RESUMEN
X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina , Regulación hacia Abajo , Citometría de Flujo , Enfermedades Genéticas Ligadas al Cromosoma X , Selectina L , Monocitos , Proteína Inhibidora de la Apoptosis Ligada a X , Humanos , Citometría de Flujo/métodos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Masculino , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Monocitos/metabolismo , Monocitos/inmunología , Selectina L/genética , Selectina L/metabolismo , Femenino , Neutrófilos/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Preescolar , Adulto , Niño , Trastornos LinfoproliferativosRESUMEN
BACKGROUND: Inborn Errors of Immunity (IEI) comprise several genetic anomalies that affect different components of the innate and adaptive responses, predisposing to infectious diseases, autoimmunity and malignancy. Different studies, mostly in adults, have reported a higher prevalence of cancer in IEI patients. However, in part due to the rarity of most of these IEI subtypes (classified in ten categories by the Primary Immunodeficiency Committee of the International Union of Immunological Societies), it is difficult to assess the risk in a large number of patients, especially during childhood. OBJECTIVE: To document the cancer prevalence in a pediatric cohort from a single referral institution, assessing their risk, together with the type of neoplasia within each IEI subgroup. METHOD: An extensive review of clinical records from 1989 to 2022 of IEI patients who at some point developed cancer before the age of sixteen. RESULTS: Of a total of 1642 patients with IEI diagnosis, 34 developed cancer before 16 years of age, showing a prevalence (2.1%) significantly higher than that of the general age matched population (0.22). Hematologic neoplasms (mostly lymphomas) were the most frequent malignancies. CONCLUSION: This study represents one of the few reports focused exclusively in pediatric IEI cases, describing not only the increased risk of developing malignancy compared with the age matched general population (a fact that must be taken into account by immunologists during follow-up) but also the association of the different neoplasms with particular IEI subtypes, thus disclosing the possible mechanisms involved.
Asunto(s)
Neoplasias , Humanos , Niño , Prevalencia , Neoplasias/epidemiología , Neoplasias/inmunología , Neoplasias/etiología , Masculino , Femenino , Preescolar , Adolescente , Lactante , Síndromes de Inmunodeficiencia/epidemiología , Síndromes de Inmunodeficiencia/inmunología , Recién NacidoRESUMEN
CARD11-associated diseases are monogenic inborn errors of immunity involving immunodeficiency, predisposition to malignancy and immune dysregulation such as lymphoproliferation, inflammation, atopic and autoimmune manifestations. Defects in CARD11 can present as mutations that confer a complete or a partial loss of function (LOF) or contrarily, a gain of function (GOF) of the affected gene product. We report clinical characteristics, immunophenotypes and genotypes of 15 patients from our center presenting with CARD11-associated diseases. Index cases are pediatric patients followed in our immunology division who had access to next generation sequencing studies. Variant significance was defined by functional analysis in cultured cells transfected with a wild type and/or with mutated hCARD11 constructs. Cytoplasmic aggregation of CARD11 products was evaluated by immunofluorescence. Nine index patients with 9 unique heterozygous CARD11 variants were identified. At the time of the identification, 7 variants previously unreported required functional validation. Altogether, four variants showed a GOF effect as well a spontaneous aggregation in the cytoplasm, leading to B cell expansion with NF-κB and T cell anergy (BENTA) diagnosis. Additional four variants showing a LOF activity were considered as causative of CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS). The remaining variant exhibited a neutral functional assay excluding its carrier from further analysis. Family segregation studies expanded to 15 individuals the number of patients presenting CARD11-associated disease. A thorough clinical, immunophenotypical, and therapeutic management evaluation was performed on these patients (5 BENTA and 10 CADINS). A remarkable variability of disease expression was clearly noted among BENTA as well as in CADINS patients, even within multiplex families. Identification of novel CARD11 variants required functional studies to validate their pathogenic activity. In our cohort BENTA phenotype exhibited a more severe and expanded clinical spectrum than previously reported, e.g., severe hematological and extra hematological autoimmunity and 3 fatal outcomes. The growing number of patients with dysmorphic facial features strengthen the inclusion of extra-immune characteristics as part of the CADINS spectrum. CARD11-associated diseases represent a challenging group of disorders from the diagnostic and therapeutic standpoint, especially BENTA cases that can undergo a more severe progression than previously described.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Síndromes de Inmunodeficiencia , Humanos , Proteínas Adaptadoras de Señalización CARD/metabolismo , Guanilato Ciclasa/metabolismo , Heterocigoto , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , FN-kappa B/metabolismoRESUMEN
Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.