Covariance among age, spinal p38 MAP kinase activation and allodynia.

This study examined effects of age (young rats, approximately 35 days, vs mature rats, approximately 75-110 days) on spinal nerve ligation (SNL)-induced tactile allodynia and phosphorylation of p38 (as measured by phospho-p38 MAP kinase [P-p38]) in dorsal root ganglia and spinal cord. Effects of SNL combined with spinal nerve transection also were assessed. Mature rats displayed milder SNL-induced allodynia than young rats. Addition of spinal nerve transection distal to the ligation in older animals resulted in an allodynia comparable to that seen in young animals. In DRG, both groups displayed early (5 h) and late (10 days) peaks in P-p38 following surgery as compared to na?ve rats. Tight nerve ligation plus transection had no additional effect on P-p38 levels in DRG. In spinal cord, young rats had increased levels of P-p38 from 5 h to 3 days after SNL. Phosphorylated p38 levels then decreased, with a second peak at 10 days. In contrast, spinal cord from mature rats showed less early p38 phosphorylation, although they also displayed a late 10-day peak. Addition of a transection to the ligation produced restoration of the early peak along with intensification of allodynia. Alterations of spinal P-p38 at early time points thus seem to covary with intensity of tactile allodynia. PERSPECTIVE: Age and modifications to spinal nerve ligation, a common model of neuropathic pain, influence spinal p38 phosphorylation and allodynia. Early levels of spinal P-p38 seem to covary with allodynia intensity. This may mean that small variations of an injury could affect the therapeutic window of a p38 antagonist.     

SB 239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence

The anti-inflammatory/antiallergic activity of a novel second-generation p38 mitogen-activated protein kinase inhibitor, SB 239063[trans-1-(4-hydroxycyclohexyl) -4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole], was investigated in vivo and in vitro. SB 239063 had an IC(50) of 44 nM for inhibition of recombinant purified human p38alpha. In lipopolysaccharide-stimulated human peripheral blood monocytes, SB 239063 inhibited interleukin-1 and tumor necrosis factor-alpha production (IC(50) values = 0.12 and 0.35 microM, respectively). A role for p38 kinase in cytokine-associated inflammation in the mouse was shown by p38 activation in the lung and inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production by SB 239063 (ED(50) = 5.8 mg/kg p.o.). Antiallergic activity was demonstrated by essential abolition (approximately 93% inhibition) of inhaled ovalbumin (OA)-induced airway eosinophilia by SB 239063 (12 mg/kg p.o.), measured by bronchoalveolar lavage (BAL) in OA-sensitized mice. In addition, p38 kinase was found by Western analysis to be activated in guinea pig lung. Administration of SB 239063 (10 or 30 mg/kg p.o.) in conscious guinea pigs markedly reduced ( approximately 50% inhibition) OA-induced pulmonary eosinophil influx, measured by BAL 24 h after antigen. SB 239063 (10 mg/kg b.i.d. p.o.) administered after leukotriene D(4) inhalation, reduced by 60% the persistent airway eosinophilia seen at 4 days. Apoptosis of cultured eosinophils isolated from guinea pig BAL was increased by SB 239063 (1-10 microM) in the presence of interleukin-5. These results indicate that SB 239063 is a potent inhibitor of inflammatory cytokine production, inhibits eosinophil recruitment, in addition to enhancing apoptosis of these cells. Collectively, the results support the potential utility of p38 kinase inhibitors, such as SB 239063, for the treatment of asthma and other inflammatory disorders.     

艰难梭菌二元毒素A的原核表达及免疫原性分析

目的构建艰难梭菌二元毒素(Cdt)A的原核表达载体,表达并纯化His-CdtA重组蛋白,分析其免疫原性。方法以RT 027型艰难梭菌为模板,采用聚合酶链反应(PCR)扩增cdtA的全长序列,导入大肠埃希菌BL21感受态细胞,诱导His-CdtA重组蛋白表达,并用纯化后的蛋白免疫小鼠,分析小鼠产生的抗体效价。结果成功构建pET-28b-cdtA原核表达载体,诱导并纯化出高浓度的His-CdtA重组蛋白,免疫小鼠后产生高效价的抗CdtA抗体。结论纯化的His-CdtA重组蛋白免疫小鼠产生了高效价的抗CdtA抗体,为后续制备抗CdtA单克隆抗体及建立CdtA的实验室检测方法学奠定了基础。     

CCK-8 inhibits LPS-induced IL-1beta production in pulmonary interstitial macrophages by modulating PKA, p38, and NF-kappaB pathway

The neuropeptide cholecystokinin octapeptide (CCK-8) inhibits inflammation by downregulating the expression of proinflammatory cytokines, such as tumor necrosis factor alpha and interleukin (IL) 1beta during endotoxin shock. However, the signaling mechanism of CCK-8 action has not yet been clearly elucidated. In this study, we have examined the possible signaling pathways by which CCK-8 inhibits lipopolysaccharide (LPS)-induced IL-1beta production in rat pulmonary interstitial macrophages. In macrophages, LPS is known to activate p38 kinase, which, in turn, activates nuclear factor (NF)-kappaB to induce IL-1beta production. We found that the pretreatment of cells with CCK-8 blocked the LPS-induced p38 kinase, NF-kappaB activation, and IL-1beta production. Furthermore, CCK-8 treatment activated the cyclic adenosine monophosphate-protein kinase A signaling pathway and H-89 (a protein kinase A inhibitor), abrogated the inhibitory effects of CCK-8 on p38 kinase activation and NF-kappaB activation. In addition, we also demonstrate that the specific antagonist to CCK-1 receptor (CCK-1R) and CCK-2 receptor (CCK-2R) abrogate the CCK action, and that the effects of the antagonist specific to CCK-1R is more significant. These results suggest that these responses were mediated through CCK-1R and CCK-2R, and CCK-1R might be the major receptor responsible for the anti-inflammatory effect of CCK-8. Taken together, our results indicate that the stimulation of cyclic adenosine monophosphate-protein kinase A signaling pathway by CCK-8 through CCK-1R and CCK-2R inhibits the LPS-induced activation of p38 kinase and NF-kappaB to block the IL-1beta production in rat pulmonary interstitial macrophages.     

Regulation of macrophage IL-10 production postinjury via beta2 integrin signaling and the P38 MAP kinase pathway

Although LPS receptor (CD14) signaling is mediated in part by beta2 integrins, the role of beta2 integrins in macrophage LPS signaling postinjury remains unknown. To study this, splenic macrophages were isolated from mice 7 days postburn, and inflammatory mediator production was determined. Macrophages isolated from injured mice produced higher levels of PGE2, TNF-alpha, IL-6, and IL-10 and lower levels of IL-12 in response to LPS stimulation than did cells from sham-treated mice. Blockade of beta2 integrin signaling by addition of antibodies against the CD11b (alphaCD11b) to the cultures increased IL-10 production by macrophages from injured mice without affecting other mediators. In contrast, sham macrophage responses to LPS were unaffected by alphaCD11b. Inhibition of p38 MAP kinase activity attenuated IL-10 production and abrogated the enhanced IL-10 response induced by alphaCD11b, whereas ERK 1/2 inhibition had no effect. Burn injury was associated with increased levels of total and phosphorylated p38 MAP kinase. These findings indicate that LPS signaling via beta2 integrins acts to attenuate the exaggerated induction of IL-10 by macrophages postinjury. Moreover, this effect of beta2 integrin signaling postinjury appears to be downstream of the p38 MAP kinase pathway and is independent of other markers of macrophage hyperactivity.     

Effect of ceftobiprole treatment on growth of and toxin production by Clostridium difficile in cecal contents of mice

Ceftobiprole and ceftobiprole medocaril did not promote growth of or toxin production by Clostridium difficile in mouse cecal contents, whereas ceftazidime, cefoxitin, ceftriaxone, cefotaxime, and ertapenem did. The relatively low propensity of ceftobiprole to promote C. difficile was attributable to inhibitory activity against C. difficile and sparing of anaerobic microflora.     

Effect of antibiotic treatment on growth of and toxin production by Clostridium difficile in the cecal contents of mice

In mice, subcutaneous administration of antibiotics that disrupt the anaerobic microflora (i.e., clindamycin, piperacillin-tazobactam, and ceftriaxone) facilitated in vitro growth of and toxin production by Clostridium difficile in cecal contents, whereas antibiotics that cause minimal disruption of the anaerobic microflora (i.e., levofloxacin, cefepime, and aztreonam) did not.     

Selective activation of the mitogen-activated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes.

Previous studies suggested that tyrosine kinase activation is an important signal transduction event in the IL-1 response of chondrocytes. The present study identifies the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 as major tyrosine phosphorylated proteins in IL-1 stimulated chondrocytes. Kinase assays on immunoprecipitates with myelin basic protein as substrate showed that ERK-1 and ERK-2 activation was detectable within 5 min after IL-1 stimulation and decreased to baseline within 60 min. Analysis of other members of the MAP kinase family showed that chondrocytes also express c-Jun NH2 terminal kinase (JNK)-1, JNK-2, and p38 proteins. These kinases were time-dependently activated by IL-1. Among other chondrocyte activators tested, only TNF activated all three of the MAP kinase subgroups. JNK and p38 were not activated by any of the other cytokines and growth factors tested. However, ERK was also activated by PDGF, IGF-1, and IL-6. Phorbol 12-myristate 13-acetate, calcium ionophore, and cAMP analogues only increased ERK activity but had no significant effects on JNK or p38. These results suggest differential activation of MAP kinase subgroups by extracellular stimuli. ERK is activated in response to qualitatively diverse extracellular stimuli and various second messenger agonists. In contrast, JNK and p38 are only activated by IL-1 or TNF, suggesting that these kinases participate in the induction of the catabolic program in cartilage.     

Performance of two rapid, single-use immunoassays for the detection of Clostridium difficile toxin A

Two rapid, single-use immunoassays for C. difficile toxin A, the Clearview C. DIFF A (Wampole Laboratories, Cranbury, N.J.) and the ImmunoCard Toxin A assays (Meridian Diagnostics Inc., Cincinnati, Ohio) were compared to the cytotoxin assay for their ability to detect C. difficile toxin in fecal specimens. A total of 537 specimens were tested and 47 (8.8%) were positive by the cytotoxin assay. The sensitivity, specificity, positive predictive value, and negative predictive value of the toxin A assays were as follows: 70.2% (95% CI, 57.1 to 83.3), 98.8% (95% CI, 97.8 to 99.8), 84.6% (95% CI, 73.3 to 95.9), and 97.2% (95% CI, 95.7 to 98.6) respectively for the Clearview assay; and 74.5% (95% CI, 62.0 to 86.9), 99.0% (95% CI, 98.1 to 99.9), 87.5% (95% CI, 77.3 to 97.8), and 97.6% (95% CI, 96.2 to 98.9) respectively for the ImmunoCard assay. Both toxin A assays are less sensitive than the cytotoxin assay, however, these assays offer a rapid and easy-to-perform test that may be used in conjunction with the cytotoxin assay for laboratory confirmation of C. difficile-associated disease.     

二烯丙基二硫诱导HL-60细胞G2/M期生长阻滞的分子机制研究

目的　探讨二烯丙基二硫 (DADS)诱导HL 6 0细胞G2 /M期细胞生长阻滞的分子机制。方法　用不同浓度的DADS处理HL 6 0细胞 ,分别作用 0 ,6 ,1 2 ,2 4 ,4 8h后用MTT法测定细胞增殖活性 ;用流式细胞术和有丝分裂指数测定细胞增殖周期 ;用Westernblot检测 p38丝裂原活化蛋白 (p38MAPK)及Cdc2 5B和Cdc2激酶的表达和磷酸化水平 ;用RT PCR检测p38MAPKmRNA的表达。结果　DADS抑制HL 6 0细胞增殖呈浓度依赖性 ,且DADS(2 0 μmol/L)与ATRA(1 0nmol/L)对HL 6 0细胞增殖活性影响相近 (P >0 .0 5 ) ;DADS 2 0 μmol/L作用HL 6 0细胞 1 2h后能引起G2 /M期细胞百分数增高并达到最大值 ,而此时有丝分裂指数明显下降 (P <0 .0 5 ) ;同时 ,磷酸化p38MAPK蛋白及p38MAPKmRNA的表达达到高峰 (P <0 .0 5 ) ,并引起Cdc2 5B和Cdc2磷酸化水平的相应变化。而p38特异性抑制剂SB2 0 2 1 90 (1 0 μmol/L)能阻断DADS对HL 6 0细胞增殖的抑制作用 (P <0 .0 5 )。结论　DADS能启动HL 6 0细胞G2 /M控制点 ,它的激活可能与磷酸化 p38MAPK的活化有关     

A p38 MAP kinase regulates the expression of the Aedes aegypti defensin gene in mosquito cells

An Aedes aegypti p38 (Aap38) mitogen-activated protein kinase was isolated and characterized in this study. The 1761 bp long full-length Aap38 cDNA encodes an open reading frame of 358 amino acids, exhibiting characteristics of Thr/Tyr dual kinase specificities. We showed that bacteria activate both the kinase activity of Aap38 and the expression of the Aedes aegypti defensin A (AaDefA) gene, which is inhibited by a p38 kinase inhibitor SB203580 and dsRNA interference of Aap38. A similar result was obtained by a reporter construct containing the AaDefA regulatory region linked to Ds-Red. The lipopolysaccharide-activated reporter gene was inhibited by SB203580. In addition, Aap38 translocated to the nucleus after lipopolysaccharide induction. Our findings suggest that the p38 protein kinase pathway is involved in the antibacterial peptide synthesis in mosquitoes.     

Effects of piperacillin/tazobactam on Clostridium difficile growth and toxin production in a human gut model

OBJECTIVES: Clostridium difficile infection (CDI) is a major cause of morbidity in the nosocomial environment. Antimicrobial agents such as the third-generation cephalosporins, lincosamides and aminopenicillins are well known for their propensity to induce CDI, but the definitive reasons why remain to be elucidated. Despite their broad spectrum of activity against both aerobic and anaerobic bacteria, the ureidopenicillins remain a class of antimicrobials infrequently associated with the development of CDI. METHODS: We used a triple-stage chemostat model that simulates the human gut to study the effects of the ureidopenicillin/beta-lactamase inhibitor combination piperacillin/tazobactam on gut bacterial populations and C. difficile. RESULTS: Piperacillin/tazobactam rapidly reduced all enumerated gut bacterial populations (including bacteroides, bifidobacteria and lactobacilli) below the limits of detection by the end of the piperacillin/tazobactam instillation period. Despite such widespread disruption of gut bacterial populations, C. difficile populations remained principally as spores, with no sustained proliferation or high-level cytotoxin production observed. CONCLUSIONS: Factors other than reduced colonization resistance must be responsible for determining whether CDI develops following antimicrobial administration. We believe the gut model is a promising approach for the study of C. difficile pathogenesis reflecting in vivo events likely to occur in CDI.     

spo0A对艰难梭菌毒素A/B蛋白表达的调节作用

目的 研究spo0A基因对艰难梭菌毒素A和毒素B蛋白表达的调控作用.方法 采用ClosTron基因敲除技术构建艰难梭菌C25菌株spo0A基因突变模型.实时定量反转录PCR方法测不同生长时期(培养后5、12、24、48 h)突变株和亲代株的毒素基因tcdA、tcdB和毒素调控基因tcdC、tcdR、tcdE的表达量,分...     

The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile.

Enterotoxin A is one of the major virulence factors of Clostridium difficile, and the causative agent of antibiotic-associated pseudomembranous colitis. In cell culture (NIH-3T3, rat basophilic leukemia cells) toxin A inhibits Clostridium botulinum ADP-ribosyltransferase C3 (C3)-catalyzed ADP-ribosylation of the low molecular mass GTP-binding Rho proteins. Rho participates in the regulation of the microfilament cytoskeleton. Decrease in ADP-ribosylation of Rho occurs in a time- and concentration-dependent manner and precedes the toxin A-induced destruction of the actin cytoskeleton. Action of toxin A is not due to proteolytical degradation of Rho or to an inherent ADP-ribosyltransferase activity of toxin A. Toxin A-induced decrease in ADP-ribosylation is observed also in cell lysates and with recombinant RhoA protein. A heat stable low molecular mass cytosolic factor is essential for the toxin effect on Rho. Thus, the enterotoxin (toxin A) resembles the effects of the C. difficile cytotoxin (toxin B) on Rho proteins (Just, I., G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbath, and C. Von Eichel-Streiber. 1994. J. Biol. Chem. 269:10706-10712). The data indicate that despite different in vivo effects, toxin A and toxin B act on the same cellular target protein Rho to elicit their toxic effects.     

Effects of antibiotics and other drugs on toxin production in Clostridium difficile in vitro and in vivo.

In an attempt to understand more completely why patients treated with phenothiazines (chlorpromazine and cyamemazine), methotrexate, and certain antibiotics such as clindamycin have an increased risk of developing pseudomembranous colitis, the production of toxins A and B by Clostridium difficile in the presence of these drugs was measured in vitro as well as in vivo by using axenic mice. None of the drugs tested increased the production of toxins either in vitro or in vivo.     

抑制钠氢交换蛋白下调IL-8表达和促进p38磷酸化

我们前期的研究证明,抑制钠氢交换蛋白1(NHE1)可以减少VEGF表达从而抑制K562细胞诱导的血管生成。本研究探讨抑制NHE1后其他可能的血管生成因子变化及相关机制。用NHE1特异性抑制剂卡立泊来德10μmol/L处理K562细胞;蛋白芯片筛选NHE1抑制后血管生成因子的表达变化,实时定量PCR验证卡立泊来德处理后白介素-8(IL-8)的表达水平;构建K562稳定干扰NHE1细胞株,实时定量PCR验证干扰NHE1后IL-8的表达变化,Western blot检测卡立泊来德处理后细胞内p38磷酸化水平;p38抑制剂SB203580处理K562细胞,实时定量检测IL-8表达变化。蛋白芯片筛选结果显示,卡立泊来德处理后K562细胞中IL-8表达显著下调;实时定量结果进一步验证了这种抑制效应;卡立泊来德处理后p38磷酸化水平显著上调;卡立泊来德处理后加入SB203580抑制p38,IL-8表达可以部分恢复。结论:抑制NHE1可能通过促进p38磷酸化,下调促血管生成因子IL-8的表达。     

Extracellular signal-regulated kinase activation mediates mitochondrial dysfunction and necrosis induced by hydrogen peroxide in renal proximal tubular cells

Although tubular necrosis in acute renal failure is associated with excessive production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), the mechanism of ROS-induced cell necrosis remains poorly understood. In this study, we examined the role of the extracellular signaling-regulated kinase (ERK) pathway in H2O2-induced necrosis of renal proximal tubular cells (RPTC) in primary culture. Exposure of 60 to 70% confluent RPTC to 1 mM H2O2 for 3 h resulted in 44% necrotic cell death, as measured by trypan blue uptake, and inactivation of mitogen-activated protein kinase kinase (MEK), the upstream activator of ERK, by either 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) or 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059) or overexpression of dominant-negative mutant of MEK1, inhibited cell death. In contrast, overexpression of active MEK1 enhanced H2O2-induced cell death. H2O2 treatment led to the loss of mitochondrial membrane potential (MMP) in RPTC, which was decreased by U0126 and PD98059. Furthermore, inhibition of the MEK/ERK pathway decreased oxidant-mediated ERK1/2 activation and mitochondrial swelling in isolated renal cortex mitochondria. However, treatment with cyclosporin A (CsA), a mitochondrial permeability transition blocker, did not suppress RPTC necrotic cell death, loss of MMP, and mitochondrial swelling. We suggest that ERK is a critical mediator of mitochondrial dysfunction and necrotic cell death of renal epithelial cells following oxidant injury. Oxidant-induced necrotic cell death was mediated by a CsA-insensitive loss of MMP that is regulated by the ERK pathway.     

Clostridium difficile toxin A stimulates intracellular calcium release and chemotactic response in human granulocytes.

Clostridium difficile, a common enteric pathogen, mediates tissue damage and intestinal fluid secretion by release of two protein exotoxins: toxin A, an enterotoxin, and toxin B, a cytotoxin. Because toxin A elicits an intense inflammatory reaction in vivo, we studied the effects of highly purified C. difficile toxins on activation of human granulocytes. Toxin A at concentrations of 10(-7) to 10(-6) M, but not toxin B, elicited a significant chemotactic and chemokinetic response by granulocytes that was comparable with that induced by the chemotactic factor N-FMLP (10(-7) M). Neither toxin stimulated release of superoxide anion from granulocytes. Toxin A produced a rapid, transient rise in cytosolic [Ca2+]i, as measured by quin 2 fluorescence. Pertussis toxin and depletion of intra- and extracellular calcium blocked the toxin A effect on cytosolic [Ca2+]i. These findings suggest that the inflammatory effects of C. difficile toxin A in the intestine may be related to its ability to mobilize intracellular Ca2+ and elicit a chemotactic response by granulocytes.