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Background: We aim to investigate the effects of the Bruton’s tyrosine kinase (BTK) inhibitor remibrutinib on human in vitro cell activation induced by serum obtained from chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIndU) patients. Methods: 22 patients with CSU (mean age 52 years, 27% women) and 22 with CIndU (46 years, 27% women) were included. A sex-equivalent control group was enrolled. Total IgE serum levels and the FcεR1a expression on blood basophils were determined before the initiation of omalizumab therapy. Patients were then classified as responders or non-responders to anti-IgE therapy, according to their clinical data and IgE and FcεR1a receptor expression. Mast cell generation and stimulation and basophil stimulation were performed. Flow cytometry was used to analyze samples. Percentage of activation, degranulation and inhibition in basophils and mast cells are studied. Results: Main results show that remibrutinib inhibits mast cells and basophil degranulation induced by IgE cross-linking. Also, serum from patients induced a greater degranulation of mast cells and basophils compared to controls. We found that activation of mast cells and basophils by patient sera was not related to omalizumab responsiveness. Conclusion: Our results show that remibrutinib is able to inhibit the degranulation of human basophils and mast cells in vitro, independently of the patient’s expression of FcεR1 expression and response to omalizumab.

Objective To evaluate the spontaneous progression of cervical intraepithelial neoplasia grade 2 (CIN2) in accordance with Chlamydia trachomatis (chlamydia) serology. Methods A prospective observational study included women diagnosed with CIN2 by cervical biopsy and managed conservatively for 24 months at Hospital del Mar, Barcelona, between December 2011 and October 2013. Serum anti-chlamydia immunoglobulin G (IgG), previous cytology, and high-risk human papillomavirus (HPV) genotyping were recorded at baseline. The outcome was regression, persistence, or progression of CIN2. Results Overall, 93 women aged 18-56 years were enrolled. Spontaneous regression was observed for 61 (66%) women, and 21 (23%) progressed to CIN3. Eight (9%) women had chlamydia seropositivity at baseline. Multivariate analysis showed that anti-chlamydia IgG seropositivity (odds ratio [OR], 19.1; 95% confidence interval [CI], 1.9-189.7), previous high-grade squamous intraepithelial lesion cytology (OR, 5.0; 95% CI, 1.7-14.6), and HPV16 (OR, 4.8; 95% CI, 1.7-13.7) increased the risk of CIN2 persistence or progression. Conclusion Women with CIN2 and chlamydia IgG seropositivity had increased risk of progression to CIN2+ and immediate treatment may be recommended for these women. Larger clinical studies are needed to confirm the results, but chlamydia serology might be introduced into CIN2 management to better individualize treatment.

mRNA-based vaccines effectively induce protective neutralizing antibody responses against SARS-CoV-2, the etiological agent of COVID-19. The specific compositional patterns of these responses remain largely unknown. We found that SARS-CoV-2-naïve individuals receiving the first dose of an mRNA vaccine developed a SARS-CoV-2-specific antibody response with a subclass profile comparable to that induced by the natural infection, except IgA2, which did not increase. SARS-CoV-2-naïve subjects also mounted a robust virus-specific recall response after receiving the second dose. This response increased all IgG subclasses, but boosted neither IgM nor IgA1 and IgA2 subclasses. In contrast, individuals recovered from COVID-19 mounted peak virus-specific antibody responses upon primary immunization and did not further augment such responses following secondary immunization. Remarkably, compared to SARS-CoV-2-naïve subjects, individuals with pre-existing immunity showed increased levels of all virus-specific antibodies but IgG3 following primary vaccination. By dissecting the heterogeneity of mRNA vaccine-induced humoral responses to SARS-CoV-2, our findings indicate that the induction of optimal immune protection may require the development of personalized vaccination programs.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the E¿-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic E¿-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors., The J.Y. laboratory is funded by the Spanish Ministerio de Economía, Industria y Competitividad (grant SAF2017-83565-R), Spanish Ministerio de Ciencia e Innovación (grant PID2020-112526RB-I00), and Fundación Científica de la Asociación Española Contra el Cáncer (grant PROYEI6018YÉLA). Work in the J.E.S. laboratory is supported by a core grant to the Laboratory of Molecular Biology from the Medical Research Council (U105178808). The F.D. laboratory is supported by a Laboratory of Excellence grant (ANR-10-LABX-0034_Medalis) to Strasbourg University, Centre National de la Recherche Scientifique. The P.N. laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness/Instituto de Salud Carlos III–Fondo Europeo de Desarrollo Regional (FEDER; PI17/00199 and PI20/00625) and the Generalitat de Catalunya (2017-SGR-225). The P.M. laboratory acknowledges support from Centres de Recerca de Catalunya/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for core support, the Spanish Ministry of Economy and Competitiveness (grant SAF-2019-108160-R), the Fundación Uno entre Cienmil, the Obra Social La Caixa (grant LCF/PR/HR19/52160011), and the German Josep Carreras Leukamie Stiftung. Work at the G.R. and P.M. laboratories are cofinanced by the European Regional Development Fund through the Interreg V-A Spain-France-Andorra Program (project PROTEOblood; grant EFA360/19). The O.F.-C. laboratory is funded by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00; cofinanced with European FEDER funds) and the European Research Council (ERC-617840). T.V.-H. was supported by a Marie Sklodowska Curie fellowship (GA792923). The A.B. laboratory is supported by the Spanish Ministry of Economy and Competitiveness (grant PID2019-104695RB-I00).

Background Cardiac magnetic resonance (CMR) constitutes a gold standard in the diagnosis of cardiomyopathies. Regardless of CMR advantages, the method is time-consuming, high-cost, with limited availability in some European regions. Purpose To assess the availability and applicability of CMR for establishing the diagnosis in different populations of patients with cardiomyopathies. Methods Overall, 3208 adult patients with cardiomyopathy (1119 / 34.9% females; median age at diagnosis: 49.0 years): 1260 with dilated (DCM), 1739 with hypertrophic (HCM), 66 with restrictive (RCM) and 143 with arrhythmogenic right ventricular cardiomyopathy (ARVC) enrolled in EURObservational Research Programme (EORP) – Cardiomyopathy/Myocarditis Long-Term Registry were analysed. Results CMR scan was performed as a baseline diagnostic method in 29.4% of patients; CMR was a single diagnostic method in 0.9% of patients and in 28.6% of patients CMR was used along with transthoracic echocardiography (TTE). In 67.6% of patients TTE was at the baseline the single diagnostic imaging method. Prevalence of CMR use in different cardiomyopathies was as follows: 20.6% in DCM, 33.8% in HCM, 36.4% in RCM and 51.1% in ARVC (p The population with CMR use was younger, less symptomatic, with decreased prevalence of other cardiovascular risk factors and of associated cardiovascular diseases as compared to the population diagnosed without CMR scanning (p Abnormal CMR results were present in 93.4% of patients with the highest percentage in RCM (95.8%) and HCM (94.9%) followed by DCM (91.5%) and the lowest abnormal CMR scan ratio in ARVC (87.7%) (p=0.030). The majority of CMR examinations comprised the assessment of late gadolinium enhancement (LGE, 93.3% at baseline). Presence of CMR LGE was observed in 69.3% of all patients: 59.1% in DCM, 73.8% in HCM, in 63.9% in ARVC and with the highest prevalence in RCM (83.3%) (p Conclusion The study reveals real-life data on the low availability and applicability of CMR in adult patients with cardiomyopathies. The analysis shows the advantages of CMR imaging but also identifies the gaps between recommendations and clinical practice. Improvement regarding access, training and reimbursement is necessary to offer CMR to cardiomyopathy patients in accordance with the ESC guidelines. Funding Acknowledgement Type of funding source: None

Use of adoptive immunotherapy with virus-specific T cells (VST) in patients with inborn errors of immunity prior to hematopoietic stem cell transplantation (HSCT) has been reported in few patients. We report our experience, reviewing all the cases previously reported. We report four children with inborn errors of immunity who received VST infusion in a pre-HSCT setting in two reference centers in Spain and review all inborn errors of immunity cases previously reported. Taking into account our four cases, nine children have been reported to receive VST prior to HSCT to date: 3 severe combined immunodeficiency, 2 CTPS1 deficiency, 1 dyskeratosis congenital, 1 ORAI1 deficiency, 1 Rothmund-Thomson syndrome, and 1 combined immunodeficiency without confirmed genetic defect. In four patients, immunotherapy resulted in clinical improvement, allowing to proceed to HSCT. In these cases, the infusion was started closely to viral diagnosis [mean time 28 days (IQR; 17–52 days)], and the VST was followed shortly thereafter by HSCT [mean time 28 days (IQR; 10–99 days)]. Viremia was controlled after HSCT in two cases (performed 7 and 36 days after the infusion). Multiple infusions were required in many cases. Five out of nine patients died before receiving HSCT. These patients presented with a prolonged and uncontrolled infection before VST administration [mean time from viral diagnosis to VST infusion was 176 days (IQR; 54–1687)]. In patients with inborn errors of immunity, the efficacy of VST for treating disseminated viral infections in pre-transplant settings seems to have a limited efficacy. However, this therapy could be used in a pre-emptive setting before severe viral disease occurs or closely to HSCT.

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient's B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation. This work has been partially funded by the following: CERCA Programme/Generalitat de Catalunya (JIA), SAF2015-68472-C2-1-R grant from the Spanish Ministry of Economy and Competitiveness co-financed by European Regional Development Fund (ERDF) (JIA), RTI2018-096824-B-C21 grant from the Spanish Ministry of Science, Innovation and Universities co-financed by ERDF (JIA), AC15/00027 grant from the Instituto de Salud Carlos III/Transnational Research Projects on Rare Diseases (JIA), PI14/00405 grant from the Instituto de Salud Carlos III co-financed by ERDF (RC), PI13/00174 grant from the Spanish Ministry of Economy and Competitiveness co-financed by ERDF (PP), SAF2017-88276-R grant from the Spanish Ministry of Economy, Industry and Competitiveness co-financed by ERDF (PP), ERC-2013-CoG project 614578 from the European Research Council (PP), 20859/PI/18 grant from Fundación Séneca (PP), SAF2015-68472-C2-2-R grant from the Spanish Ministry of Economy and Competitiveness co-financed by ERDF (FC) and RTI2018-096824-B-C22 grant from the Spanish Ministry of Science, Innovation and Universities co-financed by ERDF (FC). MK acknowledges support from CRUK (A16567) and MRC (P028160). H M-B is a Rio Hortega fellowship from Instituto de Salud Carlos III (CM14/00008) and DB acknowledges support from the UCL Impact Studentship.

Background The knowledge about chronic spontaneous urticaria (CSU) phenotypes is based on its clinical characteristics, associated comorbidities, course of the disease and its response to the available effective drugs. Genotype expression and its further correlation with CSU phenotypes are still unknown. We describe the cutaneous transcriptome of patients suffering a severely active CSU refractory to antihistamine treatment. Methods Through the bioinformatic analysis of the whole Human Genome with Oligo Microarrays and Quantitative real-time polymerase chain reaction (qPCR), relevant genes expressed in non-lesional [NLS-CSU] and lesional skin [LS-CSU]) and peripheral blood were identified in 20 patients suffering from severely active CSU and 10 healthy controls (HCs) Results From 39 genes differentially expressed in NLS-CSU when compared with HCs, 31 (79.48%) were confirmed by qPCR corresponding to genes involved in epidermal homeostasis and dermal repair. From the analysis comparing LS-CSU with NLS-CSU, a selection of 142 genes was studied with qPCR, and 103 (72.53%) were confirmed. Differentially expressed genes in the phenomenon of wheal development are involved in a variety of biological functions as, epidermal differentiation, intracellular signal function, transcriptional factors cell cycle differentiation, inflammation or coagulation. Differentially expressed genes that uniformly increase or decrease along the skin worsening until the wheal appearance are shown. Conclusion The skin of CSU patients with a severely active disease shows an overall immunological skin involvement showing a peculiar gene profile. This article is protected by copyright. All rights reserved.

Recent research has proven the existence of statistical relation among fragmented QRS and several highly prevalence diseases, such as cardiac sarcoidosis, acute coronary syndrome, arrythmogenic cardiomyopathies, Brugada syndrome, and hypertrophic cardiomyopathy. One out of five hundred people suffer from hypertrophic cardiomyopathies. The relation among the fragmentation and arrhythmias drives the objective of this work, which is to propose a valid method for QRS fragmentation detection. With that aim, we followed a two-stage approach. First, we identified the features that better characterize the fragmentation by analyzing the physiological interpretation of multivariate approaches, such as principal component analysis (PCA) and independent component analysis (ICA). Second, we created an invariant transformation method for the multilead electrocardiogram (ECG), by scrutinizing the statistical distributions of the PCA eigenvectors and of the ICA transformation arrays, in order to anchor the desired elements in the suitable leads in the feature space. A complete database was compounded incorporating real fragmented ECGs, surrogate registers by synthetically adding fragmented activity to real non-fragmented ECG registers, and standard clean ECGs. Results showed that the creation of beat templates together with the application of PCA over eight independent leads achieves 0.995 fragmentation enhancement ratio and 0.07 dispersion coefficient. In the case of ICA over twelve leads, the results were 0.995 fragmentation enhancement ratio and 0.70 dispersion coefficient. We conclude that the algorithm presented in this work constructs a new paradigm, by creating a systematic and powerful tool for clinical anamnesis and evaluation based on multilead ECG. This approach consistently consolidates the inconspicuous elements present in multiple leads onto designated variables in the output space, hence offering additional and valid visual and non-visual information to standard clinical review, and opening the door to a more accurate automatic detection and statistically valid systematic approach for a wide number of applications. In this direction and within the companion paper, further developments are presented applying this technique to fragmentation detection.

BACKGROUND: The high-affinity IgE receptor (FcεRI) expression on effector cells has been poorly characterized in patients with chronic urticaria (CU) to date. OBJECTIVES: To investigate the FcεRI expression on blood basophils in a large cohort of patients with CU and its potential relationship with relevant features of the disease. METHODS: Basophil FcεRI expression was measured by flow cytometry in 287 patients with CU (192 with chronic spontaneous urticaria and 95 with chronic inducible urticaria) at their initial evaluation in our department. A control group of healthy nonatopic individuals was included to provide reference data, and the effect of antihistamine and anti-IgE therapy on the basophil FcεRI expression was also evaluated in a cohort of patients with CU. RESULTS: The median FcεRI expression was found significantly higher in patients with CU compared with healthy controls (P < .0001). A positive correlation was found between serum IgE levels and basophil FcεRI expression (R = 0.422; P < .001). Significantly higher FcεRI levels on basophils were detected in patients with CU who presented with concomitant atopic features (P = .003), negative autologous serum skin test (P = .002), negative autologous plasma skin test (P = .009), or undetected levels of antithyroid antibodies (P = 0.01). Baseline FcεRI expression was not related to the activity and duration of the disease, and was not significantly modified during antihistamine therapy; however, it correlated with the clinical response to omalizumab (P = .003). CONCLUSIONS: Although further multicenter studies are needed to corroborate these findings, the assessment of basophil FcεRI levels might be relevant in daily clinical practice supporting an autoimmune pathogenesis and predicting response to anti-IgE treatment.

The aim of the study was to assess drug levels, immunogenicity and sacroiliitis on MRI in patients with axial spondyloarthritis under biologic tapering strategy. Consecutive patients with axial spondyloarthritis who remained in low disease activity more than 1 year after dose tapering of infliximab and adalimumab were included. Plasma drug concentrations of TNF inhibitors and anti-drug antibodies were determined, and MRI of sacroiliac joints was evaluated. Of twenty patients included, eighteen had therapeutic drug levels, no patient had anti-drug antibodies, and no patient had active sacroiliitis on MRI. These data could support the biologic tapering strategy and their maintenance over time.

Hypertrophic cardiomyopathy (HCM) is a myocardial disorder that affects 0.2% of the population and it is genetically transmitted. Several ECG findings have been related to the presence of fibrosis in other cardiac diseases, but data for HCM in this setting are lacking. Our hypothesis is that fibrosis affects the electrical cardiac propagation in patients with HCM in a relatively specific way and that this effect may be detected with suitable postprocessing applied to the ECG signals. We used 43 standard 12-lead ECGs from patients with previous clinical diagnosis of HCM. Principal Component Analysis (PCA) was applied by combining the ECG-leads oriented to different anatomic regions, hence assessing the potential fibrosis effects in the resulting leads for postprocessing convenience. Linear classifier of Support Vector Machine type were used with several statistics extracted from the resulting PCA-components, including normalized power, standard deviation, kurtosis, skewness, and local maxima. Results reached 75.0% sensitivity, 80.0% specificity, 85.7% positive predictive value, 66.7% negative predictive value, and 76.9% accuracy in our database. There is evidence that myocardial fibrosis can be detected in patients with HCM by postprocessing their ECG signals.

Cytomegalovirus encephalitis is a challenging life-threatening complication following hematopoietic stem cell transplantation for which medical treatment is usually ineffective or toxic. However, in recent years, adoptive T-cell therapy has been reported to provide a significant chance of cure for patients with viral infections. Herein, two cases of pediatric patients successfully treated with third-party donor-derived virus-specific T cells for CMV meningoencephalitis are reported.

The CD45RA T cell depletion (TCD) method has been used to deplete naive T cells, preventing graft-versus-host disease (GVHD) but preserving memory cells, providing immediate functional T cells with anti-infection, antileukemia, and antirejection effects. We describe a series of 25 consecutive high-risk patients with leukemia who received haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with CD45RA TCD. Each patient received 2 cell products: 1 created by CD34 positive selection and the other through CD45RA depletion from the CD34 negative fraction by a CliniMACS device. CD45RA-depleted haplo-HSCT was well tolerated, with rapid engraftment and low risk of severe acute GVHD and chronic GVHD. Although this treatment achieved a good control of viral reactivations, such as cytomegalovirus and adenovirus, we observed an unexpectedly high rate of limbic encephalitis due to human herpesvirus-6 (HHV-6; 8 cases). Characteristically, the infection appeared early in almost all patients, just after the engraftment. Although no patient died from encephalitis, 1 patient showed neuropsychological sequelae, and another experienced secondary graft failure just after the HHV-6 reactivation. (C) 2018 American Society for Blood and Marrow Transplantation.

Objectives Atrial fibrillation (AF) is associated with increased morbidity and mortality in patients with hypertrophic cardiomyopathy (HCM). The primary aim of this study (HCM Risk-AF) was to determine the predictors of AF in a large multicentre cohort of patients with HCM. Exploratory analyses were performed to investigate the association between AF and survival and the efficacy of antiarrhythmic therapy in maintaining sinus rhythm (SR). Methods A retrospective, longitudinal cohort of patients recruited between 1986 and 2008 in seven centres was used to develop multivariable Cox regression models fitted with preselected predictors. HCM was defined as unexplained hypertrophy (maximum left ventricular wall thickness of ≥15 mm or in accordance with published criteria for the diagnosis of familial disease). 28% of patients (n=1171) had coexistent hypertension. The primary end point was paroxysmal, permanent or persistent AF detected on ECG, Holter monitoring or implantable device interrogation. Results Of the 4248 patients with HCM without pre-existing AF, 740 (17.4%) reached the primary end point. Multivariable Cox regression revealed an association between AF and female sex, age, left atrial diameter, New York Heart Association (NYHA) class, hypertension and vascular disease. The proportion of patients with cardiovascular death at 10 years was 4.9% in the SR group and 10.9% in the AF group (difference in proportions=5.9%; 95% CI (4.1% to 7.8%)). The proportion of patients with non-cardiovascular death at 10 years was 3.2% in the SR group and 5.9% in the AF group (difference in proportions=2.8%; 95% CI (0.1% to 4.2%)). An intention-to-treat propensity score analysis demonstrated that β-blockers, calcium channel antagonists and disopyramide initially maintained SR during follow-up, but their protective effect diminished with time. Amiodarone therapy did not prevent AF during follow-up. Conclusion This study shows that patients with HCM who are at risk of AF development can be identified using readily available clinical parameters. The development of AF is associated with a poor prognosis but there was no evidence that antiarrhythmic therapy prevents AF in the long term.

This is an open-access article distributed under the terms of the Creative Commons Attribution License,.-- et al., In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy., This work was funded by grants from the Spanish Ministry of Science and Innovation/ Instituto de Salud Carlos III FEDER (PS09/00973) and from the AGAUR (2009 SGR 1409). IHM is an investigator at the Miguel Servet program (Instituto de Salud Carlos III).

Previous work by our group showed that transferring bone marrow cells transduced with an autoantigen into nonmyeloablated mice with experimental autoimmune encephalomyelitis induced immune tolerance and improved symptoms of the disease. Because this effect occurred in the absence of molecular chimerism, we hypothesized that the cells responsible did not have repopulating ability and that they were not mediating central but peripheral tolerance mechanisms. In the present study, we analyzed the immunophenotype of the cells that are generated in the transduction cultures and we evaluated the immunosuppressive activity of the main cell subpopulations produced. We show that both granulocytic (CD11b+Gr-1hi) and monocytic (CD11b+Gr-1lo) myeloid-derived suppressor cells (G- and M-MDSCs, respectively) are generated during standard 4-day γ-retroviral transduction cultures (representing about 25% and 40% of the total cell output, respectively) and that the effectively transduced cells largely consist of these two cell types. A third cell population representing about 15% of the transduced cells did not express CD45 or hematopoietic lineage markers and expressed mesenchymal stromal cell markers. Transduced total bone marrow cells and sorted M-MDSCs expressed arginase and inducible nitric oxide synthase activities, produced reactive oxygen species, and inhibited antigen-induced T-cell proliferation in vitro. Transgene-expressing MDSCs could be exploited therapeutically to induce tolerance in autoimmune diseases and in gene therapy protocols.

Hypertrophic cardiomyopathy, according to its prevalence, is a comparatively common disease related to the risk of suffering sudden cardiac death, heart failure and stroke. This illness is characterized by the excessive deposition of collagen among healthy myocardium cells. This situation, which is medically known as fibrosis, constitutes effective conduction obstacles in the myocardium electrical path, and when severe enough, it can be outlined as additional peaks or notches in the QRS, clinically entitled as fragmentation. Nowadays, the fragmentation detection is performed by visual inspection, but the fragmented QRS can be confused with the noise present in the electrocardiogram (ECG). On the other hand, fibrosis detection is performed by magnetic resonance imaging with late gadolinium enhancement, the main drawback of this technique being its cost in terms of time and money. In this work, we propose two automatic algorithms, one for fragmented QRS detection and another for fibrosis detection. For this purpose, we used four different databases, including the subrogated database described in the companion paper and incorporating three additional ones, one compounded by more accurate subrogated ECG signals and two compounded by real and affected subjects as labeled by expert clinicians. The first real-world database contains QRS fragmented records and the second one contains records with fibrosis and both were recorded in Hospital Cl&iacute, nico Universitario Virgen de la Arrixaca (Spain). To deeply analyze the scope of these datasets, we benchmarked several classifiers such as Neural Networks, Support Vector Machines (SVM), Decision Trees and Gaussian Na&iuml, ve Bayes (NB). For the fragmentation dataset, the best results were 0.94 sensitivity, 0.88 specificity, 0.89 positive predictive value, 0.93 negative predictive value and 0.91 accuracy when using SVM with Gaussian kernel. For the fibrosis databases, more limited accuracy was reached, with 0.47 sensitivity, 0.91 specificity, 0.82 predictive positive value, 0.66 negative predictive value and 0.70 accuracy when using Gaussian NB. Nevertheless, this is the first time that fibrosis detection is attempted automatically from ECG postprocessing, paving the way towards improved algorithms and methods for it. Therefore, we can conclude that the proposed techniques could offer a valuable tool to clinicians for both fragmentation and fibrosis diagnoses support.

The simplest and most extended definition of myeloid-derived suppressor cells (MDSC) refers to them as immature myeloid cells with the ability to downregulate adaptative immune responses, a definition that reflects both their origin and function. Although initially described in experimental models and patients with cancer, accumulations of MDSC have also been found in other pathological conditions such as chronic/acute infections, autoimmune diseases and different types of stress. In all these situations MDSC may play their physiological role by modulating normal immune responses, both adaptive and innate. The mechanisms of action of MDSC are diverse requiring either cell to cell contact or the release of soluble factors. A better understanding of MDSC biology will open new windows of therapeutic opportunities, either by inhibiting their function (i.e. in cancer patients), or by enhancing their suppressive effects and promoting their expansion (i.e. in inflammation or autoimmunity).

Summary We conducted a multicentre study of 1844 patients from 42 Spanish intensive care units, and analysed the clinical characteristics of brain death, the use of ancillary testing, and the clinical decisions taken after the diagnosis of brain death. The main cause of brain death was intracerebral haemorrhage (769/1844, 42%), followed by traumatic brain injury (343/1844, 19%) and subarachnoid haemorrhage (257/1844, 14%). The diagnosis of brain death was made rapidly (50% in the first 24 h). Of those patients who went on to die, the Glasgow Coma Scale on admission was ≤ 8/15 in 1146/1261 (91%) of patients with intracerebral haemorrhage, traumatic brain injury or anoxic encephalopathy; the Hunt and Hess Scale was 4–5 in 207/251 (83%) of patients following subarachnoid haemorrhage; and the National Institutes of Health Stroke Scale was ≥ 15 in 114/129 (89%) of patients with strokes. Brain death was diagnosed exclusively by clinical examination in 92/1844 (5%) of cases. Electroencephalography was the most frequently used ancillary test (1303/1752, 70.7%), followed by transcranial Doppler (652/1752, 37%). Organ donation took place in 70% of patients (1291/1844), with medical unsuitability (267/553, 48%) and family refusal (244/553, 13%) the main reasons for loss of potential donors. All life-sustaining measures were withdrawn in 413/553 of non-donors (75%).

Aims Atrial fibrillation (AF) and thrombo-embolism (TE) are associated with reduced survival in hypertrophic cardiomyopathy (HCM), but the absolute risk of TE in patients with and without AF is unclear. The primary aim of this study was to derive and validate a model for estimating the risk of TE in HCM. Exploratory analyses were performed to determine predictors of TE, the performance of the CHA2DS2-VASc score, and outcome with vitamin K antagonists (VKAs). Methods and results A retrospective, longitudinal cohort of seven institutions was used to develop multivariable Cox regression models fitted with pre-selected predictors. Bootstrapping was used for validation. Of 4821 HCM patients recruited between 1986 and 2008, 172 (3.6%) reached the primary endpoint of cerebrovascular accident (CVA), transient ischaemic attack (TIA), or systemic peripheral embolus within 10 years. A total of 27.5% of patients had a CHA2DS2-VASc score of 0, of whom 9.8% developed TE during follow-up. Cox regression revealed an association between TE and age, AF, the interaction between age and AF, TE prior to first evaluation, NYHA class, left atrial (LA) diameter, vascular disease, and maximal LV wall thickness. There was a curvilinear relationship between LA size and TE risk. The model predicted TE with a C-index of 0.75 [95% confidence interval (CI) 0.70-0.80] and the D-statistic was 1.30 (95% CI 1.05-1.56). VKA treatment was associated with a 54.8% (95% CI 31-97%, P = 0.037) relative risk reduction in HCM patients with AF. Conclusions The study shows that the risk of TE in HCM patients can be identified using a small number of simple clinical features. LA size, in particular, should be monitored closely, and the assessment and treatment of conventional vascular risk factors should be routine practice in older patients. Exploratory analyses show for the first time evidence for a reduction of TE with VKA treatment. The CHA2DS2-VASc score does not appear to correlate well with the clinical outcome in patients with HCM and should not be used to assess TE risk in this population.

Human early thymic precursors have the potential to differentiate into multiple cell lineages, including T cells and plasmacytoid dendritic cells (pDCs). This decision is guided by the induction or silencing of lineage-specific transcription factors. The ETS family member Spi-B is a key regulator of pDC development, whereas T-cell development is critically dependent on GATA-3. Here we show that triggering of the Notch1 signaling pathway by Delta-like1 controls the T/pDC lineage decision by regulating the balance between these factors. CD34+CD1a- thymic progenitor cells express Notch1, but down-regulate this receptor when differentiating into pDCs. On coculture with stromal cell lines expressing either human Delta-like1 (DL1) or Jagged1 (Jag1) Notch ligands, thymic precursors express GATA-3 and develop into CD4+CD8+TCRαβ+ T cells. On the other hand, DL1, but not Jag1, down-regulates Spi-B expression, resulting in impaired development of pDCs. The Notch1-induced block in pDC development can be relieved through the ectopic expression of Spi-B. These data indicate that DL1-induced activation of the Notch1 pathway controls the lineage commitment of early thymic precursors by altering the levels between Spi-B and GATA-3. (Blood. 2006;107:2446-2452)

Alpha/beta interferon (IFN-alpha/beta) triggers antiviral and antiproliferative responses in target cells through modulation of gene expression. The JAK-STAT pathway is the major mediator of these biological effects through the activation of the transcription factors STAT1 and STAT2, and gene ablation studies have demonstrated that both STAT1 and STAT2 are required for most antiviral responses induced by IFN-alpha/beta. However, additional signaling pathways are also activated by IFN. Here, we show that these additional pathways provoke a proliferative response in activated T lymphocytes. While activation of IFN-stimulated gene factor 3 produces a dominant inhibitory signal capable of overriding the mitogenic response, absence of either STAT1 or STAT2 leads to a proliferative response to IFN. Growth stimulation by IFN-alpha/beta is independent of other STAT proteins, particularly of STAT3, since T lymphocytes from STAT1-STAT3 double-knockout mice are growth stimulated by IFN-alpha/beta treatment. IFN-alpha/beta can cooperate with numerous T-cell mitogens, including interleukin-2 (IL-2), IL-4, IL-7, and IL-12, and can contribute to the rapid restoration of the thymus following glucocorticoid-mediated ablation. These results underscore the complexity of the cellular response to IFN and suggest that the ultimate outcome of IFN action results from a balance between growth-inhibitory and -stimulatory effects.

It is unknown how B cells that mature during a germinal center reaction 'decide' between plasma or memory cell fate. Here we describe a previously unknown subpopulation of B cells in the human germinal center that is characterized by tyrosine phosphorylated transcriptional activator STAT5. These cells had an activated centrocyte phenotype and had abundant expression of BCL6 but low expression of PRDM1, both encoding transcriptional repression proteins. Using RNA interference and ectopic expression of constitutively activated forms of STAT5, we demonstrate here a function for STAT5 in the self-renewal of B cells in vitro. STAT5b isoform seemed to directly upregulate Bcl-6, and ectopic expression of Bcl-6 in B cells resulted in self-renewal and inhibition of plasma cell differentiation. These data indicate that activation of STAT5 is involved in regulation of memory B cell differentiation.

Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into human CD34+ progenitor cells by lentivirus-mediated gene transfer, which resulted in more than 95% knockdown of p53. We adapted the human-SCID mouse model to optimize the development of hematopoietic cells, particularly of T cells. This was achieved by the intraperitoneal injection of CD34+ precursor cells into newborn Rag2-/- γc-/- mice that lack T, B, and NK cells. Robust development of T cells was observed in these mice, with peripheral T-cell repopulation 8 weeks after injection of the precursor cells. Other lymphocyte and myeloid subsets also developed in these mice. Injecting p53 siRNA-transduced CD34+ cells resulted in stable expression and down-modulation of p53 in the mature T-cell offspring. Inactivating p53 did not affect the development of CD34+ cells into various mature leukocyte subsets, including T cells, but it conferred resistance to γ-irradiation and other p53-dependent apoptotic stimuli to the T cells. (Blood. 2004;104:3886-3893)

STAT3 has been described as an essential component of G-CSF-driven cell proliferation and granulopoiesis. This notion was tested by conditional gene ablation in transgenic mice. Contrary to expectation, granulocytes developed from STAT3 null bone marrow progenitors, and STAT3 null neutrophils displayed mature effector functions. Rather than a deficit in granulopoiesis, mice lacking STAT3 in their hematopoietic progenitors developed neutrophilia, and bone marrow cells were hyperresponsive to G-CSF stimulation. These studies provide direct evidence for STAT3-independent granulopoiesis and suggest that STAT3 directs a negative feedback loop necessary for controlling neutrophil numbers, possibly through induced expression of the signaling inhibitor, SOCS3.

Major histocompatibility complex (MHC) class I antigens are constitutively expressed yet highly induced by interferon (IFN) during inflammation. We found that not only IFN-induced but also normal basal expression of MHC I required IFN receptors and signal transducer and activator of transcription (STAT)1, providing genetic evidence for continuous IFN signaling. Surprisingly, an IFN-independent requirement for STAT1 was also found, specifically in T lymphocytes, where MHC class I expression was not fully accounted for by IFN signaling. This IFN-independent pathway maintained tyrosine phosphorylation of STAT1 in T but not B lymphocytes even in the absence of IFN receptors. Interestingly, interleukin (IL)-7 selectively activated STAT1 and induced MHC class I in mature T but not B cells. These loss of function studies demonstrate an essential role of endogenous IFN and activated STAT1 for constitutive MHC class I expression in normal mice and define IL-7–dependent but IFN-independent regulation of STAT1 restricted to T lymphocytes.

It has been previously described that some proteins containing HMG boxes are able to bind more strongly to DNA modified with cis-diamminedichloroplatinum (II) (cisplatin) than to unmodified DNA. In the present study, we analyzed the interaction of cisplatin-modified DNA with the human autoantigen NOR-90 (UBF), a transcription factor that contains several HMG boxes. Using autoantibodies against NOR-90 to perform ELISA and immunoprecipitation, it was confirmed that NOR-90 (UBF) was able to bind cisplatin-modified DNA more avidly than unmodified DNA or trans-diamminedichloroplatinum(II) (transplatin) modified DNA. Moreover, by Southwestern, we observed that the 97 kDalton isoform of NOR-90 (UBF1) was able to bind cisplatin-modified DNA more strongly than the 94 kDalton isoform (UBF2); binding of unmodified DNA or transplatin-modified DNA was not detected with either isoform. Sera containing autoantibodies against NOR-90 did not inhibit, but increased the binding of NOR-90 to cisplatin-modified DNA.

Notch signals are critical for T-cell development, limiting the differentiation potential of multipotent progenitors arriving in the thymus via the bloodstream. Notch ligands Delta-like and Jagged are expressed in the bone marrow and, consequently, a role in the regulation of early events of adult hematopoiesis has been proposed. However, mice with disruptions in the Notch pathway do not show gross defects in the hematopoietic stem cell compartment, limiting Notch effects at later stages of development. In this study, we identify cord blood CD34 + CD38 − CD45RA − CD90 + cells, a recently described population of hematopoietic stem cells, as one of the earliest targets of Notch in human hematopoiesis. Upon Notch activation, CD34 + CD38 − cells are blocked in their differentiation at the CD34 + CD38 − CD45RA − CD90 + stage. Importantly, population and clonal analysis demonstrate that Delta-like–1 exposure does not affect lymphoid vs myeloid decisions. However, Notch signaling is required before lymphoid commitment to preserve T-cell potential of CD34 + CD38 − CD45RA − CD90 + cells. Our experiments also show that in terms of differentiation potential, CD34 + CD38 − CD45RA − CD90 + cells cultured in the presence of Notch signals, resemble cells directly isolated from cord blood. These results could have implications for translational efforts in the design of strategies aimed to accelerate immune reconstitution after transplantation.