Measurement of cytokine release at the single cell level using the ELISPOT assay

The enzyme-linked immunospot (ELISPOT) assay took its design concept from traditional ELISA techniques and evolved over the years from a method for detecting antibodies secreted from B cells to a method for detecting cytokines or other soluble mediators secreted from a variety of different cell types. The ELISPOT assay allows the quantitative measurement of frequency of cytokine secreting cells at the single cell level directly ex vivo without elaborate in vitro expansion or manipulation of cell populations. The function of cells can be inferred from the pattern of cytokines secreted by cells in response to diverse antigenic stimuli and thus the ELISPOT assay has become a powerful method for monitoring immune responses in health and disease. The ELISPOT assay like the ELISA assay is relatively easy to perform and it has the promise of robustness, reliability, and reproducibility of performance for use as a diagnostic tool. The history, applications, validation process, and future challenges of the ELISPOT assay are discussed in this chapter.     

Comparative use of amino acids by three auxotypes of Neisseria gonorrhoeae

Auxotypes of Neisseria gonorrhoeae are usually distinguishable by their particular requirements for growth; these requirements often include amino acids. It is possible that strains needing particular substrates to grow can be distinguished not merely by their growth requirements but also by their metabolism of these particular substrates. In this work amino acid utilization and oxidation studies were performed enabling prototype, pro- and thia-strains to be distinguished. The metabolism study also underlined the importance of proline as an energy source and pointed to the probability of distinct relationships with the metabolism of the key amino acids, glutamic and aspartic acids, for the three auxotypes.It is proposed that the specific amino acid required by the naturally occurring auxotype serves as an energy source at the site of infection and has important implications with respect to particular auxotypes at various sites.     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

Functional analysis of ori1 and repA of the R-plasmid pSJ5.6 from Neisseria gonorrhoeae

The functional ori1 of the 5.6kb gonococcal R-plasmid pSJ5.6 contains an A-T rich region followed by four 22bp direct repeats and one 19bp inverted repeat. The replication region of the plasmid also contains a gene encoding for a 39kD RepA protein. We have further assessed the functionality of the replication region in pSJ5.6, an-iteron type plasmid, using in vivo complementation assays in Escherichia coli. A 2.1kb PstI-RsaI fragment containing the ori1 and repA gene of pSJ5.6 was cloned into vector pZErO -2 to obtain pZA-MRR. The pUC origin in pZA-MRR was deleted to render the plasmid dependable on the cis-acting ori1 for replication. The resulting plasmid, pMRR, was capable of replication and maintenance in E. coli. We also cloned the ori1 and repA gene separately to obtain pA-Ori and pZG-Rep, respectively. Using in vivo complementation assays, we demonstrated that the ori1(+) plasmid (pA-Ori) was maintained only when the RepA protein was supplied in trans by the high copy number plasmid pZG-Rep.     

Generation of antiserum to specific epitopes

The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific epitopes of an outer membrane protein ofNeisseria gonorrhoeae. The MtrC AP1 sequence from the invariant mtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared to bind native MtrC proteinin situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein, including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to activate complement protein can then be tested.     

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae

Summary The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.     

Nucleotide sequence and genetic organization of the NgoPII restriction-modification system of Neisseria gonorrhoeae

Summary The NgoPII restriction endonuclease, which recognizes the sequence 5-GGCC-3, differs from its isoschizomer HaeIII in being sensitive to methylation at the external cytosine residue. The entire nucleotide sequence of a cloned 3.3 kb segment of Neisseria gonorrhoeae strain P9 chromosomal DNA which harbours the NgoPII restriction-modification system has been determined. This data, coupled with sub-cloning experiments, indicates that the restriction endonuclease (R.NgoII) and modification (M.NgoII) genes are transcribed from separate promoters but are arranged in tandem, with the R.NgoPII gene being located on the 5 side of the M.NgoPII gene. Unlike all previously reported restriction systems the 3 end of the endonuclease open reading frame overlaps the 5 end of the methylase open reading frame by 8 codons. This overlap may have implications for the regulation of the NgoPII restriction-modification system.     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.     

Cellular adhesion molecules as targets for bacterial infection

A large number of bacterial pathogens targets cell adhesion molecules to establish an intimate contact with host cells and tissues. Members of the integrin, cadherin and immunoglobulin-related cell adhesion molecule (IgCAM) families are frequently recognized by specific bacterial surface proteins. Binding can trigger bacterial internalization following cytoskeletal rearrangements that are initiated upon receptor clustering. Moreover, signals emanating from the occupied receptors can result in cellular responses such as gene expression events that influence the phenotype of the infected cell. This review will address recent advances in our understanding of bacterial engagement of cellular adhesion molecules by discussing the binding of integrins by Staphylococcus aureus as well as the exploitation of IgCAMs by pathogenic Neisseria species.     

N terminus determinants of MinC from <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> mediate interaction with FtsZ but do not affect interaction with MinD or homodimerization

While bacterial cell division has been widely studied in rod-shaped bacteria, the mechanism of cell division in round (coccal) bacteria remains largely enigmatic. In the present study, interaction between the cell division inhibitor MinC from Neisseria gonorrhoeae (MinC_Ng) and the gonococcal cell division proteins MinD_Ng and FtsZ_Ng are demonstrated. Protein truncation and site-directed mutagenic approaches determined which N-terminal residues were essential for cell division inhibition by MinC_Ng using cell morphology as an indicator of protein functionality. Truncation from or mutation at the 13th amino acid of the N terminus of MinC_Ng resulted in loss of protein function. Bioinformatic analyses predicted that point mutations of L35P and L68P would affect the α-helical conformation of the protein and we experimentally showed that these mutations alter the functionality of MinC_Ng. The bacterial two-hybrid system showed that interaction of MinC_Ng with FtsZ_Ng is abrogated upon truncation of 13 N-terminal residues while MinC_Ng-MinD_Ng interaction or MinC_Ng homodimerization is unaffected. These data confirm interactions among gonococcal cell division proteins and determine the necessity of the 13th amino acid for MinC_Ng function.     

Direct transformation ofNeisseria gonorrhoeae by gel-isolated DNA

The naturally competent organism,Neisseria gonorrhoeae, can efficiently transform a marker carried on DNA purified in low-melting-temperature agarose without prior purification or dilution. Neither the agarose or buffer components inhibit transformation frequencies, but exposure to UV irradiation completely abrogates transformation.     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

淋病奈瑟菌肽基脯氨酰异构酶的原核表达及其作为候选疫苗的初步评价

【背景】淋病是我国主要的性传播疾病之一,感染淋病奈瑟菌可促进人类免疫缺陷病毒(human immunodeficiency virus, HIV)的传播和感染。目前我国淋病发病人数呈上升趋势,随着多重耐药菌株的出现,亟须研发保护性疫苗来防治淋病的传播和感染。【目的】分析淋病奈瑟菌(Neisseria gonorrhoeae, NG)肽基脯氨酰异构酶(peptidyl-prolyl isomerase, PPIase)蛋白的高级结构和表位,探讨其作为疫苗和分子诊断靶点的潜力。【方法】利用生物信息学软件分析PPIase蛋白的极性、亲水性、柔韧性、表面可及性、二级和三级结构,以及T、B细胞表位等;用pET32a(+)质粒构建PPIase蛋白的原核表达系统并纯化蛋白,用纯化的重组蛋白和超声波破碎的NG全菌抗原分别免疫BALB/c小鼠,收获免疫血清;制备NG全细胞抗原,分别以全细胞抗原酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)和间接免疫荧光试验检测重组PPIase蛋白血清抗体与NG全细胞表面抗原的结合情况。【结果】生物信息学分析结果显示,...     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

Establishment of a human CEACAM1 transgenic mouse model for the study of gonococcal infections

Neisseria gonorrhoeae is the causative microorganism for the sexually transmitted disease (STD) gonorrhea and humans are its only natural host. An animal model would be a useful tool for gonorrhea research, therefore we developed the hCEACAM1 transgenic mice, using an eukaryotic expression vector, pCDPCAM1-GI. This construct was microinjected into the zygotes of C57BL/6 mice and 22 F0 generation transgenic mice were obtained. Four (lines 50, 53, 54, and 59) of the F0 generation were found to carry the transgene by PCR and sequence analysis, respectively. Western blotting and Fluorescence-Activated Cell Sorting Analysis demonstrated that hCEACAM1 was expressed on the cell membrane of various tissues in the line 53 transgenic mouse. To initiate the disease in the animal model, the F2 or F3 transgenic mice were inoculated with N. gonorrhoeae intravaginally. Compared with normal mice, N. gonorrhoeae can successfully infect and cause inflammation in the transgenic mice. These data suggested the feasibility of using hCEACAM1 transgenic mice as an animal model for gonococcal infections.     

Identification of two new alleles, IGHV3-23*04 and IGHJ6*04, and the complete sequence of the IGHV3-h pseudogene in the human immunoglobulin locus and their prevalences in Danish Caucasians

More than 100 variable (V), 27 diversity (D), and six joining (J) genes are encoded in the human heavy chain locus, and many of these genes exists in different allelic forms. The number of genes and the allelic differences help to create diversity in the immunoglobulin receptors, a key feature of the adaptive immune system. We here report the identification of two novel and seemingly functional alleles of human heavy chain genes. The variable IGHV3-23*04 allele is found with an allele frequency of 0.21 amongst Danish Caucasians, whereas the novel joining IGHJ6*04 allele is rare (allele frequency 0.02). We also report the full sequence of IGHV3-h. The gene exists in two allelic forms but is only found in 58% of the Danish Caucasians studied. The methionine translation initiation codon is mutated, ATG→AAG, and we therefore propose that the gene is a pseudogene incapable of being translated.     

2013至2017年4 542例泌尿生殖系感染者NG、CT和UU感染特点分析

探讨淋球菌（NG）、沙眼衣原体（CT）和解脲脲原体（UU）三种性传播疾病（STD）病原体在宁夏泌尿生殖系感染者中的感染状况,为临床防治提供参考。选取2013年1月至2017年12月在宁夏医科大学总医院就诊的患者4 542例,其中男性3 561例,女性981例,应用实时荧光PCR法检测上述病原体,并分析结果。3种病原体总感染率为26.51%（1 204/4 542）,NG、CT、UU单一病原体感染率分别为26.31%、15.21%和35.70%,男、女感染率分别为23.48%和37.51%,感染率有性别差异（P＜0.01）,NG、CT感染率男性显著高于女性,UU感染率女性显著高于男性;混合感染率为3.32%（151/4 542）,男、女混合感染率分别为3.82%和153%,主要以CT+UU为主,NG+UU次之;从年龄分布来说,男性≤20岁年龄段感染率最高,女性21～30岁年龄段感染率最高;5年来NG、CT和UU感染率呈上升趋势。泌尿生殖系感染者中NG、CT、UU感染情况严重,男性易感NG和CT,女性易感UU,同时,混合感染也较常见,提示应同时检测多种病原体以防漏检。     

The immunoglobulin <Emphasis Type="Italic">IGHD</Emphasis> gene locus in C57BL/6 mice

Four immunoglobulin heavy chain diversity (IGHD) gene subgroups (DFL16, DSP2, DQ52, and DST4) have been identified previously in BALB/c mice. Although the locations of most IGHD genes have been established based on restriction map and Southern blot analysis, a complete mouse IGHD gene locus map at the sequence level is still not available. In addition, a previous restriction fragment length polymorphism study suggested that significant difference in the IGHD gene locus exists between C57BL/6 and BALB/c mice. The author has now analyzed the C57BL/6 mouse genomic data and established a complete map of the IGHD gene locus. All four IGHD subgroups previously identified in BALB/c mice were found to be present in C57BL/6 mice. However, unlike the BALB/c mice, which have at least 13 IGHD genes, the C57BL/6 genome contains only ten IGHD genes, which include one DFL16, six DSP2, one DQ52, and two DST4 genes. There are also differences in the coding regions of the DST4 and DQ52 genes between the two mouse strains.     

Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH₂-terminal His₆Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high β-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R_H = 16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.