直肠接种卡介苗可诱导出与注射接种相似的免疫应答和保护作用

本文通过直肠途径接种卡介苗(BCG),并与肠胃外途径接种进行比较对结核分枝杆菌(结核杆菌)的免疫应答及保护作用.实验选用动物为BALB/c小鼠、远交系Hartley豚鼠和恒河猴;BCG为Pasteur株1173P2;攻击用菌种为结核杆菌H37Rv.直肠免疫小鼠、豚鼠及恒河猴时分别接种2×109、2×1010和6×1010活菌单位的BCG;皮下注射免疫小鼠时接种100 μl BCG(含108活菌单位);皮内注射免疫豚鼠和恒河猴时分别接种5×106和1×109活菌单位的BCG.在两条途径免疫后不同时间,取豚鼠和恒河猴脾细胞在96孔微孔板培养,用3H TdR掺入法测定对PPD刺激的增殖反应.     

氟康唑联合克霉唑阴道片治疗复发性外阴阴道念珠菌病临床观察

目的 探讨和分析氟康唑联合克霉唑栓阴道片治疗复发性外阴阴道念珠菌病的临床疗效.方法 选择复发性外阴阴道念珠菌病患者94例,随机分为实验组和对照组各47例.实验组分别于d1、d4口服氟康唑150 mg,同时克霉唑阴道片500 mg阴道用药;对照组于d1、d4仅用克霉唑阴道片500 mg阴道用药.两组均于月经期后重复用克霉唑阴道片500 mg单次,实验组同时口服氟康唑150 mg单次,连续使用6个月,治疗结束后1周、1个月、3个月各复查1次.结果 两组治疗后1周的总有效率无显著差异,实验组为93.62％,对照组为85.11％;但治疗后1个月实验组的复发率为0,而对照组的复发率为12.5％;治疗后3个月实验组复发率为2.27％,对照组为14.29％,两组在1个月及3个月复发率的比较均有显著差异.结论 氟康唑联合克霉唑阴道片对复发性外阴阴道念珠菌病的治疗和预防复发效果满意.     

豚鼠庆大霉素耳中毒后HSP70 mRNA的表达

为探讨热休克蛋白(heatshockprotein,HSP)70mRNA在庆大霉素(gentamicin,GM)耳中毒中的意义,本实验选用耳廓反射灵敏的健康白色红目豚鼠(200~250g)20只,雌雄不拘,随机分成两组,每组10只。实验组动物每日腹腔注射GM100mg/kg;对照组动物每日腹腔注射与GM等量的生理盐水2.5ml/kg。两组动物混合饲养,均连续用药10d。在用药前1天和停药后第1天进行听脑干反应(auditorybrainstemresponse,ABR)测试。各组豚鼠在行第二次ABR检测后,应用原位杂交及图像分析技术观察GM耳中毒后HSP70mRNA在豚鼠耳蜗中表达。结果显示:实验组耳蜗ABR阈值明显高于对照组,有显著性差异(P<0.01);实验组豚鼠耳蜗血管纹、螺旋韧带、螺旋神经节细胞HSP70mRNA表达呈强阳性,其平均灰度值较正常对照组明显减小(P<0.001),即GM能显著增强耳蜗HSP70mRNA的表达。结果提示,GM中毒后,动物可能通过增加HSP70mRNA在耳蜗的表达,起保护听力的作用。     

抗生素联合卡介苗治疗结核病的动物模型评价

目的用豚鼠模型初步评价抗生素联合卡介苗治疗结核病的可行性。方法用高剂量Mtb皮下攻击豚鼠2周后,将重组ESAT6-CFP10(EC)变态反应原皮试阳性的豚鼠随机分成4组:NS组、BCG组、抗生素组、抗生素+BCG组。抗生素+BCG组接受异烟肼(isoniazid,INH)和利福喷丁(rifapentine,RFT)的联合化疗,1次/周,共3次,给药结束后,每只豚鼠皮下免疫1/10人用剂量BCG;BCG组每只豚鼠仅皮下免疫1/10人用剂量BCG;抗生素组仅给药INH和RFT,1次/周,共3次;NS组给予生理盐水作为阴性对照。全部豚鼠于攻毒13周后安乐死解剖,评价肝、脾、肺的脏器综合病变指数,计算脾脏活菌载量(lg CFU),并对肝、脾、肺脏器做组织病理检查。结果 NS组、BCG组、抗生素组和抗生素+BCG组的脏器评分分别为83±8、81±10、45±28和33±14。其中,BCG组与NS组差异无统计学意义;抗生素组和抗生素+BCG组与NS组差异均有统计学意义(分别q=6.84,P0.001;q=9.02,P0.001),两组与BCG组比较,差异也均有统计学意义(q=6.44,P0.001;q=8.63,P0.001);但抗生素组与抗生素+BCG组差异无统计学意义。抗生素+BCG组豚鼠的脾脏活菌载量为(3.62±1.13)lg CFU,与NS组的(4.92±0.52)lg CFU和BCG组的(5.20±0.43)lg CFU比较,差异均有统计学意义(q=5.54,P0.01;q=6.72,P0.001)。抗生素组脾脏活菌载量为(4.39±0.50)lg CFU,与其他各组的差异均无统计学意义。病理组织切片显示各组病变程度由重到轻依次为BCG组NS组抗生素+BCG组≈抗生素组。结论化疗后免疫1针BCG的治疗效果较差,多针次的BCG免疫效果还需进一步研究。     

三种膀胱灌注方法对膀胱树突状细胞及尿IL-2的影响

目的:探讨联合灌注预防膀胱肿瘤复发的免疫机制.方法:将44只雌性wistat大白鼠随机分为生理盐水对照组以及BCG组、丝裂霉素组、IL-2联合BCG组,分别定期进行11次膀胱灌注并分批处死取出膀胱组织,用免疫组化的方法检测膀胱组织中DC的数量,用双抗体夹心酶免疫吸附法(ELISA)测定尿IL-2含量,观察膀胱组织DC及尿IL-2的变化.结果:DC平均数量:IL-2联合BCG组为(28.3±8.0 CDS/HPF)、BCG组为(16.6±5.9 CDS/HPF)、丝裂霉素组为(4±2.3 CDS/HPF)、对照组为(4.8±3.4CDS/HPF);尿IL-2浓度:IL-2联合BCG组为(87.5±30.4 ng/L)、BCG组为(58.7±20 ng/L)、丝裂霉素组为(35.9±9.6ng/L)、对照组为(35.7±11.3 ng/L).结论:IL-2和BCG两者间有免疫促进和协同作用.     

灵芝孢子油长期毒性试验研究

目的:观察灵芝孢子油对大鼠产生的长期毒性反应和严重程度,确定本品安全用药剂量。方法:按1.67g/kg、0.83g/kg和0.42g/kg三个剂量,连续灌胃给药4个月及停药1个月,观察测试大鼠体重、血液学、血液生化学、脏器系数及病理组织学变化。结果:大鼠体重减轻,高剂量组ALP、TG值升高,高、中剂量组MCH、CREA值升高。结论:灵芝孢子油长期用药的安全剂量建议为每日用量不大于0.42g/kg。     

环孢素A治疗系统性红斑狼疮并血小板减少临床分析

入住我院2006年8月至2009年1月确诊为系统性红斑狼疮(SLE)的10例患者,均伴有血小板(PLT)明显减少,维持在(20~50)×109/L至少3月以上。给予泼尼松0.5 mg/(kg.d)及环孢素A150 mg/d治疗,3个月后100 mg/d疗程半年,观察患者临床表现,每个月检测SLE疾病活动指数(SLEDAI)、抗ds-DNA、尿蛋白、补体C3、红细胞沉降率(血沉)、C-反应蛋白(CRP)、肝功能、肾功能、血常规等,比较患者治疗前后各项指标的变化。结果显示治疗半年后患者血小板明显升高(P0.05),抗ds-DNA转阴、补体C3上升,红细胞沉降率(血沉)、C-反应蛋白较治疗前明显下降(P0.05)。提示泼尼松联合环孢素A治疗SLE合并顽固性血小板减少的疗效明显,副作用小。     

肝移植术后早期暂时性停用CSA或FK506的个体化治疗体会

目的:总结肝移植术后早期根据不同的个体及不同的病情采用个体化免疫抑制剂治疗的经验.方法:除术后早期3例因非排斥反应原因死亡外,回顾性分析39例肝移植患者术后早期3个月内有停用免疫抑制剂3天以上者与无停用免疫抑制剂者的排斥反应发生及预后情况,即不同个体在不同病情采用不同免疫抑制方法的效果.结果:39例受体中共有16例成功停用CSA或FK506平均5.6天,与24例未停药组比较,两组病人一般特征无差异,但ASA评分呈显著差异,两组排斥反应及其他并发症无明显差异(P=0.23).随访2-6年,术后随访平均3年存活的19例中,最低CSA用量为2 mg/kg/d,最低FK506为0.02 mg/kg/d,一般FK506用量为0.04-0.06 mg/kg/d.结论:对于术后早期合并肾功能不全、严重感染、药物中毒等受体载时性停用CSA或FK506有利于受体的恢复,早期治疗中应根据受体免疫状态、术后不同时期、合并的特殊病情及对各种免疫抑制剂的个体差异等来决定免疫抑制剂使用方案及用量,有效控制排斥反应、毒副作用及相关并发症,提高移植肝存活率.     

卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响

目的:探讨卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响。方法:取20只豚鼠随机分为2组,实验组连续14d肌肉注射硫酸卡那霉素,200mg/(kg.d),对照组连续14d等量肌肉注射生理盐水,停药14d处死动物后制作耳蜗标本,处死前检测其ABR的变化,免疫组化及原位杂交法测定Bcl-2的表达。结果:豚鼠卡那霉素耳慢性中毒后,ABR阈值较对照组明显上升,Bcl-2阳性表达减低,与对照组比较差异有显著性(P<0.05)。结论:卡那霉素耳慢性损害可能与抑制Bcl-2的表达有关。     

BCG—PPD的制备及应用

用三氯醋酸(TCA)和硫酸铵(AS)综合法,由卡介菌苗(BCG)培养滤液中提纯制得卡介菌素纯蛋白衍生物(BCG—PPD)。BCG—PPD的纯度和结核菌素纯蛋白衍生物国际标准(PPD-S)及中国标准(PPD—C)相近,高于加拿大标准(PPD—CT68)和丹麦标准(PPD—RT23)。在BCG免疫豚鼠中,BCG—PPD的皮肤迟发型变态反应(DTH)大于结核菌素纯蛋白衍生物(PPD)的DTH反应。在结核菌感染豚鼠组中,BCG—PPD的DTH反应小于PPD的DTH反应.在检查333名新生儿接种BCG12周后的免疫     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Intranasal Mucosal Boosting with an Adenovirus-Vectored Vaccine Markedly Enhances the Protection of BCG-Primed Guinea Pigs against Pulmonary Tuberculosis

Background

Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. However, Ad TB vaccine remains to be evaluated in a more relevant and sensitive guinea pig model of pulmonary TB. Many vaccine candidates shown to be effective in murine models have subsequently failed to pass the test in guinea pig models.

Methods and Findings

Specific pathogen-free guinea pigs were immunized with BCG, AdAg85A intranasally (i.n), AdAg85A intramuscularly (i.m), BCG boosted with AdAg85A i.n, BCG boosted with AdAg85A i.m, or treated only with saline. The animals were then infected by a low-dose aerosol of M. tuberculosis (M.tb). At the specified times, the animals were sacrificed and the levels of infection in the lung and spleen were assessed. In separate studies, the long-term disease outcome of infected animals was monitored until the termination of this study. Immunization with Ad vaccine alone had minimal beneficial effects. Immunization with BCG alone and BCG prime-Ad vaccine boost regimens significantly reduced the level of M.tb infection in the tissues to a similar extent. However, while BCG alone prolonged the survival of infected guinea pigs, the majority of BCG-immunized animals succumbed by 53 weeks post-M.tb challenge. In contrast, intranasal or intramuscular Ad vaccine boosting of BCG-primed animals markedly improved the survival rate with 60% of BCG/Ad i.n- and 40% of BCG/Ad i.m-immunized guinea pigs still surviving by 74 weeks post-aerosol challenge.

Conclusions

Boosting, particularly via the intranasal mucosal route, with AdAg85A vaccine is able to significantly enhance the long-term survival of BCG-primed guinea pigs following pulmonary M.tb challenge. Our results thus support further evaluation of this viral-vectored TB vaccine in clinical trials.     

Delayed hypersensitivity and nonspecific cellular immunity. The role of mono- and polynuclear phagocytes

Differences in the influence produced by sensitization with BCG vaccine and Staphylococcus aureus and by the reaction of delayed hypersensitivity (DH) induced, respectively, by the injection of old tuberculin and staphylococcal phagolysate on the phagocytic activity of peritoneal macrophages and blood leukocytes in different animals were experimentally demonstrated. A considerable activation of the bactericidal and ingesting functions of macrophages was observed in animals showing a pronounced DH reaction (rabbits, guinea pigs and mice), while in Wistar rats no such activation was noted. The latter showed no DH reaction after sensitization with BCG vaccine and the injection of the specific antigen. Among different strains of mice, the activation of macrophages occurred in the animals with the most pronounced DH reaction. Sensitization with BCG vaccine led to an insignificant sensitization of macrophages, and sensitization with S. aureus even suppressed the phagocytic activity of macrophages. The treatment of mice with antimacrophagal preparations (carrageenan, silica and trypan blue, but T-lymphocyte antiserum) before and after the injection of the specific antigen into the sensitized animals abolished the stimulation of anti-infection immunity.     

Sustained QT prolongation induced by tacrolimus in guinea pigs.

Recently, clinical cases have been reported of QT prolongation and torsades de pointes associated with the use of tacrolimus (FK506). We examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs in order to evaluate the arrhythmogenicity of FK506 in comparison with quinidine (QND). FK506 (0.1 or 0.01 mg/hr/kg) or QND (30 mg/hr/kg) was intravenously infused to guinea pigs and time profiles of drug concentration in blood and QTc interval were examined during and after infusion. Both FK506 and QND evoked a significant QTc prolongation, and the dose-response relationship showed an anti-clockwise hysteresis, FK506-induced QTc prolongation persisted throughout the duration of the experiment despite a decline in the plasma FK506 concentration, whilst QND-induced QTc prolongation disappeared as plasma concentrations decreased. FK506 induced a sustained QTc prolongation in guinea pigs at drug concentrations in blood that correspond to its therapeutic range in human, suggesting that it might be of clinical significance to monitor the electrocardiogram, especially when patients have congenital or acquired QT-prolonging risk factors.     

The immunopathological effects of intracolonic injection of Mycobacterium bovis BCG cell wall emulsions in guinea pigs

Summary The immunological and pathological responses of guinea pigs to an intramural colonic injection of emulsions containing cell wall (CW) extracts of Mycobacterium bovis BCG and mineral oil were studied from week 1 to week 36 post-inoculation. The emulsions contained variable concentrations of BCG CW attached to (lipid-phase), or separate from (aqueous-phase), the mineral oil. Delayed cutaneous hypersensitivity to PPD was present throughout the course of the study in a variable percentage of guinea pigs inoculated with either type of emulsion. PPD-induced blast transformation of peripheral blood lymphocytes, studied in guinea pigs which received lipid-phase emulsion, was also detectable throughout the course of the study, with maximal response seen 2 weeks post-inoculation. The intracolonic inoculations were well tolerated, with the exception of the most concentrated lipid-phase emulsion (3 mg/ml BCG CW and 5% oil), after which one of eight guinea pigs died due to a colonic impaction and rupture at the site of inoculation. The pathological response to either type of emulsion was a focal granulomatous colitis, which tended to be more severe as the concentration of BCG CW and oil increased. Extracolonic lesions were usually limited to a granulomatous lymphadenitis of lymph nodes draining the injection site; however, the most concentrated lipid-phase emulsion occasionally produced granulomatous inflammatory foci in the liver and lungs. In general, the lesions induced by the lipid-phase emulsions were more severe than those induced by aqueous-phase emulsions, but the intensity of both types of lesions peaked at 2 or 4 weeks post-inoculation. It was concluded that the guinea pig may serve as a useful model to study BCG immunotherapy of colonic neoplasms, since intracolonic injection of BCG CW resulted in systemic immunity toward mycobacterial antigens and a localized accumulation of macrophages without untoward complications. The abbreviations used in this paper are: BCG CW, bacillus Calmette-Guérin cell walls; PPD, purified protein derivative; PBL, peripheral blood lymphocytes; cpm, counts per minute; SI, stimulation index; DCH, delayed cutaneous hypersensitivity     

Effect of a peptide leukotriene antagonist, ONO-1078 on antigen-induced airway microvascular leakage in actively sensitized guinea pigs.

We examined the effect of ONO-1078, a peptide leukotriene antagonist, on antigen-induced airway microvascular leakage in ovalbumin-sensitized guinea pigs. When guinea pigs were pretreated with mepyramine, ovalbumin challenge increased vascular permeability to Evans blue dye in trachea, main bronchi and intrapulmonary airways. Oral administration of ONO-1078 significantly reduced microvascular leakage in intrapulmonary airways at doses more than 3 mg/kg, but not in trachea. Moreover, oral administration of ONO-1078 significantly reduced SRS-A mediated microvascular leakage into all airway tissues and was more effective in intrapulmonary airways at 3 mg/kg. Simultaneously, ONO-1078 also inhibited SRS-A mediated bronchoconstriction. On the other hand, azelastine (10 mg/kg, p.o.), an anti-asthma agent, failed to inhibit microvascular leakage into the airways. These results suggest that peptide leukotrienes may be important mediators of airway microvascular leakage, and that the inhibitory effect of ONO-1078 on antigen-induced airway microvascular leakage in addition to the blockade of bronchoconstriction may have therapeutic implications for bronchial asthma.     

Influence of body temperature on histamine-induced bronchoconstriction in guinea pigs

The effects of body temperature on histamine-induced bronchoconstriction were investigated in anesthetized, paralyzed, and mechanically ventilated guinea pigs. Four groups of guinea pigs were studied with constant body temperatures of 40, 38, 35, and 32 degrees C, respectively. Histamine was infused for 5 min at a rate of 50 ng.kg-1.s-1. Body cooling from 40 to 32 degrees C augmented the bronchomotor responses to histamine, which eventually rose almost fourfold. The enhancement of histamine-induced bronchoconstriction induced by body cooling was not suppressed by pretreating guinea pigs with 5 mg/kg hexamethonium or 5 mg/kg hexamethonium plus 3 mg/kg atropine; neither was the enhancement of histamine-induced bronchoconstriction suppressed in pithed guinea pigs, demonstrating that the autonomic nervous system is not involved in potentiating bronchoconstriction at low body temperatures. These results suggest that, at low body temperatures, increased airway responsiveness to histamine may be because of some direct effect of temperature on bronchial airway smooth muscle.     

Comparison of bcg strains for delayed hypersensitivity induction tested in vivo in guinea pigs and in vitro

Previous studies performed on guinea pigs demonstrated a direct dependence of tuberculin reaction size (in vivo hypersensitivity) on immunogenicity in a number of BCG strains. The present work used an in vitro method, MIF detection, for assessing hypersensitivity and compared the results obtained with tuberculin hypersensitivity tests, correlating the data with the immunogenicity of the individual BCG strains employed. The following strains were used: the Czechoslovak BCG strain No. 725, Japanese BCG strain Tokyo, Danish BCG strain Copenhagen and Soviet BCG strain Moscow. The results obtained by the two hypersensitivity testing methods, in vivo and in vitro were in a direct correlation; a direct relationship was also demonstrated between hypersensitivity tested by the in vitro method and immunogenicity. The in vitro method of MIF detection is reproducible and comparable with the other two methods employed and may be used as an alternative approach to BCG vaccine efficacy testing. It might probably also be applicable to estimation of the status of cell-mediated immunity against intracellularly parasitizing bacteria in general.     

Intravesical BCG administration in the guinea pig. II. Immunohistochemical characterization of cellular infiltrate after one or two cycles of intravesical BCG.

The intravesical administration of bacillus Calmette-Guérin (BCG) is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The aim of this study was to characterize with monoclonal antibodies the cellular infiltrates in the bladder wall occurring after one or two cycles, each comprising six weekly intravesical BCG administrations in guinea pigs. BCG-RIVM and BCG-TICE were instilled once a week in undamaged bladders at weeks 1-6 and 11-16, respectively, and retained in the bladder for 1 h. Autopsy was performed 1 week after the last BCG administration at weeks 7 and 17. By routine histology, a single BCG course induced scattered focal infiltrates consisting of minor to severe accumulations of mononuclear cells. After two cycles of BCG, a much more severe inflammatory reaction sometimes occupying almost the total bladder mucosa was noted. The major component of this inflammation was mononuclear cells, mainly lymphocytes, while typical BCG granulomas were occasionally observed. In general, the epithelial layer of the bladder showed no visible alterations. Immunohistochemical staining showed that the majority of mononuclear cells present in the infiltrate were T-cells. Widely scattered B-cells and macrophages were also present, but were much fewer in number than the T-cells. In general, T-helper/inducer cells were more frequent than T-suppressor/cytotoxic cells. Pseudofollicles, also noted after H&E staining, consisted mainly of B-cells. The second series of BCG administrations produced pseudofollicles in the infiltrates in all animals investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method for assessing IFN-γ responses in guinea pigs during TB vaccine trials

Aims: We sought to develop a new method that enables the assessment of the immune response of guinea pigs during TB vaccine evaluation studies, without the need to cull or anaesthetize animals. Method and Results: Guinea pigs were vaccinated with five different formulations of oral BCG. One week prior to challenge with Mycobacterium bovis, blood (50–200 μl) was taken from the ears of vaccinated subjects. Host RNA was isolated and amplified following antigenic restimulation of PBMCs for 24 h with 30 μg of bovine PPD. The up‐ or down‐regulation of γ‐interferon (IFN‐γ), a key cytokine involved in protection against tuberculosis, was assessed using real‐time PCR. The relative expression of prechallenge IFN‐γ mRNA in the vaccinated groups (n = 5) correlated (P < 0·001) with protection against M. bovis challenge. Conclusion: We have demonstrated that it is possible to take blood samples and track IFN‐γ responses in guinea pigs that then go on to be exposed to M. bovis, thus providing prechallenge vaccine uptake information. Significance and Impact of the Study: This methodology will also be applicable for tracking the immune responses of vaccinated guinea pigs over time that then go on to be challenged with M. tuberculosis during human TB vaccine evaluation studies.