实验感染大、小鼠和临床疑似病例肺孢子虫mt LSU rRNA基因检测

为探讨PCR技术对实验感染大、小鼠肺孢子虫肺炎(PCP)和临床疑似病例诊断的可行性。用皮下注射地塞米松法诱导SD大鼠和ICR小鼠PCP并收集临床疑似病例24h深部痰液。受试动物解剖后,制备肺印片,经瑞姬氏复合染色镜检确定肺孢子虫(Pc)感染的阳性率;同时,以针对虫体mtLSUrRNA基因设计的引物,PCR扩增鼠肺组织和支气管灌洗液(BALF),以及痰液样本中的DNA;比较实验动物样本镜检和PCR两种方法的检测结果;测定PCR的敏感性和特异性。结果表明,SD大鼠和ICR小鼠肺印片的阳性率分别为68.8%(1116)和85.7%(1821),肺组织PCR检测的阳性率分别为75%(1216)和80.9%(1721),肺泡灌洗液PCR的阳性率分别为81.2%(1316)和23.8%(521)。肺印片镜检和PCR技术检测结果无显著性差别;可见,PCR对虫体mtLSUrRNA基因的检测具有显著的特异性和敏感性,至少可以检测出0.6pg的肺孢子虫DNA;6例临床疑似病例样本中2例显示肺孢子虫PCR阳性反应。     

双氢青蒿素对卡氏肺孢子虫肺炎大鼠肺泡巨噬细胞凋亡的影响

目的：检测双氢青蒿素对卡氏肺孢子虫肺炎（PCP）大鼠肺泡巨噬细胞凋亡的影响。方法：以醋酸可的松皮下洲Wistar大鼠建立PCP动物模型，对60mg/kg双氢青蒿素治疗实验大鼠，杀鼠取肺，用胶原酶消化法分离肺泡巨噬细胞，可PI和TUNEL法检测其凋亡，同时设有正常大鼠对照组。结果：感染组和治疗组大鼠肺泡巨噬．细胞凋亡率显著高于正常对照组，治疗组大鼠肺泡巨噬细胞凋亡率明显低于感染组。结论：卡氏肺孢子虫感染引起大鼠肺泡巨噬细胞发生凋亡，经双氢青蒿素治疗后PCP大鼠肺泡巨噬细胞凋亡降低。     

双氢青蒿素对卜氏肺孢子虫肺炎大鼠TNF—α水平的影响

目的 研究双氢青蒿素对卡氏肺孢子虫肺炎大鼠血清和肺泡巨噬细胞培养上清液TNF－α水平的影响。方法 以醋酸可的松皮下注射Wistar大鼠建立卡氏肺孢子虫肺炎动物模型，用60mg/kg双氢青蒿素治疗实验大鼠，杀鼠取肺，用胶原酶消化法分离肺泡巨噬细胞，用LPS刺培养72h，同时设有感染组和正常对照，用TNF－α水平则低于感染组。结论 卡氏肺孢子虫感染引起大鼠肺泡巨噬细胞分泌高水平的TNF－α，但双氢青蒿素治疗后PCP大鼠肺泡巨噬细胞产生TNF－α水平降低。     

双氢青蒿素对卡氏肺孢子虫肺炎大鼠TNF-α水平的影响

目的研究双氢青蒿素治疗对卡氏肺孢子虫肺炎大鼠血清和肺泡巨噬细胞培养上清液 TNF- α水平的影响。方法以醋酸可的松皮下注射 Wistar大鼠建立卡氏肺孢子虫肺炎动物模型 ,用 60 m g/ kg双氢青蒿素治疗实验大鼠 ,杀鼠取肺 ,用胶原酶消化法分离肺泡巨噬细胞 ,用 L PS刺激培养 72 h,同时设有感染组和正常对照。用 TNF- α试剂盒分别检测血清和培养上清液 TNF- α的水平。结果感染组和治疗组 TNF- α水平均高于正常对照 ,治疗组 TNF- α水平则低于感染组。结论卡氏肺孢子虫感染引起大鼠肺泡巨噬细胞分泌高水平的 TNF- α,但双氢青蒿素治疗后 PCP大鼠肺泡巨噬细胞产生 TNF- α水平降低。     

实验性卡氏肺孢子虫肺炎的病理学研究

Ｗｉｓｔａｒ大鼠皮下注射醋酸考的松诱发卡氏肺孢子虫肺炎，自第６～１２周，每周解剖病鼠２只观察，全部给药鼠均发病。肺部病变表现为：（１）肺印片查见卡氏肺孢子虫包囊和滋养体。（２）组织病理学改变：第６～８周ＨＥ染色切片呈间质性肺炎伴中度淋巴细胞浸润，肺泡内缺乏泡沫样渗出物，第９～１２周肺泡内出现泡沫样渗出物，ＰＡＳ染色该渗出物呈阳性反应，ＧＭＳ染色查见染成黑色的包囊。（３）停用醋酸考的松后４～６周，肺部炎症明显好转，提示大鼠卡氏肺孢子虫肺炎有自愈可能。电镜显示包囊、滋养体及受损肺泡上皮细胞的超微结构。     

TyrphostinA1预防大鼠油酸性肺损伤的机制研究

目的：研究JAK/STAT信号通路抑制剂tyrphostinA1对大鼠油酸性肺损伤的影响，并探讨其作用机制。方法： 以大鼠油酸型ALI为研究对象，利用动物肺功能测定仪测定肺损伤过程中吸气气道阻力（Ri）和动态肺顺应性(Cdyn)的变化，利用光镜观察肺部形态学变化，称重法计算肺指数及湿/干比（W/D），紫外分光光度法检测肺组织微血管的渗透性和肺泡灌洗液（BALF）中蛋白含量，酶联免疫吸附法（ELISA）检测肺泡灌洗液（BALF）TNF-α、IL-6和IL-10的含量。结果： TyrphostinA1明显改善大鼠肺功能，减轻肺组织病理学损伤，降低肺指数及肺湿/干重比，降低肺渗透性和肺泡灌洗液（BALF）中蛋白及TNF-α、IL-6的含量，提高肺泡灌洗液（BALF）中IL-10的含量。结论： TyrphostinA1能抑制油酸诱导的肺损伤，其作用机制可能与调节细胞因子（TNF-α、IL-6和IL-10）的合成与释放有关。     

寡核苷酸探针RNA原位杂交法检测肺孢子虫的研究

目的探讨RNA原位杂交法在肺孢子虫病理检测中的应用。方法采用小亚单位RNA位点来源的寡核苷酸探针、地高辛加尾标记、Wistar雌性大鼠皮下注射地塞米松建立肺孢子虫肺炎动物模型,取肺组织制备石蜡标本进行RNA原位杂交,并将结果与瑞氏-吉姆萨染色法进行比较。结果 RNA原位杂交法于感染后第3周检到虫体,呈游离分布;7～8周时检测到大量包囊;9～10周时滋养体大量出现;组织内肺孢子虫的检出率（32/32）高于瑞氏-吉姆萨染色法（25/32）。结论 RNA原位杂交法是一种敏感特异的肺孢子虫病理检测方法。     

环介导等温扩增技术检测卡氏肺孢子虫的研究

目的 环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法 醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性).Pc LAMP产物经电泳后呈LAMP特征性梯状条带,扩增产物经Tail限制性内切酶酶切鉴定正确,对照组均无扩增产物.LAMP可检测到虫体DNA的最低浓度是lP9/pJ,为PCR的10倍.结论 检测Pc的LAMP方法敏感、特异及简便.     

卡氏肺孢子虫肺炎大、小鼠低死亡率动物模型的建立

为建立低死亡率卡氏肺孢子虫肺炎 (PCP)SD大鼠和ICR小鼠动物模型 ,本试验将雌性SD大鼠和ICR小鼠分别随机分为实验组和对照组 ,实验组采用按体重定量皮下注射地塞米松的方法 ,免疫抑制诱导建立PCP动物模型 ,对照组注射与地塞米松等体积的生理盐水。分别制作肺印片 ,经瑞 姬氏复合染色后 ,检查卡氏肺孢子虫包囊。制作肺组织病理切片 ,经HE染色后观察肺组织病理变化。用地塞米松诱导后 ,实验组SD大鼠和ICR小鼠死亡率均为 0 ,肺印片阳性率均为 76 7% (2 3 30和 2 3 30 )。肺组织出现典型的病理变化 ,并可观察到Pc包囊。实验组SD大鼠体重下降明显 ,与对照组体重比较具有极显著性差异 (P <0 0 1)。ICR小鼠经诱导后 ,体重变化不显著。采用按体重定量皮下注射地塞米松的方法可建立低死亡率PCP动物模型     

人源肺孢子虫纯培养株的建立及基因鉴定

目的建立人源肺孢子虫（PneUmOCySaSj／rovec／，Pj）纯培养株。方法从肺孢子虫肺炎（PCP）患者的支气管肺泡灌洗液（BALF）中分离巧，用改良IMDM培养基做原代、传代和冻存复苏培养；以四胺银染色计数法观察虫体增殖情况；以线粒体rRNA大亚基特异引物扩增培养物中的目的基因，与基因库中的鼠源和人源肺孢子虫基因做序列比较。结果用添加S-腺苷甲硫氨酸（SAM）等辅助剂的IMDM培养基从2例PCP患者的BALF中分离出2个巧纯培养株。分离培养的巧可进行冷冻保存和复苏培养。培养120h的巧包囊可增殖3．2倍。基因序列分析证实分离出的巧株与鼠源性肺孢子虫的线粒体rRNA大亚基同源性为67．8％，与GenBank中巧的同源性为93．2％。结论用本法建立了2个Pj纯培养株。     

Phylogeny of Pneumocystis carinii from 18 primate species confirms host specificity and suggests coevolution

Primates are regularly infected by fungal organisms identified as Pneumocystis carinii. They constitute a valuable population for the confirmation of P. carinii host specificity. In this study, the presence of P. carinii was assessed by direct examination and nested PCR at mitochondrial large subunit (mtLSU) rRNA and dihydropteroate synthetase (DHPS) genes in 98 lung tissue samples from captive or wild nonhuman primates. Fifty-nine air samples corresponding to the environment of different primate species in zoological parks were also examined. Cystic forms of P. carinii were detected in smears from 7 lung tissue samples corresponding to 5 New World primate species. Amplifications at the mtLSU rRNA gene were positive for 29 lung tissue samples representing 18 different primate species or subspecies and 2 air samples corresponding to the environment of two simian colonies. Amplifications at the DHPS gene were positive for 8 lung tissue samples representing 6 different primate species. Direct sequencing of nested PCR products demonstrated that a specific mtLSU rRNA and DHPS sequence could be attributed to each primate species or subspecies. No nonhuman primate harbored the human type of P. carinii (P. carinii f. sp. hominis). Genetic divergence in primate-derived P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species, suggesting coevolution.     

Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs

The genus Pneumocystis contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization with Pneumocystis becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of Pneumocystis in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of Pneumocystis in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke Pneumocystis growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of Pneumocystis by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of Pneumocystis carinii f. sp. carinii. Upon receipt, 64% of rats were positive for P. carinii f. sp. carinii-specific antibodies, while P. carinii f. sp. carinii DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed P. carinii f. sp. carinii DNA in all rat lungs, illustrating widespread occurrence of Pneumocystis in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of Pneumocystis exposure.     

Latent Pneumocystis carinii infection in commercial rat colonies: comparison of inductive immunosuppressants plus histopathology, PCR, and serology as detection methods

Histopathologic evaluation combined with a period of immunosuppression has been the standard procedure for detection of Pneumocystis carinii in commercial rat colonies. Variation in induction regimens and in the sensitivity of detection methods may result in underreporting of the presence of P. carinii in breeding colonies or delay its detection. In the present study, methylprednisolone and cyclophosphamide were evaluated for the ability to induce P. carinii infection in rats from an enzootically infected commercial barrier colony. The presence of P. carinii was detected by histopathologic methods and by amplification of a targeted region of the P. carinii thymidylate synthase gene by PCR over the 8-week study period. Sera taken from rats prior to either induction regimen were evaluated for the presence of P. carinii-specific antibodies by the immunoblotting technique. Few significant differences in ability to induce organism burden or in histopathology were observed between the two immunosuppressive regimens. However, a dramatic loss of weight over the study period was observed in rats treated with methylprednisolone but not in rats treated with cyclophosphamide. Although histopathologic changes attributable to P. carinii did not appear before 2 weeks with either immunosuppressant, the presence of the organism in these animals was detected by immunoblotting and PCR. Cyst scores and the intensities of the histopathologic lesions increased during the study period, but the number of rats exhibiting evidence of P. carinii infection did not change after week 3. These results suggest that use of the PCR method on postmortem lung tissue of rats without prior induction regimens or identification of anti-P. carinii antibodies in antemortem serum samples is a sufficiently sensitive method for detection of the presence of a P. carinii carrier state in rodent breeding colonies.     

Detection of Pneumocystis carinii sequences by polymerase chain reaction: animal models and clinical application to noninvasive specimens.

Pneumocystis carinii is a eukaryotic microbe which causes fatal pneumonia in patients with AIDS. Oligonucleotide primers were used to amplify the 5S rDNA sequence of P. carinii by the polymerase chain reaction (PCR) in various clinical and animal samples. Of 35 independent lung specimens tested, PCR detected the P. carinii sequence in all 23 cases which were known to be P. carinii infected, i.e., 15 from mice, 1 from rat, 3 from human autopsy, and 4 from biopsy of AIDS patients by needle aspiration. The results were consistent with clinical and microscopic diagnosis. The detection was highly sensitive and specific. Direct sequencing of these amplified DNAs revealed homogeneity of 5S rDNA sequences of independent isolates from mice, rats, and humans. Preliminary trials manifested efficacy of the PCR method to detect P. carinii sequences in induced sputum or blood from AIDS patients, the latter case suggesting that P. carinii might enter peripheral blood via phagocytosis or direct intrusion. Development of less-invasive or noninvasive PCR diagnostic techniques to detect P. carinii infection would greatly facilitate therapeutic and prophylactic management of P. carinii pneumonia.     

Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia

Objective   To study the clinical significance of a nested polymerase chain reaction (PCR) method compared to immunofluorescence (IF) for detection of Pneumocystis carinii .
Methods   The medical records of 89 patients with 91 episodes of pneumonia were scrutinised retrospectively. The pneumonia episodes were divided into categories according to the likelihood that the patient had had clinical Pneumocystis carinii pneumonia (PCP). All respiratory tract samples from the 89 patients (34 broncho-alveolar lavage (BAL) and 57 sputa) were tested for Pneumocystis carinii by IF and nested PCR.
Results   Fifteen episodes, as diagnosed by IF, were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 60%, specificity 97%). Among the P. carinii DNA-positive episodes, detected with nested PCR, 24 were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 96%, specificity 59%), since all IF-positive samples were nested PCR positive. Only one pneumonia episode classified as a probable PCP, was negative with both methods, as applied to a BAL sample.
Conclusions   IF applied to BAL or sputum seems to be the most specific method for diagnosis of clinical PCP. Additional clinical cases can be found by nested PCR, although this then gives a high risk of detecting subclinical colonisation of P. carinii .     

Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences.

We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences.     

A single-tube nested PCR for Pneumocystis carinii f. sp. hominis.

A single-tube nested PCR which amplifies the internal transcribed spacer (ITS) regions of the rRNA genes of human Pneumocystis carinii was developed. The outer primers for the first PCR, which anneal to the 18S and the 26S rRNA genes of P. carinii, were made to have a midpoint temperature (Tm) of 74 degrees C. The inner primers for the second PCR have a Tm of 56 to 58 degrees C and are specific for human P. carinii; they anneal to an area close to the beginning of ITS1 and the junction of ITS2 and the 26S rRNA genes. The reaction mixture contained 2.5 pmol of the first-PCR primers and 25 pmol of the second-PCR primers. The first PCR was performed at an annealing temperature of 68 degrees C, which did not allow the second-PCR primers to function. Since very small amounts (2.5 pmol) of the first-PCR primers were used, they were exhausted when the first PCR was completed. The single-tube nested PCR did not amplify P. carinii isolated from rats, mice, or ferrets. All 10 bronchoalveolar lavage (BAL) specimens from patients with P. carinii pneumonia were positive, whereas all 10 BAL specimens from patients with other diseases or patients infected with several commonly found fungi were negative by PCR.     

An enzyme-linked immunosorbent assay for enumeration of Pneumocystis carinii in vitro and in vivo.

An enzyme-linked immunosorbent assay (ELISA) to quantitate Pneumocystis carinii organisms from culture supernatant and rat lung has been developed. A polyclonal antibody specific to P. carinii was produced in Sprague-Dawley rats by allowing P. carinii-infected animals to recover from infection. This antibody reacted strongly to P. carinii proteins of 50 to 55 kDa and weakly to those of 33 and 116 kDa. The ELISA used this convalescent-phase antibody to quantitate the number of P. carinii organisms in lung homogenates of infected rats and supernatants from infected tissue cultures which were used to screen drugs for P. carinii. The results of the ELISA were compared with those of direct microscopic counting of organisms, and the two methods were highly correlated (r > 0.9). Thus, the ELISA can be used as an alternative method for the quantitation of P. carinii organisms, and it is superior to the conventional microscopic method because it is easier to perform and less labor-intensive.