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PURPOSE. Caspase-associated recruitment domain-9 (CARD9) deficiency is an inborn error of immunity that typically predisposes otherwise healthy patients to single fungal infections and the occurrence of multiple invasive fungal infections is rare. It has been described as the first known condition that predisposes to extrapulmonary Aspergillus infection with preserved lungs. We present a patient that expands the clinical variability of CARD9 deficiency. MATERIALS AND METHODS. Genetic analysis was performed by Sanger sequencing. Neutrophils and mononuclear phagocyte response to fungal stimulation were evaluated through luminol-enhanced chemiluminescence and whole blood production of the proinflammatory mediator IL-6 respectively. RESULTS. We report a 56-year-old Argentinean woman, whose invasive Exophiala spinifera infection at the age of 32 years was unexplained and reported in year 2004. At the age of 49 years, she presented with chronic pulmonary disease due to Aspergillus nomius. After partial improvement following treatment with caspofungin and posaconazole, right pulmonary bilobectomy was performed. Despite administration of multiple courses of antifungals, sustained clinical remission could not be achieved. We recently found that the patient’s blood showed an impaired production of interleukin (IL)-6 when stimulated with zymosan. We also found that she is homozygous for a previously reported CARD9 loss-of-function mutation (Q289*). CONCLUSIONS. This is the first report of a patient with inherited CARD9 deficiency and chronic invasive pulmonary aspergillosis (IPA) due to A. nomius. Inherited CARD9 deficiency should be considered in otherwise healthy children and adults with one or more invasive fungal diseases.

Background Germline heterozygous mutations in human signal transducer and activator of transcription 1 (STAT1) can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases, or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis. LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil domain (CCD) and DNA-binding domain (DBD). Moreover, 6% of patients with chronic mucocutaneous candidiasis with a GOF STAT1 mutation have mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants. Objective We estimated variations in the CCD/DBD of STAT1. Methods We mutagenized 342 individual wild-type amino acids in the CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity. Results Of these 342 mutants, 201 were neutral, 30 were LOF, and 111 were GOF mutations in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in the CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the effect of 2 hypomorphic and dominant negative mutations, E157K and G250E, in the CCD of STAT1 that we found in 2 unrelated patients with Mendelian susceptibility to mycobacterial diseases. Conclusion The systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and the effect of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Background Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare, genetically heterogeneous, autosomal recessive disorder. Patients suffer from recurrent infections caused by reduced levels or absence of serum immunoglobulins. Genetically, 4 subtypes of ICF syndrome have been identified to date: ICF1 (DNMT3B mutations), ICF2 (ZBTB24 mutations), ICF3 (CDCA7 mutations), and ICF4 (HELLS mutations). Aim To study the mutation spectrum in ICF syndrome. Materials and methods Genetic studies were performed in peripheral blood lymphocyte DNA from suspected ICF patients and family members. Results We describe 7 ICF1 patients and 6 novel missense mutations in DNMT3B, affecting highly conserved residues in the catalytic domain. We also describe 5 new ICF2 patients, one of them carrying a homozygous deletion of the complete ZBTB24 locus. In a meta-analysis of all published ICF cases, we observed a gender bias in ICF2 with 79% male patients. Discussion The biallelic deletion of ZBTB24 provides strong support for the hypothesis that most ICF2 patients suffer from a ZBTB24 loss of function mechanism and confirms that complete absence of ZBTB24 is compatible with human life. This is in contrast to the observed early embryonic lethality in mice lacking functional Zbtb24. The observed gender bias seems to be restricted to ICF2 as it is not observed in the ICF1 cohort. Conclusion Our study expands the mutation spectrum in ICF syndrome and supports that DNMT3B and ZBTB24 are the most common disease genes.

The noncanonical NF-κB pathway is implicated in diverse biological and immunological processes. Monoallelic C-terminus loss-of-function and gain-of-function mutations of NFKB2 have been recently identified as a cause of immunodeficiency manifesting with common variable immunodeficiency (CVID) or combined immunodeficiency (CID) phenotypes. Herein we report a family carrying a heterozygous nonsense mutation in NFKB2 (c.809G A, p.W270*). This variant is associated with increased mRNA decay and no mutant NFKB2 protein expression, leading to NFKB2 haploinsufficiency. Our findings demonstrate that bona fide NFKB2 haploinsufficiency, likely caused by mutant mRNA decay and protein instability leading to the transcription and expression of only the wild-type allele, is associated with clinical immunodeficiency, although with incomplete clinical penetrance. Abnormal B cell development, hypogammaglobulinemia, poor antibody response, and abnormal noncanonical (but normal canonical) NF-κB pathway signaling are the immunologic hallmarks of this disease. This adds a third allelic variant to the pathophysiology of NFKB2-mediated immunodeficiency disorders.

The noncanonical NF-κB pathway is implicated in diverse biological and immunological processes. Monoallelic C-terminus loss-of-function and gain-of-function mutations of NFKB2 have been recently identified as a cause of immunodeficiency manifesting with common variable immunodeficiency (CVID) or combined immunodeficiency (CID) phenotypes. Herein we report a family carrying a heterozygous nonsense mutation in NFKB2 (c.809G > A, p.W270*). This variant is associated with increased mRNA decay and no mutant NFKB2 protein expression, leading to NFKB2 haploinsufficiency. Our findings demonstrate that bona fide NFKB2 haploinsufficiency, likely caused by mutant mRNA decay and protein instability leading to the transcription and expression of only the wild-type allele, is associated with clinical immunodeficiency, although with incomplete clinical penetrance. Abnormal B cell development, hypogammaglobulinemia, poor antibody response, and abnormal noncanonical (but normal canonical) NF-κB pathway signaling are the immunologic hallmarks of this disease. This adds a third allelic variant to the pathophysiology of NFKB2-mediated immunodeficiency disorders.

Liver disease in X-linked hyper IgM syndrome (XHIGM) is an important predictor of mortality. In case liver transplantation (LT) is required, a survival benefit is observed when LT is combined with HSCT. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:143 issue:5 pages:1952-+ ispartof: location:United States status: published

Severe combined immunodeficiency (SCID) represents the most lethal form of primary immunodeficiency, with mortality rates of greater than 90% within the first year of life without treatment. Hematopoietic stem cell transplantation and gene therapy are the only curative treatments available, and the best-known prognostic factors for success are age at diagnosis, age at hematopoietic stem cell transplantation, and the comorbidities that develop in between. There are no evidence-based guidelines for standardized clinical care for patients with SCID during the time between diagnosis and definitive treatment, and we aim to generate a consensus management strategy on the supportive care of patients with SCID. First, we gathered available information about SCID diagnostic and therapeutic guidelines, then we developed a document including diagnostic and therapeutic interventions, and finally we submitted the interventions for expert consensus through a modified Delphi technique. Interventions are grouped in 10 topic domains, including 123 "agreed" and 38 "nonagreed" statements. This document intends to standardize supportive clinical care of patients with SCID from diagnosis to definitive treatment, reduce disease burden, and ultimately improve prognosis, particularly in countries where newborn screening for SCID is not universally available and delayed diagnosis is the rule. Our work intends to provide a tool not only for immunologists but also for primary care physicians and other specialists involved in the care of patients with SCID.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:128 issue:9 pages:3957-3975 ispartof: location:United States status: published

(59.4%), granulomata (49.3%), skin infections (42%), chronic diarrhea (41.9%), otitis (29%), sepsis (23.2%), abscesses (21.7%), recurrent urinary tract infection (20.3%), and osteomyelitis (15.9%). Adverse reactions to bacillus Calmette-Gu� (BCG) vaccination were identified in 30% of the studied Latin American CGD cases. The genetic diagnoses of the 71 patients revealed 53 patients from 47 families with heterogeneous mutations in the CYBB gene (five novel mutations:p.W361G,p.C282X,p.W483R,p.R226X,andp.Q93X),16 patientswith the commondeletionc.75_76 del.GTinexon2 ofNCF1 gene, and two patients with mutations in the CYBA gene. Conclusion. The majority of Latin American CGD patients carry a hemizygous mutation in the CYBB gene. They also presented a wide range of clinical manifestations most frequently bacterial and fungal infections of the respiratory tract, skin, and lymph nodes. Thirty percent of the Latin American CGD patients presented adverse reactions to BCG, indicating that this vaccine should be avoided in these patients. # 2015 Wiley Periodicals, Inc.

Antibodies are an essential component of the adaptative immune response and hold long-term memory of the immunological experiences throughout life. Antibody defects represent approximately half of the well-known primary immunodeficiencies requiring immunoglobulin replacement therapy. In this article, the authors review the current indications and therapeutic protocols in the Latin American environment. Immunoglobulin replacement therapy has been a safe procedure that induces dramatic positive changes in the clinical outcome of patients who carry antibody defects. COL0012426

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN-γ, and the proliferation of γδ T cells induced by (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil-derived ROS. We also show that the ROS-generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi-directional cross-talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role.

Mutations in the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) were previously found to be associated with hypogammaglobulinemia in humans. It has been shown that proliferation inducing ligand (APRIL) elicits class switch recombination (CSR) by inducing recruitment of MyD88 to a TACI highly conserved cytoplasmic domain (THC). We have identified a patient with hypogammaglobulinemia carrying a missense mutation (S231R) predicted to affect the THC. Aiming to evaluate the relevance of this novel mutation of TACI in CSR induction, we tested the ability of TACI, TLR9, or/and CD40 ligands to trigger CSR in naive B cells and B-cell lines carrying S231R. IgG secretion was impaired when triggered by TACI or/and TLR9 ligands on S231R-naive B cells. Likewise, these stimuli induced less expression of activation-induced cytidine deaminase, I(γ)1-C(μ), and I(γ)1-C(μ), while induction by optimal CD40 stimulation was indistinguishable from controls. These cells also showed an impaired cooperation between TACI and TLR9 pathways, as well as a lack of APRIL-mediated enhancement of CD40 activation in suboptimal conditions. Finally, after APRIL ligation, S231R-mutated TACI failed to colocalize with MyD88. Collectively, these results highlight the requirement of an intact MyD88-binding site in TACI to trigger CSR.

Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC-autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency-was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface.

International audience; Background : Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by inborn errors of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex. From the first year of life onward, most affected patients display multiple, severe, and recurrent infections caused by bacteria and fungi. Mycobacterial infections have also been reported in some patients.Objective : Our objective was to assess the effect of mycobacterial disease in patients with CGD.Methods : We analyzed retrospectively the clinical features of mycobacterial disease in 71 patients with CGD. Tuberculosis and BCG disease were diagnosed on the basis of microbiological, pathological, and/or clinical criteria.Results : Thirty-one (44%) patients had tuberculosis, and 53 (75%) presented with adverse effects of BCG vaccination; 13 (18%) had both tuberculosis and BCG infections. None of these patients displayed clinical disease caused by environmental mycobacteria, Mycobacterium leprae, or Mycobacterium ulcerans. Most patients (76%) also had other pyogenic and fungal infections, but 24% presented solely with mycobacterial disease. Most patients presented a single localized episode of mycobacterial disease (37%), but recurrence (18%), disseminated disease (27%), and even death (18%) were also observed. One common feature in these patients was an early age at presentation for BCG disease. Mycobacterial disease was the first clinical manifestation of CGD in 60% of these patients.Conclusion : Mycobacterial disease is relatively common in patients with CGD living in countries in which tuberculosis is endemic, BCG vaccine is mandatory, or both. Adverse reactions to BCG and severe forms of tuberculosis should lead to a suspicion of CGD. BCG vaccine is contraindicated in patients with CGD.

Mutations in the Fas gene (TNFRSF6) are the most common causes of Autoimmune Lymphoproliferative Syndrome (ALPS-FAS). In Argentina almost a third of patients with ALPS-FAS present a missense mutation affecting the extracellular cysteine rich domain 2 of Fas, p.Cys107Tyr (C107Y). This change was found in homozygous state in 2 patients from a consanguineous family, and heterozygously, in 3 other patients from 3 unrelated families. In these families, 12 relatives were identified as healthy carriers of the mutation. We sought to test the hypothesis that this mutation actually represents a single haplotype of TNFRSF6. DNAs from ALPS-C107Y patients and their families, as well as from 150 Argentinean control subjects were sequenced for the known higher frequency single nucleotide polymorphisms (SNPs) of TNFRSF6. The C107Y-carriers were also genotyped at 5 microsatellites proximal to the Fas gene locus. All C107Y alleles presented a unique intragenic haplotype that could be restricted to this group. Extent of haplotype sharing and variability of microsatellite alleles in C107Y chromosomes support the presence of a single haplotype block including the mutation and encompassing 2.395 Mb. A founder effect for C107Y has been evidenced in this work and the most common recent ancestor to the patients probably lived 350 years ago. This constitutes the first report of a founder event in ALPS.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired immunoglobulin production. Mutations in the gene encoding TACI (TNFRSF13B) were previously found to be associated with CVID. Previous studies have identified a variety of sequence variants in TACI where A181E and C104R were the most common, with variable frequencies in different ethnic populations. So far, no mutations were identified in the recently reported "TACI highly conserved" (THC) cytoplasmic domain, important for the induction of class switch recombination. Our study evaluated immunological and clinical data on a cohort of 28 Argentinean pediatric CVID patients and allowed the identification of two novel mutations in TNFRSF13B, including one, S231R, affecting the highly conserved THC domain. In contrast, none of the patients presented with A181E and C104R mutations.

Early diagnosis and appropriate therapy are essential for the best prognosis and quality of life in patients with primary immunodeficiency diseases (PIDDs). Experts from several Latin American countries have been meeting on a regular basis as part of an ongoing effort to improve the diagnosis and treatment of PIDD in this region. Three programmes are in development that will expand education and training and improve access to testing facilities throughout Latin America. These programmes are: an educational outreach programme (The L-Project); an immunology fellowship programme; and the establishment of a laboratory network to expand access to testing facilities. This report provides the status of these programmes based on the most recent discussions and describes the next steps toward full implementation of these programmes.

Whole-exome sequencing reveals activating STAT1 mutations in some patients with autosomal dominant chronic mucocutaneous candidiasis disease., Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease with major diagnostic and therapeutic difficulties, basically comprising two different conditions: primary and secondary forms. Recent advances regarding molecular diagnosis may be useful to distinguish from one another, especially in sporadic cases starting in early infancy. MATERIALS AND METHODS: In this report, we evaluated three Argentinean patients with clinical suspicion of HLH, but without family history. We excluded mutations in the perforin gene but identified in the three patients a novel homozygous deletion (c. 581_584delTGCC; p.Leu194-ProfsX2) in the gene-encoding syntaxin 11 (STX11), causing a premature termination codon. RESULTS AND CONCLUSION: Each parent from the three unrelated families resulted heterozygous for this deletion confirming the diagnosis of familial hemophagocytic lymphohistiocytosis type 4. Patients shared the same single-nucleotide polymorphism profile in STX11 gene, and genotyping at ten microsatellites surrounding this gene support the presence of a single-haplotype block carrying the novel mutation.

Argentina has a large number of patients with definite diagnosis of X-linked agammaglobulinemia reported in the Latin-American registry. Forty-nine of them were seen in our referral pediatric hospital, between 1987 and 2005.A retrospective study of clinical, laboratory, and molecular data showed that respiratory tract infections were the most frequent initial clinical presentation and the most common among all manifestations prior to diagnosis (69%). Up to diagnosis, we found a high frequency of severe infections (sepsis, 14% and meningitis, 16%) and a high proportion of patients with chronic lung disease. During follow-up, the development of chronic lung disease was significantly related with age at diagnosis and inappropriate treatment.Although molecular diagnosis has been available in our center for the past 10 years, there is no doubt that awareness for early recognition of immunodeficiency should be improved through broader and more comprehensive education programs emphasizing characteristics of patients with immunodeficiencies.

Herein, we describe a combination of clinical, microbiologic, and histopathologic findings significantly associated with osteomyelitis in chronic granulomatous disease. When present, these features should raise the suspicion of underlying chronic granulomatous disease. In patients with these findings, anti-infective prophylactic measures aiming to cover highly prevalent microorganisms, as well as aggressive therapeutic measures, should be strongly encouraged.

Inmunoglobulina G Endovenosa UNC is a 5% liquid Argentine intravenous immunoglobulin obtained from South American donors. This prospective trial was designed to evaluate if the product meets the minimal efficacy requirement of the US Food and Drug Administration of

This is the second report on the continuing efforts of LAGID to increase the recognition and registration of patients with primary immunodeficiency diseases in 12 Latin American countries: Argentina, Brazil, Chile, Colombia, Costa Rica, Honduras, Mexico, Panama, Paraguay, Peru, Uruguay, and Venezuela. This report reveals that from a total of 3321 patients registered, the most common form of primary immunodeficiency disease was predominantly antibody deficiency (53.2%) with IgA deficiency reported as the most frequent phenotype. This category was followed by 22.6% other well-defined ID syndromes, 9.5% combined T- and B-cell inmunodeficiency, 8.6% phagocytic disorders, 3.3% diseases of immune dysregulation, and 2.8% complement deficiencies. All countries that participated in the first publication in 1998 reported an increase in registered primary immunodeficiency cases, ranging between 10 and 80%. A comparison of the estimated minimal incidence of X-linked agammaglobulinemia, chronic granulomatous disease, and severe combined immunodeficiency between the first report and the present one shows an increase in the reporting of these diseases in all countries. In this report, the estimated minimal incidence of chronic granulomatous disease was between 0.72 and 1.26 cases per 100,000 births in Argentina, Chile, Costa Rica, and Uruguay and the incidence of severe combined immunodeficiency was 1.28 and 3.79 per 100,000 births in Chile and Costa Rica, respectively. However, these diseases are underreported in other participating countries. In addition to a better diagnosis of primary immunodeficiency diseases, more work on improving the registration of patients by each participating country and by countries that have not yet joined LAGID is still needed.

El trasplante hepático constituye la única alternativa terapéutica para numerosas enfermedades hepáticas avanzadas. En el seguimiento post-trasplante pueden observarse complicaciones de diversa gravedad. El objetivo de este estudio fue analizar la evolución a largo plazo de los pacientes trasplantados con un seguimiento mayor de un año post quirúrgico. En el período 11/92-11/01 se realizaron 264 trasplantes en 238 pacientes; incluyéndose en este trabajo 143 niños con más de un año post-trasplante. La mediana de edad ± DS fue de 5.41 ± 5.26 años (r: 0.58-21.7 años). Catorce (9.79%) fueron retrasplantados. Todos los pacientes recibieron ciclosporina (inmunosupresión primaria). Las indicaciones de trasplante fueron: falla hepática fulminante (n: 50); atresia de vías biliares (n: 38); cirrosis (n: 37); colestasis crónica (n: 13) y otras (n: 5). Las indicaciones de retrasplante fueron: cirrosis biliar (n: 7); trombosis de la arteria hepática (n: 4) y rechazo crónico (n: 3). Fueron utilizados injertos reducidos en 73/157 trasplantes(14 donantes vivos relacionados y 11 biparticiones hepáticas). La sobrevida global fue de 93%, paciente y 86%, injerto. El retrasplante y el injerto reducido fueron las variables de mayor significación para el aumento del riesgo de muerte. El déficit de talla y masa ósea se recuperó antes de los 3años post-trasplante. La incidencia de síndrome linfoproliferativo fue del 7.69%. Se diagnosticó hepatitis B de novo en 7 pacientes (4.8%). El riesgo social no afectó la sobrevida. La prevención, detección y tratamiento precoz de las complicaciones en el seguimiento a largo plazo permitió mejorar la evolución de los pacientes.Orthotopic liver transplantation is the only definitive mode of therapy for children with end-stage liver disease. However, it remains challenging because of the necessity to prevent long-term complications. The aim of this study was to analyze the evolution of transplanted patients with more than one year of follow up. Between November 1992 and November 2001, 238 patients underwent 264 liver transplantations. A total of 143 patients with more than one year of follow up were included. The median age of patients ± SD was 5.41 years ± 5.26 (r: 0.58-21.7 years). All children received primary immunosuppression with cyclosporine. The indications for liver replacement were: fulminant hepatic failure (n: 50), biliary atresia (n: 38), cirrhosis (n: 37), chronic cholestasis (n: 13) and miscellaneous (n: 5). The indications for liver re-transplantation were: biliary cirrhosis (n: 7), hepatic artery thrombosis (n: 4) and chronic rejection (n: 3). Reduced-size liver allografts were used in73/157 liver transplants, 14 of them were from living-related donors and 11 were split-livers. Patient and graft survival rates were 93% and 86% respectively. Death risk was statistically higher in retransplanted and reduced-size grafted patients. Growth retardation and low bone density were recovered before the first 3 years post-transplant. The incidence of lymphoproliferative disease was 7.69%. De novo hepatitis B was diagnosed in 7 patients (4.8%). Social risk did not affect the outcome of our population. The prevention, detection and early treatment of complications in the long-term follow up contributed to improve the outcome.

Background Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired immunoglobulin production and usually presents with a normal quantity of peripheral B cells. Most attempts aiming to classify these patients have mainly been focused on T- or B-cell phenotypes and their ability to produce protective antibodies, but it is still a major challenge to find a suitable classification that includes the clinical and immunologic heterogeneity of these patients. Objective In this study we evaluated the late stages of B-cell differentiation in a heterogeneous population of patients with pediatric-onset CVID to clinically correlate and assess their ability to perform somatic hypermutation (SHM), class-switch recombination (CSR), or both. Methods We performed a previously reported assay, the restriction enzyme hotspot mutation assay (IgκREHMA), to evaluate in vivo SHM status. We amplified switch regions from genomic DNA to investigate the quality of the double-strand break repairs in the class-switch recombination process in vivo. We also tested the ability to generate immunoglobulin germline and circle transcripts and to upregulate the activation-induced cytidine deaminase gene through in vitro T-dependent and T-independent stimuli. Results Our results showed that patients could be classified into 2 groups according to their degree of SHM alteration. This stratification showed a significant association between patients of group A, severe alteration, and the presence of noninfectious complications. Additionally, 60% of patients presented with increased microhomology use at switched regions. In vitro activation revealed that patients with CVID behaved heterogeneously in terms of responsiveness to T-dependent stimuli. Conclusions The correlation between noninfectious complications and SHM could be an important tool for physicians to further characterize patients with CVID. This categorization would help to improve elucidation of the complex mechanisms involved in B-cell differentiation pathways. Fil: Almejún, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Campos, Bárbara Carolina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Patiño, Virginia. Instituto Pasteur; Uruguay Fil: Galicchio, Miguel. Hospital de Niños Víctor J. Vilela; Argentina Fil: Zelazko, Marta. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Oleastro, Matías. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Oppezzo, Pablo. Instituto Pasteur; Uruguay Fil: Danielian, Silvia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Different primary immunodeficiencies present increased levels of IgE and cutaneous infections of viral etiology. We report a case of a 2 y, 8 m old boy with combined immunodeficiency, dermatitis and disseminated molluscum contagiosum. The patient presented high titers of IgE, eosinophilia and pronounced TCD8 lymphopenia. Impaired proliferation assays and abnormal antibody response to vaccination were found. Normal results of ZAP-70 protein, NK function, and HLA I levels, to test quantitatives and functional defects of cytotoxic cells, lead us to suspect a mutation in DOCK8 gene. Positive result in molecular study together with clinical and immunology features in the patient confirmed the diagnosis of this new immunodeficiency, being to the authors' knowledge the first case recorded in a paediatric hospital in our country.

Different primary immunodeficiencies present increased levels of IgE and cutaneous infections of viral etiology. We report a case of a 2 y, 8 m old boy with combined immunodeficiency, dermatitis and disseminated molluscum contagiosum. The patient presented high titers of IgE, eosinophilia and pronounced TCD8 lymphopenia. Impaired proliferation assays and abnormal antibody response to vaccination were found. Normal results of ZAP-70 protein, NK function, and HLA I levels, to test quantitatives and functional defects of cytotoxic cells, lead us to suspect a mutation in DOCK8 gene. Positive result in molecular study together with clinical and immunology features in the patient confirmed the diagnosis of this new immunodeficiency, being to the authors' knowledge the first case recorded in a paediatric hospital in our country.

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN-γ, and the proliferation of γδ T cells induced by (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil-derived ROS. We also show that the ROS-generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi-directional cross-talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role.

Primary immunodeficiency diseases (PIDD) are associated with significant morbidity and mortality and result in a significant public health burden. This is in part due to the lack of appropriate diagnosis and treatment of these patients. It is critical that governments become aware of this problem and provide necessary resources to reduce this impact on health care systems. Leading physicians in their respective countries must be supported by their own governments in order to implement tools and provide education and thus improve the diagnosis and treatment of PIDD. The Latin American Society of Primary Immunodeficiencies (LASID) has initiated a large number of activities aimed at achieving these goals, including the establishment of a PIDD registry, development of educational programmes and guidelines, and the introduction of a PIDD fellowship programme. These initiatives are positively impacting the identification and appropriate treatment of patients with PIDD in Latin America. Nevertheless, much remains to be done to ensure that every person with PIDD receives proper therapy. COL0012426

WOS: 000283840300002, PubMed ID: 21057261, Interleukin-12 receptor beta 1 (IL-12R beta 1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guerin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years +/- 9.8 years (range, 0.5-46.4 yr). IL-12R beta 1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought., Rockefeller University Center for Clinical and Translational Science [5UL1RR024143-03]; Rockefeller University; ANRFrench National Research Agency (ANR); PHRC; EUEuropean Union (EU) [LHSP-CT-2005-018736]; BNP Paribas Foundation; Dana Foundation; March of DimesMarch of Dimes; Fondation pour la Recherche MedicaleFondation pour la Recherche Medicale; Howard Hughes Medical InstituteHoward Hughes Medical Institute, The St. Giles Laboratory of Human Genetics of Infectious Diseases is supported by grants from The Rockefeller University Center for Clinical and Translational Science grant number 5UL1RR024143-03, and The Rockefeller University, ANR, PHRC, EU (LHSP-CT-2005-018736), BNP Paribas Foundation, the Dana Foundation, and the March of Dimes. LDB is supported by the Fondation pour la Recherche Medicale as part of the PhD program of Pierre et Marie Curie University (Paris, France). JLC was an International Scholar of the Howard Hughes Medical Institute.

Mutations in IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1 and NEMO result in a common clinical phenotype known as Mendelian Susceptibility to Mycobacterial Diseases (MSMD). Interleukin-12 receptor 01 (IL12R beta 1) deficiency is the most common genetic etiology for MSMD. Known mutations affecting IL12RB1 are recessively inherited and are associated with null response to both IL-12 and IL-23. Mutation IL12RB1 1623_1624delinsTT was originally described in 5 families from European origin (2 from Germany: I from Cyprus, France and Belgium). Interestingly, this same mutation was found in an unexpectedly high prevalence among IL-12R beta 1 deficient patients in Argentina: 5-out-of-6 individuals born to unrelated families carried this particular change. To determine whether mutation 1623_1624delinsTT represents a DNA mutational hotspot or a founder effect, 34 polymorphic markers internal or proximal to IL12RB1 were studied in the Argentinean and the Belgian patients. A common haplotype spanning 1.45-3.51 Mb was shared by all chromosomes carrying mutation 1623_1624delinsTT, and was not detected on 100 control chromosomes. Applying a modified likelihood-based method the age of the most recent common ancestor carrying mutation 1623_1624delinsTT was estimated in 475 years (95% CI, 175-1275), which is the time when the Spaniards initiated the colonization of the Americas. Mutation 1623_1624delinsTT represents the first founder effect described on IL-12R beta 1, the most frequently affected gene in MSMD, and affecting patients with European ancestors. The reason(s) behind the persistency of this mutation across multiple generations, its relative high prevalence, and any potential selective advantage are yet to be established.

The original article to which this Erratum refers was published in Journal of Pediatric Pulmonology, 42(9) 2007, 851–852. DOI: 10.1002/ppul.20653.

CD40 ligand (CD40L) deficiency is an X-linked combined immunodeficiency characterized by impaired class switch recombination. We analyzed clinical and molecular findings in 11 Argentinian patients from seven unrelated families. The mean age at onset of symptoms was 1.1 years (0.5–3.0 years) and the 10 alive patients have a median age of 17 years. We identified two nonsense mutations, including R11X reported as a “hypomorphic” defect, four missense mutations, and one point deletion. Although R11X was associated herein with parvovirus B19-anemia and higher Igs levels as previously described, histoplasmosis and Pneumocystis jiroveci pneumonia were also present. Other so-called “milder” mutation, T254M, was present in three related patients clinically and immunologically undistinguishable from the rest of the cohort. Furthermore, 10 of the 11 patients, having heterogeneous mutations, never had persistent neutropenia, none presented Cryptosporidium sp. infection nor developed liver-biliary tract disease, highlighting the debatable concept of “milder” mutations.

STAT5 proteins are components of the common growth hormone and interleukin 2 family of cytokines' signaling pathway. Mutations in the STAT5b gene, described in 2 patients, lead to growth hormone insensitivity that resembles Laron syndrome. Clinical immunodeficiency was also present, although immunologic defects have not been well characterized thus far. Here we describe a 16-year-old girl who suffered generalized eczema and recurrent infections of the skin and respiratory tract since birth. She also suffered severe chronic lung disease and multiple episodes of herpetic keratitis. Clinical features of congenital growth hormone deficiency were observed, such as persistently low growth rate, severely delayed bone age, and postnatal growth failure resulting from growth hormone resistance. This combined phenotype of growth hormone insensitivity and immunodeficiency was attributable to a homozygous C→T transition that resulted in a nonsense mutation at codon 152 in exon 5 of the STAT5b gene. This novel mutation determined a complete absence of protein expression. The main immunologic findings were moderate T-cell lymphopenia (1274/mm3), normal CD4/CD8 ratio, and very low numbers of natural killer (18/mm3) and γδ T (5/mm3) cells. T cells presented a chronically hyperactivated phenotype. In vitro T-cell proliferation and interleukin 2 signaling were impaired. CD4+ and CD25+ regulatory T cells were significantly diminished, and they probably contributed to the signs of homeostatic mechanism deregulation found in this patient. This new case, in accordance with 2 previously reported cases, definitely demonstrates the significant role of the STAT5b protein in mediating growth hormone actions. Furthermore, the main immunologic findings bring about an explanation for the clinical immunodeficiency features and reveal for the first time the relevant role of STAT5b as a key protein for T-cell functions in humans.

BACKGROUND Chronic granulomatous disease (CGD) is a primary immunodeficiency due to absent or decreased NADPH oxidase activity in phagocytic cells. The X-linked form of the disease (X-CGD) arises from mutations in the CYBB gene, which encodes the 91-kD glycoprotein gp91(phox), the largest component of the oxidase. METHODS The authors recently started the molecular characterization of X-CGD in 18 patients reported to the Argentinean Registry of Primary Immunodeficiency Diseases. The authors reviewed data from clinical records to examine the relationship of clinical presentation and the type of mutations responsible for the genotype. RESULTS The frequency and type of infections present in these patients were similar to prior reports. However, pulmonary tuberculosis was observed in the group as well as unusual complications such as eosinophilic cystitis, hepatic abscess with cholangitis, and chronic orchitis. Eleven different mutations in the CYBB gene were identified, and seven of them were novel. The types of mutations were intronic, single-nucleotide substitution resulting in nonsense or missense codons and one or two nucleotide deletions resulting in frameshifts. Molecular studies of 18 mothers revealed X-CGD carrier status in all but 2. CONCLUSIONS No correlation existed between the type of mutation and the clinical phenotype of the disease: the molecular defects identified resulted in no expression of the flavocytochrome b558 in patients' neutrophils, leading to the X91(o)-CGD phenotype. The lack of gp91(phox) protein could explain the early onset and the severity of the clinical manifestations of CGD in this group of patients from Argentina.

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.

Wiskott-Aldrich syndrome (WAS), is an X-linked immunodeficiency disease caused by mutations of the WAS protein (WASP) gene, characterized by thrombocytopenia, eczema and recurrent infections. X-linked thrombocytopenia (XLT) is a milder form with only platelet abnormalities. Cumulative mutation data have revealed that WASP genotypes are highly variable among WAS patients. By SSCP analysis, we determined the location of the mutation in 23 WAS patients from 17 unrelated families with variable clinical phenotypes. Direct sequence analysis of genomic DNA showed 9 novel mutations (Q52H, G70W, 393del7, Ex 7 Ex11del, IVS 8+1G-->C, 925delG, 959ins38, 1380del8, and IVS 2+2T-->C) and 8 known mutations distributed throughout the WAS gene. This is the first report of WAS gene mutations from a Latin American country.

Abstract 164FN2 Somatic gene therapy for X-linked severe combined immunodeficiency (X-SCID) using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (MFG-γc) resulted in excellent immunologic reconstitution but also in insertional oncogenesis. In 5/20 treated children, T cell leukemia developed, with insertional activation of LMO2 proto-oncogene in 4 of the 5. We reasoned that replacing the virus-derived promoter and enhancer elements with a weaker cellular promoter would result in improved safety yet retain efficacy. We therefore generated the pSRS11.EFS.IL2RG.pre* self-inactivating (SIN) gammaretroviral vector in which expression of γc is controlled by an intronless EF1-α promoter, in a MLV vector devoid of the LTR U3 (enhancer/promoter) region. In preclinical studies we determined ‘relative safety' using several surrogate assays. In a reporter assay, the pSRS11.EFS.IL2RG.pre* vector when inserted into the oncogenic LMO2 locus induced LMO2 expression 6–90-fold less than the parent MFG vector (MFG-γc). pSRS11.EFS.IL2RG.pre* had lower activity in a murine in vitro immortalization assay (0.2 clones per 10e5 cells, fitness score 0.00007), compared to MFG-γc (0.54 clones per 10e5 cells, fitness score 0.00025). Peripheral blood and bone marrow from C57BL6 mice transplanted with murine bone marrow transduced with pSRS11.EFS.IL2RG.pre* and followed in primary recipients over 4 months and in secondary recipients over 1 year in vivo showed no evidence of vector associated leukemias. Deep sequencing demonstrated 8 of 3621 insertions into the MDS-associated gene Evi1 in mice transplanted with MFG-γc-transduced cells while there were none (0 of 2690 insertions into Evi1) in mice transplanted with pSRS11.EFS.IL2RG.pre* vector transduced cells (P=0.025). In preclinical efficacy studies, circulating T and B lymphocytes were detectable in the peripheral blood of 7/7 γc-deficient mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells while 4/4 mice repopulated with SFFV-eGFP transduced cells remained alymphoid. Experimental animals were sacrificed approximately five months post-transplant for analysis of immune reconstitution. Flow cytometric analysis of the spleens and bone marrow revealed restoration of mature B220+IgM+ B cells and NK cell populations in all mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells. CD4+ and CD8+ T cells were also detected in both tissues and in thymi recovered from transplanted animals. T cells in these mice proliferated in response to mitogenic stimuli. Immunoglobulin subclasses IgG1 and IgG2a detected in the plasma from pSRS11.EFS.IL2RG.pre* reconstituted mice also indicated restored B cell function in these animals. Human preclinical studies also supported the correction of XSCID cellular defects using this vector. Based on these data, a multi-institutional phase I/II trial was initiated with the pSRS11.EFS.IL2RG.pre* vector using an identical clinical protocol as in the previous X-SCID trials, to determine efficacy and safety compared with the MFG-γc vector. The first patient was treated in December 2010. Six months post-gene therapy, he has attained CD3 T cell count of >800, normal proliferation to mitogens, and normal NK cell numbers. He has cleared medically-resistant oral ulcers, and a rotavirus infection acquired post-gene therapy. Nearly all of the circulating T cells (86%) and 41% of his NK cells express γc, albeit at modestly lower density than normal, as expected. However, the early kinetics of T cell reconstitution was comparable to several subjects treated with MFG-γc. These data suggest that the improved safety profile demonstrated with numerous surrogate preclinical studies is associated with efficacious transgene expression and functional immune recovery in the initial human patient treated. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells. Baum:Patent office: Patents & Royalties.

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling. © 2003 Wiley-Liss, Inc.