产单核细胞李斯特菌减毒候选菌株分子生物学特性研究

目的为了确定用于减毒的产单核细胞李斯特菌菌株,对初筛入选的血清型为1/2a菌株Lm1、Lm2、Lm3和Lm4的分子生物学特性进行了比较研究。方法利用PCR技术均成功地扩增出4株菌株的hly基因及actA和plcB基因片段。以LD50的测定试验作为菌株毒力指征,对4株菌株的毒力进行了测定。结果4株菌株均为强毒菌株,其中菌株Lm4的毒力最强,用BALB/c小鼠测得的LD50为1.47×104CFU。结论确定以Lm4作为候选菌株。     

用PCR检测六类食品中单核细胞增生李斯特氏菌

目的　调查福建省食品中李斯特氏菌的污染状况。方法　用GB4789 30 - 94单核细胞增生李斯特氏菌 (Listeriamonocytogenes简称Lm)的检验方法分离菌株 ,用PCR检测李斯特氏菌的两种致病因子。结果　从福建采集 2 6 5份样品中分离出 17株李斯特氏菌 ,其中 ,2株Lm含有李氏溶血素O (ListerialysinO ,Hly)和内化素基因 (Internalin Inl) ,15株英诺克李斯特氏菌 (Listeria innocua)中 8株含有内化素基因 ,7株两种基因均为阴性。结论　从生鸡肉和生牛肉检出 2株Lm ,表明我省存在李斯特氏菌病的可能性 ,有必要进行深入研究。     

单核细胞增生性李斯特菌PCR快速检测方法建立及应用

目的　通过扩增hly基因PCR法建立快速检测单核细胞增生性李斯特氏菌 (Lm )方法。 方法 :用PCR法扩增hly基因来检测标准菌株及临床分离的Lm的纯培养物和模拟污染的生猪肉、水和牛奶 ,并应用于实际食品检验工作中进行验证。结果　 34株Lm的PCR结果呈阳性 ,而其它李斯特菌 ,包括英诺克李斯特菌、绵羊李斯特菌、西尔李斯特菌、威儿李斯特菌和格氏李斯特菌以及非李斯特菌均未出现特异性的片段。表明所扩增的hly基因 3’末段 82 7bp的DNA片段对Lm具有较高的特异性。PCR法对Lm纯培养物的检测限达 10 5CFU/ml,对模拟污染生猪肉、水和牛奶的 30℃ 16h改良LEB增菌培养液用PCR法检测 ,结果检测限达 10CFU/ 2 5 g(ml)。进一步研究表明该PCR扩增体系能检测出 6 .5pg的LmDNA ,整个检测过程只需 2 4h。采用该法对扬州市区 16 9份食品样品检测 ,8份样品Lm呈阳性且结果与传统的生化鉴定方法完全一致。结论　扩增hly基因的PCR法对Lm的鉴定具有特异性强、灵敏度高、速度快和易操作等优点 ,可用于食品的Lm的分离     

4b型单核细胞增生李斯特菌内化素基因NTSN_0462的功能初探

目的单核细胞增生性李斯特菌(Listeria monocytogenes,Lm)对宿主的粘附、侵袭作用与其多种内化素蛋白密切相关。本研究以4b型菌株LmNTSN的內化素基因NTSN_0462为研究对象,初步探究其致病作用。方法利用同源重组技术构建该基因缺失的突变株,研究0462基因对NTSN在生长、细胞侵袭和体内定植中的作用。人结肠腺癌细胞Caco-2、人肝癌细胞HepG2的侵袭率,以及BALB/c小鼠体内肝、脾脏中定植能力的差异。结果缺失株的生长曲线结果显示,NTSN_0462基因对Lm在BHI培养基中的生长及代谢未造成明显影响。以人结肠腺癌细胞Caco-2、人肝癌细胞HepG2进行的体外试验表明,缺失株的侵袭能力低于野生株(P0.001)。以BALB/c小鼠进行的体内试验显示,缺失株在肝脏中定殖的能力显著低于野生株(P0.001),在脾脏中无统计学差异。结论 NTSN_0462基因在LmNTSN入侵肝脏和定植中发挥重要作用,是侵袭相关的重要毒力因子。     

3例单增李斯特菌感染的病原学、临床及流行病学特征分析北大核心CSCD

目的分析四川省自贡市2014年3例单增李斯特菌感染的临床特征及其病原学特征。方法搜集临床病例信息;采用血清分型,脉冲场凝胶电泳技术和多位点序列分型方法对单增李斯特菌进行分子分型。结果 3例患者均为免疫力低下人群,有发热和败血症症状;3株单增李斯特菌中2株为1/2a血清型、ST8型,1株为1/2b血清型、ST778型,3株PFGE带型均不同。结论免疫力低下人群易受单增李斯特菌侵袭性感染。通过与国内外食品及病人单增李斯特菌相关流行学资料比较,提示国外流行克隆群以及国内某些流行型菌株可引起国内李斯特菌病散发,甚至成为将来暴发的隐患。     

1例产后哺乳期腹泻患者分离单增李斯特菌的生物学特征分析

目的 分析1例单增李斯特菌所致产后哺乳期腹泻患者的临床特征、病原学、菌株的毒力基因携带及分子特征。方法 搜集病例的临床相关信息;采用选择性增菌和显色培养基分离单增李斯特菌,用VITEK- 2 COMPACT30 全自动细菌分析仪鉴定分离菌株,采用单增李斯特菌诊断血清对分离株进行血清分型,使用脉冲场凝胶电泳技术及多位点序列分型技术进行分子分型,应用PCR检测毒力基因,并用E-test方法检测分离菌株对5种抗生素的药物敏感性。结果 产后哺乳期患者食用甜瓜和冰箱冷藏的酸奶后引起不能自限的腹泻,检测结果证实病原为4b血清型、ST145序列型单增李斯特菌。分离菌株携带毒力基因plcB、act、hly、iap、prfA、inlA,对青霉素、氨苄西林、美罗培南、红霉素和复方磺胺均敏感。结论 单增李斯特菌引起的腹泻患者容易漏诊,加强孕妇等高危人群腹泻患者粪便标本中单增李斯特菌的检测,对于单增李斯特菌病病人的及时治疗,预防不良预后具有重要的公共卫生意义,尤其对这一特殊型别单增李斯特菌在我国引起的病例感染应引起重视,其可能的感染来源和致病风险值得进一步的研究。     

3例单增李斯特菌感染的病原学、临床及流行病学特征分析

目的 分析四川省自贡市2014年3例单增李斯特菌感染的临床特征及其病原学特征。方法 搜集临床病例信息;采用血清分型,脉冲场凝胶电泳技术和多位点序列分型方法对单增李斯特菌进行分子分型。结果 3例患者均为免疫力低下人群,有发热和败血症症状;3株单增李斯特菌中2株为1/2a血清型、ST8型,1株为1/2b血清型、ST778型,3株PFGE带型均不同。结论 免疫力低下人群易受单增李斯特菌侵袭性感染。通过与国内外食品及病人单增李斯特菌相关流行学资料比较,提示国外流行克隆群以及国内某些流行型菌株可引起国内李斯特菌病散发,甚至成为将来暴发的隐患。     

合肥市市售猪肉产单核细胞李斯特菌分离及hly基因序列分析

目的了解市售猪肉中产单核细胞李斯特菌(Lm)的污染状况及Lm分离菌株hly基因部分序列的分析比较。方法菌株分离鉴定应用多聚酶链反应(PCR);PCR产物直接测序,对780bp片段作序列分析。结果市售猪肉产单核细胞李斯特菌的污染率为1.17%(2/170);分离菌株WLD1和FCY1与国内外14个参考菌株的同源性分别为96.4～98.6%和95.0%～97.2%,而两者之间的同源性仅为95.9%。结论合肥市市售猪肉中有一定程度产单核细胞李斯特菌的污染,且存在李斯特菌病的可能性和食物中毒的潜在危险;分离菌株WLD1和FCY1,尤其是FCY1分化程度较高,Lmhly基因核苷酸序列差异的距离与地理分布、菌株的样品来源无一定的相关性。     

鉴定单增李斯特菌4b血清型亚型的多重PCR方法的建立

目的利用多重PCR（multiplex PCR）技术,建立针对单增李斯特菌4b血清型亚型（ECI、ECII、ECIV,non-EC）的鉴定方法。方法以LMOf2365—2798、LMOh7858—0487、0RF2110和gtcA作为靶基因设计引物,建立同时鉴别ECI、ECII、ECIV、non-EC的多重PCR反应体系,并对16株4b血清型单增李斯特菌进行亚型鉴定。结果16株4b血清型单增李斯特菌可用本方法进行分型：其中ECI亚型11株,ECII亚型1株,ECIV亚型2株,非流行克隆群（non—EC）2株。结论本方法能准确鉴定单增李斯特菌4b血清型菌株的4个亚型,可用于食品、临床标本的病原学诊断和疫情暴发的溯源调查。     

中国单增李斯特菌的基因组特征研究

目的 分析我国146株单增李斯特菌的基因组特征,了解不同家系和克隆群菌株遗传特征的差异。方法 从NCBI数据库收集我国146株不同来源、省份和分离时间的单增李斯特菌基因组数据,利用生物信息学分析软件分别对其进行基因组注释、系统发育树构建和遗传元件分析。结果 本研究中相同家系、血清型和克隆群的单增李斯特菌在系统发育进化树上聚在一起,这种分布与菌株的来源和分离省份无关,提示单增李斯特菌基因组结构具有一定的稳定性。单增李斯特菌均携带毒力岛LIPI-1,部分家系Ⅰ菌株(如CC3和CC87)携带毒力岛LIPI-3和LIPI-4,具有潜在的致病风险。大多数家系Ⅱ菌株携带质粒和环境抗性基因,有助于提高对不良环境的耐受力。食品来源的菌株中inlA基因提前终止突变发生率较高,可能减弱菌株的毒力。结论 我国单增李斯特菌主要以家系Ⅰ和家系Ⅱ菌株为主,两者在毒力基因、环境抗性基因和质粒携带方面均具有差异,显示不同亚型菌株的毒力和环境适应性存在差异,为我国单增李斯特菌的监测和李斯特菌病的防控提供了参考数据。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.