Molecular characterization of metallo-beta-lactamase-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korea: identification of two new integrons carrying the bla(VIM-2) gene cassettes

Carbapenem-resistant Acinetobacter spp. used to be rare, but are increasingly isolated in Korea. Among 28 isolates of imipenem-resistant Acinetobacter spp. found in a Korean hospital in 1998 and 1999, 14 produced metallo-beta-lactamases. The bla(VIM-2) gene was detected, by PCR, in 11 and two isolates of Acinetobacter baumannii and Acinetobacter genomospecies 3, respectively, and bla(IMP-1) in one isolate of A. baumannii. The MICs of imipenem for the isolates were 8-32 mg/L. PFGE analysis of SmaI-digested genomic DNA gave identical patterns in eight of 11 bla(VIM-2)-positive A. baumannii isolates from respiratory specimens of ICU patients. The bla(VIM-2) gene cassettes in the isolates are identical to those from Pseudomonas aeruginosa isolates in Europe, but are inserted into new class I integrons In105 and In106. The attC site of the last cassette of the array in In106 is interrupted by the insertion of a putative class II intron. This is the first report of VIM-2 beta-lactamase-producing A. baumannii and Acinetobacter genomospecies 3. Production of the VIM-2 enzyme presents an emerging threat of carbapenem resistance among Acinetobacter spp. in Korea.     

Activated human monocytes inhibit the intracellular multiplication of legionnaires’ disease bacteria

We have examined the interaction between virulent egg yolk-grown L. pneumophila, Philadelphia 1 strain, and in vitro-activated human monocytes, under antibiotic-free conditions. Freshly explanted human monocytes activated by incubation with concanavalin A (Con A) and human lymphocytes inhibited the intracellular multiplication of L. pneumophila. Both Con A and lymphocytes were required for activation. Con A was consistently maximally effective at greater than or equal to 4 μg/ml. Monocytes activated by incubation with cell-free filtered supernatant from Con A-sensitized mononuclear cell cultures also inhibited the intracellular multiplication of L. pneumophil a. The most potent supernatant was obtained from mononuclear cell cultures incubated with greater than or equal to 15 μg/ml Con A for 48 h. The degree of monocyte inhibition of L. pneumophila multiplication was proportional to the length of time monocytes were preincubated with supernatant (48 {greater than} 24 {greater than} 12 h) and to the concentration of supernatant added (40 percent {greater than} 20 percent {greater than} 10 percent {greater than} 5 percent). Monocytes treated with supernatant daily were more inhibitory than monocytes treated initially only. With time in culture, monocytes progressively lost a limited degree of spontaneous inhibitory capacity and also lost their capacity to respond to supernatant with inhibition of L. pneumophila multiplication. Supernatant-activated monocytes inhibited L. pneumophila multiplication in two ways. They phagocytosed fewer bacteria, and they slowed the rate of intracellular multiplication of bacteria that were internalized. As was the case with nonactivated monocytes, antibody had no effect on the rate of intracellular multiplication in supernatant-activated monocytes. Neither supernatant-activated nor nonactivated monocytes killed L. pneumophila in the absence of antibody. Both killed a limited proportion of these bacteria in the presence of antibody and complement. We have previously reported that anti-L, pneumophila antibody and complement neither promote effective killing of L. pneumophila by human polymorphonuclear leukocytes and monocytes nor inhibit the rate of L. pneumophila multiplication in monocytes. These findings and our present report that activated monocytes do inhibit L. pneumophila multiplication indicate that cell-mediated immunity plays a major role in host defense against Legionnaires’ disease.     

Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery.

Currently, there is a need for therapeutic vaccines that are effective in inducing robust T helper type 1 (Th1) immune responses capable of mediating viral clearance in chronic hepatitis B infection. Hepatitis B therapeutic vaccines were designed and formulated by loading the hepatitis B core antigen (HBcAg) into poly(D,L-lactic-acid-co-glycolic acid) (PLGA) nanoparticles with or without monophospholipid A (MPLA), a Th1-favoring immunomodulator. These particles were around 300 nm in diameter, spherical in shape and had approximately 50% HBcAg encapsulation efficiency. A single immunization with a vaccine formulation containing (MPLA+HBcAg) coformulated in PLGA nanoparticles induced a stronger Th1 cellular immune response with a predominant interferon-gamma (IFN-gamma) profile than those induced by HBcAg alone, free (HBcAg+MPLA) simple mixture or HBcAg-loaded nanoparticles in a murine model. More importantly, the level of HBcAg-specific IFN-gamma production could be increased further significantly by a booster immunization with the (HBcAg+MPLA)-loaded nanoparticles. In summary, these results demonstrated that codelivery of HBcAg and MPLA in PLGA nanoparticles promoted HBcAg-specific Th1 immune responses with IFN-gamma production. These findings suggest that appropriate design of the vaccine formulation and careful planning of the immunization schedule are important in the successful development of effective HBV therapeutic vaccines.     

流式细胞术分析户尘螨致敏的嗜碱性粒细胞活化试验及其临床意义

目的建立流式细胞术(FCM)分析嗜碱性粒细胞活化试验(BAT)的方法并评估其在过敏性疾病诊断中的意义。方法以CD63/CD203c/CD45抗体组合,建立用FCM分析户尘螨致敏的嗜碱性粒细胞脱颗粒的方法。以皮肤点刺试验(SPT)作为金标准方法,将FCM检测活化嗜碱性粒细胞与荧光酶联免疫吸附试验(FELISA)测定特异性IgE(sIgE)结果作比较,分析其临床应用价值。结果以CD45和CD203c联合设门,可以得到纯的嗜碱性粒细胞;CD63是活化嗜碱性粒细胞的最佳标志;Spearman相关系数显示,sIgE的Unicap分级与活化嗜碱性粒细胞呈中度相关;FCM检测活化嗜碱性粒细胞与FELISA测定sIgE结果在户尘螨过敏性疾病诊断中的差异无统计学意义(P>0.05),但前者的敏感度、符合率、阳性预测值以及阳性似然比各项指标都优于后者。结论通过FCM分析CD63的表达来确定活化嗜碱性粒细胞是诊断速发型超敏反应的一种有效、安全的体外检测方法。