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1.
Cell Mol Gastroenterol Hepatol ; 17(1): 119-130, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-37714427

RESUMEN

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) causes a marked increase in the number of T cells in the intestinal mucosa. Debate exists about whether these excess cells arise from local clonal proliferation or recruitment from the periphery. METHODS: CD8+ T cells were sorted from colon biopsy specimens and blood for T-cell receptor (TCR) ß-chain sequencing. Biopsy specimens from inflamed or uninflamed colon from ulcerative colitis or Crohn's disease cohorts were compared with colon biopsy specimens from people without IBD, as well as with autologous blood α4ß7+, α4ß7- effector/memory, terminal effector/memory CD45RA+ T cell, and mucosal-associated invariant T-cell CD8 subpopulations. RESULTS: CD8 TCR diversity in mucosa and blood did not correlate with inflammation. Repertoire overlap between any 2 distinct locations of a given person's colon was consistently high, although often lower between inflamed and uninflamed sites. CD8 TCR repertoires overlapped between the colon and each peripheral blood subpopulation studied, with the highest overlap seen for integrin α4ß7+ T cells. Inflamed tissue consistently overlapped more than uninflamed tissue with each blood subpopulation. CONCLUSIONS: CD8 T-cell clones are spread homogenously throughout the length of the colon. Although TCR repertoire overlap is greater within than between inflamed and uninflamed colon segments, a similar TCR diversity in both argues against local clonal expansion being the main source of excess cytotoxic T cells in inflamed mucosa. Rather, the increased TCR overlap observed between blood and inflamed mucosa supports the significance of T-cell trafficking in IBD pathogenesis, particularly concerning α4ß7+ T-cell populations.


Asunto(s)
Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Enfermedades Inflamatorias del Intestino/patología , Enfermedad de Crohn/patología , Receptores de Antígenos de Linfocitos T alfa-beta/genética
2.
J Crohns Colitis ; 2023 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-37855324

RESUMEN

BACKGROUND: Janus kinase (JAK) inhibitors (JAKinibs) are effective small molecule therapies for treating Crohn's Disease (CD) and Ulcerative Colitis (UC), collectively known as inflammatory bowel disease (IBD). By preventing JAKs from phosphorylating signal transducer and activator of transcription proteins, JAKinibs disrupt cytokine signaling pathways that promote inflammation. Despite considerable overlap in the JAKs they target, first and second generation JAKinibs display different clinical efficacies in CD and UC. METHODS: We conducted a comparative phosflow study of four JAKinibs (filgotinib, upadacitinib, tofacitinib, and deucravacitinib) to observe subtle mechanistic differences that may dictate their clinical behavior. Resected mesenteric lymph node (MLN) cells from 19 patients (9 CD, 10 UC) were analyzed by flow cytometry in the presence or absence of different cytokine stimuli and titrated JAKinibs. RESULTS: We found a higher potency of the JAK 1/3-preferential inhibitor, tofacitinib, for JAK 3-dependent cytokine signaling pathways in comparison to filgotinib, but a higher potency of the JAK 1-preferential inhibitors, filgotinib and upadacitinib, for JAK 3-independent cytokine signaling pathways. Deucravacitinib, a TYK2-preferential inhibitor, demonstrated a much narrower selectivity by inhibiting only IL-10 and IFN-ßpathways, albeit more potently than the other JAKinibs . Additionally, we found some differences in the sensitivity of immune cells from CD versus UC and patients with versus without a CD-associated NOD2 polymorphism to phosphorylate signal transducer and activator of transcriptions in response to specific cytokine stimulation. CONCLUSIONS: Despite their similarities, differences exist in the relative potencies of different JAKinibs against distinct cytokine families to explain their clinical efficacy.

3.
J Nat Prod ; 74(4): 567-73, 2011 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-21348461

RESUMEN

The natural product austocystin D was identified as a potent cytotoxic agent with in vivo antitumor activity and selectivity for cells expressing the multidrug resistance transporter MDR1. We sought to elucidate the mechanism of austocystin D's selective cytotoxic activity. Here we show that the selective cytotoxic action of austocystin D arises from its selective activation by cytochrome P450 (CYP) enzymes in specific cancer cell lines, leading to induction of DNA damage in cells and in vitro. The potency and selectivity of austocystin D is lost upon inhibition of CYP activation and does not require MDR1 expression or activity. Furthermore, the pattern of cytotoxicity of austocystin D was distinct from doxorubicin and etoposide and unlike aflatoxin B(1), a compound that resembles austocystin D and is also activated by CYP enzymes to induce DNA damage. Theses results suggest that austocystin D may be of clinical benefit for targeting or overcoming chemoresistance.


Asunto(s)
Aflatoxina B1/farmacología , Aflatoxinas/aislamiento & purificación , Aflatoxinas/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Aspergillus/química , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Aflatoxinas/química , Antineoplásicos/química , Sistema Enzimático del Citocromo P-450/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular
4.
BMC Rheumatol ; 5(1): 15, 2021 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-33781343

RESUMEN

BACKGROUND: B cells are critical mediators of systemic lupus erythematosus (SLE) and lupus nephritis (LN), and antinuclear antibodies can be found in the serum of approximately 98% of patients with SLE. Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that mediates signaling from immunoreceptors, including the B cell receptor. Active, phosphorylated SYK has been observed in tissues from patients with SLE or cutaneous lupus erythematosus, and its inhibition is hypothesized to ameliorate disease pathogenesis. We sought to evaluate the efficacy and characterize the mechanism of action of lanraplenib, a selective oral SYK inhibitor, in the New Zealand black/white (NZB/W) murine model of SLE and LN. METHODS: Lanraplenib was evaluated for inhibition of primary human B cell functions in vitro. Furthermore, the effect of SYK inhibition on ameliorating LN-like disease in vivo was determined by treating NZB/W mice with lanraplenib, cyclophosphamide, or a vehicle control. Glomerulopathy and immunoglobulin G (IgG) deposition were quantified in kidneys. The concentration of proinflammatory cytokines was measured in serum. Splenocytes were analyzed by flow cytometry for B cell maturation and T cell memory maturation, and the presence of T follicular helper and dendritic cells. RESULTS: In human B cells in vitro, lanraplenib inhibited B cell activating factor-mediated survival as well as activation, maturation, and immunoglobulin M production. Treatment of NZB/W mice with lanraplenib improved overall survival, prevented the development of proteinuria, and reduced blood urea nitrogen concentrations. Kidney morphology was significantly preserved by treatment with lanraplenib as measured by glomerular diameter, protein cast severity, interstitial inflammation, vasculitis, and frequency of glomerular crescents; treatment with lanraplenib reduced glomerular IgG deposition. Mice treated with lanraplenib had reduced concentrations of serum proinflammatory cytokines. Lanraplenib blocked disease-driven B cell maturation and T cell memory maturation in the spleen. CONCLUSIONS: Lanraplenib blocked the progression of LN-like disease in NZB/W mice. Human in vitro and murine in vivo data suggest that lanraplenib may be efficacious in preventing disease progression in patients with LN at least in part by inhibiting B cell maturation. These data provide additional rationale for the use of lanraplenib in the treatment of SLE and LN.

5.
Dev Cell ; 8(1): 53-64, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15621529

RESUMEN

Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein MEI-S332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. MEI-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. We elucidated a mechanism for controlling release of MEI-S332 from centromeres via phosphorylation by POLO kinase. We demonstrate that POLO antagonizes MEI-S332 cohesive function and that full POLO activity is needed to remove MEI-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. POLO phosphorylates MEI-S332 in vitro, POLO and MEI-S332 bind each other, and mutation of POLO binding sites prevents MEI-S332 dissociation from centromeres.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Sitios de Unión , Western Blotting/métodos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Células Cultivadas , Ciclina B/metabolismo , Drosophila , Proteínas de Drosophila/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , Embrión no Mamífero , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Inmunoprecipitación/métodos , Indoles/metabolismo , Larva , Masculino , Meiosis/fisiología , Microscopía Confocal/métodos , Mitosis/fisiología , Modelos Biológicos , Mutagénesis/fisiología , Fosforilación , Espermatocitos/metabolismo , Testículo/metabolismo , Xenopus/metabolismo
6.
Mol Biol Cell ; 17(4): 1744-57, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16436512

RESUMEN

Among acetyltransferases, the MYST family enzyme Esa1p is distinguished for its essential function and contribution to transcriptional activation and DNA double-stranded break repair. Here we report that Esa1p also plays a key role in silencing RNA polymerase II (Pol II)-transcribed genes at telomeres and within the ribosomal DNA (rDNA) of the nucleolus. These effects are mediated through Esa1p's HAT activity and correlate with changes within the nucleolus. Esa1p is enriched within the rDNA, as is the NAD-dependent protein deacetylase Sir2p, and the acetylation levels of key Esa1p histone targets are reduced in the rDNA in esa1 mutants. Although mutants of both ESA1 and SIR2 have enhanced rates of rDNA recombination, esa1 effects are more modest yet result in distinct structural changes of rDNA chromatin. Surprisingly, increased expression of ESA1 can bypass the requirement for Sir2p in rDNA silencing, suggesting that these two enzymes with seemingly opposing activities both contribute to achieve optimal nucleolar chromatin structure and function.


Asunto(s)
ADN de Hongos/genética , ADN Ribosómico/genética , Silenciador del Gen , Histona Acetiltransferasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Núcleo Celular/química , Núcleo Celular/enzimología , Cromatina/metabolismo , Cromosomas Fúngicos/metabolismo , ADN de Hongos/metabolismo , ADN Ribosómico/metabolismo , Dosificación de Gen , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Mutación , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2 , Sirtuinas/genética , Sirtuinas/metabolismo , Secuencias Repetidas en Tándem , Telómero/enzimología , Transcripción Genética
7.
Thromb Res ; 170: 109-118, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30172129

RESUMEN

INTRODUCTION: Spleen tyrosine kinase (SYK) mediates signal transduction in multiple hematopoietic cells, including platelets. SYK signals downstream of immunoreceptors and SYK inhibition may ameliorate disease pathology in multiple autoimmune disorders; however, the impact of SYK inhibition in platelets and its potential relevance to bleeding is not fully understood. These studies evaluated the effect of an oral SYK inhibitor, GS-9876, on platelets in vitro and in vivo, and the impact of GS-9876 plus non-steroidal anti-inflammatory drugs (NSAIDs) on platelet aggregation. MATERIAL AND METHODS: The effect of GS-9876 on platelet activation, aggregation, and binding was characterized by western blotting, aggregometry, fluorescence-activated cell sorting, and microscopy techniques. The effect of GS-9876 on in vivo bleeding time (BT) was determined in cynomolgus monkeys and humans. RESULTS: GS-9876 inhibited glycoprotein VI (GPVI)-induced phosphorylation of linker for activation of T cells and phospholipase Cγ2, platelet activation and aggregation in human whole blood, and platelet binding to collagen under arterial flow. Ex vivo, GPVI-stimulated platelet aggregation was inhibited in GS-9876-treated monkeys without a concomitant increase in BT. Similarly, orally administered GS-9876 did not increase BT in humans. No in vitro additive effects on inhibition of platelet aggregation were observed with GS-9876 plus NSAIDs in human blood. CONCLUSIONS: GS-9876 inhibited SYK activity in platelets via the GPVI receptor without prolonging BT in monkeys or humans. Furthermore, GS-9876 did not increase inhibition of platelet aggregation by NSAIDs in vitro, suggesting that these agents can potentially be combined without increasing bleeding risk in humans.


Asunto(s)
Plaquetas/metabolismo , Hemostasis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasa Syk/antagonistas & inhibidores , Adolescente , Adulto , Humanos , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Adulto Joven
8.
Oncotarget ; 5(4): 908-15, 2014 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-24659719

RESUMEN

Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Indazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Purinas/farmacología , Pirazinas/farmacología , Quinazolinonas/farmacología , Apoptosis/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Indazoles/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Linfocítica Crónica de Células B/enzimología , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Purinas/administración & dosificación , Pirazinas/administración & dosificación , Quinazolinonas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Quinasa Syk
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