Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

Adherence to Internet-Based and Face-to-Face Cognitive Behavioural Therapy for Depression: A Meta-Analysis

Background

Internet-based cognitive behavioural therapy (iCBT) is an effective and acceptable treatment for depression, especially when it includes guidance, but its treatment adherence has not yet been systematically studied. We conducted a meta-analysis, comparing the adherence to guided iCBT with the adherence to individual face-to-face CBT.

Methods

Studies were selected from a database of trials that investigate treatment for adult depression (see www.evidencebasedpsychotherapies.org), updated to January 2013. We identified 24 studies describing 26 treatment conditions (14 face-to-face CBT, 12 guided iCBT), by means of these inclusion criteria: targeting depressed adults, no comorbid somatic disorder or substance abuse, community recruitment, published in the year 2000 or later. The main outcome measure was the percentage of completed sessions. We also coded the percentage of treatment completers (separately coding for 100% or at least 80% of treatment completed).

Results

We did not find studies that compared guided iCBT and face-to-face CBT in a single trial that met our inclusion criteria. Face-to-face CBT treatments ranged from 12 to 28 sessions, guided iCBT interventions consisted of 5 to 9 sessions. Participants in face-to-face CBT completed on average 83.9% of their treatment, which did not differ significantly from participants in guided iCBT (80.8%, P  =  .59). The percentage of completers (total intervention) was significantly higher in face-to-face CBT (84.7%) than in guided iCBT (65.1%, P < .001), as was the percentage of completers of 80% or more of the intervention (face-to-face CBT: 85.2%, guided iCBT: 67.5%, P  =  .003). Non-completers of face-to-face CBT completed on average 24.5% of their treatment, while non-completers of guided iCBT completed on average 42.1% of their treatment.

Conclusion

We did not find studies that compared guided iCBT and face-to-face CBT in a single trial. Adherence to guided iCBT appears to be adequate and could be equal to adherence to face-to-face CBT.     

Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4⁺ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4⁺ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4⁺ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38⁺ CD4⁺ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38⁺ CD4⁺ T cells could be generated in vitro from CD38^- CD4⁺ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38⁺ CD4⁺ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38⁺ CD4⁺ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense.

Trial Registration

ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752     

Bioavailability of Orally Administered rhGM-CSF: A Single-Dose,Randomized, Open-Label,Two-Period Crossover Trial

Background

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is usually administered by injection, and its oral administration in a clinical setting has been not yet reported. Here we demonstrate the bioavailability of orally administered rhGM-CSF in healthy volunteers. The rhGM-CSF was expressed in Bombyx mori expression system (BmrhGM-CSF).

Methods and Findings

Using a single-dose, randomized, open-label, two-period crossover clinical trial design, 19 healthy volunteers were orally administered with BmrhGM-CSF (8 µg/kg) and subcutaneously injected with rhGM-CSF (3.75 µg/kg) respectively. Serum samples were drawn at 0.0h, 0.5h ,0.75h,1.0h,1.5h,2.0h ,3.0h,4.0h,5.0h,6.0h,8.0h,10.0h and 12.0h after administrations. The hGM-CSF serum concentrations were determined by ELISA. The AUC was calculated using the trapezoid method. The relative bioavailability of BmrhGM-CSF was determined according to the AUC ratio of both orally administered and subcutaneously injected rhGM-CSF. Three volunteers were randomly selected from 15 orally administrated subjects with ELISA detectable values. Their serum samples at the 0.0h, 1.0h, 2.0h, 3.0h and 4.0h after the administrations were analyzed by Q-Trap MS/MS TOF. The different peaks were revealed by the spectrogram profile comparison of the 1.0h, 2.0h, 3.0h and 4.0h samples with that of the 0.0h sample, and further analyzed using both Enhanced Product Ion (EPI) scanning and Peptide Mass Fingerprinting Analysis. The rhGM-CSF was detected in the serum samples from 15 of 19 volunteers administrated with BmrhGM-CSF. Its bioavailability was observed at an average of 1.0%, with the highest of 3.1%. The rhGM-CSF peptide sequences in the serum samples were detected by MS analysis, and their sizes ranging from 2,039 to 7,336 Da.

Conclusions

The results demonstrated that the oral administered BmrhGM-CSF was absorbed into the blood. This study provides an approach for an oral administration of rhGM-CSF protein in clinical settings.

Trial Registration

www.chictr.org ChiCTR-TRC-00000107     

Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

Genomic Grade Index (GGI): Feasibility in Routine Practice and Impact on Treatment Decisions in Early Breast Cancer

Purpose

Genomic Grade Index (GGI) is a 97-gene signature that improves histologic grade (HG) classification in invasive breast carcinoma. In this prospective study we sought to evaluate the feasibility of performing GGI in routine clinical practice and its impact on treatment recommendations.

Methods

Patients with pT1pT2 or operable pT3, N0-3 invasive breast carcinoma were recruited from 8 centers in Belgium. Fresh surgical samples were sent at room temperature in the MapQuant Dx™ PathKit for centralized genomic analysis. Genomic profiles were determined using Affymetrix U133 Plus 2.0 and GGI calculated using the MapQuant Dx® protocol, which defines tumors as low or high Genomic Grade (GG-1 and GG-3 respectively).

Results

180 pts were recruited and 155 were eligible. The MapQuant test was performed in 142 cases and GGI was obtained in 78% of cases (n=111). Reasons for failures were 15 samples with <30% of invasive tumor cells (11%), 15 with insufficient RNA quality (10%), and 1 failed hybridization (<1%). For tumors with an available representative sample (≥ 30% inv. tumor cells) (n=127), the success rate was 87.5%. GGI reclassified 69% of the 54 HG2 tumors as GG-1 (54%) or GG-3 (46%). Changes in treatment recommendations occurred mainly in the subset of HG2 tumors reclassified into GG-3, with increased use of chemotherapy in this subset.

Conclusion

The use of GGI is feasible in routine clinical practice and impacts treatment decisions in early-stage breast cancer.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01916837","term_id":"NCT01916837"}}NCT01916837, http://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT01916837","term_id":"NCT01916837"}}NCT01916837     

Distinct Kinetics in the Frequency of Peripheral CD4+ T Cells in Patients with Ulcerative Colitis Experiencing a Flare during Treatment with Mesalazine or with a Herbal Preparation of Myrrh,Chamomile, and Coffee Charcoal

Background

We found the first evidence of the efficacy of a herbal treatment with myrrh, dry extract of chamomile flowers, and coffee charcoal for ulcerative colitis (UC). However, the impact of the herbal treatment on the CD4⁺ T-cell compartment, which is essential for both the induction of UC and the maintenance of tolerance in the gut, is not well understood.

Aim

To analyze the frequency and functional phenotype of CD4⁺ T cells and of immune-suppressive CD4+CD25^high regulatory T cells (Tregs) in healthy control subjects, patients with UC in remission, and patients with clinical flare of UC.

Methods

Patients in clinical remission were treated with either mesalazine or the herbal preparation for 12 months. The frequencies of whole CD4⁺ T cells, CD4⁺CD25^med effector T cells, and Tregs and the expression of Foxp3 within the CD4⁺CD25^hig Tregs were determined by flow cytometry at 6 time points. We determined the suppressive capability of Tregs from healthy control subjects and from patients in remission or clinical flare.

Results

A total of 79 patients (42 women, 37 men; mean age, 48.5 years; 38 with clinical flare) and 5 healthy control subjects were included in the study. At baseline the frequencies of whole CD4⁺ T cells, CD4⁺CD25^med effector cells, and Tregs did not differ between the two treatment groups and the healthy control subjects. In addition, patients with UC in sustained clinical remission showed no alteration from baseline after 1, 3, 6, 9, or 12 months of either treatment. In contrast, CD4⁺ T cells, CD4⁺CD25^medeffector T cells, and Tregs demonstrated distinctly different patterns at time points pre-flare and flare. The mesalazine group showed a continuous but not statistically significant increase from baseline to pre-flare and flare (p = ns). In the herbal treatment group, however, the percentage of the CD4⁺ T cells was lower at pre-flare than at baseline. This decrease was completely reversed after flare, when a significant increase was seen (CD4⁺CD25^med pre-flare/flare p = 0.0461; CD4⁺CD25^high baseline/flare p = 0.0269 and pre-flare/flare p = 0.0032). In contrast, no changes in the expression of Foxp3 cells were detected within the subsets of CD4⁺CD25^high regulatory T cells. Of note, no alterations were detected in the suppressive capability of CD4⁺CD25^high regulatory T cells isolated from the peripheral blood of healthy donors, from patients in remission, or from patients with clinical flare.

Conclusions

In patients with UC experiencing acute flare, the CD4⁺ T compartment demonstrates a distinctly different pattern during treatment with myrrh, chamomile extract, and coffee charcoal than during treatment with mesalazine. These findings suggest an active repopulation of regulatory T cells during active disease.

Trial Registration

EU Clinical Trials Register 2007-007928-18/DE     

Phased cell division in natural and laboratory populations of marine planktonic diatoms

Phytoplankton samples were collected every 2 h and examined for percentage of dividing cells (doublets) during different times of the day. Thalassiothrix nitzschioids Grunow and Tropidoneis antarctica Grunow, var. polyplasta Gran, showed diurnal peaks in cell division at 1700. Thalassiosira rotula Meunier showed a nocturnal peak in cell division at 0300, while Chaetoceros vanheurckii Gran consistently exhibited low cell division rates with no apparent peaks. A culture of T. rotula kept under a similar L:D cycle to that present in the field (16:8 L:D) showed a diurnal peak in cell division at 1700. It appears from these data and recent literature that several factors are involved in the regulation of phased cell division in phytoplankton, including temperature, L:D cycle, nutrients, and size selective grazing by zooplankton.     

Evaluation of an on-farm method to assess colostrum IgG content in sows

The objective of this work was to investigate the evaluation of swine colostrum immunoglobulin G (IgG) concentration using the Brix refractometer. Colostrum samples were collected across all teats, from 124 sows of mixed parities. According to sampling time, three categories were created: samples available from 9 h before the onset of parturition until the first piglet was born were classified as before farrowing; samples collected after the first birth until 4 h later were classified as during farrowing; and finally samples collected from this point until 14 h after parturition, were classified as after farrowing. Samples were drawn and divided into three portions; one was immediately analyzed, a second was refrigerated and the third was frozen at −20°C. Fresh and refrigerated colostrum samples were analyzed at the farm with a Brix refractometer. IgG content of frozen samples was analyzed using a Brix refractometer, with a subset of 42 samples also tested with a commercially available radial immune diffusion (RID) kit. The Brix percentage ranged from 18.3% to 33.2%. Brix percentage repeatability, assessed by the intraclass correlation coefficient (ICC), was very strong (fresh ICC=0.98, refrigerated ICC=0.88 and frozen ICC=0.99). One-way repeated-measures ANOVA showed that storage temperature did not affect BRIX percentage of colostrum IgG (P>0.05). ANOVA results show a significant effect of sampling time on colostrum immunoglobulin concentration, measured with both Brix and RID (Brix: P<0.003; RID: P<0.05). Immunoglobulin G concentration measured by RID ranged from 13.27 to 35.08 mg/ml. Pearson correlation coefficient revealed that Brix percentage was positively correlated (r=0.56, P<0.001) with RID results (regression equation: RID=1.01 (±0.2) Brix −1.94 (±5.66); R²=0.31). The results of this study indicate that the Brix refractometer provides a simple, fast and inexpensive estimation of colostrum IgG in sows.     

Genetic Polymorphisms in Monoamine Systems and Outcome of Cognitive Behavior Therapy for Social Anxiety Disorder

Objective

The role of genetics for predicting the response to cognitive behavior therapy (CBT) for social anxiety disorder (SAD) has only been studied in one previous investigation. The serotonin transporter (5-HTTLPR), the catechol-o-methyltransferase (COMT) val158met, and the tryptophan hydroxylase-2 (TPH2) G-703Tpolymorphisms are implicated in the regulation of amygdala reactivity and fear extinction and therefore might be of relevance for CBT outcome. The aim of the present study was to investigate if these three gene variants predicted response to CBT in a large sample of SAD patients.

Method

Participants were recruited from two separate randomized controlled CBT trials (trial 1: n = 112, trial 2: n = 202). Genotyping were performed on DNA extracted from blood or saliva samples. Effects were analyzed at follow-up (6 or 12 months after treatment) for both groups and for each group separately at post-treatment. The main outcome measure was the Liebowitz Social Anxiety Scale Self-Report.

Results

At long-term follow-up, there was no effect of any genotype, or gene × gene interactions, on treatment response. In the subsamples, there was time by genotype interaction effects indicating an influence of the TPH2 G-703T-polymorphism on CBT short-term response, however the direction of the effect was not consistent across trials.

Conclusions

None of the three gene variants, 5-HTTLPR, COMTval158met and TPH2 G-703T, was associated with long-term response to CBT for SAD.

Trial Registration

ClinicalTrials.gov (ID-NCT0056496)     

Sex Distribution of Study Samples Reported in American Society of Biomechanics Annual Meeting Abstracts

Background

Study samples should be appropriately selected to maximize generalizability of results. Excluding one sex from studies of conditions that affect both sexes is problematic and has received attention as a public policy issue in the United States, resulting in legislation and recommendations made by the National Institutes of Health to address this deficiency of study designs. It is unknown to what extent biomechanical studies have inappropriately excluded one sex. The objective of this study was to provide objective data on this question.

Methods

A retrospective review of random samples of abstracts presented at American Society of Biomechanics annual meetings from 1983 to 2013 was conducted to assess reporting of sex of study samples and whether the study samples were approximately balanced with respect to sex.

Findings

We did not find a statistically significant increasing trend in the percentage of abstracts reporting sex over time. However, increasing trends were noted in the percentage of abstracts including both sexes (p < 0.05) and percentage of abstracts having an “approximately balanced” study sample containing 50 ± 20% females (p > 0.05). In 2013 the percentage of abstracts reporting studies having approximately balanced study samples was only 28%, far from the ideal level of 100%.

Interpretation

While there has been modest change since 1983, there remains significant room for improvement in the reporting and composition of experimental studies reported at American Society of Biomechanics annual meetings.     

Immunomodulatory Effects in a Phase II Study of Lenalidomide Combined with Cetuximab in Refractory KRAS-Mutant Metastatic Colorectal Cancer Patients

This study assessed the immunomodulatory effects in previously treated KRAS-mutant metastatic colorectal cancer patients participating in a phase II multicenter, open-label clinical trial receiving lenalidomide alone or lenalidomide plus cetuximab. The main findings show the T cell immunostimulatory properties of lenalidomide as the drug induced a decrease in the percentage CD45RA⁺ naïve T cells 3-fold while increasing the percentage HLA-DR⁺ activated T helper cells and percentage total CD45RO⁺ CD8⁺ memory T cytotoxic cells, 2.6- and 2.1-fold respectively (p<0.0001). In addition, lenalidomide decreased the percentage of circulating CD19⁺ B cells 2.6-fold (p<0.0001). Lenalidomide increased a modest, yet significant, 1.4-fold change in the percentage of circulating natural killer cells. Our findings indicate that lenalidomide significantly activates T cells, suggestive of an immunotherapeutic role for this drug in settings of maintenance therapy and tumor immunity. Furthermore, reported for the first time is the effect of lenalidomide in combination with cetuximab on T cell function, including increases in circulating naïve and central memory T cells. In summary, lenalidomide and cetuximab have significant effects on circulating immune cells in patients with colorectal carcinoma.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01032291","term_id":"NCT01032291"}}NCT01032291     

Measurements of Diel Rates of Bacterial Secondary Production in Aquatic Environments

Measurements of bacterial secondary production were carried out during 13 diel studies at one coastal marine station and in five lakes differing with respect to nutrient concentration and primary production. Bacterial secondary production was measured in situ every 3 to 5 h by [³H]thymidine incorporation into DNA. In some of the diel studies, these results were compared with results obtained from dark ¹⁴CO₂ uptake and frequency of dividing cells. Only minor diel changes were observed. The rate of [³H]thymidine incorporation into DNA and the frequency of dividing cells varied from 23 to 194% of the diel mean. The dark CO₂ uptake rate varied from 12 to 259% of the diel mean. An analysis of variance demonstrated that no specific time periods during 24 h showed significantly different production rates, supporting the idea that bacterial activities in natural assemblages are controlled by a variety of events. The best correction (r² = 0.74) was obtained between the [³H]thymidine incorporation and frequency of dividing cells procedures from the lake water samples. The actual production rates calculated by [³H]thymidine incorporation into DNA were appreciably lower than those obtained by the frequency of dividing cells and the dark CO₂ uptake techniques. Diel rates of bacterial production are discussed in relation to sampling frequency, statistical errors, and choice of method.     

Efficacy and safety of once-daily fluticasone furoate 50 mcg in adults with persistent asthma: a 12-week randomized trial

Background

Fluticasone furoate (FF) is a novel, once-daily inhaled corticosteroid (ICS) that has been shown to improve lung function vs. placebo in asthma patients. This study evaluated the efficacy and safety of FF 50 mcg compared with placebo in asthma patients uncontrolled by non-ICS therapy.

Methods

This 12-week, multicentre, randomized, double-blind, placebo-controlled, parallel-group, phase III study randomized 248 patients (aged ≥12 years) to once-daily FF 50 mcg administered via the ELLIPTA™^a dry powder inhaler or placebo. The primary endpoint was change from baseline in pre-dose evening trough forced expiratory volume in one second (FEV₁). Secondary endpoints were change from baseline in percentage of rescue-free 24-h periods (powered), evening and morning peak expiratory flow, symptom-free 24-h periods and withdrawals due to lack of efficacy. Other endpoints included Asthma Control Test™, Asthma Quality of Life Questionnaire and ELLIPTA ease of use questions. Safety was assessed throughout the study.

Results

There was a significant difference in evening trough FEV₁ between FF 50 mcg and placebo (treatment difference: 120 mL; p = 0.012). There was also a significant difference in rescue-free 24-h periods (11.6%; p = 0.004) vs. placebo. There were numerically greater improvements with FF vs. placebo for all remaining secondary endpoints. The incidence of adverse events was lower with FF (31%) than with placebo (38%); few were treatment-related (FF 50 mcg: n = 1, <1%; placebo: n = 4, 3%).

Conclusion

FF 50 mcg once daily significantly improved FEV₁ and percentage of rescue-free 24-h periods experienced over 12 weeks vs. placebo, and was well tolerated.

Trial registration

www.clinicaltrials.gov, registration number: {"type":"clinical-trial","attrs":{"text":"NCT01436071","term_id":"NCT01436071"}}NCT01436071     

Antiprion compounds that reduce PrPSc levels in dividing and stationary-phase cells

During prion diseases, a normally benign, host protein, denoted PrP^C, undergoes alternative folding into the aberrant isoform, PrP^Sc. We used ELISA to identify and confirm hits in order to develop leads that reduce PrP^Sc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration–effect relationships in dividing cells to determine potency (EC₅₀), 102 had EC₅₀ values between 1 and 10 μM and 50 had EC₅₀ values of <1 μM; none affected cell viability. We observed an excellent correlation between EC₅₀ values determined by ELISA and Western immunoblotting for 28 representative compounds in dividing cells (R² = 0.75; p <0.0001). Of the 55 confirmed SPC hits in stationary-phase cells, 23 were piperazine, indole, or urea leads. The EC₅₀ values of one indole in stationary-phase and dividing ScN2a-cl3 cells were 7.5 and 1.6 μM, respectively. Unexpectedly, the number of hits in stationary-phase cells was ～10% of that in dividing cells. The explanation for this difference remains to be determined.     

Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa

Background

Next generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR.

Methods

The Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.)

Results

In a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers.

Conclusions

Using the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions.     

Anti-TNF Therapy Reduces Serum Levels of Chemerin in Rheumatoid Arthritis: A New Mechanism by Which Anti-TNF Might Reduce Inflammation

Background

Chemerin is a specific chemoattractant for macrophages and dendritic cells (DC). In addition, it can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules and is able to activate fibroblast-like synoviocytes (FLS), suggesting a role in the pathogenesis of rheumatoid arthritis (RA). Chemerin is also an adipocytokine that has been related to the inflammatory state of endothelial cells and as such could be involved in the changes in endothelial cells in RA and perhaps increased cardiovascular morbidity. We investigated whether anti-Tumor Necrosis Factor (TNF) treatment affects chemerin levels.

Materials and Methods

49 patients with active RA (disease activity score evaluated in 28 joints (DAS28) ≥3.2) were started on adalimumab therapy. Blood was drawn from patients while fasting at baseline and 16 weeks after initiation of treatment. Chemerin serum levels were measured by ELISA and related to disease activity, mediators of inflammation and known risk factors for cardiovascular disease.

Results

Adalimumab therapy reduced chemerin serum levels, which was correlated with the reduction in DAS28 (r = 0.37, p = 0.009). In addition, the decrease in chemerin serum levels after anti-TNF treatment was associated with the decrease in serum levels of IL-6 (r = 0.39, p = 0.033) and macrophage migration inhibitory factor (MIF) (r = 0.31, p = 0.049). Baseline chemerin serum levels were not related to traditional risk factors for atherosclerosis, except perhaps for smoking (p = 0.07).

Conclusions

This exploratory study shows that adalimumab therapy lowers chemerin levels, which is associated with the reduction in disease activity parameters, and inflammatory mediators IL-6 and MIF. This suggests a possible involvement of chemerin in the migration/retention of macrophages in the synovium.

Trial Registration Nederlands Trial Register

NTR 857     

Effects of a One Year Reusable Contraceptive Vaginal Ring on Vaginal Microflora and the Risk of Vaginal Infection: An Open-Label Prospective Evaluation

Background

A contraceptive vaginal ring (CVR) containing Nestorone® (NES) and ethinyl estradiol (EE) that is reusable for 1- year (13 cycles) is under development. This study assessed effects of this investigational CVR on the incidence of vaginal infections and change in vaginal microflora.

Methods

There were 120 women enrolled into a NES/EE CVR Phase III trial and a microbiology sub-study for up to 1- year of cyclic product use. Gynecological examinations were conducted at baseline, the first week of cycle 6 and last week of cycle 13 (or during early discontinuation visits). Vaginal swabs were obtained for wet mount microscopy, Gram stain and culture. The CVR was removed from the vagina at the last study visit and cultured. Semi-quantitative cultures for Lactobacillus, Gardnerella vaginalis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, anaerobic gram negative rods (GNRs), Candida albicans and other yeasts were performed on vaginal and CVR samples. Vaginal infections were documented throughout the study.

Results

Over 1- year of use, 3.3% of subjects were clinically diagnosed with bacterial vaginosis, 15.0% with vulvovaginal candidiasis, and 0.8% with trichomoniasis. The detection rate of these three infections did not change significantly from baseline to either Cycle 6 or 13. Nugent scores remained stable. H₂O₂-positive Lactobacillus dominated vaginal flora with a non-significant prevalence increase from 76.7% at baseline to 82.7% at cycle 6 and 90.2% at cycle 13, and a median concentration of 10⁷ colony forming units (cfu) per gram. Although anaerobic GNRs prevalence increased significantly, the median concentration decreased slightly (10⁴ to 10³cfu per gram). There were no significant changes in frequency or concentrations of other pathogens. High levels of agreement between vaginal and ring surface microbiota were observed.

Conclusion

Sustained use of the NES/EE CVR did not increase the risk of vaginal infection and was not disruptive to the vaginal ecosystem.

Trial Registration

ClinicalTrials.gov NCT00263341, NCT00455156     

Evaluation of Xpert? MTB/RIF Assay in Induced Sputum and Gastric Lavage Samples from Young Children with Suspected Tuberculosis from the MVA85A TB Vaccine Trial

Objective

Diagnosis of childhood tuberculosis is limited by the paucibacillary respiratory samples obtained from young children with pulmonary disease. We aimed to compare accuracy of the Xpert^® MTB/RIF assay, an automated nucleic acid amplification test, between induced sputum and gastric lavage samples from young children in a tuberculosis endemic setting.

Methods

We analyzed standardized diagnostic data from HIV negative children younger than four years of age who were investigated for tuberculosis disease near Cape Town, South Africa [2009–2012]. Two paired, consecutive induced sputa and early morning gastric lavage samples were obtained from children with suspected tuberculosis. Samples underwent Mycobacterial Growth Indicator Tube [MGIT] culture and Xpert MTB/RIF assay. We compared diagnostic yield across samples using the two-sample test of proportions and McNemar’s χ² test; and Wilson’s score method to calculate sensitivity and specificity.

Results

1,020 children were evaluated for tuberculosis during 1,214 admission episodes. Not all children had 4 samples collected. 57 of 4,463[1.3%] and 26 of 4,606[0.6%] samples tested positive for Mycobacterium tuberculosis on MGIT culture and Xpert MTB/RIF assay respectively. 27 of 2,198[1.2%] and 40 of 2,183[1.8%] samples tested positive [on either Xpert MTB/RIF assay or MGIT culture] on induced sputum and gastric lavage samples, respectively. 19/1,028[1.8%] and 33/1,017[3.2%] admission episodes yielded a positive MGIT culture or Xpert MTB/RIF assay from induced sputum and gastric lavage, respectively. Sensitivity of Xpert MTB/RIF assay was 8/30[26.7%; 95% CI: 14.2–44.4] for two induced sputum samples and 7/31[22.6%; 11.4–39.8] [p = 0.711] for two gastric lavage samples. Corresponding specificity was 893/893[100%;99.6–100] and 885/890[99.4%;98.7–99.8] respectively [p = 0.025].

Conclusion

Sensitivity of Xpert MTB/RIF assay was low, compared to MGIT culture, but diagnostic performance of Xpert MTB/RIF did not differ sufficiently between induced sputum and gastric lavage to justify selection of one sampling method over the other, in young children with suspected pulmonary TB.

Trial Registration

ClinicalTrials.gov NCT00953927