地塞米松抑制卡氏肺孢子菌肺炎β2-防御素和Dectin-1受体的表达

目的研究卡氏肺孢子菌肺炎（Pneumocystis carinii pneumonia,PCP）鼠肺Dectin-1和β2-防御素的表达变化,探讨地塞米松对Deetin-1和B2-防御素的影响与疾病发生的相互关系。方法实验分4组：正常对照组、Pc刺激组、PCP模型组以及PCP模型恢复组。免疫抑制方法建立PCP动物模型,改良四胺银（Groeoti’s methenamine—silver nitrate method,GMS）染色检测Pc包囊;肺组织切片HE染色观察肺组织病理变化;实时荧光定量和Westernblot检测Deetin-1和B2-防御素的mRNA以及蛋白的表达。结果Pc刺激组的Dectin．1和β2-防御素的mRNA以及蛋白的表达明显高于正常对照组（P〈0．05）;Pc刺激组和PCP恢复组的Dectin-1和β2-防御素的mRNA以及蛋白显著高于PCP组（P〈0．05）,而PCP恢复组与PCP组肺部炎症无明显差别。结论对免疫功能正常宿主,Dectin-1受体和p2-防御素可能在防Pc感染中起重要作用;地塞米松抑制了鼠肺Deetin-1和β2-防御素的表达,这可能与PCP疾病的发生和发展有关。     

Immunologic comparisons of Pneumocystis carinii strains obtained from rats, ferrets, and mice using convalescent sera from the same sources.

Pneumocystis carinii (Pc) infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or trans-tracheal inoculation of Pc obtained from infected lungs of the homologous species (rat, mouse). Convalescent antisera were obtained by stopping dexamethasone treatment after 2-4 wk and allowing 5-8 wk for recovery. Parasites from infected lungs were purified by differential filtration, solubilized in loading buffer, subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and blotted to polyvinylidene fluoride sheets for Western analysis. Antisera from each animal species were reacted on Western blots of antigens from rat, ferret, and mouse. Each combination of antigen and antibody from the same species of animal showed reaction with 5 or more bands of Pc antigen. Convalescent mouse antibody did not react with rat or ferret antigens. Convalescent rat antibody reacted with a mouse antigen at about 66 kDa but not with ferret antigen, and convalescent ferret antibody showed minimal, probably non-specific reactions with both rat and mouse antigens. Variations in reactions indicate antigenic differences in Pc strains infecting these animals.     

Plasminogen activator production in a rat model of Pneumocystis carinii pneumonia

Several studies have indicated that the serine protease urokinase-plasminogen-activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid-induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase-PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell-associated uPA was observed in rats with PCP. This increase appears to be produced in response to P carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone-treated animals. Our results suggest that healthy AMs respond to the presence of P carinii with an increase in uPA production and that this response in immunodepressed rat-AMs is partially impaired.     

Pneumocystis carinii infection causes lung lesions historically attributed to rat respiratory virus

Idiopathic lung lesions characterized by dense perivascular cuffs of lymphocytes and a lymphohistiocytic interstitial pneumonia have been noted in research rats since the 1990s. Although the etiology of this disease has remained elusive, a putative viral etiology was suspected and the term 'rat respiratory virus' (RRV) has been used in reference to this disease agent. The purpose of this study was to determine whether Pneumocystis carinii infection in immunocompetent rats can cause idiopathic lung lesions previously attributed to RRV. In archived paraffin-embedded lungs (n = 43), a significant association was seen between idiopathic lung lesions and Pneumocystis DNA detected by PCR. In experimental studies, lung lesions of RRV developed in 9 of 10 CD rats 5 wk after intratracheal inoculation with P. carinii. No lung lesions developed in CD rats (n = 10) dosed with a 0.22-μm filtrate of the P. carinii inoculum, thus ruling out viral etiologies, or in sham-inoculated rats (n = 6). Moreover, 13 of 16 CD rats cohoused with immunosuppressed rats inoculated with P. carinii developed characteristic lung lesions from 3 to 7 wk after cohousing, whereas no lesions developed in rats cohoused with immunosuppressed sham-inoculated rats (n = 7). Both experimental infection studies revealed a statistically significant association between lung lesion development and exposure to P. carinii. These data strongly support the conclusion that P. carinii infection in rats causes lung lesions that previously have been attributed to RRV.     

Proliferation patterns of latent Pneumocystis carinii in rat organs during progressive stages of immunosuppression

Pneumocystis carinii is known to proliferate mainly in the lung of an immunocompromised host. In AIDS and other immune disorders sporadic extrapulmonary presence of this organism has been documented. Occasionally, P. carinii does not appear to infect the lung. These observations have been based on the detection of P. carinii by conventional staining techniques. We have sought to determine the extent of these infections by the polymerase chain reaction (PCR) in a rat model. Harlan Sprague-Dawley rats weighing between 110 and 130 g were immunosuppressed with dexamethasone (1.2 mg/l) in drinking water. During progressive stages of immunosuppression 2 rats were sacrificed at 2, 3, 4 and 5 wk, and the lung, liver, kidney, spleen and bone marrow were taken. Sonicated crude extracts of the tissues were used as template DNA for the amplification of the dihydrofolate reductase (DHFR) gene of P. carinii. All the PCR products were analyzed by Southern hybridization with radiolabelled DHFR DNA. These analyses revealed a general trend of P. carinii proliferation first in bone marrow at 2 wk, followed by liver at 3 wk, and lung at 5 wk on immunosuppression. Kidney and spleen infections were infrequent. Although P. carinii appears to proliferate in the lung at later stages of immunosuppression, the degree of proliferation is several-fold greater than in extrapulmonary organs. The extrapulmonary proliferation of P. carinii, however small, may possibly suppress hematopoietic stem cell differentiation in bone marrow, and may also contribute to the pathology present in various organs.     

Treatment and prevention of Pneumocystis carinii pneumonia and further elucidation of the P. carinii life cycle with 1,3-beta-glucan synthesis inhibitor L-671,329.

Two different classes of 1,3-beta-glucan synthesis inhibitors, the echinocandins and papulacandins, have anti-Pneumocystis activity in an immunosuppressed rat model for acute P. carinii pneumonia (PCP). This activity combined with potent anti-Candida activity makes the echinocandins attractive agents for treating both Pneumocystis and candidiasis in the immunocompromised patient. Natural product echinocandin L-671,329 rapidly eliminates greater than 99% of the P. carinii cysts after 4 days of treatment at a dose of 1 mg/kg twice daily while 2-3 weeks of therapy with trimethoprimsulfamethoxazole (TMP-SMZ) or pentamidine was required to achieve the same degree of cyst clearance. Effects of L-671,329, TMP-SMZ and pentamidine on the trophozoite stage of P. carinii were also explored using a P. carinii-specific DNA probe to quantitate organism load. Although L-671,329 was not as effective as the known agents against the trophozoite stage, prophylactic use of L-671,329 at a daily dose of 1 mg/kg prevented the development of cysts and trophozoites in the rat model. The foamy exudate commonly seen in lungs of animals with PCP is also absent in rats receiving L-671,329 prophylaxis. In addition to demonstrating the potential of L-671,329 as a prophylactic agent these studies also help in elucidating the life cycle of P. carinii. The observation that L-671,329 prophylaxis prevents the appearance of trophozoites, while acute therapy does not directly affect trophozoites, provides the first evidence that the cyst stage is required for trophozoite proliferation. The rapid elimination of cysts by L-671,329 in animals with acute PCP also indicates that all cysts are turning over within 4 days since it is the development of new cysts which is prevented with this compound.     

Occurrence of Pneumocystis carinii in Canine Distemper

Pneumocystis carinii is a eukaryotic opportunistic pathogen causing pneumonia (PCP) in immunosuppressed patients. It is best known in human medicine as a pathogen of AIDS pa-tients and in immunosuppressed transplant and cancer patients (Waltzer 1993).     

Pneumocystis antigen appearance during immunosuppression

The sequential appearance of Pneumocystis carinii (Pc) antigens during the progression of immunosuppression in rats was studied using the immunoblotting technique and specific immunologic probes. Putative Pc soluble antigens, with molecular weights of approximately 70 and 90 kd, were detected as early as 2 wk after initiation of immunosuppression in rats using a pool of monoclonal antibodies produced to Pc isolated from lungs using enzymatic digestion. Monoclonal antibodies produced to Pc isolated by massaging the lung tissue using a Stomacher apparatus and infection-derived sera did not detect soluble antigens until at least the 6th wk of immunosuppression. Analysis of Pc pellets obtained from Stomacher- and lavage-processed lungs revealed that the lower molecular weight antigens (less than or equal to 40, 45 and possibly 55-60 kd) were recognized early during the immunosuppression process.     

Pneumocystis carinii pneumonia in the rat model.

Groups of barrier-raised but not certified virus-free Sprague-Dawley rats, obtained from the same source over the course of several years, were placed on an identical immunosuppressive regimen. This caused reactivation of latent Pneumocystis carinii infection, manifest as P. carinii pneumonia (PCP) of varying severity. Rats were euthanized after 9-12 wk of immunosuppression. An assessment of the severity of the induced PCP was made, based on the total number of organisms extracted from the lungs and their ability to proliferate in short-term cell culture. Serum samples obtained at sacrifice were tested by indirect immunofluorescence for antibodies to coronavirus, parvovirus, Sendai virus, pneumonia virus of mice (PVM) and Mycoplasma pulmonis. A total of 60 rats were examined. Thirty-four of these (57%) developed moderate or severe PCP. No antibodies were detected to either coronavirus or Mycoplasma pulmonis in any of the rats. Although antibodies were detected to parvovirus in 13/60 (22%), to PVM in 29/60 (48%), and to Sendai virus in 47/60 (78%), there was no apparent correlation between the presence or absence of antibodies to these agents and the severity of PCP. Sequential observations during the course of immunosuppression are needed to clarify the role of concomitant infections in the development of PCP.     

Vitamin B3 confers resistance to sulfa drugs in Saccharomyces cerevisiae

Sulfa drugs are ubiquitous antibiotics used to treat bacterial infections and diseases caused by eukaryotes, such as Pneumocystis carinii, the leading cause of pneumonia (PCP) in HIV patients. A daily regimen of sulfonamides and multivitamins including vitamin B3 is also recommended for persons with HIV. We show that exogenous vitamin B3 (nicotinate) confers resistance to sulfa drugs in Saccharomyces cerevisiae, a model for P. carinii. We propose a model of metabolic rerouting in which increased nicotinate leads to increased intracellular concentration of p-aminobenzoate, thus leading to sulfonamide resistance.     

Pneumocystis Antigen Appearance during Immunosuppression

The sequential appearance of Pneumocystis carinii (Pc) antigens during the progression of immunosuppression in rats was studied using the immunobloiting technique and specific immunologic probes. Putative Pc soluble antigens, with molecular weights of approximately 70 and 90 kd. were delected as early as 2 wk after initiation of immunosuppression in rats using a pool of monoclonal antibodies produced to I c isolated from lungs using enzymatic digestion. Monoclonal antibodies produced to Pc isolated by massaging the lung tissue using a Stomacher apparatus and infection-derived sera did not detect soluble antigens until at least the 6th wk of immunosuppression. Analysis of Pc pellets obtained from Stomacher- and lavage-processcd lungs revealed that the lower molecular weight antigens (≤ 40, 45 and possibly 55–60 kd) were recognized early during the immunosuppression process.     

Immunopathogenesis of Pneumocystis carinii pneumonia

Pneumocystis carinii pneumonia (PCP) is a life-threatening infection that occurs in immunocompromised individuals, particularly those with advanced human immunodeficiency virus (HIV) infection. Interestingly, morbidity and mortality is related to the underlying cause of immunosuppression, with AIDS patients faring better than oncology patients for example. In addition, the prognosis of PCP has been correlated with markers of inflammation rather than with organism numbers. There is now increasing evidence that lung damage occurring during PCP is a result of the type and extent of the host inflammatory response to P. carinii rather than a result of direct damage by the organism. This review will discuss the experimental and clinical data demonstrating how the host-mediated inflammatory response to infection with P. carinii determines the ultimate outcome of PCP. A better understanding of the pathophysiology of PCP should lead to the development of improved therapies for the treatment of PCP.     

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.     

Effect of nicotine on lung S-adenosylmethionine and development of Pneumocystis pneumonia

Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 microg of R-(+) nicotine kg(-1) h(-1) intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 microg kg(-1) h(-1) into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet.     

Treatment of experimental Pneumocystis carinii pneumonia with 1,3-di(4-imidazolino-2-methoxyphenoxy)propane lactate.

An analogue of pentamidine, 1,3-di(4-imidazolino-2-methoxyphenoxy)propane (DMP) lactate, was tested against rat Pneumocystis carinii pneumonia (PCP). The drug was found to be highly active in the treatment of rat PCP at a dose of 1.75 mg/kg (parent molecule) when administered by intravenous (i.v.) injection (daily for 2 wk). The compound was also active against PCP when given orally, however, significantly higher doses of DMP were necessary when compared to the i.v. dosing regimen. Prophylactic doses (i.v.) of the drug also proved highly effective in preventing PCP.     

Survival and Infectivity of Pneumocystis carinii Outside the Mammalian Host.

SUMMARY Air blower prefilters servicing HEPA-filtered Biobubble housing Pneumocystis carinii -infected rats were stored for up to five months at -80°C to room temperature. After storage, 76% of immunosuppressed rats exposed to these prefilters developed P. carinii infections. In contrast, only 4% of control immunosuppressed rats exposed to autoclaved filters had P. carinii infections. These observations indicate that the organism has a dormant form that remains infective for at least several months outside the mammalian host.     

Generalized immune response to Pneumocystis carinii infection in the lung.

We studied inflammatory cells retrieved by bronchoalveolar lavage (BAL) from immunocompromised patients with or without Pneumocystis carinii pneumonia (PCP). Twenty-four patients with PCP, and 20 patients without PCP underwent lavages of both an uninvolved lobe and the lobe involved in pulmonary infection. Patients without P. carinii, had a significant increase (p less than 0.02) in the percentages of neutrophils (22 +/- 7.1%, mean +/- SEM) and lymphocytes (16 +/- 3.8%) in the involved lobe compared to those in the uninvolved area (neutrophils: 9 +/- 4.8%; lymphocytes: 10 +/- 2.4%). Patients with PCP, had no differences between the % neutrophils or % lymphocytes in the involved vs. uninvolved lobes. Patients with PCP had more (p less than 0.01) P. carinii in the upper lobe (23 +/- 4.6 P. carinii clusters/500 cells) than the middle lobe (11 +/- 3.6). In PCP, despite regional infections, there was a diffuse inflammatory response.     

Pneumocystis carinii--animal production perspective.

Pneumocystis carinii is an important cause of pneumonia in immunocompromised human patients. The organism is also found as a saprophyte in the lungs of many species of animals. Animal models have been used as a source of P. carinii organisms for study of the disease. The rat model has been especially useful. Initially, the infection was latent in most colonies, and P. carinii pneumonia readily developed when animals were immunosuppressed. Today, many barrier raised rodent colonies are free of adventitious viruses, bacteria, Mycoplasma sp., and parasites, including P. carinii. Variability is now seen in the rat model. The use of cultured organisms to experimentally infect rats and mice prior to immunosuppression has met the need for some investigators, however, latent-infected, barrier-raised and isolator-raised rodents are still required. Colonies specifically infected with P. carinii can provide latent-infected animals and are better protected from potentially interfering organisms than barrier-raised animals. The development of these colonies is feasible as investigators and animal producers work together to define and develop this resource.     

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。     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.