RESUMO
BACKGROUND/AIM: Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor prognosis. ARID1A gene mutations, commonly found in ovarian clear cell carcinoma (OCCC), contribute to its pathogenesis. Recent data revealed that the ARID1A mutation is related to better outcomes of cancer immunotherapy. Thus, this study aimed to investigate the immunotherapy treatment susceptibility of OCCC bearing ARID1A mutations. MATERIALS AND METHODS: Expression of ARID1A was analyzed using western blotting in ovarian cancer cell lines. OCCC cell lines JHOC-9 and RMG-V were engineered to overexpress NY-ESO-1, HLA-A*02:01, and ARID1A. Sensitivity to chemotherapy and T cell receptor-transduced T (TCR-T) cells specific for NY-ESO-1 was assessed in ARID1A-restored cells compared to ARID1A-deficient wild-type cells. RESULTS: JHOC-9 cells and RMG-V cells showed no expression of ARID1A protein. Overexpression of ARID1A in JHOC-9 and RMG-V cells did not impact sensitivity to gemcitabine. While ARID1A overexpression decreased sensitivity to cisplatin in RMG-V cells, it had no such effect in JHOC-9 cells. ARID1A overexpression reduced the reactivity of NY-ESO-1-specific TCR-T cells, as observed by the IFNγ ESLIPOT assay. CONCLUSION: Cancer immunotherapy is an effective approach to target ARID1A-deficient clear cell carcinoma of the ovary.
Assuntos
Adenocarcinoma de Células Claras , Proteínas de Ligação a DNA , Neoplasias Ovarianas , Linfócitos T Citotóxicos , Fatores de Transcrição , Humanos , Feminino , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Adenocarcinoma de Células Claras/patologia , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/imunologia , Adenocarcinoma de Células Claras/metabolismo , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Tumoral , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Proteínas de MembranaRESUMO
We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24+ 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and ß constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24+ 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with in vivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45+ T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8+ T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Animais , Camundongos , Linfócitos T CD8-Positivos , Antígeno HLA-A24 , DNA Complementar/metabolismo , Antígeno Ki-67/metabolismo , Linfócitos T Citotóxicos , Peptídeos , Osteossarcoma/genética , Epitopos/metabolismo , Neoplasias Ósseas/metabolismo , Receptores de Antígenos de Linfócitos TRESUMO
Eribulin inhibits microtubule polymerization and improves the overall survival of patients with recurrent metastatic breast cancer. A subgroup analysis revealed a low neutrophil to lymphocyte ratio (NLR) (<3) to be a prognostic factor of eribulin treatment. We thus hypothesized that eribulin might be related to the immune response for breast cancer cells and we analyzed the effects of eribulin on the immune system. Immunohistochemical staining revealed that human leukocyte antigen (HLA) class I expression was increased in clinical samples after eribulin treatment. In vitro assays revealed that eribulin treatment increased HLA class I expression in breast cancer line cells. RNA-sequencing demonstrated that eribulin treatment increased the expression of the NOD-like family CARD domain-containing 5 (NLRC5), a master regulator of HLA class I expression. Eribulin treatment increased the NY-ESO-1-specific T-cell receptor (TCR) transduced T (TCR-T) cell response for New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) overexpressed breast cancer cells. The eribulin and TCR-T combined therapy model revealed that eribulin and immunotherapy using TCR-T cells has a synergistic effect. In summary, eribulin increases the expression of HLA class 1 via HLA class 1 transactivatior NLRC5 and eribulin combination with immunotherapy can be effective for the treatment of breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas NLR , Domínio de Ativação e Recrutamento de Caspases , Recidiva Local de Neoplasia , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Neoplasias , Antígenos HLA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Evasion from immunity is a major obstacle to the achievement of successful cancer immunotherapy. Hybrids derived from cell-cell fusion are theoretically associated with tumor heterogeneity and progression by conferring novel properties on tumor cells, including drug resistance and metastatic capacity; however, their impact on immune evasion remains unknown. Here, we investigated the potency of tumor-macrophage hybrids in immune evasion. Hybrids were established by co-culture of a melanoma cell line (A375 cells) and type 2 macrophages. The hybrids showed greater migration ability and greater tumorigenicity than the parental melanoma cells. The hybrids showed heterogeneous sensitivity to New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T-cell receptor-transduced T (TCR-T) cells and two out of four hybrid clones showed less sensitivity to TCR-T compared with the parental cells. An in vitro tumor heterogeneity model revealed that the TCR-T cells preferentially killed the parental cells compared with the hybrids and the survival rate of the hybrids was higher than that of the parental cells, indicating that the hybrids evade killing by TCR-T cells efficiently. Analysis of a single-cell RNA sequencing dataset of patients with melanoma revealed that a few macrophages expressed RNA encoding melanoma differentiation antigens including melan A, tyrosinase, and premelanosome protein, which indicated the presence of hybrids in primary melanoma. In addition, the number of potential hybrids was correlated with a poorer response to immune checkpoint blockade. These results provide evidence that melanoma-macrophage fusion has a role in tumor heterogeneity and immune evasion. © 2023 The Pathological Society of Great Britain and Ireland.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Melanoma , Humanos , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Melanoma/metabolismo , Macrófagos/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de NeoplasiasRESUMO
BACKGROUND/AIM: Malignant melanoma is a fatal skin cancer and is among the most immunogenic malignancies expressing melanoma-differentiation antigens and neoantigens. SRY-related HMG-box 10 (SOX10) is a transcription factor and a neural-crest differentiation marker that is used as a diagnostic marker for melanoma whilst playing a role in melanoma initiation through activation of the SOX10-MITF axis. SOX10 was shown to play a role in melanoma initiation by inducing expression of immune checkpoint molecules (e.g., HVEM and CEACAM1). In this study, we aimed to investigate the relationship between SOX10 and the expression an immune checkpoint molecule, programmed death-1 ligand 1 (PD-L1). MATERIALS AND METHODS: SOX10 overexpression and knockdown was performed using SOX10 gene transfection and SOX10 siRNA transfection into A375 melanoma cells. PD-L1 expression was assessed by flow cytometry and western blotting. T cell response was evaluated using NY-ESO-1 specific TCR-transduced T (TCR-T) cells by IFNγ ELISPOT assay. RESULTS: SOX10 overexpression increased the expression of PD-L1, whereas SOX10 knockdown, using siRNA, decreased its expression. IFNγ ELISPOT assay revealed that overexpression of SOX10 decreased the susceptibility of cells to NY-ESO-1-specific TCR-T cells. CONCLUSION: SOX10 has a role in the intrinsic immune suppressive mechanisms of melanoma through expression of PD-L1.
Assuntos
Proteínas de Checkpoint Imunológico , Melanoma , Humanos , Linfócitos T/metabolismo , Antígeno B7-H1/genética , Ligantes , Receptor de Morte Celular Programada 1/metabolismo , Melanoma/metabolismo , Receptores de Antígenos de Linfócitos T , Fatores de Transcrição SOXE/genéticaRESUMO
Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Bexiga Urinária , Humanos , Linhagem Celular Tumoral , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/terapia , Cisplatino/uso terapêutico , Imunoterapia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação para Cima , Linfócitos T Citotóxicos/citologia , Células-Tronco Neoplásicas/efeitos dos fármacosRESUMO
Immune checkpoint inhibitor-based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo-radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death-ligand 1 (PD-L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD-L1 on OSCC cell lines, the gene and total protein expression levels of PD-L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP-induced PD-L1 attenuated the target-specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum-based chemotherapy for OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Antígeno B7-H1/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Microambiente TumoralRESUMO
The association between type 2 diabetes mellitus and prostate cancer is still under investigation, and the relationship between hyperinsulinemia and prostate cancer stem-like cells (CSCs) is elusive. Here, we investigated the function of insulin/AKT signaling in prostate CSCs. We isolated prostate CSCs as aldehyde dehydrogenase 1-high (ALDH1high) cells from the human prostate cancer 22Rv1 cell line using an ALDEFLUOR assay and established several ALDH1high and ALDH1low clones. ALDH1high clones showed high ALDH1 expression which is a putative CSC marker; however, they showed heterogeneity regarding tumorigenicity and resistance to radiation and chemotherapy. Interestingly, all ALDH1high clones showed lower phosphorylated AKT (Ser473) (pAKT) levels than the ALDH1low clones. PI3K/AKT signaling is a key cell survival pathway and we analyzed radiation resistance under AKT signaling activation by insulin. Insulin increased pAKT levels in ALDH1high and ALDH1low cells; the fold increase rate of pAKT was higher in ALDH1high cells than in ALDH1low cells. Insulin induced resistance to radiation and chemotherapy in ALDH1high cells, and the increased levels of pAKT induced by insulin were significantly related to radiation resistance. These results suggest that ALDH1 suppresses baseline pAKT levels, but AKT can be activated by insulin, leading to treatment resistance.
Assuntos
Aldeído Desidrogenase/metabolismo , Insulina/farmacologia , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação , Transdução de Sinais , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: The therapeutic benefits of mesenchymal stromal cells (MSCs) include treatment of chronic inflammation. However, given the short-lived engraftment of these cells in vivo, their therapeutic efficacy remains mysterious. Transient induction of cellular senescence contributes to activation of immune cells, which promotes clearance of damaged cells during tissue remodelling. This may occur in tissue-resident mesenchymal progenitor cells during regeneration. Elucidation of the role of senescence in tissue-resident mesenchymal progenitor cells during regeneration would provide insight into the profile of therapeutic MSCs for treatment of chronic inflammatory disease. METHODS: We evaluated multipotent mesenchymal progenitor cells, termed fibro/adipogenic progenitors (FAPs), and immune cells in acute muscle injury (AMI) model mice and mice with myosin-induced experimental autoimmune myositis, a model of chronic inflammatory myopathy (CIM). Human bone marrow MSCs were optimised for the treatment of CIM using placental extract. FINDING: FAPs in AMI transiently expressed p16INK4A on days 1 and 2 after injury and recruited phagocytic immune cells, whereas in CIM, p16INK4A expression in FAPs was low. Cellular senescence occurs during the natural maturation of the placenta. Therefore, we used human placental extract to induce p16INK4A expression in therapeutic human bone marrow MSCs in culture. Treatment of CIM with p16INK4A-expressing MSCs promoted tissue remodelling by transiently increasing the abundance of engrafted MSCs, inducing cellular senescence in innate FAPs, and recruiting phagocytic immune cells. INTERPRETATION: MSCs may exert their effect by remodelling the chronic inflammatory environment via senescence-related regenerative processes.
Assuntos
Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Citofagocitose/genética , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular/genética , Miosite/etiologia , Animais , Biomarcadores , Proliferação de Células , Senescência Celular/imunologia , Doença Crônica , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Citocinas/metabolismo , Citofagocitose/imunologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Camundongos , Miosite/metabolismo , Miosite/patologia , Regeneração , Medicina RegenerativaRESUMO
Increasing evidence suggests that an enriched environment (EE) ameliorates cognitive impairment by promoting repair of brain damage. However, the mechanisms by which this occurs have not been determined. To address this issue, we investigated whether an EE enhanced the capability of endogenous bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to prevent hippocampal damage due to diabetes by focusing on miRNA carried in BM-MSC-derived exosomes. In diabetic streptozotocin (STZ) rats housed in an EE (STZ/EE), cognitive impairment was significantly reduced, and both neuronal and astroglial damage in the hippocampus was alleviated compared with STZ rats housed in conventional cages (STZ/CC). BM-MSCs isolated from STZ/CC rats had functional and morphological abnormalities that were not detected in STZ/EE BM-MSCs. The miR-146a levels in exosomes in conditioned medium of cultured BM-MSCs and serum from STZ/CC rats were decreased compared with non-diabetic rats, and the level was restored in STZ/EE rats. Thus, the data suggest that increased levels of miR-146a in sera were derived from endogenous BM-MSCs in STZ/EE rats. To examine the possibility that increased miR-146a in serum may exert anti-inflammatory effects on astrocytes in diabetic rats, astrocytes transfected with miR-146a were stimulated with advanced glycation end products (AGEs) to mimic diabetic conditions. The expression of IRAK1, NF-κB, and tumor necrosis factor-α was significantly higher in AGE-stimulated astrocytes, and these factors were decreased in miR-146a-transfected astrocytes. These results suggested that EEs stimulate up-regulation of exosomal miR-146a secretion by endogenous BM-MSCs, which exerts anti-inflammatory effects on damaged astrocytes and prevents diabetes-induced cognitive impairment.
Assuntos
Disfunção Cognitiva/prevenção & controle , Diabetes Mellitus Experimental/patologia , MicroRNAs/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células da Medula Óssea/citologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Células Cultivadas , Disfunção Cognitiva/etiologia , Diabetes Mellitus Experimental/complicações , Exossomos/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Masculino , Aprendizagem em Labirinto , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/sangue , Estresse Oxidativo , Ratos , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Although the cognitive impairment in Alzheimer's disease (AD) is believed to be caused by amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs), several postmortem studies have reported cognitive normal subjects with AD brain pathology. As the mechanism underlying these discrepancies has not been clarified, we focused the neuroprotective role of astrocytes. After examining 47 donated brains, we classified brains into 3 groups, no AD pathology with no dementia (N-N), AD pathology with no dementia (AD-N), and AD pathology with dementia (AD-D), which represented 41%, 21%, and 38% of brains, respectively. No differences were found in the accumulation of Aß plaques or NFTs in the entorhinal cortex (EC) between AD-N and AD-D. Number of neurons and synaptic density were increased in AD-N compared to those in AD-D. The astrocytes in AD-N possessed longer or thicker processes, while those in AD-D possessed shorter or thinner processes in layer I/II of the EC. Astrocytes in all layers of the EC in AD-N showed enhanced GLT-1 expression in comparison to those in AD-D. Therefore these activated forms of astrocytes with increased GLT-1 expression may exert beneficial roles in preserving cognitive function, even in the presence of Aß and NFTs.
Assuntos
Doença de Alzheimer/patologia , Astrócitos/enzimologia , Astrócitos/patologia , Encéfalo/patologia , Transtornos Cognitivos/patologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/análise , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/análise , Transportador 2 de Aminoácido Excitatório , Feminino , Humanos , Masculino , Emaranhados Neurofibrilares/patologiaRESUMO
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the most valuable source of autologous cells for transplantation and tissue regeneration to treat osteoporosis. Although BM-MSCs are the primary cells responsible for maintaining bone metabolism and homeostasis, their regenerative ability may be attenuated in postmenopausal osteoporosis patients. Therefore, we first examined potential abnormalities of BM-MSCs in an oestrogen-deficient rat model constructed by ovariectomy (OVX-MSCs). Cell proliferation, mobilisation, and regulation of osteoclasts were downregulated in OVX-MSCs. Moreover, therapeutic effects of OVX-MSCs were decreased in OVX rats. Accordingly, we developed a new activator for BM-MSCs using human umbilical cord extracts, Wharton's jelly extract supernatant (WJS), which improved cell proliferation, mobilisation and suppressive effects on activated osteoclasts in OVX-MSCs. Bone volume, RANK and TRACP expression of osteoclasts, as well as proinflammatory cytokine expression in bone tissues, were ameliorated by OVX-MSCs activated with WJS (OVX-MSCs-WJ) in OVX rats. Fusion and bone resorption activity of osteoclasts were suppressed in macrophage-induced and primary mouse bone marrow cell-induced osteoclasts via suppression of osteoclast-specific genes, such as Nfatc1, Clcn7, Atp6i and Dc-stamp, by co-culture with OVX-MSCs-WJ in vitro. In this study, we developed a new activator, WJS, which improved the functional abnormalities and therapeutic effects of BM-MSCs on postmenopausal osteoporosis.
Assuntos
Densidade Óssea/efeitos dos fármacos , Misturas Complexas/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Osteoclastos/metabolismo , Osteoporose/terapia , Cordão Umbilical/química , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Técnicas de Cocultura , Misturas Complexas/isolamento & purificação , Modelos Animais de Doenças , Feminino , Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoclastos/citologia , Osteoporose/etiologia , Osteoporose/genética , Osteoporose/patologia , Ovariectomia/efeitos adversos , Células RAW 264.7 , Ratos , Ratos Wistar , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismo , Tomografia Computadorizada por Raios X , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Geleia de Wharton/químicaRESUMO
Bone marrow-derived mesenchymal stem cells (BM-MSC) has been applied as the most valuable source of autologous cell transplantation for various diseases including diabetic complications. However, hyperglycemia may cause abnormalities in intrinsic BM-MSC which might lose sufficient therapeutic effects in diabetic patients. We demonstrated the functional abnormalities in BM-MSC derived from both type 1 and type 2 diabetes models in vitro, which resulted in loss of therapeutic effects in vivo in diabetic nephropathy (DN). Then, we developed a novel method to improve abnormalities in BM-MSC using human umbilical cord extracts, namely Wharton's jelly extract supernatant (WJs). WJs is a cocktail of growth factors, extracellular matrixes and exosomes, which ameliorates proliferative capacity, motility, mitochondrial degeneration, endoplasmic reticular functions and exosome secretions in both type 1 and type 2 diabetes-derived BM-MSC (DM-MSC). Exosomes contained in WJs were a key factor for this activation, which exerted similar effects to complete WJs. DM-MSC activated by WJs ameliorated renal injury in both type 1 and type 2 DN. In this study, we developed a novel activating method using WJs to significantly increase the therapeutic effect of BM-MSC, which may allow effective autologous cell transplantation.
Assuntos
Nefropatias Diabéticas/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Medula Óssea , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/terapia , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Exossomos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Ratos Endogâmicos OLETF , Ratos Sprague-Dawley , Geleia de Wharton/químicaRESUMO
Bone marrow-derived mesenchymal stem cells (MSCs) have contributed to the improvement of diabetic nephropathy (DN); however, the actual mediator of this effect and its role has not been characterized thoroughly. We investigated the effects of MSC therapy on DN, focusing on the paracrine effect of renal trophic factors, including exosomes secreted by MSCs. MSCs and MSC-conditioned medium (MSC-CM) as renal trophic factors were administered in parallel to high-fat diet (HFD)-induced type 2 diabetic mice and streptozotocin (STZ)-induced insulin-deficient diabetic mice. Both therapies showed approximately equivalent curative effects, as each inhibited the exacerbation of albuminuria. They also suppressed the excessive infiltration of BMDCs into the kidney by regulating the expression of the adhesion molecule ICAM-1. Proinflammatory cytokine expression (e.g., TNF-α) and fibrosis in tubular interstitium were inhibited. TGF-ß1 expression was down-regulated and tight junction protein expression (e.g., ZO-1) was maintained, which sequentially suppressed the epithelial-to-mesenchymal transition of tubular epithelial cells (TECs). Exosomes purified from MSC-CM exerted an anti-apoptotic effect and protected tight junction structure in TECs. The increase of glomerular mesangium substrate was inhibited in HFD-diabetic mice. MSC therapy is a promising tool to prevent DN via the paracrine effect of renal trophic factors including exosomes due to its multifactorial action.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/terapia , Túbulos Renais/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Albuminúria , Animais , Células da Medula Óssea/patologia , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Exossomos/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos Endogâmicos LewRESUMO
The incidence of dementia is higher in diabetic patients, but no effective treatment has been developed. This study showed that rat bone marrow mesenchymal stem cells (BM-MSCs) can improve the cognitive impairments of STZ-diabetic mice by repairing damaged neurons and astrocytes. The Morris water maze test demonstrated that cognitive impairments induced by diabetes were significantly improved by intravenous injection of BM-MSCs. In the CA1 region of the hippocampus, degeneration of neurons and astrocytes, as well as synaptic loss, were prominent in diabetes, and BM-MSC treatment successfully normalized them. Since a limited number of donor BM-MSCs was observed in the brain parenchyma, we hypothesized that humoral factors, especially exosomes released from BM-MSCs, act on damaged neurons and astrocytes. To investigate the effectiveness of exosomes for treatment of diabetes-induced cognitive impairment, exosomes were purified from the culture media and injected intracerebroventricularly into diabetic mice. Recovery of cognitive impairment and histological abnormalities similar to that seen with BM-MSC injection was found following exosome treatment. Use of fluorescence-labeled exosomes demonstrated that injected exosomes were internalized into astrocytes and neurons; these subsequently reversed the dysfunction. The present results indicate that exosomes derived from BM-MSCs might be a promising therapeutic tool for diabetes-induced cognitive impairment.
Assuntos
Astrócitos/fisiologia , Disfunção Cognitiva/terapia , Complicações do Diabetes/terapia , Exossomos/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neurônios/fisiologia , Administração Intravenosa , Animais , Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Camundongos , Ratos , Resultado do TratamentoRESUMO
UNLABELLED: The role of the cytokine, macrophage migration inhibitory factor (MIF), and its receptor, CD74, was assessed in autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC). Two MIF promoter polymorphisms, a functional -794 CATT5-8 microsatellite repeat (rs5844572) and a -173 G/C single-nucleotide polymorphism (rs755622), were analyzed in DNA samples from over 500 patients with AIH, PBC, and controls. We found a higher frequency of the proinflammatory and high-expression -794 CATT7 allele in AIH, compared to PBC, whereas lower frequency was found in PBC, compared to both AIH and healthy controls. MIF and soluble MIF receptor (CD74) were measured by enzyme-linked immunosorbent assay in 165 serum samples of AIH, PBC, and controls. Circulating serum and hepatic MIF expression was elevated in patients with AIH and PBC versus healthy controls. We also identified a truncated circulating form of the MIF receptor, CD74, that is released from hepatic stellate cells and that binds MIF, neutralizing its signal transduction activity. Significantly higher levels of CD74 were found in patients with PBC versus AIH and controls. CONCLUSIONS: These data suggest a distinct genetic and immunopathogenic basis for AIH and PBC at the MIF locus. Circulating MIF and MIF receptor profiles distinguish PBC from the more inflammatory phenotype of AIH and may play a role in pathogenesis and as biomarkers of these diseases.
Assuntos
Hepatite Autoimune/genética , Hepatite Autoimune/fisiopatologia , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/fisiologia , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/fisiopatologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/fisiologia , Biomarcadores/metabolismo , Biópsia , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Frequência do Gene/genética , Hepatite Autoimune/patologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/patologia , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
OBJECTIVE: To study the effect of the innate cytokine macrophage migration inhibitory factor (MIF) on the susceptibility and severity of systemic lupus erythematosus (SLE) in a multinational population of 1,369 Caucasian and African American patients. METHODS: Two functional polymorphisms in the MIF gene, a -794 CATT(5-8) microsatellite repeat (rs5844572) and a -173 G/C single-nucleotide polymorphism (rs755622), were assessed for association with SLE in 3,195 patients and healthy controls. We also measured MIF plasma levels in relation to genotypes and clinical phenotypes, and assessed Toll-like receptor 7 (TLR-7)-stimulated MIF production in vitro. RESULTS: Both Caucasians and African Americans with the high-expression MIF haplotype -794 CATT(7)/-173*C had a lower incidence of SLE (in Caucasians, odds ratio [OR] 0.63, 95% confidence interval [95% CI] 0.53-0.89, P = 0.001; in African Americans, OR 0.46, 95% CI 0.23-0.95, P = 0.012). In contrast, among patients with established SLE, reduced frequencies of low-expression MIF genotypes (-794 CATT(5)) were observed in those with nephritis, those with serositis, and those with central nervous system (CNS) involvement when compared to patients without end-organ involvement (P = 0.023, P = 0.005, and P = 0.04, respectively). Plasma MIF levels and TLR-7-stimulated MIF production in vitro reflected the underlying MIF genotype of the studied groups. CONCLUSION: These findings suggest that MIF, which has both proinflammatory properties and macrophage and B cell survival functions, exerts a dual influence on the immunopathogenesis of SLE. High-expression MIF genotypes are associated with a reduced susceptibility to SLE and may contribute to an enhanced clearance of infectious pathogens. Once SLE develops, however, low-expression MIF genotypes may protect from ensuing inflammatory end-organ damage.
Assuntos
Predisposição Genética para Doença/genética , Lúpus Eritematoso Sistêmico/genética , Fatores Inibidores da Migração de Macrófagos/genética , Polimorfismo de Nucleotídeo Único/genética , Índice de Gravidade de Doença , Adulto , Negro ou Afro-Americano/etnologia , Estudos de Casos e Controles , Estudos Transversais , Feminino , Genótipo , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/etnologia , Fatores Inibidores da Migração de Macrófagos/sangue , Masculino , Pessoa de Meia-Idade , Fenótipo , Fator de Necrose Tumoral alfa/sangue , População Branca/etnologiaRESUMO
We generated an evolutionary computer program that generates complementary peptide (C-pep) sequences, with the potential to interact with a target peptide, by comparing several physico-chemical parameters of each pair of the complementary peptides being analyzed. We generated C-peps to target several molecules. About 30% of synthesized C-peps interfered with the function of their targets. C5a stimulates generation of TNFα and other inflammatory cytokines. Inhibition of C5a should be effective against sepsis, which impairs the status of cancer-bearing patients. One of the inhibitory C-peps of C5a, termed AcPepA, was effective in Cynomolgus monkeys intravenously infused with a lethal dose of bacterial LPS (4 mg/kg) destined to die. The monkeys were rescued by intravenous administration of 2 mg/kg/h of AcPepA. The excellent therapeutic effect of AcPepA is likely to be due to restriction of high mobility group box 1 (HMGB1) surge induced by the effect of C5a on C5L2, which is the second C5a receptor, since the released HMGB1 has the capacity to stimulate TLR4 as an endogeneous ligand resulting in further activation of inflammatory cells to release inflammatory cytokines forming a positive feedback circuit of inflammation.
Assuntos
Terapia de Alvo Molecular , Biblioteca de Peptídeos , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Complemento C5a/antagonistas & inibidores , Citocinas/metabolismo , Evolução Molecular Direcionada , Avaliação Pré-Clínica de Medicamentos , Endotoxemia/tratamento farmacológico , Endotoxemia/patologia , Endotoxemia/fisiopatologia , Retroalimentação Fisiológica , Proteína HMGB1/fisiologia , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Lipopolissacarídeos/toxicidade , Pulmão/patologia , Macaca fascicularis , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Receptor da Anafilatoxina C5a/fisiologia , Software , Relação Estrutura-Atividade , Receptor 4 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/análiseRESUMO
We examined the role of macrophage migration inhibitory factor (MIF) in the generation of the Th2 response using MIF-deficient mice in a model of epicutaneous sensitization to ovalbumin. Lymph node cells from sensitized MIF-deficient mice produce lower levels of Th2 cytokines after antigen challenge when compared to their wild-type counterparts. Sensitized mice lacking MIF show less pulmonary inflammation after intranasal antigen exposure. Mice deficient in CD74, the MIF receptor, also are unable to generate an inflammatory response to epicutaneous sensitization. Examination of the elicitation phase of the atopic response using DO11.10 OVA TCR transgenic animals shows that T cell proliferation and IL-2 production are strongly impaired in MIF-deficient T cells. This defect is most profound when both T cells and antigen-presenting cells are lacking MIF. These data suggest that MIF is crucial both for the sensitization and the elicitation phases of a Th2-type immune response in allergic disease.