Essential eukaryotic core

The AIDs-related fungal pathogen Pneumocystis carinii is unusual in having a remarkably compact genome of 7.7 megabase pairs (mbp) whose small size presents the opportunity to identify the essential eukaryotic core of genes. The essential eukaryotic core is defined to be a collection of essential genes shared by all eukaryotes. Sequencing the 3' ends of more than 5500 cDNAs from P. carinii allowed us to identify about 200 genes shared with its nearest known but distant relative, Schizosaccharomyces pombe and also Saccharomyces cerevisiae, and with homologs known to be essential in S. pombe or S. cerevisae. As the cDNA library contains about one half of the P. carinii genes, the size of the essential eukaryotic core (approximately 400) is slightly larger than the prokaryotic core (265-350) being identified by studies of the bacterial pathogen Mycoplasma genitalium. The collection of genes in the essential eukaryotic core may prove useful in identifying new broad spectrum antifungal drug targets.     

Genotyping of Pneumocystis carinii f. sp. hominis isolates in Japan based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes

Genotyping of Pneumocystis carinii (Pc) isolated from 24 bronchoalveolar lavage (BAL) fluid specimens in Japan was examined based on nucleotide sequence variations in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2, respectively) of rRNA genes. We found 11 ITS1 genotypes including 2 novel ones and 11 ITS2 genotypes including 3 new ones. Combining the ITS1 and ITS2 genotypes resulted in 30 ITS genotypes, of which 10 are newly described in this report. Two or more genotypes in ITS regions in a specimen were observed in 16 of 24 patients. Our results will be of help for the epidemiological investigation of Pc infection.     

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。     

Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4⁺ and CD8⁺ T cells and an increase of activated T cells expressing IL-2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-γ plays an important role in the protection of mice from P. carinii infection.     

Genes Encoding Antigenic Surface Glycoproteins in Pneumocystis from Humans

Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS-associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal-derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross-react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human-derived Pneumocystis. The cloned human-derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human-derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.     

Evaluation of the Sensitivity, Specificity, and Predictive Value of Monoclonal Antibody 3f6 For the Detection of Pneumocystis Carinii Pneumonia In Bronchoalveolar Lavage Specimens and Induced Sputum

The sensitivity and specificity of different staining procedures for the detection of Pneumocystis carinii organisms were compared. Three conventional stains (Papanicolaou, Giemsa and Grocott's) and one immunocytochemical stain using 3F6 antibody were used on smears prepared from the same specimen. Bronchoalveolar lavage (BAL) and induced sputum (IS) specimens were used for this purpose. One hundred and sixty-five episodes from 142 patients were investigated by the four different staining techniques. Cysts of P. carinii were detected in 64 episodes from 63 patients. Immunocytochemical staining with 3F6 was found to be slightly more sensitive at detecting the cysts than Grocott's, Giemsa, or Papanicolaou stain. La sensibilité et la spécificité des différentes méthodes de coloration pour la détection de Pneumocystis carinii ont été comparées. Trois colorations conventionnelles (Papanicolaou, Giemsa et Grocott) et un immunomarquage avec l'anticorps 3F6 ont été utilisés sur des frottis d'un même échantillon. Le liquide de lavage bronchoalvéolaire (LBA) et l'expectoration provoquée (EP) ont constitué le matériel d'étude. Cent soixante cinq échantillons provenant de 142 patients ont étéétudiés avec ces 4 techniques de coloration. Les kystes de P. carinii ont été détectés dans 64 échantillons chez 63 malades. L'immunocytochimie avec l'anticorps 3F6 est légèrement plus sensible pour la détection des kystes que le Grocott, le Giemsa ou le Papanicolaou. Die Sensitivität und Spezifität von verschiedenen Färbeverfahren wurden hinsichtlich des Nachweises von Pneumocystis carinii verglichen. Hierbei wurden Papanicolaou-, Giemsa- und Grocott-Färbung sowie der immunocytochemische Nachweis mit dem Antikörper 3F6 am selben Material verglichen, wobei sowohl BAL als auch induziertes Sputum verwandt wurden. 165 Episoden von 142 Patienten wurden mit den 4 Darstellungsverfahren untersucht. P. carinii-Cysten wurden in 64 Proben von 63 Patienten gefunden. Der immuncytochemische nachweis mit 3F6 erwies sich als etwas sensitiver als Grokott-, Giemsa- oder Papanicolaou-Färbung.     

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.     

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.