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1.
Medicine (Baltimore) ; 98(47): e18067, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31764838

RESUMO

Osteoporosis is a complication of type 2 diabetes mellitus (T2DM). Blockade of receptor activator of nuclear factor kappa-B ligand (RANKL) improves osteoporosis, but might also improve glucose tolerance through reduction of hepatic insulin resistance. However, the effect of denosumab (a human monoclonal antibody of RANKL) upon glycemic and metabolic parameters is controversial. We revealed the effect of denosumab upon glycemic and metabolic parameters for 52 weeks. We evaluated 20 individuals diagnosed with both osteoporosis (male and female: postmenopausal) and T2DM. We measured glycemic and metabolic parameters before and 26/52 weeks after administration of denosumab (60 mg per 26 weeks) without changing any other medication each patient was taking. All patients completed the study without complications and the T-score (lumbar spine and femoral neck) improved significantly from baseline to 52 weeks after denosumab administration (P < .001, .001, respectively). None of the glycemic parameters changed significantly from baseline to 26 weeks after denosumab administration, but levels of glycated hemoglobin and homeostasis model assessment of insulin resistance improved significantly from baseline to 52 weeks after administration (P = .019, .008, respectively). The levels of liver enzymes did not change significantly from baseline to 26 weeks after denosumab administration, but levels of aspartate transaminase and alanine aminotransferase improved significantly from baseline to 52 weeks after administration (P = .014, .004, respectively). None of the markers of lipid metabolism and body mass index changed significantly from baseline to 26/52 weeks after denosumab administration. These data demonstrated that denosumab is useful for T2DM patients with osteoporosis for glycemic control via improvement of insulin resistance. Also, the effect of denosumab might be due to improvement of hepatic function.


Assuntos
Glicemia/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Conservadores da Densidade Óssea/uso terapêutico , Denosumab/farmacologia , Denosumab/uso terapêutico , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Osteoporose/metabolismo , Idoso , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Masculino , Osteoporose/etiologia , Ligante RANK/imunologia
2.
Inflamm Res ; 68(10): 889-900, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372663

RESUMO

OBJECTIVE: To investigate the participation of canonical Wnt and NF-κB signaling pathways in an experimental model of chronic arthritis induced by methylated bovine serum albumin (mBSA) in rat temporomandibular joint (TMJ). MATERIALS AND METHODS: Wistar rats were sensitized by mBSA+Complete Freund Adjuvant (CFA)/Incomplete Freund Adjuvant (IFA) on the first 14 days (1 ×/week). Subsequently, they received 1, 2 or 3 mBSA or saline solution injections into the TMJ (1 ×/week). Hypernociceptive threshold was assessed during the whole experimental period. 24 h after the mBSA injections, the TMJs were removed for histopathological and immunohistochemical analyses for TNF-α, IL-1ß, NF-κB, RANKL, Wnt-10b, ß-catenin and DKK1. RESULTS: The nociceptive threshold was significantly reduced after mBSA injections. An inflammatory infiltrate and thickening of the synovial membrane were observed only after mBSA booster injections. Immunolabeling of TNF-α, IL-1ß and Wnt-10b was increased in the synovial membrane in arthritic groups. The immunoexpression of nuclear ß-catenin was significantly higher only in the group that received 2 booster TMJ injections. However, NF-κB, RANKL and DKK1 immunoexpression were increased only in animals with 3 mBSA intra-articular injections. CONCLUSION: Our results suggest that canonical Wnt and NF-κB signaling pathways participate in the hypernociception and inflammatory response in TMJ synovial membrane during the development of rheumatoid arthritis in rats.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Hiperalgesia/imunologia , NF-kappa B/imunologia , Articulação Temporomandibular/imunologia , Via de Sinalização Wnt , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Adjuvante de Freund , Hiperalgesia/patologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-1beta/imunologia , Lipídeos , Masculino , Ligante RANK/imunologia , Ratos Wistar , Soroalbumina Bovina , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Articulação Temporomandibular/patologia , Fator de Necrose Tumoral alfa/imunologia , Proteínas Wnt/imunologia
3.
Cancer Immunol Immunother ; 68(7): 1187-1194, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31187176

RESUMO

BACKGROUND: PD-1 inhibition (PD-1i) is the standard of care in melanoma and other malignancies. In patients with bone metastases of solid tumors, the monoclonal antibody denosumab directed against RANKL is approved for the prevention of skeletal-related events. However, RANKL is not only relevant in osteoclastogenesis, but also has immunological effects. Hence, we aimed at investigating, whether the combination of PD-1i and denosumab produces synergistic effects in metastatic melanoma treatment. METHODS: We retrospectively collected and analyzed clinical data of metastatic melanoma patients with bone metastases, who received PD-1i and denosumab therapy. RESULTS: 29 patients were identified with a median age of 60.7 years: 20 were male and 9 were female. 20 patients (69%) were in stage IV M1c and 9 (31%) in stage IV M1d; 52% had an increased serum LDH. 24 patients (83%) received PD-1i as first-line therapy and five patients (17%) as second- or third-line therapy. 13 patients received the triple combination nivolumab, ipilimumab and denosumab (N + I+D), 16 patients received PD-1i and denosumab (PD-1i + D). Within a median follow-up time of 19.8 months, 17 patients progressed with a median time to progression of 6 months. The objective response rate was 54% in the N + I + D group and 50% in the PD-1i + D group. Recalcification of bone metastases was radiologically observed in 18 (62%) patients. No unexpected treatment-related adverse events emerged. CONCLUSIONS: The combination therapy of metastatic melanoma with PD-1i and denosumab was feasible without unexpected safety issues and showed a promising efficacy signal. Further investigation in prospective studies is needed.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Denosumab/uso terapêutico , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/secundário , Denosumab/farmacologia , Feminino , Humanos , Ipilimumab/farmacologia , Ipilimumab/uso terapêutico , Estimativa de Kaplan-Meier , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/secundário , Pessoa de Meia-Idade , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Intervalo Livre de Progressão , Ligante RANK/antagonistas & inibidores , Ligante RANK/imunologia , Estudos Retrospectivos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia
4.
Int J Mol Sci ; 20(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052546

RESUMO

RANK ligand (RANKL) is a member of the tumor necrosis factor alpha superfamily of cytokines. It is the only known ligand binding to a membrane receptor named receptor activator of nuclear factor-kappa B (RANK), thereby triggering recruitment of tumor necrosis factor (TNF) receptor associated factor (TRAF) adaptor proteins and activation of downstream pathways. RANK/RANKL signaling is controlled by a decoy receptor called osteoprotegerin (OPG), but also has additional more complex levels of regulation. The existing literature on RANK/RANKL signaling in cervical cancer was reviewed, particularly focusing on the effects on the microenvironment. RANKL and RANK are frequently co-expressed in cervical cancer cells lines and in carcinoma of the uterine cervix. RANKL and OPG expression strongly increases during cervical cancer progression. RANKL is directly secreted by cervical cancer cells, which may be a mechanism they use to create an immune suppressive environment. RANKL induces expression of multiple activating cytokines by dendritic cells. High RANK mRNA levels and high immunohistochemical OPG expression are significantly correlated with high clinical stage, tumor grade, presence of lymph node metastases, and poor overall survival. Inhibition of RANKL signaling has a direct effect on tumor cell proliferation and behavior, but also alters the microenvironment. Abundant circumstantial evidence suggests that RANKL inhibition may (partially) reverse an immunosuppressive status. The use of denosumab, a monoclonal antibody directed to RANKL, as an immunomodulatory strategy is an attractive concept which should be further explored in combination with immune therapy in patients with cervical cancer.


Assuntos
Ligante RANK/imunologia , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Neoplasias do Colo do Útero/imunologia , Animais , Colo do Útero/imunologia , Colo do Útero/patologia , Feminino , Humanos , Imunoterapia/métodos , Ligante RANK/análise , Receptor Ativador de Fator Nuclear kappa-B/análise , Transdução de Sinais , Microambiente Tumoral , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/terapia
5.
Orthod Fr ; 90(1): 55-63, 2019 Mar.
Artigo em Francês | MEDLINE | ID: mdl-30994449

RESUMO

INTRODUCTION: Recent observations performed in the orthodontic department of La Pitié-Salpêtrière hospital in Paris reported an increase of non-familial eruption defects of permanent molars. Our recent data have evidenced the involvement of osteoclasts (OC) in both the eruption and the dental retention processes through the RANKL/RANK/OPG signaling pathway. These facts are at the origin of the hypothesis of the existence of an environmental etiology for those eruption defects that would correspond to the perturbation of cellular autocrine/paracrine signaling pathways as the RANKL/ RANK/OPG. MATERIALS AND METHODS: C57BL/6 mice were submitted to repeated injections with anti-RANKL neutralizing antibody during the nine days following birth. A phenotypic comparison with transgenic mice overexpressing RANK was performed for the functional characterization of the RANKL/RANK/OPG pathway. The dento-alveolar complex was analyzed using micro-CT for bone density and Masson's trichrome staining for histological examination. RESULTS: The RANKL transient invalidation of RANKL stopped the molar root development and tooth eruption contrary to transgenic mice overexpressing RANK. The recruitment and the OC activity were strongly impacted. DISCUSSION: This research is of direct clinical interest in understanding the pathology of eruption as indirect in establishing orthodontic treatment protocols for particular cases.


Assuntos
Anticorpos Monoclonais/farmacologia , Dente Molar/efeitos dos fármacos , Ligante RANK/imunologia , Erupção Dentária/efeitos dos fármacos , Raiz Dentária/efeitos dos fármacos , Animais , Anticorpos Monoclonais/administração & dosagem , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Dente Molar/diagnóstico por imagem , Dente Molar/crescimento & desenvolvimento , Ligante RANK/antagonistas & inibidores , Raiz Dentária/diagnóstico por imagem , Raiz Dentária/crescimento & desenvolvimento , Microtomografia por Raio-X
6.
J Immunol ; 202(7): 2035-2043, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30737274

RESUMO

Locally produced osteoclastogenic factor RANKL plays a critical role in the development of bone resorption in periradicular periodontitis. However, because RANKL is also required for healthy bone remodeling, it is plausible that a costimulatory molecule that upregulates RANKL production in inflammatory periradicular periodontitis may be involved in the pathogenic bone loss processes. We hypothesized that macrophage migration inhibitory factor (MIF) would play a role in upregulating the RANKL-mediated osteoclastogenesis in the periradicular lesion. In response to pulp exposure, the bone loss and level of MIF mRNA increased in the periradicular periodontitis, which peaked at 14 d, in conjunction with the upregulated expressions of mRNAs for RANKL, proinflammatory cytokines (TNF-α, IL-6, and IL-1ß), chemokines (MCP-1 and SDF-1), and MIF's cognate receptors CXCR4 and CD74. Furthermore, expressions of those mRNAs were found significantly higher in wild-type mice compared with that of MIF-/- mice. In contrast, bacterial LPS elicited the production of MIF from ligament fibroblasts in vitro, which, in turn, enhanced their productions of RANKL and TNF-α. rMIF significantly upregulated the number of TRAP+ osteoclasts in vitro. Finally, periapical bone loss induced in wild-type mice were significantly diminished in MIF-/- mice. Altogether, the current study demonstrated that MIF appeared to function as a key costimulatory molecule to upregulate RANKL-mediated osteoclastogenesis, leading to the pathogenically augmented bone resorption in periradicular lesions. These data also suggest that the approach to neutralize MIF activity may lead to the development of a therapeutic regimen for the prevention of pathogenic bone loss in periradicular periodontitis.


Assuntos
Reabsorção Óssea/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Periodontite Periapical/metabolismo , Animais , Reabsorção Óssea/imunologia , Modelos Animais de Doenças , Inflamação/imunologia , Inflamação/metabolismo , Fatores Inibidores da Migração de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Periodontite Periapical/imunologia , Ligante RANK/imunologia , Ligante RANK/metabolismo
7.
Crit Rev Oncol Hematol ; 133: 85-91, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30661662

RESUMO

Binding between the receptor activator of nuclear factor-kB (RANK) and its ligand (RANKL) triggers recruitment of TNF receptor associated factor (TRAF) adaptor proteins and activation of downstream pathways. RANK/RANKL signaling is controlled by a decoy receptor called osteoprotegerin (OPG) which interacts with RANKL. Additional networks regulating RANK/RANKL signaling are active in a context specific manner. RANK/RANKL signaling is essential for the differentiation of bone-resorbing osteoclasts, and is deregulated in pathological processes such as postmenopausal osteoporosis or cancer induced bone destruction. Cells expressing RANK and RANKL are commonly found in the tumor microenvironment. The RANKL/RANK pathway is often overexpressed in tumors of the breast, prostate, endometrium, cervix, stomach, oesophagus and bladder, thyroid and correlated with poor prognosis. RANK signaling plays an important role in the innate and adaptive immune response as it generates regulatory T (Treg) cells and increases production of cytokines. RANK expression induces chemoresistance in vitro through the activation of multiple signal transduction pathways. RANKL blockade improves the efficacy of anti-CTLA-4 monoclonal antibodies against solid tumors and experimental metastases. As RANK inhibition enhances the immune response there is an increasing interest in combining it with immune therapy in an attempt to sensitize immune resistant tumors to immune therapies. Several studies are ongoing to assess this concept. The role of RANK/RANKL inhibition should be further pursued as an immunomodulatory strategy in combination with other treatment modalities.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Denosumab/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Ligante RANK/antagonistas & inibidores , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Denosumab/farmacologia , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Ligante RANK/imunologia , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
8.
Nihon Yakurigaku Zasshi ; 153(1): 11-15, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30643086

RESUMO

Discovery of RANKL (receptor activator of NF-κB ligand) gave a great impact on identification of the mechanisms regulating osteoclast differentiation and function, establishment of research field bridging bone and mineral research and immunology (osteoimmunology), and development of a fully human anti-RANKL monoclonal neutralizing antibody (denosumab). Denosumab has been clinically available for treatment of osteoporosis and cancer-induced bone diseases in the US, Europe and many countries including Japan. Denosumab is a so-called blockbuster of which sales amount was 3.9 billion US dollars in 2017. Because RANKL is the absolute factor for osteoclast differentiation, anti-RANKL antibody is very effective and its application is good news for many patients. Recent topics are the identification of importance of RANKL on osteoblasts in regulation of osteogenesis and the demonstration of RANKL-RANK (the receptor of RANKL) dual signaling in coupling between bone resorption and bone formation. RANKL reverse signaling that we had hypothesized was demonstrated at last. In this review I describe the mechanism of anti-RANKL antibody in the treatment of metabolic bone diseases including osteoporosis. I also suggest possible applications of anti-RANKL antibody to the treatment of cancer patients.


Assuntos
Reabsorção Óssea , Denosumab/farmacologia , Osteoporose/terapia , Ligante RANK/imunologia , Anticorpos Monoclonais Humanizados , Humanos , Osteoclastos/citologia
10.
Oral Health Prev Dent ; 16(5): 457-465, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30460359

RESUMO

PURPOSE: To evaluate the clinical, biochemical, and microbiological reactions to nanocomposite containing amorphous calcium phosphate (ACP) in comparison to a traditional composite restorative material in early childhood caries. MATERIALS AND METHODS: Eighteen teeth were restored with the test material (ACP-containing resin) and 18 teeth were restored with the control material (traditional composite, TC) in fourteen paediatric patients using a split-mouth design. One caries- and restoration-free intact tooth in each patient was selected as the healthy control. Gingival crevicular fluid (GCF) and supragingival plaque samples were collected at baseline before the treatment and also on days 1, 7, 14 and 30 after treatment. Unstimulated whole saliva samples were obtained from each patient at baseline, and 1 and 6 months after restoration. GCF and saliva samples were assayed for IL-17A, IL-17F IL-17A/F, IL-17E, OPG and RANKL levels by ELISA, and plaque composition was assessed using RT-PCR. RESULTS: Clinical evaluation indicated no statistically significant differences between the two restorative materials according to the FDI criteria surface lustre, material retention and marginal adaptation properties. Pro-inflammatory IL-17 levels decreased statistically significantly at 6 months compared to baseline and 1-month values (p < 0.05). The baseline pro-inflammatory IL-17 cytokine levels in GCF samples around the carious teeth were higher than those obtained around the healthy teeth (p < 0.05), but similar in GCF from the ACP-test and TC teeth. Microbiological findings were similar in the ACP and T groups. CONCLUSION: It may be suggested that both ACP-containing and traditional resin composites show similar antimicrobial and biochemical effects in early childhood caries.


Assuntos
Fosfatos de Cálcio/uso terapêutico , Resinas Compostas/uso terapêutico , Cárie Dentária/terapia , Placa Dentária/microbiologia , Líquido do Sulco Gengival/imunologia , Saliva/imunologia , Criança , Pré-Escolar , Cárie Dentária/imunologia , Cárie Dentária/microbiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fusobacterium nucleatum/genética , Humanos , Interleucina-17/imunologia , Masculino , Osteoprotegerina/imunologia , Ligante RANK/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus mutans/genética , Streptococcus sanguis/genética
11.
Nat Rev Clin Oncol ; 15(11): 676-693, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30232468

RESUMO

Recognizing that the transformative effects of immunotherapy are currently limited to a minority of patients with cancer, research efforts are increasingly focused on expanding and enhancing clinical responses by combining immunotherapies; the repurposing of existing drugs is an attractive approach, given their well-characterized safety and pharmacokinetic profiles. Receptor activator of nuclear factor-κB (RANK) and the RANK ligand (RANKL) were initially described in the context of T cell-dendritic cell interactions; however, the discovery of an obligate role of RANK signalling in osteoclastogenesis led to the development of the anti-RANKL antibody denosumab for antiresorptive indications, including bone metastases. Randomized clinical trials and post-marketing surveillance studies have established the acceptable safety profile of denosumab. More recently, several case reports involving patients with advanced-stage melanoma have described remarkable responses following concurrent treatment with denosumab and immune-checkpoint inhibitors. Randomized trials assessing similar combinations in patients with melanoma or renal cell carcinoma are now underway. Herein, we discuss the hallmark clinical trials of denosumab in light of possible immunological effects of this agent. We highlight the role of immune cells as sources of RANK and RANKL in the tumour microenvironment and review data on RANKL inhibition in mouse models of cancer. Finally, we describe hypothetical immune-related mechanisms of action, which could be assessed in clinical trials of immune-checkpoint inhibitors and denosumab in patients with cancer.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Melanoma/tratamento farmacológico , Ligante RANK/imunologia , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Carcinoma de Células Renais/imunologia , Denosumab/uso terapêutico , Humanos , Melanoma/imunologia , Osteogênese/imunologia , Ligante RANK/antagonistas & inibidores , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Microambiente Tumoral/imunologia
12.
Int Immunopharmacol ; 64: 326-332, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30243068

RESUMO

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis, bone atrophy, and subsequent progressive destruction of articular tissue. Targeted inhibition of receptor activator of NF-kB ligand (RANKL) has been highly successful in preventing RA-mediated bone erosion in animal models and patients, suggesting that development of a RANKL vaccine might be of therapeutic value. Our previous study has shown that the recombinant RANKL vaccine Y234pNO2Phe, generated by replacement of a single tyrosine residue (Tyr234) in murine RANKL (mRANKL) with p-nitrophenylalanine (pNO2Phe), induces a high titer antibody response and prevents ovariectomy (OVX)-induced bone loss in mice. This aim of this study was to further evaluate the vaccine's preventive effects in a murine model of collagen-induced arthritis. The results of this study showed that Y234pNO2Phe not only induced a high titer antibody response and inhibited osteoclastogenesis but also significantly prevented bone erosion and ameliorated the severity of a collagen-induced arthritis (CIA) model in mice. Moreover, use of the vaccine improved the clinical situations of the CIA mice. These results suggest a potential application of an anti-RANKL vaccine in the treatment of RA-induced bone erosion.


Assuntos
Artrite Experimental/prevenção & controle , Ligante RANK/imunologia , Animais , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Osteoclastos/citologia , Osteoprotegerina/sangue , Ligante RANK/sangue , Vacinas Sintéticas/imunologia
13.
MAbs ; 10(7): 951-959, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30130443

RESUMO

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.


Assuntos
Conservadores da Densidade Óssea/química , Denosumab/química , Osteoporose/tratamento farmacológico , Mapeamento de Peptídeos , Peptídeos/química , Proteômica/métodos , Animais , Conservadores da Densidade Óssea/uso terapêutico , Cromatografia de Fase Reversa , Denosumab/uso terapêutico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Ligante RANK/imunologia , Eletricidade Estática , Espectrometria de Massas em Tandem
14.
Int Immunopharmacol ; 62: 147-154, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30015235

RESUMO

Periodontitis is a bacteria-induced disease that often leads to alveolar bone damage. We sought to determine the role and mechanism of switched memory B cells in alveolar bone destruction during periodontitis. Sensitized B cells were sorted and cultured, then their expression of receptor activator for nuclear factor-κB ligand (RANKL), interleukin-6 (IL-6), and interleukin-12 (IL-12) was detected. Using these cells, we prepared adoptive transfer models in which we induced periodontitis. We found that switched memory B cells produced more RANKL in terms of both protein and mRNA levels than other subpopulations. Switched memory B cells expressed more IL-6 and IL-12 mRNA than other subpopulations, but differences in respective protein levels were not significant. Moreover, we found that switched memory B cell transfer resulted in increased alveolar bone loss and periodontal osteoclastogenesis. Moreover, switched memory B cell transfer increased the proportion of Th1 and Th17 cells as well as the expression of RANKL, osteoprotegerin (OPG), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-1ß, IL-6, IL-17A in gingiva, and cervical lymph nodes (CLNs). The outcomes of the present study indicate that switched memory B cells regulate alveolar bone homeostasis via enhancing cytokine expression and increasing proliferation of Th1 and Th17 cells.


Assuntos
Transferência Adotiva , Perda do Osso Alveolar/imunologia , Linfócitos B/imunologia , Memória Imunológica , Periodontite/imunologia , Porphyromonas gingivalis/imunologia , Perda do Osso Alveolar/microbiologia , Animais , Linfócitos B/enzimologia , Técnicas de Cultura de Células , Modelos Animais de Doenças , Interleucina-12/imunologia , Interleucina-6/imunologia , Masculino , Osteogênese/imunologia , Ligante RANK/imunologia , Ratos Sprague-Dawley
15.
Immunity ; 48(6): 1208-1219.e4, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29858011

RESUMO

While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor κB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity.


Assuntos
Imunidade Inata/imunologia , Subpopulações de Linfócitos/imunologia , Ligante RANK/imunologia , Animais , Citocinas/biossíntese , Citocinas/imunologia , Subpopulações de Linfócitos/metabolismo , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Ligante RANK/metabolismo , Receptores CCR6/imunologia
16.
Neuroimmunomodulation ; 25(1): 23-33, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29920500

RESUMO

OBJECTIVES: Our study focused on the RANKL (receptor activator of nuclear factor-κB ligand)/RANK/OPG (osteoprotegerin) axis and selected proinflammatory/immunoregulatory upstream mediators in the peripheral blood (PBL) and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. METHODS: PBL and CSF were collected from healthy controls (n = 35) and MS patients at the clinical onset of the disease (n = 33). In addition, PBL samples were obtained from relapse-remitting (RR)-MS patients (n = 30). Patients were assessed by means of the expanded disability status scale (EDSS) and routine laboratory parameters. Soluble (s)RANKL and OPG were measured in the CSF and plasma; gene expression was detected for RANKL, RANK, OPG, and selected cytokines/chemokines (interleukin [IL]-4, IL-10, IL-17, CCL2, and CXCL12) in PBL mononuclear cells. RESULTS: The OPG level in the CSF was lower in MS patients at clinical onset than in controls. Moreover, the sRANKL/OPG ratio was higher in the CSF of MS patients at clinical onset and in the plasma of RR-MS patients than in controls. Gene expression of RANKL/RANK/OPG in PBL mononuclear cells was higher only in RR-MS patients. IL-4, CCL2, and CXCL12 were positively correlated and IL-10 was negatively correlated with RANKL/RANK expression. OPG was negatively correlated with EDSS and alkaline phosphatase level. CONCLUSION: Our study revealed that changes of RANKL/RANK/OPG axis are associated with MS, particularly the decreased OPG level in the CSF at disease onset. Therefore, these factors may serve as disease biomarkers and molecular targets of novel therapeutic approaches.


Assuntos
Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/diagnóstico , Osteoprotegerina/líquido cefalorraquidiano , Ligante RANK/líquido cefalorraquidiano , Receptor Ativador de Fator Nuclear kappa-B/líquido cefalorraquidiano , Adulto , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/imunologia , Osteoprotegerina/imunologia , Ligante RANK/imunologia , Receptor Ativador de Fator Nuclear kappa-B/imunologia
17.
Biochem Biophys Res Commun ; 501(2): 547-555, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29746861

RESUMO

Osteolytic diseases are closely associated with osteocyte fate, indicating a more efficient and crucial role of osteocyte-targeting strategy in inhibiting osteoclastogenesis. Here, we investigated the effects of lenalidomide (Lena) on osteocyte fate in order to regulate osteoclastogenesis via effective cascade-controlling response. Our data revealed that lenalidomide treatment notably rescued IL-1ß induced loss of osteocyte viability by inhibiting osteocyte apoptosis with decreased osteoclast-related factors, RANKL and Sclerostin, as demonstrated by the restricted osteoclast formation and reduced bone resorption. Additionally, iTRAQ assay revealed that IL-1ß induced activation of NF-κB inhibitor α/ß were remarkably downregulated by lenalidomide, showing that lenalidomide impaired NF-κB signaling in osteocytes for inhibiting the expression of osteoclast specific genes in osteoclasts, which was further confirmed by KEGG pathway analysis and Western blot. More interestingly, the in vivo analysis of osteocyte apoptosis and osteoclastogenesis in osteoarthritis mice model indicated a role of lenalidomide in the regulation of osteocyte fate and the consequent inhibition of RANKL-induced osteoclastogenesis. Together, these results suggest that lenalidomide regulates osteocyte fate by attenuating IL-1ß/NF-κB signaling, thereby inhibiting RANKL expression for the attenuated osteoclastogenesis both in vitro and vivo, indicating a more efficient remedy among future anti-osteoclastogenesis approaches.


Assuntos
Fatores Imunológicos/farmacologia , Interleucina-1beta/imunologia , NF-kappa B/imunologia , Osteócitos/efeitos dos fármacos , Ligante RANK/imunologia , Transdução de Sinais/efeitos dos fármacos , Talidomida/análogos & derivados , Animais , Linhagem Celular , Células Cultivadas , Lenalidomida , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/imunologia , Osteócitos/citologia , Osteócitos/imunologia , Osteogênese/efeitos dos fármacos , Talidomida/farmacologia
19.
Food Funct ; 9(5): 2653-2660, 2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29666861

RESUMO

Osteoporosis can be classified as an inflammatory disease and the crosstalk between the immune system and bone growth should be considered. The effects of bovine lactoferrin on bone by osteoimmunology were investigated in the present study. Ten week old female BALB/c mice were ovariectomized (OVX) and fed for 12 weeks with a control diet or lactoferrin (2, 20 and 100 mg kg-1 d-1). The results showed that following 12 weeks of treatment after surgery, OVX resulted in bone loss, but treatment with lactoferrin preserved bone homeostasis. Lactoferrin significantly improved bone mineral density, and increased the serum levels of alkaline phosphatase, while it decreased the serum levels of tartrate-resistant acid phosphatase. Furthermore, according to micro-computed tomography (micro-CT), the bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were elevated. The results indicated that the structure model index (SMI) was reduced in the OVX + LF groups. Additionally, lactoferrin enhanced the Max-Load and Max-Stress values of the femur, and increased the content of Ca and P. However, lactoferrin suppressed the RANKL/OPG ratio in OVX mice. Moreover, interferon-γ, interleukin-5 and interleukin-10 were elevated significantly in the OVX + LF groups. Lactoferrin had a positive effect on the bone micro-environment and might be a pleiotropic protein for the prevention and treatment of estrogen-dependent bone loss via the osteoimmunology pathway.


Assuntos
Lactoferrina/administração & dosagem , Osteoporose/prevenção & controle , Ligante RANK/imunologia , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Animais , Densidade Óssea , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/imunologia , Bovinos , Feminino , Homeostase/efeitos dos fármacos , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Osteoporose/genética , Osteoporose/imunologia , Osteoporose/fisiopatologia , Ovariectomia , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética
20.
J Vasc Surg ; 68(6S): 48S-59S.e1, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29685509

RESUMO

OBJECTIVE: Osteoclastogenic activation of macrophages (OCG) occurs in human abdominal aortic aneurysms (AAAs) and in calcium chloride-induced degenerative AAAs in mice, which have increased matrix metalloproteinase activity. As the activity of OCG in dissecting aneurysms is not clear, we tested the hypothesis that OCG contributes to angiotensin II (Ang II)-induced dissecting aneurysm (Ang II-induced AAA) in apolipoprotein E knockout mice. METHODS: AAAs were produced in apolipoprotein E knockout mice via the administration of Ang II. Additionally, receptor activator of nuclear factor kB ligand (RANKL)-neutralizing antibody (5 mg/kg) was administered to one group of mice 7 days prior to Ang II infusion. Aneurysmal sections were probed for presence of RANKL and tartrate-resistant acid phosphatase via immunohistochemistry and immunofluorescence staining. Mouse aortas were also examined for RANKL and matrix metalloproteinase 9 expression via Western blot. In vitro murine vascular smooth muscle cells (MOVAS) and murine macrophages (RAW 264.7) were analyzed for the expression of osteogenic factors via Western blot, qPCR, and flow cytometry in response to Ang II or RANKL stimulation. The signaling pathway that mediates Ang II-induced RANKL expression in MOVAS cells was also investigated via application of TG101348, a Janus kinase 2 (JAK2) inhibitor, and Western blot analysis. RESULTS: Immunohistochemical staining of Ang II-induced AAA sections revealed OCG as evidenced by increased RANKL and tartrate-resistant acid phosphatase expression compared with control mice. Immunofluorescence staining of AAA sections revealed co-localization of vascular smooth muscle cells and RANKL, revealing vascular smooth muscle cells as one potential source of RANKL. Systemic administration of RANKL-neutralizing antibody suppressed Ang II-induced AAA, with significant reduction of the maximum diameter of the abdominal aorta compared with vehicle controls (1.5 ± 0.4 mm vs 2.2 ± 0.2 mm). Ang II (1 µM) treatment induced a significant increase in RANKL messenger RNA expression levels in MOVAS cells compared with the vehicle control (1.0 ± 0.2 vs 2.8 ± 0.2). The activities of JAK2 and signal transducer and activator of transcription 5 (STAT5) were also significantly increased by Ang II treatment. Inhibition of JAK2/STAT5 suppressed Ang II-induced RANKL expression, suggesting the involvement of the JAK2/STAT5 signaling pathway. CONCLUSIONS: OCG with increased RANKL expression was present in Ang II-induced AAA, and neutralization of RANKL suppressed AAA formation. As neutralization of RANKL has been used clinically to treat osteoporosis and other osteoclast-related diseases, additional study of the effectiveness of RANKL neutralization in AAA is warranted.


Assuntos
Aneurisma Dissecante/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Transdiferenciação Celular , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/metabolismo , Aneurisma Dissecante/induzido quimicamente , Aneurisma Dissecante/patologia , Aneurisma Dissecante/prevenção & controle , Angiotensina II , Animais , Anticorpos Neutralizantes/farmacologia , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/prevenção & controle , Transdiferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Janus Quinase 2/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout para ApoE , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Ligante RANK/antagonistas & inibidores , Ligante RANK/genética , Ligante RANK/imunologia , Células RAW 264.7 , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais
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