康乐霉素A体外抗结核活性的初步研究

目的发现新安莎类抗生素,康乐霉素A体外抗结核活性。方法应用色谱技术从地中海诺卡氏菌康乐变株1747-64发酵液中分离康乐霉素A,并采用试管二倍稀释法,进行康乐霉素A对耻垢分枝杆菌（ATCC14468）,结核分枝杆菌H₃₇Rv（ATCC27294）,H₃₇Ra（ATCC25177）,临床分离的氧氟沙星（OFLX）敏感结核分枝杆菌以及临床分离的耐氧氟沙星结核分枝杆菌最低抑制浓度（Minimal Inhibitor Concentration,MIC）的试验。结果康乐霉素A对耻垢分枝杆菌活性弱,MIC为64μg/ml,对结核分枝杆菌H₃₇Rv,H₃₇Ra的MIC为8μg/ml,对临床分离的氧氟沙星敏感结核分枝杆菌菌株的MIC范围为18μg/ml,临床分离的耐氧氟沙星结核分枝杆菌菌株的MIC范围为116μg/ml。结论新安莎类抗生素,康乐霉素A体外具有明显的抗结核活性,有望成为新抗结核药物或先导化合物。     

Rv1508c调节和促进分枝杆菌对链霉素药物敏感性的研究北大核心CSCD

目的通过检测抗结核药物-利福平、异烟肼以及链霉素对结核分枝杆菌RD区(Region of Differences,RD)基因的IC50(half maximal inhibitory concentration,IC50)值,筛选对3种抗结核药物敏感或耐药的毒性结核分枝杆菌RD区基因。方法将结核分枝杆菌RD6、RD9和RD15区基因与穿梭质粒pMV261连接,连接产物电转化耻垢分枝杆菌中。分别将对数生长期的耻垢分枝杆菌以及重组耻垢分枝杆菌中加入浓度倍比稀释的异烟肼(0.031 25~4μg/ml)、利福平(0.031 25~4μg/ml)和链霉素(0.031 25~4μg/ml)100μl,37℃培养2d后,用酶标仪测A600值。用Graphpad Prism5计算IC50值并进行统计学分析。结果成功构建RD6、RD9和RD15区基因重组质粒,电转化入耻垢分枝杆菌后通过检测利福平、异烟肼以及链霉素对以上RD区基因的IC50值,成功筛选出对链霉素敏感的RD6区基因Rv1508c。同时对Rv1508c在分枝杆菌(毒性结核分枝杆菌H37Rv、减毒结核分枝杆菌H37Ra、BCG、耻垢分枝杆菌、鸟分枝杆菌、胞内分枝杆菌以及海洋分枝杆菌)中的分布做了鉴定,确定其只存在于H37Rv和H37Ra中。结论结核分枝杆菌RD6区基因Rv1508c能促进分枝杆菌对链霉素的敏感,使链霉素杀伤分枝杆菌作用增强,Rv1508c作为链霉素一个靶点,在今后研究中,Rv1508c可作为抗结核药物增敏剂的应用。     

Rv1508c调节和促进分枝杆菌对链霉素药物敏感性的研究

目的通过检测抗结核药物-利福平、异烟肼以及链霉素对结核分枝杆菌RD区(Region of Differences,RD)基因的IC50(half maximal inhibitory concentration,IC50)值,筛选对3种抗结核药物敏感或耐药的毒性结核分枝杆菌RD区基因。方法将结核分枝杆菌RD6、RD9和RD15区基因与穿梭质粒pMV261连接,连接产物电转化耻垢分枝杆菌中。分别将对数生长期的耻垢分枝杆菌以及重组耻垢分枝杆菌中加入浓度倍比稀释的异烟肼(0.031 25~4μg/ml)、利福平(0.031 25~4μg/ml)和链霉素(0.031 25~4μg/ml)100μl,37℃培养2d后,用酶标仪测A600值。用Graphpad Prism5计算IC50值并进行统计学分析。结果成功构建RD6、RD9和RD15区基因重组质粒,电转化入耻垢分枝杆菌后通过检测利福平、异烟肼以及链霉素对以上RD区基因的IC50值,成功筛选出对链霉素敏感的RD6区基因Rv1508c。同时对Rv1508c在分枝杆菌(毒性结核分枝杆菌H37Rv、减毒结核分枝杆菌H37Ra、BCG、耻垢分枝杆菌、鸟分枝杆菌、胞内分枝杆菌以及海洋分枝杆菌)中的分布做了鉴定,确定其只存在于H37Rv和H37Ra中。结论结核分枝杆菌RD6区基因Rv1508c能促进分枝杆菌对链霉素的敏感,使链霉素杀伤分枝杆菌作用增强,Rv1508c作为链霉素一个靶点,在今后研究中,Rv1508c可作为抗结核药物增敏剂的应用。     

单链构象多态性与核酸杂交联合检测耐氧氟沙星的结核分枝杆菌

目的探讨聚合酶链反应-单链构象多态性分析（PCR-SSCP）并Southern杂交检测耐氧氟沙星（OFLX）结核分枝杆菌的可行性，并探索结核分枝杆菌对OFLX敏感与耐药的界限。方法（1）体外筛选人工诱变的耐OFLX的耐药突变株，并检测OFLX对这些耐药株的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。（2）扩增结核分枝杆菌的gyrA基因，包括H37 Rv1株、H37 Ra 1株、临床分离的OFLX敏感株10株及人工诱变的耐OFLX H37 Rv（17株）、H37Ra（17株）及临床分离菌株（51株）共计85株，并对此扩增产物进行SSCP分析及Southern杂交，对比观察耐OFLX结核分枝杆菌的核酸单链条带位移的变化。为了验证PCR-SSCP并Southem杂交结果的可重复性，对36株临床分离的耐OFLX菌株的耐药界限进行初步检测。结果经PCR-SSCP及Southern杂交，85株人工诱变的耐OFLX菌株，全部检测到gyrA基因突变。在OFLX 10μg／ml这一点上，明显地存在着一个结核分枝杆菌对OFLX敏感与耐药并存的交叉区。对36株临床分离的OFLX耐药菌的PCR-SSCP合并Southem杂交检测结果显示，对比标准株H37Ra，在临床分离的耐OFLX菌株间观察到了类似的因单链构象变化而引起的单链位移的结果。结论PCR-SSCP合并Southern杂交可以清晰地区分对OFLX敏感与耐药的菌株，以100μg／m1为界作为判别结核分枝杆菌对OFLX是否已经产生耐药的标准是适宜的。     

耐氧氟沙星的结核分枝杆菌临床分离株的gyrA基因点突变特点

目的研究结核分枝杆菌临床分离株的gyrA基因点突变位点和突变频率,了解目前结核分枝杆菌临床分离株耐喹诺酮类药物的基本特点。方法以我室保藏H_(37)Rv(ATCC272294),H37Ra (ATCC251 77)菌株以及由我所参比实验室提供的具有在改良罗氏培养基上获取的、对氧氟沙星     

耻垢分枝杆菌中DNA损伤与SOS反应的实验研究

目的探讨基因毒性物质介导的DNA损伤引起分枝杆菌SOS反应的作用。方法选择耻垢分枝杆菌为分枝杆菌模式细菌,通过紫外线损伤试验检测耻垢分枝杆菌对紫外线的敏感性,通过2倍稀释法测定利福平和氧氟沙星的最小抑菌浓度（MIC）。分别用紫外线和次抑菌浓度（1/2MIC）抗生素处理耻垢分枝杆菌,在不同时间点收集菌液,以看家基因sigA为内参,采用实时定量PCR相对定量方法检测dnaE2、recA和recX等SOS基因表达水平的变化。结果紫外线暴露45s后耻垢分枝杆菌的存活率为50%,利福平和氧氟沙星的MIC分别是32μg/ml和1μg/ml。耻垢分枝杆菌dnaE2和recX等SOS基因表达水平在紫外线下暴露45s后即开始上升,recA的表达水平在紫外线暴露3h后显著上升。耻垢分枝杆菌dnaE2和recX基因表达水平在1/2MIC的利福平作用后1h或在1/2MIC的氧氟沙星作用0h后上升,recA的表达水平在1/2MIC的利福平或氧氟沙星作用3h时显著升高。结论紫外线、抗生素等基因毒性物质可以导致耻垢分枝杆菌DNA损伤及SOS基因表达水平上升。     

PCR-SSOP并Southern杂交检测实验室筛选耐氧氟沙星结核分枝杆菌的研究

目的 探讨PCR-SSCP并Southern杂交检测耐氧氟沙星(OFLX)结核分枝杆菌的可行性，并探索结核分枝杆菌对OFLX敏感与耐药的界限。方法 ①体外诱导筛选耐OFLX的自然突变株，并检测OFLX对这些耐药株的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)；②扩增结核分枝杆菌的gyrA基因(包括H37Rv、H37Ra、临床分离的OFLX敏感株及体外诱导筛选的耐OFLX的菌株)并对此扩增产物进行单链构象多态性(SSCP)分析及Southern杂交，以判定应用该技术的效果。结果经PCR-SSCP及Southern杂交检测，85株体外诱导的自然耐OFLX菌株，均检测到gyrA基因突变。研究发现．在OFLX 10μg／ml这一点上，明显地存在着一个耐OFLX结核分枝杆菌对OFLX敏感与耐药并存的交叉区。结论PCR-SSCP合并Southern杂交可清晰地区分OFLN敏感与耐药株。本研究结果显示，以10μg／ml为界作，为判别耐OFLX的标准是适宜的。     

39株对克拉霉素耐药的结核分枝杆菌临床分离株23SrRNA检测结果分析

目的 分析对克拉霉素耐药的结核分枝杆菌临床分离株23S rRNA的A2058位点的变化。 方法 选择我院菌株库的结核分枝杆菌临床分离株64株,其中10株为对全部抗结核药物敏感的结核分枝杆菌临床分离株;14株为单耐克拉霉素的结核分枝杆菌临床分离株;15株为耐多药,同时耐克拉霉素的结核分枝杆菌临床分离株;15株为耐多药,同时对克拉霉素敏感的结核分枝杆菌临床分离株;10株为广泛耐药菌株,同时对克拉霉素耐药的结核分枝杆菌临床分离株;此外,还有结核分枝杆菌标准株H37Rv 1株。对结核分枝杆菌23S rRNA行PCR检测和测序。 结果 经检测,H37Rv标准株没有A2058突变,只有1株广泛耐药临床分离株检测有A2058A-G的突变,其他临床分离株均没有突变,在耐克拉霉素的结核分枝杆菌临床分离株中占2.56%（1/39）,在广泛耐药结核分枝杆菌临床分离株中占1/10。 结论 结核分枝杆菌临床分离株对克拉霉素耐药的机制中, A2058突变可能不是产生对克拉霉素耐药的主要机制。结核分枝杆菌产生对克拉霉素耐药的机制有待进一步研究。     

氯法齐明抗结核分枝杆菌的体内外活性研究

目的 评价氯法齐明(clofazimine,CLF)在体外及体内抗结核分枝杆菌的活性,为临床应用提供依据.方法 应用微孔板指示剂法,测定氯法齐明对结核分枝杆菌标准株H37Rv及临床分离耐多药结核分枝杆菌(30株)的MIC;建立小鼠静脉感染H37Rv的结核病模型(105 CFU/只),根据氯法齐明的不同剂量,分为3个治疗组:20 mg/kg,每周给药5次(CLF-1组);10 mg/kg,每周给约5次(CLF-2组);20 mg/kg,每周给药2次(CLF-3组);治疗30 d后进行肺、脾组织活菌计数,应用单冈素方差分析,比较氯法齐明在小鼠体内的抗结核分枝杆菌活性.结果 氯法齐明对结核分枝杆菌H37Rv的MIC为0.12～0.24 μg/ml;对30株耐多药结核分枝杆菌临床分离株的MIC为0.12～1.92 μg/ml.在小鼠结核病治疗模型中,3个治疗组的小鼠肺活菌计数分别较空白对照组降低2.92、1.78和1.39 lg CFU,均具有统计学意义(F=74.09,P＜0.01).结论 氯法齐明具有较好的体外及体内抗结核分枝杆菌活性,值得进一步研究.     

结核分枝杆菌对利福喷汀与利福平交叉耐药的实验研究

目的 观察利福喷汀对结核分枝杆菌的杀菌效力及其与利福平的交叉耐药性，探讨利福喷汀对结核分枝杆菌的实验室耐药界限，为临床应用利福喷汀治疗结核病，包括治疗对利福平耐药的结核病提供依据。方法 用7H9液体培养基、苏通液体培养基、改良罗氏固体培养基测定利福喷汀和利福平对结核分枝杆菌标准株(H37Rv)及临床分离的利福平敏感株、利福平耐药株的最低抑菌浓度(MIC)。结果 在7H9液体培养基上对临床分离的19株利福平敏感株分别测定利福平和利福喷汀的MIC，有80％利福平敏感株利福平的MIC≥0．32μg／ml，而利福喷汀的MIC多分布在0．02～0．32μg／ml之间(占84％)；无论标准株H37Rv、利福平敏感株(19株)或是利福平耐药株(45株)，利福喷汀对结核分枝杆菌的MIC普遍比利福平低2～4倍；利福喷汀敏感株和耐药株的MIC差别较大，在MIC为5～10μg／ml处能最大限度地区分敏感菌和耐药菌。结论 利福喷汀与利福平存在着交叉耐药，但利福喷汀比利福平有更强的杀菌效力，部分结核分枝杆菌对利福平达到临床耐药时，仍未达到利福喷汀临床耐药，对利福平耐药的部分结核分枝杆菌对利福喷汀仍具有一定程度的敏感性，提示临床上对利福平耐药的结核病患者使用利福喷汀可能有一定效果；实验结果对利福喷汀临界耐药界限进行了初步筛查，为利福喷汀常规药敏试验的开展提供了基本试验数据。     

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.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.