RESUMO
BACKGROUND: Although early survival from sepsis has improved with timely resuscitation and source control, survivors frequently experience persistent inflammation and develop chronic critical illness. We examined whether increased copy number of endogenous alarmins, mitochondrial DNA, and nuclear DNA are associated with the early "genomic storm" in blood leukocytes and the development of chronic critical illness in hospitalized patients with surgical sepsis. METHODS: A prospective, observational, cohort study of critically ill septic patients was performed at a United States tertiary health care center. Blood samples were obtained at multiple time points after the onset of sepsis. Droplet Digital polymerase chain reaction (Bio-Rad Laboratories, Hercules, CA) was performed to quantify RHO (nuclear DNA) and MT-CO2 (mitochondrial DNA) copies in plasma. Leukocyte transcriptomic expression of 63 genes was also measured in whole blood. RESULTS: We enrolled 112 patients with surgical sepsis. Two experienced early death, 69 recovered rapidly, and 41 developed chronic critical illness. Both mitochondrial DNA and nuclear DNA copy number were increased in all sepsis survivors, but early nuclear DNA, and not mitochondrial DNA, copy number was further increased in patients who developed chronic critical illness. Cell-free DNA copy number was associated with in-hospital but not long-term (180-day and 365-day) mortality and were only weakly correlated with leukocyte transcriptomics. CONCLUSION: Increased cell-free DNA copy number persists in survivors of sepsis but is not strongly associated with leukocyte transcriptomics. Nuclear DNA but not mitochondrial DNA copy number is associated with adverse, short-term, clinical trajectories and outcomes.
Assuntos
Alarminas/imunologia , Ácidos Nucleicos Livres/genética , Dosagem de Genes/imunologia , Sepse/imunologia , Sobreviventes , Idoso , Alarminas/genética , Ácidos Nucleicos Livres/sangue , Doença Crônica/mortalidade , Doença Crônica/terapia , Estado Terminal/mortalidade , Estado Terminal/terapia , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , DNA Mitocondrial/imunologia , Feminino , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sepse/sangue , Sepse/mortalidade , Sepse/terapia , Centros de Atenção Terciária/estatística & dados numéricos , Fatores de Tempo , Resultado do TratamentoRESUMO
Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.
Assuntos
Anticorpos/genética , Formação de Anticorpos/genética , Células Clonais/imunologia , Genômica , Animais , Anticorpos/imunologia , Células CHO , Efeitos da Posição Cromossômica/genética , Cricetulus , Dosagem de Genes/genética , Dosagem de Genes/imunologia , Genoma/genética , Humanos , Plasmídeos/genética , TransgenesRESUMO
BACKGROUND: Tumor cells express programmed death ligand-1 (PD-L1) through several biological processes, thereby having different clinical significance depending on the underlying mechanism of expression. Currently, mechanisms leading to PDL1 gene expression in colorectal cancer (CRC) are not fully understood. METHODS: We investigated 98 Indonesia CRC patients to determine PD-L1 protein expressions and their correlations with PD-L1 gene copy number status, tumor infiltrating lymphocytes (TILs), tumor mutational profile, as well as clinicopathologic features. RESULTS: Our investigation demonstrated that 18% of patients positively expressed PD-L1. Further analysis on PD-L1 copy number revealed that all PD-L1+ tumors had normal copy number, indicating that the expression of PD-L1 was not a consequence of genetic amplification of PD-L1. From TILs analysis, there was a significant increase of CD8 in all tumor cells expressing PD-L1 (P=0.0051), indicating that the inducible PD-L1 expression was the prominent mechanism occurred in CRC. Furthermore, the expression of PD-L1 in this CRC population was significantly associated with high frequency of MSI compared to the remainder PD-L1- tumors (P=0.0001), suggesting the natural immunogenicity of tumors via MSI status plays role in attracting immune response. On the other hand, p53 mutations which were frequently observed within Indonesian CRCs (76.5%), they were not associated with PD-L1 expression (p=0.1108), as well as KRAS gene (29.6%; p=0.5772) and BRAF gene mutations (5%; p=0.2171). CONCLUSION: Our study demonstrated that PD-L1 expressions in CRC were predominantly found as a consequence of infiltrating CD8 T lymphocytes that in part arise in the setting of microsatellite instability. Taken together, our findings further support the role of adaptive immune resistance to drive PD-L1 induction in tumor microenvironment and may provide important rationale for strategy implementation of immunotherapy for CRC cases.
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Assuntos
Imunidade Adaptativa/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Microambiente Tumoral/imunologia , Imunidade Adaptativa/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Neoplasias Colorretais/genética , Feminino , Dosagem de Genes/genética , Dosagem de Genes/imunologia , Humanos , Indonésia , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação/genética , Mutação/imunologia , Microambiente Tumoral/genética , Adulto JovemRESUMO
Reticuloendotheliosis virus (REV), an avian retrovirus is able to infect a variety of birds and can cause immunosuppression. The aim of this study was to investigate the relationship of thymic lymphocytes apoptosis, proliferation and T cell subtype with immunosuppression. In this study, a hundred and twenty one-day old SPF chickens were randomly divided into control groups (group C) and a REV infection groups (group I). The chickens of group I received intraperitoneal injections of REV with 104.62/0.1 ml TCID50. On day 14, 21, 28 and 35 post-inoculation, the chickens of C group and I group were sacrificed by cardiac puncture blood collection, and the thymic lymphocytes was sterile collected. The proliferation ability of lymphocytes was tested by Cell Counting Kit-8. Flow cytometry was performed to detect apoptosis, cell cycle stage and the change in T cell subtype. The RNA genome copy numbers of REV virus were detected using real-time PCR. Real-time PCR and western blotting were performed to analyze the expression of CyclinD1 and Bcl-2. Our results showed that REV genome copy number steadily declined, the proliferation potential of thymic lymphocytes was inhibited, lymphocytes apoptosed, the ratio of CD4+/CD8+ decreased and the expression of CyclinD1 and Bcl-2 were firstly inhibited, then rapidly recovered. Thus, immunosuppression lead by REV is closely related to the change of T cell subtype, apoptosis, and proliferation of thymic lymphocytes.
Assuntos
Apoptose/imunologia , Proliferação de Células/fisiologia , Galinhas/imunologia , Vírus da Reticuloendoteliose/imunologia , Infecções por Retroviridae/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ciclo Celular/imunologia , Galinhas/virologia , Ciclina D1/imunologia , Dosagem de Genes/imunologia , Genoma Viral/imunologia , Terapia de Imunossupressão/métodos , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Infecções por Retroviridae/virologia , Subpopulações de Linfócitos T/virologia , Timo/virologiaRESUMO
Women develop stronger immune responses than men, with positive effects on the resistance to viral or bacterial infections but magnifying also the susceptibility to autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, the dosage of the endosomal Toll-like receptor 7 (TLR7) is crucial. Murine models have shown that TLR7 overexpression suffices to induce spontaneous lupus-like disease. Conversely, suppressing TLR7 in lupus-prone mice abolishes SLE development. TLR7 is encoded by a gene on the X chromosome gene, denoted TLR7 in humans and Tlr7 in the mouse, and expressed in plasmacytoid dendritic cells (pDC), monocytes/macrophages, and B cells. The receptor recognizes single-stranded RNA, and its engagement promotes B cell maturation and the production of pro-inflammatory cytokines and antibodies. In female mammals, each cell randomly inactivates one of its two X chromosomes to equalize gene dosage with XY males. However, 15 to 23% of X-linked human genes escape X chromosome inactivation so that both alleles can be expressed simultaneously. It has been hypothesized that biallelic expression of X-linked genes could occur in female immune cells, hence fostering harmful autoreactive and inflammatory responses. We review here the current knowledge of the role of TLR7 in SLE, and recent evidence demonstrating that TLR7 escapes from X chromosome inactivation in pDCs, monocytes, and B lymphocytes from women and Klinefelter syndrome men. Female B cells where TLR7 is thus biallelically expressed display higher TLR7-driven functional responses, connecting the presence of two X chromosomes with the enhanced immunity of women and their increased susceptibility to TLR7-dependent autoimmune syndromes.
Assuntos
Cromossomos Humanos X , Dosagem de Genes/imunologia , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico , Caracteres Sexuais , Receptor 7 Toll-Like , Inativação do Cromossomo X/imunologia , Animais , Cromossomos Humanos X/genética , Cromossomos Humanos X/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologiaRESUMO
Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota.
Assuntos
Colite Ulcerativa , Ativação do Complemento , Complemento C4b , Doença de Crohn , Microbioma Gastrointestinal/imunologia , Dosagem de Genes/imunologia , Adolescente , Criança , Pré-Escolar , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Complemento C4b/genética , Complemento C4b/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Feminino , Humanos , Masculino , Yersinia pseudotuberculosis/imunologiaRESUMO
Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Proteína Cofatora de Membrana/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/imunologia , Dosagem de Genes/imunologia , Humanos , Imunoconjugados/imunologia , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/imunologia , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The majority of autoimmune diseases have a strong gender bias, affecting mostly females. Gender-specific factors like sex-hormones, the presence or absence of a second X chromosome, and gender-specific gut microbiota may contribute to this bias. In this review we will discuss the role of the X chromosome encoded toll-like receptor 7 (TLR7) and interferon gamma (IFNγ) in the development of autoimmunity. We will also review recent data indicating how these factors may affect an immune response in a gender-dependent manner.
Assuntos
Doenças Autoimunes/genética , Subpopulações de Linfócitos B/imunologia , Interferon gama/genética , Proteínas com Domínio T/genética , Receptor 7 Toll-Like/genética , Animais , Doenças Autoimunes/imunologia , Antígeno CD11c/biossíntese , Feminino , Dosagem de Genes/imunologia , Humanos , Camundongos , Caracteres SexuaisRESUMO
Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall's corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.
Assuntos
Cromossomos Humanos Par 21/imunologia , Síndrome de Down/imunologia , Dosagem de Genes/imunologia , Timo/imunologia , Fatores de Transcrição/imunologia , Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Síndrome de Down/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Exossomos/imunologia , Exossomos/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Insulina/imunologia , Masculino , Fenótipo , RNA Mensageiro/imunologia , Receptores Nicotínicos/imunologia , Timo/patologia , Proteína AIRERESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease with a high incidence in females and a complex phenotype. Using 564Igi mice, a model of SLE with knock-in genes encoding an autoreactive anti-RNA Ab, we investigated how expression of Toll-like receptors (TLRs) in B cells and neutrophils affects pathogenesis. We established that TLR signaling through MyD88 is necessary for disease. Autoantibody was produced in mice with single deletions of Tlr7, Tlr8, or Tlr9 or combined deletions of Tlr7 and Tlr9. Autoantibody was not produced in the combined absence of Tlr7 and Tlr8, indicating that TLR8 contributes to the break in tolerance. Furthermore, TLR8 was sufficient for the loss of B-cell tolerance, the production of class-switched autoantibody, heightened granulopoiesis, and increased production of type I IFN by neutrophils as well as glomerulonephritis and death. We show that dosage of X-linked Tlr8 plays a major role in the high incidence of disease in females. In addition, we show that the negative regulation of disease by TLR9 is exerted primarily on granulopoiesis and type I IFN production by neutrophils. Collectively, we suggest that individual TLRs play unique roles in the pathogenesis of systemic lupus erythematosus, suggesting new targets for treatment.
Assuntos
Dosagem de Genes/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Caracteres Sexuais , Receptor 8 Toll-Like/imunologia , Cromossomo X/imunologia , Animais , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Feminino , Dosagem de Genes/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mielopoese/genética , Mielopoese/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/genética , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Cromossomo X/genéticaRESUMO
Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.
Assuntos
Dosagem de Genes/imunologia , Células Precursoras de Linfócitos B/imunologia , Transativadores/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dosagem de Genes/genética , Regulação da Expressão Gênica/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-7/genética , Interleucina-7/imunologia , Interleucina-7/metabolismo , Camundongos , Camundongos Mutantes , Mutação , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/imunologia , Fator de Transcrição PAX5/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Group 2 innate lymphoid cells (ILC2s; also called nuocytes, innate helper cells, or natural helper cells) provide protective immunity during helminth infection and play an important role in influenza-induced and allergic airway hyperreactivity. Whereas the transcription factor GATA binding protein 3 (Gata3) is important for the production of IL-5 and -13 by ILC2s in response to IL-33 or -25 stimulation, it is not known whether Gata3 is required for ILC2 development from hematopoietic stem cells. Here, we show that chimeric mice generated with Gata3-deficient fetal liver hematopoietic stem cells fail to develop systemically dispersed ILC2s. In these chimeric mice, in vivo administration of IL-33 or -25 fails to expand ILC2 numbers or to induce characteristic ILC2-dependent IL-5 or -13 production. Moreover, cell-intrinsic Gata3 expression is required for ILC2 development in vitro and in vivo. Using mutant and transgenic mice in which Gata3 gene copy number is altered, we show that ILC2 generation from common lymphoid progenitors, as well as ILC2 homeostasis and cytokine production, is regulated by Gata3 expression levels in a dose-dependent fashion. Collectively, these results identify Gata3 as a critical early regulator of ILC2 development, thereby extending the paradigm of Gata3-dependent control of type 2 immunity to include both innate and adaptive lymphocytes.
Assuntos
Fator de Transcrição GATA3/genética , Interleucina-13/genética , Interleucina-5/genética , Linfócitos/imunologia , Animais , Asma/genética , Asma/imunologia , Fator de Transcrição GATA3/imunologia , Dosagem de Genes/genética , Dosagem de Genes/imunologia , Homeostase/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Interleucina-13/imunologia , Interleucina-33 , Interleucina-5/imunologia , Interleucinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
PURPOSE: Common Variable Immunodeficiency Disorder (CVID) is a complex disorder that predisposes patients to recurrent and severe infections. The C104R mutation in the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) is the most frequent mutation identified in patients with CVID. We carried out a detailed immunological and molecular study in a family with a C104R mutation. METHODS: We have undertaken segregation analysis of a kindred with C104R mutations of the TACI gene. Detailed immunological and molecular investigations were carried out for this kindred and the clinical phenotype was compared to the genotype. RESULTS: Segregation analysis of our kindred showed that inheriting single or double copy of the C104R mutation does not consign an individual to CVID. All heterozygotes in the family were phenotypically different, ranging from asymptomatic to ill-health. A family member with a wild type TACI variant had CVID-related phenotype including IgA deficiency and type 1 diabetes. Interestingly, a family member with the homozygous C104R/C104R variant did not meet the criteria for CVID because he had excellent, albeit unsustained, vaccine responses to T cell dependent and T cell independent vaccine antigens despite profound hypogammaglobulinemia. CONCLUSION: The C104R mutation does not correlate with the clinical phenotypes in this family.
Assuntos
Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Variação Genética/imunologia , Mutação Puntual , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Adulto , Idoso de 80 Anos ou mais , Criança , Segregação de Cromossomos/genética , Segregação de Cromossomos/imunologia , Imunodeficiência de Variável Comum/diagnóstico , Feminino , Dosagem de Genes/genética , Dosagem de Genes/imunologia , Predisposição Genética para Doença , Genótipo , Humanos , Imunofenotipagem/métodos , Masculino , Pessoa de Meia-Idade , Linhagem , Mutação Puntual/genética , Mutação Puntual/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismoRESUMO
Human cytomegalovirus (HCMV) has been reported to reshape the NK-cell receptor (NKR) distribution, promoting an expansion of CD94/NKG2C(+) NK and T cells. The role of NK cells in congenital HCMV infection is ill-defined. Here we studied the expression of NKR (i.e., NKG2C, NKG2A, LILRB1, CD161) and the frequency of the NKG2C gene deletion in children with past congenital infection, both symptomatic (n = 15) and asymptomatic (n = 11), including as controls children with postnatal infection (n = 11) and noninfected (n = 20). The expansion of NKG2C(+) NK cells in HCMV-infected individuals appeared particularly marked and was associated with an increased number of LILRB1(+) NK cells in cases with symptomatic congenital infection. Increased numbers of NKG2C(+), NKG2A(+), and CD161(+) T cells were also associated to HCMV infection. The NKG2C deletion frequency was comparable in children with congenital HCMV infection and controls. Remarkably, the homozygous NKG2C(+/+) genotype appeared associated with increased absolute numbers of NKG2C(+) NK cells. Moreover, HCMV-infected NKG2C(+/+) children displayed higher absolute numbers of NKG2A(+) and total NK cells than NKG2C(+/-) individuals. Our study provides novel insights on the impact of HCMV infection on the homeostasis of the NK-cell compartment in children, revealing a modulatory influence of NKG2C copy number.
Assuntos
Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Regulação da Expressão Gênica/imunologia , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Citomegalovirus/genética , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/genética , Feminino , Deleção de Genes , Dosagem de Genes/imunologia , Regulação da Expressão Gênica/genética , Homeostase/genética , Homeostase/imunologia , Humanos , Lactente , Recém-Nascido , Células Matadoras Naturais/metabolismo , Contagem de Linfócitos , Masculino , Subfamília C de Receptores Semelhantes a Lectina de Células NK/biossínteseRESUMO
Increased susceptibility to recurrent viral and bacterial respiratory infections in children and young adults is not well understood. To evaluate the role of complement factor C4 in the defense against respiratory infections, we studied complement factor C4 allotypes C4A and C4B and copy numbers of C4A and C4B genes in 84 children and young adults with recurrent acute otitis media, sinusitis, or pneumonia and in 74 healthy controls. The occurrence of C4A gene deficiency was significantly higher in patients compared with controls (26% vs 14%, p = 0.048). Girls predominated in the group of patients with C4A deficiency (73% girls and 27% boys, p = 0.004). The lectin pathway of complement was more often functionally impaired in patients with C4A deficiency than in patients with no C4A deficiency (41% vs 13%, p = 0.033). Classical and alternative pathways were normal in individuals with C4 null alleles. C4A deficiency is 1 of the minor defects of the innate immunity that may predispose children and young adults to recurrent respiratory infections. C4 gene testing should be added to the list of investigations when the cause for recurrent acute otitis media, maxillary sinusitis, or pneumonia in children and young adults is sought.
Assuntos
Complemento C4a/imunologia , Imunidade Inata/genética , Otite Média/genética , Pneumonia/genética , Sinusite/genética , Doença Aguda , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Complemento C4a/deficiência , Complemento C4a/genética , Complemento C4b/genética , Complemento C4b/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , Feminino , Dosagem de Genes/imunologia , Frequência do Gene , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Otite Média/imunologia , Pneumonia/imunologia , Recidiva , Fatores Sexuais , Sinusite/imunologiaRESUMO
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous non-Hodgkin's lymphoma that may variably involve the skin, lymph nodes, and peripheral blood. Malignant burden ranges from cutaneous patches and plaques with little evidence of blood involvement to erythroderma often in association with frank leukemia, as in Sézary syndrome. Toward a better understanding of the pathogenesis of this CD4+ T-cell malignancy, we conducted a high-resolution genomic analysis combining DNA (23 samples) and mRNA (12 samples) data of peripheral blood isolates from CTCL patients across a spectrum of stages. Strikingly, even patients with limited involvement, e.g., normal CD4 counts, contained significant copy-number alterations. Defining genomic characteristics of CTCL blood involvement included gains on 8q and 17q, and deletions on 17p and chromosome 10. A consensus analysis of 108 leukemic CTCL samples demonstrated global similarities among patients with varied blood involvement, narrowing 38 of 62 loci. Toward an annotated framework for in vitro testing, we also characterized genomic alterations in five CTCL cell lines (HH, HUT78, PNO, SeAx, and Sez4), revealing intact core features of leukemic CTCL. Together, these studies produce the most comprehensive view of the leukemic CTCL genome to date, with implications for pathogenesis, molecular classification, and potential future therapeutic developments.
Assuntos
Dosagem de Genes/genética , Genômica , Leucemia/genética , Linfoma Cutâneo de Células T/genética , Neoplasias Cutâneas/genética , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Dosagem de Genes/imunologia , Genes Supressores de Tumor , Genoma Humano , Humanos , Leucemia/imunologia , Linfoma Cutâneo de Células T/imunologia , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Oncogenes/genética , Neoplasias Cutâneas/imunologiaRESUMO
Antigen-independent B-cell development occurs in several stages that depend on the expression of Ig heavy and light chain. We identified a line of mice that lacked mature B cells in the spleen. This mouse line carried approximately 11 copies of a transgene of the murine heavy chain constant region locus, and B-lineage cells expressed excessive amounts of the intracellular µ heavy chain. B-cell development failed in the bone marrow at the pro/pre B-cell transition, and examination of other lines with various copy numbers of the same transgene suggested that deficiencies in B-cell development increased with increased transgene copy number. Expression of a transgenic (Tg) light chain along with the Tg µ heavy chain led to minimal rescue of B-cell development in the bone marrow and B cells in the spleen. There are several potential mechanisms for the death of pro/pre B cells as a consequence of excess heavy chain expression.
Assuntos
Linfócitos B/metabolismo , Sistema Imunitário/patologia , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias mu de Imunoglobulina/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Medula Óssea/imunologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Dosagem de Genes/genética , Dosagem de Genes/imunologia , Sistema Imunitário/embriologia , Sistema Imunitário/crescimento & desenvolvimento , Imunocompetência/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/imunologia , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/patologia , Baço/patologia , Transgenes/genéticaRESUMO
In the absence of early B-cell factor 1 (EBF1), B-cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 and its DNA-binding partner Runx1 by evaluating B lymphopoiesis in single (EBF1(het) and Runx1(het)) and compound haploinsufficent (Ebf1(+/-) Runx1(+/-), ER(het)) mice. Here, we demonstrate that decreased Ebf1 gene dosage results in the inappropriate expression of NK-cell lineage-specific genes in B-cell progenitors. Moreover, prolonged expression of Ly6a/Sca-1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre-B and all immature B cells of ER(het) mice. Enforced EBF1 expression repressed promiscuous transcription in pro-B cells of ER(het) mice and in Ebf1(-/-) Pax5(-/-) fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B-cell identity by directing repression of non-B-cell-specific genes.
Assuntos
Linfócitos B/metabolismo , Linhagem da Célula , Linfopoese , Células Precursoras de Linfócitos B/metabolismo , Transativadores/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Dosagem de Genes/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Linfopoese/genética , Linfopoese/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Fator de Transcrição PAX5/genética , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/patologia , Transativadores/genética , Transativadores/imunologiaRESUMO
PURPOSE: Loss of mitochondrial DNA (mtDNA) has been described in whole blood samples from a small number of patients with sepsis, but the underlying mechanism and clinical implications of this observation are not clear. We have investigated the cellular basis of the mtDNA depletion in sepsis, and determined clinical correlates with mtDNA depletion. METHODS: Whole blood samples were obtained from 147 consecutive patients with severe sepsis admitted to a General Critical Care Unit in a University Hospital and 83 healthy controls. In a separate study of 13 patients with severe sepsis, blood was obtained for immediate cell sorting by flow cytometry. MtDNA content was determined in whole blood DNA by PCR methods, and subsequently in the 13 samples where white cell subtypes were separated. RESULTS: The mtDNA content of peripheral blood in human subjects was lower in patients with sepsis than controls (P < 0.0001). By studying leukocyte subsets in a subgroup of 13 patients, we showed that this was largely due to an increase in the proportion of circulating neutrophils, which contained approximately 3-fold less mtDNA than mononuclear leukocytes. However, isolated monocytes (P = 0.041) and lymphocytes (P = 0.021) from septic patients showed clear evidence of mtDNA depletion, which correlated with the APACHE II score (P = 0.015). CONCLUSIONS: In severe sepsis much of the apparent whole blood mtDNA depletion is due to a change in the differential leukocyte count. However mtDNA depletion in mononuclear cells occurs in patients with sepsis and correlates with disease severity.