Salim Merali

S-adenosylmethionine concentrations in diagnosis of Pneumocystis carinii pneumonia

Pneumocystis carinii is unable to synthesise S-adenosylmethionine and thus scavenges this intermediate. We aimed to test whether measurement of concentrations of this metabolic intermediate in plasma could provide a new method for rapid diagnosis of Pneumocystis carinii pneumonia (PCP). We measured S-adenosylmethionine plasma concentrations in 12 healthy controls, 16 patients with confirmed or suspected PCP, and 36 patients with other infections. Median concentration in healthy controls was 106 nmol/L (range 86-128), but the protein was undetectable in eight patients with histologically proven and seven with suspected PCP, and was 8 nmol/L in another confirmed case (p<0.0001). In 36 patients with other infections, S-adenosylmethionine concentrations were much the same as in controls: 18 had bacterial pneumonia, two tuberculosis, five cryptococcal meningitis, three had other infections, and eight had asymptomatic HIV-1 infection. After treatment for PCP, S-adenosylmethionine concentrations rose rapidly in all but one patient who died of the disease. Measurement of plasma S-adenosylmethionine concentrations could prove useful for diagnosis of PCP and assessment of patients response to treatment.

Polyamine analysis using N-hydroxysuccinimidyl-6-aminoquinoyl carbamate for pre-column derivatization

N-Hydroxysuccinimidyl-6-aminoquinoyl carbamate (AccQ.Fluor) was used as a polyamine pre-column derivatization reagent prior to HPLC analysis using a 5-micron C8 reversed-phase column. The fluorescence detector excitation wavelength was set at 250 nm and emission at 395 nm. Quantitation, reproducibility, linearity, recovery and stability were demonstrated. The lower limit of detection was 660 fmol. This method is 45 and 61 times more sensitive than those using the pre-column derivatizing agents dansyl chloride and orthophthalaldehyde, respectively. Applicability to biological samples was demonstrated by analyses of polyamines in extracts of mouse erythrocytes and Trypanosoma brucei brucei.

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 μg 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 μg 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.

Response of rat model of Pneumocystis carinii pneumonia to continuous infusion of deferoxamine.

The iron-chelating drug deferoxamine mesylate (DFO) is active against Pneumocystis carinii in vitro and in rat and mouse models of P. carinii pneumonia. Because DFO has a short half-life, daily divided or continuous dosage was expected to improve the dose response, as is the case with DFO treatment of malaria. Therefore, results of single daily intraperitoneal injections were compared with results of an evenly divided four-times-daily dosage and the efficacy of delivery with implanted infusion pumps. The highest bolus dosage (1,000 mg kg-1 of body weight day-1) was as effective as the standard combination of trimethoprim with sulfamethoxazole. Unexpectedly, very little improvement was observed with the divided or continuous dosage, and several mechanisms that could account for this are discussed.

Pneumocystis carinii Polyamine Catabolism

dl-α-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogenPneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 ± 0.5 pmol min−1 mg protein−1 for SSAT, similar to mammalian cells, and 6.42 ± 0.8 pmol min−1 mg protein−1 for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSATK m value of 25 μm spermidine is 20% of that of mammalian cells, and the PAO K m value of 14 nm N 1-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.

Subpopulations of Pneumocystis carinii Separated by a Percoll Gradient

Percoll (Pharmacia, Uppsala, Sweden), a colloidal suspension of polyvinylpyrrolidon-coated silica particles, is designed for separation of cells and subcellular organelles by isopycnotic density gradient centrifugation. This medium was reported as useful for isolating P. curinii from infected rat lungs [3] but the cells did not band at a single density, an observation confiied by us [unpublished]. Since this dispersion of P. cunnii in the Percoll gradient indicates a heterogeneity in the population of organisms obtained from rat lungs, we used Percoll to separate P. carinii cells that had already been purified by another method to isolate putative subpopulations. A shallow Percoll gradient produced two widely-separated sub-populations of cells isolated from rat lungs and these differed in morphological and physiological characteristics. MATERIALS AND METHODS. Rats were protected f7om bacterial infection with antibiotics, immunosuppressed by the addition of dexamethasone to the drinking water at 1.5 mg liter- and inoculated by instillation into the trachea of a 0.2 ml volume of lung homogenate prepared from a P. cunnii-infected rat. Development of P. cunnii pneumonia occurred within 3 4 weeks and the lungs were harvested. P. curinii cells were isolated as previously described [I]. Briefly, the method involved homogenization in buffer (1.34 mM KCl, 0.735 mM KHzP04,25.5 mMNazHPO4,3.72 mMNaHzP04, 31 mM NaCI, 0.025 mh4 CaC12, 0.025 mM MgC12, 50 m M dithiothreitol, pH 7.4 P I ) , differential Centrifugation, lysis of host cells with 0.85% m C 1 , treatment with DNase I, and washing in NKP. A 10 ml volume of isolated P. curinii was mixed with 30 ml of a solution containing Percoll at a concentration 20% of that supplied by Pharmacia; salts and dithiothreitol were added to match those of N U . This suspension was centrifuged at 18,000 g for 90 min. in a swinging bucket rotor thus forming a gradient and effecting an isopycnotic separation. After centrifugation, either visible bands or 1 ml fractions were collected, depending on the experiment. Percoll was washed from the fractions by diluting at least 3 fold with NKP and sedimenting the P. curinii cells by centrifugation at 5000 g and this wash was repeated once. The isolated fractions were suspended in NKP. RESULTS AND DISCUSSION. Figure 1 presents the distribution of P. curinii in the gradient and the gradient densities; note that no cells were found in the fractions between the bands. Figure 2 is a series of photomicrographs of the initially isolated cells and cells in the upper and lower bands after Percoll gradient centrifugation. Among 16 Percoll gradient separations done to date, the upper band contained from 0.21% up to 28% of the total trophozoites depending on the particular preparation. The proportion of trophozoites in the top band seemed to decline with increasing duration of infection. The lower band contained the balance of the trophozoites and all of the cysts. The mean size (kstd. dev) of the trophozoites of the upper band was 3.7 (M.7) pm by 4.3 (M.8) pm and those of the lower band 2.7 (M.6) by 3.0 (M.9) pm. The cytoplasm of the upper band trophozoites was frequently less intensely stained by Giemsa than that of the lower band. EM examination revealed that the upper band trophozoites had many more tubular extensions from the cell surface than trophozoites of the lower band (not shown). The ratio of polyamine content (nmoles polyamine [mg protein]-) of the upper band cells to that of lower band cells was 18.3 for putrescine, 7.1 for spermidine and 7.2 for spermine. The specific ornithine decarboxylase activity of the upper band was 3.38 times that of the lower band. Spermidine and spermine synthesis measured in cell-free extracts of the upper band and lower bands were 5.24 and 5.18 higher, respectively, in the upper band. The higher polyamine content and polyamine biosynthetic activity of the upper band cells suggest that these cells mav be more active in cell division than those of the lower band since increased polyamine content and polyamine biosynthesis is associated with actively cycling cells [2, 41. This is an initial report of separable subpopulations of trophozoites isolated from rat lungs and fiuther work is necessary to provide an explanation of the significance of this observation.

Pneumocystis carinii Infection Presenting as an Intra-Abdominal Cystic Mass in a Child with Acquired Immunodeficiency Syndrome

We describe the case of a pediatric patient with acquired immunodeficiency syndrome (AIDS) with an unusual large, fluid-filled intra-abdominal cystic lesion in which Pneumocystis carinii trophozoites were identified. Extrapulmonary P. carinii infection should be considered in the differential diagnosis of an intra-abdominal cystic mass in a child with AIDS.

Ubiquinone Synthesis and its Regulation in Pneumocystis carinii

ABSTRACT. The opportunistic pathogen Pneumocystis causes a type of pneumonia in individuals with defective immune systems such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), is effective in clearing mild to moderate cases of the infection. Rat‐derived Pneumocystis carinii was the first organism in which CoQ synthesis was clearly demonstrated to occur in both mitochondrial and microsomal subcellular fractions. Atovaquone inhibits microsomal CoQ synthesis with no effect on mitochondrial CoQ synthesis. We here report on additional studies evaluating CoQ synthesis and its regulation in the organism. Buparvaquone also inhibited CoQ synthesis and it reduced the synthesis of all four CoQ homologs in the microsomal but not the mitochondrial fraction. Glyphosate, which inhibits a reaction in the de novo synthesis of the benzoquinone moiety of CoQ reduced cellular ATP levels. Bacterial and plant quinones, and several chemically synthesized phenolics, flavanoids, and naphthoquinones that inhibit electron transport in other organisms were shown to reduce CoQ synthesis in P. carinii. The inhibitory action of naphthoquinone compounds appeared to depend on their molecular size and structural flexibility rather than redox potential. Results of experiments examining the synthesis of the polyprenyl chain of CoQ were consistent with negative feedback control of CoQ synthesis. These studies on P. carinii suggest that cellular sites and the control of CoQ synthesis in different organisms and cell types might be more diverse than previously thought.

Catecholamines inEntamoebae: Recent (re)discoveries

Free-living and enteric amoebae have similar two-stage life cycles, and both organisms depend on being able to monitor environmental conditions to determine whether to continue multiplying as trophozoites, or to differentiate into dormant or transmissible cysts. Conditions that support high trophozoite densities might also be expected to select for mechanisms of information exchange between these cells. We recently determined that trophozoites of at least one species ofEntamoeba release and respond to catecholamine compounds during differentiation from the trophozoite stage into the cyst stage. It turns out that this is not an entirely novel finding, as a number of previous studies have demonstrated parts of this story in free-living or enteric amoebae. We briefly review here major points of the previous studies and describe some of our recent results that have extended them.

Summary of Pneumocystis Research Presented at the 8th International Workshop on Opportunistic Protists

Pneumocysris pneumonia continues to represent a significant cause of morbidity and mortality in immune compromised patients with AIDS, hematological and solid malignancies, following organ transplantation, and in the setting of chronic immune suppression for inflammatory disorders [ 11. The International Workshops on Opportunistic Protists are convened every few years to present current research on basic and clinical aspects of this infection, and to bring together investigators for critical discussions of key aspects of the biology of these intractable organisms. The initial workshop was held in Bristol, England in 1988 and the 71h gathering occurred in June 2001 in Cincinnati, Ohio [2]. The gth International Workshop on Opportunistic Protists was held July 25-29, 2003 in Hilo, Hawaii. More than 80 scientific papers were presented in platform and poster foimat, with lively discussion and considerable insights being provided. Brief summaries of the major Pneumocystis research areas that were discussed are presented below. Colonization and carriage of Pneumocystis. Accumulating evidence was presented further supporting the concept that humans serve as a reservoir for Pneumocystis infection. Data from San Francisco documented that over 2/3 of patients with HIV, but with no microscopic evidence of Pneumocystis, had evidence of Pneumocystis jirovecii carriage or colonization by PCR. None of these individuals were treated and none developed Pneumocystis pneumonia (PcP) over the subsequent six weeks. Other individuals without suppressed immunity can also be colonized with Pneumocystis. Immunocompetent patients from Portugal with chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis were found to harbor P. jirovecii without overt PcP disease, as detected by PCR. Some of these individuals also carried mutations of the dihydropteroate synthase gene, irrespective of whether or not they had received sulfa-containing drugs. An additional study further documented asymptomatic carriage of Pneumocystis in patients with COPD, which appeared to correlate with the severity of obstruction. More severely obstructed individuals were found to have greater carriage of the organisms. A high incidence of R jirovecii colonization was also demonstrated in patients with cystic fibrosis. Finally, long-term colonization was observed in health care workers in an inpatients setting, who were studied by PCR analysis of oropharyngeal washings. Consistent with earlier observations that neonates are likely colonized with Pneumocystis shortly after birth [3], additional studies further supported the role of colonization of immune competent human infants and children. Using nasopharyngeal aspirates and PCR analysis, P. jirovecii was significantly more likely to be detected during periods of childhood respiratory infections, than during periods of good health. Colonization or carriage in household contacts was not consistently demonstrated. Epidemiology and genotyping of the organisms. The widespread availability of PCR has made further studies of the genotypes of P. jirovecii organisms derived from clinical specimens possible. Multilocus genotyping of P. jiivvecii was used to demonstrate that up to 13% of infection in HIV-infected patients with Pneumocystis are afflicted with mixed infections of the organism. In addition, it was demonstrated