Rat Pneumocystis Carinii Research Articles

Gene Cloning, Expression, and Immunological Characterization of Kexin in RatPneumocystis carinii

Objective: Pneumocystis is an opportunistic pathogen that often causes fatal pneumonia in immunocompromised patients. It remains an important cause of morbidity and mortality in immunocompromised hosts. Therefore, the development of a vaccine is needed. Kexin is a furin-like protease which may be involved in the proteolytic processing of other proteins of Pneumocystis. We focused on the cellular and humoral responses to the kexin antigen fragment of Pneumocystis in our study. Methods: The full-length DNA sequence of the kexin gene was amplified from rat Pneumocystis carinii (P. carinii). The recombinant plasmid pET-kexin was constructed, and the recombinant antigen Hiskexin was purified, respectively. Then the mice were immunized with His-kexin. To ensure the isolated protein used in the next experiments was purified and of the right molecular weight, Western blotting analysis was performed. Then the specific humoral immunity was measured by ELISA. The specific cellular immunity was measured by the proliferation response of spleen lymphocytes. Results: The full-length Pneumocystis kexin gene was cloned successfully. The sera could recognize another recombinant antigen GSTkexin containing the identical amino acid sequence of kexin fragment with His-kexin, as identified by Western blotting and ELISA. The splenocytes collected from the mice immunized with His-kexin had significantly proliferative responses to GST-kexin compared to the control animals. Conclusions: These findings showed that kexin was the potential antigen to induce immune defense against P. carinii infection. The result would provide basis for the further vaccine study in the development of immunoprophylaxis and immunotherapy against P. carinii infection.

Antitumor and Anti-Pneumocystis Carinii Activities of Novel Bisbenzamidines

Among a library of 17 bisbenzamidines connected with various linkers, compounds with a flexible pentanediamide (10) or hexanediamide (12) linker were the most potent derivatives against rat Pneumocystis carinii (IC50 values of 3 and 2 nM, respectively) and had the highest selectivity index ratios (GI50 of human tumor cells/IC50 of rat P. carinii cells) of >104. Seven compounds caused 50% growth inhibition (GI50) of tumor cells at concentrations of <100 μM while the remaining ten were not cytotoxic. DNA binding affinity (ΔTm) of the tested compounds did not correlate with either their anti-P. carinii activity or cytotoxicity.

Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli

Pneumocystis pneumonia or PCP is caused by Pneumocystis jirovecii, an obligate parasite of the human lung. In this study P. jirovecii genomic sequence encoding FAS, a trifunctional protein including dihydroneopterin aldolase (DHNA), hydroxymethyldihydropterin pyrophosphokinase (PPPK) and dihydropteroate synthase (DHPS) were identified by PCR amplification from fixed broncheolar lavage samples from patients having Pneumocystis pneumonia. The P. jirovecii trifunctional DHNA–PPPK–DHPS genes (PjFAS) showed a high degree of conservation with the rat Pneumocystis carinii and P. carinii f. sp. macaca sequences. To test the functionality of the PjFAS sequences introns were removed followed by cloning and expression of PjFAS sequences in a DHPS-disrupted Escherichia coli strain. Complementation depended on the presence of N-terminal FAS sequences in addition to a glutathione S- transferase tag to the N-terminus of PjFAS. Functional complementation allowed evaluation of DHPS mutations implicated with sulfa drug resistance.

Detection of Pneumocystis carinii by in situ hybridization in the lungs of immunosuppressed rats.

In situ hybridization was performed to detect rat Pneumocystis carinii in the lung sections. Rats were immunosuppressed by weekly subcutaneous injection of 10 mg/kg methylprednisolone. On the 6th, 8th and 9th week of immunosuppression, the lungs were removed and fixed in 10% neutral formalin. A 22 base oligonucleotide probe complementary to P. carinii 5S ribosomal RNA was commercially synthesized and its 3' terminal was labeled with biotin. In situ hybridization was performed utilizing manual capillary action technology on the Microprobe system. P. carinii were detected along the luminal surface of alveolar pneumocytes, in exudate of alveolar cavities, and also in secretory material of bronchioles. In the 6th week group, positive reaction was observed focally in the peripheral region of the lung sections, but the reaction was observed diffusely in the 8th or 9th week groups. In comparison with Grocott's methenamine silver stain, in situ hybridization technique can detect the organism rapidly, and can detect trophic forms very well. Furthermore, no nonspecific reaction with other pathogenic fungi and protozoa was recognized. Therefore, in situ hybridization can be a good technique to detect P. carinii in the lungs of infected rats.

Serial indium-111-labelled IgG biodistribution in ratPneumocystis carinii pneumonia: a tool to monitor the course and severity of the infection

To study the effect of new therapeutic strategies, we developed an animal model to monitor the course and severity of experimental Pneumocystis carinii pneumonia (PCP) in rats. P. carinii density scores in Giemsa-stained impression smears were used to follow P. carinii load. Indium-111 labelled IgG scintigraphy and biodistribution, histology of paraffin-embedded tissue sections, lung/body weight (L/B wt) ratio and cell count and differentiation of broncho-alveolar lavage (BAL) fluid were used as parameters of host inflammatory response. Statistically significant differences in L/B wt ratio, number of neutrophils in BAL fluid, P. carinii density score, histological extent of inflammation and 111In-IgG accumulation in the lung were seen between the rats sacrificed at various time points. 111In-IgG accumulation in the lung correlated well with L/B wt ratio and P. carinii density score and correlated moderately with number of neutrophils in BAL fluid and with the histological extent of inflammation.

Occurrence of Specific Sterols in Pneumocystis-carinii

We have examined the sterol composition and biosynthesis of rat Pneumocystis carinii. We found a number of lipid components among the nonsaponifiable fraction that appear unique to P. carinii. These lipids are present in P. carinii purified preparations and P. carinii -infected whole lungs but absent from control lungs. They show chromatographic properties typical of sterols on GC and HPLC; they are present in free and esterified sterol fractions isolated by thin layer chromatography, they are precipitated by digitonin and they are labeled upon incubation of purified P. carinii cell suspensions with [ 3H] mevalonate and [ 3H] squalene. The finding of these sterols opens new opportunities for the identification of chemotherapeutic targets against P. carinii.

Patent Evaluation : WO9307274-: General Hospital Corp: Cloning ofPneumocystis cariniisurface antigens and their therapeutic uses

Novelty: The genes encoding Pneumocystis carinii surface antigens have been cloned. These genes could be used to design probes for diagnostic purposes, the preparation of vaccines and the production of targeted therapeutic methods for treating Pneumocystis carinii infections.Biology: A cDNA expression library derived from rat Pneumocystis carinii was screened with monoclonal antibodies active against the gp116 major surface antigen of rat. A sequence containing a 1197 bp open reading frame was obtained and sequenced. This sequence was cloned into a Vaccinia vector.

Proliferation of rat Pneumocystis carinii on cells sheeted on microcarrier beads in spinner flasks.

A method of growing rat Pneumocystis carinii with human embryonic lung fibroblasts (HEL-299 cells) sheeted onto microcarrier beads has been developed. This method allows production of large quantities of P. carinii organisms with very little contamination of host cells. A fivefold increase in the numbers of organisms was achieved, as determined by organism count, antigen detection, and DNA quantification. The majority of organisms produced by this method are trophozoites.

Novel pentamidine analogs in the treatment of experimental Pneumocystis carinii pneumonia

We have recently demonstrated that substitution of imidazoline moieties for the amidine groups of pentamidine produces a molecule that is effective against rat Pneumocystis carinii pneumonia and that is apparently less toxic than pentamidine. For this reason, 10 novel imidazoline substituted compounds were evaluated for their effect against rat P. carinii pneumonia. While several of the new compounds were observed to have advantages over pentamidine in the treatment of disease in the rat model, only one compound stood out as a potential new clinical agent. Treatment for 2 weeks with intravenous (i.v.) doses of 1,3-di(4-imidazolino-2-methoxyphenoxy)propane (DIMP) at 1 mg/kg per day produced an anti-P. carinii pneumonia effect equivalent to i.v. doses of pentamidine at 10 mg/kg per day. Although pentamidine and one of the test drugs, 1,3-di(4-imidazolinophenoxy)propane, showed no activity against P. carinii pneumonia when administered per os, DIMP exhibited potent anti-P. carinii pneumonia activity when given by daily gavage doses of 40 and 25 mg/kg. DIMP retained significant activity when given every other day by a gavage dose of 25 mg/kg. No toxicity was observed with the drug at any of the dose levels or by either of the routes of administration. However, the low solubility of the drug prevented testing at higher i.v. doses. Our conclusion is that DIMP has the potential of providing a safer and more effective alternative to pentamidine for the treatment of P. carinii pneumonia.

Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies.

A combination of three monoclonal antibodies, two prepared against human and one against rat Pneumocystis carinii, was used in an indirect fluorescent-antibody stain (IFA) to diagnose P. carinii in both bronchoalveolar lavage and lung biopsy specimens. This combination of monoclonal antibodies was specific for P. carinii and yielded bright fluorescence of both P. carinii cysts and trophozoites. A total of 126 specimens from 93 patients were stained for P. carinii by a toluidine blue O stain and the monoclonal IFA. Forty-five (35.7%) of these were positive for P. carinii by toluidine blue O, and 43 (34.1%) were positive by IFA. There was 98.4% agreement between both methods, with no false-positive and two false-negative occurrences by IFA. An IFA procedure with monoclonal antibodies such as those used in these studies can provide a simple, fast, and sensitive method for diagnosing P. carinii pneumonia by microbiology laboratories.

A comparison of the antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting.

The antigenic characteristics of rat Pneumocystis carinii obtained from infected lungs and grown in tissue culture were compared with the properties of human P. carinii obtained from the lungs of AIDS and non-AIDS patients by the immunoblotting technique, using different sources of antibody. Major immunoreactive bands of 45, 50, and 116 kd were found in both lung and tissue culture-derived rat P. carinii, suggesting the organism retains its antigenic characteristics in short-term culture. The principal immunoreactive bands in human P. carinii included a band of 40 kd, and to a lesser extent, a band of 66 kd; these antigens were found in the lungs of six and seven AIDS patients but in only one of eight non-AIDS patients with pneumocystosis. The rat and human P. carinii antigens reacted with sera from immunized rabbits, from rats with pneumocystosis and prolonged environmental exposure to the organism, from AIDS and non-AIDS P. carinii patients, and from healthy blood donors. Reactivity of these antigens could be removed by adsorption of antisera with P. carinii-infected lungs but not with normal lungs or lungs infected with bacteria and fungi. We conclude that rat and human P. carinii have shared, as well as species-specific, antigenic determinants, which should be useful for a variety of studies with this organism.

Analyses of rat Pneumocystis carinii antigens recognized by human and rat antibodies by using western immunoblotting.

The major Pneumocystis carinii antigens inducing antibody responses in infected hosts were identified by Western immunoblotting techniques. The biochemical nature of these antigens was also elucidated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by protein staining revealed a major component with a molecular weight (MW) of greater than 205,000. This major component disappeared and a new major protein staining component of approximately 110,000 to 116,000 MW appeared when electrophoresis was done in the presence of beta-mercaptoethanol. Periodic acid-Schiff staining revealed that this major component contains carbohydrate moieties. A major component in the 55,000- to 60,000-MW region was visible with periodic acid-Schiff stain, but not with a protein stain, after electrophoresis in the presence of beta-mercaptoethanol. The majority of sera tested from humans with diagnosed pneumocystosis and from rats allowed to recover from steroid-induced pneumocystosis reacted strongly with 110,000- to 116,000-, and 55,000- to 60,000-MW components. These sera often, but not always, detected antigens with MWs of approximately 170,000, 125,000, and 30,000 to 32,000. The data suggest that the antigenic composition of P. carinii is relatively complex and that rat and human P. carinii probably share antigenic determinants. Competitive studies between infection-derived human and rat antisera for the major rat P. carinii components revealed competition; rat antisera appeared to recognize a greater range of antigenic epitopes than did human antisera. Protease treatment of the antigenic components that had been immobilized on nitrocellulose paper destroyed their antigenic reactivity with rat antibody. Treatment with sodium periodate decreased reactivity of this 110,000- to 116,000-MW component and completely destroyed the reactivity of the 55,000- to 60,000-MW component with rat antibody.

Method of testing the susceptibility of Pneumocystis carinii to antimicrobial agents in vitro.

Rat Pneumocystis carinii grown on lung-derived cell lines in tissue culture flasks and multiwell plates was tested for susceptibility to four antimicrobial agents currently being used in the treatment of human pneumocystosis. Standard criteria for organism quantitation, replication, viability, and inoculum size were established. Trimethoprim-sulfamethoxazole inhibited P. carinii growth at a concentration ratio of 1:19 microgram/ml, and pentamidine isethionate was active at 0.1 microgram/ml. alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, inhibited P. carinii at a concentration of 1 mM once erythrocytes (which are high in polyamine content) were removed from the inoculum; this effect could be overcome by the polyamine putrescine. Dapsone suppressed P. carinii replication at a dose of 0.1 microgram/ml, but this effect was lost after 72 h in culture. Overall, the reduction in P. carinii numbers with these drugs was relatively modest (45 to 84%), which is consistent with their lack of lethal effects on the organism in vivo. Thus, the system presented here should be helpful in developing new anti-P. carinii agents and in elucidating their mechanism of action.

Pneumocystis carinii: Growth variables and estimates in the A549 and WI-38 VA13 human cell lines

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.