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It is crucial to assess the levels of protection generated by natural infection or SARS-CoV-2 vaccines, mainly in individuals professionally exposed and in vulnerable groups. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection. Our aim was to assess the feasibility of a validated assay of T-cell responses.Twenty health-care-workers (HCW) were included. Antibody test to SARS-CoV-2 N and S-proteins in parallel with a commercially available whole-blood-interferon-gamma-release-assay (IGRA) to S-peptides and two detection methods, CLIA and ELISA were determined.IGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. The correlation by the two detection methods was very high (Whole-blood-IGRA-tests amenable to automation and constitutes a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become a valuable correlate of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health-care-workers.Es fundamental evaluar los niveles de protección inmune en infectados o tras la vacunación frente a SARS-CoV-2. La cuantificación de la respuesta inmune celular T puede complementar la determinación de anticuerpos. Evaluamos la viabilidad de un ensayo comercial validado de respuesta celular T específica frente a SARS-CoV-2.Se incluyeron veinte trabajadores sanitarios (TS). Medimos anticuerpos contra las proteínas N y S de SARS-CoV-2 y realizamos el ensayo de liberación de interferón-gamma (IFNγ) en sangre completa (IGRA) frente a péptidos de la proteína S. IFNγ se determinó mediante dos métodos de detección: CLIA y ELISA.IGRA detectó respuesta celular T en TS tanto infectados como vacunados. La correlación de los dos métodos de detección de IFNγ fue muy alta (R 0,8) y la sensibilidad y la especificidad variaron entre 100 y 86% y 100-73% respectivamente. Hubo una concordancia muy alta entre los niveles de anticuerpos específicos y el ensayo IGRA aunque la correlación cuantitativa fue relativamente baja. En el grupo de infectados, una dosis de vacuna fue suficiente para alcanzar el «plateau» de respuesta inmune. IGRA fue claramente positivo en un profesional vacunado inmunosuprimido que presentaba anticuerpos contra la proteína S negativos.IGRA frente a péptidos de la proteína-S es susceptible de automatización y constituye una herramienta prometedora para medir la respuesta inmune celular frente a SARS-CoV-2; es aplicable a un gran número de muestras y puede servir para valorar la protección, particularmente en los grupos vulnerables en riesgo de volver a exponerse a la infección, como los TS.

Objective To assess the potential diagnostic utility of advanced lymphocyte profiling to differentiate between primary Sjögren’s Syndrome (pSS) and non-Sjögren Sicca syndrome. Methods Distribution of peripheral lymphocyte subpopulations was analysed by flow cytometry in 68 patients with pSS, 26 patients with sicca syndrome and 23 healthy controls. The ability to discriminate between pSS and sicca syndrome was analysed using the area under the curve (AUC) of the receiver operating characteristic curve of the different lymphocyte subsets. Results The ratio between naïve/memory B cell proportions showed an AUC of 0.742 to differentiate pSS and sicca syndrome, with a sensitivity of 76.6% and a specificity of 72% for a cut-off value of 3.4. The ratio of non-switched memory B cells to activated CD4+ T cells percentage (BNSM/CD4ACT) presented the highest AUC (0.840) with a sensitivity of 83.3% and specificity of 81.7% for a cut-off value Conclusion Patients with pSS show a profound imbalance in the distribution of circulating T and B lymphocyte subsets. The ratio BNSM/CD4ACT is useful to discriminate between pSS and sicca syndrome.

BackgroundAlmost two years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted, nor new tests identified to improve the prediction and management of SARS-CoV-2 infection.MethodsRetrospective observational analysis of the predictive performance of clinical parameters and laboratory tests in hospitalised patients with COVID-19. Outcomes were 28-day survival and maximal severity in a cohort of 1,579 patients and two validation cohorts of 598 and 434 patients. A pilot study conducted in a patient subgroup measured 17 cytokines and 27 lymphocyte phenotypes to explore additional predictive laboratory tests.Findings1) Despite a strong association of 22 clinical and laboratory variables with the outcomes, their joint prediction power was limited due to redundancy. 2) Eight variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the statistical predictive power. 3) The interpretation of clinical and laboratory variables was improved by grouping them in categories. 4) Age and organ damage-related tests were the best predictors of survival, and inflammatory-related tests were the best predictors of severity. 5) The pilot study identified several immunological tests (including chemokine ligand 10, chemokine ligand 2, and interleukin 1 receptor antagonist), that performed better than currently used tests.ConclusionsCurrently used tests for clinical management of COVID-19 patients are of limited predictive value due to redundancy, as all measure aspects of two major processes: inflammation, and organ damage. There are no independent predictors based on the quality of the nascent adaptive immune response. Understanding the limitations of current tests would improve their interpretation and simplify clinical management protocols. A systematic search for better biomarkers is urgent and feasible.This study was funded by Instituto de Salud Carlos III, Madrid, Spain, grants COV20/00416, Cov20/00654 and COV20/00388 to R.P–B, ATS and JBM respectively and co–financed by the European Regional Development Fund (ERDF). DÁ–S is recipient of a doctoral fellowship from the Vall d’Hebron Research Institute, Barcelona, Spain. ASM was supported by a postdoctoral grant “Juan Rodés” (JR18/00022) from Instituto de Salud Carlos III through the Ministry of Economy and Competitiveness, Spain

Endotype; Organ dysfunction; Sepsis Endotipo; Disfunción de órganos; Sepsis Endotip; Disfunció d'òrgans; Sèpsia Sepsis is a heterogeneous disease with variable clinical course and several clinical phenotypes. As it is associated with an increased risk of death, patients with this condition are candidates for receipt of a very well-structured and protocolized treatment. All patients should receive the fundamental pillars of sepsis management, which are infection control, initial resuscitation, and multiorgan support. However, specific subgroups of patients may benefit from a personalized approach with interventions targeted towards specific pathophysiological mechanisms. Herein, we will review the framework for identifying subpopulations of patients with sepsis, septic shock, and multiorgan dysfunction who may benefit from specific therapies. Some of these approaches are still in the early stages of research, while others are already in routine use in clinical practice, but together will help in the effective generation and safe implementation of precision medicine in sepsis.

It is known that the immunoregulatory networks in human Chagas disease play a key role in parasitemia control during the acute phase. However, little is known regarding the control of parasitemia during the chronic phase. The aim of the study was to describe the serum cytokine profile of Trypanosoma cruzi chronically infected patients and to evaluate its relationship with the presence or absence of parasitemia in peripheral blood. This is a prospective observational study where adult Chagas disease patients were included. Patients previously treated for Chagas disease, pregnant women, and immunosuppressed patients were excluded. Demographic and clinical information was collected, and T. cruzi real-time polymerase chain reaction (RT-PCR) and serum cytokine profile were determined in peripheral blood. Forty-five patients were included. Trypanosoma cruzi RT-PCR in peripheral blood resulted positive in 19 (42.2%) patients. No differences in the serum cytokine profile were found depending on cardiac or digestive involvement. However, patients with positive T. cruzi RT-PCR had a higher median concentration of IL-10 and IL-1beta and a lower median concentration of IL-8 than those with negative T. cruzi PCR. These results reinforce the key role that this anti-inflammatory cytokine (IL-10) plays in parasitemia control.

The presence of mutations in PRF1, UNC13D, STX11 and STXBP2 genes in homozygosis or compound heterozygosis results in immune deregulation. Most such cases lead to clinical manifestations of haemophagocytic lymphohistiocytosis (HLH). In the present study, we analyzed degranulation and cytotoxicity in a pediatric patient with a late presentation of HLH associated with Epstein-Barr virus infection. Remarkably, the results of the degranulation assay showed reduction of CD107a median fluorescence intensity (MFI) and absent cytotoxicity. Genetic analysis identified compound heterozygous mutations in STXBP2 gene: a previously reported splicing defect in exon 15 (c.1247-1G>C, p.V417LfsX126) and a novel missense mutation in exon 9 (c.728T>G, p.L243R). Transfection experiments of STXBP2-L243R or STXBP2-WT constructs showed an undetectable protein expression of the STXBP2-L243R mutation. The residue L243 is highly preserved evolutionarily; moreover, computational analysis of its structure revealed its participation in the rich network of interactions that stabilizes domains 2 and 3 of the protein. Altogether, we demonstrated by molecular and in silico analysis that the new L243R mutation in STXBP2 plays a pathogenic role that, together with the p.Val417Leufsc mutation, shows the synergistic negative effect of these two mutations on STXBP2 function, leading to a decrease of degranulatory activity in vivo.

Recent studies have shown a suboptimal humoral response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in patients diagnosed with hematologic malignancies; however, data about cellular immunogenicity are scarce. The aim of this study was to evaluate both the humoral and cellular immunogenicity 1 month after the second dose of the mRNA-1273 vaccine. Antibody titers were measured by using the Elecsys and LIAISON anti–SARS-CoV-2 S assays, and T-cell response was assessed by using interferon-γ release immunoassay technology. Overall, 76.3% (184 of 241) of patients developed humoral immunity, and the cellular response rate was 79% (184 of 233). Hypogammaglobulinemia, lymphopenia, active hematologic treatment, and anti-CD20 therapy during the previous 6 months were associated with an inferior humoral response. Conversely, age >65 years, active disease, lymphopenia, and immunosuppressive treatment of graft-versus-host disease (GVHD) were associated with an impaired cellular response. A significant dissociation between the humoral and cellular responses was observed in patients treated with anti-CD20 therapy (the humoral response was 17.5%, whereas the cellular response was 71.1%). In these patients, B-cell aplasia was confirmed while T-cell counts were preserved. In contrast, humoral response was observed in 77.3% of patients undergoing immunosuppressive treatment of GVHD, whereas only 52.4% had a cellular response. The cellular and humoral responses to the SARS-CoV-2 mRNA-1273 vaccine in patients with hematologic malignancies are highly influenced by the presence of treatments such as anti-CD20 therapy and immunosuppressive agents. This observation has implications for the further management of these patients., The authors also thank the Cellex Foundation for providing research facilities and equipment and the CERCA Programme/Generalitat de Catalunya for institutional support.

Newborn screening; Severe combined immunodeficiency Cribatge nounat; Immunodeficiència combinada severa Cribado neonato; Inmunodeficiencia combinada grave Purine nucleoside phosphorylase (PNP) deficiency is a rare inherited disorder, resulting in severe combined immunodeficiency. To date, PNP deficiency has been detected in newborn screening only through the use of liquid chromatography tandem mass spectrometry. We report the first case in which PNP deficiency was detected by TREC analysis. This research was funded by Jeffrey Modell Foundation.

Background SARS-CoV-2 infection portends a broad range of outcomes, from a majority of asymptomatic cases to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty, and co-morbidities such as obesity, diabetes, and cardiovascular disease. A precise knowledge of the molecular and biological mechanisms that may explain the association of severe disease with male sex is still lacking. Here, we analyzed the relationship of serum testosterone levels and the immune cell skewing with disease severity in male COVID-19 patients. Methods Biochemical and hematological parameters of admission samples in 497 hospitalized male and female COVID-19 patients, analyzed for associations with outcome and sex. Longitudinal (in-hospital course) analyses of a subcohort of 114 male patients were analyzed for associations with outcome. Longitudinal analyses of immune populations by flow cytometry in 24 male patients were studied for associations with outcome. Results We have found quantitative differences in biochemical predictors of disease outcome in male vs. female patients. Longitudinal analyses in a subcohort of male COVID-19 patients identified serum testosterone trajectories as the strongest predictor of survival (AUC of ROC = 92.8%, p < 0.0001) in these patients among all biochemical parameters studied, including single-point admission serum testosterone values. In lethal cases, longitudinal determinations of serum luteinizing hormone (LH) and androstenedione levels did not follow physiological feedback patterns. Failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and augmented circulating classical monocytes. Conclusions Recovery or failure to reinstate testosterone levels is strongly associated with survival or death, respectively, from COVID-19 in male patients. Our data suggest an early inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by full recovery in survivors or a peripheral failure in lethal cases. These observations are suggestive of a significant role of testosterone status in the immune responses to COVID-19 and warrant future experimental explorations of mechanistic relationships between testosterone status and SARS-CoV-2 infection outcomes, with potential prophylactic or therapeutic implications., This study was funded by grants from the Ministerio de Ciencia e Innovación (RTI2018-096055-B-I00), Consejo Superior de Investigaciones Científicas’ COVID-19 Research Fund (CSIC-COV19-006, CSIC-COV19-201), Agència de Gestió d’Ajuts Universitaris i de Recerca (2020PANDE00048 and 2017SGR 1411 GRC), and Plan Nacional de I+D (PID-107139RB-C21) and Instituto Nacional de la Salud Carlos III (PI18/00346 and COVID-19_00416).

Limfòcits b; Metilació de l'ADN; Genoma Linfocitos b; Metilación de ADN; Genoma B-lymphocytes; DNA methylation; Genome Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns. Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R to E.B.]; cofunded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe. E.B is supported by Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program); P.L. and C.P. are supported by the German Cancer Aid project CO-CLL [70113869]; B.G. is funded by the Deutsche Forschungsgemeinschaft [GR1617/14-1/iPAD, SFB1160/2_B5, RESIST–EXC 2155–Project ID 390874280, CIBSS–EXC-2189–Project ID 390939984]; BMBF [GAIN 01GM1910A]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R].

Background Clinical trials on the different vaccines to SARS-CoV-2 have demonstrated protection efficacy, but it is urgent to assess the levels of protection generated with real-world data, especially in individuals professionally exposed. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection but there are not validated T cell response applicable to large number of samples. Objective To assess the feasibility of using T-cell responses to SARS-CoV-2 S peptides by commercially available whole blood interferon-gamma release assays (IGRA) as a correlate of protection. Patients Twenty health care workers before and after vaccination. Methods Antibody test to SARS-CoV-2 N and S proteins in parallel with one IGRA assay and two detection techniques than can be automated. Results IGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. the correlation by the two detection methods, CLIA and ELISA, very high (R>0.9) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between antibody and the IGRA assay in the ability to detect immune response to SARS-CoV-2 there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one Ig (S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA test. Conclusion Whole blood IGRA tests amenable to automation, as the one here reported, constitute a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become valuable correlates of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health care workers. Clinical Implications Commercial kits of whole blood Interferon-gamma release assay (IGRA) constitute an reliable method for clinical laboratories to assess T-cell response after natural infection by SARS-CoV-2 and after BNT162b2 mRNA vaccination and are suitable for large scale application. Key Messages Commercial kits of whole blood interferon-gamma release assay (IGRA) are potentially very useful tools to measure the T cell response to SARS-CoV-2 after COVID-19 and after SARS-CoV-2 vaccination. One vaccine dose restores T cell response in COVID recovered patients, but the vaccination boost was required for naive participants to attain a comparable response. T cell response seem to decay in COVID recovered subjects after the boost second vaccination dose. Capsule Summary Measuring T cell responses by commercially available whole blood interferon gamma release assays (IGRA) provide a promising additional correlate of protection to COVID and may be useful to reassure vulnerable group professionals at risk of being exposed to SARS-CoV-2 infection.

Monoallelic loss-of-function mutations in NFKB1 were recently recognized as the most common monogenic cause of common variable immunodeficiency (CVID). The prototypic clinical phenotype of NFKB1-deficient patients includes common CVID features, such as hypogammaglobulinaemia and sinopulmonary infections, plus other highly variable individual manifestations. Here, we describe a patient with a profound CVID phenotype and severe gastrointestinal manifestations, including chronic and recurrent diarrhoea. Using an NGS customized panel of 323 genes related to primary immunodeficiencies, we identified a novel monoallelic loss-of-function mutation in NFKB1 leading to a truncated protein (c.1149delT/p.Gly384Glu ∗ 48). Interestingly, we also found a rare variant in NOD2 previously associated with Crohn's disease (p.His352Arg). Our patient had hypogammaglobulinaemia with a small number of B cells, most of which were naive. The most noteworthy findings included marked skewing towards a Th1 phenotype in peripheral blood T cells and excessive production of proinflammatory cytokines (IL-1β, TNFα). The patient's 6-year-old daughter, a carrier of the NFKB1 mutation, is clinically asymptomatic, but has started to show cellular and molecular changes. This case of NFKB1 deficiency appears to be a combination of immunodeficiency and a hyperinflammatory state. The current situation of the patient's daughter provides a glimpse of the preclinical phase of the condition.

Purpose Germline heterozygous mutations of GATA2 underlie a variety of hematological and clinical phenotypes. The genetic, immunological, and clinical features of GATA2-deficient patients with mycobacterial diseases in the familial context remain largely unknown. Methods We enrolled 15 GATA2 index cases referred for mycobacterial disease. We describe their genetic and clinical features including their relatives. Results We identified 12 heterozygous GATA2 mutations, two of which had not been reported. Eight of these mutations were loss-of-function, and four were hypomorphic. None was dominant-negative in vitro, and the GATA2 locus was found to be subject to purifying selection, strongly suggesting a mechanism of haploinsufficiency. Three relatives of index cases had mycobacterial disease and were also heterozygous, resulting in 18 patients in total. Mycobacterial infection was the first clinical manifestation in 11 patients, at a mean age of 22.5 years (range: 12 to 42 years). Most patients also suffered from other infections, monocytopenia, or myelodysplasia. Strikingly, the clinical penetrance was incomplete (32.9% by age 40 years), as 16 heterozygous relatives aged between 6 and 78 years, including 4 older than 60 years, were completely asymptomatic. Conclusion Clinical penetrance for mycobacterial disease was found to be similar to other GATA2 deficiency-related manifestations. These observations suggest that other mechanisms contribute to the phenotypic expression of GATA2 deficiency. A diagnosis of autosomal dominant GATA2 deficiency should be considered in patients with mycobacterial infections and/or other GATA2 deficiency-related phenotypes at any age in life. Moreover, all direct relatives should be genotyped at the GATA2 locus., INSERM, University of Paris; Rockefeller University; St. Giles Foundation; National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Allergy & Infectious Diseases (NIAID) [R37AI095983]; French National Research Agency (ANR) under the "Investments for the future" programFrench National Research Agency (ANR) [ANR-10-IAHU-01, ANR13-ISV3-0001-01, ANR-16-CE17-0005-01]; ECOS-NORD [C19S01-63407, SRC2017]; ANRHGDIFDFrench National Research Agency (ANR) [ANR-14-CE15-006-01]; ANR-IFNGPHOX [ANR-13-ISV3-0001-01]; GENMSMD [ANR-16-CE17-0005-01]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2012/11757-2, 2010/51814-0, 2012/51094-2]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ)National Council for Scientific and Technological Development (CNPq) [303809/2010-8]; Instituto de Salud Carlos IIIInstituto de Salud Carlos IIIEuropean Commission [PI11/01086, PI14/00405]; European Regional Development Fund (ERDF)European Commission; Colombia-France (ECOS-NORD/COLCIENCIAS/MEN/ICETEX) [619-2013]; Diana Garcia de Olarte foundation PID; ColcienciasDepartamento Administrativo de Ciencia, Tecnologia e Innovacion Colciencias [713-2016, 111574455633]; "Poste d'accueil" INSERMInstitut National de la Sante et de la Recherche Medicale (Inserm); Imagine Institute, The Laboratory of Human Genetics of Infectious Diseases is supported in part by institutional grants from INSERM, University of Paris, The Rockefeller University and the St. Giles Foundation, the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) (R37AI095983), and grants from the French National Research Agency (ANR) under the "Investments for the future" program (ANR-10-IAHU-01) and IFNGPHOX (ANR13-ISV3-0001-01 for JB and ACN), GENMSMD (ANR-16-CE17-0005-01 for JB) grants, ECOS-NORD (C19S01-63407 for JB and JFR), and SRC2017 (for JB). CO-Q is supported by ANRHGDIFD (ANR-14-CE15-006-01). AG was supported by the ANR-IFNGPHOX (ANR-13-ISV3-0001-01), GENMSMD (ANR-16-CE17-0005-01), and the Imagine Institute. AC-N and EBO-J are supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP grants 2012/11757-2, 2010/51814-0, and 2012/51094-2) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ grant 303809/2010-8). MMG and RC are supported by Instituto de Salud Carlos III, grants PI11/01086 and PI14/00405, co-financed by the European Regional Development Fund (ERDF). JFR and AAA are supported by Colombia-France (ECOS-NORD/COLCIENCIAS/MEN/ICETEX; 619-2013, Diana Garcia de Olarte foundation PID and Colciencias grant 713-2016 #111574455633). JR was supported by "Poste d'accueil" INSERM and Imagine Institute.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15–25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk–benefit profile was maintained for ≤96 wk.

The Catalonian Newborn Screening Program (CNSP) began in 1969, in Barcelona. It was promoted by Dr. Juan Sabater Tobella and supported by Barcelona Provincial Council and Juan March Foundation. That is how the Institute of Clinical Biochemistry was born, whose aims were diagnosis, research and teaching, along with the spirit of contributing to the prevention of mental retardation. The CNSP began with the detection of phenylketonuria (PKU), and, in 1982, the Program was expanded with the inclusion of congenital hypothyroidism detection. Towards 1990, the Program covered almost 100% of all newborns (NB) in Catalonia. In 1999, the CNSP was expanded with the incorporation of cystic fibrosis. It took fourteen years, until 2013, to make the largest expansion so far, with the incorporation of 19 metabolic diseases to the screening panel. The detection of sickle cell disease began in 2015 and in 2017 the detection of severe combined immunodeficiency was included. Currently, the CNSP includes 24 diseases in its main panel. Since 1969, 2,787,807 NBs have been screened, of whom 1,724 have been diagnosed with any of these diseases, and 252 of other disorders by differential diagnosis with those included in the main panel. The global prevalence is 1: 1,617 NBs affected by any of the diseases included in the CNSP and 1: 1,140 NBs if incidental findings diagnosed through the CNSP are included.El Programa de Cribado Neonatal de Cataluña (PCNC) se inició en el año 1969, en Barcelona, impulsado por el Dr. Juan Sabater Tobella y apoyado por la Diputación de Barcelona y la Fundación Juan March. Así nació el Instituto de Bioquímica Clínica para acometer funciones asistenciales, de investigación y docencia, con el espíritu de contribuir a la prevención del retraso mental. El PCNC se inició con la detección de la fenilcetonuria (PKU) y en el año 1982 se amplió con la detección del hipotiroidismo congénito. Hacia el año 1990 la cobertura territorial llegó casi al 100% de todos los recién nacidos en Cataluña. En 1999 se amplió el PCNC con la incorporación de la fibrosis quística y tras catorce años, en 2013, se realizó la ampliación más numerosa hasta ahora, con la incorporación de la detección de 19 enfermedades metabólicas hereditarias. En el año 2015 comenzó la detección de la enfermedad de células falciformes y en el 2017 la detección de la inmunodeficiencia combinada grave. Actualmente, el PCNC incluye la detección de 24 enfermedades. Desde su inicio en el año 1969, se han cribado 2.787.807 recién nacidos, de los cuales 1.724 han sido diagnosticados de alguna de las 24 enfermedades que componen nuestro panel principal y 252 por diagnóstico diferencial de las primeras. En total la prevalencia global es de 1:1.617 RN afectos de alguna de las enfermedades incluidas en el PCNC y de 1:1.140 RN si se incluyen los hallazgos incidentales encontrados.

Microbial challenges, such as widespread bacterial infection, induce endotoxin tolerance. This state of hyporesponsiveness to subsequent infections is mainly displayed by monocytes and macrophages. Endotoxin tolerance is generally acquired following a septic episode. In this study, we investigated DNA methylation changes during the acquisition of in vitro tolerance. We identified a set of TET2-mediated demethylation events that are specific to toll-like receptor (TLR) 2 and TLR4 stimulation. Lipopolysaccharide (LPS)-specific demethylation occurs at genomic sites that have low accessibility in quiescent monocytes, concomitantly with the transcriptional activation of many inflammation-related genes, and they are enriched in binding motifs for several signal transducer and activator of transcription (STAT) family members. Indeed, STAT1, STAT3 and STAT5, elements of the JAK2 pathway, are phosphorylated in association with the acquisition of endotoxin tolerance. Inhibition of the JAK2 pathway impairs the activation of tolerized genes on the first encounter with LPS. This is evidence of a crucial role for this pathway in determining the initial response of these genes to bacterial antigens and provides a pharmacological target to prevent exacerbated responses, allowing regulated responses upon subsequent challenges. Finally, we assess the pathological relevance of the JAK2 pathway in monocytes from patients with sepsis.

Severe combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TREC) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first three years and a half of experience (January 2017 - June 2020) are shown here, using EnLite Neonatal TREC kit (Perkin Elmer) with 20 copies/µL as TREC detection cutoff. Of 222,857 newborns screened, 48 tested positive: three patients were diagnosed with SCID (incidence 1:74,285); 17 patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 13,109 newborns; twenty two patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TREC between 3 and 6 months of life; one case had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months; and five patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Even longer follow-up could be necessary to define the exact incidence of SCID in Catalonia.La inmunodeficiencia combinada grave (IDCG) es la forma más grave de inmunodeficiencia primaria, que afecta sobre todo a los linfocitos T, y puede ser detectada al nacer mediante la cuantificación de los círculos de escisión del receptor de linfocitos T (TREC) en una muestra de sangre impregnada en papel (DBS). La detección precoz de esta enfermedad permite establecer de forma temprana un tratamiento adecuado en el paciente, permitiendo así su curación. El cribado neonatal de IDCG comenzó en Cataluña en enero de 2017, siendo la primera región española y europea en incluirla oficial y universalmente en su programa. En el presente trabajo se presentan los resultados obtenidos durante los tres primeros años y medio de experiencia (enero 2017 – junio 2020) empleando el kit EnLite Neonatal TREC (Perkin Elmer), con un cutoff de detección de TREC de 20 copias/µL. De 222.857 recién nacidos analizados, cuarenta y ocho fueron detecciones positivas: tres casos de IDCG (incidencia de 1:74.285); diecisiete casos de linfopenia T no IDCG (incidencia de 1:13.109); veintidós casos falsos positivos (recuento de linfocitos inicialmente normal, con normalización de TREC entre los tres y seis meses de vida); un caso con linfopenia transitoria (con un recuento de linfocitos inicialmente bajo, que se normaliza en los meses siguientes); y cinco pacientes se encuentran todavía en estudio. Los resultados obtenidos aportan evidencias de los beneficios que supone incluir esta enfermedad en los programas de cribado neonatal. Podría ser necesario un seguimiento todavía más prolongado para acabar de definir la incidencia exacta de IDCG en Cataluña.

Contains fulltext : 229571.pdf (Publisher’s version ) (Closed access) BACKGROUND: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. OBJECTIVE: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. METHODS: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. RESULTS: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. CONCLUSIONS: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.

Background and aims Identification of SARS-CoV-2-infected patients at high-risk of poor prognosis is crucial. We aimed to establish predictive models for COVID-19 pneumonia severity in hospitalized patients. Methods Retrospective study of 430 patients admitted in Vall d’Hebron Hospital (Barcelona) between 03-12-2020 and 04-28-2020 due to COVID-19 pneumonia. Two models to identify the patients who required high-flow-oxygen-support were generated, one using baseline data and another with also follow-up analytical results. Calibration was performed by a 1000-bootstrap replication model. Results 249 were male, mean age 57.9 years. Overall, 135 (31.4%) required high-flow-oxygen-support. The baseline predictive model showed a ROC of 0.800 based on: SpO2/FiO2 (adjusted Hazard Ratio-aHR = 8), chest x-ray (aHR = 4), prior immunosuppressive therapy (aHR = 4), obesity (aHR = 2), IL-6 (aHR = 2), platelets (aHR = 0.5). The cut-off of 11 presented a specificity of 94.8%. The second model included changes on the analytical parameters: ferritin (aHR = 7.5 if ≥200ng/mL) and IL-6 (aHR = 18 if ≥64pg/mL) plus chest x-ray (aHR = 2) showing a ROC of 0.877. The cut-off of 12 exhibited a negative predictive value of 92%. Conclusions SpO2/FiO2 and chest x-ray on admission or changes on inflammatory parameters as IL-6 and ferritin allow us early identification of COVID-19 patients at risk of high-flow-oxygen-support that may benefit from a more intensive disease management.

Summary In addition to their detection in typical X-linked severe combined immunodeficiency, hypomorphic mutations in the interleukin (IL)-2 receptor common gamma chain gene (IL2RG) have been described in patients with atypical clinical and immunological phenotypes. In this leaky clinical phenotype the diagnosis is often delayed, limiting prompt therapy in these patients. Here, we report the biochemical and functional characterization of a nonsense mutation in exon 8 (p.R328X) of IL2RG in two siblings: a 4-year-old boy with lethal Epstein–Barr virus-related lymphoma and his asymptomatic 8-month-old brother with a TlowB+natural killer (NK)+ immunophenotype, dysgammaglobulinemia, abnormal lymphocyte proliferation and reduced levels of T cell receptor excision circles. After confirming normal IL-2RG expression (CD132) on T lymphocytes, signal transducer and activator of transcription-1 (STAT-5) phosphorylation was examined to evaluate the functionality of the common gamma chain (γc), which showed partially preserved function. Co-immunoprecipitation experiments were performed to assess the interaction capacity of the R328X mutant with Janus kinase (JAK)3, concluding that R328X impairs JAK3 binding to γc. Here, we describe how the R328X mutation in IL-2RG may allow partial phosphorylation of STAT-5 through a JAK3-independent pathway. We identified a region of three amino acids in the γc intracellular domain that may be critical for receptor stabilization and allow this alternative signaling. Identification of the functional consequences of pathogenic IL2RG variants at the cellular level is important to enable clearer understanding of partial defects leading to leaky phenotypes.

Background The current scenario of newborn screening is changing as DNA studies are being included in the programs of several countries. Severe combined immunodeficiency (SCID) disorders can be detected using quantitative PCR assays to measure T‐cell receptor excision circles (TRECs), a byproduct of correct T‐cell development. However, in addition to SCID, other T‐cell‐deficient phenotypes such as 22q11.2 deletion syndrome 22q11.2 duplication syndrome, CHARGE syndrome, and trisomy 21 are detected. Methods We present our experience with the detection of 22q11.2 deletion syndrome and 22q11.2 duplication syndrome in a series of 103,903 newborns included in the newborn screening program of Catalonia (Spain). Results Thirty newborns tested were positive (low TREC levels) and five were found to have copy number variations at the 22q11 region (4 deletions and 1 duplication) when investigated with array comparative genomic hybridization technology and MLPA. Conclusion Newborn screening for SCID enables detection of several conditions, such as 22q syndromes, which should be managed by prompt, proactive approaches with adequate counseling for families by a multidisciplinary team., Newborn screening (NBS) using T‐cell receptor excision circle (TREC) assays to detect severe combined immunodeficiency (SCID) has been established in several geographic areas. In addition to SCID, other T‐cell deficient conditions, such as 22q11.2 deletion syndrome (DS), CHARGE syndrome, and trisomy 21, can also be detected through NBS. An early diagnosis of 22q11.2 DS is associated with a better clinical outcome, mainly because of the possibility of early protocolled care, including rehabilitation and adapted school programs, an appropriate vaccination schedule, and adequate genetic counseling.

Background: Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders. The lack of comprehensive disease-specific mutation databases may hinder or delay classification of the genetic variants found in samples from these patients. This is especially true for familial hemophagocytic lymphohistiocytosis (FHL), a life-threatening PID classically considered an autosomal recessive condition, but with increasingly demonstrated genetic heterogeneity. Objective: The aim of this study was to build an open-access repository to collect detailed information on the known genetic variants reported in FHL. Methods: We manually reviewed more than 120 articles to identify all reported variants related to FHL. We retrieved relevant information about the allelic status, the number of patients with the same variant, and whether functional assays were done. We stored all the data retrieved in a PostgreSQL database and then built a website on top of it, using the Django framework. Results: The database designed (FHLdb) (https://www.biotoclin.org/FHLdb) contains comprehensive information on reported variants in the 4 genes related to FHL (PRF1, UNC13D, STXBP2, STX11). It comprises 240 missense, 69 frameshift, 51 nonsense, 51 splicing, 10 in-frame indel, 7 deep intronic, and 5 large rearrangement variants together with their allelic status, carrier(s) information, and functional evidence. All genetic variants have been classified as pathogenic, likely pathogenic, uncertain significance, likely benign or benign, according to the American College of Medical Genetics guidelines. Additionally, it integrates information from other relevant databases: clinical evidence from ClinVar and UniProt, population allele frequency from ExAC and gnomAD, and pathogenicity predictions from well-recognized tools (e.g., PolyPhen-2, SIFT). Finally, a diagram depicts the location of the variant relative to the gene exon and protein domain structures. Conclusion: FHLdb includes a broad range of data on the reported genetic variants in familial HLH genes. It is a free-access and easy-to-use resource that will facilitate the interpretation of molecular results of FHL patients, and it illustrates the potential value of disease-specific databases for other PIDs., This study was funded by Instituto de Salud Carlos III, grants PI17/00660 and PI18/00346, cofinanced by the European Regional Development Fund (ERDF). LB-M is supported by a Formació de Personal Investigador (FPI) fellowship from Generalitat de Catalunya (2018_FI_B00072). FC was funded by grants SAF2015-68472-C2-2-R from the Ministerio de Economía y Competitividad (Spain) and FEDER (EU) and RTI2018- 096824-B-C22 from the Spanish Ministry of Science, Innovation and Universities co-financed by ERDF (FC), and by Direcció General de Recerca, Generalitat de Catalunya (2014SGR-866 and 2017SGR-702).

Mutations in activation induced deaminase (AID) lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated cytosine deamination has been proposed as mediating active demethylation, although evidences both support and cast doubt on such a role. We here made use of HIGM2 B cells to investigate direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation defects, of which approximately 25% of these defects correspond to active events. For genes that undergo active demethylation that is impaired in HIGM2 individuals, we did not observe AID involvement but a participation of TET enzymes. DNA methylation alterations in HIGM2 naïve B cells are related to premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data supports a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.

Background In a previous in-depth phenotypic analysis of PBLs in JIA, we detected differences in cell subpopulations dependent on the age and also some cellular changes that could be often associated with an exhausted phenotype. Objectives To analyze the expression of co-inhibitory and co-stimulatory molecules in different cellular subpopulations in Oligoarticular JIA patients stratified by age and treatment. Methods Exploratory cross-sectional study. 53 patients fulfilling Oligoarticular JIA (ILAR) criteria and 22 controls were included. All of them were children or young adults (2 to 35 years of age). JIA patients were either not treated (n=14) or treated with MTX (n=14), anti-TNF (n=8), or a combination of both (n=16). JIA disease activity was clinically and biologically assessed. Four flow cytometry panels were designed for the analysis of co-stimulatory/co-inhibitory markers, some typical of “exhausted” cells (PD1, TIM 3, TIGIT, CD226, CD137, HVEM, LIGHT, BTLA), assessed in memory, effector and naive CD4+ and CD8+ T lymphocytes, and also in B lymphocytes and NK cells. Statistical analysis was performed, and FDR correction was applied for p values. Results No correlation was found among the different exhaustion/activation markers and age neither in JIA nor in controls. Interestingly, age-related differential trends were observed in the CD8+ compartment in controls, while in JIA patients were detected in the B cell compartment. We didn’t observe, after correcting for treatment, any significant difference in lymphocyte subpopulations defined by the above co-inhibitory and co-stimulatory markers in JIA patients in relation to indexes of disease activity. However, there was a tendency towards a higher expression of CD137 in several CD8+ subpopulations of active patients as compared to inactive. Conclusion No clear differences were found in PBL subpopulations of oligoarticular-JIA patients regarding co-stimulatory/co-inhibitory molecules. This finding could be explained, at least in part, by the good therapeutic control achieved in this patient population, or by the particular characteristics of this group of patients, in which prevails a more localized expression of the disease. It could be of interest to carry out complementary analysis to further define the differential tendencies observed. Acknowledgement Instituto de Salud Carlos III (ISCIII), Sociedad Espanola Reumatologia (SER) Disclosure of Interests None declared

[Background]: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients., [Methods]: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes., [Results]: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening., [Conclusion]: Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed., Funded by grants PI14–01495 and PI17–01401 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds) and by one ACCI project from CIBERER and one grant to the FPI cohort from CIBERES., We acknowledge support of the publication fee by the CSIC Open Access Publication Support initiative through its Unit of Information Resources for Research (URICI).