Improved HUMARA for the Detection of X-Linked Agammaglobulinemia Carriers.

Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.

Coronavirus disease 2019, allergic diseases, and allergen immunotherapy: Possible favorable mechanisms of interaction.

Background: Both, allergen immunotherapy (AIT) and SARS-COV-2 infection cause a set of immunologic changes that respectively vary during the course of the treatment or the disease. Objective: To review immune changes brought along by each of these entities and how they might interrelate. Methods: We start presenting a brief review of the structure of the new coronavirus and how it alters the functioning of the human immune system. Subsequently, we describe the immune changes induced by AIT and how these changes could be favorable or unfavorable in the allergic patient infected with SARS-CoV-2 at a particular point of time during the evolving infection. Results: We describe how a healthy immune response against SARS-CoV-2 develops, versus an immune response that is initially suppressed by the virus, but ultimately overactivated, leading to an excessive production of cytokines (cytokine-storm-like). These changes are then linked to the clinical manifestations and outcomes of the patient. Reviewing the immune changes secondary to AIT, it becomes clear how AIT is capable of restoring a healthy innate immunity. Investigators have previously shown that the frequency of respiratory infections is reduced in allergic patients treated with AIT. On the other hand it also increases immunoregulation. Conclusion: As there are many variables involved, it is hard to predict how AIT could influence the allergic patient's reaction to a SARS-CoV-2 infection. In any case, AIT is likely to be beneficial for the patient with allergic rhinitis and/or allergic asthma in the context of the SARS-CoV-2 pandemic as controlling allergic diseases leads to a reduced need for contact with healthcare professionals. The authors remind the reader that everything in this article is still theoretical, since at the moment, there are no published clinical trials on the outcome of COVID-19 in allergic patients under AIT.

Universal influenza vaccination: a Mexican Expert Position Paper.

OBJECTIVE: Influenza is a costly disease for the population. It is a cause of seasonal morbidity and mortality, epidemics and pandemics or syndemics. Given the variability of the virus, surveillance systems are implemented in order to update the strains and include them in the annual influenza vaccine. This vaccine is currently recommended in some high-risk groups. However, universal vaccination remains controversial. To evaluate the evidence and describe the position of a panel of experts on the relevance of universal vaccination against influenza virus. MATERIAL AND METHODS: Five clinical questions were asked, whereby a systematic search of the literature in electronic sources and a Delphi panel were carried out. The evidence was analyzed, and recommendations were issued by the experts. RESULTS: The group of experts recommends vaccinating the population starting at six months of age and include people who live with egg protein allergy, with comorbidities (diabetes, obesity, cancer), health workers and pregnant women. CONCLUSIONS: Vaccination, starting with vulnerable groups, is a necessary, ethical and cost-effective strategy. However, expanding the coverage to achieve universal vaccination could reduce the transmission of the disease and its consequences in the population.

OBJETIVO: La influenza es una enfermedad costosa para la población. Es causa de morbimortalidad estacional, epidemias y pandemias o sindemias. Debido a la variabilidad del virus, se implementan sistemas de vigilancia para actualizar las cepas e incluirlas en la vacuna antiinfluenza anual. Actualmente se recomienda esta vacuna en algunos grupos de alto riesgo. Sin embargo, la vacunación universal es aún controvertida. Evaluar la evidencia y describir la posición de un panel de expertos sobre la pertinencia de la vacunación universal contra el virus de influenza. MATERIAL Y MÉTODOS: Se realizaron cinco preguntas clínicas, con las que se realizó una búsqueda sistemática de la literatura en fuentes electrónicas y un panel Delphi. Se analizó la evidencia y se emitieron recomendaciones por los expertos. RESULTADOS: El grupo de expertos recomienda vacunar a la población desde los seis meses de edad e incluir a personas que viven con alergia a la proteína del huevo, con comorbilidades (diabetes, obesidad, cáncer), trabajadores de la salud y embarazadas. CONCLUSIONES: La vacunación, iniciando con los grupos vulnerables, es una estrategia necesaria, ética y costo-efectiva. Sin embargo, extender la cobertura para lograr la vacunación universal podría disminuir la transmisión de la enfermedad y sus consecuencias en la población.

Genetic, Immunological, and Clinical Features of the First Mexican Cohort of Patients with Chronic Granulomatous Disease.

PURPOSE: Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by an inability of phagocytes to produce reactive oxygen species, impairing their killing of various bacteria and fungi. We summarize here the 93 cases of CGD diagnosed in Mexico from 2011 to 2019. METHODS: Thirteen Mexican hospitals participated in this study. We describe the genetic, immunological, and clinical features of the 93 CGD patients from 78 unrelated kindreds. RESULTS: Eighty-two of the patients (88%) were male. All patients developed bacterial infections and 30% suffered from some kind of fungal infection. Fifty-four BCG-vaccinated patients (58%) presented infectious complications of BCG vaccine. Tuberculosis occurred in 29%. Granulomas were found in 56% of the patients. Autoimmune and inflammatory diseases were present in 15% of patients. A biological diagnosis of CGD was made in 89/93 patients, on the basis of NBT assay (n = 6), DHR (n = 27), and NBT plus DHR (n = 56). The deficiency was complete in all patients. The median age of biological diagnosis was 17 months (range, 0-186 months). A genetic diagnosis was made in 83/93 patients (when material was available), corresponding to CYBB (n = 64), NCF1 (n = 7), NCF2 (n = 7), and CYBA (n = 5) mutations. CONCLUSIONS: The clinical manifestations in these Mexican CGD patients were similar to those in patients elsewhere. This cohort is the largest in Latin America. Mycobacterial infections are an important cause of morbidity in Mexico, as in other countries in which tuberculosis is endemic and infants are vaccinated with BCG. X-linked CGD accounted for most of the cases in Mexico, as in other Latin American countries. However, a significant number of CYBA and NCF2 mutations were identified, expanding the spectrum of known causal mutations.

Failing to Make Ends Meet: The Broad Clinical Spectrum of DNA Ligase IV Deficiency. Case Series and Review of the Literature.

DNA repair defects are inborn errors of immunity that result in increased apoptosis and oncogenesis. DNA Ligase 4-deficient patients suffer from a wide range of clinical manifestations since early in life, including: microcephaly, dysmorphic facial features, growth failure, developmental delay, mental retardation; hip dysplasia, and other skeletal malformations; as well as a severe combined immunodeficiency, radiosensitivity, and progressive bone marrow failure; or, they may present later in life with hematological neoplasias that respond catastrophically to chemo- and radiotherapy; or, they could be asymptomatic. We describe the clinical, laboratory, and genetic features of five Mexican patients with LIG4 deficiency, together with a review of 36 other patients available in PubMed Medline. Four out of five of our patients are dead from lymphoma or bone marrow failure, with severe infection and massive bleeding; the fifth patient is asymptomatic despite a persistent CD4+ lymphopenia. Most patients reported in the literature are microcephalic females with growth failure, sinopulmonary infections, hypogammaglobulinemia, very low B-cells, and radiosensitivity; while bone marrow failure and malignancy may develop at a later age. Dysmorphic facial features, congenital hip dysplasia, chronic liver disease, gradual pancytopenia, lymphoma or leukemia, thrombocytopenia, and gastrointestinal bleeding have been reported as well. Most mutations are compound heterozygous, and all of them are hypomorphic, with two common truncating mutations accounting for the majority of patients. Stem-cell transplantation after reduced intensity conditioning regimes may be curative.

Patients with Primary Immunodeficiencies Are a Reservoir of Poliovirus and a Risk to Polio Eradication.

Immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) have been isolated from primary immunodeficiency (PID) patients exposed to oral poliovirus vaccine (OPV). Patients may excrete poliovirus strains for months or years; the excreted viruses are frequently highly divergent from the parental OPV and have been shown to be as neurovirulent as wild virus. Thus, these patients represent a potential reservoir for transmission of neurovirulent polioviruses in the post-eradication era. In support of WHO recommendations to better estimate the prevalence of poliovirus excreters among PIDs and characterize genetic evolution of these strains, 635 patients including 570 with primary antibody deficiencies and 65 combined immunodeficiencies were studied from 13 OPV-using countries. Two stool samples were collected over 4 days, tested for enterovirus, and the poliovirus positive samples were sequenced. Thirteen patients (2%) excreted polioviruses, most for less than 2 months following identification of infection. Five (0.8%) were classified as iVDPVs (only in combined immunodeficiencies and mostly poliovirus serotype 2). Non-polio enteroviruses were detected in 30 patients (4.7%). Patients with combined immunodeficiencies had increased risk of delayed poliovirus clearance compared to primary antibody deficiencies. Usually, iVDPV was detected in subjects with combined immunodeficiencies in a short period of time after OPV exposure, most for less than 6 months. Surveillance for poliovirus excretion among PID patients should be reinforced until polio eradication is certified and the use of OPV is stopped. Survival rates among PID patients are improving in lower and middle income countries, and iVDPV excreters are identified more frequently. Antivirals or enhanced immunotherapies presently in development represent the only potential means to manage the treatment of prolonged excreters and the risk they present to the polio endgame.

Multifocal Recurrent Osteomyelitis and Hemophagocytic Lymphohistiocytosis in a Boy with Partial Dominant IFN-γR1 Deficiency: Case Report and Review of the Literature.

Mutations in the genes coding for cytokines, receptors, second messengers, and transcription factors of interferon gamma (IFN-γ) immunity cause Mendelian susceptibility to mycobacterial disease (MSMD). We report the case of a 7-year-old male patient with partial dominant (PD) IFN-γ receptor 1 deficiency who had suffered from multifocal osteomyelitis attributable to bacille Calmette-Guérin vaccination since the age of 18 months. He developed hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory complication, and died with multiorgan dysfunction, despite having been diagnosed and treated relatively early. Patients with PD IFN-γR1 deficiency usually have good prognosis and might respond to human recombinant subcutaneous IFN-γ. Several monogenic congenital defects have been linked to HLH, a catastrophic "cytokine storm" that is usually ascribed to lymphocyte dysfunction and thought to be triggered by interferon gamma. This is the sixth patient with both MSMD and HLH of whom we are aware. The fact that patients with macrophages that cannot respond to IFN-γ still develop HLH, bring these assumptions into question.

Primary Immunodeficiency Diseases in Aguascalientes, Mexico: Results from an Educational Program.

PURPOSE: Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders characterized mainly by recurrent infections. Late diagnosis remains as one of the main issues to solve. We aimed to increase PID diagnosis in Aguascalientes, a 1.3 million inhabitants state in the center of Mexico, and to describe the clinical features of such patients. METHODS: We developed an educational program for health personnel and general public; patients with possible PID were referred to a State University clinical center from December 2011 to December 2012. The patients were evaluated at the clinic and their definitive diagnosis pursued through laboratory, molecular and genetic assays. We describe the findings of those patients and analyze the impact of the program in terms of number of referrals. RESULTS: After 41 talks and 12 media appearances 151 patients were referred for evaluation. Fifteen (9.9%) were diagnosed with PID: five (33%) had antibody deficiencies, seven (47%) Well-defined syndromes, two (13%) Severe combined Immunodeficiency (SCID) and one case (7%) of an innate immune deficiency. All of the 15 PID patients had been referred by physicians, as opposed to the public. We estimated a "number needed to teach" of 75 physicians to get one PID patient referral. CONCLUSION: Educational programs are a fundamental part of the global efforts to increase PID diagnosis and care. To be successful, such programs should include public relations, reach for first-contact physicians, and aim to develop an efficient referral network with molecular diagnostic capability. Enhancing medical knowledge on PID is a successful strategy to improve early diagnosis and treatment.

Clinical Features, Non-Infectious Manifestations and Survival Analysis of 161 Children with Primary Immunodeficiency in Mexico: A Single Center Experience Over two Decades.

PURPOSE: The hallmark of Primary immunodeficiencies (PID) is unusual infection, although other immunological non-infectious manifestations such as autoimmunity, allergy and cancer are often present. Most published reports focus on one disease or defect groups, so that a global prevalence of non-infectious manifestations of PID is hard to find. We aimed to describe the clinical features of our pediatric patients with PID, as well as the frequency and evolution of allergy, cancer and autoimmunity. METHODS: We reviewed all the available charts of patients being followed for PID from 1991 to the spring of 2012 at the National Institute of Pediatrics, Mexico City, to describe their demographic, clinical and laboratory features. Their diagnoses were established by pediatric immunologists in accordance to ESID criteria, including routine immunological workup and specialized diagnostic assays. We divided patients by decade of diagnosis to analyze their survival curves. RESULTS: There were 168 charts available, from which we excluded one duplicate and six equivocal diagnoses. We studied the charts of 161 PID patients (68% male, 86% alive), mostly from the center of the country, with a positive family history in 27% and known consanguinity in 11%. Eighty percent of the patients were diagnosed during the last decade. Current median age was 124 months; median age at onset of infections, 12 months; median age at diagnosis, 52 months; median age at death, 67.5 months. Severe infection and bleeding were the cause of 22 deaths. Eighty-six percent of all patients had at least one infection, while non-infectious manifestations had a global prevalence of 36%, namely: autoimmunity 19%, allergies 17%, and cancer 2.4%. Survival curves were not significantly different when compared by decade of diagnosis. CONCLUSIONS: Compared to other registry reports, we found a lower prevalence of antibody defects, and of associated allergy and cancer. We could only locate two isolated IgA deficiencies and four cases of cancer among our PID patients. Although antibody defects are the most prevalent group (30%), the distribution we found is similar to that reported in Iran, Kuwait, Egypt and Taiwan, with a close 27% share for phagocyte defects, and 26% for the formerly called "well-defined" syndromes. Of note, autoimmune and inflammatory complications are high among our patients with chronic granulomatous disease, as has been reported in both the United States and Japan, but not in Europe.

Next generation sequencing reveals skewing of the T and B cell receptor repertoires in patients with wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is due to mutations of the WAS gene encoding for the cytoskeletal WAS protein, leading to abnormal downstream signaling from the T cell and B cell antigen receptors (TCR and BCR). We hypothesized that the impaired signaling through the TCR and BCR in WAS would subsequently lead to aberrations in the immune repertoire of WAS patients. Using next generation sequencing (NGS), the T cell receptor ß and B cell immunoglobulin heavy chain (IGH) repertoires of eight patients with WAS and six controls were sequenced. Clonal expansions were identified within memory CD4(+) cells as well as in total, naïve and memory CD8(+) cells from WAS patients. In the B cell compartment, WAS patient IGH repertoires were also clonally expanded and showed skewed usage of IGHV and IGHJ genes, and increased usage of IGHG constant genes, compared with controls. To our knowledge, this is the first study that demonstrates significant abnormalities of the immune repertoire in WAS patients using NGS.

Gastric adenocarcinoma in the context of X-linked agammaglobulinemia: case report and review of the literature.

The hallmarks of X-linked Agammaglobulinemia (XLA) are panhypogammaglobulinemia, absent B-cells, and recurrent sinopulmonary and gastrointestinal infections starting at an early age, as well as other infections like cellulitis, meningitis, arthritis and sepsis. A number of non-infectious complications have been reported in these patients, including autoimmune diseases and malignancy, especially lymphomas. Here, we report the case of a 30-year old man who developed gastric adenocarcinoma in the context of XLA. Previous reports of, and hypotheses addressing the development of cancer in patients with XLA, are also summarized. Solid cancer in XLA affects mainly the gastrointestinal tract and seems to be related to chronic infection. A natural evolution can be traced back from gastric adenocarcinoma to megaloblastic anemia due to achlorhydria in the context of chronic infection; periodic endoscopy thus seems justified to detect and treat carcinoma in early stages.

IL-12Rß1 deficiency: mutation update and description of the IL12RB1 variation database.

IL-12Rß1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL-12Rß1 is a receptor chain of both the IL-12 and the IL-23 receptor and deficiency of IL-12Rß1 thus abolishes both IL-12 and IL-23 signaling. IL-12Rß1 deficiency is caused by bi-allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL-12Rß1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL-12Rß1 protein. In addition to disease-causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database (www.LOVD.nl/IL12RB1). In this article, we review the function of IL-12Rß1 and molecular genetics of human IL12RB1.

Increased pro-inflammatory cytokine production after lipopolysaccharide stimulation in patients with X-linked agammaglobulinemia.

PURPOSE: To evaluate the lipopolysaccharide (LPS)-induced pro-inflammatory cytokine response by peripheral blood mononuclear cells (PBMCs) from XLA patients. METHODS: Thirteen patients with XLA were included in the study. LPS-induced TNF-α, IL-1ß, IL-6, and IL-10 production was determined in PBMCs from patients and matched healthy controls by ELISA. Cytokine production was correlated with the severity of mutation, affected domain and clinical characteristics. RESULTS: In response to LPS, PBMCs from XLA patients produced significantly higher amounts of pro-inflammatory cytokines and IL-10 compared to controls, and this production was influenced neither by the severity of the mutation nor the affected domain. PBMCs from patients with a history of more hospital admissions before their diagnosis produced higher levels of TNF-α. PBMCs from patients with lower serum IgA levels showed a higher production of TNF-α and IL-1ß. Less severe (punctual) mutations in the Btk gene were associated with higher serum IgG levels at diagnosis. CONCLUSIONS: Our results demonstrate a predominantly inflammatory response in XLA patients after LPS stimulation and suggest a deregulation of TLR signaling in the absence of Btk. This response may be influenced by environmental factors.

Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

[Effect of transfer factor on treatment with glucocorticoids in a group of pediatric patients with persistent moderate allergic asthma]. / Efecto del factor de transferencia en el tratamiento con glucocorticoides en un grupo de pacientes pediátricos con asma alérgica moderada persistente.

BACKGROUND: Inhaled glucocorticoids are the most effective and potent drugs used to control the inflammatory bronchial reaction in patients with asthma. There are several research projects evaluating the use of immune modulators in the treatment of the asthma related inflammatory process. OBJECTIVE: To evaluate the effect of transfer factor in the treatment of pediatric patients with moderate persistent allergic asthma in terms of inhaled glucocorticoid dosing and time of using. PATIENTS AND METHODS: Randomized, double blind, placebo controlled pilot clinical trial in a cohort of pediatric patients (6-17 years old) with moderate persistent allergic asthma. Two groups were formed. Group one received transfer factor and group two was given placebo. Both groups received conventional therapy with inhaled budesonide and formoterol. Daily respiratory symptoms (cough during day, or at night, and wheezing episodes) were recorded in a personal diary. Spirometric evaluations were performed before enrolling patients, and at 1, 3 and 6 months after. RESULTS: Eleven patients were enrolled in each group. Patients in the transfer factor group showed a statistical significant reduction in the inhaled glucocorticoid doping since month 3, and this difference was maintained until the end of study. Patients on TF group showed also a non statistical significant improvement in spirometrical findings and also showed a better asthma control. CONCLUSIONS: Transfer factor helps to reduce inhaled glucocorticoids dose in patients with allergic rhinitis; however, studies with a larger number of patients should be done in order to obtain better results.