CD20免疫反应阳性细胞在人胎阑尾的发育

目的从形态学的角度探讨人胎阑尾内CD20阳性B细胞在各胎龄段的发育。方法收集13～28周人胎22例,测量各胎儿顶臀长（CRL）。按Patten法确定胎龄,取其阑尾,经HE染色、免疫组织化学sP法染色,光镜观察。结果13～16周阑尾上皮已经完全演变成为单层柱状,胎儿阑尾绒毛出现,结构分层逐渐明显,淋巴细胞散在分布,淋巴小结雏形逐渐形成;14周CD20免疫反应阳性细胞出现,数量少,单个分布于结缔组织内;17～20周CD20阳性反应细胞增多,成群分布;23—28周粘膜固有层内CD20阳性细胞参与构成淋巴小结。统计学处理证实随胎龄增加,CD20阳性细胞数量逐渐增多（P〈0．01）。结论14周CD20阳性细胞出现,随胎龄增加数量增多,主要参与构成淋巴小结。     

胎儿腭扁桃体T细胞亚群CD4和CD8阳性细胞的发育

目的:探讨胎儿腭扁桃体T细胞亚群CD4和CD8阳性细胞的发育.方法:收集9～28周因故终止妊娠胎儿腭扁桃体,免疫组织化学SP法显色显示CD4阳性细胞和CD8阳性细胞.结果:12周,腭扁桃体固有层内见少量CD4阳性细胞及CD8阳性细胞.14周,CD8阳性细胞数量较CD4阳性细胞增多,隐窝上皮内始见少量CD8阳性细胞和CD4阳性细胞浸润.22周,CD4阳性细胞免疫反应增强,数量仍较CD8阳性细胞少.至28周,两种阳性细胞数量逐渐增多,但以CD8阳性细胞为主,两者在固有层内散在分布或集聚成小群落,隐窝上皮内多呈单个分布.结论:12周CD4和CD8亚群已迁入胎儿腭扁桃体;随胎龄增长,两个亚群T细胞数量逐渐增多,但CD8亚群多于CD4亚群;两个亚群的T细胞发育时序基本一致.     

人胎肠系膜淋巴结组织发生及相关T、B细胞的发育

目的:从形态学的角度探讨人胎肠系膜淋巴结组织发生及相关T、B细胞的发育.方法:收集人胎33例,测量顶臀长,按Patten法确定胎龄.采用常规组织学H-E染色,免疫组织化学法,观察人胎肠系膜淋巴结组织发生及相关T、B细胞的发育.结果:9周,人胎肠系膜淋巴结原基形成,IgM阳性细胞出现,散在分布;11周,出现CD3阳性细胞,原基中有高内皮微静脉;15周,形成早期髓质;23周,IgM阳性细胞集聚形成小结状.连续切片显示,CD3阳性细胞分布在小结深面形成薄层副皮质区,可辨认皮质和髓质;至28周时,淋巴小结内均未发现生发中心.结论:9周淋巴结原基出现.15周早期髓质形成,髓质形成早于皮质.23周皮、髓质明显可辨,皮质内出现淋巴小结,但至28周时小结仍为初级淋巴小结;9周出现B细胞;11周出现T细胞.     

CD3和CD20阳性细胞在人胎腭扁桃体的发育方法

目的 探讨T、B细胞在人胎儿腭扁桃体内的发育情况.方法 收集9～20周因故终止妊娠胎儿腭扁桃体22例,采用免疫组织化学方法,以细胞表面分化群(CD)抗体(CD3和CD20)染色分别显示T细胞和B细胞,用BioMiaspro图像分析软件对免疫反应阳性细胞进行计数,有关数据做统计学分析.结果 妊娠9周时,胎儿腭扁桃体原基内...     

免疫磁珠体外纯化人胎脑中CD133阳性干细胞的实验研究

为了探讨免疫磁珠体外纯化人胎儿脑内 CD13 3阳性细胞的可行性 ,本研究取人胎儿脑室下区并制备单细胞悬液 ,采用磁珠分选法选择 CD13 3阳性细胞群 ,台盼蓝染色观察活力并采用流式细胞仪鉴定纯化前后 CD13 3的阳性表达率。结果显示 ,分选后所得细胞纯度为 ( 85 .5 7± 1.66) % ,回收率为 ( 62 .3± 18) % ,纯化前后细胞活力无显著性差异 ( P>0 .0 5 )。结论 :联合应用CD13 3表面标志及免疫磁珠分选系统可有效地从人胎儿脑组织中直接分离得到高纯度的 CD13 3阳性干细胞 ,细胞活力不受影响     

人胎海马生长抑素和神经生长因子免疫阳性神经元的研究

本文采用免疫细胞化学ABC法对20例胎龄为16～36周的人胎儿海马进行了观察。证明人胎海马存在着生长抑素和神经生长因子免疫阳性神经元，两者均散在分布于海马皮质的深层，为多极神经元；免疫反应阳性颗粒均匀地分布于神经元胞质中，生长抑素阳性细胞中的颗粒比神经生长因子阳性细胞中的颗粒粗大。本文结果提示：人胎海马中存在的上述两类阳性神经元可能在中枢神经系统的发育中起着重要作用。     

人胎视网膜超氧化物歧化酶和波形蛋白免疫阳性细胞的发育

目的：观察人胎视网膜超氧化物歧化酶（SOD）和波形蛋白（VIM）免疫阳性细胞的分布发育。方法：不同孕龄的人胎16例,ABC免疫细胞化学方法显示视网膜SOD和VIM免疫阳性细胞,结果：（1）SOD免疫阳性细胞：E15w节细胞层开始出现SOD免疫阳性细胞;D20W和E28W SOD免疫阳性细胞排列较整齐,分布于视网膜的外核层,内核层,节细胞层,其数量增多,其中内核层SOD免疫阳性细胞增多明显。（2）VIM免疫阳性细胞的发育：E15w内界膜开始出现Muller细胞的VIM免疫阳性终足,并见VIM免疫阳性突起伸向外界膜;E20 人VIM免疫阳性物质集中于内界膜,并见VIM免疫阳性突起伸向外界膜;E28wVIM免疫阳性物质的数量较E20w以前各孕龄明显增多,除色素上皮和视杆视锥层外均有VIM免疫阳性物质出现,除伸向外界膜的VIM免疫阳性突起外,还内网层,内核层,节细胞层和神经纤维层还可见水平走行的细胞突起,结论：（1）视网膜发育基本成熟后,视网膜SOD可能主要来源于内核层的SOD免疫阳性细胞。（3）视网膜神经纤维髓鞘是从内向外逐渐形成的。     

人胎儿脑室区神经前体细胞的分离纯化、培养及鉴定

目的建立有效的人胎儿神经前体细胞分离及纯化系统。方法取人自然流产胎儿脑室区(VZ)并制备单细胞悬液,采用免疫磁珠法分离CD133阳性及CD133阴性细胞群,体外培养并比较两者增殖能力。用免疫荧光化学方法检测CD133阳性细胞群中神经上皮干细胞蛋白(nestin)的表达及诱导分化后的多向分化潜能。结果纯化后CD133阳性细胞群体外扩增能力强,在无血清培养体系中能形成神经球并可连续传代,免疫荧光化学法检测表明其nestin抗原阳性,诱导分化后可分化为神经元、星形胶质细胞及少突胶质细胞。而CD133阴性细胞在培养体系中多呈单个分散状态,神经球形成数目与CD133阳性细胞有显著性差异(P<0.05)。结论免疫磁珠法可有效分离人胎儿脑内CD133阳性细胞,且该细胞在体外培养体系中具有神经前体细胞的特征。     

胎儿脊神经节发育过程中P2X3和降钙素基因相关肽受体的表达

目的:观察人胎儿脊神经节发育过程中P2X3和降钙素基因相关肽(CGRP)受体的表达变化,探讨在神经系统发育过程中P2X3和CGRP受体的作用和意义.方法:运用尼氏染色、ABC免疫组织化学显色的方法,观察胎儿脊神经节发育过程中P2X3和CGRP受体的表达规律.结果:在人胎儿脊神经节的神经元内P2X3和CGRP受体均有不同程度的表达.在胎儿第16～32周脊神经节中P2X3受体免疫阳性细胞数逐渐减少.在胎儿第16～32周脊神经节的神经元中CGRP受体阳性细胞数逐渐增多,阳性表达逐渐增强.结论:在胎儿脊神经节的发生、发育过程中,P2X3和CGRP受体呈不同程度的表达,表明其在神经系统的发育过程中发挥着重要的作用.     

胎儿胸腺发育过程中T细胞抗原受体的表达

目的:检测不同胎龄胎儿胸腺组织内T细胞抗原受体(TCR)的表达及发育规律.方法:18～40周胎儿胸腺组织,采用免疫组织化学SP法检测TCRαβ和TCRγδ的表达. 结果:胚胎21周,胸腺组织开始出现TCRαβ和TCRγδ阳性细胞.TCRαβ阳性细胞主要分布被膜下及小叶间隔中,围绕血管成群分布.TCRγδ阳性细胞的分布与TCRαβ阳性细胞相似, 但细胞数量较TCRαβ阳性细胞少.38周以后胎儿胸腺内TCR阳性细胞主要分布在皮髓质交界处,被膜下及小叶间隔中的TCR阳性细胞较早期少.结论:TCR阳性细胞在胚胎早期开始出现,其分布部位提示局部微环境有利于这些细胞的分化发育.     

Differential expression of leukocyte common antigen in human fetal lymphoid organs.

To investigate the differential expression of various types of leukocyte common antigen (LCA) isoforms during development, we analyzed human fetal lymphoid organs, including the thymus, liver, spleen, and bone marrow from 14 weeks to 29 weeks of gestational age by immunohistochemical and flow cytometric methods. In fetal thymus, over 90% of thymocytes throughout the entire fetal life expressed CD45RO and CD45RB, while CD45RA was expressed only in less than 5% of thymocytes. This expression pattern of LCA isoforms was established by a gestational age of 14 weeks or earlier, and persisted throughout the fetal period. The tissue distribution was different from each isoform; CD45RO-positive thymocytes were found in both the cortex and medulla at the 14th week with low intensity, but was localized in the cortex with increasing fetal age. CD45RB-positive thymocytes distributed mainly in the medulla from early gestational age. Among extrathymic lymphoid organs, a small portion of lymphoid cells expressing leukocyte common antigens appeared first in the liver at 10-12 weeks of gestational age and was followed by a small number in the spleen and bone marrow by 13-15 weeks. All lymphoid cells in these extrathymic lymphoid organs at this stage were CD19+ B cells. The number of these CD19+ cells increased abruptly during the early period of mid-gestational age. The pattern of tissue distribution of each LCA isoform in the fetal liver and spleen correlated well with the patterns of quantitative analysis by flow cytometry. In summary we found that different LCA isoforms expressed in cell-type-specific pattern and showed different tissue distribution during the period of fetal development, and that LCA was the earliest antigen expressed by lymphocytes in the thymus and extrathymic lymphoid organs in our series.     

Pulmonary histiocytic sarcoma mimicking pulmonary Langerhans cell histiocytosis in a young adult presenting with spontaneous pneumothorax: a potential diagnostic pitfall

We present a case of a histiocytic sarcoma incidentally detected in peripheral lung tissue resected for a spontaneous pneumothorax. Furthermore, we discuss the practical approach to pulmonary Langerhans cell histiocytosis, the main differential diagnosis of this lesion in the lung, based on morphological and immunohistochemical features. A 23-year-old male patient presented with recurrent pneumothoraces. The pulmonary tissue showed a single round granuloma-like lesion measuring 4 mm in diameter in close neighbourhood to a bronchial wall. The granuloma consisted of histiocytic cells with enlarged pale nuclei, plasma cells, lymphocytes and scanty eosinophilic granulocytes giving the impression of a granuloma of pulmonary Langerhans cell histiocytosis on haematoxylin and eosin (H&E) stains. Immunohistochemically, the histiocytic cells were negative for CD1a and S-100. They were positive for CD68, HLA-DR, CD14, CD4, CD11c, CD45LCA and lysozyme. MIB1 (Ki67) showed a nuclear staining of approximately 10% of the histiocytic cells. In summary, these findings were in keeping with a histiocytic sarcoma, a rare haematopoetic neoplasm. By demonstrating this particular case, we emphasise the importance of proving the diagnosis of pulmonary Langerhans cell histiocytosis by means of immunohistochemistry. In case of a negative CD1a reaction in a histiocytic lesion, further immunohistochemical studies have to be performed in order not to misdiagnose a malignant haematopoetic lesion.     

Sequestration of inhaled particulate antigens by lung phagocytes. A mechanism for the effective inhibition of pulmonary cell-mediated immunity.

Dendritic cells (DCs) have emerged as the dominant antigen-presenting cells (APCs) of the lung, playing a vital role in the induction of cell-mediated immunity to inhaled antigens. We have previously demonstrated that an airway challenge with the soluble antigen hen egg lysozyme yields rapid acquisition of specific antigen-presenting cell activity by purified pulmonary DCs and a cell-mediated immune response in the lung upon secondary challenge. To examine how a particulate antigen leads to a cell-mediated response in vivo, graded concentrations of heat-killed Listeria (HKL) were injected intratracheally into Lewis rats. The bacteria were rapidly ingested by lung macrophages and polymorphonuclear leukocytes. The ability of purified pulmonary DCs pulsed in vivo by an airway challenge with HKL to subsequently stimulate HKL-specific responses ex vivo showed a threshold response, requiring a dose in excess of 10(9) organisms/rat. By contrast, all dosages of HKL yielded specific sensitization of lymphocytes in the draining bilar nodes. Pulmonary DCs purified from rats after a secondary in vivo airway challenge with HKL at day 14 were ineffective antigen-presenting cells except at high dosages of antigen. The generation of cell-mediated pulmonary inflammation paralleled the antigen-presenting cell activity of pulmonary DCs and was observed only at high antigen dosages. Hen egg lysozyme immobilized onto polystyrene beads and injected intratracheally yielded comparable results to those observed with HKL. We suggest that a pulmonary cellular immune response is generated to an inhaled particulate antigen when the protective phagocytic capacities of the lung are exceeded and antigen is able to interact directly with interstitial DCs. The diversion of particulate antigens by pulmonary phagocytes may help to limit undesirable pulmonary inflammation while allowing the generation of antigen-specific immune lymphocytes in vivo.     

Vaccine immunity to coccidioidomycosis occurs by early activation of three signal pathways of T helper cell response (Th1, Th2, and Th17)

We have previously reported that C57BL/6 mice vaccinated with a live, attenuated mutant of Coccidioides posadasii, referred to as the ΔT vaccine, are fully protected against pulmonary coccidioidomycosis. This model was used here to explore the nature of vaccine immunity during the initial 2-week period after intranasal challenge. Elevated neutrophil and eosinophil infiltration into the lungs of nonvaccinated mice contrasted with markedly reduced recruitment of these cells in vaccinated animals. The numbers of lung-infiltrated macrophages and dendritic cells showed a progressive increase in vaccinated mice and corresponded with reduction of the lung infection. Concentrations of selected inflammatory cytokines and chemokines were initially higher in lung homogenates of vaccinated mice but then generally decreased at 14 days postchallenge in correlation with containment of the organism and apparent dampening of the inflammation of host tissue. Profiles of cytokines detected in lung homogenates of ΔT-vaccinated mice were indicative of a mixed T helper 1 (Th1)-, Th2-, and Th17-type immune response, a conclusion which was supported by detection of lung infiltration of activated T cells with the respective CD4(+) gamma interferon (IFN-γ)(+), CD4(+) interleukin-5 (IL-5)(+), and CD4(+) IL-17A(+) phenotypes. While Th1 and Th2 immunity was separately dispensed of by genetic manipulation without loss of ΔT vaccine-mediated protection, loss of functional Th17 cells resulted in increased susceptibility to infection in immunized mice. Characterization of the early events of protective immunity to Coccidioides infection in vaccinated mice contributes to the identification of surrogates of immune defense and provides potential insights into the design of immunotherapeutic protocols for treatment of coccidioidomycosis.     

严重急性呼吸综合征的临床病理及发病机制研究

目的　研究严重急性呼吸综合征 (SARS)的病理学特征及临床治疗的病理学基础 ,并探讨SARS的发病机制。方法　采用光、电镜观察 ,对 2例SARS系统尸检病例和 4例多器官多部位穿刺标本进行病理学观察 ;应用免疫组化标记并分析肺组织及免疫器官中各淋巴亚群的分布及数量变化 ;核酸原位杂交结合电镜观察 ,作SARS冠状病毒 (SARS CoV)在体病原学定位及定量检测。结果　 6例SARS肺组织均呈弥漫性肺泡上皮损伤 ,2例尸检肺组织呈急性间质性炎和区域性肺水肿 ,2例尸检和1例穿刺肺组织中肺泡腔内透明膜形成 ,1例尸检和 2例穿刺肺组织呈脱屑性终末细支气管炎及肺泡炎 ,2例穿刺病例见早期肺纤维化及肺泡腔机化。SARS肺外器官 ,2例病程 <12dSARS病例免疫器官呈较广泛的出血坏死性炎 ,组织细胞及单核细胞样免疫母细胞反应性增生 ,骨髓组织内单核 粒细胞系统相对抑制 ,而 4例病程 >2 1dSRAS病例脾脏中央动脉周围T淋巴细胞增生 ,骨髓像大致正常。体内SARS CoV存在多种感染靶细胞和靶器官 ,其中肺脏为主要靶器官 ,支气管、肾、肾上腺、心肌、胃肠道、淋巴组织及睾丸等也为靶器官。肺组织内以CD8+ 细胞浸润为主 ,杂以少数CD4 + 细胞 ;淋巴结及脾脏中CD3+ 、CD4 + 、CD8+ 和CD2 0 + 淋巴细胞亚群呈不同程度减少及比例失衡 ,而     

Lung-resident CD4+ T cells are sufficient for IL-4Rα-dependent recall immunity to Nippostrongylus brasiliensis infection

Immunity to Nippostrongylus brasiliensis reinfection requires pulmonary CD4⁺ T-cell responses. We examined whether secondary lymphoid recruited or pre-existing lung CD4⁺ T-cell populations coordinated this immunity. To do this, we blocked T-cell egress from lymph nodes using Fingolimod (FTY720). This impaired host ability to resolve a primary infection but did not change effectiveness of recall immunity. Associated with this effective recall immunity was the expansion and T helper type 2 polarization of a pre-existing pulmonary CD4⁺ T-cell population. LTβR-Ig (lymphotoxin beta-receptor fusion protein)-mediated disruption of stromal cell organization of immune cells did not disrupt this recall immunity, suggesting that protection was mediated by a pulmonary interstitial residing CD4⁺ T-cell population. Adoptive transfer of N. brasiliensis-experienced pulmonary CD4⁺ T cells from FTY720-treated wild-type or T-cell interleukin (IL)-4Rα-deficient mice demonstrated protection to be IL-4Rα dependent. These results show that pre-existing CD4⁺ T cells can drive effective recall immunity to N. brasiliensis infection independently of T-cell recruitment from secondary lymphoid organs.     

多器官功能障碍综合征小鼠肺间质树突状细胞的病理学观察

目的探讨肺间质树突状细胞在多器官功能障碍综合征（MODS）免疫紊乱及脏器损伤机制中的变化与作用。方法C57BL／6小鼠腹腔注射酵母多糖复制MODS模型,分为正常、3—6h（致伤早期）、12～48h[失控性全身炎性反应（SIRS）期]、5～7d（恢复期）和10～12d（MODS期）组。光镜与电镜观察各组小鼠肺及间质树突状细胞的病变;运用免疫组织化学方法检测间质树突状细胞表面标记物CD11c和CD205,共刺激分子CD80和CD86在肺中的表达水平;逆转录-聚合酶链反应法检测趋化因子SLC及其受体CCR7在肺中的表达情况;流式细胞术检测MODS各期小鼠外周血CD4^＋与CD8^＋的T淋巴细胞数量与比值。结果致伤早期,肺间质树突状细胞显著增生,共刺激分子CD80和CD86低水平表达,趋化因子SLC及其受体CCR7在肺组织中表达水平开始上升,外周血T淋巴细胞CD4^＋／CD8^＋比值下降;SIRS期,间质树突状细胞继续增生,CD80和CD86标记阳性细胞数显著上升（与正常组比较均P〈0．01）,SLC与CCR7在肺组织中表达明显高于正常组（均P〈0．01）,外周血T淋巴细胞CD4^＋／CD8^＋比值明显下降（与正常组比较P〈0．01）;MODS期,肺间质树突状细胞高度增生,但CD80和CD86表达显著减少（与SIRS期比较P〈0．01）,肺组织中SLC表达水平继续上升,而CCR7表达水平明显下降（与SIRS期比较P〈0．01）,外周血T淋巴细胞CD4^＋／CD8^＋比值显著下降。结论肺间质树突状细胞在MODS中的变化可能参与并影响了MODS病程中的免疫失衡与免疫抑制过程。CCR7的表达水平可以作为估价间质树突状细胞迁移活性和机体免疫应答水平的一个指标。     

Pulmonary interstitial glycogenosis in Birt-Hogg-Dubé syndrome-associated lung cysts: A new insight into the pathogenesis?

Multiple lung cysts are one of the major features of Birt-Hogg-Dubé syndrome (BHD), but little is known about their nature and pathogenesis. We report a case of a woman diagnosed with BHD lung cysts who exhibited pulmonary interstitial glycogenosis (PIG), a mesenchymal abnormality hitherto undescribed in this disease, in specimens resected at 14 and 29 years of age. Histopathologically, oval to spindle clear cells were seen in the subepithelial interstitial tissue of septal structures and the walls of the cysts. They had abundant periodic acid–Schiff-positive cytoplasmic glycogen. Immunohistochemically, these cells were positive for a few markers of mesenchymal stem cell-like lineage, including vimentin, CD44, and CD10, and negative for markers of epithelial or specific mesenchymal differentiation; these results were consistent with the reported immunophenotype of PIG cells. These PIG cells were more abundant in her specimen at age 14 years than in the second specimen from adulthood. The present case suggests that BHD lung cysts belong to a group of pulmonary developmental disorders characterized by combined PIG and alveolar simplification/cystic change. Disorders with PIG may persist until adulthood and may be of clinical and pathological significance.     

Pulmonary sarcoidosis: A clinico-pathological study.

The light and electron microscopic changes in biopsy tissue from the lung of a 30-year-old housewife severeley incapacitated by diffuse pulmonary sarcoidosis with pulmonary hypertension are presented. The lung tissue was distorted by numerous granulomas in the interstitial tissues and within alveoli. Many pulmonary blood vessels including arteries were damaged by the granulomas. The ultrastructural features of the epithelioid cells were found to be distinctive and probably specific. The giant cells which accompanied the epithelioid cells contained two types of inclusion body: one appeared to be related to the Schaumann body but the nature and origin of the second type was not clear. Many of the granulomas were surrounded by avascular fibrous tissue which contained, in addition to mature fibroblasts, myofibroblasts and a primitive form of cell that appeared to be a fibroblast precursor. It was conjectured that the myofibroblasts, through their contractile powers, might increase the distortion of the lung architecture and thereby the patient's disability. The alveolar walls were thickened by a diffuse infiltrate of macrophages and epithelioid cells but there was no excess of collagen and elastic fibres. The evidence suggested that the epithelioid cells developed from macrophages. From the cellular nature of the diffuse infiltration of the alveolar walls and the absence of fibrosis it seemed that the disease was still at an early and active stage, a conclusion strengthened by the fact that treatment with corticosteroids led to marked and sustained clinical improvement.     

T lymphocyte phenotypic profile in lung segments affected by cavitary and non-cavitary tuberculosis

Clinical manifestations of pulmonary tuberculosis (TB) may depend on a complex interaction between the host and the pathogen. Clinical outcomes of pulmonary tuberculosis are variable, ranging from asymptomatic lifelong infection to parenchymal lung destruction, resulting in cavitary lesions. To investigate the hypothesis that local cellular immune response may affect presentation and outcome in tuberculosis, we performed bronchoalveolar lavage (BAL) in lung segments affected by cavitary and non-cavitary tuberculosis. We then correlated the type of cellular response at the level of the involved lung segments with clinical evolution in terms of cavity formation. We found alveolar lymphocytosis in patients with both cavitary and non-cavitary pulmonary tuberculosis, with increased CD4+ lymphocytes in patients with non-cavitary pulmonary tuberculosis. A predominant Th1 immune response has been observed in non-cavitary patients, while cavitary involved segments exhibit the presence of Th2 lymphocyte subsets. These data, while confirming the importance of Th1-type CD4+ cells and IFN-gamma in effective cellular immunity in active pulmonary tuberculosis, also suggest that the presence of Th2 lymphocytes may contribute to tissue necrosis phenomena associated with cavitary evolution of pulmonary tuberculosis. Our observations indicate the importance of the type of local immune response at the site of disease in the development of different clinical characteristics and outcome in pulmonary tuberculosis.