结核分枝杆菌低剂量感染小鼠模型的建立与分析

目的 建立结核分枝杆菌低剂量感染的小鼠模型,分析感染小鼠组织荷菌量、组织病理随感染时间的动态变化。 方法 以100 菌落形成单位(CFU)的结核分枝杆菌标准株H37Rv经尾静脉途径感染雌性C57BL/6J小鼠40只,于感染后1、3、5、8、12、16、20、24周取材,每个时间点5只小鼠,脾、肺组织匀浆培养检测脾脏组织的荷菌量,脾脏、肺脏及肝脏组织经HE染色分析病理变化。 结果 小鼠感染后3周脾脏荷菌量达到（4.97±0.19）lg CFU,在感染后5周下降至（3.64±0.22）lg CFU,至感染后8周降到最低的（2.75±0.23）lg CFU;感染后3周肝、脾、肺等脏器出现病理改变,感染5周时病变加重,感染8周时病变自然减轻。 结论 成功建立了结核分枝杆菌低剂量感染小鼠模型,为结核分枝杆菌慢性持续感染或潜伏感染的研究可提供有用的工具。     

结核分枝杆菌感染小鼠动物模型的建立及研究进展

小鼠动物模型是结核分枝杆菌(MTB)感染研究中应用最为广泛的动物模型,能不同程度地模拟人体结核病发展过程,并且可以较为准确地评价结核病的病理损伤、存活时间以及免疫学机制,为研究感染状态下MTB生理特性,抗原表达情况及解释MTB感染的发生、维持、活化提供病原体方面的信息。成功建立MTB感染的小鼠动物模型对MTB感染研究工作非常重要。     

DNA疫苗治疗小鼠耐多药结核病的研究

目的研究结核分枝杆菌DNA 疫苗治疗小鼠耐多药结核病的效果。方法用结核分枝杆菌耐利福平和异烟肼临床分离株HB361尾静脉注射60只雌性BALB/c小鼠后,将小鼠随机均匀地分为6组,感染后第3d开始,分别用生理盐水（A组）、pVAX1载体（B组）、利福平（C组）、HSP65 DNA疫苗（D组）、Ag85A DNA 疫苗（E组）、Ag85A/ESAT6嵌合DNA疫苗（F组）治疗60d,DNA疫苗每隔15d肌肉注射1次,共5次。治疗结束后3周,分别取肺和脾观察病理改变、称取重量、做菌落计数。结果治疗结束后3周,与对照组比较,D组、E组和F组肺脏病变有不同程度减轻,病变局限,2/3区域可见正常的肺泡结构,肺泡轮廓相对清晰,细胞分布均匀。与A组相比,D组、E组和F组肺脏菌落数分别减少了0.34、0.50 和0.26 logs;脾脏菌落数依次减少了0.37、0.46 和0.28 logs（P<0.05,P<0.01）。结论Ag85A DNA 疫苗治疗小鼠耐多药结核病效果优于HSP65 DNA和Ag85A/ESAT6嵌合DNA疫苗。     

结核分枝杆菌感染小鼠模型的建立与评价

目的建立结核分枝杆菌感染的小鼠模型,分析感染小鼠重量指数、组织荷菌量、组织病理改变随感染时间的变化。方法以1.1×10^5菌落形成单位（CFU）的结核分枝杆菌标准株H37Rv经尾静脉感染50只雌性BALB/c小鼠,于感染后1、2、3、4、8周杀鼠,每个时间点剖杀10只,观察肺组织病理改变并称重,计算重量指数,同时做肺和肝菌落计数。结果感染1、2、3、4、8周小鼠的肺脏器重量指数分别为0.0112±0.0036、0.0101±0.0071、0.0112±0.0046、0.0151±0.0057和0.0198±0.0069,差异有统计学意义（F=4.24,P〈0.01）;肝脏器重量指数分别为0.0558±0.0059、0.0591±0.0094、0.0569±0.0076、0.0582±0.0030和0.0619±0.0079,差异无统计学意义（F=0.86,P〉0.05）;脾脏器重量指数分别为0.0107±0.0034、0.0146±0.0060、0.018±0.0034、0.0174±0.0026和0.0204±0.0066,差异有统计学意义（F=4.01,P〉0.05）;肺菌落数分别为（4.472±0.504）log、（5.539±0.429）log、（6.294±0.478）log、（6.250±0.315）log和（6.836±0.196）log,差异有统计学意义（F=43.90,P〈0.01）。感染后1周小鼠肺组织可见病理改变,且随着感染时间的增加病变范围逐渐扩大,病变程度逐渐严重。结论成功建立了结核分枝杆菌感染小鼠模型,该模型可用于结核疫苗和药物研究。     

结核分枝杆菌IgG和IgM抗体检测试剂临床应用价值的研究

目的 评估结核分枝杆菌IgG和IgM抗体检测试剂的临床价值。方法 评估实验分2次进行:(1)于2014年5月选择于解放军第三〇九医院就诊的患者,经病历回顾性分析分为肺结核组(92例)和非结核对照组(99例),同时,选取94名健康对照者。(2)于2016年12月至2017年3月共选取解放军第三〇九医院就诊的118例患者血清标本,经病历回顾性分析分为结核病组(62例)和非结核对照组(56例)。对痰和支气管灌洗液进行涂片检查,收集研究对象血清标本应用结核分枝杆菌IgG和IgM抗体检测试剂盒(胶体金免疫层析法)进行检测。结果 第1次实验:应用结核分枝杆菌IgG和IgM抗体检测试剂盒检测研究对象血清中抗结核抗体的敏感度为64.1%(59/92),特异度为89.1%(172/193),阳性预测值为73.8%(59/80),阴性预测值为83.9%(172/205),一致率为81.1%(231/285)。92例肺结核患者中,IgG抗体检测的阳性率为64.1%(59/92),其中涂阳肺结核患者的阳性率为73.8%(31/42),涂阴肺结核患者的阳性率为56.0%(28/50),差异无统计学意义(χ ²=3.15,P=0.076);而IgM检测均为阴性。第2次实验:应用结核分枝杆菌IgG和IgM抗体检测试剂盒检测118例研究对象血清中不同抗结核抗体的敏感度为45.2%(28/62),特异度为78.6%(44/56),阳性预测值为70.7%(29/41),阴性预测值57.1%(44/77),一致率为61.9%(73/118)。62例结核病患者中,IgG抗体检测的阳性率为45.2%(28/62),其中,涂阳肺结核患者的阳性率为56.3%(18/32),明显高于涂阴肺结核患者的29.2%(7/24),差异有统计学意义(χ ²=4.07,P=0.044);而IgM检测只有1例阳性,阳性率为1.6%(1/62)。 结论 结核分枝杆菌IgG和IgM抗体检测试剂中,IgG检测对活动性结核病具有较好的辅助诊断价值,而IgM检测的临床价值尚需进一步评估。     

B细胞免疫应答与抗结核分枝杆菌感染的研究进展

已有大量的研究阐释了细胞免疫机制在抵御结核分枝杆菌方面的重要性。然而,最近的研究表明,B淋巴细胞通过与细胞免疫应答产生多种相互作用扮演了一个曾被低估的角色,形成宿主防御结核分枝杆菌的一个重要方面。因此,作者提倡在结核病免疫学中应有一种进步性的视角,即细胞免疫和体液免疫并不是相互排斥的。本文对最近支持B细胞在结核分枝杆菌感染方面的重要作用进行了综述。     

Fluctuations in viremia,aminotransferases and IgM antibody to hepatitis B core antigen in chronic hepatitis B patients with disease exacerbations

We studied the relationships between the serum levels of viremia, aminotransferases and IgM anti-HBc, measured by monthly quantitative assays, in 52 untreated chronic hepatitis B patients (41 anti-HBe+, 11 HBeAg+) followed up for 12–20 months. Forty hepatitis exacerbations were observed in 17/41 anti-HBe+ (41.5%) and in 6/11 HBeAg+ patients (54.5%) (p = NS); all but one were clinically asymptomatic. We analyzed the fluctuations in the serum levels of the three parameters before, during and after the hepatitis exacerbations and found this chronological sequence of events in 96.2% of them: HBV-DNA increase→ALT flare→IgM anti-HBc increase. These results suggest that both antiviral immune reactions and ALT flares were triggered by quantitative variations in viremia. HBV-DNA baseline levels before flares were lower in anti-HBe+ (3.9±1.2 pg/ml) than in HBeAg+ patients (35.3±5.4 pg/ml) (p<0.0001) and there was an inverse correlation between basal values and viremia level increases at the time of disease exacerbations (p< 0.001). This suggests that for a hepatitis exacerbation to occur, low basal viremia needed to increase markedly, while moderate increases in HBV-DNA serum levels were sufficient to trigger ALT flares in patients with elevated basal viremia. In conclusion, asymptomatic hepatitis B exacerbations are frequent in the natural history of chronic HBV infection, and monthly monitoring of HBV-DNA, ALT and IgM anti-HBc appears to be a suitable method to evaluate their frequencies and entities. This method can be a helpful guide for clinical and therapeutic decision-making in the single patient with chronic hepatitis B.     

Recruitment of Mycobacterium tuberculosis specific CD4+ T cells to the site of infection for diagnosis of active tuberculosis

Context. Accurate and early diagnosis of active tuberculosis (TB) is problematic as current diagnostic methods show low sensitivity (acid‐fast bacilli smears), are time‐consuming (culture of biological samples) or show variable results [Mycobacterium tuberculosis (MTB)‐specific PCR]. Objectives. In the course of infection, MTB‐specific T cells clonally expand at the site of infection and may thus be used as diagnostic marker for active disease. Design. In this cohort study, the frequency of MTB‐specific, interferon (IFN)‐γ expressing CD4⁺ T cells obtained from peripheral blood and the site of disease in 25 patients with suspected TB was assessed (n = 11, bronchoalveolar lavage; n = 7, pleural fluid; n = 1, ascites; n = 1, joint fluid; n = 5, cerebrospinal fluid). Results. Amongst 15 patients who showed proven active TB infection, a striking increase of MTB‐specific T cells was detected at the site of infection compared with peripheral blood (median increase: 28.5‐fold, range: 7.25–531 fold; median of IFN‐γ‐producing CD4⁺ T cells from blood: 0.02%, range: 0–0.52%; median of IFN‐γ‐producing CD4⁺ T cells from the site of infection: 1.81%, range: 0.29–6.55%, P < 0.001). Main outcome measure. Recruitment of MTB‐specific T cells to the site of infection yielded a sensitivity of 100% and specificity of 90%, irrespective of the compartment affected. Conclusions. The accumulation of MTB‐specific T cells at the site of infection may prove as useful diagnostic marker for an accurate and rapid diagnosis of active TB.     

Diagnosis and treatment of latent infection with Mycobacterium tuberculosis

In clinical practice, latent infection with Mycobacterium tuberculosis is defined by the presence of an M. tuberculosis‐specific immune response in the absence of active tuberculosis. Targeted testing of individuals from risk groups with the tuberculin skin test or an interferon‐γ release assay is currently the best method to identify those with the highest risk for progression to tuberculosis. Positive predictive values of the immunodiagnostic tests are substantially influenced by the type of test, the age of the person who is tested, the prevalence of tuberculosis in the society and the risk group to which the person belongs. As a general rule, testing should only be offered when preventive chemotherapy will be accepted in the case of a positive test result. Preventive chemotherapy can effectively protect individuals at risk from the development of tuberculosis, although at least 3 months of combination therapy or up to 9 months of monotherapy are required, and overall acceptance rate is low. Improvements of the current generation of immunodiagnostic tests could substantially lower the number of individuals that need to be treated to prevent a case of tuberculosis. If shorter treatment regimens were equally effective than those currently recommended, acceptance of preventive chemotherapy could be much improved.     

Vivax infection alters peripheral B‐cell profile and induces persistent serum IgM

B cell–mediated humoral responses are essential for controlling malarial infection. Studies have addressed the effects of Plasmodium falciparum infection on peripheral B‐cell subsets but not much is known for P. vivax infection. Furthermore, majority of the studies investigate changes during acute infection, but not after parasite clearance. In this prospective study, we analysed peripheral B‐cell profiles and antibody responses during acute P. vivax infection and upon recovery (30 days post‐treatment) in a low‐transmission area in India. Dengue patients were included as febrile‐condition controls. Both dengue and malaria patients showed a transient increase in atypical memory B cells during acute infection. However, transient B cell‐activating factor (BAFF)–independent increase in the percentage of total and activated immature B cells was observed in malaria patients. Naïve B cells from malaria patients also showed increased TLR4 expression. Total IgM levels remained unchanged during acute infection but increased significantly at recovery. Serum antibody profiling showed a parasite‐specific IgM response that persisted at recovery. A persistent IgM autoantibody response was also observed in malaria but not dengue patients. Our data suggest that in hypoendemic regions acute P. vivax infection skews peripheral B‐cell subsets and results in a persistent parasite‐specific and autoreactive IgM response.     

注射用母牛分枝杆菌预防性治疗潜伏性结核感染的细胞免疫机制

目的评价母牛分枝杆菌（微卡）在预防性治疗潜伏性结核感染中的细胞免疫学作用机理。方法对入学的大学新生进行PPD筛检和X线透视检查，确定潜伏结核感染人群，随机分为对照组、化学药物治疗组和微卡治疗组，每组中各随机选择30例，于用药前、用药后1个月、用药后6个月分别采集血样，采用流式细胞术检测符项细胞免疫学指标，动态观察三组免疫前后细胞免疫学指标的变化情况。结果治疗1个月后，微卡组外周血中CD3^＋、CD4^＋、CD8^＋、NK、NKT和γδT各细胞亚群的比例较之对照组普遍升高，各组IL4^＋、IFNγ^＋表达也显著升高；治疗6个月后，微卡组CD4^＋T细胞比例显著高于同期的对照组和化疗组；CD8^＋、γδT各细胞亚群的比例与用药后一个月相比呈下降趋势，其中CD8^＋T细胞与同期对照组比较下降显著；微卡组IL4^＋表达与同期化疗组、对照组比较显著性降低，同时IFNγ^＋表达较用药前水平显著升高。结论微卡能显著提高潜伏结核感染者固有和获得性细胞免疫水平，尤其是增高并维持CD4^＋细胞的比例，降低CD8^＋细胞的比例，并能够调节Th1／Th2平衡，提示微卡对潜伏性结核感染有预防性免疫治疗作用，避免潜伏结核感染者的发病。     

Semiquantitative assessment of IgM antibody to hepatitis B core antigen and prediction of the severity of chronic hepatitis B

Controversial results have been reported concerning the correlation between serum levels of IgM antibodies to hepatitis B core antigen (IgM HBcAb) and the histological activity of chronic hepatitis B. In this study, paired serum samples and liver biopsies were collected from 200 consecutive chronic hepatitis B patients (mean age 39.2 +/- 0.8 years; M:F 154:46; 41 hepatitis B e antigen (HBeAg) positive) and tested for IgM HBcAb using a semiquantitative highly sensitive assay (IMx CORE-M(R)). The severity of liver disease was assessed by the Ishak score, grading the necroinflammatory activity (by using the histology activity index, HAI) and staging the fibrosis. The index values of IgM HBcAb were significantly different among patients with mild (HAI < or = 6), moderate (HAI 7-12) and severe (HAI > or = 13) necroinflammatory activity but the stage of fibrosis was unrelated to the IgM HBcAb. According to the index value of IgM HBcAb, we selected three groups of patients: Group A included 36 patients with an index value below 0.200; Group B, 99 patients with an index value between 0.200 and 0.500; and Group C, 65 patients with an index value over 0.500. The mean HAI grading in Group A was 5.3 +/- 0.4, in Group B it was 7.4 +/- 0.3 and in Group C it was 8.9 +/- 0.4 (f = 16.5, P < 0.0001). A mild HAI grading was observed in 77.8% of Group A, in 47.5% of Group B and in 23.1% of Group C patients; conversely, severe grading was detected in 0% of Group A, in 11.1% of Group B and in 18.5% of Group C patients (P < 0.0001). An index value of IgM HBcAb below 0.200 was 75% predictive of a mild necroinflammatory activity (29% sensitivity and 91.6% specificity) and ruled out a severe activity. Therefore, the quantitative assessment of IgM HBcAb appears to be a useful clinical tool in the prediction of the necroinflammatory activity of chronic hepatitis B. A serum index value of IgM HBcAb consistently below 0.200 could be considered a surrogate marker of remission of hepatitis B virus-induced liver disease.     

慢性乙型病毒性肝炎治疗现状和展望

HBV感染是导致肝硬化和肝癌的主要原因,在过去20余年里,慢性乙型肝炎的治疗药物主要为干扰素和核苷(酸)类似物两大类,它们虽然能有效抑制病毒复制,但仍面临HBsAg清除率极低,安全停药难等问题,难以实现"临床治愈(或功能性治愈)"。新型的血清标志物的出现为指导临床如何安全停用抗病毒药物开辟了新的途径,同时,治疗慢性乙型肝炎的新型药物和生物制剂的崛起也给慢性乙型肝炎患者带来福音。     

Influence of HLA-DRB1 alleles on Th1 and Th2 cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis

The influence of human leukocyte antigens (HLA) on the immune response is well established. We investigated the regulatory role of HLA-DRB1 alleles on cytokine response to live M. tuberculosis and its culture filtrate antigen (CFA) in normal healthy subjects (NHS) and pulmonary tuberculosis (PTB) patients. Th1 (IFN-gamma and IL-12p40), Th2 (IL-4 and IL-5), pro-inflammatory (IL-6 and IL-8) and anti-inflammatory (TGF-beta and IL-10) cytokines were measured by ELISA in 72-h-old peripheral blood mononuclear cell culture supernatants from 58 NHS and 48 PTB patients. HLA-DRB1 genotyping was carried out by polymerase chain reaction and dot-blot hybridization with biotinylated sequence-specific oligonucleotide probes and detection by chemiluminescence. In response to live M. tuberculosis and CFA, significantly increased levels of IL-6, IL-8 and TGF-beta and decreased IFN-gamma, IL-12p40 and IL-10 were seen in PTB patients compared to NHS. We observed a significantly increased IFN-gamma response in HLA-DRB1*03-positive NHS (p=0.03) and decreased IFN-gamma response in HLA-DRB1*15-positive patients (p=0.04) than respective allele-negative individuals. An increased level of IL-12p40 in DRB1*10 (p=0.02) and IL-10 in DRB1*12- (p=0.03) positive NHS and an increased level of IL-6 in DRB1*04- (p=0.02) positive patients were observed. The study suggests that HLA-DRB1 alleles differentially modulate the various cytokine responses to M. tuberculosis antigens, which may influence the cellular and humoral immune responses to M. tuberculosis infection in a susceptible host.     

气溶胶感染小鼠急性结核病模型的建立及应用

目的建立气溶胶感染小鼠急性结核病模型,应用该模型评价抗结核新化合物的体内活性。方法应用气溶胶感染装置对BALB/C系小鼠进行不同浓度的H37Rv雾化感染,10d后应用抗结核药及新化合物开始治疗,给予15个剂量,在感染后3d、10d及30d分别进行小鼠解剖、肺活菌计数,统计分析该感染模型及药物的有效性。结果感染106cfu/ml的菌量可以较好的作为模型感染剂量,肺活菌计数和组织病理学结果均表明成功建立气溶胶感染小鼠急性结核病模型。INH治疗后肺活菌计数比较空白对照组可以降低5.95lgcfu,应用该模型筛选到新的抗结核活性化合物JYS-17。结论气溶胶感染小鼠急性模型适合抗结核新药早期活性筛选和评价。     

结核分枝杆菌DNA疫苗对小鼠结核病免疫治疗作用的实验研究

目的探讨结核分枝杆菌DNA疫苗pcD85B、pcDMPT64以及小鼠白细胞介素12(IL-12)真核表达质粒psIL12对结核分枝杆菌感染的疗效与机制。方法将结核分枝杆菌H37Rv感染的C57BL/J6小鼠100只随机分成生理盐水对照组、pcDNA3.1对照组,psIL12治疗组,pcD85B、pcDMPT64、pcD85B+pcDMPT64、pcD85B+psIL12DNA疫苗治疗组。感染4周后分别给予生理盐水、空质粒、psIL-12及DNA疫苗,第1次治疗后2个月处死小鼠,检测器官荷菌量、脾淋巴细胞特异性γ干扰素(IFN-γ)、白细胞介素4(IL4)、肿瘤坏死因子-α(TNF-α)的分泌水平,并于第1次治疗后2个月、5个月观察小鼠肺、脾组织病理改变情况。结果pcD85B治疗组肺组织荷菌量(lg-1CFU/g)为6.99±0.40,比生理盐水对照组(8.15±0.37)、pCDNA3.1对照组(8.19±0.29)显著降低(P<0.01);脾组织荷菌量(lg-1CFU/g,x±s)为5.17±0.33,比生理盐水对照组(5.76±0.16)及空质粒对照组(5.88±0.21)显著降低(P<0.05)。psIL12治疗组的肺(7.41±0.50)、脾(5.31±0.21)荷菌量比对照组显著降低(P<0.05);pcD85B+psIL12治疗组肺、脾荷菌量与pcD85B组比较差别无统计学意义。pcD85B组脾淋巴细胞IFN-γ、TNFα水平比对照组显著升高(P<0.05),各组间IL4水平差异无统计学意义。生理盐水对照组、pCDNA3.1对照组肺组织     

Active tuberculosis and Mycobacterium tuberculosis latent infection in patients with HIV/AIDS

Objectives

Tuberculosis (TB) remains an important disease associated with HIV infection and AIDS in Brazil, even in a setting of free access to antiretroviral therapy (ART) and TB treatment. In previous studies, isoniazid therapy (IT) for latent infection with Mycobacterium tuberculosis (LIMTb) was found to reduce the risk of TB by 62% in patients with a tuberculin test (TT)>5 mm. The objectives of this study were to investigate the occurrence of TB, the prevalence of LIMTb and the coverage of the TT and IT, and to estimate the number of missed opportunities to prevent TB in patients with HIV/AIDS.

Methods

A random sample of patients with HIV/AIDS was selected; data from the medical files were obtained, and a TT was performed in consenting subjects.

Results

In the 203 subjects included in the study, TB occurrence was 13.3%, LIMTb prevalence was 20% and the coverage of the TT and IT was 59.2 and 55%, respectively. Patients with TB had a lower nadir CD4 cell count, but their CD4 recovery was comparable to that of non‐TB patients. Patients with LIMTb always had a higher CD4 cell count.

Conclusions

By expanding the coverage of the TT and IT to nearly 100%, we could more than double the number of prevented cases of TB. TB prevention programmes must be reinforced to reduce the number of missed opportunities for diagnosis, and IT must be improved to reduce TB among patients with HIV/AIDS. Empowering patients with knowledge about TB, the preventive role of IT and the need for an annual TT may be the best way of lowing rates of TB in patients with HIV/AIDS.     

病原体特异性IgM抗体检测在儿童呼吸道急性感染病原快速诊断中的价值研究

目的:研究病原体特异性IgM抗体检测在儿童呼吸道急性感染病原快速诊断中的价值。方法:本研究为前瞻性研究。选取朝阳市第二医院2018年10月至2018年12月住院的急性呼吸道感染患儿482例作为研究对象,采用间接免疫荧光法检测呼吸道合胞病毒(RSV)、流感病毒A(INFA)、流感病毒B(INFB)等11种病原体特异性Ig...