A high burden of cardiac disease was found among 110 consecutive adolescents with vertically-acquired human immunodeficiency virus infection in Zimbabwe: 37% had an NYHA score =2:echocardiography showed two thirds had LVH, 24% had LV diastolic dysfunction and 31% had RV dilatation.
Background. At least one-third of human immunodeficiency virus (HIV)–infected infants survive to adolescence even without antiretroviral therapy (ART), but are at high risk of complications including cardiac disease. We investigated the characteristics of cardiac disease among adolescents with HIV infection diagnosed in late childhood who were receiving ambulatory HIV care in Harare, Zimbabwe.
Methods. Consecutive adolescents with vertically acquired HIV attending 2 HIV outpatient treatment clinics were studied. Assessment included clinical history and examination, and 2-dimensional, M-mode, pulsed- and continuous-wave Doppler echocardiography.
Results. Of 110 participants (47% male; median age, 15 years; interquartile range, 12–17 years), 78 (71%) were taking ART. Exertional dyspnea, chest pain, palpitations, and ankle swelling were reported by 47 (43%), 43 (39%), 10 (9%), and 7 (6%), respectively. The New York Heart Association score was ≥2 in 41 participants (37%). Echocardiography showed that 74 participants (67%) had left ventricular (LV; septal and/or free wall) hypertrophy and 27 (24%) had evidence of impaired LV relaxation or restrictive LV physiology. The estimated pulmonary artery systolic pressure (ePASP) was >30 mm Hg in 4 participants (3.6%); of these 2 also had right ventricular (RV) dilatation. Another 32 participants (29%), without elevated ePASP, had isolated RV dilatation.
Conclusions. A significant burden of cardiac disease was seen among adolescents with vertically acquired HIV infection. More than half were asymptomatic yet had significant echocardiographic abnormalities. These findings highlight the need to screen this population in order to better define the geography, natural history, etiopathogenic mechanisms, and management (including the timing and choice of optimal therapeutic ART and cardiac drug interventions) to prevent development and/or progression of HIV-associated cardiac disease.
adolescent; Africa; cardiac disease; cardiomyopathy; vertically-acquired HIV
We demonstrate that Pneumocystis reaches a >90% prevalence
peak at 3–5 months of age and associates with increased mucus (MUC5AC), suggesting
airway epithelium stimulation in infants during this age range. Host ability to clear
mucus would determine pathogenic expression.
Background. Pneumocystis without
obvious accompanying pathology is occasionally reported in autopsied infant lungs. Its
prevalence and significance are unknown. Interestingly, this mild infection induces a
strong activation of mucus secretion–related genes in young immunocompetent rodents
that has not been explored in infants. Excess mucus is induced by multiple airway
offenders through nonspecific pathways and would explain a cofactor role of
Pneumocystis in respiratory disease. We undertook characterization of
the prevalence of Pneumocystis and associated mucus in infant lungs.
Methods. Samples from 128 infants (mean age, 101 days)
who died suddenly and unexpectedly in Santiago during 1999–2004 were examined for
Pneumocystis using nested polymerase chain reaction (nPCR)
amplification of the P. jirovecii mtLSU ribosomal RNA gene and
immunofluorescence microscopy (IF). Pneumocystis-negative infants 28 days
and older and their age-closest positives were studied for MUC5AC expression and
Pneumocystis burden by Western blot and quantitative PCR,
Results. Pneumocystis DNA was
detected by nPCR in 105 of the 128 infants (82.0%) and
Pneumocystis organisms were visualized by IF in 99 (94.3%) of
the DNA-positive infants. The infection was commonest at 3–4 months with 40 of 41
(97.6%) infants of that age testing positive. MUC5AC was significantly increased in
Pneumocystis-positive tissue specimens (P =
.013). Death was unexplained in 113 (88.3%) infants; Pneumocystis
was detected in 95 (84.0%) of them vs 10 of 15 (66.7%) with explained death
(P = .28).
Conclusions. A highly focal
Pneumocystis infection associated to increased mucus expression is
almost universally present in the lungs of infants dying unexpectedly in the community
regardless of autopsy diagnosis.
immunocompetent; non-specific immune response; autopsy; MUC5AC; Sudden Infant Death Syndrome (SIDS)
Southern Africa is witnessing the emergence of an epidemic of long-term survivors of vertically- acquired HIV infection presenting with untreated HIV as adolescents. Dermatologic conditions, common in both HIV-infected adults and children, have not been described in this age-group. We investigated the prevalence and spectrum of skin conditions in adolescents admitted to hospitals in Zimbabwe.
Three hundred and one consecutive adolescents admitted to two central Harare hospitals, underwent a dermatologic examination. Clinical history, HIV serology and CD4 lymphocyte counts were obtained. HSV-2 serology was used as a surrogate marker for sexual activity.
One hundred and thirty-nine (46%) patients were HIV-1 antibody positive, of whom only 2 (1.4%) were HSV-2 antibody positive. The prevalence of any skin complaint among HIV-infected and uninfected participants was 88% and 14%, respectively (OR 37.7, 95% CI=19.4-72). The most common HIV-related conditions were pruritic papular eruptions (42%) and plane warts >5% of body area (24%). Having three or more skin conditions, a history of recurrent skin rashes and angular cheilitis were each associated with CD4 counts <200 cells/μl (p<0.03, p<0.01 and p<0.05, respectively).
Skin disease was a common and striking feature of underlying HIV-infection in hospitalized HIV- infected adolescents in Zimbabwe. In resource-poor settings with maturing epidemics, the presence of skin disease should be regarded as a strong indication for HIV testing and especially as it may reflect advanced immunosuppression. The high frequency of multiple plane warts has not previously been described, and may be a feature that distinguishes vertically-infected from horizontally-infected adolescents.
HIV infection; adolescents; skin disease
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).
acquired immune deficiency syndrome; HIV; Pneumocystis; Pneumocystis pneumonia; dihydropteroate synthase
Experiments were carried out in the retina of the tiger salamander (Ambystoma tigrinum) to evaluate the importance of d-serine synthesis on light-evoked N-methyl d-aspartate (NMDA) receptor-mediated components of ganglion cells and contributions to the proximal negative field potential. We blocked the synthesis of d-serine through brief exposures of the retina to phenazine ethosulfate and validated the changes in the tissue levels of d-serine using capillary electrophoresis methods to separate and measure the amino acid enantiomers. Ten minute exposures to phenazine ethosulfate decreased d-serine levels in the retina by about 50% and significantly reduced the NMDA receptor contribution to light responses of the inner retina. This is the first report of a linkage between d-serine synthesis and NMDA receptor activity in the vertebrate retina.
d-serine; ganglion cells; glia; NMDA receptors; retina; serine racemase
A high burden of chronic lung disease (CLD) was found among 116 consecutive adolescents with vertically acquired human immunodeficiency virus in Zimbabwe. The main cause of HIV-associated CLD appears to be obliterative bronchiolitis, which has not previously been recognized among this patient group.
Background. Long-term survivors of vertically acquired human immunodeficiency virus (HIV) infection are reaching adolescence in large numbers in Africa and are at high risk of delayed diagnosis and chronic complications of untreated HIV infection. Chronic respiratory symptoms are more common than would be anticipated based on the HIV literature.
Methods. Consecutive adolescents with presumed vertically acquired HIV attending 2 HIV care clinics in Harare, Zimbabwe, were recruited and assessed with clinical history and examination, CD4 count, pulmonary function tests, Doppler echocardiography, and chest radiography (CXR). Those with suspected nontuberculous chronic lung disease (CLD) were scanned using high-resolution computed tomography (HRCT).
Results. Of 116 participants (43% male; mean age, 14 ± 2.6 years, mean age at HIV diagnosis, 12 years), 69% were receiving antiretroviral therapy. Chronic cough and reduced exercise tolerance were reported by 66% and 21% of participants, respectively; 41% reported multiple respiratory tract infections in the previous year, and 10% were clubbed. More than 40% had hypoxemia at rest (13%) or on exercise (29%), with pulmonary hypertension (mean pulmonary artery pressure >25 mm Hg) in 7%. Forced expiratory volume in 1 second (FEV1) was <80% predicted in 45%, and 47% had subtle CXR abnormalities. The predominant HRCT pattern was decreased attenuation as part of a mosaic attenuation pattern (31 of 56 [55%]), consistent with small airway disease and associated with bronchiectasis (Spearman correlation coefficient (r2 = 0.8) and reduced FEV1 (r2 = −0.26).
Conclusions. Long-term survivors of vertically acquired HIV in Africa are at high risk of a previously undescribed small airway disease, with >40% of unselected adolescent clinic attendees meeting criteria for severe hypoxic CLD. This condition is not obvious at rest. Etiology, prognosis, and response to treatment are currently unknown.
In this descriptive case series, 80 soldiers from Fort Campbell, Kentucky, with inhalational exposures during service in Iraq and Afghanistan were evaluated for dyspnea on exertion that prevented them from meeting the U.S. Army's standards for physical fitness.
The soldiers underwent extensive evaluation of their medical and exposure history, physical examination, pulmonary-function testing, and high-resolution computed tomography (CT). A total of 49 soldiers underwent thoracoscopic lung biopsy after noninvasive evaluation did not provide an explanation for their symptoms. Data on cardiopulmonary-exercise and pulmonary-function testing were compared with data obtained from historical military control subjects.
Among the soldiers who were referred for evaluation, a history of inhalational exposure to a 2003 sulfur-mine fire in Iraq was common but not universal. Of the 49 soldiers who underwent lung biopsy, all biopsy samples were abnormal, with 38 soldiers having changes that were diagnostic of constrictive bronchiolitis. In the remaining 11 soldiers, diagnoses other than constrictive bronchiolitis that could explain the presenting dyspnea were established. All soldiers with constrictive bronchiolitis had normal results on chest radiography, but about one quarter were found to have mosaic air trapping or centrilobular nodules on chest CT. The results of pulmonary-function and cardiopulmonary-exercise testing were generally within normal population limits but were inferior to those of the military control subjects.
In 49 previously healthy soldiers with unexplained exertional dyspnea and diminished exercise tolerance after deployment, an analysis of biopsy samples showed diffuse constrictive bronchiolitis, which was possibly associated with inhalational exposure, in 38 soldiers.
The NMDA receptor coagonist D-serine is important in a number of different processes in the central nervous system, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major coagonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated D-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an AMPA receptor dependent mechanism. AMPA-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity.
Retina; D-serine; NMDA receptor; AMPA receptor; capillary electrophoresis; glia
First-line therapy for Pneumocystis jirovecii pneumonia (PCP) is trimethoprim/sulfamethoxazole. Few data exist to guide the choice of second-line therapy for patients failing or developing toxicity to first-line therapy.
A case note review of 1122 patients with 1188 episodes of HIV-associated PCP from three observational cohorts in Copenhagen, London and Milan, between 1989 and 2004, was conducted.
Trimethoprim/sulfamethoxazole (962 PCP episodes, 81%) was the most frequently used first-line therapy, followed by intravenous pentamidine (87 episodes, 7%), clindamycin/primaquine (72 episodes, 6%) and ‘other’ (atovaquone, dapsone/pyrimethamine, trimetrexate or inhaled pentamidine; 67 episodes, 6%). Rates of unchanged therapy were trimethoprim/sulfamethoxazole = 79%, clindamycin/primaquine = 65% and pentamidine = 60% (P < 0.001). First-line therapy was changed because of failure in 82 (7%) episodes and because of toxicity in 198 (17%) episodes. Three month survival rates were trimethoprim/sulfamethoxazole = 85%, clindamycin/primaquine = 81% and pentamidine = 76% (P = 0.09). After adjustment for possible confounders, pentamidine was associated with a significantly greater risk of death at 3 months [hazard ratio (HR) = 2.0, 95% confidence interval (CI) = 1.2–3.4]. Second-line therapy survival rates differed: trimethoprim/sulfamethoxazole = 85%; clindamycin/primaquine = 87%; and pentamidine = 60% (P = 0.01). Multivariable time-updated Cox regression analysis showed a greater risk of death associated with pentamidine (HR = 3.3, 95% CI = 2.2–5.0), but not for clindamycin/primaquine, when both were compared with trimethoprim/sulfamethoxazole.
Pentamidine was associated with a greater risk of death when used as first- and second-line therapy for HIV-associated PCP, and was associated with more treatment changes. Clindamycin/primaquine appeared superior to pentamidine as second-line therapy for PCP in patients failing or developing toxicity with trimethoprim/sulfamethoxazole. In patients failing first-line treatment with non-trimethoprim/sulfamethoxazole regimens, second-line therapy should be trimethoprim/sulfamethoxazole.
PCP; pneumocystis; therapy; adverse drug reactions; HIV-1
In this study, we demonstrate that D-serine interacts with N-methyl-D-aspartate receptor (NMDAR) coagonist sites of retinal ganglion cells of the tiger salamander retina by showing that exogenous D-serine overcomes the competitive antagonism of 7-chlorokynurenic acid for this site. Additionally, we show that exogenous D-serine was more than 30 times as effective at potentiating NMDAR currents compared with glycine. We thus examined the importance of glycine transport through the application of selective antagonists of the GlyT1 (NFPS) and GlyT2 (ALX-5670) transport systems, while simultaneously evaluating the degree of occupancy of the NMDAR coagonist binding sites. Analysis was carried out with electrophysiological recordings from the inner retina, including whole-cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative field potential. Blocking the GlyT2 transport system had no effect on the light-evoked NMDAR currents or on the sensitivity of these currents to exogenous D-serine. In contrast, when the GlyT1 system was blocked, the coagonist sites of NMDARs showed full occupancy. These findings clearly establish the importance of the GlyT1 transporter as an essential component for maintaining the coagonist sites of NMDARs in a non-saturated state. The normal, unsaturated state of the NMDAR coagonist binding sites allows modulation of the NMDAR currents, by release of either D-serine or glycine. These results are discussed in light of contemporary findings which favor D-serine over glycine as the major coagonist of the NMDARs found in ganglion cells of the tiger salamander retina.
GlyT1; PNFP; retinal ganglion cell; whole-cell recording
We examined the role of GlyT1, the high-affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N-methyl-D-aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1−/+) and compared those results with wild-type (WT) littermate controls (GlyT1+/+). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N-[3-([1,1-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1−/+ retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole-cell recordings from ganglion cells while bath applying D-serine or D-serine + NMDA. The interpretation of these studies was simplified by blocking post-synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D-serine in the GlyT1−/+ mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1−+ animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D-serine as the endogenous coagonist.
coagonist binding sites; GlyT1 glycine transporters; N-methyl-D-aspartic acid receptors; retina
The cellular innate immune response to HIV-1 is poorly characterised. In view of HIV-1 tropism for macrophages, which can be activated via pattern recognition receptors (PRR) to trigger antimicrobial defenses, we investigated innate immune responses to HIV-1 by monocyte derived macrophages.
In a model of productive HIV-1 infection, cellular innate immune responses to HIV-1 were investigated, at the level of transcription factor activation, specific gene expression and genome-wide transcriptional profiling. In addition, the viral determinants of macrophage responses and the physiological effect of innate immune cellular activation on HIV-1 replication were assessed.
Productive HIV-1 infection did not activate NF-κB and IRF3 transcription factors or interferon gene expression (IFN), and caused remarkably small changes to the host cell transcriptome, with no evidence of inflammatory or IFN signatures. Evasion of IFN induction was not dependent on HIV-1 envelope mediated cellular entry, inhibition by accessory proteins or reverse transcription of ssRNA that may reduce innate immune cellular activation by viral RNA. Furthermore, IFNβ priming did not sensitize responses to HIV-1. Importantly, exogenous IFNβ or stimulation with the RNA analogue poly I:C to simulate innate immune activation invoked HIV-1 restriction.
We conclude that macrophages lack functional PRRs for this virus and that HIV-1 tropism for macrophages helps to establish a foothold in the host without triggering innate immune cellular activation which would otherwise block viral infection effectively.
HIV-1; Innate immunity; Macrophages; Interferons; Pattern recognition receptors
Previous health economic studies recommend either a dual screening strategy [tuberculin skin test (TST) followed by interferon-γ-release assay (IGRA)] or a single one [IGRA only] for latent tuberculosis infection (LTBI), the former largely based on claims that it is more cost-effective. We sought to examine that conclusion through the use of a model that accounts for the additional costs of adverse drug reactions and directly compares two commercially available versions of the IGRA: the Quantiferon-TB-Gold-In-Tube (QFT-GIT) and T-SPOT.TB.
A LTBI screening model directed at screening contacts was used to perform a cost-effectiveness analysis, from a UK healthcare perspective, taking into account the risk of isoniazid-related hepatotoxicity and post-exposure TB (2 years post contact) using the TST, QFT-GIT and T-SPOT.TB IGRAs.
Examining costs alone, the TST/IGRA dual screening strategies (TST/T-SPOT.TB and TST/QFT-GIT; £162,387 and £157,048 per 1000 contacts, respectively) cost less than their single strategy counterparts (T-SPOT.TB and QFT-GIT; £203,983 and £202,921 per 1000 contacts) which have higher IGRA test costs and greater numbers of persons undergoing LTBI treatment. However, IGRA alone strategies direct healthcare interventions and costs more accurately to those that are truly infected.
Subsequently, less contacts need to be treated to prevent an active case of TB (T-SPOT.TB and QFT-GIT; 61.7 and 69.7 contacts) in IGRA alone strategies. IGRA single strategies also prevent more cases of post-exposure TB. However, this greater effectiveness does not outweigh the lower incremental costs associated with the dual strategies. Consequently, when these costs are combined with effectiveness, the IGRA dual strategies are more cost-effective than their single strategy counterparts. Comparing between the IGRAs, T-SPOT.TB-based strategies (single and dual; £39,712 and £37,206 per active TB case prevented, respectively) were more cost-effective than the QFT-GIT-based strategies (single and dual; £42,051 and £37,699 per active TB case prevented, respectively). Using the TST alone was the least cost-effective (£47,840 per active TB case prevented). Cost effectiveness values were sensitive to changes in LTBI prevalence, IGRA test sensitivities/specificities and IGRA test costs.
A dual strategy is more cost effective than a single strategy but this conclusion is sensitive to screening test assumptions and LTBI prevalence.
Pneumocystis jirovecii pneumonia (PCP) remains the leading cause of opportunistic infection among human immunodeficiency virus (HIV)–infected persons. Previous studies of PCP that identified case-fatality risk factors involved small numbers of patients, were performed over few years, and often focused on patients who were admitted to the intensive care unit.
The objective of this study was to identify case-fatality risk factors present at or soon after hospitalization among adult HIV-infected patients admitted to University College London Hospitals (London, United Kingdom) from June 1985 through June 2006.
Patients and Methods
We performed a review of case notes for 494 consecutive patients with 547 episodes of laboratory-confirmed PCP.
Overall mortality was 13.5%. Mortality was 10.1% for the period from 1985 through 1989, 16.9% for the period from 1990 through June 1996, and 9.7% for the period from July 1996 through 2006 (P = .142). Multivariate analysis identified factors associated with risk of death, including increasing patient age (adjusted odds ratio [AOR], 1.54; 95% confidence interval [CI], 1.11–2.23; P = .011), subsequent episode of PCP (AOR, 2.27; 95% CI, 1.14–4.52; P = .019), low hemoglobin level at hospital admission (AOR, 0.70; 95% CI, 0.60–0.83; P < .001), low partial pressure of oxygen breathing room air at hospital admission (AOR, 0.70; 95% CI, 0.60–0.81; P < .001), presence of medical comorbidity (AOR, 3.93; 95% CI, 1.77–8.72; P = .001), and pulmonary Kaposi sarcoma (AOR, 6.95; 95% CI, 2.26–21.37; P =.001). Patients with a first episode of PCP were sicker (mean partial pressure of oxygen at admission ± standard deviation, 9.3 ± 2.0 kPa) than those with a second or third episode of PCP (mean partial pressure of oxygen at admission ± standard deviation, 9.9 ± 1.9 kPa; P =.008), but mortality among patients with a first episode of PCP (12.5%) was lower than mortality among patients with subsequent episodes of PCP (22.5%) (P = .019). No patient was receiving highly active antiretroviral therapy before presentation with PCP, and none began highly active antiretroviral therapy during treatment of PCP.
Mortality risk factors for PCP were identifiable at or soon after hospitalization. The trend towards improved outcome after June 1996 occurred in the absence of highly active antiretroviral therapy.
Macrophages contribute to HIV-1 infection at many levels. They provide permissive cells at the site of inoculation, augment virus transfer to T cells, generate long-lived viral reservoirs and cause bystander cell apoptosis. A body of evidence suggests that the role of macrophages in cellular host defence is also compromised by HIV-1 infection. In this respect, macrophages are potent cells of the innate immune system that initiate and regulate wide-ranging immunological responses. This study focuses on the effect of HIV-1 infection on innate immune responses by macrophages at the level of signal transduction, whole genome transcriptional profiling and cytokine secretion. We show that in an ex vivo model, M-CSF differentiated monocyte-derived macrophages (MDM) uniformly infected with replicating CCR5 tropic HIV-1, without cytopathic effect, exhibit selective attenuation of the NF-κB activation pathway in response to TLR4 and TLR2 stimulation. However, functional annotation clustering analysis of genome-wide transcriptional responses to lipopolysaccharide stimulation suggests substantial preservation of gene expression changes at the systems level, with modest attenuation of a subset of up-regulated LPS responsive genes, and no effect on a selection of inflammatory cytokine responses at the protein level. These results extend existing reports of inhibitory interactions between HIV-1 accessory proteins and NF-κB signalling pathways, and whole genome expression profiling provides comprehensive assessment of the consequent effects on immune response gene expression. Unexpectedly, our data suggest innate immune responses are broadly preserved with limited exceptions, and pave the way for further study of the complex relationship between HIV-1 and immunological pathways within macrophages.
Monocytes/Macrophages; AIDS; Lipopolysaccharide; Cell Activation; Signal Transduction
nomenclature; Pneumocystis; parasitology; letter
PCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions.
When copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted.
These findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.
Quantitative measurement of NF-κB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-κB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-κB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-κB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-κB activation reporter cell line.
Nuclear factor-kappa B; Confocal immunofluoresence; Image analysis; Macrophages
Purpose: A proteomics approach is warranted to further elucidate the molecular steps involved in lung tumor development. We asked whether we could classify preinvasive lesions of airway epithelium according to their proteomic profile.
Experimental Design: We obtained matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiles from 10-μm sections of fresh-frozen tissue samples: 25 normal lung, 29 normal bronchial epithelium, and 20 preinvasive and 36 invasive lung tumor tissue samples from 53 patients. Proteomic profiles were calibrated, binned, and normalized before analysis. We performed class comparison, class prediction, and supervised hierarchic cluster analysis. We tested a set of discriminatory features obtained in a previously published dataset to classify this independent set of normal, preinvasive, and invasive lung tissues.
Results: We found a specific proteomic profile that allows an overall predictive accuracy of over 90% of normal, preinvasive, and invasive lung tissues. The proteomic profiles of these tissues were distinct from each other within a disease continuum. We trained our prediction model in a previously published dataset and tested it in a new blinded test set to reach an overall 74% accuracy in classifying tumors from normal tissues.
Conclusions: We found specific patterns of protein expression of the airway epithelium that accurately classify bronchial and alveolar tissue with normal histology from preinvasive bronchial lesions and from invasive lung cancer. Although further study is needed to validate this approach and to identify biomarkers of tumor development, this is a first step toward a new proteomic characterization of the human model of lung cancer tumorigenesis.
early detection; mass spectrometry; preneoplasia; profiling
Isolates of Pneumocystis jiroveci from sulfa-exposed and nonexposed patients from London, United Kingdom, and Harare, Zimbabwe, were genotyped. At the dihydropteroate synthase (DHPS) locus, there was evidence of selection pressure from sulfa drug exposure, and reversal of DHPS genotype ratios occurred when selection pressure was absent or was removed.
The disease known as Pneumocystis carinii pneumonia (PCP) is a major cause of illness and death in persons with impaired immune systems. While the genus Pneumocystis has been known to science for nearly a century, understanding of its members remained rudimentary until DNA analysis showed its extensive diversity. Pneumocystis organisms from different host species have very different DNA sequences, indicating multiple species. In recognition of its genetic and functional distinctness, the organism that causes human PCP is now named Pneumocystis jiroveci Frenkel 1999. Changing the organism’s name does not preclude the use of the acronym PCP because it can be read “Pneumocystis
pneumonia.” DNA varies in samples of P. jiroveci, a feature that allows reexamination of the relationships between host and pathogen. Instead of lifelong latency, transient colonization may be the rule.
Pneumocystis; nomenclature; human; epidemiology; genetics
A mother and her 4.5-week-old infant had Pneumocystis carinii pneumonia contemporaneously. Genotyping of P. carinii f. sp. hominis DNA at three independent loci showed the same genotype in samples from mother and infant. These data suggest transmission of P. carinii organisms from the mother to her infant.
The possible transmission of Pneumocystis carinii f. sp. hominis from patients with P. carinii pneumonia to asymptomatic health care workers (HCW), with or without occupational exposure to human immunodeficiency virus (HIV)-infected patients with P. carinii pneumonia, was examined. HCW in a specialist inpatient HIV-AIDS facility and a control group in the general medical-respiratory service in the same hospital provided induced sputum and/or nasal rinse samples, which were analyzed for the presence of P. carinii f. sp. hominis DNA by using DNA amplification (at the gene encoding the mitochondrial large subunit rRNA [mt LSU rRNA]). P. carinii f. sp. hominis DNA was detected in some HCW samples; those with the closest occupational contact were more likely to have detectable P. carinii DNA. P. carinii DNA was detected in one HCW who carried out bronchoscopy over a 2-year period. P. carinii-positive samples were genotyped by using DNA sequence variations at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, along with bronchoalveolar lavage samples from patients with P. carinii pneumonia hospitalized at the same time. Genotyping identified 31 different P. carinii f. sp. hominis ITS genotypes, 26 of which were found in the patient samples. Five of the eight ITS genotypes detected in HCW samples were not observed in the patient samples. The results suggested that HCW in close occupational contact with patients who had P. carinii pneumonia may have become colonized with P. carinii. Carriage was asymptomatic and did not result in the development of clinical disease.
The tuberculin skin test (TST) is a model of integrated innate and adaptive human immune responses to Mycobacterium tuberculosis, but the component processes that are involved in this model have not previously been defined in vivo. We used transcriptional profiling to study these responses within the TST at molecular and system levels. Skin biopsies from TST injection sites were examined in subjects classified as TST+ or TST− by clinical and histological criteria. Genome-wide expression arrays showed evolution of immune responses reflecting T-cell activation and recruitment with uniquely Th1-polarized responses and cytotoxic T cells (CTLs). In addition, distinct innate immune and IFN-γ-stimulated gene expression signatures were identified, under the regulation of NF-κB and STAT1 transcriptional control. These were highly enriched for chemokines and MHC class II molecules providing a potential mechanism for paracrine amplification of inflammatory responses in the TST, by supporting cellular recruitment and enhancing antigen presentation. The same repertoire of innate and adaptive immune responses was evident in TST+ and TST− subjects alike, clinically positive TSTs being distinguished only by quantitatively greater differences. These data provide new insights into complex multifaceted responses within the TST, with much greater sensitivity than previous clinical or histological assessments.
Delayed-type hypersensitivity; Gene expression profiling; Innate immunity; Th1 type responses; Tuberculin skin test
Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM.