艾滋病合并肺孢子菌肺炎1例

本文报道1例通过肺组织活检明确诊断的艾滋病合并肺孢子菌肺炎(Pneumocystis carinii pneumonia,PCP)病例,结合文献复习,分析艾滋病合并PCP的病理学特点及临床诊治措施。本例患者经实验室检查确诊为艾滋病,通过气管镜肺活检取得肺组织标本,组织病理学诊断为PCP,给予复方磺胺甲唑治疗后病情好转。PCP多见于艾滋病等免疫缺陷患者,临床上表现为间质性肺炎,提高对该病的认识并尽早进行病原学检测是确诊的关键。尽早使用复方磺胺甲唑等有效药物是改善预后的主要措施。     

Multi-Gene Family of Major Surface Glycoproteins of Pneumocystis carinii: Full-Size cDNA Cloning and Expression

The major surface glycoprotein (MSG) of Pneumocystis cariniiplays a crucial role in the fatal pneumonia caused by this organismin AIDS patients. A cDNA encoding a full-length MSG polypeptidewas isolated from a phage library of rat-derived P. cariniicDNAs. The deduced MSG, referred to as the MSG5 subtype, isa 120,765-Da protein composed of 1,076 amino acids and containsan anchoring hydrophobic sequence at the C-terminus of the protein.Sequence analyses of cloned MSG-cDNAs revealed an MSG-gene familywith 70% protein sequence identity between subtypes. P. cariniikaryotype hybridization analyses indicated that the MSG genefamily members are scattered throughout most of the P. cariniichromosomes. These recombinant MSG proteins reacted with theantiserum from P. carinii-infected rats, as expected, and antiserumgenerated against P. carinii-infected mice, indicating the existenceof common determinants in MSG polypeptides. The family of MSGproteins is rich in cysteine residues and these cysteine arehighly conserved in all MSG subtypes regardless of species specificity,suggesting the structural and/or functional importance of thesecysteine. The pathobiological significance of the MSG gene familyand its sequence diversity in P. carinii is discussed.     

Analysis of Pneumocystis carinii Cyst Wall I. Evidence for an Outer Surface Membrane

ABSTRACT. It has long been thought that the cyst form of Pneumocystis carinii , which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii . This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a β-1–3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.     

Genetic Diversity of Pneumocystis carinii Derived from Infected Rats, Mice, Ferrets, and Cell Cultures

ABSTRACT. The degree of strain and/or species diversity among Pneumocystis carinii isolates is unknown. As a first approach to the study of P. carinii genetic relatedness, we compared the pulsed field gel electrophoretic karyotypes of P. carinii derived from lung homogenates of three immunosuppressed host animals: rats transtracheally inoculated with P. carinii -infected rat lung; mice transtracheally inoculated with P. carinii -infected mouse lung; and ferrets which developed reactivated latent P. carinii pneumonia. Rat P. carinii propagated on HEL299 cells was also examined. Karyotypes of P. carinii DNA from both rat lung homogenate and cell culture were identical (14 bands, 315–680 kb). In contrast, mouse and ferret P. carinii DNA karyotypes were each distinctly different from the rat P. carinii samples (mouse P. carinii 15 bands, 315–610 kb; ferret P. carinii nine bands, 410–760 kb). Three distinct rat P. carinii gene probes reacted with both Southern-transferred rat and mouse P. carinii DNA but not with ferret P. carinii DNA. Thus, P. carinii from rat, mouse, and ferret are genetically diverse. The results are consistent with recently reported antigenic and nucleic acid sequence differences among P. carinii isolates recovered from different hosts.     

Separation of Pneumocystis carinii from the lung of the steriod-suppressed rat

Pneumocystis carinii pneumonia was induced in rats by chronic corticosteroid immunosuppression. The parasite was separated from virtually all contaminating host cells using the technique of unit gravity sedimentation. Cellular contamination was routinely below 0.02%. The same technique allowed partial separation of the cyst from the trophozoite stage.     

Regulation of the plasma membrane potential in Pneumocystis carinii

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.     

A Tandem Repeat of Rat-derived Pneumocystis carinii Genes Encoding the Major Surface Glycoprotein

ABSTRACT. A fragment from the genome of rat-derived Pneumocystis carinii was found to contain two MSG genes arranged as a direct repeat. The sequences from one gene (MSG B), the region between the two genes, and part of the second gene (MSG A) were determined. The two MSG genes were not identical in sequence. The open reading frames of MSG A and MSG B encode non-identical proteins, both of which are similar to that encoded by a previously published cDNA. The MSG B gene sequence showed no evidence of introns. The 5'and 3'untranslated regions of the MSG gene pair were highly conserved, but the regions immediately upstream of the open reading frames of MSG A and B were different from the region upstream of a previously characterized MSG cDNA. Primers designed to extend upstream of the 5'end of MSG and downstream of the 3'end of MSG were used in a polymerase chain reaction with total genomic P. carinii DNA as template. Presumptive intergenic amplification products from this reaction were cloned and sequenced. The sequences of these regions were similar but distinct, indicating that tandem arrangement of MSG genes is a common organizational motif.     

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Summary— The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouseor rat-derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn-binding receptor is present at the surface of mouse-derived P carinii organisms.     

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.     

Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae

The Pneumocystis carinii gene encoding the enzyme dihydrofolate synthase (DHFS), which is involved in the essential biosynthesis of folates, was isolated from clones of the Pneumocystis genome project, and sequenced. The deduced P. carinii DHFS protein shares 38% and 35% identity with DHFS of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. P. carinii DHFS expressed from a plasmid functionally complemented a S. cerevisiae mutant with no DHFS. Comparison of available DHFSs with highly similar folylpolyglutamate synthases allowed the identification of potential signatures responsible for the specificities of these two classes of enzymes. The results open the way to experimentally analyse the structure and function of P. carinii mono-functional enzyme DHFS, to investigate a possible role of DHFS in the resistance to antifolates of P. jirovecii, the species infecting specifically humans, and to develop a new class of antifolates.     

中药补骨脂及鸦胆子对大鼠卡氏肺孢子虫肺炎的防治研究

目的探讨中药补骨脂及鸦胆子对大鼠卡氏肺孢子虫肺炎的防治效果。方法用地塞米松皮下注射建立大鼠卡氏肺孢子虫肺炎动物模型,用补骨脂、鸦胆子及两者合剂治疗实验鼠,观察其肺脑组织病理、肺脑组织和血清中MDA(丙二醛)、XOD(黄嘌呤氧化酶)、GSH(谷胱甘肽)等指标变化。结果补骨脂、鸦胆子及合剂治疗实验大鼠体重明显回升,与模型对照差异有显著性(P<005);各治疗组的包囊减少率均高于60%,合剂治疗组不优于各单独治疗组,鸦胆子及合剂治疗组清除氧自由基能力较强。结论鸦胆子及补骨脂对大鼠卡氏肺孢子虫肺炎具有一定疗效。     

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott’s methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.     

High-Speed Cell Sorting of Infectious Trophic and Cystic Forms of Pneumocystis carinii

ABSTRACT. The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6±0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.