Development and Evaluation of an NTM-IGRA to Guide Pediatric Lymphadenitis Diagnosis

Nontuberculous mycobacteria; Tuberculosis; GlycopeptidolipidsMicobacterias no tuberculosas; Tuberculosis; GlicopeptidolípidosMicobacteris no tuberculosos; Tuberculosi; GlicopeptidolípidsBackground: Diagnosis of nontuberculous mycobacteria (NTM) infections remains a challenge. In this study, we describe the evaluation of an immunological NTM-interferon (IFN)-γ release assay (IGRA) that we developed using glycopeptidolipids (GPLs) as NTM-specific antigens. Methods: We tested the NTM-IGRA in 99 samples from pediatric patients. Seventy-five were patients with lymphadenitis: 25 were NTM confirmed, 45 were of unknown etiology but compatible with mycobacterial infection and 5 had lymphadenitis caused by an etiologic agent other than NTM. The remaining 24 samples were from control individuals without lymphadenitis (latently infected with M. tuberculosis, uninfected controls and active tuberculosis patients). Peripheral blood mononuclear cells were stimulated overnight with GPLs. Detection of IFN-γ producing cells was evaluated by enzyme-linked immunospot assay. Results: NTM culture-confirmed lymphadenitis patient samples had a significantly higher response to GPLs than the patients with lymphadenitis of unknown etiology but compatible with mycobacterial infection (P < 0.001) and lymphadenitis not caused by NTM (P < 0.01). We analyzed the response against GPLs in samples from unknown etiology lymphadenitis but compatible with mycobacterial infection cases according to the tuberculin skin test (TST) response, and although not statistically significant, those with a TST ≥5 mm had a higher response to GPLs when compared with the TST <5 mm group. Conclusions: Stimulation with GPLs yielded promising results in detecting NTM infection in pediatric patients with lymphadenitis. Our results indicate that the test could be useful to guide the diagnosis of pediatric lymphadenitis. This new NTM-IGRA could improve the clinical handling of NTM-infected patients and avoid unnecessary misdiagnosis and treatments

E-cigarettes : effects in phagocytosis and cytokines response against Mycobacterium tuberculosis

Cigarette smoking and tuberculosis are a significant cause of death worldwide. Several epidemiological studies have demonstrated cigarette smoking is a risk factor for tuberculosis. Electronic cigarettes have recently appeared as a healthier alternative to conventional smoking, although their impact in tuberculosis is not well understood. The aim of this study was to explore the effect of electronic cigarettes in phagocytosis of Mycobacterium tuberculosis and cytokines production. In vitro infection was carried out by exposing THP-1 macrophages to four electronic vapor extracts and the intracellular burden of M. tuberculosis was determined. The percentage of infection was evaluated by confocal microscopy and the cytokine production by Luminex. A reduction of intracellular M. tuberculosis burden in THP- 1 macrophages was found after its exposure to electronic vapor extract; the same trend was observed by confocal microscopy when Mycobacterium bovis BCG-GFP strain was used. Electronic cigarettes stimulate a pro-inflammatory cytokine response. We conclude that electronic cigarettes impair the phagocytic function and the cytokine response to M. tuberculosis

Tobacco Smoking and Second-Hand Smoke Exposure Impact on Tuberculosis in Children

Mycobacterium tuberculosis; Child; Passive smokingTuberculosis micobacteriana; Niño; Tabaquismo pasivoTuberculosi micobacteriana; Infant; Tabaquisme passiuLittle is known about whether second-hand smoke (SHS) exposure affects tuberculosis (TB). Here, we investigate the association of cigarette smoke exposure with active TB and latent TB infection (LTBI) in children, analyzing Interferon-Gamma Release Assays’ (IGRAs) performance and cytokine immune responses. A total of 616 children from contact-tracing studies were included and classified regarding their smoking habits [unexposed, SHS, or smokers]. Risk factors for positive IGRAs, LTBI, and active TB were defined. GM-CSF, IFN-γ, IL-2, IL-5, IL-10, IL-13, IL-22, IL-17, TNF-α, IL-1RA and IP-10 cytokines were detected in a subgroup of patients. Being SHS exposed was associated with a positive IGRA [aOR (95% CI): 8.7 (5.9–12.8)] and was a main factor related with LTBI [aOR (95% CI): 7.57 (4.79–11.94)] and active TB [aOR (95% CI): 3.40 (1.45–7.98)]. Moreover, IGRAs’ sensitivity was reduced in active TB patients exposed to tobacco. IL-22, GM-CSF, IL-5, TNF-α, IP-10, and IL-13 were less secreted in LTBI children exposed to SHS. In conclusion, SHS is associated with LTBI and active TB in children. In addition, false-negative IGRAs obtained on active TB patients exposed to SHS, together with the decrease of specific cytokines released, suggest that tobacco may alter the immune response.This research was supported by: (i) a grant from the Instituto de Salud Carlos III (PI 13/01546, PI16/01912, and PI18/00411), integrated in the Plan Nacional I+D+I and co-funded by the ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER) and CERCA Programme/Generalitat de Catalunya; and (ii) a grant from the Sociedad Española de Neumología y Cirugía Torácica (SEPAR; Barcelona, Spain) (Smoking Integrated Research Programme Call)

Study of CD27, CD38, HLA-DR and Ki-67 immune profiles for the characterization of active tuberculosis, latent infection and end of treatment

Mycobacterium tuberculosis; T-cells; Immune responseTuberculosis micobacteriana; Células T; Respuesta inmuneTuberculosi micobacteriana; Cèl·lules T; Resposta immuneBackground: Current blood-based diagnostic tools for TB are insufficient to properly characterize the distinct stages of TB, from the latent infection (LTBI) to its active form (aTB); nor can they assess treatment efficacy. Several immune cell biomarkers have been proposed as potential candidates for the development of improved diagnostic tools. Objective: To compare the capacity of CD27, HLA-DR, CD38 and Ki-67 markers to characterize LTBI, active TB and patients who ended treatment and resolved TB. Methods: Blood was collected from 45 patients defined according to clinical and microbiological criteria as: LTBI, aTB with less than 1 month of treatment and aTB after completing treatment. Peripheral blood mononuclear cells were stimulated with ESAT-6/CFP-10 or PPD antigens and acquired for flow cytometry after labelling with conjugated antibodies against CD3, CD4, CD8, CD27, IFN-γ, TNF-α, CD38, HLA-DR, and Ki-67. Conventional and multiparametric analyses were done with FlowJo and OMIQ, respectively. Results: The expression of CD27, CD38, HLA-DR and Ki-67 markers was analyzed in CD4+ T-cells producing IFN-γ and/or TNF-α cytokines after ESAT-6/CFP-10 or PPD stimulation. Within antigen-responsive CD4+ T-cells, CD27− and CD38+ (ESAT-6/CFP-10-specific), and HLA-DR+ and Ki-67+ (PPD- and ESAT-6/CFP-10-specific) populations were significantly increased in aTB compared to LTBI. Ki-67 demonstrated the best discriminative performance as evaluated by ROC analyses (AUC > 0.9 after PPD stimulation). Data also points to a significant change in the expression of CD38 (ESAT-6/CFP-10-specific) and Ki-67 (PPD- and ESAT-6/CFP-10-specific) after ending the anti-TB treatment regimen. Furthermore, ratio based on the CD27 median fluorescence intensity in CD4+ T-cells over Mtb-specific CD4+ T-cells showed a positive association with aTB over LTBI (ESAT-6/CFP-10-specific). Additionally, multiparametric FlowSOM analyses revealed an increase in CD27 cell clusters and a decrease in HLA-DR cell clusters within Mtb-specific populations after the end of treatment. Conclusion: Our study independently confirms that CD27−, CD38+, HLA-DR+ and Ki-67+ populations on Mtb-specific CD4+ T-cells are increased during active TB disease. Multiparametric analyses unbiasedly identify clusters based on CD27 or HLA-DR whose abundance can be related to treatment efficacy. Further studies are necessary to pinpoint the convergence between conventional and multiparametric approaches.This research was supported by a grant from the Instituto de Salud Carlos III (PI18/00411, PI19/01408 and CP20/00070), integrated in the Plan Nacional de I + D + I and cofunded by the ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER); a grant from the Sociedad Española de Neumología y Cirugía Torácica (project 25/2016; SEPAR; Barcelona, Spain); and from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement no. 823854 (INNOVA4TB). JD and IL are researchers from the Miguel Servet programme. AS-M is supported by a Juan Rodés (JR18/00022) postdoctoral fellowship from ISCIII

A systems biology approach to the characterization of stress response in Dermacentor reticulatus tick unfed larvae

[Background]: Dermacentor reticulatus (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in D. reticulatus unfed larvae, with special emphasis on stress response. [Principal Findings]: The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as D. reticulatus with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in D. reticulatus unfed larvae and a Rickettsia sp. similar to R. raoultii was identified in these ticks. [Significance]: The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in D. reticulatus ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens.This research was supported by grants BFU2011-23896 and the EU FP7 ANTIGONE project number 278976. M. Popara is an Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). N. Ayllón and R.C. Galindo were funded by MEC, Spain.Peer Reviewe

Discovery of circulating miRNAs as biomarkers of chronic Chagas heart disease via a small RNA-Seq approach

Circulating miRNAs; Biomarkers; Chronic Chagas heart diseasemiARN circulantes; Biomarcadores; Enfermedad cardíaca crónica de ChagasmiRNAs circulants; Biomarcadors; Malaltia cardíaca crònica de ChagasChagas disease affects approximately 7 million people worldwide in Latin America and is a neglected tropical disease. Twenty to thirty percent of chronically infected patients develop chronic Chagas cardiomyopathy decades after acute infection. Identifying biomarkers of Chagas disease progression is necessary to develop better therapeutic and preventive strategies. Circulating microRNAs are increasingly reliable biomarkers of disease and therapeutic targets. To identify new circulating microRNAs for Chagas disease, we performed exploratory small RNA sequencing from the plasma of patients and performed de novo miRNA prediction, identifying potential new microRNAs. The levels of the new microRNAs temporarily named miR-Contig-1519 and miR-Contig-3244 and microRNAs that are biomarkers for nonchagasic cardiomyopathies, such as miR-148a-3p and miR-224-5p, were validated by quantitative reverse transcription. We found a specific circulating microRNA signature defined by low miR-Contig-3244, miR-Contig-1519, and miR-148a-3 levels but high miR-224-5p levels for patients with chronic Chagas disease. Finally, we predicted in silico that these altered circulating microRNAs could affect the expression of target genes involved in different cellular pathways and biological processes, which we will explore in the future.The funding was supported by Agencia Nacional e Promoción Científica y Tecnológica, PICT 2013-1892, Secretaría de Ciencia y Tecnología, Universidad Nacional de Rosario, 1MED410, Ministerio de Economía y competitividad and Fondo Europeo de Desarrollo Regional, SAF2016-75988-R (MINECO/FEDER), SAF2015-63868-R (MINECO/FEDER), Red de Investigación de Centros de Enfermedades Tropicales, RICET RD12/0018/0004, Consejería de Sanidad, Comunidad de Madrid, S-2010/BMD-2332, Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional, PGC2018-096132-B-I00

Molecular and Cellular Mechanisms of M. tuberculosis and SARS-CoV-2 Infections-Unexpected Similarities of Pathogenesis and What to Expect from Co-Infection

Tuberculosis is still an important medical and social problem. In recent years, great strides have been made in the fight against M. tuberculosis, especially in the Russian Federation. However, the emergence of a new coronavirus infection (COVID-19) has led to the long-term isolation of the population on the one hand and to the relevance of using personal protective equipment on the other. Our knowledge regarding SARS-CoV-2-induced inflammation and tissue destruction is rapidly expanding, while our understanding of the pathology of human pulmonary tuberculosis gained through more the 100 years of research is still limited. This paper reviews the main molecular and cellular differences and similarities caused by M. tuberculosis and SARS-CoV-2 infections, as well as their critical immunological and pathomorphological features. Immune suppression caused by the SARS-CoV-2 virus may result in certain difficulties in the diagnosis and treatment of tuberculosis. Furthermore, long-term lymphopenia, hyperinflammation, lung tissue injury and imbalance in CD4+ T cell subsets associated with COVID-19 could propagate M. tuberculosis infection and disease progression

Residential radon and characteristics of chronic obstructive pulmonary disease

It is not known whether residential radon exposure may be linked to the development of chronic obstructive pulmonary disease (COPD) and/or have an influence on the functional characteristics or exacerbations of COPD. The aim of this study was therefore to ascertain whether there might be an association between residential radon concentrations and certain characteristics of COPD. We analyzed COPD cases drawn from a case–control study conducted in an area of high radon exposure. Data were collected on spirometric pulmonary function variables, hospital admissions, and smoking. Radon measurements were taken using alpha-track-type CR-39 detectors individually placed in patients’ homes. All statistical analyses were performed using the IBM SPSS v22 computer software program. The study included 189 COPD cases (79.4% men; median age 64 years). The median radon concentration was 157 Bq/m3. No differences were found between radon concentration and sex, age or severity of breathing obstruction as measured by FEV1%. It should be noted, however, that 48.1% of patients with FEV1%  600 Bq/m3 exhibited no different characteristics in lung function. Exposure to radon does not appear to have an influence on the clinical characteristics of smokers and ex-smokers with COPD. As exposure to residential radon increases, there is no trend towards a worsening of FEV1%. Further studies are thus needed to analyze this possible association in never-smokers with COPDSpanish Society of Pneumology & Thoracic Surgery (Sociedad Española de Neumología y Cirugía Torácica—SEPAR), competitive Project Number 439, call 2018S

A model based on the combination of ifn-γ, ip-10, ferritin and 25-hydroxyvitamin d for discriminating latent from active tuberculosis in children

Altres ajuts: This work was supported by grants from: (i) the Isolana Foundation, (ii) the Maria Francisca de Roviralta Foundation expedient AT/MA 1-19/07/2017, AT/MA 70-27/04/2016, and AT/MA 3-22/10/2014 and (vi) Fundació Recerca i Docència Mútua Terrassa.In recent years, pediatric research on tuberculosis (TB) has focused on addressing new biomarkers with the potential to be used as immunological non-sputum-based methods for the diagnosis of TB in children. The aim of this study was to characterize a set of cytokines and a series of individual factors (ferritin, 25-hydroxyvitamin D [25(OH)D], parasite infections, and nutritional status) to assess different patterns for discriminating between active TB and latent TB infection (LTBI) in children. The levels of 13 cytokines in QuantiFERON-TB Gold In-Tube (QFT-GIT) supernatants were analyzed in 166 children: 74 with active TB, 37 with LTBI, and 55 uninfected controls. All cytokines were quantified using Luminex or ELISA. Ferritin and 25(OH)D were also evaluated using CLIA, and Toxocara canis Ig-G antibodies were detected with a commercial ELISA kit. The combination of IP-10, IFN-γ, ferritin, and 25(OH)D achieved the best diagnostic performance to discriminate between active TB and LTBI cases in children in relation to the area under receiver operating characteristic (ROC) curve 0.955 (confidence interval 95%: 0.91-1.00), achieving optimal sensitivity and specificity for the development of a new test (93.2 and 90.0%, respectively). Children with TB showed higher ferritin levels and an inverse correlation between 25(OH)D and IFN-γ levels. The model proposed includes a combination of biomarkers for discriminating between active TB and LTBI in children to improve the accuracy of TB diagnosis in children. This combination of biomarkers might have potential for identifying the onset of primary TB in children

Expression of early growth response gene-2 and regulated cytokines correlate with recovery from Guillain-Barré syndrome

Guillain-Barré syndrome (GBS) is an immune-mediated peripheral neuropathy. The goal of this research was the identification of biomarkers associated with recovery from GBS. In this study, we compared the transcriptome of PBMCs from a GBS patient and her healthy twin to discover possible correlates of disease progression and recovery. The study was then extended using GBS and spinal cord injury unrelated patients with similar medications and healthy individuals. The early growth response gene-2 (EGR2) was upregulated in GBS patients during disease recovery. The results provided evidence for the implication of EGR2 in GBS and suggested a role for EGR2 in the regulation of IL-17, IL-22, IL-28A, and TNF-ß cytokines in GBS patients. These results identified biomarkers associated with GBS recovery and suggested that EGR2 overexpression has a pivotal role in the downregulation of cytokines implicated in the pathophysiology of this acute neuropathy.This work was supported partially by European Union Framework Program 7 Antigone Project 278976. L.M.-H. was supported by a fellowship from the University of Castilla La Mancha (UCLM). M.V. was supported by the research plan of the UCLM.Peer Reviewe