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1.
Artigo em Inglês | MEDLINE | ID: mdl-31568798

RESUMO

Genetic testing has become an integral component of the diagnostic evaluation of patients with suspected primary immunodeficiency diseases. Results of genetic testing can have a profound effect on clinical management decisions. Therefore clinical providers must demonstrate proficiency in interpreting genetic data. Because of the need for increased knowledge regarding this practice, the American Academy of Allergy, Asthma & Immunology Primary Immunodeficiency Diseases Committee established a work group that reviewed and summarized information concerning appropriate methods, tools, and resources for evaluating variants identified by genetic testing. Strengths and limitations of tests frequently ordered by clinicians were examined. Summary statements and tables were then developed to guide the interpretation process. Finally, the need for research and collaboration was emphasized. Greater understanding of these important concepts will improve the diagnosis and management of patients with suspected primary immunodeficiency diseases.

2.
Clin Immunol ; 207: 40-42, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31301515

RESUMO

Mutations in MYD88 cause susceptibility to invasive bacterial infections through impaired signaling downstream of toll-like receptors (TLRs) and IL-1 receptors. We studied a patient presenting with neutropenia, delayed umbilical cord separation, BCG adenitis, andP. aeruginosapneumonia. Next-generation DNA sequencing identified a novel homozygous truncation mutation in MYD88 that abolishes MyD88 expression. The patient's dermal fibroblasts had severely impaired IL-6 production after stimulation with ligands for the MyD88-dependent receptors TLR2, TLR4 and IL-1R, while responses to ligands for the MyD88-independent receptors TLR3 and TNF-α were preserved. Notably, secretion of TNF-α, which is essential for BCG control, was also impaired after LPS stimulation. In this first report of BCG infection in MyD88 deficiency, data suggest that MyD88-dependent TNF-α production contributes to control of mycobacterial disease.

5.
J Allergy Clin Immunol ; 144(2): 574-583.e5, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30872117

RESUMO

BACKGROUND: The tumor TNF receptor family member 4-1BB (CD137) is encoded by TNFRSF9 and expressed on activated T cells. 4-1BB provides a costimulatory signal that enhances CD8+ T-cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. The ligand for 4-1BB is expressed on antigen-presenting cells and EBV-transformed B cells. OBJECTIVE: We investigated the genetic basis of recurrent sinopulmonary infections, persistent EBV viremia, and EBV-induced lymphoproliferation in 2 unrelated patients. METHODS: Whole-exome sequencing, immunoblotting, immunophenotyping, and in vitro assays of lymphocyte and mitochondrial function were performed. RESULTS: The 2 patients shared a homozygous G109S missense mutation in 4-1BB that abolished protein expression and ligand binding. The patients' CD8+ T cells had reduced proliferation, impaired expression of IFN-γ and perforin, and diminished cytotoxicity against allogeneic and HLA-matched EBV-B cells. Mitochondrial biogenesis, membrane potential, and function were significantly reduced in the patients' activated T cells. An inhibitory antibody against 4-1BB recapitulated the patients' defective CD8+ T-cell activation and cytotoxicity against EBV-infected B cells in vitro. CONCLUSION: This novel immunodeficiency demonstrates the critical role of 4-1BB costimulation in host immunity against EBV infection.

6.
Pediatr Rheumatol Online J ; 17(1): 7, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30764840

RESUMO

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) were historically thought to be distinct entities, often managed in isolation. In fact, these conditions are closely related. A collaborative approach, which incorporates expertise from subspecialties that previously treated HLH/MAS independently, is needed. We leveraged quality improvement (QI) techniques in the form of an Evidence-Based Guideline (EBG) to build consensus across disciplines on the diagnosis and treatment of HLH/MAS. METHODS: A multidisciplinary work group was convened that met monthly to develop the HLH/MAS EBG. Literature review and expert opinion were used to develop a management strategy for HLH/MAS. The EBG was implemented, and quality metrics were selected to monitor outcomes. RESULTS: An HLH/MAS clinical team was formed with representatives from subspecialties involved in the care of patients with HLH/MAS. Broad entry criteria for the HLH/MAS EBG were established and included fever and ferritin ≥500 ng/mL. The rheumatology team was identified as the "gate-keeper," charged with overseeing the diagnostic evaluation recommended in the EBG. First-line medications were recommended based on the acuity of illness and risk of concurrent infection. Quality metrics to be tracked prospectively based on time to initiation of treatment and clinical response were selected. CONCLUSION: HLH/MAS are increasingly considered to be a spectrum of related conditions, and joint management across subspecialties could improve patient outcomes. Our experience in creating a multidisciplinary approach to HLH/MAS management can serve as a model for care at other institutions.


Assuntos
Linfo-Histiocitose Hemofagocítica/diagnóstico , Síndrome de Ativação Macrofágica/diagnóstico , Algoritmos , Consenso , Citocinas/sangue , Diagnóstico Diferencial , Medicina Baseada em Evidências/métodos , Humanos , Linfo-Histiocitose Hemofagocítica/terapia , Síndrome de Ativação Macrofágica/terapia , Guias de Prática Clínica como Assunto , Melhoria de Qualidade
7.
JCI Insight ; 3(16)2018 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-30135305

RESUMO

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.

8.
J Allergy Clin Immunol ; 141(4): 1450-1458, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28916186

RESUMO

BACKGROUND: Combined immunodeficiencies (CIDs) are diseases of defective adaptive immunity with diverse clinical phenotypes. Although CIDs are more prevalent in the Middle East than Western countries, the resources for genetic diagnosis are limited. OBJECTIVES: This study aims to characterize the categories of patients with CIDs in Iran clinically and genetically. METHODS: Clinical and laboratory data were obtained from 696 patients with CIDs. Patients were subdivided into those with syndromic (344 patients) and nonsyndromic (352 patients) CIDs. Targeted DNA sequencing was performed on 243 (34.9%) patients. RESULTS: The overall diagnostic yield of the 243 sequenced patients was 77.8% (189 patients). The clinical diagnosis of hyper-IgE syndrome (P < .001), onset of disease at greater than 5 years (P = .02), and absence of multiple affected family members (P = .04) were significantly more frequent in the patients without a genetic diagnosis. An autosomal recessive disease was found in 62.9% of patients, reflecting the high rate of consanguinity in this cohort. Mutations impairing VDJ recombination and DNA repair were the most common underlying causes of CIDs. However, in patients with syndromic CIDs, autosomal recessive mutations in ataxia-telangiectasia mutated (ATM), autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3), and microdeletions in 22q11.21 were the most commonly affected genomic loci. Patients with syndromic CIDs had a significantly lower 5-year survival rate rather than those with nonsyndromic CIDs. CONCLUSIONS: This study provides proof of principle for the application of targeted next-generation sequencing panels in countries with limited diagnostic resources. The effect of genetic diagnosis on clinical care requires continued improvements in therapeutic resources for these patients.

9.
J Allergy Clin Immunol ; 141(3): 1050-1059.e10, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28601686

RESUMO

BACKGROUND: LPS-responsive beige-like anchor protein (LRBA) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) deficiencies give rise to overlapping phenotypes of immune dysregulation and autoimmunity, with dramatically increased frequencies of circulating follicular helper T (cTFH) cells. OBJECTIVE: We sought to determine the mechanisms of cTFH cell dysregulation in patients with LRBA deficiency and the utility of monitoring cTFH cells as a correlate of clinical response to CTLA4-Ig therapy. METHODS: cTFH cells and other lymphocyte subpopulations were characterized. Functional analyses included in vitro follicular helper T (TFH) cell differentiation and cTFH/naive B-cell cocultures. Serum soluble IL-2 receptor α chain levels and in vitro immunoglobulin production by cultured B cells were quantified by using ELISA. RESULTS: cTFH cell frequencies in patients with LRBA or CTLA4 deficiency sharply decreased with CTLA4-Ig therapy in parallel with other markers of immune dysregulation, including soluble IL-2 receptor α chain, CD45RO+CD4+ effector T cells, and autoantibodies, and this was predictive of favorable clinical responses. cTFH cells in patients with LRBA deficiency were biased toward a TH1-like cell phenotype, which was partially reversed by CTLA4-Ig therapy. LRBA-sufficient but not LRBA-deficient regulatory T cells suppressed in vitro TFH cell differentiation in a CTLA4-dependent manner. LRBA-deficient TFH cells supported in vitro antibody production by naive LRBA-sufficient B cells. CONCLUSIONS: cTFH cell dysregulation in patients with LRBA deficiency reflects impaired control of TFH cell differentiation because of profoundly decreased CTLA4 expression on regulatory T cells and probably contributes to autoimmunity in patients with this disease. Serial monitoring of cTFH cell frequencies is highly useful in gauging the clinical response of LRBA-deficient patients to CTLA4-Ig therapy.

10.
Clin Immunol ; 183: 142-144, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28822832

RESUMO

RASGRP1 is a guanine-nucleotide-exchange factor essential for MAP-kinase mediated signaling in lymphocytes. We report the second case of RASGRP1 deficiency in a patient with a homozygous nonsense mutation in the catalytic domain of the protein. The patient had epidermodysplasia verruciformis, suggesting a clinically important intrinsic T cell function defect. Like the previously described patient, our proband also presented with CD4+ T cell lymphopenia, impaired T cell proliferation to mitogens and antigens, reduced NK cell function, and EBV-associated lymphoma. The severity of the disease and the development of EBV lymphoma in both patients suggest that hematopoietic stem cell transplantation should be performed rapidly in patients with RASGRP1 deficiency.


Assuntos
Proteínas de Ligação a DNA/genética , Epidermodisplasia Verruciforme/genética , Infecções por Vírus Epstein-Barr/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Linfoma Difuso de Grandes Células B/genética , Linfopenia/genética , Linfócitos T CD4-Positivos , Criança , Códon sem Sentido , Consanguinidade , Epidermodisplasia Verruciforme/complicações , Infecções por Vírus Epstein-Barr/complicações , Evolução Fatal , Feminino , Homozigoto , Humanos , Linfoma Difuso de Grandes Células B/complicações , Linfoma Difuso de Grandes Células B/virologia , Linfopenia/complicações , Índice de Gravidade de Doença
11.
Front Pharmacol ; 8: 262, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28553230

RESUMO

The dialysis of human and mouse neutrophils in patch clamp experiments in the conventional whole-cell mode induces the emergence of a chloride (Cl-) current that appeared to be primarily regulated by cytoplasmic ionic strength. The characteristics of this current resembled that of the classical, and ubiquitous volume-sensitive outwardly rectifying Cl- current: strong outward rectification, selectivity sequence of the Eisenman1 type, insensitivity to external pH and strong inhibition by tamoxifen, DCPIB and WW781. We show that this current is essentially supported by the leucine rich repeat containing 8 A (LRRC8A); the naturally occurring LRRC8A truncation mutant in ebo/ebo mice drastically reduced Cl- conductance in neutrophils. Remarkably, the residual component presents a distinct pharmacology, but appears equally potentiated by reduced ionic strength. We have investigated the role of the LRRC8A-supported current in the ionic homeostasis of the phagosomal compartment. The vacuolar pH, measured using SNARF-1 labeled Candida albicans, normally rises because of NADPH oxidase activity, and this elevation is blocked by certain Cl- channel inhibitors. However, the pH rise remains intact in neutrophils from the ebo/ebo mice which also demonstrate preserved phagocytic and respiratory burst capacities and normal-sized vacuoles. Thus, the LRRC8A-dependent conductance of neutrophils largely accounts for their "swell activated" Cl- current, but is not required for homeostasis of the phagosomal killing compartment.

12.
J Allergy Clin Immunol ; 140(6): 1651-1659.e1, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28192143

RESUMO

BACKGROUND: Leucine-rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine-rich repeats (LRRs) at its C-terminal end and is an essential component of the volume-regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T- and B-cell development in mice. Lrrc8a-/- mice have severely defective T-cell development and function. It is not known whether the T- and B-cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity. OBJECTIVE: We sought to determine whether VRAC activity is required for normal T-cell development and function. METHODS: VRAC activity was examined by using patch-clamp analysis. Flow cytometry was used to examine T-cell development. T-cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques. RESULTS: We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2-bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8aebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a-/- mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a-/- mice, ebo/ebo mice have normal T-cell development and function and intact antibody response to T-dependent antigen. CONCLUSION: LRRC8A-dependent VRAC activity is dispensable for T-cell development and function.


Assuntos
Agamaglobulinemia/genética , Proteínas de Transporte de Ânions/metabolismo , Linfócitos B/fisiologia , Proteínas de Membrana/metabolismo , Linfócitos T/fisiologia , Animais , Anticorpos/sangue , Diferenciação Celular , Proliferação de Células , Tamanho Celular , Células Cultivadas , Humanos , Transporte de Íons/genética , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes , Deleção de Sequência/genética
15.
Pediatr Allergy Immunol Pulmonol ; 29(2): 91-94, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27458538

RESUMO

Rituximab is a monoclonal antibody (mAb) primarily used to treat oncologic and autoinflammatory conditions. Although hypersensitivity reactions (HSRs) and desensitization protocols to mAbs have been well described in adults, the experience in the pediatric population is very limited. We sought to determine the safety and efficacy of desensitization to rituximab in the pediatric population at our institution. We retrospectively reviewed the experience with HSRs and desensitization to rituximab during a 5-year period in our tertiary care pediatric center, including reaction evaluation, premedication regimens, and desensitization procedures and protocols. A total of 17 desensitizations to rituximab were performed in three patients. A 14-year-old patient underwent successful desensitization to rituximab using a published adult protocol without incident. Two younger patients (ages 7 years and 23 months) experienced significant reactions during initial desensitization attempts. Therefore, we designed a modified desensitization protocol to rituximab, with particular attention to the rate of infusion as mg/kg/h. This new patient weight-based protocol was successfully used in a total of 13 desensitizations in these two patients. Desensitization to rituximab was a safe and effective procedure in our pediatric population. We present a new patient weight-based desensitization protocol for pediatric patients who develop HSRs to rituximab, with particular usefulness for younger pediatric patients and potential utility in pediatric patients with HSRs to other mAbs.

16.
Proc Natl Acad Sci U S A ; 107(9): 4287-92, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20142498

RESUMO

In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is down-regulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly down-regulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection.


Assuntos
Células Dendríticas/imunologia , Endocitose , Receptores Imunológicos/imunologia , Animais , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Fagocitose
17.
Mol Biochem Parasitol ; 137(2): 229-38, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15383293

RESUMO

The fused dihydrofolate reductase/thymidylate synthase gene of Toxoplasma gondii contains ten exons spanning approximately 8 kb of genomic DNA. We have examined the ends of DHFR-TS transcripts within this gene, and find a complex pattern including two discrete 5' termini and multiple polyadenylation sites. No TATAA box or other classical promoter motif is evident in 1.4 kb of genomic DNA upstream of the coding region, but transcript mapping by RNase protection and primer extension reveals two prominent 5' ends at positions -369 and -341 nt relative to the ATG initiation codon. Upstream genomic sequences include GC-rich regions and the (opposite strand) WGAGACG motif previously identified in other T. gondii promoters. Mutagenesis of recombinant reporter plasmids demonstrates that this region is essential for efficient transgene expression. Sequencing the 3' ends from multiple independent mRNA clones demonstrates numerous polyadenylation sites, distributed over >650 nt of genomic sequence beginning approximately 250 nt downstream of the stop codon. Within this region, certain sites seem to be preferred: 14 different positions were found among the 32 polyadenylated transcripts examined, but approximately 40% of the transcripts map to two loci. The 3' noncoding region is rich in A and T nucleotides, and contains an imperfect 50 nt direct repeat, but no obvious poly(A) addition signal was identified.


Assuntos
Genes de Protozoários , Complexos Multienzimáticos/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Toxoplasma/enzimologia , Toxoplasma/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Mapeamento Cromossômico , Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA de Protozoário/genética , Transcrição Genética
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