Chemico-enzymatic synthesis of Salmonella newington O-specific polysaccharide and its modified derivatives

Treatment of the DMan (beta 1-4) LRha (alpha 1-3) D [3H]Gal-derivative of moraprenyl pyrophosphate with the cell envelope preparation from S. newington results in the formation of polysaccharide with beta 1-6 linkage between the trisaccharide units (polymerization degree approximately 8). The synthetic derivatives of moraprenyl pyrophosphate which contain D-talose, 4-deoxy-D-xylo-hexose, LRha (alpha 1-3) DGlc or LRha (alpha 1-3) DGlc (alpha 1-6) DGal were found to serve as substrates for the biosynthesis of the corresponding modified polysaccharides.     

宋内氏痢疾杆菌O-SP-TT结合疫苗的制备及其免疫学特性的研究

宋内氏痢疾杆菌（S.Sonnei)的一个重要保护性抗原是菌体抗原（O抗原）即细菌细胞壁中的脂多糖（LPS）成分,已证实血清中足够水平的抗LPS IgG抗体抗体即能对该菌提供免疫保护作用。本文选用Westphal热酚法提纯宋内氏痢疾菌LPS,经酸水解法进一步脱毒处理后得到无毒但却具弱免疫原性的O-特异性多糖（O－SP）,然后采用化学方法即通过连接剂己二酸二肼（ADH）将其共价结合到破伤风类毒素（TT）上,共制备得到了三批宋内氏痢疾杆菌O－SP－TT结合疫苗。经生化和免疫学方法检测证实我们提纯的LPOS,OSP及其合成的L－SP－AH衍生物,O－SP－TT结合物均具有宋内氏痢疾杆菌O抗原特异性,同时其核酸和杂蛋白质含量低（≤2%),表明纯度较高。同时经小鼠免疫原性试验证实三批结合物免疫小鼠后产生的抗LPS IgG抗体滴度均比单一O－SP免疫后产生的要高出10倍以上,且结合物再次注射后存在加强应答。补体介导的体外杀菌力试验表明结合物免疫小鼠后诱导产生的血清抗LPSIgG抗体对宋内氏痢疾杆菌具特异性杀菌活性。本文结果表明宋内氏痢疾杆菌O-SP-TT结合疫苗可望作为一种有效的侯选痢疾疫苗做进一步的大规模人体观察。     

福氏2a痢疾杆菌O—特异性多糖与破伤风类毒素结合疫苗的制备及其免疫学特性

通过己二酸二肼（ADH）将福氏2a痢疾杆菌脂多糖（LPS）经酸水解脱毒纯化后得到的O－SP和破伤风类毒素（TT）蛋白共价结合,制备了福氏2a痢疾杆菌结合物。以2.5μg多糖或含2.5μg多糖的结合物经皮下注射免疫NIH品系雌性小鼠,同时以25μg多糖或含25μg多糖的结合物经耳缘静脉注射家兔。用ELISA方法分别检测小鼠和家兔血清中抗脂多糖抗体水平及抗TT水平,并用豚鼠血清补体介导进行了体外杀菌实验。结果显示：用本实验方法提取的多糖,合成的多糖衍生物,多糖－蛋白结合物都具有福氏2a痢疾杆菌O－抗原特异性,其化学组成及结构特异性和国外文献报道基本一致。单独用多糖免疫小鼠和家兔未能诱导LPS抗体,而结合物免疫小鼠和家兔的血清诱导出了较高的抗LPS IgG抗体及抗TT抗体,并有较强的体外杀菌活性。产生的抗体存在着免疫记忆性,可以产生再次免疫应答加强反应。     

Effect of tumor necrosis factor-alpha on infection with Salmonella typhimurium in a mouse model

Mice, inoculated with Salmonella typhimurium, either intraperitoneally (i.p.) or intragastrically (i.g.), developed a systemic infection with a high death rate within a few days after inoculation. Pretreatment of the mice with moderate concentrations of i.p. administered TNF-alpha 24 h before the administration of bacteria reduced the establishment of intracellular infection in the intestinal epithelial cells, and development of bacteremia. The mortality rate was reduced, and the survival time was extended by the same treatment. This effect of TNF-alpha was more pronounced against i.g. than against i.p. inoculated bacteria. The effect was dose dependent, thus concentrations above or below the optimal dose had less effect. No synergistic effect was seen if TNF-alpha was given in combination with interferon-gamma. These results indicate, that TNF-alpha may have a physiological effect in the host defence against facultatively intracellular Gram-negative bacteria.     

志贺氏I型痢疾杆菌O-特异性多糖与破伤风类毒素结合疫苗的制备及其在小鼠体内的免疫原性

从志贺氏I型痢疾杆菌LPS中分离纯化出O SP半抗原 ,以ADH为连接剂将其与TT结合形成O SP TT结合疫苗 ,并用此结合疫苗免疫NIH小鼠 ,结果显示单独使用O SP免疫后 ,小鼠血清中没有抗LPS抗体产生 ,而用O SP TT免疫后小鼠血清中产生了抗LPSIgG和IgM抗体 ,且IgG抗体水平高于IgM抗体 ;O SP TT免疫组第二次和第三次免疫后IgG抗体水平均有显著的升高 (P <0 0 1) ,但第二次和第三次免疫后血清IgM抗体虽有升高 ,但与前一次免疫相比均无显著差异 (P >0 0 5 ) ,表明O SP TT结合疫苗具有加强免疫应答效应。补体介导的体外杀菌活性试验结果证明 ,O SP TT免疫血清在 1∶16 0倍稀释后仍对志贺氏I型痢疾杆菌具有特异性杀菌活性。     

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.     

Chemico-enzymatic synthesis of O-specific polysaccharide of Salmonella anatum and its analogs using partially purified polymerase preparation

A partial purified polymerase from S. anatum was used for the synthesis of polysaccharide [-6) [14C]Man(beta 1-4)Rha(alpha 1-3)Gal(alpha 1-]n and its analogues containing D-glucose residue instead of D-galactose or D-mannose. Structures of these polymers were confirmed by methylation analysis and radioimmunochemical tests.     

Down-regulation of IgE and IgG4 antibodies to tetanus toxoid and diphtheria toxoid by covaccination with cellular Bordetella pertussis vaccine

Pertussis (P) toxin acts as adjuvant for IgE formation against simultaneously administered Ags in animal models. P vaccination may also have an adjuvant impact on IgE formation against coadministered diphtheria (D) and tetanus (T) Ags in humans. Sera of 103 D-T-P-immunized and 319 D-T-immunized children aged 2 years were analyzed for IgE, IgG4, and IgG to D and T (radioallergosorbent test), total IgE and IgE against common inhalant allergens (CAP radioallergosorbent test fluoroenzyme immunoassay). Fewer D-T-P- than D-T-immunized children had sera positive for T-IgE (12.6 vs 53.6%, p < 0.001), T-IgG4 (71.6 vs 89.2%, p < 0.001), D-IgE (31.0 vs 70.5%, p < 0.001), and D-IgG4 (85.2 vs 93.4%, p = 0.039). Suppression of T-IgE was not dependent on the cutoff chosen for a positive test result, but was dependent on the proportion of D-T immunizations given with P. The risk for sensitization to common environmental allergens did not differ (odds ratio 0.953, 95% confidence interval 0.815-1.114). No significant differences between D-T- and D-T-P-immunized children were found with regard to T-IgG or D-IgG. In summary, IgE and IgG4 (but not IgG) serum levels to coadministered D- and T-Ags are suppressed among P-immunized children as compared with nonimmunized children. These results suggest that the presence of a microbial product during Ag exposure can down-regulate an IgE/IgG4 response in humans.     

Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharide

A series of polyprenyl phosphates with modified structure of polyprenyl residue was prepared through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile was found to represent a very efficient, simple and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepared were studied as substrates for the enzymes of Salmonella anatum O-specific polysaccharide biosynthesis. Correct stereochemistry of alpha- and beta-isoprenic units was found to be essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.     

肺炎链球菌18C型糖蛋白结合物的制备及其免疫原性

制备肺炎链球菌18C型荚膜多糖－破伤风类毒素结合物（CPS－TT）,测定结合物的理化性质,抗原特异性及其在动物中的免疫原性。结果显示,结合物能与相应的多糖和破伤风抗血清形成明显的沉淀线,蛋白／多糖比率为1．86,结合物分子大小（Kd值）为0．058。注射小鼠后可诱导明显的抗体应答,而且随着注射针次的增加,抗体反应水平明显增高,显示加强效应。结果表明,制备的肺炎链球菌糖蛋白结合物抗原性良好,具有胸腺依赖性的特性,在小鼠中显示较好的免疫原性。     

Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium

Toxic and mutagenic activities of formaldehyde were studied in Salmonella typhimurium strain TM677, using forward mutation to 8-azaguanine (8-AG) resistance both in the absence and in the presence of Aroclor-induced rat-liver postmitochondrial supernatant (PMS). The results showed that formaldehyde was toxic and mutagenic to the bacteria in both systems, but toxicity and mutagenicity were reduced in the presence of PMS. The minimum concentration required to induce toxicity and mutagenicity was 0.17 mM in the absence of PMS and 0.33 mM in the presence of PMS.     

Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies.

Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells.     

Mutagenicity of acrylamide and its analogues in Salmonella typhimurium

Acrylamide and its 15 analogues have been tested for mutagenicity in 5 TA strains of Salmonella typhimurium. Acrylamide, N-tert-butylacrylamide, crotonamide, diacetone acrylamide, N,N-diethylacrylamide, N,N-dimethylacrylamide, N-hydroxymethylacrylamide, N-methylacrylamide, N-isobutoxymethylacrylamide, N-isopropylacrylamide, methacrylamide, N,N'-methylene-bis-acrylamide and N-tert-octylacrylamide appeared not to be mutagenic in the standard Ames assay both with and without Aroclor 1254-induced S9, and in both the plate incubation and liquid preincubation procedures. Three epoxide analogues, i.e., glycidamide, N,N-diglycidyl acrylamide and glycidyl methacrylamide showed mutagenicity in one or two strains both with and without the S9.     

Antitumor therapeutic effects of Salmonella typhimurium containing Flt3 Ligand expression plasmids in melanoma-bearing mouse

An attenuated strain of Salmonella typhimurium has been tested in animals and clinically as an anticancer agent due to its in vivo tumor-targeting and tumoricidal properties. We exploited a genetically-engineered S. typhimurium harboring Flt3 Ligand (Flt3L) expression vectors as a tumoricidal agent to enhance its therapeutic efficacy. Flt3L showed tumoricidal effects when expressed in tumor cells in vitro. When melanoma-bearing mice were treated locally with Salmonella, S. typhimurim with Flt3L expression vectors inhibited tumor growth more than Salmonella controls (50% vs. 0% in tumor regression rates). Moreover, it prolonged survivals of animals without induction of memory antitumor protective responses to a parental tumor re-challenge (50% vs. 0% in survival rates). These results suggest that a genetically engineered S. typhimurium with Flt3L expression vectors has the potential to be applicable as a safer and more effective tumor-targeting and tumoricidal agent.     

Mutagenicity of cadmium in Salmonella typhimurium and its synergism with two nitrosamines

Cadmium chloride (CdCl2) at concentrations of 0.5 mM was significantly mutagenic in Salmonella typhimurium tester strains and reverted histidine auxotrophy due either to missense (TA1975 and TA1535) or to frameshift (TA1537) mutations. It also induced forward mutations to 8-azaguanine resistance in each strain, but failed to increase mutation frequencies in strain TA100. More importantly, CdCl2 increased the mutagenicity of two common nitrosamines in synergistic fashion, at a level up to 30-fold greater than expected from simple additivity. The mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine was increased about 10-fold in the presence of 0.5 mM CdCl2. This synergism was seen both in the induction of 8-azaguanine resistance and the reversion of histidine auxotrophy and was observed in the repair-proficient strain TA1975 as well as its repair-defective (uvrB-) derived strain TA1535. The synergism was dependent upon Cd concentration and was much reduced at 0.25 mM CdCl2. The strongest synergism was observed in the reversion of histidine auxotrophy in TA1975 by 180 microM methylnitrosourea and 0.5 mM CdCl2. In contrast to mutagenicity, there was no evidence for synergism in the toxicity of CdCl2. These data suggest that cadmium might interfere with the repair of both spontaneous and nitrosamine-induced mutations. They also raise the possibility that cadmium and nitrosamines may have synergistic effects as environmental carcinogens.     

Mapping of rfa Genes in Salmonella typhimurium by ES18 and P22 Transduction and by Conjugation

Loci termed rfa, determining biosynthesis of somatic lipopolysaccharide core, have been mapped in Salmonella typhimurium LT2. The smooth-specific phage P22 co-transduced two leaky rfa alleles with cysE and with pyrE; one of the leaky alleles is perhaps rfaG, and the other is an unidentified gene concerned with synthesis of the heptose-containing part of the core. The lipopolysaccharide-indifferent phage ES18 (or its variant ES18.h1) co-transduced rfaF, rfaG, rfaL, rfa(R-res-1), and rfa(R-res-2) alleles with cysE and with pyrE, at rates indicating the order cysE–rfaF–(rfa[R-res-1], rfa[r-res-2], rfaL)–rfaG–pyrE. One proven (and two suspected) rfaE alleles and five proven rfaH alleles were not co-transduced with cysE or pyrE. Hfr crosses indicated that the proven rfaE allele mapped between serA and strA.