人类微孢子虫检测方法研究进展

微孢子虫(microsporidia)是一类专性细胞内寄生的单细胞真核生物。是引起微孢子虫病的真菌类病原。在已知并被命名的1500多种微孢子虫中,共有9个属中的17个虫种可以感染人。人类微孢子虫可侵染包括肠道、肝、肺、脑等部位,引起慢性腹泻、肝炎、角膜炎、脑炎、血液系统性感染等,严重影响人类健康。研究开发快速高效的人类微孢子虫诊断方法成为当前病原微生物检测领域研究的热点。人类微孢子虫的发现历史实际上是伴随检测方法的不断进步而逐渐进行的。这些检测方法包括,透射电镜(transmission electron microscopy)、苏木精-伊红染色(hematoxylin-eosin stain,HE)、亚甲蓝染色(methylene blue)、吉姆萨染色(giemsa)、革兰氏染色(gram stain)、韦伯氏改良三色染色法(Weber’s chromotrope-based staining)、荧光增白剂染色法(calcofluor white staining)、抗原检测、抗体检测、实时荧光定量PCR (quantitative real-time PCR,q PCR)、环介导等温扩增(loop-mediated isothermal amplification,LAMP)、DNA点杂交模型等。随着技术的进步以及更多微孢子虫的检出,使人类能够更好地认识微孢子虫、并制定微孢子虫准确快速检测方法和防控策略。     

改良银染色法鉴定卡氏肺孢子虫的研究

本文采用碘酸钠作氧化剂对卡氏肺孢子虫的包囊进行银染色，并与铬酸氧化的结果作了比较。在同一批次小鼠肺涂片，氧化时间相同时，过碘酸钠氧化可显示较多的含括号状结构的包囊，这将利于临床实验室的鉴别诊断，实验结果表明过碘酸钠作氧化剂的银染色效果优于铬酸。     

小鼠结核分枝杆菌耐药模型的建立与评价

目的 建立小鼠结核分枝杆菌耐利福平株静脉感染小鼠模型.方法 30只BALB/c雌性小鼠经尾静脉感染结核分枝杆菌耐利福平株每只106 CFU,观察小鼠一般状况,并分别在感染后6周、10周、14周处死小鼠,进行脾、肺组织病理切片、抗酸染色、计脏器荷菌数.结果 感染后小鼠体重呈逐渐上升趋势,脏器病理变化随时间推移由急性炎症逐...     

不同品系小鼠感染甲型流感病毒肺小血管内血栓形成

目的本实验旨在观察不同品系小鼠感染甲型流感病毒后肺组织内血栓形成的情况。方法使用H1N1病毒A/California/7/2009（CA7）株和H3N2病毒A/Brisbane/10/07株,对BALB/C小鼠、Scid小鼠、NOD/LTJ小鼠、BALB/C-nu小鼠、NOD-Scid小鼠和icosl-KO小鼠经乙醚麻醉后进行滴鼻攻毒。检测小鼠感染后肺组织病毒拷贝数并观察肺组织病理学改变。结果 H1N1和H3N2滴鼻攻毒的各组小鼠均染毒,病理表现为程度略有差异的间质性肺炎。13只H1N1病毒感染小鼠和6只H3N2感染小鼠在肺组织中观察到多个小血管内有血栓形成,血栓成分主要为纤维素和血小板。结论各品系小鼠感染H1N1和H3N2流感病毒后均可能出现肺组织内血栓形成。     

兰州地区实验动物肺支原体感染调查和分离鉴定

目的了解兰州地区啮齿类实验动物的肺支原体感染情况和感染菌株.方法用分离培养法对兰州地区640只啮齿类实验动物肺支原体的感染情况分春夏秋冬进行调查,并对分离株进行克隆纯化,从形态学,生化特性和血清学方面进行鉴定.结果肺支原体在普通级小鼠中的感染率为23%,普通级豚鼠、地鼠、大鼠和清洁级小鼠未发现有感染,且感染率与季节无明显相关性.分离株经鉴定均为支原体科支原体属肺支原体.     

嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性研究

目的观察不同来源的嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性.方法取源于野鼠、实验大鼠和小鼠的嗜肺巴氏杆菌3株,对30只受试大鼠和小鼠进行交叉人工感染,并于感染后不同时期取咽拭子分离培养,对感染前后菌株,应用RAPD-PCR、SDS-PAGE和Western blot进行基因型、蛋白和抗原成份比较,以及生物学特性的比较.结果受试实验动物对3株嗜肺巴氏杆菌均易感,被接种的动物能稳定携带嗜肺巴氏杆菌直到试验结束,重新分离的嗜肺巴氏杆菌在生物学特性、蛋白成份、抗原性和基因型方面无明显改变.结论同一株嗜肺巴氏杆菌能在实验大鼠和小鼠中相互传染.     

关于肺孢子虫的命名与分类

早在20世纪初,Chagas和Carinii就首次从感染克氏、路氏锥虫的豚鼠肺组织和大鼠肺组织中发现肺孢子虫,数年后,Delano夫妇进一步证实它是一种新的病原体,并为纪念Carinii学者而将其命名为卡氏肺孢子虫(pneumocystis carinii)。到了20世纪50……     

甲型H1N1流感病毒感染小鼠的发病机制

目的甲型H1N1流感病毒A/California/7/2009感染BALB/c小鼠,研究甲型H1N1流感病毒病毒性肺炎发病机制。方法 4～6周龄雌性BALB/c小鼠60只,随机分为2组,实验组和对照组,每组30只。CA7流感病毒滴鼻制备甲流病毒感染小鼠模型。攻毒后第5天解剖实验和对照组小鼠,取肺组织,测定肺组织中IL-2,IL-6,TNF-α含量。结果结果实验组肺组织中IL-6,TNF-α,水平明显高于对照组,IL-2水平明显低于对照组,差异均有显著性（P〈0.05）。结论 IL-6、TNF-α、IL-2这3种细胞因子在感染甲流病毒后的显著性变化与病毒感染后的肺组织病理损伤有密切的关系。     

山苍子油对小鼠系统性新生隐球菌感染的实验研究

目的研究山苍子油治疗小鼠系统性新生隐球菌感染的疗效。方法建立小鼠系统性新生隐球菌感染模型,观察给药后小鼠的中位生存时间,检测小鼠肾脏及肺菌落形成单位计数。结果山苍子油不仅能够显著延长感染小鼠的中位生存时间,提高其生存率,而且可显著增加感染小鼠肾脏及肺菌落清除率。结论山苍子油对系统性新生隐球菌感染小鼠具有治疗作用。     

艾滋病与机会性致病原虫感染

艾滋病（ＡＩＤＳ）是当前世界上严重威胁人类健康的最疑难病症,危及ＡＩＤＳ病人生命的机会性致病原虫感染,已引起高度重视。卡氏肺孢子虫可引起子卡氏肺孢子虫肺炎,是ＡＩＤＳ病人最危险机会性感染病原体之一,刚地弓形虫主要成弓形虫脑病,是导致ＡＩＤＳ病人死亡的重要原因之一,隐孢子虫和贾第虫可引起严重腹泻,患者可因体质消耗而死亡,对这４种原虫感染做出早期诊断,及时治疗,是减少ＡＩＤＳ病人死亡率的重要措施之一。     

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120).

Pneumocystis carinii gp120 can elicit a specific T-cell proliferative response in mice after immunization with a gp120 preparation or with a crude P. carinii homogenate. It can also elicit a proliferative response from SCID mice after recovery from natural infection with P. carinii, implicating this glycoprotein as an important antigen in the host's response to P. carinii infection.     

Pneumocystis carinii infection sensitizes lung to radiation-induced injury after syngeneic marrow transplantation: role of CD4+ T cells

A murine model of bone marrow transplant (BMT)-related lung injury was developed to study how infection sensitizes lung to the damaging effects of total body irradiation (TBI) at infectious and TBI doses that individually do not cause injury. Mice infected with Pneumocystis carinii exhibited an asymptomatic, rapid, and transient influx of eosinophils and T cells in bronchoalveolar lavage fluid (BALF). In contrast, mice infected with P. carinii 7 days before receiving TBI and syngeneic BMT (P. carinii/TBI mice) exhibited severe pulmonary dysfunction, surfactant aggregate depletion, and surfactant activity reductions at 17 days post-BMT. BALF from P. carinii/TBI mice contained a disproportionate initial influx of CD4(+) T cells (CD4(+):CD8(+) ratio of 2.7) that correlated with progressive lung injury (from 8 to 17 days post-BMT). Levels of TNF-alpha in BALF were significantly increased in P. carinii/TBI mice compared with mice given either insult alone, with peak values found at 11 days post-BMT. In vivo depletion of CD4(+) T cells in P. carinii/TBI mice abrogated pulmonary dysfunction and reduced TNF-alpha levels in BALF, whereas depletion of CD8(+) T cells did not affect lung compliance or TNF-alpha. Lung injury was not attributable to direct P. carinii damage, since CD4-depleted P. carinii/TBI mice that exhibited no injury had higher average lung P. carinii burdens than either mice given P. carinii alone or undepleted P. carinii/TBI mice. Together, these results indicate that P. carinii infection can sensitize the lung to subsequent TBI-mediated lung injury via a process dependent on non-alloreactive CD4(+) T cells.     

Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice

Innate immunity plays an important role in pulmonary host defense against Pneumocystis carinii, an important pathogen in individuals with impaired cell-mediated immunity. We investigated the role of GM-CSF in host defense in a model of P. carinii pneumonia induced by intratracheal inoculation of CD4-depleted mice. Lung GM-CSF levels increased progressively during the infection and were significantly greater than those in uninfected controls 3, 4, and 5 wk after inoculation. When GM-CSF gene-targeted mice (GM-/-) depleted of CD4+ cells were inoculated with P. carinii, the intensities of infection and inflammation were increased significantly compared with those in CD4-depleted wild-type mice. In contrast, transgenic expression of GM-CSF directed solely in the lungs of GM-/- mice (using the surfactant protein C promoter) dramatically decreased the intensity of infection and inflammation 4 wk after inoculation. The concentrations of surfactant proteins A and D were greater in both uninfected and infected GM-/- mice compared with those in wild-type controls, suggesting that this component of the innate response was preserved in the GM-/- mice. However, alveolar macrophages (AM) from GM-/- mice demonstrated impaired phagocytosis of purified murine P. carinii organisms in vitro compared with AM from wild-type mice. Similarly, AM production of TNF-alpha in response to P. carinii in vitro was totally absent in AM from GM-/- mice, while GM-CSF-replete mice produced abundant TNF in this setting. Thus, GM-CSF plays a critical role in the inflammatory response to P. carinii in the setting of impaired cell-mediated immunity through effects on AM activation.     

Clearance of Pneumocystis carinii in mice is dependent on B cells but not on P carinii-specific antibody

Both CD4(+) T cells and B cells are critical for defense against Pneumocystis carinii infection; however, the mechanism by which B cells mediate protection is unknown. We show that P. carinii-specific IgM is not sufficient to mediate clearance of P. carinii from the lungs since CD40-deficient mice produced normal levels of specific IgM, but were unable to clear the organisms. Using chimeric mice in which the B cells were deficient in CD40 (CD40KO chimeras) we found that clearance of P. carinii infection is delayed compared with wild-type controls. These CD40KO chimeric mice produced normal levels of P. carinii-specific IgM, but did not produce class-switched IgG or IgA. Similarly, clearance of P. carinii was delayed in mice deficient in FcgammaRI and III (FcgammaRKO), indicating that P. carinii-specific IgG partially mediates opsonization and clearance of P. carinii. Opsonization of organisms by complement did not compensate for the lack of specific IgG or FcgammaR, since C3-deficient and C3-depleted FcgammaRKO mice were still able to clear P. carinii. Finally, micro MT and CD40KO chimeric mice had reduced numbers of activated CD4(+) T cells in the lungs and lymph nodes compared with wild-type mice, suggesting that B cells are important for activation of T cells in response to P. carinii. Together these data indicate that P. carinii-specific IgG plays an important, but not critical, role in defense against P. carinii. Moreover, these data suggest that B cells also mediate host defense against P. carinii by facilitating CD4(+) T cell activation or expansion.     

T cell costimulatory molecule function determines susceptibility to infection with Pneumocystis carinii in mice

Loss of T cell number and function during HIV infection or secondary to pharmacologic immunosuppression renders individuals susceptible to opportunistic infections, including Pneumocystis carinii pneumonia. Because costimulatory receptors are critical for optimal T cell function, we hypothesized that these proteins would regulate susceptibility to opportunistic infections. We found that despite normal T cell numbers, mice deficient in the costimulatory molecules CD2 and CD28 spontaneously developed P. carinii pneumonia. In experiments using intratracheal injection of P. carinii organisms to induce infection, the loss of CD28 alone was sufficient to render mice susceptible to acute infection; however, the organism was eventually cleared. Examination of inflammatory responses to P. carinii revealed that mice deficient in both CD2 and CD28 accumulated CD8(+) T cells in their lungs in response to infection and demonstrated markedly reduced specific Ab titers. Analysis of cytokine profiles suggested that regulation of IL-10 and IL-15 may be important elements of the response to this pathogen. Thus, costimulatory molecule function is critical in determining the initial susceptibility to infection with P. carinii. Analysis of immunologic responses in these mice may provide important insights into the defects that render individuals susceptible to opportunistic infection, and provide opportunities for novel immunologically based therapies.     

Neonatal T cells in an adult lung environment are competent to resolve Pneumocystis carinii pneumonia

Initiation of the pulmonary inflammatory response to Pneumocystis carinii is delayed by 3 wk in mice infected as neonates compared with adults. There was no difference in the proliferative response of draining lymph node T cells from mice infected as neonates compared with adults when stimulated in vitro with either Con A or anti-CD3 mAB: However, TNF-alpha and IFN-gamma mRNA expression in the lungs of P. carinii-infected neonates was significantly lower than in adults indicating a lack of appropriate activation signaling in the local environment. This may have been due to active suppression because TGF-beta mRNA expression was significantly elevated in neonatal lungs compared with adults. To determine whether T cells from 10-day-old mice would effect resolution of P. carinii if harbored in an adult lung environment, cells were adoptively transferred to SCID mice with established P. carinii infections. There was no difference in the kinetics of T cell migration into the lungs or of clearance of P. carinii organisms when SCID mice were reconstituted with splenocytes from young mice as compared with adult mice. Furthermore, splenocytes from young mice stimulated both TNF-alpha and IFN-gamma mRNA expression to levels that were similar to that in the lungs of SCID mice reconstituted with adult cells. These data indicate that neonatal lymphocytes are competent to resolve P. carinii infection when harbored in an adult lung environment, suggesting that the neonatal lung environment, and not the T cells, is ineffective at responding to P. carinii infection.     

Inoculated mouse model of Pneumocystis carinii pneumonia.

A transtracheally inoculated mouse model of Pneumocystis carinii has been developed using BALB/c mice, a widely available strain free of latent P. carinii infection. The mean infectivity score of untreated inoculated mice was 4.1 compared to the mean infectivity score of 0.1 for trimethoprim/sulfamethoxazole (50/250 mg/kg) treated inoculated mice, approximately a four-log difference. An inoculated mouse model of P. carinii infection provides both a source of organisms from a different host and an animal model for study of drugs for therapy and prophylaxis which is less costly than rats and which requires less drug than required for rats.     

Introduction of Pneumocystis carinii in a colony of SCID mice.

Pneumocystis carinii-free SCID mice were housed closely exposed to corticosteroid-treated non-SCID mice in a conventional area of our laboratory animal facilities. A one-day exposure was sufficient for P. carinii transmission. The lung infection increased thereafter. Irradiation or splenectomy of SCID mice at the beginning of the exposure resulted in a marked increase of parasite multiplication. Extrapulmonary foci of pneumocystosis were detected in heart and spleen of SCID mice infected by P. carinii via air transmission.     

A survey of Pneumocystis carinii infection in research mouse colonies in Japan.

To determine the frequency of Pneumocystis carinii infection in mouse colonies maintained for biomedical research in medical colleges or medical faculties in universities in Japan, 409 nu/nu mice were sent to 43 animal facilities from a P. carinii-free colony. The animals were housed for 6 months in groups of 3 to 10 animals per room, and examined for the presence of parasites and infection. Colonies in 10 (24.4%) of 41 facilities were positive for the infection. Of 383 animals in 69 rooms, the organism was detected in 66 (17.2%) animals in 13 (18.8%) rooms. The difference in the proportion of rooms where mice were positive for P. carinii is clearly seen among these three groups; SPF mouse rooms (4 of 38 rooms, 10.5%), SPF mouse rooms with breeding units (5 of 25 rooms, 20.0%) and conventional mouse rooms (4 of 6 rooms, 66.7%). The survey indicates that strict housing arrangements and husbandry techniques are necessary to keep SPF mice free from P. carinii infection.     

Establishment of Pneumocystis carinii in various mouse strains using natural transmission to initiate infection.

A mouse model for Pneumocystis carinii has now been established in several strains of mice: C3Heb/FeJ, C3HeN, Balb/c, DBA/2N and athymic. In lieu of using invasive methods for initiating P. carinii infections, mice infected with P. carinii (seed mice) transmitted the disease to mice without latent infection via short term co-habitation. Acute infections in recipient mice developed approximately 5-6 wk after C3Heb/FeJ seeds were removed, while control unseeded litter-mates remained uninfected. This approach allows investigators to consistently transmit P. carinii to mice and to select the strain of mouse desired for use in a particular study.