Effects of freezing/thawing on the biomechanical properties of human tendons

Abstract Numerous biomechanical studies using osteoarticular complex need frozen cadaveric specimens. Some of these studies deal with the resistance of the tendinous structures, for example the resistance of some autografts, such as the patellar ligament and the semitendinosus and gracilis tendons for reconstruction of the anterior cruciate ligament. The aim of this study was the in-vitro evaluation of the mechanical modifications induced by freezing/thawing on human tendons. The long head of the biceps brachii tendon was used as the reference. Eight pairs of tendons of the long head of the biceps brachii were taken from eight fresh cadavers. After drawing lots, one was tested immediately, the other was deep-frozen and then thawed. With an Instron material-testing machine, we performed a relaxation test and a uniaxial tensile test, to estimate the ultimate tensile failure and the elastic modulus of each pair of tendons. Freezing had no influence on the tendinous relaxation, but altered significantly the ultimate tensile failure and Young’s modulus of the tendons.     

Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin.

The enterotoxic material in cell-free growth preparations of Klebsiella pneumoniae serotype 5 was purified by sequential ultrafiltration and gel filtration (GF) procedures and the fractions were assayed for enterotoxic activity by determining their ability to induce in vivo net water secretion in the rat jejunum. Whole-cell lysates were inactive. Anaerobic broth culture conditions yielded a 10-fold increase in toxin production over aerobic conditions. Enterotoxic activity was absent in the UM-10 retentate of the broth filtrate but present in both the retentate and filtrate of the UM-2 membrane. GF of the two UM-2 ultrafiltration fractions through a Sephadex G-25 column yielded an active eluate, whose potency was increased by 10- or 200-fold, in or adjacent to the void volume. When subsequently passed through a G-50 column, these pools eluted at a Kav of between 0.4 and 0.6 and were further increased in potency by two- or fivefold. A second equally potent fraction was also recovered in the void volume of the G-50 eluate of the UM-2 filtrate; this may represent a polymer. Progressive purification by GF was associated with an increased protein and decreased carbohydrate content of the most active fractions. The most active G-50 eluate of the UM-2 retentate had a minimal effective enterotoxic dose of 5 mug/ml and that of the filtrate was less than 0.1 mug/ml. Heating the active GF eluates to 100 C for 30 min did not abolish enterotoxic activity and lowering the pH to 1 or incubation with either Pronase or trypsin had no effect on activity. These observations indicate that K. pneumoniae heat-stable enterotoxin is probably a single toxin with an apparent molecular weight in the range of 5,000. The elution characteristics during GF as well as the chemical composition of the most purified enterotoxin fractions indicate that the toxin is not associated with endotoxin.     

Surfactant protein D enhances phagocytosis and killing of unencapsulated phase variants of Klebsiella pneumoniae

Pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin (collectin) that is secreted into the alveoli and distal airways of the lung. We have studied the interactions of SP-D and alveolar macrophages with Klebsiella pneumoniae, a common cause of nosocomial pneumonia. SP-D does not agglutinate encapsulated K. pneumoniae but selectively agglutinates spontaneous, unencapsulated phase variants, such as Klebsiella strain K50-3OF, through interactions with their lipopolysaccharides (LPS). These effects are calcium dependent and inhibited with maltose but not lactose, consistent with involvement of the SP-D carbohydrate recognition domain. Precoating of K50-3OF with SP-D enhances the phagocytosis and killing of these organisms by rat alveolar macrophages in cell culture and stimulates the production of nitric oxide by the NR-8383 rat alveolar macrophage cell line. SP-D similarly enhances the NO response to K50-3OF LPS adsorbed to Latex beads under conditions where soluble LPS or SP-D, or soluble complexes of SP-D and LPS, do not stimulate NO production. Our studies demonstrate that interactions of SP-D with exposed arrays of Klebsiella LPS on a particulate surface can enhance the host defense activities of alveolar macrophages and suggest that activation of macrophages by SP-D requires binding to microorganisms or other particulate ligands. Because unencapsulated phase variants are likely to be responsible for the initial stages of tissue invasion and infection, we speculate that SP-D-mediated agglutination and/or opsonization of K. pneumoniae is an important defense mechanism for this respiratory pathogen in otherwise healthy individuals.     

Effects of freezing and storage temperature on MS2 viability

Monitoring human enteric virus levels in domestic wastewater effluent is crucial to protecting human health. Occasionally, during intensive sampling, wastewater samples must be stored for later viral analysis. Little data exist regarding how enteric viruses survive during storage at different temperatures in secondary treated wastewater. During a field-scale study assessing pathogen removal performance by various onsite treatment technologies, the MS2 bacteriophage, an indicator of enteric viruses, was inoculated into septic tank (STE), sand filter, peat filter and constructed wetland (CW) effluents to determine virus decay at various storage temperatures. Virus stored at temperatures > or =10 degrees C and at -20 degrees C decayed nearly twice as fast as those stored at 4 degrees C or -80 degrees C. Decreased water quality decreased viral decay rates at 4 degrees C and -80 degrees C, with slowest decay occurring in STE and the fastest in sterile PBS and low pH peat effluent. In CW effluent after 8 days, less MS2 was inactivated when stored at 4 degrees C (20%) compared to -80 degrees C (58%); however, during extended storage (approximately 300 days), less MS2 was inactivated at -80 degrees C (75%) compared to 4 degrees C (93%). We recommend that viruses in wastewater be stored in the dark at 4 degrees C unless storage for >40 days is necessary.     

Thermal and rheological properties of poly(vinyl alcohol) hydrogels prepared by repeated cycles of freezing and thawing

Differential scanning calorimetry and scanning electron microscopy were carried out in order to study the relation between the structure and properties of poly(vinyl alcohol) hydrogels prepared by repeated cycles of freezing-thawing. The effect of the number N of repeated cycles of freezing-thawing, of the degree of saponification (DS), and of the degree of polymerization (DP) on structure and properties was examined. The elastic modulus and the endothermic enthalpy, estimated from the area surrounded by the endothermic peak, increase with increasing N, DS, and DP. The melting temperature T_m shifts to higher temperatures with increasing DS and DP, but it is almost independent of N for gels with high DS or DP. In PVA gels with lower DP or DS, T_m shifts to higher temperatures with increasing N. The temperature dependence of the elastic modulus is discussed on the basis of a theory on thermoreversible gels.     

Comparison of pyogenic liver abscess caused by non-Klebsiella pneumoniae and Klebsiella pneumoniae.

From January 1996 to April 2002, a total of 248 patients with pyogenic liver abscess were enrolled in this study. Abscesses caused by Klebsiella pneumoniae accounted for 69% (171) of cases. Abscesses caused by K. pneumoniae were more strongly associated with diabetes mellitus or impaired fasting glucose than liver abscesses caused by non-K. pneumoniae (70.2% vs 32.5%). Solitary abscess and monomicrobial isolates were more frequent in the K. pneumoniae group than that in the non-K. pneumoniae group. A total of 42 patients were treated with antibiotics alone. Antibiotics treatment was combined with other procedures, including single aspiration in 23 patients, percutaneous drainage in 176 and surgical drainage in 7. A higher incidence of metastatic infections occurred in the K. pneumoniae group than in the non-K. pneumoniae group (14.6% vs 3.8%). By contrast, the mortality rate of the K. pneumoniae group was lower than that of non-K. pneumoniae group (4.1% vs 20.8%). There was no significant difference in the relapse rate between these 2 groups (6.5% vs 6.4%). We also found that the presence of respiratory symptoms (including cough, dyspnea, or chest distress), size of abscess > or =5 cm in diameter and non-K. pneumoniae pathogens were significant prognostic factors for mortality.     

Identification of the major adherence ligand of Klebsiella pneumoniae in the receptor for coliphage T7 and alteration of Klebsiella adherence properties by lysogenic conversion.

We have studied the adherence of both laboratory and wild-type Klebsiella pneumoniae strains, isolated from sputum, urine, and stool samples, to human buccal and intestinal and urinary tract epithelial cells. Of 32 unencapsulated strains, 30 adhered to all epithelial cells tested. Four K. pneumoniae strains lysogenic for AP3, a phage which causes conversion to resistance of coliphages T3, T7, and phi I, were all unable to adhere to epithelial cells. One of these strains was cured from phage infection and became capable of adhering, Spontaneous mutants resistant to coliphage T7, as well as K. pneumoniae K59-sensitive cells preadsorbed with inactivated T7 particles, did not adhere to epithelial cells. All strains capable of adhering were able to adsorb coliphage T7 and T3, whereas all nonadhesive strains were not. AP3-like prophages were induced from 7 of 12 nonadhesive Klebsiella strains. A laboratory strain which was able to adhere was lysogenized with 2 of these phages. In both cases, the strain lost its ability to adsorb coliphages T3, T7, and phi I and to adhere to human epithelial cells. All K. pneumoniae adhesive strains agglutinated yeast cells, whereas the nonadhesive strains did not. Competition studies have shown that D-mannose and concanavalin A prevented adherence to human epithelial cells, yeast agglutination, and adsorption of coliphage T7 to K. pneumoniae cells. It is concluded that in K. pneumoniae adherence to epithelial cells is mediated by the receptor for coliphages T7 (and T3), which in turn recognizes D-mannose in the receptors it binds.     

尿路致病性大肠埃希菌FYUA基因敲除株的构建及其生物学特性

目的构建尿路致病性大肠埃希菌FYUA基因敲除株,比较野生株和敲除株之间生物学特性的改变。方法利用λRed同源重组系统构建FYUA基因敲除株△FYUA;测定A600绘制CFT073和△FYUA在LB液体培养基和无菌尿液中的增殖曲线;平板菌落计数法比较CFT073和△FYUA菌落形成能力;利用96孔板结晶紫染色法比较CFT073和△FYUA生物膜形成能力。结果成功构建CFT073 FYUA基因敲除株△FYUA;CFT073和△FYUA在LB液体培养基中具有相似的增殖曲线,△FYUA在无菌尿液中的增殖速率明显低于CFT073(P0.05);CFT073和△FYUA菌落形成能力无明显区别;△FYUA的生物膜形成能力低于CFT073(P0.05)。结论 FYUA基因对于菌株在无菌尿液中的生长繁殖以及生物膜形成可能发挥重要作用。     

Discrimination of Klebsiella pneumoniae and Klebsiella oxytoca phylogenetic groups and other Klebsiella species by use of amplified fragment length polymorphism

Bacteria of the genus Klebsiella are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. The two clinically and epidemiologically most important species, Klebsiella pneumoniae and K. oxytoca, have recently been shown to be subdivided into three and two phylogenetic groups, respectively. The aim of this study was an in depth evaluation of the amplified fragment length polymorphism (AFLP) genetic characterization method for epidemiological and phylogenic analyzes of Klebsiella isolates. First, we investigated the variability of AFLP patterns for Klebsiella strains within and between different outbreaks. Second, by use of carefully characterized phylogenetically representative strains, we examined whether different Klebsiella species and phylogenetic groups can be discriminated using AFLP. Twenty-four strains originating from seven presumed outbreaks and 31 non-associated strains were investigated. The AFLP fingerprints of all epidemiologically associated strains showed three or fewer fragment differences, whereas unrelated strains differed by at least four fragments. Cluster analysis of the AFLP data revealed a very high concordance with the phylogenetic assignation of strains based on the gyrA sequence and ribotyping data. The species K. pneumoniae, K. oxytoca, K. terrigena and the possibly synonymous pair K. planticola/K. ornithinolytica each formed a separate cluster. Similarly, strains of the phylogenetic groups of K. pneumoniae and K. oxytoca fell into their corresponding clusters, with only two exceptions. This study provides a preliminary cut-off value for distinguishing epidemiologically non-related Klebsiella isolates based on AFLP data; it confirms the sharp delineation of the recently identified phylogenetic groups, and demonstrates that AFLP is suitable for identification of Klebsiella species and phylogenetic groups.     

黄连素对肺炎克雷伯菌生长曲线的影响

目的 探讨黄连素与环丙沙星对肺炎克雷伯菌（Kpn）生长曲线的影响.方法 2012年1月至2012年7月本院细菌室收集的42株临床Kpn菌株,药敏试验筛选出环丙沙星和黄连素均有效的敏感株6株,命名为S1～S6;耐药株6株,命名为R1 ～R6.每个菌株分为4个实验小组：空白对照组、环丙沙星组、黄连素组、环丙沙星＋黄连素组.空白对照组不进行任何处理;根据前期单用环丙沙星与黄连素测定的最低抑菌浓度（MIC）,黄连素浓度均使用500 mg/L,环丙沙星的浓度≤1/2 MIC.利用比浊法测定6株耐药株和6株敏感株Kpn的各实验小组在培养时间为0、1、2、3、4、5、6、7、8、9、10h的吸光度（A600nm值）并绘制生长曲线.结果 生长曲线显示黄连素500 mg/L对临床耐药和敏感Kpn的生长曲线均有影响,当黄连素与环丙沙星联合应用时对其生长的抑制效应加大.临床耐药和敏感Kpn的生长曲线并无明显差异.结论 黄连素能增加临床耐药Kpn对环丙沙星的敏感性.     

种属和发育期及不同实验条件对卵裂期胚胎冷冻复苏结局的影响

背景：人卵裂期胚胎冷冻复苏的研究中,不同的实验条件及实验动物模型是否与人类胚胎具有相同的敏感性从而反映出实验方案的优劣值得探讨。 目的：观察胚胎种属和发育期及不同冷冻条件对卵裂期胚胎冷冻复苏结局的影响。 方法：将人卵裂期胚胎作为对照组,将KM小鼠胚分为2细胞,4细胞,8细胞组。各组胚胎随机采用以下实验方案：①冷冻操作环境温度18~20 ℃、24~26 ℃和37 ℃。②慢速程序化方案、自制straw叶片玻璃化方案和CPS玻璃化方案。③与玻璃化液接触时间< 40 s、40~60 s和60~90 s。各组胚胎比较不同实验条件下胚胎复苏率和培养24 h发育率。 结果与结论：①对照组24~26 ℃冷冻操作环境温度复苏率高于37 ℃冷冻操作环境(P < 0.05)。②对照组和4细胞鼠胚组采用自制straw叶片玻璃化方案复苏率高于慢速程序化方案(P < 0.05),培养24 h胚胎发育率差异无显著性意义(P > 0.05)。③各组胚胎与冷冻保护剂接触不同时间胚胎复苏率差异无显著性意义(P > 0.05)。表明卵裂期胚胎玻璃化冷冻复苏效果优于慢速程序化,24~26 ℃操作环境、减少冷冻保护剂剂量和缩短接触时间可改善玻璃化冷冻复苏结局;相同冷冻条件下,胚胎种属和发育阶段对冷冻复苏结局有影响,4细胞鼠胚更适合作为研究人类卵裂期胚胎冷冻复苏的实验模型。     

Two Cases of Pyomyositis Caused by Klebsiella pneumoniae and Review of the Literature

Two patients with diabetes mellitus developed Klebsiella pneumoniae pyomyositis involving multiple muscles and other metastatic foci associated with bacteraemia. The cases are presented here, and the 22 cases of gram-negative pyomyositis reported previously in the literature are reviewed. As gram-positive cocci cause 99% of pyomyositis, it is postulated that the pathogenesis of pyomyositis may be associated with the ability of the bacteria to adhere to muscles by various cell surface adhesins that are not present in gram-negative bacilli. It is also postulated that a high serum glucose concentration may facilitate the growth and formation of the Klebsiella pneumoniae capsule, hence increasing its virulence and causing serious disseminated Klebsiella infections in diabetic patients. Electronic Publication     

影响肺炎克雷伯菌粘附上皮细胞作用的研究

目的:研究不同理化及生物因素对肺炎克雷伯菌粘附宿主细胞的影响,探讨其粘附宿主细胞的可能机制。方法:用肺炎克雷伯菌临床分离株K.pnueumoniae 03183(K.p03183)建立粘附宿主细胞模型,通过结晶紫染色及扫描电镜观察其粘附效率;以不同理化及生物因素预处理K.p03183,平板菌落计数法观察上述因素对K.p03183粘附细胞的影响;用基因芯片技术,检测HMGN2蛋白预处理细菌对于其粘附相关基因表达的影响。结果:K.p03183可粘附至A549、T24、HeLa等3种上皮细胞系,其粘附率与E.coli 25992接近;氯化锂和盐酸胍、高碘酸钠及热预处理可明显降低K.p03183对于A549和T24的粘附率(P0.05);同时,胃蛋白酶及HMGN2蛋白预处理K.p03183,也可抑制其对A549和T24的粘附(P0.01);但胰蛋白酶预处理却能增加细菌对于A549细胞的粘附(P0.05);同时,HMGN2还可通过上调dksA基因表达,从而干扰细菌粘附细胞。结论:肺炎克雷伯菌可能借助细胞壁及其表面蛋白和碳水化合物等成分粘附至宿主上皮细胞,通过非共价键结合表面蛋白或消除碳水化合物成分,抑制细菌粘附至宿主细胞。     

Challenge of evolving Klebsiella pneumoniae infection in patients on hemodialysis: from the classic strain to the carbapenem-resistant hypervirulent one

Loss of renal function may render hemodialysis patients more susceptible to infectious diseases, which is the second of all-causes mortality in this population. In addition to infection caused by the classic Klebsiella pneumoniae (cKp), however, hemodialysis staffs are now facing new challenge with growing prevalence of the carbapenem-resistant Kp (CR-Kp) and hypervirulent Kp (hvKp) as they are respectively associated with increased drug-resistance and virulence. We therefore chose to share our recent experience in treating severe infections either caused (cKp, CR-Kp, hvKp) or complicated (CR-hvKp) by these strains in hemodialysis patients. Based upon yet beyond published works, we further came up with the detection of intracranial lesion, novel diagnostic approach using unique biomarkers followed by selection of appropriate antibiotics, management of metastasic abscesses and bracing for the most lethal scenario in the order of cKp, CR-Kp, hvKp and CR-hvKp, respectively. Since reports of complicated hvKp infection in hemodialysis patients were rare, we also discussed in details this clinical entity focusing on its epidemiology, mechanism of increased virulence and involvement of the arteriovenous fistula as insidious source of persistent septicemia. By covering the full spectrum of clinically relevant Kp stains specifically from the viewpoint of nephrology, our work had highlighted the importance of infection control in uremic state and vice versa. As such, it may greatly raise the awareness of dialysis staffs against the challenge of evolving Klebsiella pneumoniae infection in hemodialysis patients and expeditiously reach a higher degree of readiness which was proved to be the key determinant of ultimate survival.