Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema

Background

Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated.

Objective

To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis.

Methods

Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively.

Results

We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-α responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-α responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-α but not IL-4 in undepleted PBMC.

Conclusions

Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE.     

DAG Expression: High-Throughput Gene Expression Analysis of Real-Time PCR Data Using Standard Curves for Relative Quantification

Background

Real-time quantitative PCR (qPCR) is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available.

Results

The recently developed commercial microarrays allow for the drawing of standard curves of multiple assays using the same n-fold diluted samples. Data Analysis Gene (DAG) Expression software has been developed to perform high-throughput gene-expression data analysis using standard curves for relative quantification and one or multiple reference genes for sample normalization. We discuss the application of DAG Expression in the analysis of data from an experiment performed with Fluidigm technology, in which 48 genes and 115 samples were measured. Furthermore, the quality of our analysis was tested and compared with other available methods.

Conclusions

DAG Expression is a freely available software that permits the automated analysis and visualization of high-throughput qPCR. A detailed manual and a demo-experiment are provided within the DAG Expression software at http://www.dagexpression.com/dage.zip.     

MendeLIMS: a web-based laboratory information management system for clinical genome sequencing

Background

Large clinical genomics studies using next generation DNA sequencing require the ability to select and track samples from a large population of patients through many experimental steps. With the number of clinical genome sequencing studies increasing, it is critical to maintain adequate laboratory information management systems to manage the thousands of patient samples that are subject to this type of genetic analysis.

Results

To meet the needs of clinical population studies using genome sequencing, we developed a web-based laboratory information management system (LIMS) with a flexible configuration that is adaptable to continuously evolving experimental protocols of next generation DNA sequencing technologies. Our system is referred to as MendeLIMS, is easily implemented with open source tools and is also highly configurable and extensible. MendeLIMS has been invaluable in the management of our clinical genome sequencing studies.

Conclusions

We maintain a publicly available demonstration version of the application for evaluation purposes at http://mendelims.stanford.edu. MendeLIMS is programmed in Ruby on Rails (RoR) and accesses data stored in SQL-compliant relational databases. Software is freely available for non-commercial use at http://dna-discovery.stanford.edu/software/mendelims/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-290) contains supplementary material, which is available to authorized users.     

CD57 Expression and Cytokine Production by T Cells in Lesional and Unaffected Skin from Patients with Psoriasis

Background

The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.

Methodology

We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.

Principal Findings

We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.

Conclusions/Significance

These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.     

Methotrexate in chronic-recurrent calcium pyrophosphate deposition disease: no significant effect in a randomized crossover trial

Introduction

Calcium pyrophosphate deposition (CPPD) may cause severe arthropathy, major joint destruction and treatment options are limited. The aim of this study was to test the therapeutic efficacy of methotrexate (MTX) in chronic or recurrent CPPD arthropathy.

Methods

Patients with CPPD arthropathy were randomized to receive either weekly subcutaneous injections of 15 mg/week of MTX or placebo (PBO) for three months, in a double-blind, crossover randomized controlled trial. Inclusion criteria comprised definite CPPD disease, recurrent arthritis or persistent polyarthritis, and an insufficient response to NSAIDs, glucocorticoids or colchicine. The primary outcome was an improvement in the disease activity scores based on 44 joints (DAS44). The analysis was performed on an intent-to-treat basis.

Results

We randomized 26 patients, and compared 25 treatment periods on MTX with 21 treatment periods on PBO. Baseline characteristics were balanced between the groups. The evolution of the DAS44 was not statistically significantly different between groups (median DAS44 decreased by −0.08 on MTX versus −0.13 on PBO, after three months, P = 0.44). Furthermore, pain levels remained stable in both groups (median change in VAS Pain −1 unit on MTX and 0 on PBO, P = 0.43), and none of the secondary outcomes was significantly different between the two groups. Minor adverse events (AE) did not differ in frequency between the groups, but the only serious AE occurred on MTX (bicytopenia).

Conclusions

The results of this trial with MTX in this older population with chronic or recurrent CPPD arthropathy suggest no strong effect of MTX on disease activity.

Trial registration

EudraCT No: 2007-003479-37. Registered 26 April 2008     

An evidence-based approach to identify aging-related genes in Caenorhabditis elegans

Background

Extensive studies have been carried out on Caenorhabditis elegans as a model organism to elucidate mechanisms of aging and the effects of perturbing known aging-related genes on lifespan and behavior. This research has generated large amounts of experimental data that is increasingly difficult to integrate and analyze with existing databases and domain knowledge. To address this challenge, we demonstrate a scalable and effective approach for automatic evidence gathering and evaluation that leverages existing experimental data and literature-curated facts to identify genes involved in aging and lifespan regulation in C. elegans.

Results

We developed a semantic knowledge base for aging by integrating data about C. elegans genes from WormBase with data about 2005 human and model organism genes from GenAge and 149 genes from GenDR, and with the Bio2RDF network of linked data for the life sciences. Using HyQue (a Semantic Web tool for hypothesis-based querying and evaluation) to interrogate this knowledge base, we examined 48,231 C. elegans genes for their role in modulating lifespan and aging. HyQue identified 24 novel but well-supported candidate aging-related genes for further experimental validation.

Conclusions

We use semantic technologies to discover candidate aging genes whose effects on lifespan are not yet well understood. Our customized HyQue system, the aging research knowledge base it operates over, and HyQue evaluations of all C. elegans genes are freely available at http://hyque.semanticscience.org.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0469-4) contains supplementary material, which is available to authorized users.     

Interplay of filaggrin loss-of-function variants, allergic sensitization, and eczema in a longitudinal study covering infancy to 18 years of age

Background

Immune specific genes as well as genes regulating the formation of skin barrier are major determinants for eczema manifestation. There is a debate as to whether allergic sensitization and filaggrin gene (FLG) variants lead to eczema or FLG variants and eczema increase the risk of allergic sensitization. To investigate the time-order between eczema and allergic sensitization with respect to FLG variants, data from a large prospective study covering infancy to late adolescence were analyzed.

Methodology/Principal Findings

Repeated measurements of eczema and allergic sensitization (documented by skin prick tests) at ages 1, 2, 4, 10, and 18 years were ascertained in the Isle of Wight birth cohort (n = 1,456). Three transition periods were analyzed: age 1-or-2 to 4, 4 to 10, and 10 to 18 years. FLG variants were genotyped in 1,150 participants. Over the three transition periods, in temporal sequence analyses of initially eczema-free participants, the combined effect of FLG variants and allergic sensitization showed a 2.92-fold (95% CI: 1.47–5.77) increased risk ratio (RR) of eczema in subsequent examinations. This overall risk was more pronounced at a younger age (transition period 1-or-2 to 4, RR = 6.47, 95% CI: 1.96–21.33). In contrast, FLG variants in combination with eczema showed a weaker, but significant, risk ratio for subsequent allergic sensitization only up to 10 years of age.

Conclusions/Significance

Taking the time order into account, this prospective study demonstrates for the first time, that a combination of FLG variants and allergic sensitization increased the risk of eczema in subsequent years. Also FLG variants interacted with eczema and increased the risk of subsequent allergic sensitization, which, was limited to the younger age. Hence, early restoration of defective skin barrier could prevent allergic sensitization and subsequently reduce the risk of eczema development.     

Impact of Anthelminthic Treatment in Pregnancy and Childhood on Immunisations,Infections and Eczema in Childhood: A Randomised Controlled Trial

Background

Helminth infections may modulate immune responses to unrelated pathogens and allergens; these effects may commence prenatally. We addressed the hypothesis that anthelminthic treatment in pregnancy and early childhood would improve responses to immunisation and modulate disease incidence in early childhood with both beneficial and detrimental effects.

Methods and Findings

A randomised, double-blind, placebo-controlled trial was conducted in Entebbe, Uganda [ISRCTN32849447]. In three independent randomisations, 2507 pregnant women were allocated to receive single-dose albendazole or placebo, and praziquantel or placebo; 2016 of their offspring were randomised to receive quarterly single-dose albendazole or placebo from age 15 months to 5 years. Primary outcomes were post-immunisation recall responses to BCG and tetanus antigens, and incidence of malaria, diarrhoea, and pneumonia; incidence of eczema was an important secondary outcome. Analysis was by intention-to-treat. Of 2345 live births, 1622 (69%) children remained in follow-up at age 5 years. 68% of mothers at enrolment, and 11% of five-year-olds, had helminth infections. Maternal hookworm and Schistosoma mansoni were effectively treated by albendazole and praziquantel, respectively; and childhood hookworm and Ascaris by quarterly albendazole. Incidence rates of malaria, diarrhoea, pneumonia, and eczema were 34, 65, 10 and 5 per 100 py, respectively. Albendazole during pregnancy caused an increased rate of eczema in the children (HR 1.58 (95% CI 1.15–2.17), p = 0.005). Quarterly albendazole during childhood was associated with reduced incidence of clinical malaria (HR 0.85 (95% CI 0.73–0.98), p = 0.03). There were no consistent effects of the interventions on any other outcome.

Conclusions

Routine use of albendazole in pregnancy may not always be beneficial, even in tropical developing countries. By contrast, regular albendazole treatment in preschool children may have an additional benefit for malaria control where helminths and malaria are co-endemic. Given the low helminth prevalence in our children, the effect of albendazole on malaria is likely to be direct.

Trial registration

Current Controlled Trials ISRCTN32849447     

First-in-Man Open Clinical Trial of a Combined rdESAT-6 and rCFP-10 Tuberculosis Specific Skin Test Reagent

Background

Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity.

Methodology

In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1∶1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 µg or 0.1 µg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN γ response just below the Quantiferon® cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded.

Conclusion

The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00793702","term_id":"NCT00793702"}}NCT00793702     

Type I Interferon: Potential Therapeutic Target for Psoriasis?

Background

Psoriasis is an immune-mediated disease characterized by aberrant epidermal differentiation, surface scale formation, and marked cutaneous inflammation. To better understand the pathogenesis of this disease and identify potential mediators, we used whole genome array analysis to profile paired lesional and nonlesional psoriatic skin and skin from healthy donors.

Methodology/Principal Findings

We observed robust overexpression of type I interferon (IFN)–inducible genes and genomic signatures that indicate T cell and dendritic cell infiltration in lesional skin. Up-regulation of mRNAs for IFN-α subtypes was observed in lesional skin compared with nonlesional skin. Enrichment of mature dendritic cells and 2 type I IFN–inducible proteins, STAT1 and ISG15, were observed in the majority of lesional skin biopsies. Concordant overexpression of IFN-γ and TNF-α–inducible gene signatures occurred at the same disease sites.

Conclusions/Significance

Up-regulation of TNF-α and elevation of the TNF-α–inducible gene signature in lesional skin underscore the importance of this cytokine in psoriasis; these data describe a molecular basis for the therapeutic activity of anti–TNF-α agents. Furthermore, these findings implicate type I IFNs in the pathogenesis of psoriasis. Consistent and significant up-regulation of type I IFNs and their associated gene signatures in psoriatic skin suggest that type I IFNs may be potential therapeutic targets in psoriasis treatment.     

Oral Doxycycline Reduces Pterygium Lesions; Results from a Double Blind,Randomized, Placebo Controlled Clinical Trial

Purpose

To determine whether oral doxycycline treatment reduces pterygium lesions.

Design

Double blind, randomized, placebo controlled clinical trial.

Participants

98 adult patients with primary pterygium.

Methods

Patients were randomly assigned to receive 100 mg oral doxycycline twice a day (49 subjects), or placebo (49 subjects), for 30 days. Photographs of the lesion were taken at the time of recruitment and at the end of the treatment. Follow-up sessions were performed 6 and 12 months post-treatment. Statistical analyses for both continuous and categorical variables were applied. p values of less than 0.05 were considered to indicate statistical significance.

Main Outcome Measures

The primary endpoint was the change in lesion size after 30 days of treatment.

Results

The primary endpoint was not met for the whole population but subgroup analysis showed that doxycycline was effective in patients of Caucasian origin while other ethnicities, mostly Hispanic, did not respond to the treatment. Moreover, there was a correlation between age and better response (p = 0.003). Adverse events were uncommon, mild, and in agreement with previous reports on short doxycycline treatments.

Conclusions

Oral doxycycline was superior to placebo for the treatment of primary pterygia in older Caucasian patients. These findings support the use of doxycycline for pterygium treatment in particular populations.

Trial Registration

European Union Clinical Trials Register EudraCT 2008-007178-39     

Prenatal Vitamin D Supplementation and Child Respiratory Health: A Randomised Controlled Trial

Background

Observational studies suggest high prenatal vitamin D intake may be associated with reduced childhood wheezing. We examined the effect of prenatal vitamin D on childhood wheezing in an interventional study.

Methods

We randomised 180 pregnant women at 27 weeks gestation to either no vitamin D, 800 IU ergocalciferol daily until delivery or single oral bolus of 200,000 IU cholecalciferol, in an ethnically stratified, randomised controlled trial. Supplementation improved but did not optimise vitamin D status. Researchers blind to allocation assessed offspring at 3 years. Primary outcome was any history of wheeze assessed by validated questionnaire. Secondary outcomes included atopy, respiratory infection, impulse oscillometry and exhaled nitric oxide. Primary analyses used logistic and linear regression.

Results

We evaluated 158 of 180 (88%) offspring at age 3 years for the primary outcome. Atopy was assessed by skin test for 95 children (53%), serum IgE for 86 (48%), exhaled nitric oxide for 62 (34%) and impulse oscillometry of acceptable quality for 51 (28%). We found no difference between supplemented and control groups in risk of wheeze [no vitamin D: 14/50 (28%); any vitamin D: 26/108 (24%) (risk ratio 0.86; 95% confidence interval 0.49, 1.50; P = 0.69)]. There was no significant difference in atopy, eczema risk, lung function or exhaled nitric oxide between supplemented groups and controls.

Conclusion

Prenatal vitamin D supplementation in late pregnancy that had a modest effect on cord blood vitamin D level, was not associated with decreased wheezing in offspring at age three years.

Trial Registration

Controlled-Trials.com ISRCTN68645785     

Safety and Immunogenicity of DNA and MVA HIV-1 Subtype C Vaccine Prime-Boost Regimens: A Phase I Randomised Trial in HIV-Uninfected Indian Volunteers

Study Design

A randomized, double-blind, placebo controlled phase I trial.

Methods

The trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos.

Results

Both vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1^st and 2^nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination.

Conclusions

Although DNA priming resulted in enhancement of immune responses after 1^st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting.

Trial Registration

Clinical Trial Registry CTRI/2009/091/000051