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1.  Outbreak of Pneumocystis Pneumonia in Renal and Liver Transplant Patients Caused by Genotypically Distinct Strains of Pneumocystis jirovecii1 
Transplantation  2013;96(9):10.1097/TP.0b013e3182a1618c.
An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used.
P. jirovecii isolates from 22 transplant-cases, 2 colonized RTRs and 19 Pneumocystis-control samples were genotyped by restriction fragment length polymorphism and multi-locus sequence typing analysis. Contact tracing were used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients.
Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs and a colonized RTR in 3 distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis-control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only CMV viremia was consistently associated with PCP (P = 0.03; P = 0.009). Mycophenolate mofetile was associated with PCP risk only in the RTR population (P = 0.04).
We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by inter-human transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months post-transplantation. Since patients at risk cannot be identified clinically and outbreaks cannot be predicted, six months of PCP chemoprophylaxis should be considered for all renal and liver transplant recipients.
PMCID: PMC3826781  PMID: 23903011
Pneumocystis jirovecii; pneumocystis pneumonia; liver transplant; renal transplant; outbreak
2.  Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae 
The Journal of Infectious Diseases  2013;208(1):170-179.
The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine–rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine–rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with β-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg.
PMCID: PMC3666137  PMID: 23532098
Pneumocystis jirovecii; major surface glycoprotein; upstream conserved sequence; antigenic variation; GPI anchored protein
3.  Influence of Low-dose Ritonavir With and Without Darunavir on the Pharmacokinetics and Pharmacodynamics of Inhaled Beclomethasone 
To identify an alternative inhaled corticosteroid to fluticasone propionate that can be safely coadministered with HIV protease inhibitors (PIs), we assessed the safety and pharmacokinetics of beclomethasone dipropionate (BDP) and its active metabolite, 17-monopropionate (17-BMP), in combination with ritonavir (RTV) and darunavir/ritonavir (DRV/r).
Open label, prospective, randomized pharmacokinetic and pharmacodynamic study in healthy volunteers.
Thirty healthy volunteers received inhaled BDP 160 mcg BID for 14 days and were then randomized (1:1:1) into three groups: Group 1 (control) remained on BDP alone for 28 days; Group 2 received BDP + RTV 100 mg BID for 28 days, and Group 3 received BDP + DRV/r 600/100 mg BID for 28 days. Pharmacokinetic sampling for 17-BMP was performed on days 14 and 28, and pharmacokinetic parameter values were compared within patients and between groups. Cortisol stimulation testing was also performed on days 1, 14, 28, and 42 and compared within and between groups.
Geometric mean ratios (day 28:day 14) (90% CI) for 17-BMP area under the concentration-time curve in groups 1, 2, and 3, respectively, were 0.93 (0.81–1.06, p=0.27), 2.08 (1.52–2.65; p=0.006), and 0.89 (0.68–1.09; p=0.61). There were no significant reductions in serum cortisol levels within or between groups (p>0.05).
DRV/r did not increase 17-BMP exposure, while RTV alone produced a statistically significant but clinically inconsequential 2-fold increase in 17-BMP exposure. Adrenal suppression was not observed in any of the study groups. These data suggest BDP can be safely coadministered with DRV/r and likely other RTV-boosted PIs.
PMCID: PMC3683093  PMID: 23535292
beclomethasone; darunavir; ritonavir; drug interactions; adrenal function
4.  Quantitative Determinations of Anti-Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Antibody Levels in Men Who Have Sex with Men 
Infection with Kaposi’s Sarcoma-Associated Herpesvirus (KSHV/HHV-8) is common among men who have sex with men (MSM). Here quantitative anti-KSHV antibody levels were measured using Luciferase Immunoprecipitation Systems (LIPS) in a MSM cohort with and without HIV from the NIH Clinical Center. Antibodies were detected using a mixture of four KSHV antigens in the MSM cohort and in Kaposi Sarcoma (KS) patients. Along with HIV status, these results were compared with K8.1 and ORF73 ELISA, PCR virus detection, and additional LIPS testing. LIPS revealed that 25% (76/307) of the MSM cohort were KSHV seropositive, including 59 HIV+ and 17 HIV− subjects. The anti-KSHV antibody levels detected by LIPS were not statistically different between the KSHV+/HIV+ and KSHV+/HIV− subgroups, but were lower than the KS patients (P<0.0001). ELISA analysis of the MSM cohort detected a 35.5% frequency of KSHV infection and showed agreement with 81% of the samples evaluated by LIPS. Further LIPS testing with v-cyclin, a second ORF73 fragment and ORF38 reconciled some of the differences observed between LIPS and the ELISA immunoassays and the revised LIPS seroprevalence in the MSM cohort was increased to 31%. Additional quantitative antibody analysis demonstrated statistically lower KSHV antibody levels in MSM compared to KS patients, but no difference was found between KSHV infected with and without HIV coinfection. These findings also suggest that antibodies against v-cyclin and ORF38 are useful for identifying patients with asymptomatic KSHV infection.
PMCID: PMC3634905  PMID: 23541691
antibodies; Kaposi’s Sarcoma-Associated Herpesvirus (KSHV); MSM
5.  Characterization of Pneumocystis Major Surface Glycoprotein Gene (msg) Promoter Activity in Saccharomyces cerevisiae 
Eukaryotic Cell  2013;12(10):1349-1355.
Major surface glycoprotein (Msg), the most abundant cell surface protein of Pneumocystis, plays an important role in the interaction of this opportunistic pathogen with host cells, and its potential for antigenic variation may facilitate evasion of host immune responses. In the present study, we have identified and characterized the promoter region of msg in 3 species of Pneumocystis: P. carinii, P. jirovecii, and P. murina. Because Pneumocystis cannot be cultured, promoter activity was measured in Saccharomyces cerevisiae, a related fungus, using a yeast vector modified to utilize the gene coding for Renilla luciferase as a reporter gene. The 5′-flanking sequences of msg from all three Pneumocystis species showed considerable promoter activity, with increases in luciferase activity up to 15- to 44-fold above baseline. Progressive deletions helped define an ∼13-bp sequence in each Pneumocystis species that appears to be critical for promoter activity. Electrophoretic mobility shift analysis using P. carinii-specific msg promoter sequences demonstrated binding of nuclear proteins of S. cerevisiae. The 144-bp 5′-flanking region of P. murina msg showed 72% identity to that of P. carinii. The 5′-flanking region of P. jirovecii msg showed 58 and 61% identity to those of P. murina and P. carinii, respectively. The msg promoter is a good candidate for inclusion in a construct designed for genetic manipulation of Pneumocystis species.
PMCID: PMC3811333  PMID: 23893080
6.  HIV Populations Are Large and Accumulate High Genetic Diversity in a Nonlinear Fashion 
Journal of Virology  2013;87(18):10313-10323.
HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.
PMCID: PMC3754011  PMID: 23678164
8.  Altered Antibody Profiles against Common Infectious Agents in Chronic Disease 
PLoS ONE  2013;8(12):e81635.
Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons.  We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-γ autoantibodies (IFN-γ AAB), HIV and Sjögren’s syndrome (SjS) to determine if their antibody profiles differed from control subjects.  The IFN-γ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005),   and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-γ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls.  The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.
PMCID: PMC3847058  PMID: 24312567
9.  A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS 
PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.
PMCID: PMC3811116  PMID: 24179701
PacBio; CCS read; quality control (QC); pass number; quality value (QV); SVM regression; assembly
10.  HIV Infection and Antiretroviral Therapy Have Divergent Effects on Mitochondria in Adipose Tissue 
The Journal of Infectious Diseases  2012;205(12):1778-1787.
Background. Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited.
Methods. Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)–based ART, and HIV-negative controls.
Results. The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+/HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes.
Conclusions. HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.
PMCID: PMC3357134  PMID: 22476717
11.  Outbreaks of Pneumocystis Pneumonia in 2 Renal Transplant Centers Linked to a Single Strain of Pneumocystis: Implications for Transmission and Virulence 
By restriction fragment length polymorphism analysis, 2 outbreaks of Pneumocystis pneumonia in renal transplant patients in Europe were shown to be caused by the same strain of Pneumocystis; another outbreak in Japan was caused by a different strain.
Background. There have been numerous reports of clustered outbreaks of Pneumocystis pneumonia (PCP) at renal transplant centers over the past 2 decades. It has been unclear whether these outbreaks were linked epidemiologically to 1 or several unique strains, which could have implications for transmission patterns or strain virulence.
Methods. Restriction fragment length polymorphism (RFLP) analysis was used to compare Pneumocystis isolates from 3 outbreaks of PCP in renal transplant patients in Germany, Switzerland, and Japan, as well as nontransplant isolates from both human immunodeficiency virus (HIV)–infected and uninfected patients.
Results. Based on RFLP analysis, a single Pneumocystis strain caused pneumonia in transplant patients in Switzerland (7 patients) and Germany (14 patients). This strain was different from the strain that caused an outbreak in transplant patients in Japan, as well as strains causing sporadic cases of PCP in nontransplant patients with or without HIV infection.
Conclusions. Two geographically distinct clusters of PCP in Europe were due to a single strain of Pneumocystis. This suggests either enhanced virulence of this strain in transplant patients or a common, but unidentified, source of transmission. Outbreaks of PCP can be better understood by enhanced knowledge of transmission patterns and strain variation.
PMCID: PMC3404729  PMID: 22431811
12.  Galleria mellonella are resistant to Pneumocystis murina infection 
Mycopathologia  2010;171(4):273-277.
Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.
PMCID: PMC3634573  PMID: 20922567
Galleria mellonella; infection model; Pneumocystis murina
13.  Fatigue-Related Gene Networks Identified in CD14+ Cells Isolated From HIV-Infected Patients—Part II: Statistical Analysis 
Biological research for nursing  2011;15(2):152-159.
In limited samples of valuable biological tissues, univariate ranking methods of microarray analyses often fail to show significant differences among expression profiles. In order to allow for hypothesis generation, novel statistical modeling systems can be greatly beneficial. The authors applied new statistical approaches to solve the issue of limited experimental data to generate new hypotheses in CD14+ cells of patients with HIV-related fatigue (HRF) and healthy controls.
We compared gene expression profiles of CD14+ cells of nucleoside reverse transcriptase inhibitor (NRTI)-treated HIV patients with low versus high fatigue to healthy controls (n = 5 each). With novel Bayesian modeling procedures, the authors identified 32 genes predictive of low versus high fatigue and 33 genes predictive of healthy versus HIV infection. Sparse association and liquid association networks further elucidated the possible biological pathways in which these genes are involved.
Relevance for nursing practice
Genetic networks developed in a comprehensive Bayesian framework from small sample sizes allow nursing researchers to design future research approaches to address such issues as HRF.
Implication for practice
The findings from this pilot study may take us one step closer to the development of useful biomarker targets for fatigue status. Specific and reliable tests are needed to diagnosis, monitor and treat fatigue and mitochondrial dysfunction.
PMCID: PMC3546222  PMID: 22084402
CD14; genetic network; Bayesian inference; liquid association; HIV
14.  Influence of Panax ginseng on Cytochrome P450 (CYP)3A and P-glycoprotein (Pgp) Activity in Healthy Subjects 
Journal of clinical pharmacology  2011;52(6):10.1177/0091270011407194.
A number of herbal preparations have been shown to interact with prescription medications secondary to modulation of cytochrome P450 (CYP) and/or P-glycoprotein (P-gp). The purpose of this study was to determine the influence of Panax ginseng on CYP3A and P-gp function using the probe substrates midazolam and fexofenadine, respectively. Twelve healthy subjects (8 males) completed this open label, single sequence pharmacokinetic study. Healthy volunteers received single oral doses of midazolam 8 mg and fexofenadine 120 mg, before and after 28 days of P. ginseng 500 mg twice daily. Midazolam and fexofenadine pharmacokinetic parameter values were calculated and compared pre-and post P. ginseng administration. Geometric mean ratios (post-ginseng/pre-ginseng) for midazolam area under the concentration vs. time curve from zero to infinity (AUC0-∞), half life (T1/2), and maximum concentration (Cmax) were significantly reduced at 0.66 (0.55 – 0.78), 0.71 (0.53 – 0.90), and 0.74 (0.56 – 0.93), respectively. Conversely, fexofenadine pharmacokinetics were unaltered by P. ginseng administration. Based on these results, Panax ginseng appeared to induce CYP3A activity in the liver and possibly the gastrointestinal tract. Patients taking Panax ginseng in combination with CYP3A substrates with narrow therapeutic ranges should be monitored closely for adequate therapeutic response to the substrate medication.
PMCID: PMC3523324  PMID: 21646440
HIV; Panax ginseng; cytochrome P450; drug interaction; midazolam; herb
15.  LIPS arrays for simultaneous detection of antibodies against partial and whole proteomes of HCV, HIV and EBV 
Molecular bioSystems  2011;7(5):1453-1462.
For many infectious agents, the detection of antibodies is critical for diagnosis, monitoring and understanding vaccine responses. To facilitate the highly quantitative and simultaneous analysis of antibodies against multiple proteins from infectious agents, we have developed Luciferase Immunoprecipitation Systems (LIPS) arrays. By configuring microtiter plates with multiple antigens and testing control and infected serum samples at one time in solution, LIPS arrays provided highly reproducible antibody titers to panels of antigens with a wide dynamic range of detection. While all serum samples showed similar positive and negative immunoreactivity with internal control antigens derived from Influenza and Renilla luciferase-alone protein, respectively, antibody titers to many HCV and HIV antigens were generally 10 to over 400-fold higher in the infected versus uninfected samples. Additional screening of 18 proteins from the EBV proteome with serum samples from healthy EBV-infected individuals showed statistically significant antibody titers to 50% of the proteins tested. Antibody titers for the different EBV antigens in the healthy EBV-infected individuals were markedly heterogeneous highlighting the complexity of host humoral responses. These results suggest that LIPS arrays offer a highly discriminating platform for simultaneously profiling a wide spectrum of antibodies associated with many infectious agents.
PMCID: PMC3430135  PMID: 21336381
16.  The Cancer-Associated Virus Landscape in HIV Patients with Oral Hairy Leukoplakia, Kaposi's Sarcoma, and Non-Hodgkin Lymphoma 
AIDS Research and Treatment  2012;2012:634523.
Although HIV-positive patients are at higher risk for developing a variety of infection-related cancers, the prevalence of infections with the seven known cancer-associated viruses has not been studied. Luciferase immunoprecipitation systems were used to evaluate antiviral antibodies in four 23-person groups: healthy blood donors and HIV-infected patients with oral hairy leukoplakia (OLP), Kaposi's sarcoma (KS), or non-Hodgkin lymphoma (NHL). Antibody profiling revealed that all HIV-positive individuals were strongly seropositive for anti-gp41 and antireverse transcriptase antibodies. However, anti-p24 HIV antibody levels were highly variable and some OLP and KS patients demonstrated weak or negative responses. Profiling two EBV antigens revealed no statistical difference in antibody levels among the three HIV-infected groups. A high frequency of KSHV infection was detected in HIV patients including 100% of KS, 78% of OLP, and 57% of NHL patients. Most HIV-infected subjects (84%) showed anti-HBV core antibodies, but only a few showed antibodies against HCV. MCV seropositivity was also common (94%) in the HIV-infected individuals and KS patients showed statistically higher antibody levels compared to the OLP and NHL patients. Overall, 68% of the HIV-infected patients showed seropositivity with at least four cancer-associated viruses. Antibody profiles against these and other infectious agents could be useful for enhancing the clinical management of HIV patients.
PMCID: PMC3423820  PMID: 22924124
17.  Echinacea Purpurea Significantly Induces Cytochrome P450 3A (CYP3A) but does not alter Lopinavir-Ritonavir Exposure in Healthy Subjects 
Pharmacotherapy  2010;30(8):797-805.
Study Objective
To determine the influence of Echinacea purpurea on the pharmacokinetics of lopinavir-ritonavir, and on CYP3A and P-glycoprotein (P-gp) activity using the probe substrates midazolam, and fexofenadine, respectively.
Open label, single-sequence pharmacokinetic study.
Outpatient clinic in a Federal Government research hospital.
Thirteen (8 males) healthy volunteers (median age: 31 yrs).
Measurements and main results
Healthy volunteers received lopinavir-ritonavir (400/100 mg) twice daily for 30 days. On study day 16, subjects began taking Echinacea purpurea 500 mg three times daily, which they continued for four weeks, the first two weeks in combination with lopinavir-ritonavir. On days 15 and 30 of lopinavir-ritonavir administration (pre and post-Echinacea, respectively), serial blood samples were collected over 12 hrs to determine lopinavir and ritonavir concentrations and subsequent pharmacokinetic parameters using non-compartmental methods. Study subjects also received single doses of midazolam (8 mg orally) and fexofenadine (120 mg orally) before- and after 28 days of Echinacea purpurea to assess CYP3A and P-glycoprotein (P-gp) activity, respectively. Neither lopinavir nor ritonavir pharmacokinetics were significantly altered by 2 weeks of Echinacea coadministration. The geometric mean ratios (GMR, 90% CI) for lopinavir area under the concentration vs. time curve from zero to 12 hrs (AUC0–12) and maximum concentration (post-Echinacea/pre-Echinacea) were 0.96 (0.83, 1.10) and 1.00 (0.88, 1.12), respectively (P > 0.05). Conversely, GMRs (90% CIs) for midazolam AUC from time zero to infinity (AUC0-∞) and oral clearance were 0.73 (0.61, 0.85) (P = 0.008) and 1.37 (1.10, 1.63) (P = 0.02), respectively. Fexofenadine pharmacokinetics did not significantly differ pre- and post-echinacea administration (P > 0.05).
Echinacea purpurea induced CYP3A activity but did not alter lopinavir concentrations, most likely due to the presence of the potent CYP3A inhibitor, ritonavir. Echinacea purpurea is unlikely to alter the pharmacokinetics of ritonavir-boosted protease inhibitors but may cause modest decreases in plasma concentrations of other CYP3A substrates.
PMCID: PMC3407958  PMID: 20653355
HIV; protease inhibitors; lopinavir; ritonavir; Echinacea purpurea; herb; cytochrome P450; P-glycoprotein; drug interaction
18.  Lack of Compartmentalization of HIV-1 Quasispecies Between the Gut and Peripheral Blood Compartments 
The Journal of Infectious Diseases  2011;204(2):309-314.
Compartmental differences in human immunodeficiency virus type 1 (HIV-1) between the gut and peripheral blood and within the gut were examined. Biopsy specimens from the colon and ileum and peripheral blood samples were collected from chronically HIV-1–infected individuals. HIV-1 envelope sequences were examined from cell-associated DNA and RNA and virion RNA. Phylogenetic analysis revealed no evidence of compartmentalization of HIV-1 between the gut and peripheral blood and within the gut (colon and ileum). HIV-1 sequences detected in the gut were transcriptionally active and were also found in peripheral blood from matching time points, providing evidence of ongoing virus production in the gut and equilibrium of HIV-1 between the gut and peripheral blood compartments.
PMCID: PMC3114472  PMID: 21673043
19.  Discordant antibody and cellular responses to Pneumocystis major surface glycoprotein variants in mice 
BMC Immunology  2012;13:39.
The major surface glycoprotein (Msg) of Pneumocystis is encoded by approximately 50 to 80 unique but related genes. Msg diversity may represent a mechanism for immune escape from host T cell responses. We examined splenic T cell proliferative and cytokine as well as serum antibody responses to recombinant and native Pneumocystis antigens in immunized or Pneumocystis-infected mice. In addition, immune responses were examined in 5 healthy humans.
Proliferative responses to each of two recombinant Msg variant proteins were seen in mice immunized with either recombinant protein, but no proliferation to these antigens was seen in mice immunized with crude Pneumocystis antigens or in mice that had cleared infection, although the latter animals demonstrated proliferative responses to crude Pneumocystis antigens and native Msg. IL-17 and MCP-3 were produced in previously infected animals in response to the same antigens, but not to recombinant antigens. Antibody responses to the recombinant P. murina Msg variant proteins were seen in all groups of animals, demonstrating that all groups were exposed to and mounted immune responses to Msg. No human PBMC samples proliferated following stimulation with P. jirovecii Msg, while antibody responses were detected in sera from 4 of 5 samples.
Cross-reactive antibody responses to Msg variants are common, while cross-reactive T cell responses are uncommon; these results support the hypothesis that Pneumocystis utilizes switching of Msg variant expression to avoid host T cell responses.
PMCID: PMC3411419  PMID: 22788748
Antigenic variation; Immune response; Major surface glycoprotein; Pneumocystis
20.  HIV-Associated Pneumocystis Pneumonia 
During the past 30 years, major advances have been made in our understanding of HIV/AIDS and Pneumocystis pneumonia (PCP), but significant gaps remain. Pneumocystis is classified as a fungus and is host-species specific, but an understanding of its reservoir, mode of transmission, and pathogenesis is incomplete. PCP remains a frequent AIDS-defining diagnosis and is a frequent opportunistic pneumonia in the United States and in Europe, but comparable epidemiologic data from other areas of the world that are burdened with HIV/AIDS are limited. Pneumocystis cannot be cultured, and bronchoscopy with bronchoalveolar lavage is the gold standard procedure to diagnose PCP, but noninvasive diagnostic tests and biomarkers show promise that must be validated. Trimethoprim-sulfamethoxazole is the recommended first-line treatment and prophylaxis regimen, but putative trimethoprim-sulfamethoxazole drug resistance is an emerging concern. The International HIV-associated Opportunistic Pneumonias (IHOP) study was established to address these knowledge gaps. This review describes recent advances in the pathogenesis, epidemiology, diagnosis, and management of HIV-associated PCP and ongoing areas of clinical and translational research that are part of the IHOP study and the Longitudinal Studies of HIV-associated Lung Infections and Complications (Lung HIV).
PMCID: PMC3132788  PMID: 21653531
acquired immune deficiency syndrome; HIV; Pneumocystis; Pneumocystis pneumonia; dihydropteroate synthase
21.  Constitutional telomerase mutations are genetic risk factors for cirrhosis 
Hepatology (Baltimore, Md.)  2011;53(5):1600-1607.
Some patients with liver disease progress to cirrhosis, but the risk factors for cirrhosis development are unknown. Dyskeratosis congenita, an inherited bone marrow failure syndrome associated with mucocutaneous anomalies, pulmonary fibrosis, andcirrhosis, is caused by germ-line mutations of genesin the telomerase complex. We examined whether telomerase mutations also occurred in sporadic cirrhosis. One hundred thirty-four patients with cirrhosis of common etiologies treated at the Liver Research Institute, University of Arizona, between May 2008 and July 2009, and 528healthy subjects were screened for variation in the TERT and TERC genes by direct sequencing; an additional 1472 controls were examined for the most common genetic variation observed in patients. Telomere length of leukocytes was measured by quantitative polymerase chain reaction. Functional effects of genetic changes were assessed by transfection of mutation-containing vectors into telomerase-deficient cell lines, and telomerase activity was measured in cell lysates. Nine of the 134 patients with cirrhosis (7%) carried a missense variant in TERT, resulting in a cumulative carrier frequency significantly higher than in controls (P=0.0009). One patient was homozygous and eight were heterozygous. The allele frequency for the most common missense TERT variant was significantly higher in cirrhotic patients (2.6%) than in 2000 controls (0.7%; P=0.0011). One additional patient carried a TERC mutation. The mean telomere length of leukocytesin cirrhotic patients, including six mutant cases, was shorter than in age-matched controls(P=0.0004). Most TERT gene variants reduced telomerase enzymatic activity in vitro. Loss-of-function telomerase gene variants associated with short telomeres are risk factors for sporadic cirrhosis.
PMCID: PMC3082730  PMID: 21520173
telomere; regeneration; dyskeratosis congenital; aplastic anemia
22.  The Effect of Intermittent IL-2 Therapy on CD4 T Cells in the Gut in HIV-1 Infected Patients 
We sought to determine the effects of interleukin-2 administered in combination with antiretroviral therapy (ART) on CD4+ T cells in the gut. Lymphocytes from whole blood, colon and terminal ileum of HIV infected adults treated with interleukin-2 and ART or ART alone were examined. There were no differences between groups in the proportion of CD4+ T cells or in expression of CD25 or Ki67 by CD4+T cells in the gut. Although IL-2 administration leads to expansion of peripheral blood CD4+ T cells, there is no alteration in the proportion or activation of CD4+ T cells in the gut mucosa.
PMCID: PMC3073743  PMID: 21350367
gastrointestinal tract; mucosa; IL-2; HIV; CD4
23.  Characterization of the Meiosis-specific Recombinase Dmc1 of Pneumocystis 
The Journal of infectious diseases  2010;202(12):1920-1929.
The life cycle of Pneumocystis, which causes life-threatening pneumonia in immunosuppressed patients, remains poorly defined. In the present study, we have identified and characterized an orthologue of dmc1, a gene specific for meiotic recombination in yeasts, in 3 species of Pneumocystis. dmc1 is a single copy gene that is transcribed as an ~1.2 kb mRNA, which encodes a protein of 336–337 amino acids. Pneumocystis Dmc1 was 61 to 70% identical to those from yeast. Based on confocal microscopy, the expression of Dmc1 is primarily confined to the cyst form of Pneumocystis. By sequence analysis of 2 single copy regions of the human P. jirovecii genome, we can infer multiple recombination events, which are consistent with meiotic recombination in this primarily haploid organism. Taken together, these studies support the occurrence of a sexual phase in the life cycle of Pneumocystis.
PMCID: PMC3058675  PMID: 21050123
Pneumocystis; recombinase; recombination; sexual reproduction
24.  Characterization of the Meiosis-Specific Recombinase Dmc1 of Pneumocystis 
The Journal of Infectious Diseases  2010;202(12):1920-1929.
The life cycle of Pneumocystis, which causes life-threatening pneumonia in immunosuppressed patients, remains poorly defined. In the present study, we have identified and characterized an orthologue of dmc1, a gene specific for meiotic recombination in yeast, in 3 species of Pneumocystis. dmc1 is a single-copy gene that is transcribed as ∼1.2-kb messenger RNA, which encodes a protein of 336–337 amino acids. Pneumocystis Dmc1 was 61%–70% identical to those from yeast. Confocal microscopy results indicated that the expression of Dmc1 is primarily confined to the cyst form of Pneumocystis. By sequence analysis of 2 single-copy regions of the human Pneumocystis jirovecii genome, we can infer multiple recombination events, which are consistent with meiotic recombination in this primarily haploid organism. Taken together, these studies support the occurrence of a sexual phase in the life cycle of Pneumocystis.
PMCID: PMC3058675  PMID: 21050123
25.  CD4+ T Cells, Including Th17 and Cycling Subsets, Are Intact in the Gut Mucosa of HIV-1-Infected Long-Term Nonprogressors▿ 
Journal of Virology  2011;85(12):5880-5888.
During acute human immunodeficiency virus (HIV) infection, there is a massive depletion of CD4+ T cells in the gut mucosa that can be reversed to various degrees with antiretroviral therapy. Th17 cells have been implicated in mucosal immunity to extracellular bacteria, and preservation of this subset may support gut mucosal immune recovery. However, this possibility has not yet been evaluated in HIV-1-infected long-term nonprogressors (LTNPs), who maintain high CD4+ T cell counts and suppress viral replication in the absence of antiretroviral therapy. In this study, we evaluated the immunophenotype and function of CD4+ T cells in peripheral blood and gut mucosa of HIV-uninfected controls, LTNPs, and HIV-1-infected individuals treated with prolonged antiretroviral therapy (ART) (VL [viral load]<50). We found that LTNPs have intact CD4+ T cell populations, including Th17 and cycling subsets, in the gut mucosa and a preserved T cell population expressing gut homing molecules in the peripheral blood. In addition, we observed no evidence of higher monocyte activation in LTNPs than in HIV-infected (HIV−) controls. These data suggest that, similar to nonpathogenic simian immunodeficiency virus (SIV) infection, LTNPs preserve the balance of CD4+ T cell populations in blood and gut mucosa, which may contribute to the lack of disease progression observed in these patients.
PMCID: PMC3126290  PMID: 21471231

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