抗淋球菌IgA蛋白酶单克隆抗体特性的初步研究

本文对以重组淋球菌ＩｇＡ蛋白酶为抗原制备的７株特异性ＭｃＡｂ的特性进行了初步研究。结果有５株（１Ｃ８、１Ｅ５、１Ｆ１１、１Ｇ３和２Ｅ６）能中和淋球菌ＩｇＡ蛋白酶活性。经相加试验初步证明，其中１Ｆ１１、２Ｇ３与其它３株ＭｃＡｂ的作用位点不同。因此，淋球菌ＩｇＡ蛋白酶至少存在３个中和表位。     

产青霉素酶淋病奈瑟球菌耐药质粒的提取与酶切分析

目的 了解产青霉素酶淋病奈瑟球菌(PPNG)在本地区的分布状况及其耐药质粒的限制性核酸内切酶长度多态性分析。方法 用碘量法筛选PPNG株，碱变性法进行质粒抽提，回收7．4kb及5．4kb质粒进行酶切分析。结果 68株临床分离株筛选出PPNG菌3株，经限制性核酸内切酶长度多态性分析，PPNG菌7、4kb及5．4kb质粒含有BamHⅠ的双酶切位点，7.4kb质粒含有PstⅠ及HindⅡ单酶切位点。结论 PPNG菌株的筛选及耐药质粒的酶切分析为追踪耐药菌株的流行趋势提供了流行病学信息。     

Increasing prevalence of penicillinase‐producing Neisseria gonorrhoeae and the emergence of high‐level, plasmid‐mediated tetracycline resistance among gonococcal isolates in The Gambia

Summary One hundred and three strains of Neisseria gonorrhoeae isolated from a periurban STD clinic in The Gambia were studied for antimicrobial susceptibility, plasmid profile, and serogroup using standard procedures. Seventy-nine (77%) were penicillinase producers (PPNG) and fully resistant to penicillin (MIC ≥8 mg/l). One isolate showed chromosomally induced resistance to penicillin (MIC 2 mg/l). None of the isolates was sensitive to tetracycline; 16 (16%) showed intermediate resistance (MICs 1–8 mg/l) and 87 (84%) showed high-level plasmid-mediated resistance (TRNG) (MICs >10 mg/l). This is the first report of TRNG in The Gambia. Only 6 (6%) strains were fully sensitive to trimethoprim-sulphamethoxazole (MIC <8 mg/l); 78 (76%) showed intermediate level resistance (MICs 8–16 mg/l) and 19 (18%) were fully resistant (MIC >32 mg/l). Indications of an increase in MIC to ciprofloxacin and ceftriaxone were found in 6 (6%) and 1 (1%) strains, respectively, although all remained fully sensitive (MICs 0.004–0.03 mg/1 and 0.001–0.015 mg/l). All PPNG and TRNG strains carried the 3.2 MDa and 25.2 MDa plasmids, respectively. All isolates carried the 2.6 MDa cryptic plasmid and 9 (3 PPNG and 6 non-PPNG) carried the 24.5 MDa conjugative plasmid. Forty-four (43%) strains were typed group W1, 58 (56%) W11/111 and 1 had cross-reacting antigens. Because PPNG are frequently encountered and high-level TRNG is now prevalent, the newer cephalosporins and quinolones must now be considered as first-line drugs for the treatment of gonorrhoea in The Gambia.     

几种性传播疾病病原体检测芯片的制备

目的 :为了同时多样本检测和鉴别淋病奈瑟球菌、沙眼衣原体和解脲脲支原体 3种重要的性传播疾病病原体 ,制备了寡核苷酸检测芯片。方法 :针对 3种病原体和荧光素酶基因设计特异的引物和寡核苷酸探针 ,采用硫代和氨基双功能探针修饰技术制备寡核苷酸芯片 ,以荧光标记多重不对称PCR技术为基础 ,通过将单链PCR产物与芯片杂交实现对性传播疾病病原体的检测。结果 :对 10种与待检病原体无关的菌及定量有限稀释的荧光素酶和 3种病原体基因质粒模板进行芯片检测 ,结果表明芯片对待检病原体特异 ,其检测 4种基因的灵敏度均为 5×10 3 拷贝质粒。对 2 4份性传播疾病患者标本进行芯片检测 ,沙眼衣原体感染率为 10 0 % ,与淋病奈瑟球菌混合感染率为 83.3% (2 0 / 2 4 ) ,与传统PCR诊断结果完全一致。在 2 4份标本中 ,淋病奈瑟球菌、沙眼衣原体和解脲脲支原体三重感染病例芯片诊断为 3例 ,混合感染率为 12 .5 % (3/ 2 4 ) ;而传统PCR诊断为 4例 ,混合感染率为 16 .7% (4/2 4 ) ,两种方法的符合率为 75 %。结论 :该芯片是一种可靠检测 3种病原体的方法 ,它可快速提供有关患者混合感染的情况 ,因而为指导个性化治疗提供及时可靠的诊断依据。     

Antibiotic resistance in Staphylococcus aureus colonising the intestines of Swedish infants

Staphylococcus aureus has become a frequent coloniser of the intestinal tract of infants, but the health effects of such colonisation are not clear. In this study, the antibiotic resistance patterns of 116 S. aureus strains from the commensal intestinal microflora were determined. The strains were obtained from 81 Swedish infants who had been followed with regular stool samples and registration of antibiotic usage during their first year of life. The faecal population levels of the individual strains and the duration of their persistence in the microflora had been determined previously. The prevalence of antibiotic resistance among the 116 strains was modest: methicillin, 0%; penicillin G, 78%; erythromycin A, 3%; tetracycline, 2%; clindamycin, 0.9%; and fusidic acid, 0.9%. Colonisation by antibiotic-resistant strains was unrelated to antibiotic consumption by individual infants. Antibiotic-resistant strains were as capable of persisting in the intestinal microflora and reaching high faecal population levels as fully susceptible strains. No strain lost or acquired resistance during the colonisation period. Thus, antibiotic-resistant strains of S. aureus seem to be as fit for competition in the large bowel microflora as susceptible strains, even in the absence of selective pressure from antibiotics. This may aggravate the ecological consequences of antibiotic resistance development.     

A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B

We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-κ (in the standard) and each anti-IgG subclass-specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing κ chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5–6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation.     

Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Germ-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-gamma, IL-12, TNF, IL-10 and TGF-beta production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-gamma, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-beta. T cell depletion abolished IFN-gamma and decreased IL-12 production, but did not affect IL-10 and TGF-beta production. Conversely, neither IL-10 nor TGF-beta was produced in cultures of B cell-depleted MLN. In addition, CD4(+) T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-gamma when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-beta, but not IFN-gamma, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-gamma-producing CD4(+) T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.     

我国脑膜炎奈瑟菌孔蛋白PorA、PorB的多态性分析

目的以蛋白质组学研究为基础,分析2003—2005年我国流脑暴发流行期间C群脑膜炎奈瑟菌菌株特征,建立以致病性相关蛋白多态性为目标的新分型方法。方法利用双向电泳和MALDI-TOF质谱鉴定分析2003—2005年流脑流行期内分离的66株C群脑膜炎奈瑟菌菌株及2株参考菌株的蛋白表达,重点分析与致病性相关的蛋白多态性特征。结果根据孔蛋白PorA、PorB在双向电泳中的多态性,建立了12个特征菌型。安徽菌株的PorA和PorB蛋白电泳谱型显示出了高度的一致性,而其他地区的菌株则显示出高度的多态性。结论PorA和PorB蛋白2-DE电泳谱型可以作为一种新的菌株分型方法,可能在菌型变迁检测和流脑暴发预警中具有重要应用前景。     

Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS)

Tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-6 production by human monocytes in response to a clinical strain of the Gram-negative encapsulated bacteria Neisseria meningitidis and an isogenic lpxA- strain deficient in LPS was investigated. Wild-type N. meningitidis at concentrations between 105 and 108 organisms/ml and purified LPS induced proinflammatory cytokine production. High levels of these cytokines were also produced in response to the lpxA- strain at 107 and 108 organisms/ml. The specific LPS antagonist bactericidal/permeability-increasing protein (rBPI21) inhibited cytokine production induced by LPS and wild-type bacteria at 105 organisms/ml but not at higher concentrations, and not by LPS-deficient bacteria at any concentration. These data show that proinflammatory cytokine production by monocytes in response to N. meningitidis does not require the presence of LPS. Therapeutic strategies designed to block LPS alone may not therefore be sufficient for interrupting the inflammatory response in severe meningococcal disease.