RESUMO
BACKGROUND: Despite recent advances in the treatment of squamous cell skin cancer (SCSC), the disease persists, and treatment resistance develops. Thus, identifying new targets and developing new therapeutic approaches showing low vulnerability to drug resistance is highly needed. PURPOSE: This study aimed to reveal a novel targeted phytotherapeutic strategy for SCSC treatment alone or in combination with standard targeted anticancer molecules. STUDY DESIGN: A library of natural products was utilized to identify molecules that inhibit the growth of skin cancer cells. The anticancer potential of the selected compound was evaluated in human skin squamous carcinoma models, in vitro and in vivo. A comprehensive ingenuity pathway analysis (IPA) strategy and molecular biology technology was adopted to investigate the therapeutic mechanisms in human SCSC. METHODS: The Matrigel invasion chamber, foci formation and soft agar colony formation assays were employed to study the cells invasion and migration potential in vitro. In vivo antitumor effects were evaluated in DMBA/TPA-induced skin papilloma and A431 human skin squamous carcinoma xenograft tumor models. An integrative IPA was employed to identify mechanisms and protein targets in human SCSC.Compounds synergies were determined by the bliss model and evaluated using human SCSC cell lines and xenograft tumors. Histological staining, immunofluorescence imaging, real-time PCR, Western blots, and flow cytometric analyses were employed to analyze apoptosis and cell signaling mechanisms. RESULTS: We identified (+)-cyanidan-3-ol (CD-3) as a selective compound for inhibiting the growth of SCSC cell lines. CD-3 inhibited tumor growth and burden without apparent toxicity and prolonged the survival of tumor-bearing mice. CD-3 inhibitory effects on SCSC growth are mediated via cell cycle arrest and caspase-dependent apoptosis induction. Mechanistic studies showed that CD-3 activates PP2A via inhibiting CIP2A and produces tumor growth inhibitory effects via promoting dephosphorylation of oncogenic AKT/mTOR signaling proteins in SCSC cells and xenograft tumors in a PP2A dependent manner. Furthermore, the combination of CD-3 and mTOR inhibitors (mTORi) synergistically reduced oncogenic phenotypes. CONCLUSIONS: Our study suggests that PP2A activation is an effective strategy for SCSC treatment and the CD-3 and mTORi combination may serve as a promising treatment for SCSC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Cutâneas , Animais , Humanos , Camundongos , Apoptose , Autoantígenos/genética , Autoantígenos/metabolismo , Autoantígenos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Male haploid cells, spermatids and spermatozoa, that appear after the establishment of immune tolerance express novel cell surface and intracellular proteins that can be recognized as foreign antigens by the self-immune system. However, these germ cells do not normally evoke a pathological immune response. The immune-privileged micro-circumstance in testis involving the blood-testis-barrier formed by Sertoli cells protects these germ cells from autoimmune attack. We recently found that immunization with heat shock protein family A member 4-like (HSPA4L), one of the new differentiation antigens of haploid cells, induced experimental autoimmune orchitis (EAO) in A/J male mice. In this study, we focused on G protein-coupled receptor kinase interacting protein-1 (GIT1), another haploid cell-specific differentiation antigen, to investigate whether GIT1 is a target autoantigen for EAO induction. GIT1 emulsified with complete Freund's adjuvant was injected subcutaneously into the mice inguinal region once on day 0 and again on day 14, and the optimum condition of EAO induction was determined. Mice immunized with 200 µg GIT1 showed significantly higher incidence of EAO than that of immunization with other concentrations. In particular, significant lymphocytic inflammation and extensive aspermatogenesis were observed in these mice at 120 days after the first immunization. These findings indicate that GIT1 is also a target antigen that induces EAO, like HSPA4L.
Assuntos
Autoantígenos , Doenças Autoimunes , Animais , Autoantígenos/farmacologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/patologia , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase , Masculino , Camundongos , Espermátides/metabolismo , Espermatogênese , Testículo/metabolismoRESUMO
BACKGROUND & AIMS: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. METHODS: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. RESULTS: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. CONCLUSION: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. LAY SUMMARY: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.
Assuntos
Colangite/etiologia , Doença Relacionada a Imunoglobulina G4/complicações , Fatores de Proteção , Idoso , Anexinas/farmacologia , Anexinas/uso terapêutico , Autoantígenos/farmacologia , Autoantígenos/uso terapêutico , Biópsia/métodos , Biópsia/estatística & dados numéricos , Colangite/fisiopatologia , Feminino , Humanos , Doença Relacionada a Imunoglobulina G4/fisiopatologia , Fígado/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The presence and clinical significance of IL-17 and IL-17-expressing cells have been studied for several cancers, although their correlation with tumor development remains controversial. Peripheral blood was collected from healthy donors and glioma patients to isolate peripheral blood mononuclear cells (PBMCs). The percentage of IL-17-expressing cells and the production of inflammatory cytokines in PBMCs and tissues were measured. Human IL17 cDNA was then inserted into the pEGFPN1 plasmid and transfected into the glioma U87MG cell line, and tumstatin was used to block the effect of the IL-17 overexpression. Stem cell transcription factors were evaluated in each group using qRT-PCR and western blotting, and proliferation and migration were detected using colony formation and wound-healing assays. The cells were then subcutaneously inoculated into nude mice to evaluate the growth of glioma. Compared with healthy donors, the PBMCs from glioma patients showed a significant accumulation of IL-17-expressing T cells. Th17 cell differentiation-related cytokines (IL-23, TGF-ß and IL-6) were increased in the tumor microenvironment. IL17 transfection increased the mRNA and protein expression of stem cell transcription factors in U87MG cells in vitro. The proliferation and migration of U87MG cells were also increased. Moreover, the pEGFPN1IL17U87MG cells grew more rapidly than other cells. However, tumstatin-treated U87MG cells showed significantly inhibited the effects of IL-17 overexpression. Tumstatin effectively suppressed IL-17-derived U87MG cell growth by downregulating stem cell maintenance factors and inducing proliferation and migration. These findings indicated that IL-17 represents a potential prognostic marker for glioma, while tumstatin has potential in the treatment for glioma.
Assuntos
Antineoplásicos/farmacologia , Autoantígenos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Colágeno Tipo IV/farmacologia , Glioma/tratamento farmacológico , Interleucina-17/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células Th17/metabolismo , Adulto , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Glioma/imunologia , Glioma/metabolismo , Glioma/patologia , Humanos , Interleucina-17/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Th17/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The human cationic anti-microbial peptide LL-37 is a T cell self-antigen in patients with psoriasis, who have increased risk of cardiovascular events. However, the role of LL-37 as a T cell self-antigen in the context of atherosclerosis remains unclear. The objective of this study was to test for the presence of T cells reactive to LL-37 in patients with acute coronary syndrome (ACS). Furthermore, the role of T cells reactive to LL-37 in atherosclerosis was assessed using apoE-/- mice immunized with the LL-37 mouse ortholog, mCRAMP. Peripheral blood mononuclear cells (PBMCs) from patients with ACS were stimulated with LL-37. PBMCs from stable coronary artery disease (CAD) patients or self-reported subjects served as controls. T cell memory responses were analyzed with flow cytometry. Stimulation of PBMCs with LL-37 reduced CD8+ effector T cell responses in controls and patients with stable CAD but not in ACS and was associated with reduced programmed cell death protein 1 (PDCD1) mRNA expression. For the mouse studies, donor apoE-/- mice were immunized with mCRAMP or adjuvant as controls, then T cells were isolated and adoptively transferred into recipient apoE-/- mice fed a Western diet. Recipient mice were euthanized after 5 weeks. Whole aortas and hearts were collected for analysis of atherosclerotic plaques. Spleens were collected for flow cytometric and mRNA expression analysis. Adoptive transfer experiments in apoE-/- mice showed a 28% reduction in aortic plaque area in mCRAMP T cell recipient mice (P < 0.05). Fifty six percent of adjuvant T cell recipient mice showed calcification in atherosclerotic plaques, compared to none in the mCRAMP T cell recipient mice (Fisher's exact test P = 0.003). Recipients of T cells from mice immunized with mCRAMP had increased IL-10 and IFN-γ expression in CD8+ T cells compared to controls. In conclusion, the persistence of CD8+ effector T cell response in PBMCs from patients with ACS stimulated with LL-37 suggests that LL-37-reactive T cells may be involved in the acute event. Furthermore, studies in apoE-/- mice suggest that T cells reactive to mCRAMP are functionally active in atherosclerosis and may be involved in modulating plaque calcification.
Assuntos
Síndrome Coronariana Aguda/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Aorta/imunologia , Doenças da Aorta/imunologia , Aterosclerose/imunologia , Autoantígenos/imunologia , Leucócitos Mononucleares/imunologia , Linfócitos T/imunologia , Calcificação Vascular/imunologia , Síndrome Coronariana Aguda/metabolismo , Transferência Adotiva , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Autoantígenos/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Humanos , Memória Imunológica , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Masculino , Camundongos Knockout para ApoE , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/transplante , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/prevenção & controle , CatelicidinasRESUMO
Homeostatic antigen presentation by hepatic antigen-presenting cells, which results in tolerogenic T-cell education, could be exploited to induce antigen-specific immunological tolerance. Here we show that antigens modified with polymeric forms of either N-acetylgalactosamine or N-acetylglucosamine target hepatic antigen-presenting cells, increase their antigen presentation and induce antigen-specific tolerance, as indicated by CD4+ and CD8+ T-cell deletion and anergy. These synthetically glycosylated antigens also expanded functional regulatory T cells, which are necessary for the durable suppression of antigen-specific immune responses. In an adoptive-transfer mouse model of type-1 diabetes, treatment with the glycosylated autoantigens prevented T-cell-mediated diabetes, expanded antigen-specific regulatory T cells and resulted in lasting tolerance to a subsequent challenge with activated diabetogenic T cells. Glycosylated autoantigens targeted to hepatic antigen-presenting cells might enable therapies that promote immune tolerance in patients with autoimmune diseases.
Assuntos
Acetilgalactosamina/imunologia , Acetilgalactosamina/farmacologia , Acetilglucosamina/imunologia , Acetilglucosamina/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/efeitos dos fármacos , Transferência Adotiva , Animais , Apresentação de Antígeno/imunologia , Autoantígenos/farmacologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Feminino , Fígado/efeitos dos fármacos , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Baço , Linfócitos T/efeitos dos fármacosRESUMO
Ischemia/reperfusion (I/R)-induced oxidative stress is a serious clinical problem in the reperfusion therapy for ischemic diseases. Tumstatin is an endogenous bioactive peptide cleaved from type IV collagen α3 chain. We previously reported that T3 peptide, an active subfragment of tumstatin, exerts cytoprotective effects on H2O2-induced apoptosis through the inhibition of intracellular reactive oxygen species (ROS) production in H9c2 cardiomyoblasts. In this study, we investigated whether T3 peptide has cardioprotective effects against I/R injury by using in vitro and ex vivo experimental models. H9c2 cardiomyoblasts were stimulated with oxygen and glucose deprivation (OGD) for 12 h followed by reoxygenation for 1-8 h (OGD/R; in vitro model). The cells were treated with T3 peptide (30-1000 ng/ml) during OGD. Ten minutes after the pre-perfusion of T3 peptide (300 ng/ml), Langendorff perfused rat hearts were exposed to ischemia for 30 min followed by reperfusion for 1 h (ex vivo model). T3 peptide inhibited OGD/R-induced apoptosis through the inhibition of mitochondrial ROS production and dysfunction in H9c2 cardiomyoblasts. T3 peptide also prevented I/R-induced cardiac dysfunction, arrhythmia and myocardial infarction in the perfused rat heart. In conclusion, we for the first time demonstrated that T3 peptide exerts cardioprotective effects against I/R injury.
Assuntos
Apoptose/efeitos dos fármacos , Autoantígenos/administração & dosagem , Cardiotônicos/administração & dosagem , Colágeno Tipo IV/administração & dosagem , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Autoantígenos/química , Autoantígenos/farmacologia , Cardiotônicos/farmacologia , Linhagem Celular , Colágeno Tipo IV/química , Colágeno Tipo IV/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/complicações , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Myelosuppression is a major and frequently dose-limiting side effect of anticancer therapy and is responsible for most treatment-related morbidity and mortality. In addition, repeated cycles of DNA damage and cell death of hematopoietic stem and progenitor cells, followed by compensatory proliferation and selection pressure, lead to genomic instability and pave the way for therapy-related myelodysplastic syndromes and secondary acute myeloid leukemia. Protection of hematopoietic stem and progenitor cells from chemo- and radiotherapy in patients with solid tumors would reduce both immediate complications and long-term sequelae. Epidermal growth factor (EGF) and prostaglandin E2 (PGE2) were reported to prevent chemo- or radiotherapy-induced myelosuppression in mice. We tested both molecules for potentially protective effects on human CD34+ cells in vitro and established a xenograft mouse model to analyze stress resistance and regeneration of human hematopoiesis in vivo EGF was neither able to protect human stem and progenitor cells in vitro nor to promote hematopoietic regeneration following sublethal irradiation in vivo PGE2 significantly reduced in vitro apoptotic susceptibility of human CD34+ cells to taxol and etoposide. This could, however, be ascribed to reduced proliferation rather than to a change in apoptosis signaling and BCL-2 protein regulation. Accordingly, 16,16-dimethyl-PGE2 (dmPGE2) did not accelerate regeneration of the human hematopoietic system in vivo Repeated treatment of sublethally irradiated xenograft mice with known antiapoptotic substances, such as human FLT3L and thrombopoietin (TPO), which suppress transcription of the proapoptotic BCL-2 proteins BIM and BMF, also only marginally promoted human hematopoietic regeneration in vivo.
Assuntos
Autoantígenos/farmacologia , Dinoprostona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Hematopoese/efeitos dos fármacos , Iodeto Peroxidase/farmacologia , Proteínas de Ligação ao Ferro/farmacologia , Proteínas de Membrana/farmacologia , Animais , Avaliação de Medicamentos , Humanos , Camundongos , Camundongos KnockoutRESUMO
Tumstatin7 (CNYYSNS) is an antitumor peptide derived from the NC1 domain of Type IV collagen that has been associated with tumor angiogenesis. In this work, we generated a peptide composed of tumstatin7 fused to TAT, a cell-internalizing peptide consisting of 11 amino acids. Tumstatin7-TAT was internalized by cells and triggered cell death. The new peptide was more potent in inducing B16F10 melanoma cell apoptosis in vitro than the shorter tumstatin7. Whereas tumstatin7-TAT significantly reduced tumor cell viability, tumstatin7 showed only weak effects even at the highest treatment concentration applied. Both tumstatin7-TAT and tumstatin7 inhibited cell migration in an in vitro wound healing model, and the former was more effective than the latter in inhibiting tumor growth in vivo. Combining the cell-internalizing property of TAT with the tumor-specific property of tumstatin7 may provide a useful adjunct to tumor therapy.
Assuntos
Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Produtos do Gene tat/metabolismo , Melanoma Experimental/tratamento farmacológico , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/administração & dosagem , Autoantígenos/química , Movimento Celular/efeitos dos fármacos , Colágeno Tipo IV/administração & dosagem , Colágeno Tipo IV/química , Feminino , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Peptídeos/química , Cicatrização/efeitos dos fármacosRESUMO
The present work elucidates the production of recombinant human asparaginase (rhASP) under optimized fermentation and downstream processes in Escherichia coli. The maximum biomass yield of 6.7â¯g/L was achieved with fed-batch fermentation. The highest rhASP inclusion bodies recovery yield (91%) was achieved with the optimized lysis conditions. The 8.0â¯M urea at pH 8.5 has shown efficient solubilization (94%) of rhASP inclusion bodies. The refolding efficiency of rhASP increased at pH 8.5 (84%) and temperature 25°C (86%). The diluted rhASP solution was concentrated and partially purified (92%) using cross flow filtration. A single step ion exchange chromatography is successfully achieved the maximum purity of ≥ 97%. The molecular mass of purified rhASP is confirmed as 34.1â¯kDa by mass spectrometry. The secondary structure of rhASP is characterized by FT-IR spectroscopy based on the structural elements. Finally, cell proliferative assay of purified rhASP is signifies the similar biological activity over the standard.
Assuntos
Asparaginase/biossíntese , Autoantígenos/biossíntese , Proteínas Recombinantes/biossíntese , Asparaginase/química , Asparaginase/isolamento & purificação , Asparaginase/farmacologia , Autoantígenos/química , Autoantígenos/isolamento & purificação , Autoantígenos/farmacologia , Técnicas de Cultura Celular por Lotes , Proliferação de Células/efeitos dos fármacos , Cromatografia por Troca Iônica , Escherichia coli , Fermentação , Humanos , Corpos de Inclusão/enzimologia , Redobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Cooperation between VEGFR2 and integrin αVß3 is critical for neovascularization in wound healing, cardiovascular ischemic diseases, ocular diseases, and tumor angiogenesis. In the present study, we developed a rule-based computational model to investigate the potential mechanism by which the Src-induced integrin association with VEGFR2 enhances VEGFR2 activation. Simulations demonstrated that the main function of integrin is to reduce the degradation of VEGFR2 and hence stabilize the activation signal. In addition, receptor synthesis rate and recruitment from internal compartment were found to be sensitive determinants of the activation state of VEGFR2. The model was then applied to simulate the effect of integrin-binding peptides such as tumstatin and cilengitide on VEGFR2 signaling. Further, computational modeling proposed potential molecular mechanisms for the angiogenesis-modulating activity of other integrin-binding peptides. The model highlights the complexity of the crosstalk between αVß3 integrin and VEGFR2 and the necessity of utilizing models to elucidate potential mechanisms in angiogenesis-modulating peptide therapy.
Assuntos
Autoantígenos , Colágeno Tipo IV , Células Endoteliais/metabolismo , Integrina alfaVbeta3/metabolismo , Modelos Biológicos , Neovascularização Patológica , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Autoantígenos/farmacologia , Colágeno Tipo IV/farmacocinética , Colágeno Tipo IV/farmacologia , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Venenos de Serpentes/farmacocinética , Venenos de Serpentes/farmacologiaRESUMO
Selective inhibition of T cells has been implied to prevent and/or treat autoimmune and inflammatory diseases. Some food compounds that have such immune-modulating functions may serve as nutritional approach to this purpose. In this study, we chose naringenin, a citrus fruits-derived compound with antiinflammatory property, to test this possibility. In this in vitro study, we stimulated mouse T cells with anti-CD3/CD28 (polyclonal TCR activation) or autoantigen MOG35-55 in the presence of naringenin. We found that naringenin dose-dependently suppressed anti-CD3/CD28 and MOG35-55-induced T cell proliferation, production of T cell cytokines IFN-γ, IL-17, IL-6 and TNF-α. We further showed that inhibited T cell proliferation was associated with T cell cycle arrest at G0/G1 phase, which was in turn related to delayed degradation of the cyclin-dependent kinase inhibitor p27kip1 and the down-regulation of retinoblastoma protein phosphorylation in activated T cells. Finally, it was revealed that all these T cell-suppressive effects might be related to naringenin's interference with IL-2/IL-2R-mediated signaling pathway and STAT5 phosphorylation in activated T cells. Our results confirmed T cell-suppressive activity of naringenin previously reported by us and others, but for the first time, it was shown that the working mechanism may involve its ability to modulate cell cycle progression, cell cycle-related proteins and IL-2/IL-2R signaling pathway. Together, these results further support proposed potential of naringenin being a preventive/therapeutic agent in T-cell-mediated autoimmune inflammatory disorders.
Assuntos
Flavanonas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Autoantígenos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/patologia , Feminino , Flavanonas/administração & dosagem , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-2/metabolismo , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição STAT5/metabolismo , Linfócitos T/imunologiaRESUMO
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with consecutive infarcts. However, cognitive dysfunctions representing one of the most commonest neuropsychiatric symptoms are frequently present despite the absence of any ischemic brain lesions. Data on the structural and functional basis of the neuropsychiatric symptoms are sparse. To examine the effect of APS on hippocampal neurogenesis and on white matter, we induced experimental APS (eAPS) in adult female Balb/C mice by immunization with ß2-glycoprotein 1. To investigate cell proliferation in the dentate gyrus granular cell layer (DG GCL), eAPS and control mice (n = 5, each) were injected with 5-bromo-2'-deoxyuridine (BrdU) once a day for 10 subsequent days. Sixteen weeks after immunization, eAPS resulted in a significant reduction of BrdU-positive cells in the DG GCL compared to control animals. However, double staining with doublecortin and NeuN revealed a largely preserved neurogenesis. Ultrastructural analysis of corpus callosum (CC) axons in eAPS (n = 6) and control mice (n = 7) revealed no significant changes in CC axon diameter or g-ratio. In conclusion, decreased cellular proliferation in the hippocampus of eAPS mice indicates a limited regenerative potential and may represent one neuropathological substrate of cognitive changes in APS while evidence for alterations of white matter integrity is lacking.
Assuntos
Síndrome Antifosfolipídica/induzido quimicamente , Síndrome Antifosfolipídica/patologia , Proliferação de Células , Giro Denteado/patologia , Animais , Anticorpos Antifosfolipídeos/metabolismo , Autoantígenos/farmacologia , Escala de Avaliação Comportamental , Bromodesoxiuridina/administração & dosagem , Bromodesoxiuridina/metabolismo , Diferenciação Celular/fisiologia , Corpo Caloso/ultraestrutura , Modelos Animais de Doenças , Feminino , Fluorescência , Camundongos , Camundongos Endogâmicos BALB C , Neurogênese , Radiossensibilizantes/administração & dosagem , Radiossensibilizantes/metabolismo , beta 2-Glicoproteína I/farmacologiaRESUMO
Many autoimmune diseases are associated with deranged bone metabolism. The resulting localized or systemic bone loss can compromise the quality of life of patients by causing local bone deformities or fragility fractures. There is emerging evidence that antibodies have a direct impact on key players of bone homeostasis, in particular osteoclasts. Clinical and pre-clinical studies provide insight into the function of autoantibodies related to Rheumatoid Arthritis (rheumatoid factor, anti-citrullinated protein antibodies, and anti-carbamylated protein antibodies) and their inflammation-independent interaction with bone cells. Furthermore, we summarize the current knowledge about neutralizing antibodies to the antiresorptive protein osteoprotegerin, which have been described in patients with Coeliac Disease, Rheumatoid Arthritis, and Spondyloarthritis.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/farmacologia , Doenças Ósseas Metabólicas/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/metabolismo , Animais , Anticorpos Antiproteína Citrulinada/uso terapêutico , Doenças Ósseas Metabólicas/imunologia , Reabsorção Óssea/imunologia , Osso e Ossos/imunologia , HumanosRESUMO
The extracellular matrix (ECM) creates the microenvironment of the tissue; an altered ECM in the asthmatic airway may be central in airway inflammation and remodelling. Tumstatin is a collagen IV-derived matrikine reduced in the asthmatic airway wall that reverses airway inflammation and remodelling in small and large animal models of asthma. This study hypothesized that the mechanisms underlying the broad asthma-resolving effects of tumstatin were due to autocrine remodelling of the ECM. Neutrophils and endothelial cells were seeded on decellularized ECM of non-asthmatic (NA) or asthmatic (A) airway smooth muscle (ASM) cells previously exposed to tumstatin in the presence or absence of a broad matrix metalloproteinase inhibitor, Marimastat. Gene expression in NA and A ASM induced by tumstatin was assessed using RT-PCR arrays. The presence of tumstatin during ECM deposition affected neutrophil and endothelial cell properties on both NA and A ASM-derived matrices and this was only partly due to MMP activity. Gene expression patterns in response to tumstatin in NA and A ASM cells were different. Tumstatin may foster an anti-inflammatory and anti-angiogenic microenvironment by modifying ASM-derived ECM. Further work is required to examine whether restoring tumstatin levels in the asthmatic airway represents a potential novel therapeutic approach.
Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Matriz Extracelular/efeitos dos fármacos , Metaloproteinases da Matriz/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Remodelação das Vias Aéreas , Asma/genética , Asma/metabolismo , Asma/patologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Quimiotaxia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Interleucina-8/farmacologia , Metaloproteinases da Matriz/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologiaRESUMO
Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca2+-binding capacity; thus, Syt IV plays an inhibitory role in Ca2+-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca2+-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release.
Assuntos
Neurônios/metabolismo , Proteólise , Sinaptotagminas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Autoantígenos/farmacologia , Células COS , Chlorocebus aethiops , Corpo Estriado/citologia , Exocitose/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Neural/farmacologia , Oligopeptídeos/farmacologia , Células PC12/efeitos dos fármacos , Células PC12/ultraestrutura , Inibidores de Proteassoma/farmacologia , Transporte Proteico , Proteólise/efeitos dos fármacos , Ratos , Sinaptotagminas/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Autoimmune diseases occur when the immune system incorrectly recognizes self-molecules as foreign; in the case of multiple sclerosis (MS), myelin is attacked. Intriguingly, new studies reveal toll-like receptors (TLRs), pathways usually involved in generating immune responses against pathogens, play a significant role in driving autoimmune disease in both humans and animal models. We reasoned polyplexes formed from myelin self-antigen and regulatory TLR antagonists might limit TLR signaling during differentiation of myelin-specific T cells, inducing tolerance by biasing T cells away from inflammatory phenotypes. Complexes were formed by modifying myelin peptide with cationic amino acids to create peptides able to condense the anionic nucleic-acid based TLR antagonist. These immunological polyplexes eliminate synthetic polymers commonly used to condense polyplexes and do not rely on gene expression; however, the complexes mimic key features of traditional polyplexes such as tunable loading and co-delivery. Using these materials and classic polyplex analysis techniques, we demonstrate condensation of both immune signals, protection from enzymatic degradation, and tunable physicochemical properties. We show polyplexes reduce TLR signaling, and in primary dendritic cell and T cell co-culture, reduce myelin-driven inflammation. During mouse models of MS, these tolerogenic polyplexes improve the progression, severity, and incidence of disease.
Assuntos
Autoantígenos/imunologia , Autoantígenos/uso terapêutico , Autoimunidade/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Ácidos Nucleicos Peptídicos/uso terapêutico , Receptores Toll-Like/imunologia , Animais , Autoantígenos/farmacologia , Autoimunidade/efeitos dos fármacos , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Nucleicos , Ácidos Nucleicos Peptídicos/imunologia , Ácidos Nucleicos Peptídicos/farmacologia , Transdução de Sinais , Receptores Toll-Like/antagonistas & inibidores , Resultado do TratamentoRESUMO
Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10(+)-20 µm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.
Assuntos
Asma/fisiopatologia , Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Pulmão/patologia , Remodelação Vascular/efeitos dos fármacos , Resistência das Vias Respiratórias/efeitos dos fármacos , Alérgenos/administração & dosagem , Animais , Asma/imunologia , Autoantígenos/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Doença Crônica , Colágeno Tipo IV/administração & dosagem , Dermatophagoides pteronyssinus/imunologia , Feminino , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Músculo Liso/metabolismo , Carneiro Doméstico , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The present study was aimed to investigate the effect of tumstatin on inhibition of proliferation and induction of apoptosis in Saos-2 human osteosarcoma cells and to understand the mechanism involved. Inhibition of cell proliferation was analyzed by MTT assay and induction of apoptosis through nuclear fragmentation assay. Viability of Saos-2 cells was reduced to 19% on treatment with 25 µM concentration of tumstatin after 48 h. Presence of characteristic apoptotic nuclei, rounded cell shape and shrunken size were caused by tumstatin treatment at 25 µM concentration. The level of mRNA corresponding to PTEN, FasR and FasL was increased significantly in tumstatin treated Saos-2 cells compared to untreated control. Investigation of the mechanism revealed NF-κB activation by phosphorylation on serine 536. The activated NF-κB was translocated into the nucleus from the cytoplasm on treatment with tumstatin. Degradation of the IκBα by tumstatin was found to be much slower compared to that induced by treatment with TNF-α. Thus, tumstatin inhibits proliferation and induces apoptosis in Saos-2 cells through activation of NF-κB and its translocation to the nucleus. Therefore, tumstatin can play an important role in the treatment of osteosarcoma.
Assuntos
Apoptose/efeitos dos fármacos , Autoantígenos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Colágeno Tipo IV/farmacologia , Osteossarcoma/tratamento farmacológico , Fator de Transcrição RelA/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Osteossarcoma/patologia , PTEN Fosfo-Hidrolase/genética , FosforilaçãoRESUMO
Anti-cytokine therapeutic antibodies have been demonstrated to be effective in the treatment of several auto-immune disorders. However, The problems in antibody manufacture and the immunogenicity caused by multiple doses of antibodies inspire people to use auto-cytokine as immunogen to induce anti-cytokine antibodies. Nevertheless, the tolerance for inducing immune response against self-antigen has hindered the wide application of the strategy. To overcome the tolerance, here we proposed a strategy using the inter-species cytokine as immunogen for active immunization (TISCAI) to induce anti-cytokine antibody. As a proof of concept, an inter-species cytokine RANKL was successfully used as immunogen to induce anti-RANKL immune response. Furthermore, to prevent undesirable side-effects, the human RANKL was mutated based on the crystal structure of the complex of human RANKL and its rodent counterpart receptor RANK. We found, the antibodies produced blocked the osteoclast development in vitro and osteoporosis in OVX rat models. The results demonstrated this strategy adopted is very useful for general anti-cytokine immunotherapy for different diseases settings.