RESUMO
Inherited GPI deficiencies (IGDs) are a subset of congenital disorders of glycosylation that are increasingly recognized as a result of advances in whole-exome sequencing (WES) and whole-genome sequencing (WGS). IGDs cause a series of overlapping phenotypes consisting of seizures, dysmorphic features, multiple congenital malformations, and severe intellectual disability. We present a study of six individuals from three unrelated families in which WES or WGS identified bi-allelic phosphatidylinositol glycan class S (PIGS) biosynthesis mutations. Phenotypes included severe global developmental delay, seizures (partly responding to pyridoxine), hypotonia, weakness, ataxia, and dysmorphic facial features. Two of them had compound-heterozygous variants c.108G>A (p.Trp36∗) and c.101T>C (p.Leu34Pro), and two siblings of another family were homozygous for a deletion and insertion leading to p.Thr439_Lys451delinsArgLeuLeu. The third family had two fetuses with multiple joint contractures consistent with fetal akinesia. They were compound heterozygous for c.923A>G (p.Glu308Gly) and c.468+1G>C, a splicing mutation. Flow-cytometry analyses demonstrated that the individuals with PIGS mutations show a GPI-AP deficiency profile. Expression of the p.Trp36∗ variant in PIGS-deficient HEK293 cells revealed only partial restoration of cell-surface GPI-APs. In terms of both biochemistry and phenotype, loss of function of PIGS shares features with PIGT deficiency and other IGDs. This study contributes to the understanding of the GPI-AP biosynthesis pathway by describing the consequences of PIGS disruption in humans and extending the family of IGDs.
Assuntos
Anormalidades Múltiplas/genética , Aciltransferases/genética , Artrogripose/genética , Ataxia Cerebelar/genética , Epilepsia Generalizada/genética , Linhagem Celular , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Feminino , Células HEK293 , Humanos , Deficiência Intelectual/genética , Masculino , Hipotonia Muscular/genética , Mutação , Malformações do Sistema Nervoso/genética , Linhagem , Convulsões/genética , Síndrome , Sequenciamento do Exoma/métodosRESUMO
Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.
Assuntos
Aciltransferases/genética , Atrofia/genética , Doenças Ósseas Metabólicas/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Adolescente , Adulto , Alelos , Cerebelo/patologia , Criança , Pré-Escolar , Exoma/genética , Feminino , Fibroblastos/patologia , Glicosilfosfatidilinositóis/genética , Humanos , Masculino , Hipotonia Muscular/genética , Linhagem , RNA Mensageiro/genética , Convulsões/genéticaRESUMO
Congenital disorders of glycosylation (CDGs) affect multiple systems and present a broad spectrum of clinical features, often including skeletal dysplasia. Exome sequencing has led to the identification of new CDG genes. Immune and skeletal phenotypes associated with mutations in PGM3, encoding a protein that converts N-acetyl-glucosamine-6-phosphate into N-acetyl-glucosamine-1-phosphate, were recently reported. Through exome sequencing, we identified a novel homozygous mutation (c.1135T>C; p.Phe379Leu) in PGM3 in two siblings with bone marrow failure, severe combined immunodeficiency, renal and intestinal malformations, and a skeletal dysplasia resembling Desbuquois dysplasia. Severe respiratory compromise secondary to lung hypoplasia and pulmonary hypertension, and intestinal obstruction led to their demise. We thus report the most severe phenotype described so far associated with PGM3 mutations. This CDG should be considered in the presence of skeletal dysplasia associated with severe immunodeficiency. © 2017 American Society for Bone and Mineral Research.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Doenças da Medula Óssea/genética , Anormalidades Musculoesqueléticas/genética , Mutação , Fosfoglucomutase/genética , Imunodeficiência Combinada Severa/genética , Feminino , Humanos , Recém-Nascido , MasculinoRESUMO
There are over 150 known human proteins which are tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. These proteins play a variety of important roles in development, and particularly in neurogenesis. Not surprisingly, mutations in the GPI anchor biosynthesis and remodeling pathway cause a number of developmental disorders. This group of conditions has been termed inherited GPI deficiencies (IGDs), a subgroup of congenital disorders of glycosylation; they present with variable phenotypes, often including seizures, hypotonia and intellectual disability. Here, we report two siblings with compound heterozygous variants in the gene phosphatidylinositol glycan anchor biosynthesis, class P (PIGP) (NM_153681.2: c.74T > C;p.Met25Thr and c.456delA;p.Glu153AsnFs*34). PIGP encodes a subunit of the enzyme that catalyzes the first step of GPI anchor biosynthesis. Both children presented with early-onset refractory seizures, hypotonia, and profound global developmental delay, reminiscent of other IGD phenotypes. Functional studies with patient cells showed reduced PIGP mRNA levels, and an associated reduction of GPI-anchored cell surface proteins, which was rescued by exogenous expression of wild-type PIGP. This work associates mutations in the PIGP gene with a novel autosomal recessive IGD, and expands our knowledge of the role of PIG genes in human development.
Assuntos
Hexosiltransferases/genética , Proteínas de Membrana/genética , Espasmos Infantis/genética , Anormalidades Múltiplas/genética , Adulto , Linhagem Celular , Criança , Deficiências do Desenvolvimento/genética , Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Hemoglobinúria Paroxística/genética , Hexosiltransferases/metabolismo , Humanos , Deficiência Intelectual/genética , Proteínas de Membrana/metabolismo , Hipotonia Muscular/genética , Mutação , Linhagem , Convulsões/genética , Espasmos Infantis/metabolismoRESUMO
BACKGROUND: Of our 1400 exome-studied patients, 67% originate from consanguineous families. â¼80% suffer from variable degree of intellectual disability (ID). The search for disease causing genes using homozygosity mapping was progressing slowly until 2010, then markedly accelerated by the introduction of exome analysis. OBJECTIVES: To identify the disease causing mutation(s) in three patients from two unrelated families who suffered from global developmental delay, severe ID and drug-responsive seizure disorder. METHODS: Exome analysis was performed in DNA of the three patients. The identified PIGC variants were generated and transfected into PIGC-defective mouse cells and the restoration of the surface expression of mouse CD90, CD48 and FLAER was assessed using flow cytometry. The expression of these proteins was also studied on the surface of patients' leucocytes. RESULTS: Three PIGC mutations were identified; homozygous p.L189W in one family and compound heterozygosity for p.L212P/p.R21X variants in another. PIGC participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor which tethers proteins to plasma membrane. In cells lacking PIGC protein, which were transfected with each of the PIGC variants, we detected a clear reduction of surface expression of GPI-anchored proteins. Furthermore, analyses of patients' leucocytes showed significant and constant decrease of CD16 surface expression in granulocytes, and moderate decrease of CD14, CD55, CD59 and FLAER levels. CONCLUSIONS: PIGC joins the list of genes in which mutations result in defective biosynthesis of GPI anchoring, manifesting by global developmental delay and seizure disorder. The lack of specific biomarker dictates exome sequencing as the diagnostic procedure of choice in similar patients.
Assuntos
Epilepsia/genética , Hexosiltransferases/genética , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Anormalidades Múltiplas , Sequência de Aminoácidos/genética , Animais , Deficiências do Desenvolvimento , Epilepsia/fisiopatologia , Exoma/genética , Feminino , Estudos de Associação Genética , Homozigoto , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Camundongos , Mutação , LinhagemRESUMO
Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.
Assuntos
Diferenciação Celular/genética , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Camundongos SCID , Monócitos/citologia , Monócitos/metabolismo , Cordão Umbilical/citologiaRESUMO
BACKGROUND & AIMS: Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an infant of consanguineous parents. METHODS: We performed targeted WES analysis of DNA collected from the patient and her parents. We validated our findings by a similar analysis of DNA from 150 patients with very-early-onset IBD not associated with known genetic factors analyzed in Toronto, Oxford, and Munich. We compared gene expression signatures in inflamed vs noninflamed intestinal and rectal tissues collected from patients with treatment-resistant Crohn's disease who participated in a trial of ustekinumab. We performed functional studies of identified variants in primary cells from patients and cell culture. RESULTS: We identified a homozygous variant in the tripartite motif containing 22 gene (TRIM22) of the patient, as well as in 2 patients with a disease similar phenotype. Functional studies showed that the variant disrupted the ability of TRIM22 to regulate nucleotide binding oligomerization domain containing 2 (NOD2)-dependent activation of interferon-beta signaling and nuclear factor-κB. Computational studies demonstrated a correlation between the TRIM22-NOD2 network and signaling pathways and genetic factors associated very early onset and adult-onset IBD. TRIM22 is also associated with antiviral and mycobacterial effectors and markers of inflammation, such as fecal calprotectin, C-reactive protein, and Crohn's disease activity index scores. CONCLUSIONS: In WES and targeted exome sequence analyses of an infant with severe IBD characterized by granulomatous colitis and severe perianal disease, we identified a homozygous variant of TRIM22 that affects the ability of its product to regulate NOD2. Combined computational and functional studies showed that the TRIM22-NOD2 network regulates antiviral and antibacterial signaling pathways that contribute to inflammation. Further study of this network could lead to new disease markers and therapeutic targets for patients with very early and adult-onset IBD.
Assuntos
Doença de Crohn/genética , Variação Genética , Antígenos de Histocompatibilidade Menor/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Proteínas com Motivo Tripartido/genética , Idade de Início , Austrália , Células Cultivadas , Biologia Computacional , Consanguinidade , Doença de Crohn/diagnóstico , Doença de Crohn/metabolismo , Doença de Crohn/terapia , Bases de Dados Genéticas , Inglaterra , Exoma , Feminino , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Alemanha , Homozigoto , Humanos , Recém-Nascido , Antígenos de Histocompatibilidade Menor/metabolismo , Ontário , Linhagem , Fenótipo , Mapas de Interação de Proteínas , Proteínas Repressoras/metabolismo , Índice de Gravidade de Doença , Transfecção , Proteínas com Motivo Tripartido/metabolismoRESUMO
BACKGROUND: C3 glomerulonephritis (C3GN) is a rare form of glomerulopathy that is characterized by predominant C3 deposits. Eculizumab, a humanized monoclonal C5 antibody, has recently emerged as a treatment option for C3GN. We report a C3GN patient successfully treated with eculizumab. CASE DIAGNOSIS/TREATMENT: A 5-year-old boy who presented with proteinuria, hematuria, high ASO titers, and low C3 levels was initially diagnosed with post-streptococcal GN. His first kidney biopsy confirmed this diagnosis, but complement investigations identified three alternative pathway dysregulation factors: C3 nephritic factor, complement factor I heterozygous mutation (I398L), and anti-factor H autoantibodies (4,500 AU/ml). A second biopsy performed 11 months after initial presentation (nephrotic range proteinuria) showed a C3GN suggestive of isolated C3 deposits. Despite the use of intensive immunosuppressive therapy (rituximab, corticosteroids, mycophenolate), nephrotic-range proteinuria persisted and a third kidney biopsy showed the same C3GN pattern with more endocapillary proliferation. The serum C5b-9 level was elevated. Eculizumab was initiated and resulted in a significant decline of proteinuria (5.3 to 1.3 g/day) and an improvement in pathologic features. A transient interruption of eculizumab resulted in a rapid rise in proteinuria to 9.3 g/day, which decreased to 0.8 g/day after resumption of treatment. CONCLUSIONS: The administration of anti-C5 antibodies may represent a valuable therapeutic option in patients with C3GN.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Complemento C3/imunologia , Glomerulonefrite/tratamento farmacológico , Imunossupressores/uso terapêutico , Glomérulos Renais/efeitos dos fármacos , Biomarcadores/metabolismo , Biópsia , Pré-Escolar , Complemento C3/metabolismo , Glomerulonefrite/diagnóstico , Glomerulonefrite/imunologia , Humanos , Imuno-Histoquímica , Glomérulos Renais/imunologia , Glomérulos Renais/ultraestrutura , Masculino , Microscopia Eletrônica , Proteinúria/tratamento farmacológico , Proteinúria/imunologia , Indução de Remissão , Fatores de Tempo , Resultado do TratamentoRESUMO
Hereditary multiple intestinal atresia (HMIA) is a rare cause of intestinal obstruction in humans associated with a profound combined immune deficiency. Deleterious mutations of the tetratricopeptide repeat domain-7A (TTC7A) gene lead to HMIA, although the mechanism(s) causing the disease in TTC7A deficiency has (have) not yet been clearly identified. To evaluate the consequences of TTC7A deficiency, we studied the morphology of several organs from HMIA patients at different developmental stages, as well as the expression of the TTC7A protein. We performed histological and immunohistochemical analyses on biopsies and autopsies of 6 patients and 1 fetus with HMIA. Moreover, we characterized for the first time the expression of the TTC7A protein by immunostaining it in several organs from control (including fetal samples), infants, and 1 fetus with HMIA. Besides the gastrointestinal tract, HMIA disease was associated with morphological alterations in multiple organs: thymus, lung, spleen, and liver. Moreover, we demonstrated that normal TTC7A protein was expressed in the cytoplasm of epithelial cells of the intestine, thymus, and pancreas. Surprisingly, altered TTC7A protein was highly expressed in tissues from patients, mainly in the epithelial cells. We have established that HMIA associated with a TTC7A defect is characterized by multiorgan impairments. Overall, this report suggests that TTC7A protein is critical for the proper development, preservation, and/or function of thymic and gastrointestinal epithelium.
Assuntos
Síndromes de Imunodeficiência/genética , Atresia Intestinal/genética , Mutação , Proteínas/genética , Apoptose , Atrofia , Calcinose , Canadá , Estudos de Casos e Controles , Estudos de Coortes , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Feto , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Mucosa Intestinal/patologia , Obstrução Intestinal/etiologia , Intestinos/anormalidades , Fígado/patologia , Pulmão/patologia , Macrófagos/metabolismo , Masculino , Insuficiência de Múltiplos Órgãos/etiologia , Proteínas/metabolismo , Baço/patologia , Timo/patologiaRESUMO
Plasmacytoid dendritic cells (pDCs) initiate both innate and adaptive immune responses, making them attractive targets for post-transplantation immunotherapy, particularly after cord blood transplantation (CBT). Toll-like receptor (TLR) agonists are currently studied for pDC stimulation in various clinical settings. Their efficacy depends on pDC number and functionality, which are unknown after CBT. We performed a longitudinal study of pDC reconstitution in children who underwent bone marrow transplantation (BMT) and single-unit CBT. Both CBT and unrelated BMT patients received antithymocyte globulin as part of their graft-versus-host disease prophylaxis regimen. pDC blood counts were higher in CBT patients than in healthy volunteers from 2 to 9 months after transplantation, whereas they remained lower in BMT patients. We showed that cord blood progenitors gave rise in vitro to a 500-fold increase in functional pDCs over bone marrow counterparts. Upon stimulation with a TLR agonist, pDCs from both CBT and BMT recipients upregulated T cell costimulatory molecules, whereas interferon-alpha (IFN-α) production was impaired for 9 months after CBT. TLR agonist treatment is thus not expected to induce IFN-α production by pDCs after CBT, limiting its immunotherapeutic potential. Fortunately, in vitro production of large amounts of functional pDCs from cord blood progenitors paves the way for the post-transplantation adoptive transfer of pDCs.
Assuntos
Transplante de Medula Óssea , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Células Dendríticas/imunologia , Imunoterapia , Leucemia/terapia , Oligodesoxirribonucleotídeos/uso terapêutico , Receptor Toll-Like 9/agonistas , Adolescente , Antígenos CD/genética , Antígenos CD/imunologia , Soro Antilinfocitário/uso terapêutico , Contagem de Células , Proliferação de Células , Criança , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Feminino , Expressão Gênica , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunossupressores/uso terapêutico , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/biossíntese , Leucemia/genética , Leucemia/imunologia , Leucemia/patologia , Estudos Longitudinais , Masculino , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
Primary immunodeficiencies are intrinsic defects in the immune system that result in a predisposition to infection and are frequently accompanied by a propensity to autoimmunity and/or immunedysregulation. Primary immunodeficiencies can be divided into innate immunodeficiencies, phagocytic deficiencies, complement deficiencies, disorders of T cells and B cells (combined immunodeficiencies), antibody deficiencies and immunodeficiencies associated with syndromes. Diseases of immune dysregulation and autoinflammatory disorder are many times also included although the immunodeficiency in these disorders are often secondary to the autoimmunity or immune dysregulation and/or secondary immunosuppression used to control these disorders. Congenital primary immunodeficiencies typically manifest early in life although delayed onset are increasingly recognized. The early diagnosis of congenital immunodeficiencies is essential for optimal management and improved outcomes. In this International Consensus (ICON) document, we provide the salient features of the most common congenital immunodeficiencies.
Assuntos
Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Triagem Neonatal/estatística & dados numéricos , Infecções Oportunistas/prevenção & controle , Autoimunidade , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Diagnóstico Precoce , Expressão Gênica , Humanos , Síndromes de Imunodeficiência/classificação , Síndromes de Imunodeficiência/terapia , Fatores Imunológicos/uso terapêutico , Recém-Nascido , Mutação , Triagem Neonatal/métodos , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/imunologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
BACKGROUND: Beta-hemolytic Group A Streptococcus invasive disease (iGASd) has been subject to intense research since its re-emergence in the late 1980s. In Quebec, an increase in the number of severe iGASd cases has recently been observed. Because of the inter-individual variability in the severity of iGASd, a hereditary predisposition to invasive disease can be suspected. Given that iGASd occurs in MyD88- and IRAK4-deficient patients, although rarely, the increasing frequency of iGASd in the population of French-Canadian children may be associated with a deficiency in the host's innate immune response. METHODS: In this report, we assessed the influence of: (i) bacterial genotype and virulence factors, (ii) immune-cellular features, and (iii) Myd88/IRAK4-dependent response to GAS in vitro on the susceptibility to iGASd in a paediatric cohort of 16 children: 11 French-Canadian and 5 from diverse origin. FINDINGS: GAS virulence factors and genotype are not implicated in the susceptibility toward iGASd, and cellular and MyD88/IRAK4 deficiencies are excluded in our patients. CONCLUSIONS: Although it has been shown that the MyD88/IRAK4-dependent signal is involved in the response to invasive GAS, our data indicates that a MyD88/IRAK4-mediated signalling defect is not the main factor responsible for the susceptibility to severe iGASd in a paediatric population from the province of Quebec.
RESUMO
BACKGROUND AIMS: Cytokine-induced killer (CIK) cells ex vivo-expanded from cord blood (CB) or peripheral blood (PB) have been shown to be cytotoxic against autologous and allogeneic tumor cells. We have previously shown that CD56(+) CIK cells (CD3(+)CD56(+) and CD3(-)CD56(+)) are capable of killing precursor B-cell acute lymphoblastic leukemia (B-ALL) cell lines. However, the lytic pathways used by CD56(+) PB and CB-CIK cells to kill B-ALL cell lines have not been studied. METHODS: CB and PB-CIK cells were differentiated. CD56(+) CB- and PB-CIK cells were compared for expression of different phenotypic markers and for the lytic pathways used to kill B-ALL cell lines. RESULTS: We found that cytotoxic granule proteins were expressed at higher levels in CD56(+) PB-CIK than in CD56(+) CB-CIK cells. However, CD56(+) CB-CIK cells expressed more tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) compared with CD56(+) PB-CIK cells. We observed that CD56(+) CB-CIK cells used both the NKG2D and TRAIL cytotoxic pathways and were more effective at killing REH cells than CD56(+) PB-CIK cells that used only the NKG2D pathway. In contrast, CD56(+) PB-CIK cells used both NKG2D and TRAIL pathways to kill NALM6 cells, whereas CD56(+) CB-CIK cells used only the NKG2D pathway. CONCLUSIONS: Our results suggest that both the source of CIK and the type of B-ALL cell line have an impact on the intensity of the cytolytic activity and on the pathway used. These findings may have clinical implications with respect to optimizing therapeutic efficacy, which may be dependent on the source of the CIK cells and on the target tumor cells.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células Matadoras Induzidas por Citocinas/transplante , Sangue Fetal/citologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Linhagem Celular , Citotoxicidade Imunológica , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Transplante de Células-Tronco de Sangue Periférico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Whole-exome or gene targeted resequencing in hundreds to thousands of individuals has shown that the majority of genetic variants are at low frequency in human populations. Rare variants are enriched for functional mutations and are expected to explain an important fraction of the genetic etiology of human disease, therefore having a potential medical interest. In this work, we analyze the whole-exome sequences of French-Canadian individuals, a founder population with a unique demographic history that includes an original population bottleneck less than 20 generations ago, followed by a demographic explosion, and the whole exomes of French individuals sampled from France. We show that in less than 20 generations of genetic isolation from the French population, the genetic pool of French-Canadians shows reduced levels of diversity, higher homozygosity, and an excess of rare variants with low variant sharing with Europeans. Furthermore, the French-Canadian population contains a larger proportion of putatively damaging functional variants, which could partially explain the increased incidence of genetic disease in the province. Our results highlight the impact of population demography on genetic fitness and the contribution of rare variants to the human genetic variation landscape, emphasizing the need for deep cataloguing of genetic variants by resequencing worldwide human populations in order to truly assess disease risk.
Assuntos
Suscetibilidade a Doenças , Exoma/genética , Mutação , Análise de Sequência de DNA/métodos , Canadá , Demografia , França , Frequência do Gene , Genética Populacional , Humanos , Polimorfismo de Nucleotídeo Único , População Branca/genéticaRESUMO
Fas-mediated apoptosis is one of the mechanisms used by tumor cells to escape the cytotoxicity of tumor-infiltrating lymphocytes. It has been suggested that cytokine-induced killer (CIK) cells are resistant to Fas-mediated apoptosis, thereby rendering them more attractive for use in cellular immunotherapy. Unlike what was observed by others, here we show that CIK cells are sensitive to Fas-mediated apoptosis. We have observed an increase in Fas expression in the different CIK cell subpopulations (CD3(+)CD56(-), CD3(+)CD56(+), and CD3(-)CD56(+)) isolated from both cord blood (CB) and peripheral blood (PB). We also show that the bulk, as well as the CD3(+)CD56(-) and CD56(+) CB- and PB-CIK cell subpopulations were sensitive to Fas-mediated apoptosis induced by both CH11 and APO-1 antibodies, albeit with a weaker effect for the CH11 antibody on CB-CIK cells. In addition, in the presence of the APO-1 and CH11 inducers, Fas engagement inhibited the cytotoxic activity of CB- and PB-CIK cells. This new contradictory result may help explain the variable efficacy observed with CIK cells in the clinic.
Assuntos
Apoptose/imunologia , Células Matadoras Induzidas por Citocinas/imunologia , Sangue Fetal/imunologia , Linhagem Celular , Células Matadoras Induzidas por Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , HumanosRESUMO
BACKGROUND: Combined immunodeficiency (CID) is characterized by severe recurrent infections with normal numbers of T and B lymphocytes but with deficient cellular and humoral immunity. Most cases are sporadic, but autosomal recessive inheritance has been described. In most cases, the cause of CID remains unknown. OBJECTIVE: We wanted to identify the genetic cause of CID in 2 siblings, the products of a first-cousin marriage, who experienced recurrent bacterial and candidal infections with bronchiectasis, growth delay, and early death. METHODS: We performed immunologic, genetic, and biochemical studies in the 2 siblings, their family members, and healthy controls. Reconstitution studies were performed with T cells from mucosa-associated lymphoid tissue lymphoma-translocation gene 1-deficient (Malt1(-/-)) mice. RESULTS: The numbers of circulating T and B lymphocytes were normal, but T-cell proliferation to antigens and antibody responses to vaccination were severely impaired in both patients. Whole genome sequencing of 1 patient and her parents, followed by DNA sequencing of family members and healthy controls, showed the presence in both patients of a homozygous missense mutation in MALT1 that resulted in loss of protein expression. Analysis of T cells that were available on one of the patients showed severely impaired IκBα degradation and IL-2 production after activation, 2 events that depend on MALT1. In contrast to wild-type human MALT1, the patients' MALT1 mutant failed to correct defective nuclear factor-κB activation and IL-2 production in MALT1-deficient mouse T cells. CONCLUSIONS: An autosomal recessive form of CID is associated with homozygous mutations in MALT1. If future patients are found to be similarly affected, they should be considered as candidates for allogeneic hematopoietic cell transplantation.
Assuntos
Caspases/genética , Mutação , Proteínas de Neoplasias/genética , Imunodeficiência Combinada Severa/genética , Sequência de Aminoácidos , Animais , Caspases/análise , Células Cultivadas , Criança , Pré-Escolar , Humanos , Quinase I-kappa B/metabolismo , Ativação Linfocitária , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/análise , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. METHODS: We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. RESULTS: Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. CONCLUSIONS: Based on our genetic results, TTC7A is the likely causal gene for MIA.
Assuntos
Etnicidade/genética , Exoma/genética , Atresia Intestinal/genética , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Homozigoto , Humanos , Atresia Intestinal/etnologia , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Quebeque , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
BACKGROUND: Reports of acquired immunodeficiency due to autoantibodies against interferon gamma in the adult population are increasing. The interleukin-12-dependent interferon-gamma axis is a major regulatory pathway of cell-mediated immunity and is critical for protection against a few intracellular organisms, including non-tuberculous mycobacteria and Salmonella spp. We report the first case of a fatal disseminated Mycobacterium colombiense/cytomegalovirus coinfection in an adult woman associated with the acquisition of autoantibodies against interferon-gamma. CASE PRESENTATION: A 49-year-old woman, born to nonconsanguineous parents in Laos, but who had lived in Canada for the past 30 years, presented with a 1-month history of weight loss, fatigue, cough, and intermittent low-grade fever. A thoracic computed tomography scan revealed an 8 × 7 cm irregular mass impacting the right superior lobar bronchus along with multiple mediastinal and hilar adenopathies. On the fourth day of admission, the patient developed fever with purulent expectorations. Treatment for a post-obstructive bacterial pneumonia was initiated while other investigations were being pursued. Almost every culture performed during the patient's hospitalization was positive for M. colombiense. Given the late presentation of symptoms - at the age of 49 years - and the absence of significant family or personal medical history, we suspected an acquired immunodeficiency due to the presence of anti-interferon-gamma autoantibodies. This was confirmed by their detection at high levels in the plasma and a STAT1 phosphorylation assay on human monocytes. The final diagnosis was immunodeficiency secondary to the production of autoantibodies against interferon-gamma, which resulted in a post-obstructive pneumonia and disseminated infection of M. colombiense. The clinical course was complicated by the presence of a multiresistant Pseudomonas aeruginosa post-endobronchial ultrasound mediastinitis, cytomegalovirus pneumonitis with dissemination, and finally, susceptible P. aeruginosa ventilator-associated pneumonia with septic shock and multiple organ failure, leading to death despite appropriate antibacterial and anti-mycobacterial treatment. CONCLUSIONS: Although rare, acquired immunodeficiency syndromes should be considered in the differential diagnosis of patients with severe, persistent, or recurrent infections. Specifically, severe non-tuberculous mycobacteria or Salmonella infections in adults without any other known risk factors may warrant examination of autoantibodies against interferon-gamma because of their increasing recognition in the literature.
Assuntos
Autoanticorpos/imunologia , Coinfecção , Infecções por Citomegalovirus/etiologia , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/imunologia , Interferon gama/imunologia , Infecções por Mycobacterium/etiologia , Evolução Fatal , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response.