶⁰Co γ-ray source at 24 h post-irradiation (PI). A Western blot analysis showed that the phosphorylated form of caveolin-1 (p-caveolin-1) was expressed constitutively in the normal spinal cords and was significantly higher in the spinal cord of irradiated rats at 24 h PI. The increased expression of ED1, which is a marker of activated microglia/macrophages, was matched with that of p-caveolin-1. In the irradiated spinal cords, there was a higher level of p-caveolin-1 immunoreactivity in the isolectin B4-positive microglial, ependymal, and vascular endothelial cells, in which p-caveolin-1 was weakly and constitutively expressed in the normal control spinal cords. These results suggest that total body irradiation induces activation of microglial cells in the spinal cord through the phosphorylation of caveolin-1.     

雏鸡“卷趾”麻痹症坐骨神经病变观察

１７只１日龄肉用小公鸡,饲喂低水平核黄素日粮（１．６５ｍｇ／ｋｇ）,于２２－２３日龄间有２９．４％出现其型的“卷趾”麻痹症状。光镜观察,坐骨神经纤维雪旺氏细胞肿大、增生、酸性磷酶活性增强和髓鞘变性。严重病例的坐骨神经显著肿大,神经纤维分离,间质水肿和炎性细胞浸润。电镜观察,坐骨神经有髓神经纤维的髓鞘扭曲、分层、破碎、形成同心圆层状小体,神经轴膜与髓鞘分离形成间和神经轴索变形。雪旺氏细胞胞浆中见同心     

雏鸡核黄素缺乏症的外周神经酶细胞化学

为进一步探讨核黄素缺乏时外周神经变化的发生机理,对琥珀酸脱氢酶、酸性磷酸酶、腺苷三磷酸酶、碱性磷酸酶进行了酶细胞化学研究。结果表明,核黄素不足的鸡,雪旺氏细胞和轴突中琥珀酸脱氢酶活性下降,酸性磷酸酶活性明显升高,定位于雪旺氏细胞膜上的腺苷三磷酸酶和碱性磷酸酶活性均下降。通过对琥珀酸脱氢酶、腺苷三磷酸酶、碱性磷酸酶活性变化的动态观察,证明轻症鸡自愈的现象以及雪旺氏细胞中琥珀酸脱氢酶活性下降可能是形成脱髓鞘、外周神经水肿、导致雪旺氏细胞膜上的腺苷三磷酸酶和碱性磷酸酶活性下降的原因之一,而增生的雪旺氏细胞中琥珀酸脱氢酶活性增强则可能是修复损伤的一种代偿。腺苷三磷酸酶活性下降与神经水肿密切相关。     

坐骨神经内神经诱向因子的制备与表达

用聚丙烯酰胺凝胶电泳在坐骨神经损伤侧远端提取具有神经诱向生长作用的18kDa蛋白作为抗原，通过免疫BALB／C小鼠，经杂交瘤技术与ELISA法筛选，获取稳定分泌单克隆抗体的杂交瘤细胞株。免疫组织化学法和计算机图像处理系统显示：生后1，3，7，14，21和28日龄大鼠坐骨神经中18kDa蛋白免疫反应的分布，此分布随年龄发育而逐渐减弱。     

Early steps of insulin receptor signaling in chicken and rat: apparent refractoriness in chicken muscle

The early steps of insulin receptor (IR) signaling (tyrosine phosphorylation of IR beta-subunit, IRS-1 and Shc and PI 3'-kinase activity) have been characterized in two target tissues in the chicken: liver and muscle. The signaling cascade appeared to depend on nutritional status in the liver, but not in muscle (with a possible exception for a minor tyrosine phosphorylation of the 52 kDa Shc isoform). In this study, we compared the responses of the liver and muscle to exogenous insulin (10 or 1000 mU/kg) in chickens and rats. In the liver, IRS-1 and Shc proteins were present in smaller amounts and the regulatory subunit p85 of PI 3'-kinase was present in larger amounts in chickens than in rats. In the basal state (saline injection), the level of tyrosine phosphorylation of IR was lower, and that of Shc higher, in chickens than in rats. PI 3'-kinase activity in chickens was half that in rats. Insulin activated all components of the cascade in a dose-dependent manner in both species. A different pattern was observed in the muscle. In the basal state, the levels of tyrosine phosphorylation of IR and of PI 3'-kinase activity were much higher in chickens than in rats (by factors of 2 and 30, respectively). Insulin strongly activated all components of the cascade in rats (but with no significant increase in the phosphorylation of Shc). No activation was observed in chickens (with only a slight but significant increase in the tyrosine phosphorylation of Shc). The insulin cascade therefore appears to respond normally in chicken liver but to be refractory in chicken muscle. The large amount of p85 and high levels of PI 3'-kinase activity in muscle may contribute to this situation, making chicken muscle an interesting model of insulin resistance.     

The membrane‐type estrogen receptor G‐protein‐coupled estrogen receptor suppresses lipopolysaccharide‐induced interleukin 6 via inhibition of nuclear factor‐kappa B pathway in murine macrophage cells

The female sex hormone estrogen exerts anti‐inflammatory effects. The G‐protein‐coupled estrogen receptor (GPER) has been recently identified as a novel membrane‐type estrogen receptor that can mediate non‐genomic estrogenic effects on many cell types. We previously demonstrated that GPER inhibits tumor necrosis factor alpha‐induced expression of interleukin 6 (IL‐6) through repression of nuclear factor‐kappa B (NF‐κB) promoter activity using human breast cancer cells. Although several reports have indicated that GPER suppresses Toll‐like receptor‐induced inflammatory cytokine expression in macrophages, the molecular mechanisms of the inhibition of cytokine production via GPER remain poorly understood. In the present study, we examined GPER‐mediated inhibition of IL‐6 expression induced by lipopolysaccharide (LPS) stimulation in a mouse macrophage cell line. We found that the GPER agonist G‐1 inhibited LPS‐induced IL‐6 expression in macrophage cells, and this inhibition was due to the repression of NF‐κB promoter activity by GPER. G‐1 treatment also decreased the phosphorylation of inhibitor of κB kinases. Among the mitogen‐activated protein kinases, the phosphorylation of c‐jun N‐terminal kinase (JNK) was increased by G‐1. These findings delineate the novel mechanism of the inhibition of LPS‐induced IL‐6 through GPER‐activated JNK‐mediated negative regulation of the NF‐κB pathway in murine macrophage cells, which links anti‐inflammatory effects to estrogen.     

甘草查尔酮A对LPS诱导的奶牛蹄真皮细胞炎性反应的抑制作用

采用10,50,100 mg/L甘草查尔酮A处理蹄真皮炎性细胞24 h后,测定细胞上清液中TNF-a、IL-1β、IL-6水平及SOD、MDA含量,Western blot法检测ERK、JNK、p38、IκBα及p65蛋白表达水平.结果 显示,甘草查尔酮A可显著降低TNF-a、IL-1β、IL-6水平,提高SOD活性并...     

Strain differences in the branching of the sciatic nerve in rats

The sciatic nerve in the rat is the site most often used for peripheral nerve regeneration studies. The length of sciatic nerve available for research, however, depends on the point at which the sciatic nerve divides into the peroneal and tibial nerves. In the present study, the hind limbs of 150 adult male rats of five different strains (Sprague-Dawley, Fischer 344, Wistar-Han, Lewis and Nude) were analysed with regard to femur length, the point at which the sciatic nerve divides into the tibial and peroneal nerves, and where these are surrounded by the same epineurium, and the point at which they are encased in individual epineurial sheaths. The results indicate that the lengths of sciatic nerve are fairly constant in all strains of rats. In absolute terms, they amount to about one-third of the length of the femur for stretches of undivided sciatic nerve, and up to nearly half of the femur length for stretches where the tibial and peroneal nerves are already present, but are still enclosed by the same epineurium. In 61.7% of the hind limbs examined in Fischer rats, however, no sciatic nerve could be seen as such, but only in the form of its successors surrounded by the separate epineuria. This makes it highly advisable not to use male adult Fischer rats in peripheral nerve regeneration studies with the sciatic nerve as the point of focus.     

Effect of vaccination on cell populations in lung washes from calves after infection with respiratory syncytial virus

The inflammatory response in the air-passages of the lungs of calves after intranasal inoculation with respiratory syncytial virus (RSV) was compared in RSV-vaccinated and control animals. Total cells recovered from lung washings remained the same; however, the fold by eight days after infection and the type of cells changed from a predominance (85 per cent) of macrophages to equal proportions of macrophages and neutrophils (45 per cent) during the course of infection. The absolute numbers of neutrophils rose by 15-fold. In contrast, when RSV-vaccinated calves were challenged, the total number of cells recovered from lung washings remained the same; however, the numbers of macrophages decreased and the numbers of neutrophils increased by fivefold. Cytological studies of the lung washings revealed no evidence of an exacerbated inflammatory response in RSV-vaccinated calves. Levels of virus replication were significantly reduced in RSV-vaccinated compared with control animals.     

Fas/FasL通路对镉激活PC12细胞MAPK通路的影响

旨在研究Fas/FasL通路对镉激活PC12细胞MAPK通路的影响,用10 μmol L^-1醋酸镉（CdAc₂）分别处理插入非特异性序列PC12细胞与Fas基因沉默PC12细胞12 h;用10 μmol·L^-1CdAc₂与40 μmol·L^-1 Z-IETD-FMK （caspase-8特异性抑制剂）单独或联合处理PC12细胞12 h。通过Western blot检测Fas/FasL通路相关蛋白Fas、FasL、Fas相关死亡域蛋白（FADD）、Cleaved caspase-8、死亡结构域相关蛋白（Daxx）、凋亡信号调节激酶1（ASK1）的表达与MAPK通路相关蛋白ERK1/2、JNK1/2、p-ERK1/2、p-JNK1/2的表达;Hoechst33258荧光染色检测细胞凋亡形态学变化,流式细胞术检测细胞凋亡率。结果显示,Fas shRNA慢病毒极显著抑制镉引起的PC12细胞Fas/FasL通路相关蛋白表达量和凋亡率的升高（P<0.01）,缓解镉引起的PC12细胞凋亡形态学变化;Fas shRNA与Z-IETD-FMK均能够极显著抑制镉引起的PC12细胞MAPK通路相关蛋白ERK1/2与JNK1/2磷酸化水平升高（P<0.01）。综上表明,Fas/FasL通路调控MAPK通路参与镉致PC12细胞凋亡。     

Pathology of spontaneous tumour lesions in pullets and adult chickens in commercial farms - Short communication

Twenty pullets and adult chickens, aged 100 to 403 days, from several commercial chicken farms were examined by gross and histopathology. Grossly, all chickens had white-greyish masses in the visceral organs with or without enlargement of the peripheral nerves. Histopathological examination revealed Marek's disease (MD) lymphoma, lymphoid leukosis (LL) and myeloid leukosis (ML) in 14/20, 5/20 and 1/20 of the chickens, respectively. Lesions of the sciatic nerves in chickens diagnosed as having MD lymphoma were various. No neoplastic and/or inflammatory cells were noted in the peripheral nerves of chickens diagnosed as having LL and ML. These results indicated that MD lymphoma could also develop in older chickens; thus, microscopic examination is needed to identify MD in older chickens showing lymphocyte-derived tumours.     

Apoptosis of resident and inflammatory macrophages before and during the inflammatory response of the virgin bovine mammary gland

Background

Macrophages may play a prominent role in defense of the bovine mammary gland, and their functionality is necessary for successful eradication of bacterial pathogens. In contrast to necrosis, however, apoptosis has not yet been studied in macrophages from bovine mammary glands. Therefore, the aim of this study was to confirm the occurrence of apoptosis in macrophages from resting heifer mammary glands and during the inflammatory response.

Methods

Inflammatory response was induced by phosphate buffered saline (PBS) and by lipopolysaccharide (LPS). Resident macrophages (_RESMAC) were obtained before and inflammatory macrophages (_INFMAC) 24, 48, 72 and 168 hours after inducing inflammatory response in mammary glands of unbred heifers. Cell samples were analyzed for differential counts, apoptosis and necrosis using flow cytometry.

Results

Populations of _RESMAC and _INFMAC contained monocyte-like cells and vacuolized cells. Apoptosis was detected differentially in both morphologically different types of _RESMAC and _INFMAC and also during initiation and resolution of the inflammatory response. In the _RESMAC population, approximately one-tenth of monocyte-like cells and one-third of vacuolized cells were apoptotic. In the _INFMAC population obtained 24 h after PBS treatment, approximately one-tenth of monocyte-like cells and almost one-quarter of vacuolized cells were apoptotic. At the same time following LPS, however, we observed a significantly lower percentage of apoptotic cells in the population of monocyte-like _INFMAC and vacuolized _INFMAC. Moreover, a higher percentage of apoptotic cells in _INFMAC was detected during all time points after PBS in contrast to LPS. Comparing _RESMAC and _INFMAC, we observed that vacuolized cells from populations of _RESMAC and _INFMAC underwent apoptosis more intensively than did monocyte-like cells.

Conclusions

We conclude that apoptosis of virgin mammary gland macrophages is involved in regulating their lifespan, and it is involved in the resolution process of the inflammatory response.     

Contrast-enhanced ultrasonography for evaluation of the blood perfusion of sciatic nerves in healthy dogs

Blood supply to the peripheral nerves is essential for fulfilling their structural and functional requirements. This prospective, experimental, exploratory study aimed to assess the feasibility of contrast-enhanced ultrasonography (CEUS) for evaluating blood perfusion of the sciatic nerve in normal dogs. Contrast-enhanced ultrasonography examinations were performed on the bilateral sciatic nerves after bolus injection of Sonazoid™ (0.015 mL/kg) in 12 healthy Beagles for 150 s. Then, qualitative assessment of the wash-in timing, degree and enhancement patterns, and quantitative measurement of the peak intensity and time to peak intensity were performed from the sciatic nerve. The results were compared to those obtained from the adductor muscle around the nerve and caudal gluteal artery. After contrast agent injection, the sciatic nerve was enhanced at approximately 13–14 s, immediately after wash-in of the caudal gluteal artery. The peak intensity of the sciatic nerve was significantly lower than that of the caudal gluteal artery and higher than that of the adductor muscle. The time to peak intensity was significantly slower than that of the caudal gluteal artery; but was not significantly different from that of the adductor muscle. There were no significant differences in the peak intensity and time to peak intensity between the left and right sciatic nerves. These results demonstrate the feasibility of CEUS to assess blood perfusion of the sciatic nerve in healthy dogs qualitatively and quantitatively. This result from healthy dogs could serve as a reference for further studies that evaluate the sciatic nerve under pathological conditions.     

Mycobacterial neuritis in a cat

A 2-year-old spayed female domestic shorthair cat was referred for evaluation of rapidly progressive lameness of the right hind limb, which was paralyzed. Histologic examination of biopsy specimens revealed pyogranulomatous inflammation affecting the sciatic, common peroneal, and tibial nerves, and slender, beaded, acid-fast bacilli within macrophages, nerve fibers, and degenerate axons. A diagnosis of mycobacterial neuritis was made and the cat was treated with clofazimine and enrofloxacin for extended periods. Treatment was partially effective; the goal of returning normal function to the limb was not achieved, but disease progression was halted and the cat remained a viable pet.     

GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

【目的】研究硒蛋白谷胱甘肽过氧化物酶4(glutathione peroxidases 4,GPX4)失活如何参与调控脂多糖(lipopolysaccharide, LPS)诱导的RAW264.7巨噬细胞炎症反应及其潜在的分子机制。【方法】体外培养RAW264.7巨噬细胞,以DMSO为对照,使用0.1～5.0μmol/L GPX4抑制剂FIN56处理,通过CCK-8法检测细胞活力和Western blotting检测GPX4蛋白表达水平,确定抑制剂最适浓度。将RAW264.7巨噬细胞分为4组：对照组,添加DMSO培养24 h; FIN56(GPX4抑制剂)组,添加0.5μmol/L FIN56培养24 h; DMSO-LPS组,DMSO培养24 h后使用LPS(100 ng/mL)刺激3 h; FIN56-LPS组,FIN56培养24 h后使用LPS刺激3 h。各组细胞经培养后,利用荧光探针2′,7′-二氯二氢荧光素二乙酸酯(2′,7′-dichlorodi-hydrofluorescein diacetate, H₂DCFDA)检测细胞内活性氧(reactive...     

3 Tesla magnetic resonance imaging study of the normal canine femoral and sciatic nerves

Understanding the normal course and optimizing visualization of the canine peripheral nerves of the lumbar plexus, in particular the sciatic and the femoral nerves, is essential when interpreting images of patients with suspected peripheral neuropathies such as inflammatory or neoplastic conditions. The purpose of this prospective, anatomic study was to describe the magnetic resonance imaging (MRI) anatomy of the normal canine femoral and sciatic nerves and to define the sequences in which the nerves are best depicted. A preliminary postmortem cadaver study was performed to determine optimal sequences and imaging protocol. In a second step the optimized technique was implemented on 10 healthy Beagle dogs, included in the study. The applied protocol included the following sequences: T1‐weighted, T2‐weighted, T2‐Spectral Attenuated Inversion Recovery, T1‐weighted postcontrast and T1‐Spectral Presaturated Inversion Recovery postcontrast. All sequences had satisfactory signal‐to‐noise ratio and contrast resolution in all patients. The sciatic and femoral nerves were seen in all images. They were symmetric and of homogeneous signal intensity, being iso‐ to mildly hyperintense to muscle on T2‐weighted, mildly hyperintense in T2‐Spectral Attenuated Inversion Recovery, and iso‐ to mildly hypointense in T1‐weighted images. No evidence of contrast enhancement in T1‐weighted and T1‐Spectral Presaturated Inversion Recovery postcontrast sequences was observed. The anatomic landmarks helpful to identify the course of the femoral and sciatic nerves are described in detail. This study may be used as an anatomical reference, depicting the normal canine femoral and sciatic nerves at 3 Tesla MRI.     

Peripheral neuropathy associated with dietary riboflavin deficiency in the chicken. I. Light microscopic study

Chickens fed a riboflavin-deficient diet from hatching had leg weakness and paralysis as early as 12 days of age. Signs worsened through day 16; after 35 days, recovery was evident. Sciatic nerves from affected chickens were enlarged. Significant microscopic lesions were confined to peripheral nerves and included tissue separation (suggesting interstitial edema), Schwann cell swelling, perivascular leukocytic infiltration, and segmental demyelination accompanied by accumulation of osmiophilic debris in Schwann cell cytoplasm. Axon degeneration was present, but was not a primary lesion. Acid phosphatase enzyme activity of Schwann cells was increased in affected nerves. These results demonstrate that dietary riboflavin deficiency causes a demyelinating peripheral neuropathy in young, rapidly growing chickens.     

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.