Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins: INSIGHT INTO THE ROLE OF g2-g3 LINKER IN pH/Ca2+ INSENSITIVITY OF THE FIRST HALF*

Because of its ability to rapidly depolymerize F-actin, plasma gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of gelsolin resides in its N terminus, we made several truncated versions of plasma gelsolin. The smaller versions, particularly the one composed of the first 28–161 residues, depolymerized the F-actin much faster than the native gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca²⁺ or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca²⁺ as well as low pH, whereas G1-G2 and residues 28–161 prefer collapsed states in Ca²⁺-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca²⁺-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the gelsolin-like sequence of g2-g3 linker.     

Recent advances in the biology of IL-1 family cytokines and their potential roles in development of sepsis

The IL-1 family comprises two anti-inflammatory cytokines (IL-37, IL-38), two receptor antagonists (IL-1ra, IL-36ra), and seven ligand agonists (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, IL-36γ). The members of this family exert pleiotropic effects on intercellular signaling, leading to pro- or anti-inflammatory responses. They initiate potent inflammatory and immune responses by binding to specific receptors in the IL-1 receptor family, and their activities are repressed by naturally occurring inhibitors. Various immune cells produce and are regulated by these crucial molecules, which appear to be involved in the pathogenesis of diverse diseases including cancer as well as inflammatory and autoimmune disorders. Recent decades have seen substantial progress in understanding how the IL-1 family contributes to the development of sepsis. In this review, we will briefly introduce the IL-1 family and discuss its critical role in inflammatory and immune responses. The potential significance of IL-1 members in sepsis will also be explored, together with the clinical implications for treating this dangerous condition.     

A ‘culture’ shift: Application of molecular techniques for diagnosing polymicrobial infections

With the advancement of microbiological discovery, it is evident that many infections, particularly bloodstream infections, are polymicrobial in nature. Consequently, new challenges have emerged in identifying the numerous etiologic organisms in an accurate and timely manner using the current diagnostic standard. Various molecular diagnostic methods have been utilized as an effort to provide a fast and reliable identification in lieu or parallel to the conventional culture-based methods. These technologies are mostly based on nucleic acid, proteins, or physical properties of the pathogens with differing advantages and limitations. This review evaluates the different molecular methods and technologies currently available to diagnose polymicrobial infections, which will help determine the most appropriate option for future diagnosis.     

中心静脉-动脉二氧化碳分压差与动脉-中心静脉氧含量差的比值(Pcv-aCO2/Ca-cvO2)变化率对急诊重症监护室脓毒症患者预后的评估价值

目的:探讨中心静脉动脉二氧化碳分压差/氧含量差(Pcv-aCO_2/Ca-cvO_2)变化率在急诊重症监护室(EICU)高乳酸脓毒血症患者病情及预后评估中的临床应用价值。方法:选择2017年1月到2018年9月入住急诊重症监护室的48例高乳酸(乳酸大于4 mmol/h)脓毒血症患者,均按2016年脓毒症指南进行液体复苏治疗。采集复苏前(T0h)和开始复苏后6h(T6h)、24h(T24h)的动脉血、上腔静脉血气分析以及动脉血乳酸浓度。计算并记录各时间点的乳酸,乳酸清除率,中心静脉动脉二氧化碳分压差(Pcv-aCO_2)值,中心静脉动脉二氧化碳分压差/氧含量(Pcv-aCO_2/Ca-cvO_2)值及其变化率。根据治疗24h改良SOFA评分是否改善将患者分为两组,即改良SOFA改善组和未改善组,观察和比较两组间基本临床资料及化验参数,并分析各时间点各参数之间的相关性,以及这些参数能否有效预测高乳酸脓毒血症患者病情危重程度和预后。结果:45例患者纳入最终分析,3例因为24h内死亡或者自动出院脱落。其中,17例24hSOFA改善,28例未改善;20例死亡,25例存活。两组患者复苏前各项一般临床资料指标比较差异均无统计学意义(P0.01)。24hSOFA改善组与未改善组患者Pcv-aCO_2/Ca-cvO_2(T24h)、Pcv-aCO_2/Ca-cvO_2变化率(0-24h)存在组间差异(P0.01)。45例患者的乳酸清除率(0-24h)与Pcv-aCO_2/Ca-cvO_2变化率(0-24h)呈显著相关性(r=0.906,P=0.034)。ROC分析显示Pcv-aCO_2/Ca-vO_2变化率(0-24h)能有效预测24hSOFA评分改善,同其他指标相比,曲线下面积最大(AUROC=0.851),最佳界值是0.307(30.7%),敏感度是76.5%,特异度是92.9%;Pcv-aCO_2/Ca-vO_2变化率(0-24h)也能有效预测脓毒症患者院内死亡,AUROC=0.696,AUROC较24h乳酸值小,但不存在统计学差异,最佳界值是0.181(18.1%),敏感度是65%,特异度是68%。结论:液体复苏前到开始复苏后24h的Pcv-aCO_2/Ca-cvO_2变化率可以有效预测高乳酸脓毒症患者的器官功能改善情况,也能有效预测脓毒症患者院内死亡的发生。     

罗氟司特对脓毒症炎症大鼠炎症因子及淋巴细胞的调控机制分析

摘要 目的：探究罗氟司特通过JAK/STAT途径对脓毒症炎症大鼠炎症因子及淋巴细胞的调控机制分析。方法：将54只SD大鼠随机分为正常组（n=18）,模型组（n=18）和罗氟司特组（n=18）,正常组正常喂养,模型组和罗氟司特组建立脓毒症大鼠模型。模型组和罗氟司特组腹膜内注射0.9 %的氯化钠和罗氟司特。干预7 d后,对大鼠取样。测定灌洗液中炎症细胞的数量、JAK和STAT-3的蛋白、AST和ALT、IL-6和TNF-α的mRNA表达。结果：在观察的7 d中,正常组大鼠均存活,生存率高于模型组和罗氟司特组（P<0.05）,罗氟司特组生存率高于模型组（P<0.05）。与正常组相比,模型组和罗氟司特组术后症状评分均增加（P<0.05）,罗氟司特组术后症状评分低于模型组（P<0.05）。模型组和罗氟司特组的miRNA-218表达低于正常组（P<0.05）,而罗氟司特组高于模型组（P<0.05）;模型组和罗氟司特组淋巴细胞,中性粒细胞和单核细胞总数,JAK和STAT-3的蛋白质表达,AST、ALT、IL-6和TNF-α水平高于正常组（P<0.05）,罗氟司特组上述指标低于模型组（P<0.05）。结论：罗氟司特通过JAK / STAT途径阻断miRNA-218表达,抑制促炎性细胞因子IL-6和TNF-α的表达,减轻脓毒症炎症的机制和肝脏损害并改善多发性脓毒症的生存率。     

电针对脓毒症大鼠肺炎症反应和高迁移率族蛋白B1的影响

摘要 目的：研究电针足三里对脓毒症大鼠肺脏炎症反应和病理损伤的调节,为脓毒症的临床治疗提供新的理论依据。方法：成年雄性SD大鼠40只,按照随机数字表法分为假手术组（sham组）、脓毒症（盲肠结扎穿刺术( Cecal ligation and puncture, CLP)组）、脓毒症+假电针组（非经非穴组）和脓毒症+电针足三里组（足三里组）,每组10只。除第一组外,其余大鼠均采用盲肠结扎穿孔术（CLP）制备脓毒症大鼠模型;造模前,脓毒症+假电针组选择非经非穴处电针刺激,脓毒症+电针足三里组给予足三里穴位电针处理,电针参数为疏密波,2 Hz,1 mA,持续30 min,连续5天。术后24 h,先取支气管肺泡灌洗液, 再取右肺测湿干重比,取左肺下叶观察病理学改变,取左肺上叶检测炎症因子肿瘤坏死因子-α(Tumor necrosis factor, TNF-α)、白介素-1(interleukin -1, IL-1β)、白介素-6(interleukin-6, IL-6)和HMGB1。结果：与sham组比较,CLP组大鼠肺组织出现明显病理损伤（均P＜0.05）,炎症因子明显升高（均P＜0.01）,高迁移率族蛋白1（High mobility group 1 protein, HMGB1）明显升高(P＜0.05);经过电针足三里处理,脓毒症大鼠肺组织病理损伤明显减轻（P＜0.05）,炎症因子明显降低（均P＜0.01）,HMGB1明显减少(P＜0.05)。CLP组与非经非穴组大鼠生存率、肺组织病理损伤、炎症因子和HMGB1无明显差异,差异无统计学意义（均P＞0.05）。结论：电针足三里可以减轻脓毒症大鼠肺的炎症反应和组织损伤,降低其肺脏HMGB1水平。     

Protein chip for the parallel quantification of high and low abundant biomarkers for sepsis

We present herein a protein chip for diagnosis of sepsis that combines both a sandwich and a binding inhibition format in order to quantify high (CRP) and low abundant proteins (cytokines, PCT, neopterin) in parallel. Using the combined assay format the lowest detectable concentrations for CRP, IL-6, IL-8, IL-10, TNFα, PCT, and neopterin are 3 mg/L, 15 ng/L, 26 ng/L, 65 ng/L, 40 ng/L, 78 ng/L, and 0.46 μg/L. Four different combined assay formats are tested, using separate or joint incubation steps of analytes and detection antibodies. Yet, low limit of detection (LOD) and short processing time are contradictory: while the combined assay performed in a multistep protocol is extremely sensitive (e.g., the LOD for IL-6 is 15 ng/L), but more time-consuming (4 h), the all-in-one protocol takes only 2.5 h, but suffers from lower sensitivity compared with the multistep protocol (e.g., the LOD for IL-6 is up to 40 times enhanced). Reproducibility is good in both cases (CV 5–20%).     

Protective effects of finasteride on the pulmonary immune response in a combined model of trauma-hemorrhage and polymicrobial sepsis in mice

Literature supports findings about a gender specific outcome following multiple trauma. Male sex hormones such as dihydrotestosterone (DHT) exert deleterious effects on the posttraumatic immune response whereas increased estradiol concentrations are correlated with improved outcome. Pretreatment with the 5α-reductase inhibitor finasteride resulted in an improved outcome following trauma-hemorrhage (TH) in mice. The present study tested the hypothesis that finasteride exerts beneficial effects on the posttraumatic immune response also in a combined setting of TH and sepsis when administered during the resuscitation process.

Material and Methods

Male C57BL/6N-mice were subjected to TH (blood pressure, 35 mm Hg, 60 min) followed by finasteride application and fluid resuscitation. Thereafter, finasteride was administered every 12 h. 24 h after TH, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Plasma cytokines (MIP-1α, MIP-1β, TNF-α, MCP-1, IL-6), productive capacity by alveolar macrophages (AM) and systemic estradiol levels were determined 4 h thereafter. The expression of pro-inflammatory mediators in lung tissue was evaluated by PCR. Pulmonary infiltration of PMN was determined by immunohistochemical staining.

Results

Finasteride treatment resulted in a reduced posttraumatic cytokine secretion of AM as well as in a decreased concentration of MCP-1 and MIP-1β in lung tissue. Systemic estradiol levels were increased following finasteride treatment.

Conclusion

Finasteride mediates salutary effects on the pulmonary immune response using a therapeutical approach following TH–CLP in mice. Thus, finasteride might represent a relevant therapeutic substance following major trauma also in the clinical setting.     

Therapeutic potential of plasma gelsolin administration in a rat model of sepsis

BackgroundGelsolin is an actin-binding protein found in the cytoplasm and in extracellular fluids including blood plasma. Plasma gelsolin concentration decreases after a wide range of injuries. We hypothesized that the repletion of gelsolin would limit inflammation and tissue injury in a rat model of sepsis using cecal ligation and double puncture (2CLP).MethodsHuman plasma gelsolin (pGSN, 10 mg in 1 ml saline) was administered once immediately following surgery, and control 2CLP (2CLP Alb) and sham animals were injected with 1 ml saline containing equimolar albumin. Treatments were administered intraperitoneally (IP), intravenously (IV), or subcutaneously (SC).ResultsGelsolin levels in the 2CLP Alb group were lower than in sham animals. Administration of pGSN increased levels when administered IV and SC, but not IP. Morbidity scores were significantly less severe in the 2CLP pGSN group than in the 2CLP Alb group when pGSN was administered IV and SC, but not IP. Furthermore, enzymatic activity indicative of tissue damage (lactate dehydrogenase and alanine transaminase) was significantly lower in 2CLP pGSN group when treated SC compared to 2CLP Alb group.ConclusionThese data provide further evidence that exogenous gelsolin can reduce morbidity from sepsis.     

Non-toxigenic Clostridium sordellii: clinical and microbiological features of a case of cholangitis-associated bacteremia

Toxigenic Clostridium sordellii strains are increasingly recognized to cause highly lethal infections in humans that are typified by a toxic shock syndrome (TSS). Two glucosylating toxins, lethal toxin (TcsL) and hemorrhagic toxin (TcsH) are believed to be important in the pathogenesis of TSS. While non-toxigenic strains of C. sordellii demonstrate reduced cytotoxicity in vitro and lower virulence in animal models of infection, there are few data regarding their behavior in humans. Here we report a non-TSS C. sordellii infection in the context of a polymicrobial bacterial cholangitis. The C. sordellii strain associated with this infection did not carry either the TcsL-encoding tcsL gene or the tcsH gene for TcsH. In addition, the strain was neither cytotoxic in vitro nor lethal in a murine sepsis model. These results provide additional correlative evidence that TcsL and TcsH increase the risk of mortality during C. sordellii infections.