Synthesis of Haemophilus influenzae carbohydrate surface antigens

The pathogenic bacteria Haemophilus influenzae, causing, i.a., meningitis and otitis, contain both capsular and lipopolysaccharide surface antigens. The syntheses of several oligosaccharides corresponding to native H. influenzae polysaccharide structures is outlined with an emphasis on synthetically challenging features. Hence, the synthesis of a branched inner core lipopolysaccharide tetrasaccharide structure, α- , -Hepp-(13)-[β- -Glcp-(14)]-α- , -Hepp-(15)-αKdo, containing the unusual higher carbon sugars -glycero- -manno-heptose and Kdo is described, as well as the assembly of di- and trimers of the repeating unit of the capsular polysaccharides of serotype c,[−4)-3-OAc-β- -GlcpNAc-(13)-α- -Galp-(1-PO⁻₃−] and serotype f[−3)-β- -GalpNAc-(14)-3-OAc-α- -GalpNAc-(1-PO⁻₃], both linked via anomeric phospodiester linkages. Also efforts towards the synthesis of the repeating unit of the capsular polysaccharide of serotype e,3)-β- -GlcpNAc-(14)-[β- -Fruf-(23)]-β- -ManpNAcA-(1, containing a β-fructofuranosidic residue, is discussed. All synthetic derivates are spacer-equipped to allow formation of glycoconjugates for biological applications.     

儿童肺炎链球菌和流感嗜血杆菌耐药及感染特征分析

目的 了解儿童呼吸道感染肺炎链球菌(Streptococcus pneumonia,SP)和流感嗜血杆菌(Haemophilus influenzae,Hi)的分布特征、耐药情况,及耐药菌抗生素间的相互影响,以更合理地指导临床用药.方法 对2009-2010年临床呼吸道感染患儿进行痰、咽拭子或肺泡灌洗液培养分离Hi和SP.因子需求试验鉴定Hi,头孢硝噻酚法检测β-内酰胺酶;奥普托辛和胆汁溶菌试验确认SP.两菌均采用K-B法检测常用对抗生素的耐药性.结果 收集SP 495株,Hi 515株,多见于3岁以下儿童,以呼吸科分离率最高.SP对红霉素、四环素、阿奇霉素、复方新诺明的耐药率分别为98.4％、66.1％、98％和81.6％,其对青霉素敏感性仅为9.5％.Hi有42.7％产生β-内酰胺酶,Hi对氯霉素、复方新诺明、氨苄青霉素、阿奇霉素的耐药率分别为22.1％、21.6％、36.7％和62.7％.与青霉素敏感SP和β-内酰胺酶阴性Hi相比,耐药SP和阳性Hi更易对氯霉素、四环素和复方新诺明耐药.结论 SP和Hi以婴幼儿为主,多见于呼吸科.其耐药情况严峻,青霉素耐药和产β-内酰胺酶菌株会诱导其他抗生素耐药,引发多重耐药.合理使用抗生素以及对两菌的耐药监测应引起高度重视.     

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.     

Human lactoferrin receptor activity in non-encapsulated Haemophilus influenzae

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.     

海口市流感嗜血杆菌流行病学及耐药性研究

目的:研究海口市呼吸系统疾病患儿流感嗜血杆菌流行病学及耐药性情况,为疾病的预防和治疗提供参考。方法:收集2006-2010年海口市各医院(海南医学院附属医院、海南省人民医院、海口市人民医院、海南省农垦总医院)临床送检的咽拭子、鼻咽分泌物、痰、脑脊液等标本486例,然后分离鉴定流感嗜血杆菌;采用Etest法测定对分离到的95株流感嗜血杆菌进行抗生素药敏试验检测。结果:流感嗜血杆菌总检出率19.55%,其检出情况在不同地区和不同民族之间存在差异,考虑其原因可能和社会经济条件有关,流感嗜血杆菌对常用抗生素的体外药敏结果依次为:左氧氟沙星、头孢曲松>阿莫西林/克拉维酸、阿奇霉素>氯霉素>头孢克洛>阿莫西林>氨苄西林>磺胺甲基异噁唑。结论:海口流感嗜血杆菌由于全年气温较高,其发病率无明显季节性,细菌检出率在不同地区及不同民族之间存在差别。对左氧氟沙星、头孢曲松、阿莫西林/克拉维酸、阿奇霉素、氯霉素的耐药率低,其可作为临床治疗流感嗜血杆菌感染的首选药物,而对氨苄西林和磺胺甲基异噁唑的耐药率较高,其作为临床治疗流感嗜血杆菌首选药物的传统地位受到了严重挑战。     

In vitro cytotoxicity of Haemophilus influenzae lipopolysaccharides for bovine aortal endothelial cells

The cytotoxicity of purified lipopolysaccharide (LPS) from a prototype Haemophilus influenzae type b (Hib) strain (Eagan) and three transformants, differing in their LPS phenotype, for bovine aortal endothelial cells (BAOEC) was investigated. All LPS preparations caused cell disruption and release of lactate dehydrogenase (LDH), an indicator of cytotoxicity, from BAOEC monolayers but to differing extents. There was no correlation between the cytotoxicity of purified Hib LPS to BAOEC monolayers and potential to cause bacteraemia in experimental animals.     

流感嗜血杆菌的基因组学研究进展

流感嗜血杆菌Rd型是第一个被测序的细菌.其基因组大小为1830137bp,A含量为31%,C为19%,G为19%,T为31%,GC含量为38%,TIGR确认了1473个具有重要功能的基因.基因组序列中包含有复制子、核糖体启动子、毒力基因、DNA转运系统、调节子等结构.流感嗜血杆菌的基因组研究必将对寻找疾病相关基因和抗原基因,筛选药物作用靶位,研制特异的诊断试剂、药物和疫苗具有十分重要的科学意义.对流感嗜血杆菌的基因组学研究进展进行了综述.     

The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: role in heme utilization

Haemophilus influenzae has an absolute growth requirement for heme and a heme binding lipoprotein (HbpA) has been implicated in the utilization of this essential nutrient. HbpA was identified by examining clones from an H. influenzae genomic library that caused Escherichia coli harboring the clone to bind heme. However, HbpA has not been shown to mediate heme acquisition in H. influenzae. We constructed an insertional mutation of hbpA in a nontypeable H. influenzae strain and demonstrated a role for the gene in utilization of multiple heme sources. This is the first report confirming a role for HbpA in utilization of heme.     

Virulence of non-β-lactamase-mediated ampicilin-resistant Haemophilus influenzae

We questioned whether strains of ampicillin-resistant, non-beta-lactamase-producing (AmpR NBLP) Haemophilus influenzae with lower affinity penicillin-binding proteins (PBPs) might have altered virulence. The virulence of resistant transformant strains and the susceptible recipient was compared using infant rats. Following intraperitoneal inoculation, there was a significantly lower mortality rate and incidence and magnitude of bacteremia with two of three transformants compared to the recipient strain. Reduced virulence was not associated with greater bactericidal activity of serum or human neutrophils or faster clearance of the transformant following intravenous injection. Heated rat or human plasma supported exponential growth of the recipient, but not the transformant, suggesting deficient in vivo multiplication. We conclude that H. influenzae with altered PBPs are less virulent in an infant rat model which may be related to differences in in vivo growth.     

流感嗜血杆菌的分型研究

目的分析同济医院分离的流感嗜血杆菌的生物学分型及荚膜基因分型,了解本地区分离的流感嗜血杆菌的主要流行株。方法2012年1月1日至2012年12月31日从华中科技大学同济医学院附属同济医院分离流感嗜血杆菌100株。根据脲酶、吲哚和鸟氨酸脱羧酶试验对流感嗜血杆菌进行传统的生物学分型,分为Ⅰ～Ⅷ八个生物型。回顾患者病史资料,分析生物学分型和流感嗜血杆菌所引起的疾病之间的关系。用流感嗜血杆菌荚膜编码基因（bexA）和a—f型特异性荚膜基因设计引物,采用PCR技术对流感嗜血杆菌进行荚膜基因检测。通过生物学分型和荚膜基因分型结果的比对,探讨两者之间的关联。结果分离的100株流感嗜血杆菌生物学分型结果如下：Ⅲ型6株,Ⅳ型28株,Ⅴ型1株,Ⅵ型54株,Ⅶ型11株。未分离到Ⅰ型、Ⅱ型和Ⅷ型。分析患者的临床诊断,发现主要流行株Ⅵ型流感嗜血杆菌主要引起患者肺炎（包括支气管肺炎和新生儿肺炎）和支气管炎（包括毛细支气管炎和喘息性支气管炎）。荚膜基因分型结果显示,未分离到b型和b-型流感嗜血杆菌。共分离到1株f型,其余99株均为无荚膜抗原的不可分型流感嗜血杆菌。生物学分型和荚膜分型之间无明显的相关性。结论该院分离的流感嗜血杆菌主要为生物型Ⅵ型。回顾患者病史,发现Ⅵ型主要引起肺炎和支气管炎。荚膜基因分型显示,本地区分离的流感嗜血杆菌主要为不可分型流感嗜血杆菌。生物学分型和荚膜基因分型之间无明显相关性。     

肺炎链球菌C多糖含量检测方法的建立

目的使用肺炎链球菌C多糖单克隆抗体(单抗),建立检测荚膜多糖中残留的C多糖含量的方法。方法选择BALB/c雌性小鼠,采用体内诱生单抗腹水,放大生产肺炎链球菌C多糖单抗;使用间接ELISA、抑制性ELISA对其进行特异性鉴定;用特异性和亲和力高的单抗尝试建立肺炎链球菌荚膜多糖中C多糖含量检测的速率比浊法,并对该方法的线性、精密度、特异性进行验证。结果所制4株单抗的抗体类别均为IgM,识别的抗原表位互不相同,亲和力也不同。间接ELISA、抑制性ELISA结果均显示,所获得的单抗能够作用于肺炎链球菌C多糖上的磷酸胆碱位点,具有很好的特异性。选择单抗E8建立速率比浊检测方法的线性、精密度和特异性均良好,检测结果表明肺炎链球菌23个血清型荚膜多糖中C多糖含量不同。结论利用单抗建立了检测肺炎链球菌荚膜多糖中残留的C多糖的含量的速率比浊法。     

Analysis of immunoglobulin light chain loci in sheep

A sheep kappa cDNA probe was isolated, characterized by sequence analysis and shown to have significant sequence identity to other kappa light chains. This probe and a sheep lambda light chain probe were used to estimate the extent of various sheep immunoglobulin light chain gene loci by Southern blot analysis of genomic DNA. The results showed that the sheep has a single hybridizing kappa constant gene and three to five kappa V segment bands. Segregation of three polymorphic bands at the lambda C locus indicated that they were products of separate C segments. Restriction fragment pattern variations were obtained using light chain probes on various sheep breeds, but no pattern or individual band was characteristic for a particular breed.     

Comparing domain interactions within antibody Fabs with kappa and lambda light chains

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.