幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 *

目的 利用生物信息学软件评价幽门螺杆菌(Helicobacter pylori)多价表位疫苗CWAE的抗原结构,经原核表达获得高纯度CWAE蛋白,进而鉴定多价表位疫苗CWAE的免疫学性质。方法 通过生物信息学软件分析H. pylori多价表位疫苗CWAE的抗原结构;用人工合成的H. pylori多价表位肽融合基因WAE替换重组质粒pET28a-CUE中的UE基因,构建重组质粒pET28a-CWAE。然后,将pET28a-CWAE转入大肠杆菌BL21(DE3)中,经IPTG诱导表达,并通过Ni-NTP镍离子亲和层析纯化抗原蛋白CWAE;利用GM1-ELISA鉴定CWAE中CTB组分的黏膜免疫佐剂活性。最后,通过ELISA和小鼠脾脏淋巴细胞增殖实验检测CWAE激发BALB/c小鼠产生抗H. pylori抗体体液免疫和淋巴细胞免疫应答的能力。结果 通过生物信息学软件证实H. pylori多价表位疫苗CWAE具有科学合理的结构;重组表达质粒pET28a-CWAE经PCR、双酶切和基因测序鉴定,融合基因CWAE与设计序列完全一致;重组基因工程菌株pET28a-CWAE/BL21(DE3)经IPTG诱导表达,抗原蛋白CWAE主要以包涵体形式存在,经Ni-NTP镍离子亲和层析纯化,纯度约达93.2%;GM1-ELISA实验证实,CWAE中CTB组分依旧保持有较好的黏膜佐剂活性;ELISA结果证实CWAE能够激发BALB/c小鼠产生H. pylori特异性抗体,而小鼠脾脏淋巴细胞增殖实验进一步证实CWAE能够激发针对H. pylori多种致病因子的淋巴细胞免疫反应。结论 H. pylori多价表位疫苗CWAE具有科学合理的抗原结构,经原核表达可获得高纯度抗原蛋白,能够激发BALB/c小鼠产生H. pylori特异性抗体体液免疫和淋巴细胞免疫应答。为研发防治H. pylori感染的多价表位疫苗奠定实验基础。     

Suppressed apoptosis in the inflamed gastric mucosa of Helicobacter pylori-colonized iNOS-knockout mice

Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice. Methods: iNOS-knockout mice (iNOS^−/−) and their iNOS^+/+ littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10⁸ colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Results: The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS^+/+ and iNOS^−/−strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS^+/+ mice, and not in the iNOS^−/− mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS^−/− mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS^−/− mice with H. pylori infection may be linked to preneoplastic transformation.     

A stain for demonstrating Helicobacter pylori in gastric biopsies

Helicobacter pylori continues to be a significant health care problem. It is associated with a variety of stomach disorders such as gastritis, gastric ulcer disease, gastric carcinoma and B-cell gastric lymphoma. One common method diagnosing an infection by this bacterium is microscopic examination of routine processed gastric or duodenal biopsies. With this type of specimen, it is necessary to demonstrate visually the presence of H. pylori using an appropriate staining technique. This paper presents a simple staining technique for demonstrating H. pylori in gastric biopsy specimens using carbol fuchsin staining against a contrasting background of light green.     

The role of neutrophils and inflammation in gastric mucosal injury

Gastric inflammation is a highly complex biochemical protective response to cellular/tissue injury. When this process occurs in an uncontrolled manner, the result is excessive cellular/tissue damage that results chronic inflammation and destruction of normal tissue. Current evidence suggests that Helicobacter pylori (H. pylori) infection and nonsteroidal anti-inflammatory drug (NSAID) ingestion are major causative factors in the pathogenesis of gastric mucosal injury in humans. In response to H. pylori infection or NSAID, neutrophils are recruited to the site of inflammation and generate reactive oxygen and nitrogen species and proteases. However, neutrophils are not able to kill the bacteria that live in the gastric mucus, and compounds produced by activated neutrophils themselves may be potentially harmful for normal tissue. It has been shown that leukocyte-vascular endothelial cell interaction is regulated by various cell adhesion molecules, and that this interaction is directly or indirectly modified by many factors, the origin of which is H. pylori and NSAIDs. This review describes the potential role of neutrophils and neutrophil-associated inflammation for gastric oxidative stress and injury induced by H. pylori and/or NSAID.     

The distribution of 4-hydroxynonenal-modified proteins in gastric mucosa of duodenal peptic ulcer patients

This study used monoclonal antibody specific for 4-hydroxynonenal (HNE)-histidine to evaluate immunohistochemical distribution of HNE-protein adducts in gastric mucosa biopsies of 52 peptic ulcer patients (all positive for H. pylori) and of 20 healthy volunteers (eight positive and 12 negative for H. pylori). HNE-modified proteins were present in glandular epithelium in all subjects, both patients with duodenal peptic ulcer and healthy subjects. Hence, the presence of HNE did not appear to be related to the presence of H. pylori. However, in patients with duodenal peptic ulcer accumulation of HNE-protein adducts was frequently observed also in nuclei, while in the control group such subcellular distribution of HNE was not observed at all. This study shows physiological presence of HNE in human gastric mucosa, but also suggests its role in pathology of gastric dysfunction in duodenal peptic ulcer patients manifested by accumulation of HNE-protein adducts in particular in nuclei of gastric glandular epithelium.     

Oxidative stress defense mechanisms to counter iron-promoted DNA damage in Helicobacter pylori

Iron, a key element in Fenton chemistry, causes oxygen-related toxicity to cells of most living organisms. Helicobacter pylori is a microaerophilic bacterium that infects human gastric mucosa and causes a series of gastric diseases. Exposure of H. pylori cells to air for 2 h elevated the level of free iron by about 4-fold as measured by electron paramagnetic resonance spectroscopy. H. pylori cells accumulated more free iron as they approached stationary phase growth, and they concomitantly suffered more DNA damage as indicated by DNA fragmentation analysis. Relationships between the intracellular free iron level, specific oxidative stress enzymes, and DNA damage were identified, and new roles for three oxidative stress-combating enzymes in H. pylori are proposed. Mutant cells defective in either catalase (KatA), in superoxide dismutase (SodB) or in alkyl hydroperoxide reductase (AhpC) were more sensitive to oxidative stress conditions; and they accumulated more free (toxic) iron; and they suffered more DNA fragmentation compared to wild type cells. A significant proportion of cells of sodB, ahpC, or katA mutant strains developed into the stress-induced coccoid form or lysed; they also contained significantly higher amounts of 8-oxo-guanine associated with their DNA, compared to wild type cells.     

Why Helicobacter pylori has Lewis antigens

In mimicry with human gastric epithelial cells, the lipopolysaccharide of Helicobacter pylori expresses Lewis blood group antigens. Recent data suggest that molecular mimicry does not promote immune evasion, nor does it lead to induction of autoantibodies, but that H. pylori Lewis X mediates adhesion to gastric epithelial cells and is essential for colonization.     

Mutation as an origin of genetic variability in Helicobacter pylori

The availability of two complete Helicobacter pylori genome sequences and recent studies of its population genetics have provided a detailed picture of genetic diversity in this important human gastric pathogen. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori.     

幽门螺杆菌致病岛CagL重组抗原的可溶性表达及其多克隆抗体的制备和分析*

目的:利用原核表达和蛋白质纯化技术获得高纯度的幽门螺杆菌致病岛CagL重组抗原(rCagL),利用其制备anti-CagL多克隆抗体,并分析抗体的特异性。方法:通过生物信息学软件分析rCagL的抗原结构;利用PCR长片段DNA合成技术合成不含有信号肽序列的幽门螺杆菌致病岛CagL基因,将其插入表达质粒pCzn1中,构建重组质粒pCzn1-rCagL。然后,将pCzn1-rCagL转入大肠杆菌Arctic Express中,经IPTG诱导表达后,通过Ni-IDA镍离子亲和层析纯化重组抗原rCagL,利用Western blot鉴定rCagL与His标签抗体和Anti-H. pylori抗体的免疫反应性;最后,通过rCagL辅以弗氏佐剂免疫BALB/c小鼠,制备anti-CagL多克隆抗血清,通过ELISA方法分析抗血清的特异性。结果:生物信息学软件表明重组抗原rCagL具有较好的抗原性质;重组质粒pCzn1-rCagL经双酶切和基因测序等技术鉴定,证实rCagL核苷酸序列与理论序列完全一致;基因工程菌株pCzn1-rCagL/Arctic Express在低温11℃条件经IPTG诱导表达。 SDS-PAGE实验结果证实:rCagL可实现相对高效地可溶性蛋白表达,可溶性蛋白约占包涵体的62.07%。经Ni-IDA亲和层析柱纯化,可获得高纯度rCagL,纯度约为96.6%。Western blot结果证实:重组抗原rCagL可特异性与His标签抗体和Anti-H. pylori抗体结合。ELISA结果证实:经rCagL免疫小鼠制备的多克隆抗体anti-CagL可特异性识别rCagL和H. pylori裂解物,具有较高的抗体特异性。结论:重组抗原rCagL在低温条件下可实现可溶性表达,经纯化可获得高纯度抗原蛋白;rCagL具有较好的抗原性,制备的多克隆抗体具有较好的免疫特异性,为发展H. pylori相关诊断试剂奠定了实验基础。     

Flavonoids of Helianthus series Microcephali

Ray flower and leaf flavonoids were investigated for the three species of Helianthus series Microcephali. Ray flowers of all species contain coreopsin, sulphurein, and quercetin 7-O-glucoside; those of H. microcephalus also contain quercetin 3-O-glucoside. A mixture of flavonoid aglycones, mostly methoxylated flavones, occurs in leaves of H. microcephalus, but not in H. glaucophyllus or H. laevigatus which also lack the glandular trichomes that in Helianthus are typically associated with flavonoid aglycones. The presence of compounds with the 6,8,4′ pattern of methoxylation in H. microcephalus suggests that the series is more similar in flavonoids to series Angustifolii than to series Corona-solis.     

The interaction of Trypanosoma congolense and Haemonchus contortus in Djallonké sheep

The interaction between Trypanosoma congolense and Haemonchus contortus was studied in 5 groups of 8 Djallonké sheep. Two groups received a single infection with either H. contortus or T. congolense, and 2 groups were infected with T. congolense followed by H. contortus (TH) or vice versa (HT). One group was kept as uninfected controls. Mortality due to infection was observed only in the dual infection groups. In the TH group, the effects were more acute whereas in the HT group they were more chronic. No significant differences in weight gain could be demonstrated between infected and control groups. Djallonké sheep are able to withstand a single infection with either T. congolense or H. contortus, which confirms their trypanotolerant nature and provides preliminary indication of resistance against helminth infections. However, when exposed to successive infections with both parasites, some of the animals lose this tolerance.     

Flavonoids of Helianthus series Angustifolii

Leaf and ray flower flavonoids were investigated for the seven species of Helianthus series Angustifolii. Flavone aglycones occur in small glandular trichomes located on leaf undersurfaces of H. angustifolius, H. floridanus and H. simulans. Other species lacked both glandular trichomes and flavone aglycones. Flavonol glycosides occur in low concentrations in leaves of all species but were not characterized. Anthochlors (chalkones) occur in leaves of H. heterophyllus and H. longifolius. Ray flower flavonoids include anthochlor and flavonol glycosides and occur in the basal region of the ligule producing a band of UV A around the head. Anthochlors are the predominant ray flower flavonoids in H. angustifolius, H. heterophyllus and H. longifolius, whereas they are absent and quercetin 7-glucoside is present in H. carnosus and H. floridanus. Cladistic analysis of flavonoid and morphological characters indicates that evolution in the series has been a radiation from ancestral types rather than a linear sequence of progressively more derived species.     

Flavonoids of five Hieracium species of British Columbia

Five Hieracium species were examined for flavonoid constituents: H. albertinum, H. albiflorum, H. cynoglossoides, H. gracile and H. umbellatum. The major compounds present in all species were mono- and diglycosides, of apigenin and luteolin. Chrysoeriol occurred per se in several species. Quercetin-3-O-glucoside was seen only in H. umbellatum. Species specific patterns of flavonoid glycosides were evident in all five taxa; H. albertinum and H. cynoglossoides showed the greatest similarity which reflects their morphological similarity. Limited examination of artificial hybrids showed additivity of flavonoid glycoside patterns.     

Contribution of ferrous iron to maintenance of the gastric colonization of Helicobacter pylori in miniature pigs

Our previous study showed that the colonization levels of Helicobacter pylori were higher in the stomachs of 5-day-old miniature pigs than in 2-week-old ones. As dietary factors can cause these differences, we compared two diets, i.e., Weanymilk and a similar formula with a higher concentration of Fe(II), Weanylobulin. The colonization levels in the fundic mucosa were significantly higher in 2-week-old pigs fed Weanylobulin than in those fed Weanymilk. Supplementing Weanylobulin with an iron chelator, deferoxamine mesylate, significantly lowered the bacteria counts in the gastric mucosa. Normal diets supplemented with Fe(II) in 2-month-old pigs caused significantly more sites of bacteria in the antrum compared with normal diets alone. In addition, ranitidine, an inhibitor of gastric acid secretion that reduces Fe(III) to Fe(II) in the stomach, decreased the bacteria counts in 10-month-old pigs. These results suggested that Fe(II) maintained the colonization levels of H. pylori in the stomach of the miniature pigs.     

Ultrastructural studies on experimental primary amoebic meningo-encephalitis (PAME) of mouse due to Naegler1a aerobia and Hartmannella culbertsoni

Primary amoebic meningo-encephalitis (PAME) in mouse can be easily produced by intranasal inoculation of Naegleria aerobia Singh & Das, 1970 and Hartmannella culbertsoni Singh & Das, 1970. The ultrastructural characteristics of N. aerobia and H. culbertsoni from infected mouse brain tissues showed many similar characteristics. The distinctive features between the two were the retention of phagocytic activity by N. aerobia as well as pinocytic activity in the ingestion of food as compared to H. culbertsoni where only pinocytic activity was observed. Staining characteristics of mitochondria suggested that N. aerobia was more active in the brain tissues than H. culbertsoni. Membrane-bound darkly stained bodies of unknown function were found in the brains infected with both N. aerobia and H. culbertsoni. They may have some bearing on the pathogenicity of these amoebae. Human cases of PAME so far reported are mostly due to N. aerobia and rarely with H. culbertsoni. This may be due to certain inherent characteristics present in N. aerobia which help them to survive on human brain tissue. On the other hand, the same may be lacking in H. culbertsoni. These ultrastructural differences in both N. aerobia and H. culbertsoni, as revealed by electron microscope studies of the infected brain tissues of mice, are presented in this paper.     

Settlement of the serpulid polychaete Hydroides elegans (Haswell) on the arborescent bryozoan Bugula neritina (L.): evidence of a chemically mediated relationship

The tube building polychaete Hydroides elegans Haswell was found living attached to colonies of the arborescent bryozoan Bugula neritina (L.) in Port Shelter, Hong Kong. Field data collected during the period of January through May 1996, showed that H. elegans density reached 77.6 individuals of H. elegans per g wet weight of B. neritina. Density of H. elegans on B. neritina at depths from the surface to 0.5 m was lower than that at depths below 1 m. In January–March, when there were no H. elegans settling on PVC plates or found on natural substrata, numbers on B. neritina were ca. 5 per g wet weight. H. elegans settled on B. neritina and grew rapidly as mean diameter of tubes increased from 605 μm in February to 936 μm in March. In laboratory experiments, larvae of H. elegans settled and metamorphosed on branches of B. neritina and on the bottom of dishes containing B. neritina leachate. Compounds extracted from the leachate of B. neritina induced 74% of H. elegans larvae to metamorphose at a concentration of 16 μg/ml seawater, compared to 5% in dishes containing only filtered seawater (controls). Metabolites from the leachate of B. neritina which were bound to amberlite XAD-2, indicating they are lipophilic in nature, induced over 70% metamorphosis in H. elegans larvae at 56 μg/ml seawater. A biofilm from one of four strains of bacterial isolates associated with the surface of B. neritina induced low levels of metamorphosis in H. elegans larvae, while other bacterial isolates were detrimental to the survival of juvenile H. elegans. Field experiments further demonstrated that H. elegans settled preferentially on Phytagel discs embedded with whole extracts of B. neritina over control Phytagel discs. Metabolites from B. neritina deterred feeding on alginate pellets by assemblages of local fishes in field assays. Metabolites originating from B. neritina, bacteria colonizing B. neritina, and the complex structure of B. neritina contributed to the recruitment of H. elegans to B. neritina surfaces. Hydroides elegans may gain a refuge from predation by associating with B. neritina colonies both from its structural and chemical attributes.     

Isolation and characterization of Histophilus somni (Haemophilus somnus) in semen samples of rams with epididymitis

Histophilus somni (Haemophilus somnus) has been reported as the cause of epididymitis in rams. This bacterium has also been found in the preputial mucosa of rams without epididymitis lesions. H. somni is a bacterium that is difficult to characterize, since it is a pleomorphic Gram-negative bacilli of characteristics similar to Actinobacillus seminis, which is also found in ram epididymitis lesions. The objective of this work was to determine if H. somni (H. somnus) is involved in cases of sheep epididymitis. A clinical examination was performed in 160 rams, extracting semen by electro-ejaculation of 28 of them, which had epididymal lesions. The penis was exteriorized in order to avoid prepuce contamination. The semen samples were cultivated in chocolate agar in a 10% CO₂ environment. Two strains were isolated in pure culture with a colony morphology and microscopy similar to H. somni (H. somnus). These were identified using the API 20 E system, using as a control the reference strain of H. somnus (2336ATCC). One of the isolates (129H) resulted identical to the reference strain and the other (827) presented differences in the arginine decarboxylase, H₂S, catalase and inositol reactions, although these differences have been reported (in strains isolated from different geographic origins, animal species and anatomical region). To characterize the isolates, an electrophoretic analysis of total proteins was performed (PAGE–SDS) finding identical profiles between the reference strain of H. somnus and isolate 129H and similar in relation to isolate 827. The amplification of a fragment of approximately 407 bp was observed in the 129H isolate and the ATCC strain, but not in 827. In other samples, isolations were made of Brucella ovis, Corynebacterium spp., Staphylococcus and other pleomorphic Gram-negative bacilli similar to A. seminis. Therefore, it has been confirmed that H. somni is present in the reproductive tract of rams and it could be involved in the presentation of ovine epididymitis. It is important that we underline that this is the first report of H. somni isolation in Mexico from ram semen samples.     

重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析*

目的:利用酿酒酵母表面展示技术筛选幽门螺杆菌候选疫苗,并分析其免疫原性。方法:以幽门螺杆菌的空泡型细胞毒素A(vacA)基因作为研究对象,构建重组S.cerevisiae EBY100/pYD1-VacA,通过Western blot、免疫荧光标记和流式细胞仪对S.cerevisiae EBY100/pYD1-VacA进行体外表达分析。以PBS和S.cerevisiae EBY100/pYD1为对照组,S.cerevisiae EBY100/pYD1-VacA为实验组,口服免疫SPF级BALB/c小鼠。通过ELISA分析检测口服免疫后小鼠抗VacA特异性IgG及分泌型IgA效价。结果:VacA抗原蛋白被成功地展示在S.cerevisiae EBY100表面。小鼠经口服免疫S.cerevisiae EBY100/pYD1-VacA后可诱导产生较高的VacA特异性抗体。结论:表面展示型酿酒酵母可以作为幽门螺杆菌候选疫苗的递送载体,与此同时,这也为开发其他细菌或病毒疫苗提供新思路。     

基于COI和ND5序列的中国南部唇鱼骨和间鱼骨群体遗传结构

唇鱼骨和间鱼骨均为分布较广的初级淡水鱼类,是理想的亲缘地理研究材料;且两者形态特征较为相似,不易鉴别,故两者的分布记述和物种有效性存在争议.为了解我国南部唇鱼骨和间鱼骨的群体遗传结构并探讨两者的物种有效性,本研究对8条水系的唇鱼骨和9条水系的间鱼骨共130尾个体的COI和ND5基因序列片段进行了测定,并对这两个基因的组合序列(2151 bp)进行了分析.结果表明: 在130尾个体的COI和ND5基因组合序列中,共有196个核苷酸变异位点,共检测出50个单倍型,单倍型多样性为0.964,核苷酸多样性为0.019,遗传多样性较高.基于COI和ND5基因组合序列构建的 NJ 树显示,所有种群可分为两支,支系Ⅰ包含了韩江和九龙江的全部单倍型以及瓯江的部分单倍型,余下的单倍型组成了支系Ⅱ.两支系间的遗传距离为0.036,而唇鱼骨与间鱼骨之间的遗传距离为0.027.单倍型网络图表明,韩江、九龙江种群和其他水系种群分化较大;漠阳江种群由海南岛种群扩散而来;海南岛各种群及漠阳江种群的单倍型分支与珠江水系单倍型的分支之间的亲缘关系较近,与长江水系单倍型分支之间的亲缘关系则较远;湘江、桂江和柳江之间的亲缘关系较近.AMOVA分析结果显示,地理区之间的变异约占71.2%,地理区内种群间变异约占16.6%,种群内的变异占12.2%,表明其遗传分化主要来自地理区之间.错配分析及中性检验结果显示,全部种群、唇鱼骨种群、间鱼骨种群、支系Ⅰ和支系Ⅱ在历史上均没有发生过明显的扩张.     

Encapsulation of Haematococcus pluvialis using chitosan for astaxanthin stability enhancement

Astaxanthin is receiving commercial interest due to its use as a preferred pigment in aquaculture feeds. Its antioxidant activity is approximately 100 times higher than that of β-tocopherol, and can be used as a potential agent against cancer. Astaxanthin can easily be degraded by thermal or oxidative processes during the manufacture and storage. In this study, astaxanthin and its biological activity were protected against oxidative environmental conditions by encapsulating the homogenized cells in chitosan. Haematococcus pluvialis were formed into beads, which were then coated with 5 layers of chitosan film, resulting in chitosan-algae capsules that have a mean diameter of 0.43 cm and the total film thickness of approximately 100 μm. No significant loss in the amount of astaxanthin content in H. pluvialis was found due to the process of encapsulation. However, approximately 3% loss of antioxidant activity of the H. pluvialis was observed after encapsulation. The results of stability under different storage conditions showed that although encapsulation caused 3% loss of antioxidant activity, the longer term stability of the dried algae biomass, beads, and capsules indicated that encapsulation of H. pluvailis in chitosan film was capable of protecting the algae cells from oxidative stresses.