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1.
Int J Mol Sci ; 20(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31489916

RESUMO

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis. Sodium azide induced oncosis but also classic apoptosis, which was blocked by zVAD (z-Vla-Ala-Asp(OMe)-fluoromethylketone). Oncotic cells were found to be viability+ve/caspase-3-ve/RIP3+ve/-ve (Receptor-interacting serine/threonine protein kinase 3). These two cell populations also displayed a DNA damage response (DDR) phenotype pH2AX+ve/PARP-ve, cleaved PARP induced caspase independent apoptosis H2AX-ve/PARP+ve and hyper-activation or parthanatos H2AX+ve/PARP+ve. Oncotic cells with phenotype cell viability+ve/RIP3-ve/caspase-3-ve showed increased DDR and parthanatos. Necrostatin-1 down-regulated DDR in oncotic cells and increased sodium azide induced apoptosis. This flow cytometric approach to cell death research highlights the link between ACD and the RCD processes of programmed apoptosis and necrosis.


Assuntos
Biomarcadores , Citometria de Fluxo , Imunofenotipagem , Neoplasias/metabolismo , Apoptose , Caspases/metabolismo , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/patologia
2.
Gut ; 65(4): 584-94, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25715355

RESUMO

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4ß7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.


Assuntos
Transferência Adotiva , Terapia Baseada em Transplante de Células e Tecidos/métodos , Doença de Crohn/terapia , Linfócitos T Reguladores/imunologia , Animais , Doença de Crohn/imunologia , Metilação de DNA , Ensaio de Imunoadsorção Enzimática , Fatores de Transcrição Forkhead/genética , Humanos , Técnicas In Vitro , Interleucina-17/metabolismo , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos SCID , Fenótipo , Reação em Cadeia da Polimerase , Transplante Heterólogo
3.
Gastroenterology ; 149(6): 1564-1574.e3, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26170138

RESUMO

BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents.


Assuntos
Fatores Biológicos/metabolismo , Imunoglobulina G/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Proteólise/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Adalimumab/metabolismo , Anticorpos Monoclonais Humanizados/metabolismo , Fatores Biológicos/farmacologia , Biópsia , Estudos de Casos e Controles , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colo/imunologia , Colo/metabolismo , Colo/patologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Epitopos/metabolismo , Etanercepte/metabolismo , Feminino , Humanos , Immunoblotting/métodos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Infliximab/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Masculino , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade
4.
Gastroenterology ; 149(2): 456-67.e15, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25917784

RESUMO

BACKGROUND & AIMS: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. METHODS: ILCs were isolated from colons of Tbx21(-/-) × Rag2(-/-) mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. RESULTS: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. CONCLUSIONS: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells.


Assuntos
Antígenos CD4/metabolismo , Citocinas/metabolismo , Imunidade Inata/efeitos dos fármacos , Doenças Inflamatórias Intestinais/imunologia , Interleucina-6/farmacologia , Linfócitos/efeitos dos fármacos , Animais , Complexo CD3/metabolismo , Técnicas de Cultura de Células , Colo/citologia , Colo/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-23/metabolismo , Interleucina-6/administração & dosagem , Interleucinas/metabolismo , Linfócitos/imunologia , Camundongos , Camundongos Knockout , Receptores Desencadeadores da Citotoxicidade Natural/metabolismo , Interleucina 22
5.
Gastroenterology ; 147(1): 172-83, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24704524

RESUMO

BACKGROUND & AIMS: T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients. METHODS: Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls. Isolated lamina propria mononuclear cells (LPMCs) and mucosal tissue explants were incubated with otelixizumab for 24 or 48 hours. Production of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. Levels of 36 cytokines and chemokines and phosphorylation of 39 receptor tyrosine kinases and signaling molecules were measured using protein arrays. Immunoblot analysis was used to analyze T-cell transcription factors. RESULTS: Incubation of intestinal tissues or LPMCs with otelixizumab reduced production of interferon gamma, interleukin (IL)-17A, and other inflammatory cytokines and chemokines, simultaneously increasing production of IL-10. Mucosal biopsy specimens from patients with IBD retained inflammation-associated tyrosine phosphoprotein profiles ex vivo. Incubation of the inflamed tissue with otelixizumab reduced phosphorylation of these proteins to levels observed in control tissues. Otelixizumab also markedly reduced phosphorylation of proteins associated with T-cell receptor activation. Neutralization of IL-10 blocked the anti-inflammatory effects of otelixizumab. CONCLUSIONS: We observed anti-inflammatory effects of anti-CD3 in inflamed intestinal tissues from patients with IBD. The antibody appears to down-regulate T-cell activation via IL-10.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Colo/metabolismo , Citocinas/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Fosfoproteínas/metabolismo , Adolescente , Adulto , Biópsia , Estudos de Casos e Controles , Colo/efeitos dos fármacos , Colo/patologia , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Doenças Inflamatórias Intestinais/patologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
6.
J Immunol ; 191(5): 2752-63, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23904167

RESUMO

In nonhuman primates, Vγ9Vδ2(+) (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation. Cell suspensions prepared from peripheral blood and intestinal biopsies were stimulated with microbial phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate [HDMAPP]) and analyzed by flow cytometry to determine Vδ2T cell phenotype, cytokine production, and proliferative potential. Circulating Vδ2T cells expressed gut-homing integrin α4ß7 (>70%), which was coexpressed with skin-associated cutaneous leukocyte Ag by up to 15% of the total population. However, Vδ2T cell activation with HDMAPP and exposure to retinoic acid (signaling via retinoic acid receptor α) increased α4ß7 expression and enhanced binding to mucosal addressin cell adhesion molecule-1 in vitro while simultaneously suppressing cutaneous leukocyte Ag, thereby generating a committed gut-tropic phenotype. Confocal microscopy and flow cytometry identified frequent Vδ2T cells that migrated out of human intestinal biopsies and comprised both CD103(+) and CD103(-) subsets that produced TNF-α and IFN-γ upon phosphoantigen exposure, with more frequent cytokine-producing cells in the CD103(-) population. Activated intestinal Vδ2T cells expressed CD70 and HLA-DR but were unable to drive the proliferation of allogeneic naive CD4(+) T cells. Instead, phosphoantigen-activated CD103(-) Vδ2T cells increased T-bet expression and enhanced IFN-γ production by autologous colonic αß T cells via an IFN-γ-dependent mechanism. These data demonstrate that circulating Vδ2T cells display enhanced gut-homing potential upon microbial activation and populate the human intestinal mucosa, generating functionally distinct CD103(+) and CD103(-) subsets that can promote inflammation by colonic αß T cells.


Assuntos
Interferon gama/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Subpopulações de Linfócitos T/imunologia , Citometria de Fluxo , Humanos , Mucosa Intestinal/metabolismo , Teste de Cultura Mista de Linfócitos , Microscopia Confocal , Fenótipo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/metabolismo
7.
Gastroenterology ; 140(3): 947-56, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21147106

RESUMO

BACKGROUND & AIMS: IgA contributes to homeostatic balance between host and intestinal microbiota. Mechanisms that initiate the IgA response are unclear and likely to differ between humans and animal models. We used multiple experimental approaches to investigate the origin of human intestinal plasma cells that produce IgA in the gastrointestinal tract. METHODS: Complexity of IgA-producing plasma cell populations in human gastrointestinal mucosa and bone marrow and the specific response to oral cholera vaccine were compared by analysis of immunoglobulin genes. Flow cytometry, gene expression analysis, and immunohistochemistry were used to analyze signaling pathways induced by B-cell receptor engagement in human gut-associated lymphoid tissue (GALT) and involvement of innate immunity in B-cell activation in GALT compared with nonintestinal sites. RESULTS: Human intestinal IgA-producing plasma cells appeared to be of germinal center origin; there was no evidence for the population complexity that accompanies multiple pathways of derivation observed in bone marrow. In germinal center B cells of human GALT, Btk and Erk are phosphorylated, CD22 is down-regulated, Lyn is translocated to the cell membrane, and Fos and Jun are up-regulated; these features indicate B-cell receptor ligation during germinal center evolution. No differences in innate activation of B cells were observed in GALT, compared with peripheral immune compartments. CONCLUSIONS: IgA-producing plasma cells appear to be derived from GALT germinal centers in humans. B-cell receptor engagement promotes formation of germinal centers of GALT, with no more evidence for innate immune receptor activation in the mucosa than nonintestinal immune compartments. Germinal centers in GALT should be targets of mucosal vaccinations because they are the source of human intestinal IgA response.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Inata , Imunidade nas Mucosas , Imunoglobulina A/metabolismo , Mucosa Intestinal/imunologia , Plasmócitos/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Administração Oral , Tirosina Quinase da Agamaglobulinemia , Idoso , Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Genes de Imunoglobulinas , Centro Germinativo/metabolismo , Humanos , Imunidade Inata/genética , Imunidade nas Mucosas/genética , Imunoglobulina A/genética , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Ativação Linfocitária , Pessoa de Meia-Idade , Fosforilação , Plasmócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Quinases da Família src/metabolismo
8.
J Immunol ; 185(7): 4118-27, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20833837

RESUMO

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by ß-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-ß, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer's patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.


Assuntos
Células Dendríticas/microbiologia , Escherichia coli Enteropatogênica/patogenicidade , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Fatores de Virulência/metabolismo , Western Blotting , Separação Celular , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Escherichia coli Enteropatogênica/imunologia , Escherichia coli Enteropatogênica/metabolismo , Ensaio de Imunoadsorção Enzimática , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Teste de Cultura Mista de Linfócitos , Microscopia Confocal , NF-kappa B/imunologia , Fatores de Virulência/imunologia
9.
J Am Soc Nephrol ; 21(10): 1645-56, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20724536

RESUMO

Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.


Assuntos
Aquaporina 2/metabolismo , Arginina Vasopressina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Colforsina , AMP Cíclico/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Ratos , Transcrição Gênica
10.
Sci Rep ; 11(1): 19422, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593832

RESUMO

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.


Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-23/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL
11.
Commun Biol ; 3(1): 140, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198438

RESUMO

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.


Assuntos
Anti-Inflamatórios/farmacologia , Desenho de Fármacos , Inflamação/prevenção & controle , Leucócitos Mononucleares/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/enzimologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/enzimologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Estabilidade Enzimática , Feminino , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Injeções Intravenosas , Leucócitos Mononucleares/enzimologia , Masculino , Proteólise , Ratos Sprague-Dawley , Ratos Wistar , Células THP-1 , Técnicas de Cultura de Tecidos , Ubiquitinação
12.
Front Immunol ; 10: 361, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30891036

RESUMO

The intestinal mucosa in inflammatory bowel disease (IBD) contains increased frequencies of lymphocytes and a disproportionate increase in plasma cells secreting immunoglobulin (Ig)G relative to other isotypes compared to healthy controls. Despite consistent evidence of B lineage cells in the mucosa in IBD, little is known of B cell recruitment to the gut in IBD. Here we analyzed B cells in blood of patients with Crohn's disease (CD) and ulcerative colitis (UC) with a range of disease activities. We analyzed the frequencies of known B cell subsets in blood and observed a consistent reduction in the proportion of CD27-IgD- B cells expressing all Ig isotypes in the blood in IBD (independent of severity of disease and treatment) compared to healthy controls. Successful treatment of patients with biologic therapies did not change the profile of B cell subsets in blood. By mass cytometry we demonstrated that CD27-IgD- B cells were proportionately enriched in the gut-associated lymphoid tissue (GALT) in IBD. Since production of TNFα is a feature of IBD relevant to therapies, we sought to determine whether B cells in GALT or the CD27-IgD- subset in particular could contribute to pathology by secretion of TNFα or IL-10. We found that donor matched GALT and blood B cells are capable of producing TNFα as well as IL-10, but we saw no evidence that CD27-IgD- B cells from blood expressed more TNFα compared to other subsets. The reduced proportion of CD27-IgD- B cells in blood and the increased proportion in the gut implies that CD27-IgD- B cells are recruited from the blood to the gut in IBD. CD27-IgD- B cells have been implicated in immune responses to intestinal bacteria and recruitment to GALT, and may contribute to the intestinal inflammatory milieu in IBD.


Assuntos
Subpopulações de Linfócitos B/imunologia , Colite Ulcerativa/sangue , Colite Ulcerativa/imunologia , Doença de Crohn/sangue , Doença de Crohn/imunologia , Imunoglobulina D/metabolismo , Mucosa Intestinal/imunologia , Nódulos Linfáticos Agregados/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Adulto , Anticorpos Monoclonais Humanizados/uso terapêutico , Biópsia , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Feminino , Fármacos Gastrointestinais/uso terapêutico , Humanos , Infliximab/uso terapêutico , Interleucina-10/metabolismo , Mucosa Intestinal/patologia , Masculino , Nódulos Linfáticos Agregados/patologia , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/metabolismo , Ustekinumab/uso terapêutico
13.
Sci Rep ; 9(1): 14042, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575982

RESUMO

V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD.


Assuntos
Anticorpos/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Imunoterapia/métodos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Anticorpos/análise , Anticorpos/metabolismo , Feminino , Humanos , Intestinos/química , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Fator de Necrose Tumoral alfa/imunologia
14.
Cell Rep ; 27(5): 1461-1471.e4, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31042473

RESUMO

B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma Difuso de Grandes Células B/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas F-Box/metabolismo , Feminino , Centro Germinativo/citologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Proteólise , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
15.
PLoS One ; 14(4): e0215033, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31002701

RESUMO

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn's patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1ß in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD.


Assuntos
Colite/tratamento farmacológico , Cicloexilaminas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Epóxido Hidrolases/antagonistas & inibidores , Doenças Inflamatórias Intestinais/tratamento farmacológico , Triazinas/farmacologia , Animais , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Camundongos , Camundongos Endogâmicos C57BL
16.
Cell Rep ; 27(13): 3956-3971.e6, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242426

RESUMO

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.


Assuntos
Microambiente Celular , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Comunicação Parácrina , Proteínas de Ligação a RNA/metabolismo , Feminino , Células HEK293 , Humanos , Células MCF-7 , Masculino
17.
J Med Chem ; 62(14): 6482-6494, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31265286

RESUMO

RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.


Assuntos
Benzotiazóis/farmacologia , Fosfatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Animais , Benzotiazóis/química , Benzotiazóis/farmacocinética , Benzotiazóis/uso terapêutico , Colite/tratamento farmacológico , Cães , Descoberta de Drogas , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Fosfatos/química , Fosfatos/farmacocinética , Fosfatos/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/uso terapêutico , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Suínos , Porco Miniatura
18.
Sci Rep ; 8(1): 4941, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563546

RESUMO

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.


Assuntos
Citocinas/sangue , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab , Mucosa Intestinal/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Biomarcadores/sangue , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Íleo/metabolismo , Íleo/patologia , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/patologia , Infliximab/farmacocinética , Infliximab/farmacologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Fator de Necrose Tumoral alfa/sangue
19.
Nat Commun ; 9(1): 3857, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30242242

RESUMO

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27-CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.


Assuntos
Linfócitos B , Tecido Linfoide/citologia , Humanos , Memória Imunológica , Tecido Linfoide/imunologia , Fenótipo
20.
Sci Rep ; 7(1): 10180, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860510

RESUMO

The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown. We therefore tested whether genetic variants in the IL1RL1 distal promoter are involved in UC and affect glucocorticoid-mediated ST2 expression. Serum ST2 levels and genetic variants in the IL1RL1 distal promoter were examined by ELISA and PCR sequencing in UC patients receiving corticosteroids. Glucocorticoid-mediated ST2 production was evaluated in intestinal mucosa cultures. Molecular regulation of glucocorticoid-mediated ST2 was assessed by RT-qPCR, ChIP assay and luciferase reporter assay. Dexamethasone effect on ST2 transcript expression was analyzed in leukocytes and related to IL1RL1 variants. Sequencing of a distal IL1RL1 promoter region demonstrated that SNPs rs6543115(C) and rs6543116(A) are associated with increased sST2 in UC patients on corticosteroids. Dexamethasone up-regulated sST2 transcription through interaction with the glucocorticoid-response element (GRE) carrying rs6543115(C) variant. Our data indicate that IL1RL1 SNPs rs6543115(C) confer susceptibility to UC and is contained in the GRE, which may modulate glucocorticoid-induced sST2 expression.


Assuntos
Corticosteroides/farmacologia , Colite Ulcerativa/tratamento farmacológico , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Polimorfismo de Nucleotídeo Único , Regulação para Cima , Corticosteroides/uso terapêutico , Adulto , Células Cultivadas , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise de Sequência de DNA
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