Synoviocyte-Derived Angiopoietin-Like Protein 2 Contributes to Synovial Chronic Inflammation in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis of the diarthrodial joints. Several proinflammatory cytokines derived from both infiltrating inflammatory cells and activated resident cells within the RA joint play a fundamental role in the processes that cause inflammation. However, anticytokine treatment is beneficial but not curative, the effects are only partial, and nonresponses are common. Therefore, an effort has been made to identify other key regulators of inflammation in articular structures to develop new therapies to suppress synovial inflammation and joint destruction in RA. Adipose tissue-derived angiopoietin-like protein 2 (Angptl2) activates an inflammatory cascade in endothelial cells and induces chemotaxis of monocytes/macrophages in obesity, resulting in initiation and propagation of inflammation within adipose tissues and obesity-related metabolic diseases. Angptl2 mRNA and protein are abundantly expressed in hyperplastic rheumatoid synovium of RA patients, especially in fibroblast-like and macrophage-like synoviocytes, but not in B and T lymphocytes. Angptl2 concentration in joints of RA patients was also significantly increased in comparison with patients with osteoarthritis, which in comparison with RA represents a significantly lower inflammatory grade form of arthritis. Notably, Angptl2 promoted increased chemotactic activities of CD14⁺CD16⁻ monocytes from synovial fluid of RA patients. Therefore, Angptl2 acts as an important rheumatoid synovium-derived inflammatory mediator in RA pathogenesis.Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis of the diarthrodial joints. The RA synovial membrane contains activated B and T lymphocytes, plasma cells, mast cells, and significantly activated monocytes/macrophages. The synovium is normally a relatively acellular structure with a delicate intimal lining, thus these cells are recruited via an intense neovascularization process with associated lymphangiogenesis. Hyperplasia of the intimal lining results from macrophage-like and fibroblast-like synoviocytes. These resident and infiltrating cells within the RA joint could be a source of proinflammatory cytokines that activate inflammatory pathways in a paracrine or autocrine fashion and play a fundamental role in processes underlying inflammation, articular destruction, and comorbidities associated with RA.^1,2,3,4,5,6 In fact, numerous cytokines, such as interleukin (IL)-1, IL-6, IL-15, IL-18, and tumor-necrosis factor (TNF)-α, as well as various chemokines, are active within the synovium and synovial fluid in joints of RA patients.^2,7,8 Continuous anticytokine treatment, such as through use of TNF-α and IL-1β inhibitors, is required for long-term control, and discontinuation of treatment leads to disease flare-up, indicating the importance of cytokine-related inflammation in pathogenesis of RA.² Furthermore, although such anticytokine treatment is beneficial,^{9,10,11,12,13,14} it is not curative, its effects are partial, and nonresponses are common.^2,15 These findings indicate that the mechanism of inflammation in the RA joint is more complex than previously thought, thus suggesting that new factors and mechanisms are operating that could serve as novel therapeutic targets for RA.The Angptl (Angiopoietin-like protein) family was identified as a group of proteins possessing structural similarity to angiopoietin, which contains an N-terminal coiled-coil domain functioning in oligomerization and a C-terminal fibrinogen-like domain serving as a receptor binding site.^16,17 Although Angptls were predicted to function as ligands for the angiopoietin-receptor; Tie-2 or its family member; Tie-1, they do not bind to either, strongly suggesting biological functions different from those of angiopoietins. More recently, we reported that Angptl2, a member of the Angptl family, is expressed in a variety of tissues, especially in obese adipose tissues.¹⁸ Angptl2 expression has been shown to increase by hypoxia and endoplasmic reticulum stress, both of which are commonly observed in pathological conditions. We also showed that Angptl2 signaling via integrins activated an inflammatory cascade in endothelial cells and induced chemotaxis of monocytes/macrophages. Constitutive Angptl2 activation in vivo induced inflammation of the vasculature characterized by abundant attachment of leukocytes to vessel walls and increased permeability. These findings suggest that adipocyte-derived Angptl2 acts as a key chronic inflammatory mediator in obesity, resulting in obesity-related metabolic diseases. These findings, plus the fact that its expression is not restricted to adipose tissues, suggest a possible role of Angptl2 in other chronic inflammatory diseases.The current study showed that Angptl2 mRNA and protein are abundantly expressed in the hyperplastic synoviocytes from RA patients. An in vitro culture analysis revealed that the synoviocytes from RA patients secrete Angptl2. Indeed, the concentration of Angptl2 in RA synovial fluid is significantly higher than that seen in osteoarthritis (OA), which is a lower inflammatory grade arthritis than RA. Angptl2 increases the chemotactic activities of monocytes from RA synovial fluid. Taken together, these findings establish Angptl2 as a hyperplastic rheumatoid synovium-derived inflammatory mediator in RA joints.     

Clostridium difficile-Associated diarrhoea in uremic patients

An outbreak of 94 episodes of Clostridium difficile-associated diarrhoea in 62 patients in a nephrology ward over a two-year period was investigated. Quantitative stool cultures were performed on ten uremic patients not on antibiotics and without diarrhoea and on ten healthy controls. All diarrhoeal episodes were associated with Clostridium difficile,and no other bacterial pathogens were isolated. Thirty-two relapses occurred in 16 patients, fourteen of the relapses without preceding antibiotic exposure.Clostridium difficile could not be isolated from the environment of the patients. Uremic patients, who had a significantly increased number of Clostridium spp. in their stools, are predisposed to Clostridium difficile infections.     

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80⁺ Kupffer cells, as well as in isolated nonparenchymal cells. MLK3^−/− mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.Key words: Alcoholic liver disease (ALD), Kupffer cells, Necroptosis, Toll-like receptor 4 (TLR4), Cytokines     

Nosocomial Outbreak of Clostridium difficile-Associated Diarrhoea due to a Clindamycin-Resistant Enterotoxin A-Negative Strain

A clindamycin-resistant toxin A-negative, toxin B-positive Clostridium difficile strain caused an outbreak among 24 hospitalized patients at the Department of Surgery, the Intensive Care unit, and the Department of Internal Medicine of an 800-bed academic hospital. Nineteen patients had undergone a surgical intervention and all 24 patients received at least one dose of antibiotics prior to the development of Clostridium difficile-associated diarrhoea. Twenty-seven episodes of Clostridium difficile-associated diarrhoea in 24 patients were categorized as mild (n=19), severe (n=7), or fatal (n=1). Relapses occurred in three patients. Nineteen of the 27 episodes required anti-Clostridium difficile treatment. Molecular typing performed by arbitrary primer polymerase chain reaction (PCR) and PCR amplification of rRNA intergenic spacer regions revealed that the outbreak strains recovered from culture were identical. The outbreak strain belonged to serogroup F and was resistant to erythromycin, clindamycin, and tetracycline, whereas susceptibility to chloramphenicol varied. No phenotypic activity of enterotoxin A was detected. A deletion of approximately 1.7 kb was found in the toxin A gene. Cytotoxin B had an unusual effect on cell culture assays that, at first, was not recognized as Clostridium difficile specific but could be neutralized with anti-Clostridium difficile B cytotoxin. Electronic Publication     

Toll-Like Receptor 4-Linked Janus Kinase 2 Signaling Contributes to Internalization of Brucella abortus by Macrophages

Brucella abortus is an intracellular pathogen that uses a crafty strategy to invade and proliferate within host cells, but the distinct signaling pathways associated with phagocytic mechanisms of B. abortus remain unclear. The present study was performed to test the hypothesis that Toll-like receptor 4 (TLR4)-linked signaling interacting with Janus kinase 2 (JAK2) plays an essential role in B. abortus phagocytosis by macrophages. The effects of TLR4-JAK2 signaling on B. abortus phagocytosis in murine macrophage RAW 264.7 cells were observed through an infection assay and confocal microscopy. We determined that the uptake of B. abortus was negatively affected by the dysfunction of TLR4 and JAK2. F-actin polymerization detected by flow cytometry and F-actin assay was amplified for B. abortus entry, whereas that event was attenuated by the disruption of TLR4 and JAK2. Importantly, JAK2 phosphorylation and actin skeleton reorganization were suppressed immediately after B. abortus infection in bone marrow-derived macrophages (BMDMs) from TLR4^−/− mice, showing the cooperation of JAK2 with TLR4. Furthermore, small GTPase Cdc42 participated in the intermediate pathway of TLR4-JAK2 signaling on B. abortus phagocytosis. Consequently, TLR4-associated JAK2 activation in the early cellular signaling events plays a pivotal role in B. abortus-induced phagocytic processes in macrophages, implying the pathogenic significance of JAK2-mediated entry. Here, we elucidate that this specific phagocytic mechanism of B. abortus might provide achievable strategies for inhibiting B. abortus invasion.     

Bone Morphogenic Protein-7 Contributes to Cerebral Ischemic Preconditioning Induced-Ischemic Tolerance by Activating p38 Mitogen-Activated Protein Kinase Signaling Pathway

Cerebral ischemic preconditioning (IPC), which refers to a transient and noninjurious ischemia is able to induce tolerance against the subsequent lethal ischemia, including ischemic stroke. We have previously reported that bone morphogenic protein-7 (BMP-7) contributes to the neuroprotective effects of IPC-induced ischemic tolerance, and thus ameliorates the following ischemia/reperfusion (I/R) injury in rats. Consequently, in the present study, we continued to explore the underlying regulatory mechanisms involved in BMP-7-mediated cerebral IPC in the rat model of ischemic tolerance. Male Wistar rats were preconditioned by 15-min middle cerebral artery occlusion (MCAO). After 2-day reperfusion, these animals were subjected to prolonged MCAO for 2 h. Our results showed that the phosphorylated p38 mitogen-activated protein kinase (MAPK) paralleling to BMP-7 was up-regulated by IPC in rat brain. Inactivation of p38 MAPK by pretreatment of SB203580, a p38 MAPK-specific suppressor, weakened the protective effect of IPC on CA1 neurons. Moreover, the enhanced phosphorylation of p38 MAPK induced by IPC was attenuated when the endogenous BMP-7 was inhibited by BMP-7 antagonist noggin. Besides, blockade of p38 MAPK signal transduction pathway via SB203580 abrogated the protective effects of exogenous BMP-7 against cerebral infraction. These present findings suggest that BMP-7 contributes to cerebral IPC-induced ischemic tolerance via activating p38 MAPK signaling pathway.     

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2^fl/fl conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2^fl/fl mice but not in CamKII-Cre;ERK2^fl/fl mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.The extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPKs) has been intensely studied for roles in brain development, including control of both axonal^1,2 and dendritic³ growth. Astroglial process extension is also dependent on ERK signaling.⁴ Recent work strongly implicates ERK-mediated signaling in synaptic plasticity and memory formation, and mutations in ERK2 and other ERK pathway components are known to underlie some forms of inherited human cognitive disorders.^5,6,7 ERK MAPK activation is also linked to astroglial activation in several forms of central nervous system (CNS) injury.^8,9,10To directly test the importance of ERK MAPKs in vivo, gene knockout approaches are needed. Whereas ERK1 general knockout (KO) mice are viable and have architecturally normal brains,¹¹ general KO of ERK2 is embryonic lethal, because of defects in mesoderm differentiation and placental development.^12,13,14 Thus, conditional KO (cKO) strategies are necessary to study brain-specific roles of ERK2. Two recent reports^15,16 describe aspects of the developmental phenotype resulting from CNS-specific KO of the mapk1 gene, encoding ERK2. We now present a novel aspect of the adult phenotype of a brain-specific ERK2 cKO driven by Nestin:Cre. Our findings suggest an unexpected and specific role for this classical MAPK member in the regulation of brain collagen deposition.     

丝氨酸/苏氨酸蛋白激酶AKT2的体外激酶活性分析

目的 建立蛋白激酶AKT2体外磷酸化检测体系.方法 构建携带AKT2 cDNA编码区的pLNCX2逆转录病毒重组载体,包装重组病毒,转导293A细胞,G418筛选得到稳定表达组成型活化的AKT2细胞株,应用免疫沉淀获得蛋白激AKT2;将核基质结合蛋白SATB1的1～204的氨基酸序列及其47位丝氨酸的突变体S47A、S47D,分别与GST基因融合表达载体pGEX4T-1进行重组,经测序鉴定后转化大肠埃希菌BL-21,IPTG诱导表达经亲和纯化得到GST-SATB1 1-204、GST-SATB1 1-204 S47A和GST-SATB1 1-204 S47D融合蛋白;利用免疫沉淀的AKT2磷酸化GST-SATB1融合蛋白,应用免疫印迹检测其是否被磷酸化.结果 细胞表达的蛋白激酶AKT2能高效的将野生型SATB1 1-204 磷酸化,而不能磷酸化其两种突变体.结论 成功建立了一个蛋白激酶体外磷酸化系统.     

蛋白激酶CK2与DNA断裂修复

蛋白激酶CK2(酪蛋白激酶Ⅱ)是真核细胞中普遍存在的一种信使非依赖的丝氨酸/苏氨酸蛋白激酶,它底物众多,功能广泛.DNA断裂修复是一个涉及很多种酶和蛋白的过程,CK2在其中起着很重要的作用.     

应激活化蛋白激酶与蛋白激酶p38活性测定

应激活化蛋白激酶（ＳＡＰＫ）和Ｐ＾３２是丝裂素活化蛋白激酶家系（ＭＡＰＫｓ）的主要成员。与应激和某些炎性细胞因子引起的细胞反应有关，参与心血管疾病、细胞凋亡与机体的应激反应。本文主要介绍用免疫沉淀法提取ＳＡＰＫ和ｐ３８，据ＳＡＰＫ和ｐ３８分别可以磷酸化ｃ－Ｊｕｎ和ＡＴＦ－２的原理，采用聚丙烯酰胺电泳（ＳＤＳ－ＰＡＧＥ）方法，分别测定ＳＡＰＫ和ｐ３８将＾３２Ｐ参入其特异性底物的量，反映其活性。     

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα^−/−) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα^−/− MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.     

A Conserved Apicomplexan Microneme Protein Contributes to Toxoplasma gondii Invasion and Virulence

The obligate intracellular parasite Toxoplasma gondii critically relies on host cell invasion during infection. Proteins secreted from the apical micronemes are central components for host cell recognition, invasion, egress, and virulence. Although previous work established that the sporozoite protein with an altered thrombospondin repeat (SPATR) is a micronemal protein conserved in other apicomplexan parasites, including Plasmodium, Neospora, and Eimeria, no genetic evidence of its contribution to invasion has been reported. SPATR contains a predicted epidermal growth factor domain and two thrombospondin type 1 repeats, implying a role in host cell recognition. In this study, we assess the contribution of T. gondii SPATR (TgSPATR) to T. gondii invasion by genetically ablating it and restoring its expression by genetic complementation. Δspatr parasites were ∼50% reduced in invasion compared to parental strains, a defect that was reversed in the complemented strain. In mouse virulence assays, Δspatr parasites were significantly attenuated, with ∼20% of mice surviving infection. Given the conservation of this protein among the Apicomplexa, we assessed whether the Plasmodium falciparum SPATR ortholog (PfSPATR) could complement the absence of the TgSPATR. Although PfSPATR showed correct micronemal localization, it did not reverse the invasion deficiency of Δspatr parasites, because of an apparent failure in secretion. Overall, the results suggest that TgSPATR contributes to invasion and virulence, findings that have implications for the many genera and life stages of apicomplexans that express SPATR.     

Usefulness of Simultaneous Detection of Toxin A and Glutamate Dehydrogenase for the Diagnosis of Clostridium difficile-Associated Diseases

 The aim of this study was to evaluate an immunoassay (Triage; Biosite Diagnostics, BMD, France) for detecting both a specific antigen of Clostridium difficile (glutamate dehydrogenase [GDH]) and toxin A. Evaluation of the test was carried out in 304 fecal samples from patients suspected of having Clostridium difficile-associated diseases. The results with GDH and toxin A were compared with those of a culture and cytotoxicity assay (toxin B). The prevalence rates for toxin B-positive and culture-positive fecal samples were 11.2% and 25%, respectively. The sensitivity of the Triage immunoassay was 90.8% for GDH and 79.4% for toxin A. A negative result with both toxin A and GDH was very reliably able to eliminate a diagnosis of Clostridium difficile-associated disease (negative predictive value 99.6%). Triage is a very rapid (20 min) and easy-to-perform test. It could be useful for diagnostic purposes and also for detecting nontoxigenic strains in epidemiogical studies.     

Opacity-Associated Protein A Contributes to the Binding of Haemophilus influenzae to Chang Epithelial Cells

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.     

Factors Associated with Nosocomial Diarrhea and Clostridium difficile-Associated Disease on the Adult Wards of an Urban Tertiary Care Hospital

 A prospective survey of the adult inpatient population of an urban tertiary care hospital was conducted to determine factors associated with the development of nosocomial diarrhea and the acquisition of Clostridium difficile-associated disease. During the 3-month survey, 98 patients with nosocomial diarrhea were enrolled, and 38 controls were recruited. The controls were patients without diarrhea lying in beds adjacent to the affected patients. Factors significantly associated with nosocomial diarrhea were the administration of a special diet (P=0.02) and receipt of a greater number of different antibiotics (P=0.02). Among the 98 patients with diarrhea, Clostridium difficile toxin B was identified in the stool of 13. Factors found to be associated with the presence of toxin B as compared to other causes of nosocomial diarrhea were a greater number of individual antibiotics used during hospitalization (P=0.02) and receipt of a cephalosporin (P=0.03) or, more specifically, a third-generation cephalosporin (P=0.02). Among patients with nosocomial diarrhea, those who had toxin in their stool had a significantly higher total antibiotic burden (expressed as antibiotic days) than those with diarrhea due to other causes (P=0.01).