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1.
J Immunol ; 213(1): 86-95, 2024 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-38787200

RESUMO

The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3, also called cryopyrin) inflammasome is an intracellular innate immune complex, which consists of the pattern-recognition receptor NLRP3, the adaptor apoptosis-assciated speck-like protein containing a caspase recruitment domain, and procaspase-1. Aberrant activation of the NLRP3 inflammasome causes an autoinflammatory disease called cryopyrin-associated periodic syndrome (CAPS). CAPS is caused by gain-of-function mutations in the NLRP3-encoding gene CIAS1; however, the mechanism of CAPS pathogenesis has not been fully understood. Thus, unknown regulators of the NLRP3 inflammasome, which are associated with CAPS development, are being investigated. To identify novel components of the NLRP3 inflammasome, we performed a high-throughput screen using a human protein array, with NLRP3 as the bait. We identified a NLRP3-binding protein, which we called the cryopyrin-associated nano enhancer (CANE). We demonstrated that CANE increased IL-1ß secretion after NLRP3 inflammasome reconstitution in human embryonic kidney 293T cells and formed a "speck" in the cytosol, a hallmark of NLRP3 inflammasome activity. Reduced expression of endogenous CANE decreased IL-1ß secretion upon stimulation with the NLRP3 agonist nigericin. To investigate the role of CANE in vivo, we developed CANE-transgenic mice. The PBMCs and bone marrow-derived macrophages of CANE-transgenic mice exhibited increased IL-1ß secretion. Moreover, increased autoinflammatory neutrophil infiltration was observed in the s.c. tissue of CANE-transgenic versus wild-type mice; these phenotypes were consistent with those of CAPS model mice. These findings suggest that CANE, a component of the NLRP3 inflammasome, is a potential modulator of the inflammasome and a contributor to CAPS pathogenesis.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Animais , Inflamassomos/metabolismo , Inflamassomos/imunologia , Camundongos , Humanos , Células HEK293 , Síndromes Periódicas Associadas à Criopirina/imunologia , Síndromes Periódicas Associadas à Criopirina/genética , Camundongos Endogâmicos C57BL , Interleucina-1beta/metabolismo , Camundongos Knockout
2.
JACC Basic Transl Sci ; 8(7): 862-880, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37547071

RESUMO

Histologic evaluations revealed excessive accumulations of macrophages and absence of fibroblastic interstitial cells in explanted bioprosthetic valves. Comprehensive gene and protein expression analysis and histology unveiled an accumulation of fibrinogen and plasminogen, an activator of infiltrated macrophages, from degenerated valve surfaces in the interstitial spaces. These pathologies were completely reproduced in a goat model replaced with an autologous pericardium-derived aortic valve. Further preclinical animal experiments using goats demonstrated that preventing infiltration of macrophages and circulating proteins by increasing collagen density and leaflet strength is an effective treatment option.

3.
Viruses ; 15(7)2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37515185

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) and the auxiliary receptor Neuropilin-1 (NRP1) to enter host cells. NRP1 has another isoform, NRP2, whose function in COVID-19 has seldom been reported. In addition, although patients with severe cases of COVID-19 often exhibit increased levels of proinflammatory cytokines, the relationship between these cytokines and SARS-CoV-2 proliferation remains unknown. The aim of this study is to clarify the roles of proinflammatory cytokines in Neuropilin expressions and in SARS-CoV-2 infection. To identify the expression patterns of NRP under inflamed and noninflamed conditions, next-generation sequencing (RNA-seq), immunohistochemistry, quantitative real-time PCR, and Western blotting were performed using primary cultured fibroblast-like synoviocytes, MH7A (immortalized cell line of human rheumatoid fibroblast-like synoviocytes), immortalized MRC5 (human embryonic lung fibroblast), and synovial tissues. To measure viral proliferative capacity, SARS-CoV-2 infection experiments were also performed. NRP2 was upregulated in inflamed tissues. Cytokine-stimulated human fibroblast cell lines, such as MH7A and immortalized MRC5, revealed that NRP2 expression increased with co-stimulation of tumor necrosis factor α (TNFα) and interleukin-1 beta (IL-1ß) and was suppressed with anti-TNFα antibody alone. TNFα and IL-1ß promoted SARS-CoV-2 proliferation and Spike protein binding. The viral proliferation coincided with the expression of NRP2, which was modulated through plasmid transfections. Our results revealed that proinflammatory cytokines, including TNFα, contribute to NRP2 upregulation and SARS-CoV-2 proliferation in host human cells.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Proliferação de Células , Citocinas , Interleucina-1beta , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
4.
PLoS One ; 18(2): e0281746, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36800329

RESUMO

The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/caspase-1/interleukin(IL)-1ß axis, also known as the inflammasome pathway, is indispensable for IL-1ß activation in response to various pathogens or own damages. Previously, we developed an NLRP3-inflammasome using a cell-free system and identified ASC targeting drugs; thus, examination of ASC-related histopathology in various diseases could help to provide indications for these drugs. Here, we generated mice deficient only in ASC-protein (ASC-deficient (AD) mice) using CRISPR/Cas9 technology, studied which tissues were most affected, and obtained histopathological images of lipopolysaccharide (LPS)-induced endotoxemia. C57BL/6 wild-type (WT) and (AD) mice were injected intraperitoneally with a lethal dose (50 µg/g) of LPS. Statistical analysis of the survival of C57BL/6 mice and AD mice was performed using the Kaplan-Meier method and the log-rank test. The histopathological findings of multiple tissues from these mice were compared. Acute inflammation (e.g., catarrhal inflammation), along with congestion was observed in the colon of WT mice but not in that of AD mice. Adhesion of neutrophils to capillaries, along with interstitial infiltration, were observed in multiple tissues from WT mice. In AD mice, neutrophil infiltration was less severe but remained evident in the stomach, small intestine, heart, liver, kidney, spleen, and brain. Notably, there was no difference between WT and AD mice with respect to alveolar neutrophil infiltration and interstitial edema. These findings suggest that even though ASC contributes to systemic inflammation, it is dependent on the tissue involved. Intestinal congestion and edema might be good candidates for anti-ASC-targeted therapy.


Assuntos
Endotoxemia , Inflamassomos , Animais , Camundongos , Inflamassomos/metabolismo , Lipopolissacarídeos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Endotoxemia/induzido quimicamente , Camundongos Endogâmicos C57BL , Caspase 1/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Edema
5.
Inflamm Regen ; 43(1): 2, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609460

RESUMO

BACKGROUND: Blood coagulation factor XIII (FXIII) promotes cross-linking between fibrin molecules at the final stage of the blood coagulation cascade. However, its expression in cells or tissues and function, particularly factor XIII subunit B (FXIII-B), remains controversial. Hemorrhagic FXIII deficiency following anti-interleukin-6 (IL-6) receptor antibody treatment has been reported in patients with rheumatoid arthritis (RA). Patients receiving this biologics have reduced FXIII activity when compared to the activity in those treated with other biologics. The relationship between pro-inflammatory cytokines and FXIII expression remains unknown. METHODS: To investigate the expression pattern of FXIII in synovial tissues, immunohistochemistry, RT-qPCR, and western blotting were performed. FXIII-A expressed monocyte-derived macrophages were treated with recombinant IL-6 and anti-IL-6 receptor antibody. RNA sequencing of FXIII-B-overexpressing cells was performed to clarify the function of FXIII-B. RESULTS: The immunohistochemical analysis of synovial tissues revealed that factor XIII subunit A (FXIII-A) was expressed in M2 macrophages, and FXIII-B was expressed in fibroblast-like synoviocytes. IL-6 stimulation upregulated FXIII-A expression in IL-4-induced monocyte-derived macrophages, and the anti-IL-6 receptor antibody suppressed FXIII-A expression. FXIII-B was more abundantly secreted in the supernatant of fibroblast-like synoviocytes compared with that of other cells. RNA sequencing showed that FXIII-B elevated the expression of genes associated with anti-apoptotic molecules and chemokines. CONCLUSIONS: Our findings highlight that synovial tissue is one of the sources of FXIII production. We also have demonstrated IL-6-dependent FXIII-A expression and the novel potential functions of FXIII-B.

6.
Exp Brain Res ; 240(11): 2999-3009, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36198842

RESUMO

When participants control periodic isometric force cycling between two target forces, they more accurately control force in a joint action than in an individual action. In some other studies, however, individuals tend to outperform dyads in joint action. The present study thus examined experimental conditions in which dyads outperformed individuals in a task of force produced by two people. This study consisted of two tasks with two target conditions and three force production conditions. The individual task was performed by one participant, and the joint task was performed by two participants. In absolute and relative target conditions, the participants made continuous, discrete, and periodic isometric pressing movements with the index finger. Although no difference was seen in force error between tasks in the continuous condition, the joint task had a smaller error than the individual task in the two other conditions. The joint task had a smaller variable force than the individual task in the periodic conditions, but no difference was seen in force variability between tasks in the two other conditions. Participants mainly controlled force in both tasks in the continuous condition. In the periodic or discrete condition at a prescribed interval, however, participants had to control both force and timing in the individual task, and muscle force must be mainly controlled to compensate for force errors by synchronizing interpersonal force outputs in the joint task. Thus, dyads can reduce the dimensionality of the control problem because they can synchronize their action which provides timing information.


Assuntos
Contração Isométrica , Desempenho Psicomotor , Humanos , Contração Isométrica/fisiologia , Desempenho Psicomotor/fisiologia , Movimento/fisiologia , Dedos , Análise de Variância
7.
Int J Immunopathol Pharmacol ; 36: 3946320221104554, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35615856

RESUMO

INTRODUCTION: Dialysis-related amyloidosis (DRA) caused by ß2-microgloblin (B2M) fibrils is a serious complication for patients with kidney failure on long-term dialysis. Deposition of B2M amyloid fibrils is thought to be due not only to serum extracellular B2M but also to infiltrating inflammatory cells, which may have an important role in B2M amyloid deposition in osteoarticular tissues in patients with DRA. Here, we asked whether B2M amyloid fibrils activate the inflammasome and contribute to formation and deposition of amyloid fibrils in cells. METHODS: Amyloid formation was confirmed by a thioflavin T (ThT) spectroscopic assay and scanning electron microscopy (SEM). Activation of inflammasomes was assessed by detecting interleukin (IL)-1ß in culture supernatants from human embryonic kidney (HEK) 293T cells ectopically expressing inflammasome components. IL-1ß secretion was measured by enzyme-linked immunosorbent assay. Expression and co-localization were analyzed by immunohistochemistry and dual immunofluorescence microscopy. RESULTS: B2M amyloid fibrils interacted directly with NLRP3/Pyrin and to activate the NLRP3/Pyrin inflammasomes, resulting in IL-1ß secretion. When HEK293T cells were transfected with inflammasome components NLRP3 or Pyrin, along with ASC, pro-caspase-1, pro-IL-1ß, and B2M, ThT fluorescence intensity increased. This was accompanied by IL-1ß secretion, which increased in line with the amount of transfected B2M. In this case, morphological glowing of amyloid fibrils was observed by SEM. In the absence of ASC, there was no increase in ThT fluorescence intensity or IL-1ß secretion, or any morphological glowing of amyloid fibrils. NLRP3 or Pyrin and B2M were co-localized in a "speck" in HEK293T cells, and co-expressed in infiltrated monocytes/macrophages in the osteoarticular synovial tissues in a patient with DRA. CONCLUSION: Taken together, these data suggest that inflammasome assembly is required for the subsequent triggering of intracellular formation of B2M amyloid fibrils, which may contribute to osteoarticular deposition of B2M amyloid fibrils and inflammation in patients with DRA.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Amiloide , Células HEK293 , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pirina
8.
Virchows Arch ; 480(4): 919-925, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35171325

RESUMO

Intimal sarcoma is one of the most common and well-known primary malignant neoplasms of the aorta and heart. The authors reviewed cases of intimal sarcoma from histological, immunohistochemical and genetic perspectives. Twenty cases of intimal sarcoma were retrieved. Immunohistochemistry and FISH of MDM2 and PDGFRA genes were performed. All 20 tumours were composed of spindle-shaped, stellate, oval or polygonal tumour cells with irregular hyperchromatic nuclei arranged in a haphazard pattern, accompanied by nuclear pleomorphism and frequent mitotic figures. Other histological findings were as follows: abnormal mitosis in 10 cases (50%), necrosis in 15 cases (75%), myxoid stroma in 12 cases (60%), cartilaginous formation in 1 case (5%), haemorrhage in 12 cases (60%) and fibrinous deposition in 14 cases (70%). The tumours were positive for MDM2 in 16 cases (80%), ERG in 4 cases (20%), alpha-smooth muscle actin in 6 cases (30%), desmin in 5 cases (25%) and AE1/AE3 in 4 cases (20%). Immunohistochemical positivity was focal in each case. Loss of H3K27me3 expression was noted in 2 cases (10%). MDM2 and PDGFRA gene amplifications were detected in 11 cases (55%) and 1 case (5%), respectively. Fisher's exact test revealed a significant correlation between MDM2 gene amplification and myxoid stroma (p = 0.0194). No parameters showed any association with the anatomical location of the tumours. It was suggested that myxoid histology of intimal sarcoma may be associated with MDM2 gene amplification and that intimal sarcoma may be divided into myxoid and non-myxoid types.


Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Neoplasias Vasculares , Perfil Genético , Humanos , Imuno-Histoquímica , Sarcoma/genética , Sarcoma/patologia , Neoplasias Vasculares/patologia
9.
Sci Rep ; 11(1): 22351, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34785680

RESUMO

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a key adaptor protein of inflammasomes and a proapoptotic molecule; however, its roles in signal transduction in pancreatic ductal adenocarcinoma (PDAC) cells remain unknown. Here, we clarified the role and mechanisms of action of ASC in PDAC using clinical evidence and in vitro data. ASC expression in PDAC tissues was analyzed using public tumor datasets and immunohistochemistry results of patients who underwent surgery, and PDAC prognosis was investigated using the Kaplan-Meier Plotter. ASC expression in PDAC cells was downregulated using small-interfering RNA, and gene expression was assessed by RNA sequencing. Review of the Oncomine database and immunostaining of surgically removed tissues revealed elevated ASC expression in PDAC tumors relative to non-tumor tissue, indicating poor prognosis. We observed high ASC expression in multiple PDAC cells, with ASC silencing subsequently inhibiting PDAC cell growth and altering the expression of cell cycle-related genes. Specifically, ASC silencing reduced cyclin D1 levels and stopped the cell cycle at the G1 phase but did not modulate the expression of any apoptosis-related molecules. These results show that ASC inhibited tumor progression via cell cycle modulation in PDAC cells and could be a potential therapeutic target.


Assuntos
Proteínas Adaptadoras de Sinalização CARD , Carcinoma Ductal Pancreático , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias , Neoplasias Pancreáticas , Proteínas Adaptadoras de Sinalização CARD/biossíntese , Proteínas Adaptadoras de Sinalização CARD/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Taxa de Sobrevida , Neoplasias Pancreáticas
10.
Inflamm Regen ; 41(1): 33, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635190

RESUMO

The long battle between humans and various physical, chemical, and biological insults that cause cell injury (e.g., products of tissue damage, metabolites, and/or infections) have led to the evolution of various adaptive responses. These responses are triggered by recognition of damage-associated molecular patterns (DAMPs) and/or pathogen-associated molecular patterns (PAMPs), usually by cells of the innate immune system. DAMPs and PAMPs are recognized by pattern recognition receptors (PRRs) expressed by innate immune cells; this recognition triggers inflammation. Autoinflammatory diseases are strongly associated with dysregulation of PRR interactomes, which include inflammasomes, NF-κB-activating signalosomes, type I interferon-inducing signalosomes, and immuno-proteasome; disruptions of regulation of these interactomes leads to inflammasomopathies, relopathies, interferonopathies, and proteasome-associated autoinflammatory syndromes, respectively. In this review, we discuss the currently accepted molecular mechanisms underlying several autoinflammatory diseases.

11.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638873

RESUMO

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.


Assuntos
Antígenos CD34/biossíntese , Insuficiência da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Técnicas de Cultura de Células , Regulação da Expressão Gênica , Células-Tronco/metabolismo , Valva Aórtica/patologia , Insuficiência da Valva Aórtica/patologia , Hipóxia Celular , Feminino , Humanos , Masculino , RNA Mensageiro/biossíntese , Células-Tronco/patologia
12.
Int J Immunopathol Pharmacol ; 35: 20587384211038357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34396831

RESUMO

INTRODUCTION: Nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), an intracellular pattern recognition receptor, recognizes various pathogen-associated molecular pattern and/or damage-associated molecular pattern molecules to constitute inflammasome that act as an interleukin (IL)-1ß processing platform. Injected insulin is reported to induce focal amyloidosis and the formation of subcutaneous lumps called insulin balls, but the formation of subcutaneous lumps and the underlying cytotoxic mechanism has not been elucidated. METHODS: Amyloid formation was evaluated by thioflavin T spectroscopic assay and scanning electron microscopy. Binding between insulin amyloid fibrils and NLRP3 was evaluated by immunoprecipitation followed by native polyacrylamide gel electrophoresis. Inflammasome activation was evaluated by immunofluorescence speck formation called "ASC speck" and Western blotting. IL-1ß secretion in culture supernatants of peripheral blood mononuclear cells was evaluated by enzyme-linked immunosorbent assay. Cytotoxicity was measured by lactate dehydrogenase release assay. RESULTS: Insulin amyloid fibrils interact directly with NLRP3, resulting in NLRP3 inflammasome activation and pyroptotic cell death. CONCLUSION: Insulin ball formation and cytotoxicity may be associated with NLRP3 inflammasome activation followed by pyroptotic cell death.


Assuntos
Amiloide/metabolismo , Inflamassomos/metabolismo , Insulina/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Benzotiazóis/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/metabolismo
13.
Cell Death Differ ; 28(11): 2991-3008, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33993194

RESUMO

Human death domain superfamily proteins (DDSPs) play important roles in many signaling pathways involved in cell death and inflammation. Disruption or constitutive activation of these DDSP interactions due to inherited gene mutations is closely related to immunodeficiency and/or autoinflammatory diseases; however, responsible gene mutations have not been found in phenotypical diagnosis of these diseases. In this study, we comprehensively investigated the interactions of death-fold domains to explore the signaling network mediated by human DDSPs. We obtained 116 domains of DDSPs and conducted a domain-domain interaction assay of 13,924 reactions in duplicate using amplified luminescent proximity homogeneous assay. The data were mostly consistent with previously reported interactions. We also found new possible interactions, including an interaction between the caspase recruitment domain (CARD) of CARD10 and the tandem CARD-CARD domain of NOD2, which was confirmed by reciprocal co-immunoprecipitation. This study enables prediction of the interaction network of human DDSPs, sheds light on pathogenic mechanisms, and will facilitate identification of drug targets for treatment of immunodeficiency and autoinflammatory diseases.


Assuntos
Superfamília de Domínios de Morte/genética , Imunidade/genética , Morte Celular , Humanos , Transdução de Sinais
14.
Commun Biol ; 4(1): 273, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654176

RESUMO

Cancer immunotherapy using T cells redirected with chimeric antigen receptor (CAR) has shown a lot of promise. We have established a single-chain antibody (scFv) generation system in which scFv library-expressing CAR-T cells can be screened appropriately based on their antitumor functions. A variable region library containing the variable and J regions of the human immunoglobulin light or heavy chain was fused with the variable region of a heavy or light chain encoded by an existing tumor-specific antibody to generate a new scFv library. Then, scFv library-expressing CAR-T cells were generated and stimulated with target cells to concentrate the antigen-specific population. Using this system, target-specific recognition of CAR-T cells appeared to be finely tuned by selecting a new variable region. Importantly, we have demonstrated that the newly optimized scFv-expressing CAR-T cells had better proliferation capacity and durable phenotypes, enabling superior reactivity against advanced tumors in vivo in comparison with the original CAR-T cells. Therefore, the optimization of an scFv is needed to maximize the in vivo antitumor functions of CAR-T cells. This system may allow us to adjust an immunological synapse formed by an scFv expressed by CAR-T cells and a target antigen, representing an ideal form of CAR-T-cell immunotherapy.


Assuntos
Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/metabolismo , Imunoterapia Adotiva , Linfoma/terapia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/metabolismo , Linfócitos T/transplante , Animais , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Sinapses Imunológicas , Células Jurkat , Células K562 , Linfoma/genética , Linfoma/imunologia , Linfoma/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos Quiméricos/genética , Anticorpos de Cadeia Única/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Front Cardiovasc Med ; 8: 761591, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35187100

RESUMO

OBJECTIVES: The molecular mechanisms underlying post-operative pericardial adhesions remain poorly understood. We aimed to unveil the temporal molecular and cellular mechanisms underlying tissue dynamics during adhesion formation, including inflammation, angiogenesis, and fibrosis. METHODS AND RESULTS: We visualized cell-based tissue dynamics during pericardial adhesion using histological evaluations. To determine the molecular mechanism, RNA-seq was performed. Chemical inhibitors were administered to confirm the molecular mechanism underlying adhesion formation. A high degree of adhesion formation was observed during the stages in which collagen production was promoted. Histological analyses showed that arterioles excessively sprouted from pericardial tissues after the accumulation of neutrophils on the heart surface in mice as well as humans. The combination of RNA-seq and histological analyses revealed that hyperproliferative endothelial and smooth muscle cells with dedifferentiation appeared in cytokine-exposed sprouting vessels and adhesion tissue but not in quiescent vessels in the heart. SMAD2/3 and ERK activation was observed in sprouting vessels. The simultaneous abrogation of PI3K/ERK or TGF-ß/MMP9 signaling significantly decreased angiogenic sprouting, followed by inhibition of adhesion formation. Depleting MMP9-positive neutrophils shortened mice survival and decreased angiogenic sprouting and fibrosis in the adhesion. Our data suggest that TGF-ß/matrix metalloproteinase-dependent tissue remodeling and PI3K/ERK signaling activation might contribute to unique angiogenesis with dedifferentiation of vascular smooth muscle cells from the contractile to the synthetic phenotype for fibrosis in the pericardial cavity. CONCLUSIONS: Our findings provide new insights in developing prevention strategies for pericardial adhesions by targeting the recruitment of vascular cells from heart tissues.

16.
Clin Exp Rheumatol ; 38 Suppl 127(5): 49-52, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33331265

RESUMO

OBJECTIVES: The modification and pathogenesis of MEFV exon 2 or 3 variants in familial Mediterranean fever (FMF) remains unclear. We compared the clinical and laboratory characteristics between the coexistence and noncoexistence of MEFV exon 2 or 3 variants in patients with FMF that had a heterozygous MEFV exon 10 mutation. METHODS: We excluded patients with FMF that had two MEFV exon 10 mutations in one or more alleles and/or MEFV mutations in exons other than in exons 2, 3, or 10. Finally, we reviewed 131 Japanese patients with FMF that had a heterozygous MEFV exon 10 mutation, and they were divided into the groups with and without MEFV exon 2 or 3 variants of 97 and 34, respectively. RESULTS: All patients with MEFV exon 2 variants had either E148Q and/or L110P variants, none of patients had exon 3 variants. In the univariate analysis, the group with variants had significantly earlier onset, a higher percentage of thoracic pain with febrile attacks, a higher frequency of attack, and a higher IL-18 level at remission compared to the group without variants (all, p<0.05). Importantly, multivariate analyses showed that the coexistence of MEFV exon 2 variants was independently and significantly associated with earlier onset of FMF and thoracic pain (both, p<0.05). CONCLUSIONS: Our results suggested that coexistence of MEFV exon 2 variants have additional effects on manifestations of FMF with MEFV exon 10 mutations. Our findings highlighted the modifications and pathogenesis of such MEFV variants in FMF.


Assuntos
Febre Familiar do Mediterrâneo , Inflamassomos , Éxons , Febre Familiar do Mediterrâneo/diagnóstico , Febre Familiar do Mediterrâneo/genética , Humanos , Japão , Mutação , Pirina/genética
17.
Clin Exp Rheumatol ; 38 Suppl 127(5): 35-41, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33025889

RESUMO

OBJECTIVES: We aimed to identify the whole nucleotide sequence of the Mediterranean fever (MEFV) gene in familial Mediterranean fever (FMF) and reveal novel single nucleotide variants (SNVs) associated with the susceptibility of FMF. METHODS: SeqCap capturing technique followed by Illumina next-generation sequencing have been used to assess two hundred SNVs in the whole region of MEFV in 266 Japanese patients with FMF and 288 ethnically matched controls. We performed an association analysis using these SNVs to identify genetic variants that predispose to FMF. RESULTS: We identified the two most significant SNVs [rs28940578; M694I in exon 10, odds ratio (OR) = 153, p=2.47×10-21 and rs3743930; E148Q in exon 2, OR = 1.65, p<0.0005]. Stratified analysis identified rs28940578 as a risk allele in typical FMF. Haplotype AG, defined by rs401298 and rs28940578, was the most significant and prevalent among patients with typical FMF compared with controls (22.4% vs. 0%, respectively; OR = 137, p=1.44×10-31). Haplotype GTC, defined by rs11466018, rs224231, and rs401877, was the most significant among patients with typical FMF without the rs28940578 mutation compared with controls (15.9% vs. 6%, respectively; OR = 12.4, p=0.004). CONCLUSIONS: rs28940578 is associated with the highest risk in typical FMF cases. This is consistent with results from previous studies in Japan. We found a novel MEFV gene haplotype that confers susceptibility of FMF among typical FMF without the rs28940578 mutation. There were no relevant SNVs identified in MEFV among the atypical FMF group.


Assuntos
Febre Familiar do Mediterrâneo , Febre Familiar do Mediterrâneo/diagnóstico , Febre Familiar do Mediterrâneo/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Japão , Mutação , Pirina/genética
18.
Sci Rep ; 10(1): 13562, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782316

RESUMO

NLRP3, an intracellular pattern recognition receptor, recognizes numerous pathogens and/or its own damage-associated molecules, and forms complexes with the adaptor protein ASC. These complexes constitute the NLRP3 inflammasome, a platform for processing interleukin (IL)-1ß and/or IL-18. Several NLRP3 mutations result in constitutive activation of the NLRP3 inflammasome, causing cryopyrin-associated periodic syndrome (CAPS). To the best of our knowledge, small compounds that specifically inhibit inflammasome activation through the pyrin domain (PYD) have not yet been developed. This study describes an attempt to develop small compounds targeting the NLRP3 inflammasome. A core chemical library of 9,600 chemicals was screened against reconstituted NLRP3 inflammasome in a cell-free system with an amplified luminescence proximity homogeneous assay and a cell-based assay by human peripheral blood mononuclear cells (PBMCs). Inflammasome activation was evaluated by ASC-speck formation in human PBMCs, accompanied by IL-1ß secretion and processing, and by using IL-1ß-based dual operating luciferase (IDOL) mice. The activity of these compounds was evaluated clinically using PBMCs from a patient with Muckle-Wells syndrome (MWS), a type of CAPS, with an R260W mutation in NLRP3. Screening identified KN3014, a piperidine-containing compound targeting the interaction between NLRP3 and ASC through the PYD. KN3014 reduced ASC-speck formation in human PBMCs, luminescence from IDOL mice, and auto-secretion of IL-1ß by PBMCs from the patient with MWS. These findings suggest that KN3014 may be an attractive candidate for treatment of MWS, as well as other NLRP3 inflammasomopathies.


Assuntos
Anti-Inflamatórios/farmacologia , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Leucócitos Mononucleares/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Piperidinas/farmacologia , Animais , Anti-Inflamatórios/química , Estudos de Casos e Controles , Síndromes Periódicas Associadas à Criopirina/metabolismo , Síndromes Periódicas Associadas à Criopirina/patologia , Ensaios de Triagem em Larga Escala , Humanos , Inflamassomos/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Piperidinas/química
20.
Ann Thorac Surg ; 110(1): 40-49, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31760051

RESUMO

BACKGROUND: The molecular mechanisms underlying aortic valve calcification are poorly understood. Here, we aimed to identify the master regulators of calcification by comparison of genes in valve interstitial cells (VICs) with calcified and noncalcified aortic valves. METHODS: Calcified aortic valves were surgically excised from patients with aortic valve stenosis who required aortic valve replacements. Noncalcified and calcified sections were obtained from aortic valve leaflets. Collagenase-digested tissues were seeded into dishes, and VICs adhering to the dishes were cultured for 3 weeks, followed by comprehensive gene expression analysis. Functional analyses of identified proteins were performed by in vitro calcification assays. Tissue localization was determined by immunohistochemical staining for normal (n = 11) and stenotic valves (n = 30). RESULTS: We found 87 genes showing greater than a twofold change in calcified tissues. Among these genes, 68 were downregulated and 19 were upregulated. Cyclooxygenase-1 (COX1) messenger RNA and protein levels were upregulated in VICs from calcified tissues. The COX1 messenger RNA and protein levels in VICs were also strongly increased by stimulation with osteoblast differentiation medium. These were VIC-specific phenotypes and were not observed in other cell types. Immunohistochemical staining revealed that COX1-positive VICs were specifically localized in the calcified area of aortic valve tissues. CONCLUSIONS: The VIC-specific COX1 overexpression played a crucial role in calcification by promoting osteoblast differentiation in aortic valve tissues.


Assuntos
Estenose da Valva Aórtica/enzimologia , Valva Aórtica/enzimologia , Valva Aórtica/patologia , Calcinose/enzimologia , Ciclo-Oxigenase 1/fisiologia , Fibroblastos/enzimologia , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/citologia , Valva Aórtica/metabolismo , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/cirurgia , Calcinose/cirurgia , Cálcio/metabolismo , Células Cultivadas , Meios de Cultura/farmacologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , Feminino , Perfilação da Expressão Gênica , Implante de Prótese de Valva Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/patologia , Osteogênese , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Vimentina/análise
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