Ambient Air Pollution Associated with Suppressed Serologic Responses to Pneumocystis jirovecii in a Prospective Cohort of HIV-Infected Patients with Pneumocystis Pneumonia

Background

Ambient air pollution (AAP) may be associated with increased risk for Pneumocystis pneumonia (PCP). The mechanisms underlying this association remain uncertain.

Objectives

To determine if real-life exposures to AAP are associated with suppressed IgM antibody responses to P. jirovecii in HIV-infected (HIV+) patients with active PCP, and to determine if AAP, mediated by suppressed serologic responses to Pneumocystis, is associated with adverse clinical outcomes.

Methods

We conducted a prospective cohort study in HIV+ patients residing in San Francisco and admitted to San Francisco General Hospital with microscopically confirmed PCP. Our AAP predictors were ambient air concentrations of particulate matter of < 10 µm in diameter (PM₁₀) and < 2.5 µm in diameter (PM_2.5), nitrogen dioxide (NO₂), ozone (O₃), and sulfur dioxide (SO₂) measured immediately prior to hospital admission and 2 weeks prior to admission. Our primary outcomes were the IgM serologic responses to four recombinant P. jirovecii major surface glycoprotein (Msg) constructs: MsgC1, MsgC3, MsgC8, and MsgC9.

Results

Elevated PM₁₀ and NO₂ exposures immediately prior to and two weeks prior to hospital admission were associated with decreased IgM antibody responses to P. jirovecii Msg. For exposures immediately prior to admission, every 10 µg/m³ increase in PM₁₀ was associated with a 25 to 35% decrease in IgM responses to Msg (statistically significant for all the Msg constructs), and every 10 ppb increase in NO₂ was associated with a 19-45% decrease in IgM responses to Msg (statistically significant for MsgC8 and MsgC9). Similar findings were seen with exposures two weeks prior to admission, but for fewer of the Msg constructs.

Conclusions

Real life exposures to PM₁₀ and NO₂ were associated with suppressed IgM responses to P. jirovecii Msg in HIV+ patients admitted with PCP, suggesting a mechanism of immunotoxicity by which AAP increases host susceptibility to pulmonary infection.     

Sterol composition of Pneumocystis jirovecii with blocked 14alpha-demethylase activity

Several drugs that interact with membrane sterols or inhibit their syntheses are effective in clearing a number of fungal infections. The AIDS-associated lung infection caused by Pneumocystis jirovecii is not cleared by many of these therapies. Pneumocystis normally synthesizes distinct C28 and C29 24-alkylsterols, but ergosterol, the major fungal sterol, is not among them. Two distinct sterol compositional phenotypes were previously observed in P. jirovecii. One was characterized by delta7 C28 and C29 24-alkylsterols with only low proportions of higher molecular mass components. In contrast, the other type was dominated by high C31 and C32 24-alkylsterols, especially pneumocysterol. In the present study, 28 molecular species were elucidated by nuclear magnetic resonance analysis of a human lung specimen containing P. jirovecii representing the latter sterol profile phenotype. Fifteen of the 28 had the methyl group at C-14 of the sterol nucleus and these represented 96% of the total sterol mass in the specimen (excluding cholesterol). These results strongly suggest that sterol 14alpha-demethylase was blocked in these organisms. Twenty-four of the 28 were 24-alkylsterols, indicating that methylation of the C-24 position of the sterol side chain by S-adenosyl-L-methionine:sterol C-24 methyl transferase was fully functional.     

Epidemiology of Pneumocystis jirovecii Pneumonia in Venezuela

Current Fungal Infection Reports - The aim of this work is to contribute to the knowledge of the epidemiology of pneumocystosis or Pneumocystis jirovecii pneumonia (PCP) in Venezuela, by an updated...     

Interaction Energy Analysis of Nonclassical Antifolates with Human Dihydrofolate Reductase

The x-ray structure of the PTX:NADPH:L22F human mutant DHFR ternary complex was used as a structural template to generate structural models for the following wild type DHFR complexes: PTX:DHFR:NADPH, TMP:DHFR:NADPH, EPM:DHFR:NADPH, and TMQ:DHFR:NADPH. Each of these complexes were subsequently modeled in a 60 Å cube of explicit water and minimized to a rms gradient of from 1.0-3.0·10^-5 kcal·Å^-1. For each complex, interaction energies were calculated for the antifolate interaction with each of the following: the DHFR binding site residues, the entire DHFR protein, the solvated complex (containing DHFR, NADPH, and solvent water), water alone, and NADPH. Additionally, each antifolate was subdivided into distinct substructural regions and interaction energy calculations were performed in order to evaluate their contributions to overall antifolate interaction. Each antifolate showed its most stable interaction with the solvated complex. Substructural regions which consisted of a nitrogen containing aromatic ring system contributed most to the stability of the antifolate interactions, while the hydrocarbon aromatic rings, methoxy, and ethoxy groups showed much less stable interaction energies. Since the different substructural regions of nonclassical antifolates differ in their contributions to overall antifolate binding, those substructural regions which exhibit relatively unfavorable interaction energies may constitute important targets in the design of improved DHFR inhibitors.     

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234–238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159–11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H→C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H→C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.     

Humoral Immune Responses to Pneumocystis jirovecii Antigens in HIV-Infected and Uninfected Young Children with Pneumocystis Pneumonia

Background

Humoral immune responses in human immunodeficiency virus (HIV)-infected and uninfected children with Pneumocystis pneumonia (PcP) are poorly understood.

Methods

Consecutive children hospitalized with acute pneumonia, tachypnea, and hypoxia in South Africa were investigated for PcP, which was diagnosed by real-time polymerase chain reaction on lower respiratory tract specimens. Serum antibody responses to recombinant fragments of the carboxyl terminus of Pneumocystis jirovecii major surface glycoprotein (MsgC) were analyzed.

Results

149 children were enrolled of whom 96 (64%) were HIV-infected. PcP occurred in 69 (72%) of HIV-infected and 14 (26%) of HIV-uninfected children. HIV-infected children with PcP had significantly decreased IgG antibodies to MsgC compared to HIV-infected patients without PcP, but had similar IgM antibodies. In contrast, HIV-uninfected children with PcP showed no change in IgG antibodies to MsgC, but had significantly increased IgM antibodies compared to HIV-uninfected children without PCP. Age was an independent predictor of high IgG antibodies, whereas PcP was a predictor of low IgG antibodies and high IgM antibodies. IgG and IgM antibody levels to the most closely related MsgC fragments were predictors of survival from PcP.

Conclusions

Young HIV-infected children with PcP have significantly impaired humoral immune responses to MsgC, whereas HIV-uninfected children with PcP can develop active humoral immune responses. The children also exhibit a complex relationship between specific host factors and antibody levels to MsgC fragments that may be related to survival from PcP.     

Folate Reductase and Specific Dihydrofolate Reductase Activities of the Amethopterin-Sensitive Streptococcus faecium var. durans

Preliminary evidence is presented that indicates that the dihydrofolate reductase activity of amethopterin-sensitive Streptococcus faecium var. durans ATCC 8043 is separable into two dihydrofolate reductases, one of which also reduces folate.     

Genetic Polymorphisms in Plasmodium vivax Dihydrofolate Reductase and Dihydropteroate Synthase in Isolates from the Philippines,Bangladesh, and Nepal

Genetic polymorphisms of pvdhfr and pvdhps genes of Plasmodium vivax were investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal. The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP. Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia. All samples carried mutant combination alleles of pvdhfr and pvdhps. The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively. Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles. New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.     

中国仓鼠卵细胞二氢叶酸还原酶基因在大肠杆菌中的高效表达

本工作采用PCR法将哺乳动物表达载体pSVA75上的中国仓鼠卵细胞(CHO)二氢叶酸还原酶基因扩增后插入到原核生物高效表达质料pBV221中,构建成胞内表达载体pDHFR-1,经转化大肠杆菌(E.coli DH5α)后获高效表达,表达量占总蛋白量的12.2%。利用这种方法不仅为深入研究该酶的结构和功能的关系及其新生肽链的折叠提供丰富的实验材料,而且也有希望成为较好的外源DNA原核扩增表达系统。 Abstract:The technique of PCR was used to amplify the Chinese Hamster Ovary DHFR gene in the mammal expression vector pSVA75,and the amplified fragments were inserted into the vector pBV221,to construct the vector pDHFR-1.The CHO dhfr was fransformaed into E.coli DH5α and high-level expressed.The scanning results(scanned by LKB Enhanced Laser Densitometer)showed that the expression level had reached 12.2% of the total cellular proteins.Therefore,this provides an abundant experimental material for studying the relationship between the protein construct and function,and for studying the biology of the nascent peptide chain during biosynthesis.This pathway may be a good expression system of prokaryocyte for amphifing eukaryotic gene.     

Multicenter Study of Trimethoprim/Sulfamethoxazole-Related Hepatotoxicity: Incidence and Associated Factors among HIV-Infected Patients Treated for Pneumocystis jirovecii Pneumonia

The incidence of hepatotoxicity related to trimethoprim/sulfamethoxazole (TMP/SMX) administered at a therapeutic dose may vary among study populations of different ethnicities and hepatotoxic metabolites of TMP/SMX may be decreased by drug-drug interaction with fluconazole. We aimed to investigate the incidence of hepatotoxicity and the role of concomitant use of fluconazole in HIV-infected patients receiving TMP/SMX for Pneumocystis jirovecii pneumonia. We reviewed medical records to collect clinical characteristics and laboratory data of HIV-infected patients who received TMP/SMX for treatment of P. jirovecii pneumonia at 6 hospitals around Taiwan between September 2009 and February 2013. Hepatotoxicity was defined as 2-fold or greater increase of aminotransferase or total bilirubin level from baselines. Roussel UCLAF Causality Assessment Method (RUCAM) was used to analyze the causality of drug-induced liver injuries. NAT1 and NAT2 acetylator types were determined with the use of polymerase-chain-reaction (PCR) restriction fragment length polymorphism to differentiate common single-nucleotide polymorphisms (SNPs) predictive of the acetylator phenotypes in a subgroup of patients. During the study period, 286 courses of TMP/SMX treatment administered to 284 patients were analyzed. One hundred and fifty-two patients (53.1%) developed hepatotoxicity, and TMP/SMX was considered causative in 47 (16.4%) who had a RUCAM score of 6 or greater. In multivariate analysis, concomitant use of fluconazole for candidiasis was the only factor associated with reduced risk for hepatotoxicity (adjusted odds ratio, 0.372; 95% confidence interval, 0.145–0.957), while serostatus of hepatitis B or C virus, NAT1 and NAT2 acetylator types, or receipt of combination antiretroviral therapy was not. The incidence of hepatotoxicity decreased with an increasing daily dose of fluconazole up to 4.0 mg/kg. We conclude that the incidence of TMP/SMX-related hepatotoxicity was 16.4% in HIV-infected Taiwanese patients who received TMP/SMX for pneumocystosis. Concomitant use of fluconazole was associated with decreased risk for TMP/SMX-related hepatotoxicity.     

Function of T4D Structural Dihydrofolate Reductase in Bacteriophage Infection

Various properties of the bacteriophage structural dihydrofolate reductase (DFR) have been examined to determine its function during phage infection. It has been found that a binding site for reduced nicotinamide adenine dinucleotide phosphate (NADPH), most likely on the DFR present in the phage tail plate, is required for phage viability. Attachment of adenosine diphosphoribose, an analogue of NADPH, to this site prevents phage adsorption and injection. This adenosine diphosphoribose inhibition can be competitively reversed by the addition of NADPH or oxidized nicotinamide adenine dinucleotide phosphate. It is suggested that, during phage infection, the host bacterial cell might leak compounds functionally similar to the pyridine nucleotides. These compounds have been shown to nonenzymatically change the conformation of the phage tail plate DFR which is apparently necessary for successful injection.     

Diagnosis,Burden and Mortality of Pneumocystis jirovecii Pneumonia in Venezuela

Current Fungal Infection Reports - The aim of this work is to contribute to the knowledge of diagnosis, burden, and mortality of pneumocystosis or Pneumocystis jirovecii pneumonia (PCP) in...