RESUMEN
BACKGROUND: Identification of patients with lymphocytic variant hypereosinophilic syndrome (L-HES) is challenging, and has important prognostic and therapeutic implications. OBJECTIVE: This study was undertaken to assess diagnostic tools for L-HES and to develop evidence-based diagnostic recommendations. METHODS: Biomarkers of T-cell-driven disease were compared between patients with L-HES versus idiopathic HES (I-HES) variants. Those performed routinely (serum immunoglobulin levels, T-cell phenotyping, T-cell receptor [TCR] gene rearrangement patterns) were collected from medical files, whereas others were prospectively assessed on stored blood samples (serum CCL17/thymus and activation regulated chemokine [TARC] levels, in vitro cytokine production). RESULTS: This study included 48 patients with I-HES and 20 with L-HES associated with a CD3-CD4+ T-cell subset, including 7 with less than 5% aberrant cells. Neither increased serum immunoglobulin levels nor clonal TCR gene rearrangements were sufficiently sensitive or specific for L-HES. In contrast, systematically enhanced expression of the T-cell surface antigens CD2, CD5, CD45RO, and CD95 by these cells allowed for accurate detection by flow cytometry. Serum CCL17/TARC levels were significantly higher in patients with L-HES compared with those with I-HES, and a threshold of 3000 pg/mL allowed for detection of all subjects with L-HES with 75% specificity. Quantification of intracytoplasmic cytokine production by flow cytometry is the most reliable method for detection of enhanced type 2 cytokine expression, most notably for IL-4 and IL-13. CONCLUSION: Adapting the standard of procedure for T-cell phenotyping in patients with unexplained hypereosinophilia is currently the most reliable means of identifying those with CD3-CD4+ L-HES.
Asunto(s)
Síndrome Hipereosinofílico , Complejo CD3 , Linfocitos T CD4-Positivos , Citocinas , Humanos , Síndrome Hipereosinofílico/diagnóstico , Síndrome Hipereosinofílico/genética , Linfocitos TRESUMEN
Background: Lymphocytic variant hypereosinophilic syndrome is characterized by marked over-production of eosinophilopoietic factor(s) by dysregulated T cells leading to eosinophil expansion. In most cases, these T cells are clonal and express a CD3-CD4+ phenotype. As this is a rare disorder, presenting manifestations, disease course, treatment responses, and outcome are not well-characterized. Materials and Methods: In this retrospective single-center observational study, we reviewed medical files of all patients with persistent hypereosinophilia seen between 1994 and 2019 in whom CD3-CD4+ T cells were detected. Data collection included clinical and biological findings at presentation, treatment responses, disease course, and serial CD3-CD4+ T cell counts. Results: Our cohort comprises 26 patients, including 2 with hypereosinophilia of undetermined significance. All 24 symptomatic patients had cutaneous lesions and/or angioedema, and fasciitis was present in several cases. The aberrant T cell subset represented 2% or less total lymphocytes in 11 subjects. TCR gene rearrangement patterns on whole blood were polyclonal in these cases, while they all had serum CCL17/TARC levels above 1,500 pg/ml. Disease manifestations were mild and did not require maintenance therapy in roughly one third of the cohort, while two thirds required long-term oral corticosteroids and/or second-line agents. Among these, interferon-alpha was the most effective treatment option with a response observed in 8/8 patients, one of whom was cured of disease. Treatment had to be interrupted in most cases however due to poor tolerance and/or development of secondary resistance. Anti-interleukin-5 antibodies reduced blood eosinophilia in 5/5 patients, but clinical responses were disappointing. A sub-group of 5 patients had severe treatment-refractory disease, and experienced significant disease- and treatment-related morbidity and mortality, including progression to T cell lymphoma in three. Conclusions: This retrospective longitudinal analysis of the largest monocentric cohort of CD3-CD4+ T cell associated lymphocytic variant hypereosinophilic syndrome published so far provides clinicians confronted with this rare disorder with relevant new data on patient presentation and outcome that should help tailor therapy and follow-up to different levels of disease severity. It highlights the need for novel therapeutic options, especially for the subset of patients with severe treatment-refractory disease. Future research efforts should be made toward understanding CD3-CD4+ T cell biology in order to develop new treatments that target primary pathogenic mechanisms.
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Linfocitos T CD4-Positivos/inmunología , Síndrome Hipereosinofílico/inmunología , Subgrupos de Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Complejo CD3/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
BACKGROUND: Peritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed. METHODS AND FINDINGS: We investigated for 24 patients prospectively enrolled with a possible diagnosis of peritoneal TB, the diagnostic value of the analysis of IFN-γ production by peritoneal fluid lymphocytes in response to a short in vitro stimulation with mycobacterial antigens. The patients were classified in two groups: non-TB and confirmed or highly probable TB. Diagnosis of TB was based on microbiological and histopathological criteria and/or a favorable response to anti-TB treatment. The IFN-γ production by peritoneal CD4+ T lymphocytes was analyzed by flow cytometry after an overnight in vitro stimulation with three different mycobacterial antigens, purified protein derivative (PPD), heparin-binding haemagglutinin (HBHA) or early-secreted-antigen-target-6 (ESAT-6). The percentages of PPD-, HBHA- or ESAT-6-induced IFN-γ-producing peritoneal fluid CD4+ T lymphocytes were higher in the TB group than in the non-TB group (p = 0.0007, p = 0.0004, and p = 0.0002 respectively). Based on cut-off values determined by ROC curve analysis of the results from TB and highly probable TB compared to those of non-TB patients, the sensitivity of these three tests was 100% with a specificity of 92%. CONCLUSIONS: The analysis of mycobacterial-induced IFN-γ production by peritoneal lymphocytes is a promising tool to reliably and rapidly diagnose peritoneal TB. Further studies should be performed on larger cohorts of patients in high-TB-incidence countries to confirm the clinical value of this new diagnostic approach for peritoneal TB.
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Antígenos Bacterianos/inmunología , Ascitis/metabolismo , Linfocitos T CD4-Positivos/inmunología , Interferón gamma/biosíntesis , Mycobacterium tuberculosis/inmunología , Peritoneo/patología , Tuberculosis/diagnóstico , Tuberculosis/epidemiología , Adolescente , Adulto , Anciano , Proteínas Bacterianas/inmunología , Bélgica/epidemiología , Femenino , Humanos , Incidencia , Lectinas/inmunología , Masculino , Persona de Mediana Edad , Curva ROC , Tuberculina/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.
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Linfocitos B Reguladores/inmunología , Bronquiolitis Viral/inmunología , Receptores de Quimiocina/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Linfocitos B Reguladores/virología , Bronquiolitis Viral/patología , Linfocitos T CD4-Positivos/inmunología , Receptor 1 de Quimiocinas CX3C , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Perfilación de la Expresión Génica , Humanos , Recién Nacido , Activación de Linfocitos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitiales Respiratorios , TranscriptomaRESUMEN
OBJECTIVES: To determine the role of S100A8/A9 in the pathogenesis of primary Sjögren's syndrome (pSS). METHODS: The serum levels of S100A8/A9 were determined in pSS patients and healthy controls by ELISA. The expression of S100A8/A9 in salivary glands was assessed by immunohistochemistry. The phenotype of S100A8+ and S100A9+ cells was identified using double immunofluorescence. The effects of S100A8/A9 on cytokine production by peripheral blood mononuclear cells (PBMCs) from pSS patients were determined in vitro by flow cytometry. The effects of pro-inflammatory cytokines on S100A8/A9 secretion were additionally investigated in vitro by ELISA in PBMCs from pSS patients and control subjects. RESULTS: Serum levels of S100A8/A9 were significantly increased in pSS patients compared to healthy controls. The tissular expression of S100A8 and S100A9, identified in professional phagocytes (neutrophils, monocytes and plasmacytoid dendritic cells), was increased in the salivary glands of pSS patients and correlated with focus score. In vitro, recombinant S100A8/A9 increased the production of IL-1ß, IL-6, TNF-α, IFN-γ, IL-10, IL-17A and IL-22 by PBMCs. The S100A8/A9-induced increase in TNF-α production in pSS patients was significant relative to controls. Furthermore, IL-1ß, TNF-α, IL-6, and IL-17A stimulated release of S100A8/A9 from PBMCs in pSS patients. CONCLUSIONS: S100A8/A9 is increased in pSS patients contributing to the in vitro increased production of pro-inflammatory cytokines. As such, S100A8/A9 in concert with other cytokines might contribute to the pathogenesis of pSS.
Asunto(s)
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Citocinas/metabolismo , Fagocitos/metabolismo , Glándulas Salivales/metabolismo , Síndrome de Sjögren/metabolismo , Regulación hacia Arriba , Calgranulina A/sangre , Calgranulina B/sangre , Citocinas/farmacología , Femenino , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Fagocitos/citología , Fagocitos/efectos de los fármacos , Síndrome de Sjögren/sangreRESUMEN
OBJECTIVES: To investigate the role of the interleukin (IL)-33-ST2 axis in the pathophysiology of primary Sjögren's syndrome (pSS). METHODS: Serum levels of IL-33 and sST2 were determined by ELISA. The expression of IL-33 and ST2 was investigated in salivary glands (SG) by immunohistochemistry. PBMC were isolated and stimulated with IL-33, IL-12 and IL-23 and the cytokine profile response was examined by flow cytometry. Intracellular cytokine detection of IFNγ and IL-17 was performed by flow cytometry. RESULTS: Serum IL-33 and sST2 levels were increased in pSS patients compared with controls and patients with systemic lupus erythematosus. Expression of IL-33 was upregulated in SG with Chisholm scores of 2 and 3 of pSS patients but comparable with controls for SG with Chisholm score of 4. ST2 expression in SG was downregulated in pSS patients. IL-33 at different concentrations did not increase the secretion of pro-inflammatory cytokines but acted synergistically with IL-12 and IL-23 to promote IFNγ production. NK and NKT cells were identified as main producers of IFNγ in vitro and were found in SG of pSS patients. CONCLUSIONS: IL-33 is released in pSS, and acts with IL-12 and IL-23 to favour the secretion of IFNγ by NK and NKT cells.
Asunto(s)
Interleucinas/metabolismo , Receptores de Superficie Celular/metabolismo , Glándulas Salivales/metabolismo , Síndrome de Sjögren/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Interferón gamma/efectos de los fármacos , Interferón gamma/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-12/farmacología , Interleucina-17/metabolismo , Interleucina-23/farmacología , Interleucina-33 , Interleucinas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Masculino , Persona de Mediana Edad , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/metabolismo , Síndrome de Sjögren/etiologíaRESUMEN
Mycobacterium tilburgii rarely causes disseminated disease. We describe a case of M. tilburgii infection in an otherwise healthy 33-year-old woman, who was found to carry bi-allelic mutations of the gene encoding the ß1 chain of the IL-12 receptor.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/inmunología , Receptores de Interleucina-12/deficiencia , Adulto , Alelos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/patología , Femenino , Granuloma/tratamiento farmacológico , Granuloma/genética , Granuloma/inmunología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/microbiología , Células Asesinas Naturales/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Mutación/inmunología , Mycobacterium/inmunología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Receptores de Interleucina-12/genética , RecurrenciaRESUMEN
BACKGROUND: The recent development of eosinophil-targeting agents has raised enthusiasm for management of patients with hypereosinophilic syndromes. Roughly half of anti-IL-5-treated patients with corticosteroid-responsive lymphocytic (L-HES) and idiopathic disease variants can be tapered off corticosteroids. Potential consequences of corticosteroid-withdrawal on clonal expansion of pre-malignant CD3â»CD4⺠T-cells associated with L-HES are a subject of concern. Indeed, corticosteroid treatment inhibits T-cell activation and may lower blood CD3â»CD4⺠cell counts. On the other hand, previous studies have shown that eosinophils support CD4 T-cell activation, suggesting that targeted eosinophil depletion may negatively regulate these cells. OBJECTIVES: Effects of eosinophils on CD4 T-cell activation in vitro were investigated as an indirect means of exploring whether treatment-induced eosinophil depletion may affect pathogenic T-cells driving L-HES. METHODS: Helper (CD4) T-cells and CD3â»CD4⺠cells from healthy controls and L-HES patients, respectively, were cultured in vitro in presence of anti-CD3/CD28 or dendritic cells. Effects of eosinophils on T-cell proliferation and cytokine production were investigated. RESULTS: Eosinophils enhanced CD3-driven proliferation of CD4 T-cells from healthy subjects in vitro, while inhibiting TCR-independent proliferation and IL-5 production by CD3â»CD4⺠T-cells. CONCLUSIONS: While this study confirms previous work showing that eosinophils support activation of normal helper T-cells, our in vitro findings with CD3â»CD4⺠T-cells suggest that eosinophil-depletion may favor activation and expansion of this pathogenic lymphocyte subset. With the ongoing development of eosinophil-targeted therapy for various eosinophilic conditions, the indirect consequences of treatment on the underlying immune mechanisms of disease should be investigated in detail in the setting of translational research programs.
Asunto(s)
Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/citología , Eosinófilos/citología , Activación de Linfocitos , Corticoesteroides/uso terapéutico , Proliferación Celular , Técnicas de Cocultivo , Citocinas/inmunología , Células Dendríticas/citología , Regulación de la Expresión Génica , Humanos , Síndrome Hipereosinofílico/inmunología , Interleucina-5/inmunología , Monocitos/citología , Investigación Biomédica TraslacionalRESUMEN
Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.
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Antígenos CD19/genética , Síndromes de Inmunodeficiencia/genética , Mutación Missense , Triptófano/genética , Secuencia de Aminoácidos , Antígenos CD19/química , Antígenos CD19/inmunología , Estudios de Casos y Controles , Niño , Femenino , Humanos , Síndromes de Inmunodeficiencia/inmunología , Masculino , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Linfocitos T/inmunología , Triptófano/inmunologíaAsunto(s)
Agammaglobulinemia/inmunología , Antígenos CD/inmunología , Linfocitos B/inmunología , Agammaglobulinemia/genética , Agammaglobulinemia/patología , Formación de Anticuerpos , Antígenos CD/genética , Antígenos CD19/biosíntesis , Antígenos CD19/inmunología , Linfocitos B/patología , Codón sin Sentido , Femenino , Genotipo , Humanos , Activación de Linfocitos , Cooperación Linfocítica , Masculino , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/inmunología , Tetraspanina 28 , VacunaciónRESUMEN
Interleukin-12 receptor ß1 (IL-12Rß1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guérin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years ± 9.8 years (range, 0.5-46.4 yr). IL-12Rß1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought.
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Sudunidad beta 1 del Receptor de Interleucina-12/deficiencia , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Citocinas/sangre , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Sudunidad beta 1 del Receptor de Interleucina-12/genética , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Infecciones por Mycobacterium no Tuberculosas/genética , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Micobacterias no Tuberculosas/aislamiento & purificación , Análisis de SupervivenciaRESUMEN
BACKGROUND: Mepolizumab, a monoclonal anti-IL-5 antibody, is an effective corticosteroid-sparing agent for patients with Fip1-like 1/platelet-derived growth factor receptor α fusion (F/P)-negative hypereosinophilic syndrome (HES). Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T cells. OBJECTIVE: To determine whether patients with L-HES respond to mepolizumab in terms of corticosteroid tapering and eosinophil depletion to the same extent as corticosteroid-responsive F/P-negative patients with HES and a normal T-cell profile. METHODS: Patients enrolled in the mepolizumab trial were evaluated for L-HES on the basis of T-cell phenotyping and T-cell receptor gene rearrangement patterns, and their serum thymus-and-activation-regulated chemokine (TARC) levels were measured. Response to treatment was compared in patient subgroups based on results of these analyses. RESULTS: Lymphocytic variant HES was diagnosed in 13 of 63 patients with HES with complete T-cell assessments. The ability to taper corticosteroids on mepolizumab was similar in patients with L-HES and those with a normal T-cell profile, although a lower proportion of patients with L-HES maintained eosinophil levels below 600/µL. Increased serum TARC levels (>1000 pg/mL) had no significant impact on the ability to reduce corticosteroid doses, but a lower proportion of patients with elevated TARC achieved eosinophil control on mepolizumab. CONCLUSION: Mepolizumab is an effective corticosteroid-sparing agent for patients with L-HES. In some cases however, eosinophil levels remain above 600/µL, suggesting incomplete neutralization of overproduced IL-5 or involvement of other eosinophilopoietic factors.
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Corticoesteroides/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Síndrome Hipereosinofílico/tratamiento farmacológico , Interleucina-5/metabolismo , Prednisona/administración & dosificación , Linfocitos T/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Complejo CD3/metabolismo , Antígenos CD4/metabolismo , Quimiocina CCL17/sangre , Método Doble Ciego , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Femenino , Citometría de Flujo , Humanos , Síndrome Hipereosinofílico/inmunología , Interleucina-5/inmunología , Linfocitos , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.
Asunto(s)
Antígenos CD19/fisiología , Antígenos CD/genética , Síndromes de Inmunodeficiencia/etiología , Mutación , Antígenos CD19/análisis , Subgrupos de Linfocitos B/inmunología , Niño , Femenino , Humanos , Interferón gamma/biosíntesis , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos B/fisiología , Hipermutación Somática de Inmunoglobulina , Subgrupos de Linfocitos T/inmunología , Tetraspanina 28RESUMEN
The flow cytometric basophil activation test (BAT), based on the detection of allergen-induced CD63 expression, has been proved effective in the diagnosis of various IgE-mediated allergies. However, there is not yet consensus about the suitability of CD203c expression as a specific basophil activation marker and its diagnostic reliability. The goal of the present study was to compare measurement of CD63 and CD203c expression using BAT in a model of cat allergy and to determine optimal experimental conditions for both markers. Heparinized whole blood samples from 20 cat allergic patients and 19 controls were incubated with Fel d1 (relevant allergen) or anti-FcepsilonRI (positive control) either in IL-3 or IL-3-free conditions. An optimal gating of basophils was achieved in triple staining protocols: anti-IgE PE/anti-CD45 PerCP/anti-CD63 FITC or anti-IgE FITC/anti-CD45 PerCP/anti-CD203c PE. We demonstrated that IL-3 significantly enhanced CD63-induced expression by basophils obtained from cat allergic patients in response to Fel d1. Sensitivity was found to be 100%. The CD203c protocol, when performed under IL-3-free conditions, also demonstrated 100% sensitivity. Only one of the control subjects was positive in both tests. In conclusion, using well-defined experimental conditions, the measurement of CD203c up-regulation on basophils in response to specific allergens is as reliable as CD63-BAT for the in vitro diagnosis of patients with IgE-mediated allergy.
Asunto(s)
Antígenos CD/sangre , Basófilos/inmunología , Gatos/inmunología , Citometría de Flujo/métodos , Hipersensibilidad/diagnóstico , Hidrolasas Diéster Fosfóricas/sangre , Pirofosfatasas/sangre , Regulación hacia Arriba , Animales , Antígenos CD/inmunología , Prueba de Desgranulación de los Basófilos/métodos , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Betula/inmunología , Biomarcadores/análisis , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Humanos , Hipersensibilidad/metabolismo , Interleucina-3/farmacología , Leucotrienos/sangre , Leucotrienos/metabolismo , Hidrolasas Diéster Fosfóricas/inmunología , Glicoproteínas de Membrana Plaquetaria/inmunología , Pirofosfatasas/inmunología , Tetraspanina 30RESUMEN
Although routinely used in clinical practice, skin prick tests and serum specific IgE often fail to distinguish between IgE-sensitization and symptomatic IgE-mediated allergy. There is therefore a need for new laboratory tests relating allergic symptoms to the offending agent. In this way, we evaluated the diagnostic reliability of a new whole blood quantitative real-time PCR assay for IL-4 and IL-13 mRNAs. We compared the response of cat allergic patients and non-cat allergic controls upon anti-IgE and cat allergen (Fel d1) stimulation of whole blood. Allergen addition led to a significant increase of IL-4 and IL-13 mRNAs in allergic patients compared to non-allergic controls (p<0.0001). Both cytokine mRNA levels were strongly correlated and peaked within 2 h after Fel d1 or anti-IgE addition. This rapid increase as well as purification experiments led us to the conclusion that basophils represent an important if not the main source of both transcripts in this setting. The effect was allergen-specific since not observed when stimulating blood from cat allergic patients with birch pollen. Interestingly, we found that IFN-gamma mRNA, contrary to IL-4 mRNA, reached higher levels in response to Fel d1 with control individuals than with patients allergic to cat. This study shows that whole blood real-time PCR is a valuable method for IL-4 and IL-13 mRNA measurement after in vitro allergen challenge. We suggest that it might be useful, complementing conventional markers, for the diagnosis and the follow-up of allergic diseases. Further assessments are required to evaluate the clinical potential of this technique.
Asunto(s)
Hipersensibilidad/sangre , Inmunoglobulina E/sangre , Interleucina-13/sangre , Interleucina-4/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adulto , Alérgenos/inmunología , Animales , Basófilos/metabolismo , Gatos , Humanos , Hipersensibilidad/metabolismo , Inmunoglobulina E/química , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Cinética , Leucocitos Mononucleares/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas Cutáneas , Factores de TiempoRESUMEN
BACKGROUND AND OBJECTIVES: The lymphocytic variant of hypereosinophilic syndrome (LV-HES) is an underrated disease defined by the monoclonal proliferation of interleukin-5 secreting T-cells. This disease is distinguished by a period of chronic lymphoproliferation without clinical transformation, which is frequently a precursor to T-cell lymphoma. In this study, LV-HES was used as a model of pre-malignancy to identify specific marker(s) predictive of the potential for malignant transformation. DESIGN AND METHODS: The karyotypic abnormalities detected in the abnormal CD3-CD4+ T cells were further characterized by fluorescent in situ hybridization. A multi-step retrospective analysis was performed on successive blood samples during a six-year follow up to correlate the evolution of cytogenetic changes with clinical progression. Expression array analysis was used to investigate the effect of these chromosomal aberrations on gene expression. RESULTS: A 6q deletion was detected in the two LV-HES patients during their chronic disease phase. An additional 10p deletion was found alone or in association with the 6q defect in one patient prior to the development of a CD3-CD4+ T-cell lymphoma six years after diagnosis. We show that the 6q but not the 10p deletion is both stable and persistent throughout the chronic disease, finally emerging as the predominant aberration in the lymphoma cells. Six genes mapped to the 6q-deleted region displayed altered gene expression profiles both in the chronic and malignant disease phases. INTERPRETATION AND CONCLUSIONS: Our data suggest that the 6q deletion represents an early cytogenetic marker for T-cell transformation.
Asunto(s)
Complejo CD3/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Aberraciones Cromosómicas , Cromosomas Humanos Par 6 , Síndrome Hipereosinofílico/genética , Adulto , Secuencia de Bases , Cromosomas Artificiales Bacterianos , Cromosomas Humanos Par 10 , Femenino , Eliminación de Gen , Humanos , Hibridación Fluorescente in Situ , Linfocitos/patología , Datos de Secuencia MolecularRESUMEN
BACKGROUND: Type 1 diabetes mellitus is a T-cell-mediated autoimmune disease that leads to a major loss of insulin-secreting beta cells. The further decline of beta-cell function after clinical onset might be prevented by treatment with CD3 monoclonal antibodies, as suggested by the results of a phase 1 study. To provide proof of this therapeutic principle at the metabolic level, we initiated a phase 2 placebo-controlled trial with a humanized antibody, an aglycosylated human IgG1 antibody directed against CD3 (ChAglyCD3). METHODS: In a multicenter study, 80 patients with new-onset type 1 diabetes were randomly assigned to receive placebo or ChAglyCD3 for six consecutive days. Patients were followed for 18 months, during which their daily insulin needs and residual beta-cell function were assessed according to glucose-clamp-induced C-peptide release before and after the administration of glucagon. RESULTS: At 6, 12, and 18 months, residual beta-cell function was better maintained with ChAglyCD3 than with placebo. The insulin dose increased in the placebo group but not in the ChAglyCD3 group. This effect of ChAglyCD3 was most pronounced among patients with initial residual beta-cell function at or above the 50th percentile of the 80 patients. In this subgroup, the mean insulin dose at 18 months was 0.22 IU per kilogram of body weight per day with ChAglyCD3, as compared with 0.61 IU per kilogram with placebo (P<0.001). In this subgroup, 12 of 16 patients who received ChAglyCD3 (75 percent) received minimal doses of insulin (< or =0.25 IU per kilogram per day) as compared with none of the 21 patients who received placebo. Administration of ChAglyCD3 was associated with a moderate "flu-like" syndrome and transient symptoms of Epstein-Barr viral mononucleosis. CONCLUSIONS: Short-term treatment with CD3 antibody preserves residual beta-cell function for at least 18 months in patients with recent-onset type 1 diabetes.
Asunto(s)
Complejo CD3/inmunología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Inmunoglobulina G/uso terapéutico , Insulina/uso terapéutico , Islotes Pancreáticos/efectos de los fármacos , Adolescente , Adulto , Autoanticuerpos/sangre , Glucemia/análisis , Péptido C/biosíntesis , Niño , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Femenino , Técnica de Clampeo de la Glucosa , Herpesviridae/aislamiento & purificación , Humanos , Inmunoglobulina G/efectos adversos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/fisiopatología , MasculinoRESUMEN
One of the mechanisms proposed to explain immunomodulatory actions of ultraviolet light (UV) is production of endogenous anti-inflammatory cytokines. The purpose of the present study is to evaluate how UV light affects the production of IL-10 and IL-1Ra and to provide insight as to the role of phagocytosis of apoptotic lymphocytes in this process. Cytokine production was evaluated in a coculture system consisting in UV-treated lymphocytes in the presence of autologous PBMC. The impact of phagocytosis was tested by two blocking agents cytochalasin E and anti-CD36 mAb. The apoptotic process affecting irradiated lymphocytes was progressive, culminating at 48 h. To achieve significant cytokine production, irradiated lymphocytes were incubated overnight at 37 degrees C. Coculture of apoptotic lymphocytes with autologous PBMC resulted in a significant increase of IL-1Ra mRNA (+340%; P = 0.001) and protein (+72%; P = 0.001) production. This synthesis was blocked by cytochalasin E but upregulated by CD36 receptor cross-linking. Our study shows that UV light induces lymphocyte apoptosis followed by its phagocytosis by monocyte/macrophages, a step that preferentially activates IL-1Ra.