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1.
Genes Chromosomes Cancer ; 63(3): e23229, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38481055

RESUMO

A close relationship has been demonstrated between genomic complexity and clinical outcome in uterine smooth muscle tumors. We studied the genomic profiles by array-CGH of 28 fumarate hydratase deficient leiomyomas and 37 leiomyomas with bizarre nuclei (LMBN) from 64 patients. Follow-up was available for 46 patients (from three to 249 months, mean 87.3 months). All patients were alive without evidence of disease. For 51 array-CGH interpretable tumors the mean Genomic Index (GI) was 16.4 (median: 9.8; from 1 to 57.8), significantly lower than the mean GI in LMS (mean GI 51.8, p < 0.001). We described three groups: (1) a group with FH deletion (24/58) with low GI (mean GI: 11 vs. 22,4, p = 0.02), (2) a group with TP53 deletion (17/58) with higher GI (22.4 vs. 11 p = 0.02), and (3) a group without genomic events on FH or TP53 genes (17/58) (mean GI:18.3; from 1 to 57.8). Because none of these tumors recurred and none showed morphological features of LMS we concluded that GI at the cut-off of 10 was not applicable in these subtypes of LM. By integration of all those findings, a GI <10 in LMBN remains a valuable argument for benignity. Conversely, in LMBN a GI >10 or alteration in tumor suppressor genes, should not alone warrant a diagnosis of malignancy. Nine tumors were tested with Nanocind CINSARC® signature and all were classified in low risk of recurrence. We propose, based on our observations, a diagnostic approach of these challenging lesions.


Assuntos
Leiomioma , Neoplasias Uterinas , Feminino , Humanos , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Fumarato Hidratase/genética , Leiomioma/genética , Leiomioma/patologia , Genes p53 , Genômica
2.
Mod Pathol ; 36(10): 100243, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37307879

RESUMO

Neoplasms harboring a KAT6B/A::KANSL1 fusion were initially reported as benign (leiomyomas) and malignant (leiomyosarcomas, low-grade endometrial stromal sarcomas [LG-ESSs]) uterine neoplasms. However, they may represent an emerging entity characterized by clinical aggressiveness contrasting with a rather reassuring microscopic appearance. Here, we aimed to confirm that this neoplasm is a distinct clinicopathologic and molecular sarcoma and identify criteria that should alert pathologists and lead to KAT6B/A::KANSL1 fusion testing in routine practice. Therefore, we conducted a comprehensive clinical, histopathologic, immunohistochemical, and molecular study, including array comparative genomic hybridization, whole RNA-sequencing, unsupervised clustering, and cDNA mutational profile analyses of 16 tumors with KAT6B::KANSL1 fusion from 12 patients. At presentation, patients were peri-menopausal (median, 47.5 years), and the primary tumors were located in the uterine corpus (12/12, 100%), with an additional prevesical location in 1 (8.3%) of 12 cases. The relapse rate was 33.3% (3/9). All tumors (16/16, 100%) showed morphologic and immunohistochemical features overlapping between leiomyoma and endometrial stromal tumors. A whirling recurrent architecture (resembling fibromyxoid-ESS/fibrosarcoma) was found in 13 (81.3%) of 16 tumors. All tumors (16/16, 100%) exhibited numerous arterioliform vessels, and 13 (81.3%) of 18 had large hyalinized central vessels and collagen deposits. Estrogen and progesterone receptors were expressed in 16 (100%) of 16 and 14 (87.5%) of 16 tumors, respectively. Array comparative genomic hybridization performed on 10 tumors classified these neoplasms as simple genomic sarcomas. Whole RNA-sequencing on 16 samples and clustering analysis on primary tumors found that the KAT6B::KANSL1 fusion always occurred between exons 3 of KAT6B and 11 of KANSL1; no pathogenic variant was identified on cDNA, all neoplasms clustered together, close to LG-ESS, and pathway enrichment analysis showed cell proliferation and immune infiltrate recruitment pathway involvement. These results confirm that the sarcomas harboring a KAT6B/A::KANSL1 fusion represent a distinct clinicopathologic entity, close to LG-ESS but different, with clinical aggressiveness despite a reassuring morphology, for which the KAT6B/A::KANSL1 fusion is the molecular driver alteration.

3.
Mod Pathol ; 33(6): 1041-1055, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31857685

RESUMO

Adenoid cystic carcinoma (ACC) of the breast with a predominant solid pattern is difficult to diagnose with certainty and differentiate from more common triple-negative breast cancers (TNBCs) of basal-phenotype. To better characterize solid ACC, we performed a clinical, morphological, immunohistochemical, and molecular comparative analysis of 33 ACCs of the breast comprising 17 solid variant ACCs and 16 conventional ACCs. Solid ACCs displayed basaloid morphology with an exclusive or predominant epithelial cell population associated with decreased myoepithelial differentiation, while demonstrating MYB protein overexpression similar to the more common type of ACC. Strong and diffuse MYB expression by immunochemistry was observed in 14/17 (82%) of solid ACCs while MYB rearrangements were detected by break apart fluorescence in situ hybridization (FISH) in only 3/16 (19%) of solid ACCs. Conversely, weak MYB immunohistochemical expression was observed in only 7/204 (3%) of TNBC. Solid ACCs displayed a transcriptomic profile distinct from conventional ACCs with 549 genes showing a highly significant differential expression between conventional and solid ACC [false discovery rate (FDR) < 0.01; log2FC > |1|]. EnrichR and Kegg Pathway analyses identified PI3K-Akt and focal adhesion signaling pathways as significantly overexpressed in conventional ACCs compared with solid ACCs which significantly overexpressed the nitrogen metabolism pathway. CREBBP mutations and NOTCH activating gene mutations were only present in solid ACCs, concerning 5/16 (31%) of cases for each gene. Tumors with NOTCH activating mutations displayed a strong diffuse nuclear NICD1 staining, an established marker of Notch pathway activation. Solid ACCs also differed from basal-type TNBC, with fewer TP53 mutations and a more stable genomic profile on array comparative genomic hybridization (CGH). In summary, solid-type ACC of the breast is a distinct molecular entity within the ACC family and is different from common basal-type TNBC. MYB is a diagnostically useful biomarker of solid ACC and NOTCH could be a novel potential therapeutic target in 30% of cases.


Assuntos
Neoplasias da Mama/genética , Proteína de Ligação a CREB/genética , Carcinoma Adenoide Cístico/genética , Receptores Notch/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína de Ligação a CREB/metabolismo , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Receptores Notch/metabolismo , Estudos Retrospectivos
4.
J Biol Chem ; 291(17): 8918-30, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26912654

RESUMO

Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection.


Assuntos
Imunidade Adaptativa , Colite/imunologia , Colo/imunologia , Imunidade Inata , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Poli(ADP-Ribose) Polimerases/deficiência , Animais , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/lesões , Colo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Sulfato de Dextrana/toxicidade , Mucosa Intestinal/lesões , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
5.
Eur J Immunol ; 44(8): 2489-2499, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24777831

RESUMO

Primarily defined by their antigen-presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM-CSF and IL-15 (IL-15 DCs) can be triggered with the Toll-like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL-4 (IL-4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)-γ induces the cytotoxic activity of IL-4 but not IL-15 DCs. Although the IFN-γ-STAT-1 signaling pathway is overall functional in IL-15 DCs, IFN-γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN-γ-stimulated IL-15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT-3, and can be partially restored with PIAS1 siRNA and STAT-3 inhibitors.


Assuntos
Células Dendríticas/metabolismo , Interferon gama/metabolismo , Interleucina-15/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-4/metabolismo , Ligantes , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo
6.
Pediatr Crit Care Med ; 16(1): 37-44, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25162512

RESUMO

OBJECTIVE: Children with congenital heart disease have loss of intestinal epithelial barrier function, which increases their risk for postoperative sepsis and organ dysfunction. We do not understand how postoperative cardiopulmonary support or the inflammatory response to cardiopulmonary bypass might alter intestinal epithelial barrier function. We examined variation in a panel of plasma biomarkers to reflect intestinal epithelial barrier function (cellular and paracellular) after cardiopulmonary bypass and in response to routine ICU care. DESIGN: Prospective cohort. SETTING: University medical center cardiac ICU. PATIENTS: Twenty children aged between newborn and 18 years undergoing repair or palliation of congenital heart disease with cardiopulmonary bypass. INTERVENTIONS: We measured baseline and repeated plasma intestinal fatty acid-binding protein, citrulline, claudin 3, and dual sugar permeability testing to reflect intestinal epithelial integrity, epithelial function, paracellular integrity, and paracellular function, respectively. We measured baseline and repeated plasma proinflammatory (interleukin-6, tumor necrosis factor-α, and interferon-γ) and anti-inflammatory (interleukin-4 and interleukin-10) cytokines, known to modulate intestinal epithelial barrier function in murine models of cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS: All patients had abnormal baseline intestinal fatty acid-binding protein concentrations (mean, 3,815.5 pg/mL; normal, 41-336 pg/mL). Cytokine response to cardiopulmonary bypass was associated with early, but not late, changes in plasma concentrations of intestinal fatty acid-binding protein 2 and citrulline. Variation in biomarker concentrations over time was associated with aspects of ICU care indicating greater severity of illness: claudin 3, intestinal fatty acid-binding protein 2, and dual sugar permeability test ratio were associated with symptoms of feeding intolerance (p < 0.05), whereas intestinal fatty acid-binding protein was positively associated with vasoactive-inotrope score (p = 0.04). Citrulline was associated with larger arteriovenous oxygen saturation difference (p = 0.04) and had a complex relationship with vasoactive-inotrope score. CONCLUSIONS: Children undergoing cardiopulmonary bypass for repair or palliation of congenital heart disease are at risk for intestinal injury and often present with evidence for loss of intestinal epithelial integrity preoperatively. Greater severity of illness requiring increased cardiopulmonary support rather than the inflammatory response to cardiopulmonary bypass seems to mediate late postoperative intestinal epithelial barrier function.


Assuntos
Biomarcadores/sangue , Citocinas/sangue , Cardiopatias Congênitas/cirurgia , Enteropatias/etiologia , Mucosa Intestinal/patologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Inflamação , Unidades de Terapia Intensiva Pediátrica , Enteropatias/sangue , Masculino , Período Pós-Operatório , Estudos Prospectivos
7.
Gastroenterology ; 145(3): 613-24, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23747339

RESUMO

BACKGROUND & AIMS: Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. METHODS: Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. RESULTS: Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1ß reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. CONCLUSIONS: After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation and cytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).


Assuntos
Densidade Óssea , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Colite/metabolismo , Rim/metabolismo , Processamento de Proteína Pós-Traducional , Canais de Cátion TRPV/metabolismo , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/transplante , Colite/induzido quimicamente , Colite/imunologia , Glucuronidase/metabolismo , Interferon gama/metabolismo , Proteínas Klotho , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Computadorizada por Raios X , Ácido Trinitrobenzenossulfônico , Fator de Necrose Tumoral alfa/metabolismo
8.
J Immunol ; 189(8): 3878-93, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22972928

RESUMO

In vitro data and transgenic mouse models suggest a role for TGF-ß signaling in dendritic cells (DCs) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHC class II (MHCII) and costimulatory molecules and increased expression of IDO, among others. To test whether a complete lack of TGF-ß signaling in DCs predisposes mice to spontaneous autoimmunity and to verify the mechanisms implicated previously in vitro, we generated conditional knockout (KO) mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 wk of age with multiorgan autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, costimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more proinflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated Foxp3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25(-)Foxp3(+) Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFN-γ and were not capable of directing Ag-specific Treg conversion unless in the presence of anti-IFN-γ blocking Ab. Adoptive transfer of induced Tregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGF-ß signaling in DCs is critical in the control of autoimmunity through both Treg-dependent and -independent mechanisms, but it does not affect MHCII and costimulatory molecule expression.


Assuntos
Doenças Autoimunes/prevenção & controle , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Colite/genética , Colite/imunologia , Colite/prevenção & controle , Células Dendríticas/patologia , Modelos Animais de Doenças , Tolerância Imunológica/genética , Imunofenotipagem , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Linfócitos T Reguladores/patologia
9.
Am J Physiol Gastrointest Liver Physiol ; 305(10): G667-77, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24029465

RESUMO

Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.


Assuntos
Bactérias/classificação , Colite/microbiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Colite/induzido quimicamente , Colite/genética , Sulfato de Dextrana/toxicidade , Fezes/microbiologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética
10.
Blood ; 117(5): 1555-64, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21123824

RESUMO

Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.


Assuntos
Vacinas Anticâncer/administração & dosagem , Extratos Celulares/administração & dosagem , Fatores de Transcrição Forkhead/metabolismo , Memória Imunológica/imunologia , Leucemia Experimental/terapia , Chaperonas Moleculares/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Western Blotting , Vacinas Anticâncer/imunologia , Extratos Celulares/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interferon gama/metabolismo , Leucemia Experimental/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismo , Células Tumorais Cultivadas
11.
J Immunol ; 187(12): 6310-7, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22075702

RESUMO

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.


Assuntos
Citotoxicidade Imunológica , Células Dendríticas/imunologia , Interferon gama/fisiologia , Neoplasias Mamárias Experimentais/imunologia , Melanoma Experimental/imunologia , Receptores de Interferon/fisiologia , Células Th1/imunologia , Células Th1/metabolismo , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica/genética , Células Dendríticas/metabolismo , Feminino , Interferon gama/metabolismo , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptor de Interferon gama
12.
Am J Physiol Gastrointest Liver Physiol ; 302(1): G85-96, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22038826

RESUMO

Curcumin (diferulolylmethane) is an anti-inflammatory phenolic compound found effective in preclinical models of inflammatory bowel diseases (IBD) and in ulcerative colitis patients. Pharmacokinetics of curcumin and its poor systemic bioavailability suggest that it targets preferentially intestinal epithelial cells. The intestinal epithelium, an essential component of the gut innate defense mechanisms, is profoundly affected by IFN-γ, which can disrupt the epithelial barrier function, prevent epithelial cell migration and wound healing, and prime epithelial cells to express major histocompatibility complex class II (MHC-II) molecules and to serve as nonprofessional antigen-presenting cells. In this report we demonstrate that curcumin inhibits IFN-γ signaling in human and mouse colonocytes. Curcumin inhibited IFN-γ-induced gene transcription, including CII-TA, MHC-II genes (HLA-DRα, HLA-DPα1, HLA-DRß1), and T cell chemokines (CXCL9, 10, and 11). Acutely, curcumin inhibited Stat1 binding to the GAS cis-element, prevented Stat1 nuclear translocation, and reduced Jak1 phosphorylation and phosphorylation of Stat1 at Tyr(701). Longer exposure to curcumin led to endocytic internalization of IFNγRα followed by lysosomal fusion and degradation. In summary, curcumin acts as an IFN-γ signaling inhibitor in colonocytes with biphasic mechanisms of action, a phenomenon that may partially account for the beneficial effects of curcumin in experimental colitis and in human IBD.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colo/efeitos dos fármacos , Curcumina/farmacologia , Interferon gama/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocinas/efeitos dos fármacos , Humanos , Mucosa Intestinal/imunologia , Janus Quinase 1/metabolismo , Complexo Principal de Histocompatibilidade , Camundongos , Fosforilação , Fator de Transcrição STAT1/metabolismo , Transcrição Gênica/efeitos dos fármacos
13.
Gastroenterology ; 138(4): 1384-94, 1394.e1-2, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20004202

RESUMO

BACKGROUND & AIMS: Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS: We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS: Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS: The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.


Assuntos
Colite/metabolismo , Glucuronidase/antagonistas & inibidores , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Transferência Adotiva , Animais , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Glucuronidase/genética , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-10/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Rim/metabolismo , Proteínas Klotho , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/fisiologia , Osteoporose/etiologia , Transcrição Gênica/efeitos dos fármacos
14.
Cancer Discov ; 11(9): 2248-2265, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837065

RESUMO

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.


Assuntos
Glioblastoma/tratamento farmacológico , Imunoterapia Adotiva , Interferon gama/metabolismo , Células Mieloides/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Glioblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Gastroenterology ; 137(3): 965-75, 975.e1-10, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19450596

RESUMO

BACKGROUND & AIMS: NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of interferon (IFN)-gamma, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity. METHODS: Control or dextran sulfate sodium (DSS)-treated, 6- to 8-week-old wild-type (WT) and NHE3(-/-) mice were used for the experiments. Small intestines were dissected for further analysis. RESULTS: NHE3(-/-) mice have elevated numbers of CD8alpha(+) T and natural killer cells in the intraepithelial lymphocytes and lamina propria lymphocytes compartments, representing the source of IFN-gamma. NHE3(-/-) mice display alterations in epithelial gene and protein expression patterns that predispose them to a high susceptibility to DSS, with accelerated mortality resulting from intestinal bleeding, hypovolemic shock, and sepsis, even at a very low DSS concentration. Microarray analysis and intestinal hemorrhage indicate that NHE3 deficiency predisposes mice to DSS-induced small intestinal injury, a segment never reported as affected by DSS, and demonstrate major differences in the colonic response to DSS challenge in WT and NHE3(-/-) mice. In NHE3(-/-) mice, broad-spectrum oral antibiotics or anti-asialo GM1 antibodies reduce the expression of IFN-gamma and iNOS to basal levels and delay but do not prevent severe mortality in response to DSS treatment. CONCLUSIONS: These results suggest that NHE3 participates in mucosal responses to epithelial damage, acting as a modifier gene determining the extent of the gut inflammatory responses in the face of intestinal injury.


Assuntos
Sulfato de Dextrana/toxicidade , Homeostase , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Contagem de Células Sanguíneas , Colo/metabolismo , Regulação para Baixo , Endotelina-1/metabolismo , Gangliosídeo G(M1)/metabolismo , Hemorragia Gastrointestinal/induzido quimicamente , Interferon gama/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética
16.
J Immunol ; 181(10): 6955-63, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18981115

RESUMO

Imatinib mesylate (Gleevec, STI571), a selective inhibitor of a restricted number of tyrosine kinases, has been effectively used for the treatment of Philadelphia chromosome-positive leukemias and gastrointestinal stromal tumors. Imatinib may also directly influence immune cells. Suppressive as well as stimulating effects of this drug on CD4(+) and CD8(+) T lymphocytes or dendritic cells have been reported. In the current study, we have investigated the influence of imatinib mesylate on CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg), a critical population of lymphocytes that contributes to peripheral tolerance. Used at concentrations achieved clinically, imatinib impaired Treg immunosuppressive function and FoxP3 expression but not production of IL-10 and TGF-beta in vitro. Imatinib significantly reduced the activation of the transcription factors STAT3 and STAT5 in Treg. Analysis of Treg TCR-induced signaling cascade indicated that imatinib inhibited phosphorylation of ZAP70 and LAT. Substantiating these observations, imatinib treatment of mice decreased Treg frequency and impaired their immunosuppressive function in vivo. Furthermore, imatinib mesylate significantly enhanced antitumor immune responses to dendritic cell-based immunization against an imatinib-resistant BCR-ABL negative lymphoma. The clinical applications of imatinib mesylate might thus be expanded with its use as a potent immunomodulatory agent targeting Treg in cancer immunotherapy.


Assuntos
Antineoplásicos/administração & dosagem , Células Dendríticas/transplante , Imunoterapia Ativa/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Benzamidas , Western Blotting , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/efeitos dos fármacos , Mesilato de Imatinib , Imuno-Histoquímica , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Linfócitos T Reguladores/imunologia
17.
Front Immunol ; 11: 274, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194552

RESUMO

Sarcomas are heterogeneous malignant mesenchymal neoplasms with limited sensitivity to immunotherapy. We recently demonstrated an increase in Kynurenine Pathway (KP) activity in the plasma of sarcoma patients treated with pembrolizumab. While the KP has already been described to favor immune escape through the degradation of L-Tryptophan and production of metabolites including L-Kynurenine, Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the KP, still represents an attractive therapeutic target, and its blockade had not yet been investigated in sarcomas. Using immunohistochemistry, IDO1 and CD8, expression profiles were addressed within 203 cases of human sarcomas. At a preclinical level, we investigated the modulation of the KP upon PDL1 blockade in a syngeneic model of sarcoma through mRNA quantification of key KP enzymes within the tumor. Furthermore, in order to evaluate the possible anti-tumor effect of IDO blockade in combination with PDL1 blockade, an innovative IDO inhibitor (GDC-0919) was used. Its effect was first assessed on Kynurenine to Tryptophan ratio at plasmatic level and also within the tumor. Following GDC-0919 treatment, alone or in combination with anti-PDL1 antibody, tumor growth, immune cell infiltration, and gene expression profiling were measured. IDO1 expression was observed in 39.1% of human sarcoma cases and was significantly higher in tumors with high CD8 infiltration. In the pre-clinical setting, blockade of PDL1 led to a strong anti-tumor effect and was associated with an intratumoral inflammatory cytokines signature driven by Ifng but also with a modulation of the KP enzymes including Ido1 and Ido2. IDO1 inhibition using GDC-0919 resulted in (i) a significant decrease of plasmatic Kynurenine to Tryptophan ratio and in (ii) a decrease of tumoral Kynurenine. However, GDC-0919 used alone or combined with anti-PDL1, did not show anti-tumoral activity and did not affect the tumor immune cell infiltrate. In order to elucidate the mechanism(s) underlying the lack of effect of GDC-0919, we analyzed the gene expression profile of intratumoral biopsies. Interestingly, we have found that GDC-0919 induced a downregulation of the expression of pvr and granzymes, and an upregulation of inhba and Dtx4 suggesting a potential role of the IDO pathway in the control of NK function.


Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Sarcoma/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Indolamina-Pirrol 2,3,-Dioxigenase/análise , Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Cinurenina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Sarcoma/imunologia , Sarcoma/metabolismo , Células Tumorais Cultivadas , Adulto Jovem
18.
Inflamm Bowel Dis ; 14(6): 780-93, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18200517

RESUMO

BACKGROUND: Curcumin (diferulolylmethane) has been shown to have a protective role in mouse models of inflammatory bowel diseases (IBD) and to reduce the relapse rate in human ulcerative colitis (UC), thus making it a potentially viable supportive treatment option. Trinitrobenzene sulfonic acid (TNBS) colitis in NKT-deficient SJL/J mice has been described as Th1-mediated inflammation, whereas BALB/c mice are believed to exhibit a mixed Th1/Th2 response. METHODS: We therefore investigated the effect of dietary curcumin in colitis induced in these 2 strains. RESULTS: In the BALB/c mice, curcumin significantly increased survival, prevented weight loss, and normalized disease activity. In the SJL/J mice, curcumin demonstrated no protective effects. Genomewide microarray analysis of colonic gene expression was employed to define the differential effect of curcumin in these 2 strains. This analysis not only confirmed the disparate responses of the 2 strains to curcumin but also indicated different responses to TNBS. Curcumin inhibited proliferation of splenocytes from naive BALB/c mice but not SJL/J mice when nonspecifically stimulated in vitro with concanavalin A (ConA). Proliferation of CD4(+) splenocytes was inhibited in both strains, albeit with about a 2-fold higher IC(50) in SJL/J mice. Secretion of IL-4 and IL-5 by CD4(+) lymphocytes of BALB/c mice but not SJL/J mice was significantly augmented by ConA and reduced to control levels by curcumin. CONCLUSIONS: The efficacy of dietary curcumin in TNBS colitis varies in BALB/c and SJL/J mouse strains. Although the exact mechanism underlying these differences is unclear, the results suggest that the therapeutic value of dietary curcumin may differ depending on the nature of immune dysregulation in IBD.


Assuntos
Curcumina/administração & dosagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Concanavalina A/farmacologia , Dieta , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/patologia , Linfócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Análise em Microsséries , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Ácido Trinitrobenzenossulfônico
19.
PLoS One ; 11(4): e0152044, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27050757

RESUMO

Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na+/H+ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na+/H+ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na+/H+ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2-/- and NHE3-/-/Rag2-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25-FoxP3+ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.


Assuntos
Colite/imunologia , Intestinos/microbiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Linfócitos T/imunologia , Animais , Colite/metabolismo , Camundongos , Camundongos Knockout
20.
Biomed Res Int ; 2015: 891236, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26491691

RESUMO

T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.


Assuntos
Antígenos CD/imunologia , Apirase/imunologia , Arginase/imunologia , Células Dendríticas/imunologia , Neoplasias Experimentais/imunologia , Linfócitos T/imunologia , Evasão Tumoral , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Dendríticas/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologia , Linfócitos T/patologia
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