RESUMO
Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.
Assuntos
Metilação de DNA , Epigênese Genética , Queratinócitos , Mosaicismo , Poroceratose , Regiões Promotoras Genéticas , Poroceratose/genética , Poroceratose/patologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Regiões Promotoras Genéticas/genética , Masculino , Alelos , FemininoRESUMO
Autoimmune diseases often arise from conditions where the immune system is compromised. While lymphopenia-induced proliferation (LIP) is crucial for immune system development and maturation, it is also caused by environmental insults, such as infection, and becomes a risk factor for autoimmunity in adults. We used Dsg3H1 TCR transgenic mice, whose T cells are designed to recognize desmogrein-3, a skin antigen, to explore the impact of lymphopenia on post-thymic tolerance. Dsg3H1 mice are known to delete the most highly autoreactive T cells in the thymus, and develop only subtle immune-mediated pathology in the steady state. However, we found that transient lymphopenia induced by total body irradiation (TBI) or cyclophosphamide (CY) results in massive dermatitis in Dsg3H1 mice. The symptoms included expansion and development of self-reactive T cells, their differentiation into CD44high IL-17-producing helper T cells, and severe neutrophilic inflammation. Repopulation of FOXP3+ T regulatory cells after lymphopenia normally occurred, suggesting escape of skin-reactive conventional T cells from control by regulatory T cells. Furthermore, we found that a depletion of the intestinal microbiota by antibiotics prevents CY-induced dermatitis, indicating roles of the commensal intestinal microbiota in LIP and Th17 development in vivo. The current data suggested that post-thymic tolerance of Dsg3H1 mice is established on a fragile balance in the lymphoreplete immune environment and broken by the interplay between lymphopenia and intestinal microbiota. The dynamic phenotypes observed in Dsg3H1 mice prompt a re-evaluation of opportunistic lymphopenia together with the microbiota as pivotal environmental factors, impacting individuals with genetic predispositions for autoimmune diseases.
Assuntos
Microbioma Gastrointestinal , Tolerância Imunológica , Linfopenia , Camundongos Transgênicos , Animais , Camundongos , Microbioma Gastrointestinal/imunologia , Linfopenia/imunologia , Tolerância Imunológica/imunologia , Pele/imunologia , Pele/microbiologia , Pele/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/imunologia , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: No consensus has been reached regarding the optimal chemotherapy for metastatic extramammary Paget's disease (EMPD), a rare cutaneous adenocarcinoma, because of the lack of solid evidence from prospective trials. However, the immunohistochemical profile of EMPD reportedly resembles that of breast cancer, particularly in terms of human epidermal growth factor receptor 2 (HER2) expression, suggesting that HER2 is a promising therapeutic target for advanced HER2-positive EMPD. METHODS: In this phase II single-arm trial, 13 Japanese patients received intravenous trastuzumab (loading dose of 8 mg/kg and maintenance dose of 6 mg/kg) and docetaxel (75 mg/m2) every 3 weeks for up to 2 years. The docetaxel dose was reduced or discontinued according to its toxicity. The primary trial endpoints were objective response rate (ORR) after 3 cycles of treatment and safety throughout the study period. RESULTS: All 13 patients completed 3 cycles of combination therapy. The median follow-up was 27.9 months. The ORR was 76.9% (n =â 10/13; 90% CI, 50.5-93.4). Frequently observed adverse events were neutropenia (100%), hypoalbuminemia (84.6%), and mucocutaneous infection (84.6%), all of which were well tolerated. CONCLUSION: The combination of docetaxel and trastuzumab demonstrated a favorable clinical effect and acceptable tolerability, which makes it a good treatment option for HER2-positive metastatic EMPD (ClinicalTrials.gov Identifier: UMIN000021311, jRCTs031180073).
RESUMO
Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1(hi) monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.
Assuntos
Quimiocinas/biossíntese , Folículo Piloso/imunologia , Células de Langerhans/fisiologia , Estresse Fisiológico , Alopecia , Animais , Movimento Celular , Quimiocina CCL20/biossíntese , Quimiocina CCL8/biossíntese , Quimiocinas/metabolismo , Folículo Piloso/metabolismo , Humanos , Queratinócitos/metabolismo , Células de Langerhans/imunologia , Camundongos , Camundongos Pelados , Receptores CCR2/metabolismo , Receptores CCR6/metabolismo , Receptores CCR8/metabolismo , Pele/imunologiaRESUMO
Staphylococcus aureus skin colonization is universal in atopic dermatitis and common in cancer patients treated with epidermal growth factor receptor inhibitors. However, the causal relationship of dysbiosis and eczema has yet to be clarified. Herein, we demonstrate that Adam17(fl/fl)Sox9-(Cre) mice, generated to model ADAM17-deficiency in human, developed eczematous dermatitis with naturally occurring dysbiosis, similar to that observed in atopic dermatitis. Corynebacterium mastitidis, S. aureus, and Corynebacterium bovis sequentially emerged during the onset of eczematous dermatitis, and antibiotics specific for these bacterial species almost completely reversed dysbiosis and eliminated skin inflammation. Whereas S. aureus prominently drove eczema formation, C. bovis induced robust T helper 2 cell responses. Langerhans cells were required for eliciting immune responses against S. aureus inoculation. These results characterize differential contributions of dysbiotic flora during eczema formation, and highlight the microbiota-host immunity axis as a possible target for future therapeutics in eczematous dermatitis.
Assuntos
Dermatite Atópica/imunologia , Disbiose/imunologia , Eczema/imunologia , Células de Langerhans/imunologia , Pele/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteínas ADAM/imunologia , Proteína ADAM17 , Animais , Antibacterianos/farmacologia , Corynebacterium/imunologia , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/genética , Dermatite Atópica/microbiologia , Disbiose/tratamento farmacológico , Disbiose/genética , Disbiose/microbiologia , Eczema/tratamento farmacológico , Eczema/genética , Eczema/microbiologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Integrases/genética , Integrases/imunologia , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/microbiologia , Células de Langerhans/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/imunologia , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/microbiologia , Pele/patologia , Staphylococcus aureus/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/microbiologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.
Assuntos
Desmogleína 3/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Pênfigo/genética , Receptores de IgG/genética , Adulto , Idoso , Animais , Autoimunidade/imunologia , Linfócitos B/imunologia , Desmogleína 3/imunologia , Feminino , Técnicas de Introdução de Genes , Humanos , Imunoglobulina G/genética , Imunoglobulina M/genética , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Pênfigo/imunologia , Pênfigo/patologia , Receptores de IgG/metabolismoRESUMO
BACKGROUND: Cutaneous polyarteritis nodosa (cPN) is a necrotizing arteritis of medium-sized vessels limited to the skin. Because of its rarity and the diversity of its clinical manifestations, there is no consensus treatment. Moreover, there are no established indicators that predict disease severity or its outcome. OBJECTIVES: To investigate clinico-laboratory features that predict patients requiring systemic therapy, including corticosteroids, to control the disease activity. METHODS: Thirty-six cPN patients who had not received systemic corticosteroids at the initial visit were retrospectively analysed by correlating the treatment and its response with clinico-laboratory findings. RESULTS: The major medications administered were antiplatelet agents (63.9%), vasodilators (38.9%), and prednisolone (PSL) (36.1%). In all, 23 cases achieved remission without PSL; 5 were managed with compression therapy alone or even observation; 18 received antiplatelet monotherapy or combined with vasodilator/dapsone; 13 required PSL; 10 achieved remission with PSL monotherapy or PSL and single/multiple medications and 3 with PSL and multiple drugs failed to achieve remission and underwent limb amputation. There were more skin ulcers and an elevated peripheral white blood cell (WBC) count and erythrocyte sedimentation rate (ESR) before corticosteroid induction in patients requiring PSL. Three cases with treatment failure had a markedly elevated ESR (>50). CONCLUSIONS: More than half of cPN can achieve remission without corticosteroids; an elevated WBC and the presence of skin ulcers predict the need for PSL; a high ESR before corticosteroid induction predicts treatment resistance, even with PSL.
RESUMO
The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (â¼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.
Assuntos
Morte Celular/fisiologia , Células Epidérmicas/metabolismo , Queratinócitos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular , Epiderme/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , PeleRESUMO
Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desmogleína 3/metabolismo , Pênfigo/imunologia , Abatacepte/farmacologia , Transferência Adotiva , Animais , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Desmogleína 3/genética , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Masculino , Camundongos , Camundongos Knockout , Linfócitos T Reguladores , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: In clinical research on multifactorial diseases such as atopic dermatitis, data-driven medical research has become more widely used as means to clarify diverse pathological conditions and to realize precision medicine. However, modern clinical data, characterized as large-scale, multimodal, and multi-center, causes difficulties in data integration and management, which limits productivity in clinical data science. METHODS: We designed a generic data management flow to collect, cleanse, and integrate data to handle different types of data generated at multiple institutions by 10 types of clinical studies. We developed MeDIA (Medical Data Integration Assistant), a software to browse the data in an integrated manner and extract subsets for analysis. RESULTS: MeDIA integrates and visualizes data and information on research participants obtained from multiple studies. It then provides a sophisticated interface that supports data management and helps data scientists retrieve the data sets they need. Furthermore, the system promotes the use of unified terms such as identifiers or sampling dates to reduce the cost of pre-processing by data analysts. We also propose best practices in clinical data management flow, which we learned from the development and implementation of MeDIA. CONCLUSIONS: The MeDIA system solves the problem of multimodal clinical data integration, from complex text data such as medical records to big data such as omics data from a large number of patients. The system and the proposed best practices can be applied not only to allergic diseases but also to other diseases to promote data-driven medical research.
Assuntos
Pesquisa Biomédica , Dermatite Atópica , Humanos , Dermatite Atópica/diagnóstico , Dermatite Atópica/terapia , Gerenciamento de Dados , Medicina de PrecisãoRESUMO
Various autoimmune responses increase with age, but the underlying mechanism is not clear. In this study, we used CD4+ T cells expressing a transgenic T cell receptor specific for desmoglein 3 (Dsg3), which is the target antigen of the autoimmune bullous disease pemphigus vulgaris, to examine how peripheral immunological tolerance against pathogenic autoreactive CD4+ T cells changes with age. Dsg3-specific T cells were deleted within 14 days after adoptive transfer into young mice (8 weeks old), while they escaped deletion when transferred into older mice over 42 weeks old. Dsg3-specific T cells produced higher levels of the proinflammatory cytokine IFN-γ in aged mice than in young mice. In addition, the expression levels of both OX40 and Birc5, which are important for cell survival in T cell clonal proliferation, were higher in aged than in young mice. The dysfunction in suppressing proinflammatory cytokine secretion and Birc5 upregulation in Dsg3-specific autoreactive T cells may reflect an aspect of the preliminary steps in autoimmune disease development in the aged population. Understanding this mechanism may lead to better risk evaluation of autoimmune disease development and to onset prevention.
RESUMO
Pierre-Robin sequence (PRS) is a rare, congenital defect presenting with micrognathia, glossoptosis, and airway obstruction with variable inclusion of a cleft palate. Overlapping PRS with neurofibromatosis type 2 (NF2) is a syndrome caused by a chromosome 22q12 microdeletion including NF2. We describe a patient with severe early-onset NF2 overlapping with PRS that showed micrognathia, glossoptosis, and a mild form of cleft palate. We detected a de novo chromosome 22q12 microdeletion including MN1 and NF2 in the patient. Previous cases of overlapping PRS and NF2 caused by the chromosome 22q12 microdeletions showed severe NF2 phenotypes with variable severity of cleft palate and microdeletions of varying sizes. Genotype-phenotype correlations and comparison of the size and breakpoint of microdeletions suggest that some modifier genes distal to MN1 and NF2 might be linked to the cleft palate severity.
Assuntos
Fissura Palatina , Glossoptose , Micrognatismo , Neurofibromatose 2 , Síndrome de Pierre Robin , Humanos , Síndrome de Pierre Robin/genética , Neurofibromatose 2/genética , Fissura Palatina/genética , Micrognatismo/genética , Cromossomos , Transativadores/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.
Assuntos
Anticorpos Anti-Idiotípicos/isolamento & purificação , Desmogleína 3/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/isolamento & purificação , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Química Click , Cicloparafinas/química , Desmogleína 3/genética , Desmogleína 3/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Luciferases/química , Oligonucleotídeos , Proteínas/genética , Proteínas/imunologiaRESUMO
Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/fisiologia , Proteínas de Membrana/metabolismo , Baço/citologia , Proteína ADAM10/genética , Proteína ADAM10/imunologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/imunologia , Animais , Transplante de Medula Óssea , Membrana Celular/imunologia , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Fibroblastos , Células-Tronco Hematopoéticas/fisiologia , Imunidade Celular , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Baço/imunologia , Quimeras de TransplanteRESUMO
While bulk water and hydration water coexist in cells to support the expression of biological macromolecules, how the dynamics of water molecules, which have long been only a minor role in molecular biology research, relate to changes in cellular states such as cell death has hardly been explored so far due to the lack of evaluation techniques. In this study, we developed a high-precision measurement system that can discriminate bulk water content changes of ±0.02% (0.2 mg/cm3) with single-cell-level spatial resolution based on a near-field CMOS dielectric sensor operating at 65 GHz. We applied this system to evaluate the temporal changes in the bulk water content during the cell death process of keratinocytes, called corneoptosis, using isolated SG1 (first layer of stratum granulosum) cells in vitro. A significant irreversible increase in the bulk water content was observed approximately 1 h before membrane disruption during corneoptosis, which starts with cytoplasmic high Ca2+ signal. These findings suggest that the calcium flux may have a role in triggering the increase in the bulk water content in SG1 cells. Thus, our near-field CMOS dielectric sensor provides a valuable tool to dissect the involvement of water molecules in the various events that occur in the cell.
Assuntos
Queratinócitos , Água , Morte Celular , Epiderme/metabolismo , Queratinócitos/metabolismo , Água/metabolismoRESUMO
BACKGROUND: Natto (fermented soybeans)-induced hypersensitivity is characterized by delayed symptom onset that hampers diagnosis. We aimed to clarify the clinical utility of the basophil activation test (BAT) in the diagnosis of natto-induced hypersensitivity. METHODS: Five patients with a history of anaphylaxis and chronic urticaria suspected of natto-induced hypersensitivity and seven with chronic spontaneous urticaria clinically unrelated to natto were enrolled in the patient and control groups, respectively. The BAT was performed with two incubation times, 15 min and 1 h, in combination with various concentrations of natto-mucilage extract. RESULTS: In controls, CD203c levels in basophils remained low in the 15-min incubation but were significantly increased in the 1-h incubation. In the patient group, in the 15-min condition, basophil CD203c was significantly upregulated by natto mucilage but not by soybean vs controls (P = 0.001). Low concentrations of natto mucilage were sufficient to upregulate basophil CD203c in the anaphylaxis cases, but high concentrations were required to induce the same effect in the urticaria cases. Finally, the dose-dependent pattern of the BAT results differed significantly between the anaphylaxis and urticaria cases (P = 0.006). Thus, a strong background reaction was observed in the BAT with 1 h incubation; 15 min of incubation was sufficient to identify patients with natto-induced hypersensitivity and may distinguish the clinical phenotype of natto-induced hypersensitivity, i.e., anaphylaxis or urticaria. CONCLUSIONS: The BAT with a 15-min incubation period is useful in diagnosing natto-induced hypersensitivity.
Assuntos
Teste de Degranulação de Basófilos/métodos , Hipersensibilidade Alimentar/diagnóstico , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Diester Fosfórico Hidrolases/sangue , Pirofosfatases/sangue , Alimentos de Soja/efeitos adversos , Urticária/complicaçõesRESUMO
To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-ß, VEGF, Wnt/ß-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).
Assuntos
Ciclofosfamida/administração & dosagem , Interleucina-2/administração & dosagem , Linfócitos do Interstício Tumoral/transplante , Melanoma/terapia , Vidarabina/análogos & derivados , Administração Intravenosa , Técnicas de Cultura de Células , Ciclofosfamida/uso terapêutico , Estudos de Viabilidade , Redes Reguladoras de Genes , Humanos , Inibidores de Checkpoint Imunológico , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/citologia , Masculino , Melanoma/genética , Melanoma/imunologia , Pessoa de Meia-Idade , Projetos Piloto , Condicionamento Pré-Transplante , Resultado do Tratamento , Vidarabina/administração & dosagem , Vidarabina/uso terapêuticoRESUMO
FGFR3 encodes a transmembrane receptor tyrosine kinase that has six autophosphorylation sites of tyrosine. Among them, Y770 is a negative regulatory site for the downstream signaling of FGFR3. Constitutive active mutations in FGFR3 are involved in human developmental disorders including familial acanthosis nigricans, an autosomal dominant disorder characterized by general hyperpigmentation with mild acanthosis of the epidermis. Here, we report two unrelated cases of familial acanthosis nigricans with a heterozygous c.2302G>T (p.E768*) mutation in FGFR3 (NM_000142.5). FGFR3 mRNA purified from the skin lesion neither showed aberrant splicing nor nonsense-mediated mRNA decay, indicating that the FGFR3 mutant simply lacked the C-terminal 768-806 amino acids including Y770. While all of the known pathogenic mutations were missense mutations in FGFR3 showing autosomal dominant trait, the c.2302G>T mutation of FGFR3 is a unique autosomal dominant nonsense mutation that causes familial acanthosis nigricans probably via loss of negative regulatory autophosphorylation site of FGFR3.
Assuntos
Acantose Nigricans/genética , Mutação de Sentido Incorreto , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Pré-Escolar , Transtornos Cromossômicos , Feminino , Predisposição Genética para Doença , Testes Genéticos , Heterozigoto , Humanos , LactenteRESUMO
Autoimmune bullous skin diseases, such as pemphigus and pemphigoid, may enable clarification of the mechanisms of immune regulation in the skin. Pemphigus and pemphigoid are mediated by essentially IgG autoantibodies against structural proteins of the desmosomes at cell-cell junctions and hemidesmosomes at epidermal-dermal junctions, respectively, and are characterized by blisters and erosions in the skin and/or mucous membranes. Intensive investigation over the last 3 decades has identified their target antigens and developed serological diagnostic tools as well as mouse models to help us understand their pathophysiology. Based on these advances, several new therapeutic approaches have become available, and more effective and less toxic targeted approaches are under development.