The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

Vaccination practices in pediatric transplantation: A survey among member centers of the European reference network TransplantChild.

BACKGROUND: There is considerable variation in vaccination practices between pediatric transplant centers. This study aims to evaluate active immunization attitudes and practices among ERN-TransplantChild centers and identify potential areas of improvement that could be addressed by shared evidence-based protocols. METHODS: A cross-sectional questionnaire of attitudes and practices toward immunization of pediatric SOT and HSCT candidates and recipients was sent to a representative member of multidisciplinary teams from 27 European centers belonging to the ERN-TransplantChild. RESULTS: A total of 28/62 SOT programs and 6/12 HSCT programs across 21 European centers participated. A quarter of centers did not have an on-site protocol for the immunizations. At the time of transplantation, pediatric candidates were fully immunized (80%-100%) in 57% and 33% of the SOT and HSCT programs. Variations in the time between vaccine administration and admission to the waiting list were reported between the centers, with 2 weeks for inactivated vaccines and variable time (2-4 weeks) for live-attenuated vaccines (LAVs). Almost all sites recommended immunization in the post-transplant period, with a time window of 4-8 months for the inactivated vaccines and 16-24 months for MMR and Varicella vaccines. Only five sites administer LAVs after transplantation, with seroconversion evaluated in 80% of cases. CONCLUSIONS: The immunization coverage of European pediatric transplant recipients is still inconsistent and far from adequate. This survey is a starting point for developing shared evidence-based immunization protocols for safe vaccination among pediatric transplant centers and generating new research studies.

Impact of cytomegalovirus infection prior to hematopoietic stem cell transplantation in children with inborn errors of immunity.

The presence of active viral infections has an impact on the prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT). Nevertheless, the number of reports of cytomegalovirus infection in patients with inborn errors of immunity (IEI) who undergo HSCT is relatively low. To analyze the effect of cytomegalovirus infection acquired prior to curative treatment on patient survival in 123 children with IEI. An observational and retrospective study was performed with patients younger than 18 years diagnosed with IEI who were candidates for HSCT, gene therapy, or thymus transplantation at five hospitals in Spain between 2008 and 2019. We included 123 children, 25 infected by cytomegalovirus prior to undergoing curative treatment (20.3%). At IEI diagnosis, 24 of the patients were already infected, 21 of whom had symptomatic cytomegalovirus disease (87%), while the other three patients developed disease before undergoing curative treatment. The patients with cytomegalovirus infection had higher mortality than those without (p = 0.006). Fourteen patients developed refractory cytomegalovirus infection (56%), all of whom died, while no patients with non-refractory infection died (p = 0.001) All deaths that occurred before curative treatment and three of the five after the treatment were attributed to cytomegalovirus. Patients with refractory cytomegalovirus disease had the highest pre-HSCT mortality rate (64.3%), compared with the non-infected children and those with non-refractory cytomegalovirus disease (10.1%) (p < 0.0001). CONCLUSION: Prevention and prompt control of cytomegalovirus infection, together with early HSCT/gene therapy, are crucial for improving the prognosis in children with IEI. WHAT IS KNOWN: • Cytomegalovirus is the most frequent viral infection in children with inborn errors of immunity who are candidates to hematopoietic stem cell transplantation (HSCT). • Active viral infections at the time of HSCT lead to worse prognosis. WHAT IS NEW: • In children with inborn errors of immunity and indication of HSCT, refractory cytomegalovirus disease is associated with a very high mortality rate, compared with non-infected children and those with non-refractory cytomegalovirus disease. • In patients with novel transplantation indications, the presence and treatment response of CMV infection should be considered to decide the best possible moment for HSCT.

Detection of specific RBD+ IgG+ memory B cells by flow cytometry in healthcare workers and patients with inborn errors of immunity after BNT162b2 m RNA COVID-19 vaccination.

Introduction: Inborn errors of immunity (IEI) are a heterogeneous group of diseases caused by intrinsic defects of the immune system. Estimating the immune competence of immunocompromised patients for an infection risk assessment or after SARS-CoV-2 vaccination constituted a challenge. Methods: The aim of this study was to determine the humoral responses of patients with IEI through a comprehensive analysis of specific receptor-binding domain-positive (RBD+) IgG+ memory B cells (MBCs) by flow cytometry, together with routine S-specific IgG antibodies and QuantiFERON SARS-CoV-2 (T-cell response), before the vaccine and 3 weeks after a second dose. Results and discussion: We first analyzed the percentage of specific RBD+ IgG+ MBCs in healthy healthcare workers. Within the control group, there was an increase in the percentage of specific IgG+ RBD+ MBCs 21 days after the second dose, which was consistent with S-specific IgG antibodies.Thirty-one patients with IEI were included for the pre- and post-vaccination study; IgG+ RBD+ MBCs were not evaluated in 6 patients due to an absence of B cells in peripheral blood. We detected various patterns among the patients with IEI with circulating B cells (25, 81%): an adequate humoral response was observed in 12/25, consider by the detection of positive S-specific IgG antibodies and the presence of specific IgG+ RBD+ MBCs, presenting a positive T-cell response; in 4/25, very low S-specific IgG antibody counts correlated with undetectable events in the IgG+ RBD+ MBC compartment but with positive cellular response. Despite the presence of S-specific IgG antibodies, we were unable to detect a relevant percentage of IgG+ RBD+ MBCs in 5/25; however, all presented positive T-cell response. Lastly, we observed a profound failure of B and T-cell response in 3 (10%) patients with IEI, with no assessment of S-specific IgG antibodies, IgG+ RBD+ MBCs, and negative cellular response. The identification of specific IgG+ RBD+ MBCs by flow cytometry provides information on different humoral immune response outcomes in patients with IEI and aids the assessment of immune competence status after SARS-CoV-2 mRNA vaccine (BNT162b2), together with S-specific IgG antibodies and T-cell responses.

Persistent Antiphospholipid Antibodies Are Not Associated With Worse Clinical Outcomes in a Prospective Cohort of Hospitalised Patients With SARS-CoV-2 Infection.

Objective: Patients with COVID-19 presented with an elevated prevalence of antiphospholipid antibodies (aPL) but the relationship with thrombosis is controversial. We analysed the persistence of aPL and their association with the clinical outcomes during hospitalisation in a cohort of COVID-19 patients. Patients and Methods: We conducted a prospective study including consecutive hospitalised patients with COVID-19 from Hospital Clínic of Barcelona between March 28th and April 22nd, 2020. Clinical outcomes during hospitalisation were thrombosis, intensive care unit (ICU) admission, and severe ventilatory failure. We determined both criteria and non-criteria aPL. Of note, in those patients with a positive result in the first determination, a second sample separated by at least 12 weeks was drawn to test the persistence of aPL. Results: One hundred and fifty-eight patients (59.5% men) with a mean age of 61.4 ± 14.9 years old were included. Thrombosis was present in 28 (17.7%) patients, severe respiratory failure in 47 (30.5%), and 30 (18.9%) patients were admitted to ICU. Sixteen (28.6%) patients were positive for the criteria aPL at both determinations and only two (3.6%) of them suffered from thrombosis during hospitalisations (both had aCL IgG). However, they presented with low titers of aCL. Of note, aPL were not related to thrombosis, ICU admission or severe respiratory failure. Conclusion: Although aPL were prevalent in our cohort of hospitalised COVID-19 patients and they were persistent in half of tested patients, most determinations were at low titers and they were not related to worse clinical outcomes.

Working Algorithms and Detection Methods of Autoantibodies in Autoimmune Liver Disease: A Nationwide Study.

Autoantibody detection is the cornerstone of autoimmune liver diseases (AILD) diagnosis. Standardisation of working algorithms among autoimmunity laboratories, as well as being aware of the sensitivity and specificity of various commercial techniques in daily practice, are still necessary. The aim of this nationwide study is to report the results of the 2020 Autoimmunity Workshop organised by the Autoimmunity Group of the Spanish Society of Immunology and to provide useful information to clinicians and laboratory specialists to improve the management of autoantibody detection in AILD diagnoses. Serum samples from 17 patients with liver diseases were provided by the organisers of the 2020 Autoimmunity Workshop and were subsequently analysed by the 40 participating laboratories. Each laboratory used different techniques for the detection of autoantibodies in each patients' serum sample, according to their working algorithm. Thus, almost 680 total complete patient reports were obtained, and the number of results from different autoantibody detection techniques was >3000. Up to eight different working algorithms were employed, including indirect immunofluorescence assays (IFA) and antigen-specific techniques (AgST). The IFA of HEp-2 cells was more sensitive than IFA of rat triple tissue for the study of anti-nuclear autoantibodies (ANA) associated with AILD. The IFA of a human neutrophil study for the analysis of anti-neutrophil cytoplasmic autoantibodies was not carried out systemically in all patients, or by all laboratories. AgSTs were the most sensitive methods for the detection of anti-smooth muscle/F-actin, soluble liver antigen, liver cytosol-1, M2-mitochondrial autoantibodies, and ANA associated with primary biliary cholangitis. The main differences in AMA detection were due to patients with autoantibodies against the non-dominant epitope of pyruvate dehydrogenase complex. Given that they are complementary, IFA and AgST should be performed in parallel. If there is high suspicion of AILD, AgST should always be performed.

Pediatric Chagas disease in the non-endemic area of Madrid: A fifteen-year review (2004-2018).

BACKGROUND: Chagas disease (CD) has become an emerging global health problem in association with the immigration of individuals from endemic areas (in LatinAmerica) to other countries.Spain is the country in Europe with the highest number of CD cases. Concerning pediatric CD, treatment is not only better tolerated by younger children but also has greater cure possibilities. The aim of this study was to describe clinical and epidemiological aspects of CD in a pediatric population diagnosed of 10 hospitals in the Community of Madrid during the 2004-2018 period, as well as the safety and efficacy of CD treatment on this population. METHODOLOGY/PRINCIPAL FINDINGS: A multicenter, retrospective, descriptive study was conducted. The studied population included all identified children under the age of 18 with a diagnosis of CD. Diagnosis was performed with a positive parasitological test (with subsequent confirmation) or confirmed persistence of positive serology beyond 9 months, for children younger than one year-old, and with two different positive serological tests, for children older than one. Fifty-one children were included (59% male; 50.9% born in Spain). All mothers were from Latin America. The median age at diagnosis was 0.7 months for those under one year of age, and 11.08 years for those older than one year-old. Only one case presented a symptomatic course (hydrops faetalis, haemodynamic instability at birth, ascites, anaemia). For 94% treatment was completed. Considering patients who received benznidazole (47), AE were recorded in 48,9%. Among the 32 patients older than one year-old treated with benznidazole, 18 (56.25%) had adverse events whereas in the 15 under one year, 5(33,3%) did. Eigtheen (78.2%) of the patients with benznidazole AE were older than one year-old(median age 11.4 years). Of the patients treated with nifurtimox (9), AE were reported in 3 cases (33,3%). Cure was confirmed in 80% of the children under one year-old vs 4.3% in those older (p<0.001). Loss to follow- up occurred in 35.3% of patients. CONCLUSIONS/SIGNIFICANCES: Screening programs of CD since birth allow early diagnosis and treatment, with a significantly higher cure rate in children treated before one year of age, with lower incidence of adverse events. The high proportion of patients lost to follow-up in this vulnerable population is of concern.

Beta-2-Glycoprotein-I Deficiency Could Precipitate an Antiphospholipid Syndrome-like Prothrombotic Situation in Patients With Coronavirus Disease 2019.

OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) present coagulation abnormalities and thromboembolic events that resemble antiphospholipid syndrome (APS). This work has aimed to study the prevalence of APS-related antigens, antibodies, and immune complexes in patients with COVID-19 and their association with clinical events. METHODS: A prospective study was conducted on 474 adults with severe acute respiratory syndrome coronavirus 2 infection hospitalized in two Spanish university hospitals. Patients were evaluated for classic and extra-criteria antiphospholipid antibodies (aPLs), immunoglobulin G (IgG)/immunoglobulin M (IgM) anticardiolipin, IgG/IgM/immunoglobulin A (IgA) anti-ß2-glicoprotein-I (aß2GPI), IgG/IgM antiphosphatidylserine/prothrombin (aPS/PT), the immune complex of IgA aß2GPI (IgA-aß2GPI), bounded to ß2-glicoprotein-1 (ß2GPI) and ß2GPI levels soon after COVID-19 diagnosis and were followed-up until medical discharge or death. RESULTS: Prevalence of aPLs in patients with COVID-19 was as follows: classic aPLs, 5.8%; aPS/PT, 4.6%; IgA-aß2GPI, 15%; and any aPL, 21%. When patients were compared with individuals of a control group of a similar age, the only significant difference found was the higher prevalence of IgA-aß2GPI (odds ratio: 2.31; 95% confidence interval: 1.16-4.09). No significant differences were observed in survival, thrombosis, or ventilatory failure in aPL-positive versus aPL-negative patients. ß2GPI median levels were much lower in patients with COVID-19 (15.9 mg/l) than in blood donors (168.8 mg/l; P < 0.001). Only 3.5% of patients with COVID-19 had normal levels of ß2GPI (>85 mg/l). Low levels of ß2GPI were significantly associated with ventilatory failure (P = 0.026). CONCLUSION: ß2GPI levels were much lower in patients with COVID-19 than in healthy people. Low ß2GPI-levels were associated with ventilatory failure. No differences were observed in the COVID-19 evolution between aPL-positive and aPL-negative patients. Functional ß2GPI deficiency could trigger a clinical process similar to that seen in APS but in the absence of aPLs.

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals.

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.

Current Practices on Diagnosis, Prevention and Treatment of Post-Transplant Lymphoproliferative Disorder in Pediatric Patients after Solid Organ Transplantation: Results of ERN TransplantChild Healthcare Working Group Survey.

(1) Background: Post-transplant lymphoproliferative disease (PTLD) is a significant complication of solid organ transplantation (SOT). However, there is lack of consensus in PTLD management. Our aim was to establish a present benchmark for comparison between international centers and between various organ transplant systems and modalities; (2) Methods: A cross-sectional questionnaire of relevant PTLD practices in pediatric transplantation was sent to multidisciplinary teams from 17 European center members of ERN TransplantChild to evaluate the centers' approach strategies for diagnosis and treatment and how current practices impact a cross-sectional series of PTLD cases; (3) Results: A total of 34 SOT programs from 13 European centers participated. The decision to start preemptive treatment and its guidance was based on both EBV viremia monitoring plus additional laboratory methods and clinical assessment (61%). Among treatment modalities the most common initial practice at diagnosis was to reduce the immunosuppression (61%). A total of 126 PTLD cases were reported during the period 2012-2016. According to their histopathological classification, monomorphic lesions were the most frequent (46%). Graft rejection after PTLD remission was 33%. Of the total cases diagnosed with PTLD, 88% survived; (4) Conclusions: There is still no consensus on prevention and treatment of PTLD, which implies the need to generate evidence. This might successively allow the development of clinical guidelines.

Infliximab concentrations in two non-switching cohorts of patients with inflammatory bowel disease: originator vs. biosimilar.

Biosimilars are replacing originator compounds due to their similar effectiveness, safety and pharmacokinetics. Our objective was to compare the differences in pharmacokinetics and clinical outcomes between the originator infliximab (Ifx) and the biosimilar CT-P13 in a patient cohort with inflammatory bowel disease (IBD). Our cohort study included 86 patients from a historical and a prospective cohort from the start of infliximab treatment to 22 weeks later. Serum infliximab, antidrug antibody levels and other serum biomarkers were measured at weeks 0, 2, 6, 14 and 22. Remission outcomes were evaluated at weeks 14 and 22. Drug levels were measured prospectively and analysed using MANOVA. Of the 86 patients, 44 (51%) and 42 (49%) were administered the originator and CT-P13, respectively. Originator trough levels were higher than the biosimilar trough levels (35 vs. 21, 20.1 vs. 11, 6.6 vs. 2.9 and 4.3 vs. 1.7 µg/mL at weeks 2, 6, 14 and 22, respectively). A post-hoc analysis demonstrated changes in mean serum drug levels over time (p < 0.001) and according to the drug employed (p = 0.001). At week 22, 13 (81%) patients administered the originator achieved clinical remission compared with 5 (19%) patients with the biosimilar (p = 0.02). None of the patients administered the originator withdrew from the treatment compared with 7 for the biosimilar. During the study, there were significant differences in serum infliximab levels between the originator and the CT-P13 in the patients with IBD. The clinical outcomes were influenced by the type of compound administered.

Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions.

Most monogenic disorders have a primary clinical presentation. Inherited ISG15 deficiency, however, has manifested with two distinct presentations to date: susceptibility to mycobacterial disease and intracranial calcifications from hypomorphic interferon-II (IFN-II) production and excessive IFN-I response, respectively. Accordingly, these patients were managed for their infectious and neurologic complications. Herein, we describe five new patients with six novel ISG15 mutations presenting with skin lesions who were managed for dermatologic disease. Cellularly, we denote striking specificity to the IFN-I response, which was previously assumed to be universal. In peripheral blood, myeloid cells display the most robust IFN-I signatures. In the affected skin, IFN-I signaling is observed in the keratinocytes of the epidermis, endothelia, and the monocytes and macrophages of the dermis. These findings define the specific cells causing circulating and dermatologic inflammation and expand the clinical spectrum of ISG15 deficiency to dermatologic presentations as a third phenotype co-dominant to the infectious and neurologic manifestations.

[Delayed allergic reaction to furosemide with a positive lymphocyte transformation test]. / Reacción alérgica tardía a furosemida con positividad de la prueba de transformación de linfocitos.

BACKGROUND: Furosemide is the most commonly prescribed loop diuretic worldwide. Although, its extended use, furosemide rarely induces allergic reactions. Until 2013, only 49 cases of furosemide allergy had been described. CLINICAL CASE: We have reported on a patient who developed a delayed, erythematous and pruritic skin eruption after the ingestion of furosemide. The implication of furosemide in the reaction was established by a positive lymphocyte transformation test (LTT). CONCLUSION: This is the first reported case of hypersensitivity to furosemide in which this drug was confirmed as the trigger by a positive LTT. LTT could become a decent diagnostic alternative for patients who experience delayed reactions to furosemide.

Antecedentes: La furosemida es el diurético de asa más comúnmente prescrito en todo el mundo. A pesar de su amplio uso, rara vez induce reacciones alérgicas. Hasta 2013 solo se habían descrito 49 casos de alergia a la furosemida. Caso clínico: Mujer de 68 años con hipertensión tratada con amlodipina, así como rinoconjuntivitis alérgica. Después del consumo de 40 mg de furosemida por cinco días debido a edema bilateral maleolar presentó exantema cutáneo maculopapular, eritematoso y pruriginoso. Los resultados positivos en la prueba de transformación de linfocitos confirmaron la implicación de la furosemida en la reacción. Conclusión: El caso que se reporta es el primero de hipersensibilidad a la furosemida en el que se confirma la implicación del fármaco por medio de una prueba de transformación de linfocitos positiva, la cual podría convertirse en una alternativa diagnóstica para los pacientes que experimentan reacciones adversas tardías a la furosemida.

[Delayed allergic reaction to amlodipine with a positive lymphocyte transformation test]. / Reacción alérgica tardía a amlodipino con positividad de la prueba de transformación de linfocitos.

BACKGROUND: Amlodipine is one of the third generation dihydropyridine calcium channel blockers used for hypertension. Mild, moderate and severe reactions have been reported with calcium channel blockers. Cross-reactivity among these drugs has not been established. CLINICAL REPORT: We have presented the case of a patient who developed a delayed pruritic, and erythematous maculopapular skin exanthema after the intake of amlodipine. A positive lymphocyte transformation test (LTT) confirmed the implication of amlodipine in the reaction and showed positivity to another calcium channel blocker, nifedipine, demonstrating cross reactivity. CONCLUSION: This is the first reported case of hypersensitivity to amlodipine in which the involvement of the drug is confirmed by a positive TTL. TTL could become a good diagnostic alternative for patients who experience late reactions to amlodipine and possibly cross-reactivity with nifedipine.

Antecedentes: el amlodipino es uno de los bloqueadores de los canales de calcio de dihidropiridinas de tercera generación utilizado para la hipertensión. Se han notificado reacciones leves, moderadas y graves con bloqueadores de los canales de calcio. No se ha establecido la reactividad cruzada entre estos fármacos. Caso clínico: se presenta el caso de una paciente con exantema cutáneo maculopapular, eritematoso y pruriginoso tardío tras la ingestión de amlodipino. La prueba de transformación de linfocitos mostró positividad al amlodipino, lo que confirmó la implicación de este fármaco en la reacción; también mostró positividad al nifedipino, lo que demostró la presencia de reactividad cruzada. Conclusión: este es el primer caso reportado de hipersensibilidad a amlodipino en el que se confirma la implicación del fármaco por medio de una prueba positiva de transformación de linfocitos, la cual podría convertirse en una buena opción diagnóstica en aquellos pacientes que experimentan reacciones tardías al amlodipino, así como posible reactividad cruzada con nifedipino.