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1.
J Allergy Clin Immunol Glob ; 3(4): 100296, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39148513

RESUMO

Background: Symptoms in patients with systemic mastocytosis (SM) are associated with an increase in mast cell burden and release of mast cell-derived mediators. The most frequent presentation of SM is indolent SM (ISM), with moderate symptoms and prognosis. Basophil numbers in these patients are generally normal. However, when examining basophil activation in patients with ISM, we noted an abnormal response to N-formylmethione-leucyl-phenylalanine (fMLP). Objective: Our aim was to compare basophil responsiveness to fMLP and anti-IgE in healthy volunteers and patients with ISM and relate the findings to fMLP receptor (FPR) expression. Methods: Basophils isolated from peripheral blood of 15 patients with ISM and 14 healthy volunteers were stimulated with fMLP or anti-IgE. CD63 expression to assess basophil activation and expression of FPRs were assessed by flow cytometry. Results: Baseline expression of CD63 on basophils was similar between the healthy volunteers and patients with ISM. fMLP induced higher expression of CD63 on basophils from patients with ISM, whereas responses to anti-IgE were similar between groups. Basophils from patients with ISM also had higher fMLP1 receptor (FPR1) expression, wheresas FPR2 and FPR3 were not detected. fMLP blocked the binding of anti-FPR1 antibody to FPR1, consistent with the conclusion that fMLP signals through FPR1. Conclusions: Level of fMLP-induced basophil activation is higher in patients with ISM, which is associated with an increase in FPR1 expression. Further investigation is needed to determine why FPR1 expression is elevated, whether such expression might serve as an additional surrogate marker in the diagnosis of ISM, and whether enhanced responses of basophils to fMPL might have some relationship to unexplained episodes of mediator release.

2.
Blood Adv ; 7(9): 1796-1810, 2023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-36170795

RESUMO

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT.


Assuntos
Mastocitose , Transtornos Mieloproliferativos , Humanos , Triptases/genética , Mastócitos , Valores de Referência , Procedimentos Desnecessários , Mastocitose/diagnóstico , Transtornos Mieloproliferativos/patologia
3.
Front Oncol ; 12: 871390, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35574362

RESUMO

The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/neu-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/neu-positive cancer cells in vitro and in vivo was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/neu tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/neu-positive tumor masses in vitro. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/neu IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFß signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE's.

4.
Front Immunol ; 13: 841045, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35251038

RESUMO

Mast cell hyperactivity and accumulation in tissues are associated with allergy and other mast cell-related disorders. However, the molecular pathways regulating mast cell survival in homeostasis and disease are not completely understood. As glioma-associated oncogene (GLI) proteins are involved in both tissue homeostasis and in the hematopoietic system by regulating cell fate decisions, we sought to investigate the role for GLI proteins in the control of proliferation and survival of human mast cells. GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in the tyrosine kinase receptor KIT (HMC-1.1 and HMC-1.2, and LAD2 cells, respectively), while GLI2 transcripts were only present in HMC-1.1 and HMC-1.2 cells, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both cultured human and murine mast cells, and reduced the number of peritoneal mast cells in mice. Although GLI proteins are typically activated via the hedgehog pathway, steady-state activation of GLI in mast cells occurred primarily via non-canonical pathways. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function including USP48, which promotes p53 degradation; and iASPP, which inhibits p53-induced transcription, thus leading to the induction of p53-regulated apoptotic genes. Furthermore, we found that GLI silencing inhibited the proliferation of neoplastic mast cell lines, an effect that was more pronounced in rapidly growing cells. Our findings support the conclusion that GLI1/2 transcription factors are critical regulators of mast cell survival and that their inhibition leads to a significant reduction in the number of mast cells in vitro and in vivo, even in cells with constitutively active KIT variants. This knowledge can potentially be applicable to reducing mast cell burden in mast cell-related diseases.


Assuntos
Mastócitos , Fatores de Transcrição , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Animais , Proliferação de Células , Humanos , Mastócitos/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53 , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
5.
J Allergy Clin Immunol ; 149(4): 1392-1401.e6, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34506850

RESUMO

BACKGROUND: CD25+ human mast cells (huMCs) have been reported in patients with monoclonal mast cell diseases and in rare association with inflammation. However, the regulation of CD25 expression on huMCs and the possible biologic consequences remain poorly understood. OBJECTIVE: We sought to identify conditions that would upregulate CD25 expression on huMCs and to explore possible functional implications. METHODS: huMCs were cultured from peripheral blood progenitor cells over 6 to 8 weeks. Expression of CD25 was determined by fluorescence-activated cell sorting and soluble CD25 by ELISA. Signal transducer and activator of transcription 5 (STAT5) phosphorylation induced by IL-2 in huMCs, regulatory T (Treg) cells, or in cocultured huMCs and Treg cells was examined by fluorescence-activated cell sorting. RESULTS: Addition of IL-3 to CD34+ progenitors at the initiation of huMC cultures in the presence of stem cell factor and IL-6 upregulated the expression of CD25 in developing huMCs and resulted in shedding of soluble CD25 into the media. Removal of IL-3 after the first week of culture did not affect subsequent expression of CD25. Furthermore, addition of IL-3 14 days after the initiation of the culture did not induce significant CD25 expression. Treatment with anti-IL-3 antibody or the Janus kinase inhibitor tofacitinib blocked IL-3-induced CD25 upregulation. Binding of IL-2 to CD25+ huMCs did not induce STAT5 phosphorylation. However, coincubation of Treg cells with CD25+ huMCs pretreated with IL-2 was sufficient to result in STAT5 phosphorylation in Treg cells. CONCLUSIONS: IL-3 promotes CD25 expression and shedding by huMCs. Although CD25+ huMCs do not respond to IL-2, they bind IL-2 and may act as a reservoir of IL-2 to then activate lymphocytes.


Assuntos
Interleucina-3 , Mastócitos , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Interleucina-3/metabolismo , Interleucina-3/farmacologia , Fator de Transcrição STAT5/metabolismo , Linfócitos T Reguladores , Regulação para Cima
6.
Int J Mol Sci ; 20(22)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698677

RESUMO

BACKGROUND: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. METHODS: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (ß-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. RESULTS: LADR cells were larger and more granulated as seen with Wright-Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. CONCLUSIONS: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.


Assuntos
Linhagem Celular/citologia , Mastócitos/citologia , Antígenos CD/metabolismo , Degranulação Celular , Proliferação de Células , Quimases/metabolismo , Regulação da Expressão Gênica , Infecções por HIV/patologia , Humanos , Mastócitos/fisiologia , Transdução de Sinais , Triptases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
8.
Oncotarget ; 9(73): 33866, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30333917

RESUMO

[This corrects the article DOI: 10.18632/oncotarget.457.].

9.
Front Immunol ; 9: 631, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29643855

RESUMO

Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.


Assuntos
Neoplasias Hematológicas/metabolismo , Mastócitos/fisiologia , Mastocitose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Apoptose , Carcinogênese , Proliferação de Células , Sobrevivência Celular , Reparo do DNA , Neoplasias Hematológicas/genética , Humanos , Hidrazinas/farmacologia , Mastocitose/genética , Camundongos , Camundongos Knockout , Mutação/genética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Leukoc Biol ; 103(4): 749-759, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29360169

RESUMO

BACKGROUND: Systemic Capillary Leak Syndrome (SCLS) is an extremely rare and life-threatening vascular disorder of unknown etiology. SCLS is characterized by abrupt and transient episodes of hypotensive shock and edema due to plasma leakage into peripheral tissues. The disorder has garnered attention recently because its initial presentation resembles more common vascular disorders including systemic anaphylaxis, sepsis, and acute infections with the Ebola/Marburg family of filoviruses. Although approximately 70-85% of patients with SCLS have a concurrent monoclonal gammopathy of unknown significance (MGUS), any contribution of the paraprotein to acute flares is unknown. PROCEDURE: To identify circulating factors that might trigger acute SCLS crises, we profiled transcriptomes of paired peripheral blood mononuclear cell fractions obtained from patients during acute attacks and convalescent intervals by microarray. RESULTS: This study uncovered 61 genes that were significantly up- or downregulated more than 2.5-fold in acute samples relative to respective baselines. One of the most upregulated genes was ADM, which encodes the vasoactive peptide adrenomedullin. A stable ADM protein surrogate (pro-ADM) was markedly elevated in SCLS acute sera compared to remission samples or sera from healthy controls. Monocytes and endothelial cells (ECs) from SCLS subjects expressed significantly more ADM in response to proinflammatory stimuli compared to healthy control cells. Application of ADM to ECs elicited protective effects on vascular barrier function, suggesting a feedback protective mechanism in SCLS. CONCLUSIONS: Since ADM has established hypotensive effects, differentiating between these dual actions of ADM is crucial for therapeutic applications aimed at more common diseases associated with increased ADM levels.


Assuntos
Adrenomedulina/metabolismo , Biomarcadores/metabolismo , Síndrome de Vazamento Capilar/patologia , Endotélio Vascular/patologia , Leucócitos Mononucleares/patologia , Monócitos/patologia , Doença Aguda , Idoso , Síndrome de Vazamento Capilar/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo
11.
J Immunol Methods ; 448: 105-111, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28629733

RESUMO

The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×106 vs. 2.4±0.28×106, respectively, from 1.0×108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.


Assuntos
Antígenos CD34/metabolismo , Separação Celular/métodos , Leucaférese , Mastócitos/metabolismo , Células-Tronco/metabolismo , Antígenos CD34/imunologia , Biomarcadores/metabolismo , Orçamentos , Degranulação Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Separação Celular/economia , Forma Celular , Células Cultivadas , Redução de Custos , Análise Custo-Benefício , Criopreservação , Meios de Cultura/metabolismo , Citometria de Fluxo , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Leucaférese/economia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismo , Fator de Células-Tronco/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Fatores de Tempo , Fluxo de Trabalho
13.
Proc Natl Acad Sci U S A ; 113(49): 14115-14120, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27872312

RESUMO

Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the ß-subunit of the high-affinity IgE receptor (FcεRIß) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIß expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIß is a potential approach for mast cell-specific treatment of allergic diseases.


Assuntos
Degranulação Celular/efeitos dos fármacos , Dermatite Alérgica de Contato/terapia , Mastócitos/efeitos dos fármacos , Oligonucleotídeos Antissenso/uso terapêutico , Splicing de RNA/efeitos dos fármacos , Receptores de IgE/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Anafilaxia Cutânea Passiva/genética , Receptores de IgE/genética
14.
J Allergy Clin Immunol ; 137(3): 907-18.e9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26431580

RESUMO

BACKGROUND: IL-5(+) pathogenic effector T(H)2 (peT(H)2) cells are a T(H)2 cell subpopulation with enhanced proinflammatory function that has largely been characterized in murine models of allergic inflammation. OBJECTIVE: We sought to identify phenotype markers for human peT(H)2 cells and characterize their function in patients with allergic eosinophilic inflammatory diseases. METHODS: Patients with eosinophilic gastrointestinal disease (EGID), patients with atopic dermatitis (AD), and nonatopic healthy control (NA) subjects were enrolled. peT(H)2 and conventional T(H)2 (cT(H)2) cell phenotype, function, and cytokine production were analyzed by using flow cytometry. Confirmatory gene expression was measured by using quantitative RT-PCR. Prostaglandin D2 levels were measured with ELISA. Gut T(H)2 cells were obtained by means of esophagogastroduodenoscopy. RESULTS: peT(H)2 cells were identified as chemoattractant receptor-homologous molecule expressed on T(H)2 cells-positive (CRTH2(+)), hematopoietic prostaglandin D synthase-positive CD161(hi) CD4 T cells. peT(H)2 cells expressed significantly greater IL-5 and IL-13 than did hematopoietic prostaglandin D synthase-negative and CD161(-) cT(H)2 cells. peT(H)2 cells were highly correlated with blood eosinophilia (r = 0.78-0.98) and were present in 30- to 40-fold greater numbers in subjects with EGID and those with AD versus NA subjects. Relative to cT(H)2 cells, peT(H)2 cells preferentially expressed receptors for thymic stromal lymphopoietin, IL-25, and IL-33 and demonstrated greater responsiveness to these innate pro-TH2 cytokines. peT(H)2 but not cT(H)2 cells produced prostaglandin D2. In patients with EGID and those with AD, peT(H)2 cells expressed gut- and skin-homing receptors, respectively. There were significantly greater numbers of peT(H)2 cells in gut tissue from patients with EGID versus NA subjects. CONCLUSION: peT(H)2 cells are the primary functional proinflammatory human T(H)2 cell subpopulation underlying allergic eosinophilic inflammation. The unambiguous phenotypic identification of human peT(H)2 cells provides a powerful tool to track these cells in future pathogenesis studies and clinical trials.


Assuntos
Eosinófilos/imunologia , Eosinófilos/metabolismo , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Imunidade Inata , Memória Imunológica , Imunofenotipagem , Interleucina-5/metabolismo , Camundongos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fenótipo , Receptores CCR/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/citologia
15.
Curr Protoc Immunol ; 110: 6.24.1-6.24.18, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26237012

RESUMO

Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.


Assuntos
Citocinas/metabolismo , Citometria de Fluxo , Antígenos/imunologia , Antígenos de Superfície/metabolismo , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodos , Espaço Intracelular/metabolismo , Ionomicina/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Sensibilidade e Especificidade , Coloração e Rotulagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/imunologia
16.
J Clin Cell Immunol ; 5: 1000213, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25405070

RESUMO

OBJECTIVES: The Systemic Capillary Leak Syndrome (SCLS) is a rare and potentially fatal disorder resembling systemic anaphylaxis that is characterized by transient episodes of hypotensive shock and peripheral edema. The pathogenesis of SCLS is unknown, and triggers for attacks are apparent only in a minority of patients. We introduce a clinical algorithm for the diagnosis of SCLS, and we investigated potential serum biomarkers of acute SCLS episodes. METHODS: We analyzed serum cytokines in a cohort of 35 patients with an established diagnosis of SCLS and characterized the effects of SCLS sera on endothelial cell function. We investigated the cellular source(s) of CXCL10, a chemokine that was significantly elevated in both basal and acute SCLS sera, by flow cytometry. RESULTS: Several cytokines were elevated in acute SCLS sera compared to baseline or sera from healthy controls, including CXCL10, CCL2, IL-1ß, IL-6, IL-8, IL-12 and TNFα. The majority of acute sera failed to activate endothelial cells as assessed by surface adhesion marker expression. Monocytes appear to be the major source of serum CXCL10, and the percentage of CXLC10+ monocytes in response to IFNγ stimulation was increased in SCLS subjects compared to controls. CONCLUSIONS: The presence of proinflammatory cytokines in acute SCLS sera suggests that inflammation or infection may have a role in triggering episodes. The enhanced capacity of monocytes from SCLS patients to produce CXCL10 suggests a new therapeutic avenue for SCLS.

17.
Clin Mol Allergy ; 11(1): 4, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24314292

RESUMO

BACKGROUND: Th2 cytokine responses are enhanced by all trans retinoic acid (ATRA), the bioavailable form of vitamin A. Retinoic acid receptor alpha (RARα) is the high affinity receptor for ATRA that mediates these pro-Th2 effects. We have previously characterized two major human Th2 subpopulations: IL-5- Th2 (IL-5-, IL-4+, IL-13+) and IL-5+ Th2 cells (IL-5+, IL-4+, IL-13+), which represent less and more highly differentiated Th2 cells, respectively. We hypothesized that the pro-Th2 effects of ATRA may differentially affect these Th2 subpopulations. METHODS: Specific cytokine producing Th2 subpopulations were identified using intracellular cytokine staining. Proliferation was measured using the Cell Trace Violet proliferation tracking dye. Apoptotic cells were identified using either annexin-V or active caspase 3 staining. Th2 gene expression was measured using quantitative polymerase chain reaction. RESULTS: ATRA increased the output of Th2 cells from house dust mite allergen (HDM) specific short-term cell lines, and this enhancement was limited to the IL-5+ Th2 subpopulation. Conversely, the RARα antagonist Ro415253 decreased Th2 cell output from these cultures, and this effect was again limited to the IL-5+ Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited Th2 cell proliferation, and this affect was more pronounced for the IL-5+ vs. IL-5- Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited the expression of IL5 in a significant manner, which was not found for IL4 or IL13. CONCLUSIONS: We report that the reciprocal regulation of Th2 cytokine expression and proliferation by RARα modulators are largely limited to modulation of IL-5 gene expression and to proliferation of the highly differentiated IL-5+ Th2 subpopulation. These results suggest that RARα antagonism is a potential means to therapeutically target allergic inflammation. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01212016.

19.
Oncotarget ; 3(3): 345-56, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22538444

RESUMO

The peroxisome proliferator-activated receptorγ (PPARγ) is a key regulator of metabolism, proliferation, inflammation and differentiation, and upregulates tumor suppressor genes, such as PTEN, BRCA1 and PPARγ itself. Examination of mammary carcinogenesis in transgenic mice expressing the dominant-negative Pax8PPARγ fusion protein revealed that tumors were estrogen receptorα (ER)-positive and sensitive to the ER antagonist, fulvestrant. Here we evaluated whether administration of an irreversible PPARγ inhibitor in vivo could similarly induce ER expression in otherwise ER-negative mammary tumors following induction of carcinogenesis, and sensitize them to the antitumor effects of fulvestrant. In addition, we wished to determine whether the effect of GW9662 was associated with a PPAR-selective gene expression profile. Mammary carcinogenesis was induced in wild-type FVB mice by treatment with medroxyprogesterone and dimethylbenz(a)anthracene (DMBA) that were subsequently maintained on a diet supplemented with 0.1% GW9662, and tumorigenesis and gene expression profiling of the resulting tumors were determined. Administration of GW9962 resulted in ER+ tumors that were highly sensitive to fulvestrant. Tumors from GW9662-treated animals exhibited reduced expression of a metabolic gene profile indicative of PPARγ inhibition, including PPARγ itself. Additionally, GW9662 upregulated the expression of several genes associated with the transcription, processing, splicing and translation of RNA. This study is the first to show that an irreversible PPARγ inhibitor can mimic a dominant-negative PPARγ transgene to elicit the development of ER-responsive tumors. These findings suggest that it may be possible to pharmacologically influence the responsiveness of tumors to anti-estrogen therapy.


Assuntos
Anilidas/administração & dosagem , Carcinoma/prevenção & controle , Estradiol/análogos & derivados , Neoplasias Mamárias Experimentais/prevenção & controle , PPAR gama/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno , Anilidas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Carcinógenos , Carcinoma/induzido quimicamente , Carcinoma/genética , Carcinoma/mortalidade , Quimioprevenção/métodos , Sinergismo Farmacológico , Estradiol/administração & dosagem , Estradiol/farmacologia , Antagonistas de Estrogênios/administração & dosagem , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/mortalidade , Camundongos , Análise em Microsséries , Terapia de Alvo Molecular
20.
Cancer Prev Res (Phila) ; 5(1): 51-60, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21955520

RESUMO

Stem cell antigen-1 (Sca-1, Ly6A) is a glycerophosphatidylinositol (GPI)-anchored protein that was identified as a murine marker of bone marrow stem cells. Although Sca-1 is widely used to enrich for stem and progenitor cells in various tissues, little is known about its function and associated signaling pathways in normal and malignant cells. Here, we report that the absence of Sca-1 in the mammary gland resulted in higher levels of PPARγ and PTEN, and a reduction of pSer84PPARγ, pERK1/2, and PPARδ. This phenotype correlated with markedly increased sensitivity of Sca-1 null mice to PPARγ agonist GW7845 and insensitivity to PPARδ agonist GW501516. Reduction of Sca-1 expression in mammary tumor cells by RNA interference resulted in a phenotype similar to the Sca-1 deficient mammary gland, as evidenced by increased PPARγ expression and transcriptional activity, resulting in part from a lesser susceptibility to proteasomal degradation. These data implicate Sca-1 as a negative regulator of the tumor suppressor effects of PPARγ.


Assuntos
Antígenos Ly/genética , Antineoplásicos/farmacologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , PPAR gama/metabolismo , Animais , Anticarcinógenos/farmacologia , Linhagem Celular Tumoral , Predisposição Genética para Doença , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxazóis/farmacologia , Fenótipo , Interferência de RNA , Transdução de Sinais , Tiazóis/farmacologia , Tirosina/análogos & derivados , Tirosina/farmacologia
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