Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes

Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.     

Genetic diversity among Escherichia coli O157:H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:H7 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or II. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be H7) were significantly different from other Stx-positive and -negative E. coli O157:H7 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:H7 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.     

CMV plasma DNAemia and risk of cancer among HIV-infected patients: A case–control study nested in the ANRS CO3 Aquitaine Cohort,France, 2002–2007

BackgroundCMV reactivation, which enhances immune senescence, could be associated with a higher risk of cancer.ObjectivesWe compared the prevalence of positive CMV DNAemia in HIV-infected patients with and without cancer.Study designThis case–control study, nested in the ANRS-CO3 Aquitaine Cohort, included patients with a first diagnosis of cancer (2002–2007) as cases. Two controls were matched per case.Cancer risk was estimated using conditional logistic regression models, an Odds Ratio (OR) of 2 could be detected with 80% power. The variables considered were: ≥1 positive CMV DNAemia, CD4+ and CD8+ counts, HIV plasma load. Plasma CMV DNA was retrospectively quantified within the 3-year period preceding the endpoint.ResultsThe 143 cases (93 non-AIDS-related and 50 AIDS-related cancers) and 284 controls had a median age of 47 years (IQR: 41–56). At the time of diagnosis or censorship, for cases and controls, median values were respectively, for CD4+ count: 327 cells/mm³ (IQR: 164–514) and 416 (IQR: 275–582), and for HIV plasma load: 2.6 log₁₀ copies/mL (IQR: 1.7–4.7) and 1.7 log₁₀ copies/mL (IQR: 1.7–3.3). We performed 2056 CMV PCR; 14 cases (9.8% [95% CI: 4.9–14.7]) and 19 controls (6.7% [CI: 3.8–9.6]) presented ≥1 positive PCR. CMV DNAemia was not associated with the risk of cancer (unadjusted and adjusted p-values = 0.19 and 0.54, respectively). HIV load >500 copies/mL was independently associated with a higher risk of cancer (OR = 2.02; p = 0.002; 95% CI: 1.29–3.17).ConclusionThis large case–control study did not show any differential exposure to positive CMV plasma DNAemia between cancer cases and controls.     

Parent satisfaction with the electronic medical record in an academic pediatric rheumatology practice

Background

Patient satisfaction has not been widely studied with respect to implementation of the electronic medical record (EMR). There are few reports of the impact of the EMR in pediatrics.

Objective

The objective of this study was to assess the impact of implementation of an electronic medical record system on families in an academic pediatric rheumatology practice.

Methods

Families were surveyed 1 month pre-EMR implementation and 3 months post-EMR implementation.

Results

Overall, EMR was well received by families. Compared with the paper chart, parents agreed the EMR improved the quality of doctor care (55% or 59/107 vs 26% or 26/99, P < .001). More parents indicated they would prefer their pediatric physicians to use an EMR (68% or 73/107 vs 51% or 50/99, P = .01).

Conclusions

Transitioning an academic pediatric rheumatology practice to an EMR can increase family satisfaction with the office visit.     

TBX3 and TBX5 duplication: A family with an atypical overlapping Holt-Oram/ulnar-mammary syndrome phenotype

Holt-Oram syndrome (HOS) is a rare, autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. A wide spectrum of TBX5 mutations have been reported previously, most resulting in a null allele leading to haploinsufficiency. TBX5 gene duplications have been previously reported in association with typical and atypical HOS phenotypes. Ulnar-Mammary syndrome (UMS) is a distinct rare, autosomal dominant condition caused by mutations in the TBX3 gene. TBX5 and TBX3 are physically linked in cis on human chromosome 12 and contiguous chromosome 12q24 deletions comprising both TBX5 and TBX3 genes have been previously reported but to our knowledge, duplications have never been described. We report on a large German family with at least 17 affected individuals over 6 generations bearing a duplication at 12q24.21 identified on array-CGH comprising both TBX5 and TBX3 genes. Affected patients are presenting with HOS and UMS symptoms, consisting of variable limb anomalies involving the radial and the ulnar rays and cardiac findings such as congenital heart defects, persistent arterial duct or aortic stenosis, and non-classical symptoms, such as supernumerary nipples and cardiomyopathy. Fluorescence in situ hybridisation confirmed a tandem duplication at the 12q24.21 locus. This is the first report of a contiguous TBX3/TBX5 duplication associated with HOS/UMS phenotype.     

Efficacy response of inhaled beclomethasone dipropionate in asthma is proportional to dose and is improved by formulation with a new propellant

BACKGROUND: This study tested the hypothesis that there would be improved asthma control with increasing doses of beclomethasone dipropionate (BDP) formulated in hydrofluoroalkane-134a (HFA-BDP) and the standard chlorofluorocarbon propellants (CFC-BDP). Because HFA-BDP has improved lung deposition compared with CFC-BDP, this study also tested the hypothesis that HFA-BDP would provide more effective control of asthma than CFC-BDP. METHODS: In this multicenter, randomized, parallel-group blinded study, asthmatic subjects who had deterioration in asthma control after discontinuation of inhaled corticosteroids were randomized to receive one of 6 possible treatments: 100 microg/d, 400 microg/d, or 800 microg/d of HFA-BDP or 100 microg/d, 400 microg/d, or 800 microg/d of CFC-BDP for 6 weeks. Changes in spirometry, daytime asthma symptom and nighttime asthma-related sleep disturbance scores, morning and evening peak expiratory flows, and daily use of inhaled beta-agonist for symptom control on diary cards were assessed over 6 weeks of treatment. RESULTS: Three hundred twenty-three patients were randomized to the 6 treatment groups, which had similar demographics and baseline lung function. There were significantly larger changes from baseline at week 6 in FEV(1) percent predicted with increasing doses of both HFA-BDP and CFC-BDP. The FEV(1) percent predicted dose-response curve for HFA-BDP was shifted to the left compared with the dose-response curve for CFC-BDP. By using the Finney bioassay method, it was calculated that 2.6 times as much CFC-BDP would be required to achieve the same improvement in FEV(1) percent predicted as HFA-BDP (95% confidence interval, 1.1-11.6). All treatment groups except the 100 microg/d CFC-BDP group tolerated study drug well. Ten (17%) of 59 patients in this group reported an acute asthma episode, increased asthma symptoms (6 of the 8 reports of increased asthma symptoms were classified as severe), or both, and 8 patients withdrew from the study (3 for adverse events related to asthma). CONCLUSIONS: Increasing doses of inhaled corticosteroids lead to improved lung function and asthma control. Moreover, the reformulation of BDP in HFA enables effective asthma control at much lower doses than CFC-BDP.     

Heterogeneity of NSD1 alterations in 116 patients with Sotos syndrome

Sotos syndrome is an overgrowth syndrome characterized by distinctive facial features, learning difficulties, and macrocephaly with frequent pre- and postnatal overgrowth with advanced bone age. Here, we report on our experience in the molecular diagnostic of Sotos syndrome on 116 patients. Using direct sequencing and a quantitative multiplex PCR of short fluorescent fragments (QMPSF)-based assay allowing accurate detection of both total and partial NSD1 deletions, we identified NSD1 abnormalities in 104 patients corresponding to 102 Sotos families (90%). NSD1 point mutations were detected in 80% of the index cases, large deletions removing the NSD1 gene entirely in 14%, and intragenic NSD1 rearrangements in 6%. Among the 69 detected distinct point mutations, 48 were novel. The QMPSF assay detected an exonic duplication and a mosaic partial deletion. QMPSF mapping of the 15 large deletions revealed the heterogeneity of the deletions, which vary in size from 1 to 4.5 Mb. Clinical features of NSD1-positive Sotos patients revealed that the phenotype in patients with nontruncating mutations was less severe that in patients with truncating mutations. This study confirms the heterogeneity of NSD1 alterations in Sotos syndrome and therefore the need to complete sequencing analysis by screening for partial deletions and duplications to ensure an accurate molecular diagnosis of this syndrome.     

Determinants of maximal oxygen uptake in moderate acute hypoxia in endurance athletes

The factors determining maximal oxygen consumption were explored in eight endurance trained subjects (TS) and eight untrained subjects (US) exposed to moderate acute normobaric hypoxia. Subjects performed maximal incremental tests at sea level and simulated altitudes (1,000, 2,500, 4,500 m). Heart rate (HR), stroke volume (SV), cardiac output arterialized oxygen saturation oxygen uptake ventilation ( expressed in normobaric conditions) were measured. At maximal exercise, ventilatory equivalent transport and O₂ extraction (O₂ER_max) were calculated. In TS, remained unchanged despite a significant reduction in at 4,500 m. SV_max remained unchanged. decreased in TS at 4,500 m, was lower in TS and greater at 4,500 m vs. sea level in both groups. Sa′O_2max decreased at and above 1,000 m in TS and 2,500 m in US, O₂ER_max increased at 4,500 m in both groups. decreased with altitude and was greater in TS than US up to 2,500 m but not at 4,500 m. decreased with altitude but the decrement was larger in TS at 4,500 m. In both groups in moderate hypoxia was correlated with Several differences between the two groups are probably responsible for the greater in TS at 4,500 m : (1) the relative hypoventilation in TS as shown by the decrement in at 4,500 m (2) the greater decrement in TS due to a lower Sa′O_2max and unchanged 3) the smaller increase in O₂ER_max in TS, insufficient to compensate the decrease in