Using ‘collective omics data’ for biomedical research training

Systems‐scale molecular profiling data accumulating in public repositories may constitute a useful resource for immunologists. It is for instance likely that information relevant to their chosen line of research be found among the more than 90,000 data series available in the NCBI Gene Expression Omnibus. Such ‘collective omics data’ may also be employed as source material for training purposes. This is the case when training curricula aim at the development of bioinformatics skills necessary for the analysis, interpretation or visualization of data generated on global scales. But ‘collective omics data’ may also be reused for training purposes to foster the development of the skills and ‘mental habits’ underpinning traditional reductionist science approaches. This review describes a small‐scale initiative involving investigators, for the most part immunologists, having engaged in a range of training activities relying on ‘collective omics data’.     

The CDK domain of p21 is a suppressor of IL‐1β‐mediated inflammation in activated macrophages

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21^(WAF1/CIP1), an established suppressor of cell cycle progression, is a inhibitor of IL‐1β synthesis in macrophages. Mice deficient in p21 (p21^?/?) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL‐1β. Administration of IL‐1 receptor antagonist reduces LPS‐induced lethality in p21^?/? mice. Analysis of isolated macrophages, which are one of the central producers of IL‐1β, reveals that deficiency for p21 led to more IL‐1β mRNA and pro‐protein synthesis following TLR ligation. The increase in IL‐1β pro‐protein is associated with elevated secretion of active IL‐1β by p21^?/? macrophages. siRNA‐mediated knockdown of p21 in human macrophages results in increased IL‐1β secretion as well. A peptide mapping strategy shows that the cyclin‐dependent‐kinase (CDK)‐binding domain of p21 is sufficient to reduce the secretion of IL‐1β by p21^?/? macrophages. These data suggest a novel role for p21 and specifically for the CDK‐binding domain of p21^(WAF1/CIP1) in inhibiting inflammation.     

Noninvasive assessment of fetal pulmonary blood flow in experimental pulmonary hypertension in the fetal lamb

The aim of this study was to assess pulmonary arterial blood flow changes induced by the creation of a systemic arteriovenous fistula (120 d gestation) in the fetal lamb using Doppler technique. Doppler echocardiographic assessment of the pulmonary artery blood flow performed 1, 6, and 14 d after surgery showed that mean pulmonary arterial blood flow in the left or right pulmonary artery was 224 +/- 58 mL/min at day 1 in the fistula group, significantly higher than in the control group (113 +/- 22 mL/min; p < 0.01, ANOVA test) whether no difference was found at days 6 and 14. The mean inner diameter of the left pulmonary artery measured on postmortem lung arteriograms compared favorably to the one measured on day 14 at the same level on ultrasound. The mean left pulmonary arterial blood flow, measured at birth on day 14 after surgery, using ultrasonic flow transducer, was not statistically different from the one measured by Doppler on day 14. Our data demonstrate that echocardiography allows accurate assessment of pulmonary arterial blood flow in utero, providing evidence suggesting transient high pulmonary blood flow that did not last >6 d after the creation of a systemic fistula.     

A Review of the Clinical Presentation of Dientamoebiasis

Among 750 symptomatic and asymptomatic patients, Dientamoeba fragilis was detected at a prevalence of 5.2% and more common than Giardia intestinalis. Most infected patients presented with diarrhea and abdominal pain with symptoms greater than 2 weeks duration being common. Bacterial and viral causes of infection were excluded by routine microbiological techniques. Treatment of D. fragilis infection with either iodoquinol, paromomycin, or combination therapy resulted in the eradication of the parasite and complete resolution of symptoms. Treatment failure/relapses were associated only with the use of metronidazole. Nineteen patients were examined for pin worm, no Enterobius vermicularis, a proposed vector of transmission, were detected. Intermittent shedding of D. fragilis was found to be highly variable. These studies confirm the pathogenic nature of D. fragilis and we recommend laboratories routinely test for the organism.     

Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair

There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.     

Zaleplon and zolpidem objectively alleviate sleep disturbances in mountaineers at a 3,613 meter altitude

STUDY OBJECTIVES: To assess the effects of zolpidem and zaleplon on nocturnal sleep and breathing patterns at altitude, as well as on daytime attention, fatigue, and sleepiness. DESIGN: Double-blind, randomized, placebo-controlled, cross-over trial. SETTING: 3 day and night alpine expedition at 3,613 m altitude. PARTICIPANTS: 12 healthy male trekkers. PROCEDURE: One week spent at 1,000 m altitude (baseline control), followed by 3 periods of 3 consecutive treatment nights (N1-3) at altitude, to test 10 mg zolpidem, 10 mg zaleplon, and placebo given at 21:45. MEASURES: Sleep from EEG, actigraphy and sleep logs; overnight arterial saturation in oxygen (SpO2) from infrared oximetry; daytime attention, fatigue and sleepiness from a Digit Symbol Substitution Test, questionnaires, and sleep logs; acute mountain sickness (AMS) from the Lake Louise questionnaire. RESULTS: Compared to baseline control, sleep at altitude was significantly impaired in placebo subjects as shown by an increase in the amount of Wakefulness After Sleep Onset (WASO) from 17 +/- 8 to 36 +/- 13 min (P<0.05) and in arousals from 5 +/- 3 to 20 +/- 8 (P<0.01). Slow wave sleep (SWS) and stage 4 respectively decreased from 26.7% +/- 5.8% to 20.6% +/- 5.8% of total sleep time (TST) and from 18.2% +/- 5.2% to 12.4% +/- 3.1% TST (P<0.05 and P<0.001, respectively). Subjects also complained from a feeling of poor sleep quality combined with numerous 02 desaturation episodes. Subjective fatigue and AMS score were increased. Compared to placebo control, WASO decreased by approximately 6 min (P<0.05) and the sleep efficiency index increased by 2% (P<0.01) under zaleplon and zolpidem, while SWS and stage 4 respectively increased to 22.5% +/- 5.4% TST (P<0.05) and to 15.0% +/- 3.4% TST (P<0.0001) with zolpidem only; both drugs further improved sleep quality. No adverse effect on nighttime SpO2, daytime attention level, alertness, or mood was observed under either hypnotic. AMS was also found to be reduced under both medications. CONCLUSIONS: Both zolpidem and zaleplon have positive effects on sleep at altitude without adversely affecting respiration, attention, alertness, or mood. Hence, they may be safely used by climbers.     

Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.     

Screening 34 Peanut Introductions for Allergen Content Using ELISA

Peanut is one of the most allergenic foods and reports of accidental ingestion of peanuts in unsuspected food are increasing. No information is available on the allergen content of peanut germplasm grown commercially and used in the food and confectionery industry. The objectives of this study were: (1) to determine the allergen contents of 34 peanut introductions (PI); and (2) to identify naturally occurring allergen-free and/or low or hypoallergenic peanuts germplasm. A basic ELISA protocol was utilized to detect the presence of antigens in the peanut lines using a pool of human sera from patients with documented history of peanut allergy. Two naturally occurring low, or hypo-allergenic germplasm were identified as PI 261942 and PI 338386. Both are Valencia market types with total allergen content significantly lower (P ≤0.05) than that of PI 119880 (0.550), PI 119876 (0.415) and PI 118991 (0.410) three Valencia market types and PI 262111 (0.485), a Virginia market type. No allergen-free PI was found. Allergen content of peanut lines from Bolivia and Paraguay were significantly (P ≤0.05) different to those from Venezuela. No significant difference was observed in the allergen content of the four market types.