Bacterial cell wall compounds as promising targets of antimicrobial agents II. Immunological and clinical aspects

The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.     

A simple and sensitive liquid chromatographic technique for the determination of cefotetan disodium in human plasma and its application in a pharmacokinetic study

A simple and sensitive liquid chromatographic method was developed for quantification of cefotetan disodium (CTT),a semi-synthetic cephamycin antibiotic,in human plasma.CTT and the internal standard chloramphenicol were extracted from plasma by a simple one-step protein precipitation with 35% (v/v) perchloric acid.Separation was carried out on a reverse-phase C18 column with a mobile phase of acetonitile-water containing 0.5% (v/v) phosphoric acids (20:80,v/v) at a flow rate of 1.0 mL/min.The column effluent was monitored by UV detection at 300 nm.The column temperature was maintained at 40°C.This method demonstrated good linearity in the range of 0.525-300.0 μg/mL,with correlation coefficients greater than 0.99.The limit of quantification (LOQ) was 0.525 μg/mL in human plasma.Intra-and inter-day precisions were less than 6.63% in terms of relative standard deviation (RSD).The accuracy,when expressed by the bias,ranged from 0.57% to 4.04%.The mean extraction recovery of CTT was higher than 40.94%.The method was found to be precise,accurate,and specific for CTT quantitative analysis,and was successfully applied for a pharmacokinetic study of CTT after a single intravenous dose of 1.0 g of CTT in healthy Chinese subjects.     

The development of a Chinese equivalence version of letter-number span test

The present study aimed to develop a Chinese equivalence version of the Letter-Number (LN) Span Test and to explore the preliminary construct and discriminative validity of the developed version among a group of healthy Chinese volunteers and patients with stroke. A group of 165 (73 men and 92 women) healthy participants were recruited for the validation study, most of them were undergraduates or postgraduates. Moreover, a comparison was made between nine patients with stroke and the healthy controls. For the healthy sample, the Chinese version correlated significantly with the English version in total number of correct span (r = .6, p < .00001) and the longest span (r = .5, p < .00005). The Chinese version of LN Span Test was also found to be significantly associated with memory-loaded tests but not other tests. A series of ANCOVAs controlling for age, education, and IQ indicated that stroke patients performed significantly worse than the healthy controls in LN Span total number of correct responses (p < .04), immediate recall (p < .0005), and delayed recall (p < .0005) of WMS-R, SART total number of correct response (p < .0005), PASAT dyads correct response (p < .01). The preliminary findings suggest that the Chinese version of the LN Span Test shows impressive preliminary validity among a group of healthy volunteers and an impressive clinical discriminative validity among a group of stroke cases.     

小分子化合物诱导大鼠脂肪基质干细胞向神经元的分化

目的:探讨小分子化合物诱导大鼠脂肪基质干细胞(ADSCs)向神经元的分化。方法:用Trichostatin A(TSA)、RG-108、8-BrcAMP和Rolipram 4种小分子物质对SD大鼠的ADSCs进行诱导分化,用免疫荧光形态学、免疫印迹和qRT-PCR等方法分别检测诱导前、后ADSCs神经元标志的表达。结果:诱导7 d时,ADSCs呈明显的神经元样结构,胞体呈圆形,具有多个突起,神经元样形态变化率高达95.12%±2.65%;诱导后的ADSCs nestin、β-tubulinⅢ、MAP2和ChAT在mRNA和蛋白水平的表达均有升高。结论:小分子化合物可将ADSCs诱导分化为神经元样细胞,其可作为治疗神经系统疾病及损伤的干细胞移植的种子细胞。     

CpG ODN联合乙型肝炎疫苗与乙型肝炎疫苗免疫效果对比研究

目的探讨CpG ODN乙型肝炎疫苗与市售乙型肝炎疫苗对Balb/c小鼠的免疫作用效果。方法将乙肝疫苗、乙肝疫苗+100μgCpG ODN分别肌肉注射到4～6周龄,16～18g Balb/c小鼠体内,于第1次免疫后28d以同样剂量加强免疫1次。分别于第1次免疫后28、42、63 d收集小鼠血清,用ELISA方法检测抗HBs IgG抗体。结果实验组产生的抗HBs IgG与对照组HBsIgG相比P<0.05,具有显著性差异。28、42、63 d抗HBs IgG分别是对照疫苗的114倍、4.73倍、5.41倍以上。结论 CpG ODN能够显著增强小鼠对乙肝疫苗的免疫应答,在较短时间内显著的提高乙肝抗体的产生水平,免疫效果是乙肝疫苗的5倍以上。     

Targeted Inhibition of Prostate Cancer Metastases with an RNA Aptamer to Prostate-specific Membrane Antigen

Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC.     

应变率显像对糖尿病患者左室收缩功能的评价

目的探讨应变率显像(SRI)技术评价糖尿病患者左室收缩功能的临床价值。方法获取33例糖尿病患者和26例正常人心尖左心长轴、二腔和四腔切面应变率曲线,测量左室各节段心肌收缩期峰值应变率和二尖瓣环峰值应变率(SR)。用Simpson法测量左室射血分数(LVEF)。结果与正常组相比,糖尿病组左室各节段心肌的纵向SR均明显降低(P<0.05),瓣环的SR与LVEF呈正相关(r=0.619,P<0.05)。结论SRI为定量评价糖尿病患者左室整体和局部收缩功能提供了无创的新方法。     

全程无缝隙优质护理应用于心肌梗死介入治疗患者围术期的临床效果

目的 探讨全程无缝隙优质护理应用于心肌梗死介入治疗患者围术期的临床效果.方法 选取2017年5月至2019年7月于我院接受介入治疗的200例心肌梗死患者作为研究对象,根据护理方式不同将其分为对照组(100例,常规围术期护理)和观察组(100例,全程无缝隙优质护理).比较两组护理效果.结果 观察组护理满意度高于对照组(P...     

New Antiseptic Peptides To Protect against Endotoxin-Mediated Shock

Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the “endotoxic principle” of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its “endotoxic conformation,” the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.The life-threatening clinical consequences of sepsis and septic shock arise from recognition of microbial immunostimulatory molecules by the hosts'' professional immune cells and the release of hemodynamically active mediators. The most potent immunostimulatory constituents are part of the microbial cell envelope, such as lipopolysaccharide (LPS) or lipoproteins. They are released continuously due to cell growth and division and massively liberated as a consequence of the attack of the immune system. In the case of Gram-negative bacteria, the most potent factor is LPS, which, therefore, is also called an endotoxin. LPS concentrations in blood serum as low as 1 ng/ml are able to cause sepsis. Septic shock resulting from bacterial infection remains a frequent cause of death, particularly in intensive care units, with more than 200,000 people dying each year in the United States alone. Death by septic shock can happen despite appropriate broad-range antibiotic treatment, which may kill bacteria but is not only incapable of neutralizing immunostimulatory LPS but also may promote its release into circulation (11).The response of mammalian cells to LPS is initiated by its interaction with serum proteins such as lipopolysaccharide-binding protein (LBP) and specific receptors and/or binding proteins of immune cells such as soluble CD14 (sCD14) and membrane-bound CD14 (mCD14), which finally leads to cell activation through the Toll-like receptor 4 (TLR4)-MD-2 pathway (31). The hydrophobic moiety of LPS, lipid A, anchoring LPS to the bacterial outer membrane, constitutes the “endotoxic principle” of LPS (24). Enterobacterial lipid A consists of a diglucosamine backbone phosphorylated at positions 1 and 4′, to which six acyl chains are linked at positions 2,3 and 2′,3′. The physicochemical properties of LPS suggest that cationic amphiphilic agents could effectively neutralize its lipid A moiety. A number of studies have used natural proteins, peptides, or modified variants thereof to neutralize LPS and, thus, to protect it from endotoxin-mediated immunostimulation (1, 10), and in some cases, a significant protection from LPS-induced lethality in vivo was observed (10, 19). However, these approaches required very high peptide/LPS molar ratios, precluding their use in humans due to the intrinsic cytotoxicity of the peptides at these concentrations.We have designed a completely new class of peptides—synthetic anti-LPS peptides (SALPs). SALPs were originally based on the LPS-binding domain of the Limulus anti-LPS factor (LALF) (2) but were substantially changed in length and primary sequence for optimal binding to the lipid A portion of LPS. Here, we report that these peptides are highly efficient in neutralization of LPS and blockage of its immunopathological consequences in vitro and in vivo. Our preclinical study reveals that these SALPs combine excellent selectivity for LPS, with high neutralizing activity in vitro and potent protection to septic shock using the murine model in vivo. We also demonstrate very low cytotoxicity under physiological conditions, making these SALPs promising candidates for their application as therapeutic agents for the prevention and treatment of septic shock. The molecular interaction mechanism between SALPs and LPS was studied using various biophysical approaches.