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Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ's anti-atrophic effects. Consumption of EQ (500â¯mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.
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Equisetum , Extratos Vegetais , Sarcopenia , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Camundongos , Sarcopenia/tratamento farmacológico , Sarcopenia/patologia , Células RAW 264.7 , Equisetum/química , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Ligante RANK/metabolismo , NF-kappa B/metabolismo , Osteogênese/efeitos dos fármacos , Anti-Inflamatórios/farmacologiaRESUMO
Bone is a site of distant metastases, which are a common cause of morbidity and mortality with a high socio-economic impact, for many malignant tumours. In order to engineer pharmacological therapies that are suitable for this debilitating disease, this experimental work presents injectable lipid nanoemulsions, which are endowed with a long history of safe clinical usage in parenteral nutrition, their loading with vincristine and their grafting with alendronate, with a dual purpose: merging the anticancer activity of bisphosphonates and vincristine, and enhancing bone-targeted delivery. In cell studies, alendronate synergised with the anti-migration activity of vincristine, which is important as migration plays a key role in the metastatisation process. In preliminary animal studies, carried out thanks to IVIS technology, alendronate conjugation enhanced the bone targeting of fluorescently labelled nanoemulsions. These encouraging results will drive further studies on suitable animal models of the disease.
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Alendronato , Difosfonatos , Animais , Alendronato/farmacologia , Vincristina/farmacologia , Difosfonatos/uso terapêutico , Osso e Ossos , Modelos AnimaisRESUMO
Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.
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Neoplasias Pulmonares , Melanoma Experimental , Animais , Camundongos , Septinas , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: SerpinB3 is a cysteine protease inhibitor involved in liver disease progression due to its proinflammatory and profibrogenic properties. The polymorphic variant SerpinB3-PD (SB3-PD), presents a substitution in its reactive centre loop, determining the gain of function. AIMS: To disclose the clinical characteristics of a cohort of patients with cirrhosis in relation to the presence of SB3-PD and to assess the effect of this genetic variant on fibrogenic and inflammatory cytokines in vitro. METHODS: We assessed SB3 polymorphism in 90 patients with cirrhosis, prospectively followed up in our referral centre. We used HepG2 and HuH-7 cells transfected to overexpress either wild-type SB3 (SB3-WT) or SB3-PD to assess their endogenous effect, while LX2 and THP-1 cells were treated with exogenous SB3-WT or SB3-PD proteins. RESULTS: Patients carrying SB3-PD had more severe portal hypertension and higher MELD scores, than patients carrying SB3-WT. In multivariate analysis, SB3-PD was an independent predictor of cirrhosis complications. Patients with SB3-PD polymorphism presented with more severe liver fibrosis and inflammatory features. Hepatoma cells overexpressing SB3-PD showed higher TGF-ß1 expression than controls. The addition of recombinant SB3-PD induced an up-regulation of TGF-ß1 in LX2 cells and a more prominent inflammatory profile in THP-1 cells, compared to the effect of SB3-WT protein. CONCLUSIONS: The polymorphic variant SB3-PD is highly effective in determining activation of TGF-ß1 and inflammation in vitro. Patients with cirrhosis who carry SB3-PD polymorphism may be more prone to develop severe liver disease progression. However, further validation studies are warranted to support the in vivo relevance of this polymorphism.
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Hepatopatias , Fator de Crescimento Transformador beta1 , Humanos , Progressão da Doença , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.
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Neoplasias da Mama , Humanos , Feminino , Camundongos , Animais , Nicotinamida Fosforribosiltransferase/metabolismo , Evasão da Resposta Imune , Citocinas/metabolismo , PrognósticoRESUMO
Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Sódio/metabolismo , Monensin/uso terapêutico , Linhagem Celular , ÁguaRESUMO
High-grade melanoma remains a major life-threatening illness despite the improvement in therapeutic control that has been achieved by means of targeted therapies and immunotherapies in recent years. This work presents a preclinical-level test of a multi-pronged approach that includes the loading of immunotherapeutic (ICOS-Fc), targeted (sorafenib), and chemotherapeutic (temozolomide) agents within Intralipid®, which is a biocompatible nanoemulsion with a long history of safe clinical use for total parenteral nutrition. This drug combination has been shown to inhibit tumor growth and angiogenesis with the involvement of the immune system, and a key role is played by ICOS-Fc. The inhibition of tumor growth in subcutaneous melanoma mouse models has been achieved using sub-therapeutic drug doses, which is most likely the result of the nanoemulsion's targeting properties. If translated to the human setting, this approach should therefore allow therapeutic efficacy to be achieved without increasing the risk of toxic effects.
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BACKGROUND: IBD is a spectrum of pathologies characterized by dysregulated immune activation leading to uncontrolled response against the intestine, thus resulting in chronic gut inflammation and tissue damage. Due to its complexity, the molecular mechanisms responsible for disease onset and progression are still elusive, thus requiring intense research effort. In this context, the development of models replicating the etiopathology of IBD and allowing the testing of new potential therapies is critical. METHODS: Colon from C57BL/6 or BALB/c mice was cultivated in a Gut-Ex-Vivo System (GEVS), exposed for 5 h to DNBS 1.5 or 2.5 mg/mL, in presence or absence of two probiotic formulations (P1 = Bifidobacterium breve BR03 (DSM16604) and B632 (DSM24706); P2 = Lacticaseibacillus rhamnosus LR04 (DSM16605), Lactiplantibacillus plantarum LP14 (DSM33401) and Lacticaseibacillus paracasei LPC09), and the main hallmarks of IBD were evaluated. RESULTS: Gene expression analysis revealed the following DNBS-induced effects: (i) compromised tight junction organization, responsible for tissue permeability dysregulation; (ii) induction of ER stress, and (iii) tissue inflammation in colon of C57BL/6 mice. Moreover, the concomitant DNBS-induced apoptosis and ferroptosis pathways were evident in colon from both BALB/c and C57BL/6 mice. Finally, the co-administration of probiotics completely prevented the detrimental effects of DNBS. CONCLUSIONS: Overall, we have provided results demonstrating that GEVS is a consistent, reliable, and cost-effective system for modeling DNBS-induced IBD, useful for studying the onset and progression of human disease at the molecular level, while also reducing animal suffering. Moreover, we have confirmed the beneficial effect of probiotics administration in promoting the remission of IBD.
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Recent advancements in regenerative medicine have enhanced the development of biomaterials as multi-functional dressings, capable of accelerating wound healing and addressing the challenge of chronic wounds. Hydrogels obtained from decellularized tissues have a complex composition, comparable to the native extracellular environment, showing highly interesting characteristics for wound healing applications. In this study, a bovine pericardium decellularized extracellular matrix (dECM) hydrogel was characterized in terms of macromolecules content, and its immunomodulatory, angiogenic and wound healing potential has been evaluated. The polarization profile of human monocytes-derived macrophages seeded on dECM hydrogel was assessed by RT-qPCR. Angiogenic markers expression has been evaluated by Western blot and antibody array on cell lysates derived from endothelial cells cultured on dECM hydrogel, and a murine in vivo model of hindlimb ischemia was used to evaluate the angiogenic potential. Fibroblast migration was assessed by a transwell migration assay, and an in vivo murine wound healing model treated with dECM hydrogels was also used. The results showed a complex composition, of which the major component is collagen type I. The dECM hydrogel is biocompatible, able to drive M2 phenotype polarization, stimulate the expression of angiogenic markers in vitro, and prevent loss of functionality in hindlimb ischemia model. Furthermore, it drives fibroblast migration and shows ability to facilitate wound closure in vivo, demonstrating its great potential for regenerative applications.
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Matriz Extracelular , Hidrogéis , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Bovinos , Colágeno Tipo I/metabolismo , Células Endoteliais , Matriz Extracelular/metabolismo , Humanos , Hidrogéis/metabolismo , Hidrogéis/farmacologia , CamundongosRESUMO
Inducible T cell co-stimulator (ICOS), an immune checkpoint protein expressed on activated T cells and its unique ligand, ICOSL, which is expressed on antigen-presenting cells and non-hematopoietic cells, have been extensively investigated in the immune response. Recent findings showed that a soluble recombinant form of ICOS (ICOS-Fc) can act as an innovative immunomodulatory drug as both antagonist of ICOS and agonist of ICOSL, modulating cytokine release and cell migration to inflamed tissues. Although the ICOS-ICOSL pathway has been poorly investigated in the septic context, a few studies have reported that septic patients have reduced ICOS expression in whole blood and increased serum levels of osteopontin (OPN), that is another ligand of ICOSL. Thus, we investigated the pathological role of the ICOS-ICOSL axis in the context of sepsis and the potential protective effects of its immunomodulation by administering ICOS-Fc in a murine model of sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in five-month-old male wild-type (WT) C57BL/6, ICOS-/-, ICOSL-/- and OPN-/- mice. One hour after the surgical procedure, either CLP or Sham (control) mice were randomly assigned to receive once ICOS-Fc, F119SICOS-Fc, a mutated form uncapable to bind ICOSL, or vehicle intravenously. Organs and plasma were collected 24 h after surgery for analyses. When compared to Sham mice, WT mice that underwent CLP developed within 24 h a higher clinical severity score, a reduced body temperature, an increase in plasma cytokines (TNF-α, IL-1ß, IL-6, IFN-γ and IL-10), liver injury (AST and ALT) and kidney (creatinine and urea) dysfunction. Administration of ICOS-Fc to WT CLP mice reduced all of these abnormalities caused by sepsis. Similar beneficial effects were not seen in CLP-mice treated with F119SICOS-Fc. Treatment of CLP-mice with ICOS-Fc also attenuated the sepsis-induced local activation of FAK, P38 MAPK and NLRP3 inflammasome. ICOS-Fc seemed to act at both sides of the ICOS-ICOSL interaction, as the protective effect was lost in septic knockout mice for the ICOS or ICOSL genes, whereas it was maintained in OPN knockout mice. Collectively, our data show the beneficial effects of pharmacological modulation of the ICOS-ICOSL pathway in counteracting the sepsis-induced inflammation and organ dysfunction.
Assuntos
Osteopontina , Sepse , Animais , Masculino , Camundongos , Creatinina , Citocinas/metabolismo , Proteínas de Checkpoint Imunológico , Imunidade , Imunomodulação , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Inflamassomos , Inflamação , Interleucina-10 , Interleucina-6 , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Quinases p38 Ativadas por Mitógeno , Sepse/tratamento farmacológico , Fator de Necrose Tumoral alfa , UreiaRESUMO
Multiple sclerosis (MS) is one of the most common neurodegenerative diseases in young adults, with early clinical symptoms seen in the central nervous system (CNS) myelin sheaths due to an attack caused by the patient's immune system. Activation of the immune system is mediated by the induction of an antigen-specific immune response involving the interaction of multiple T-cell types with antigen-presenting cells (APCs), such as dendritic cells (DCs). Antigen-specific therapeutic approaches focus on immune cells and autoantigens involved in the onset of disease symptoms, which are the main components of myelin proteins. The ability of such therapeutics to bind strongly to DCs could lead to immune system tolerance to the disease. Many modern approaches are based on peptide-based research, as, in recent years, they have been of particular interest in the development of new pharmaceuticals. The characteristics of peptides, such as short lifespan in the body and rapid hydrolysis, can be overcome by their entrapment in nanospheres, providing better pharmacokinetics and bioavailability. The present study describes the development of polymeric nanoparticles with encapsulated myelin peptide analogues involved in the development of MS, along with their biological evaluation as inhibitors of MS development and progression. In particular, particles of poly(lactic-co-glycolic) acid (PLGA) loaded with peptides based on mouse/rat (rMOG) epitope 35-55 of myelin oligodendrocyte glycoprotein (MOG) conjugated with saccharide residues were developed. More specifically, the MOG35-55 peptide was conjugated with glucosamine to promote the interaction with mannose receptors (MRs) expressed by DCs. In addition, a study of slow release (dissolution) and quantification on both initially encapsulated peptide and daily release in saline in vitro was performed, followed by an evaluation of in vivo activity of the formulation on mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS, using both prophylactic and therapeutic protocols. Our results showed that the therapeutic protocol was effective in reducing EAE clinical scores and inflammation of the central nervous system and could be an alternative and promising approach against MS inducing tolerance against the disease.
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Encefalomielite Autoimune Experimental , Esclerose Múltipla , Nanopartículas , Camundongos , Ratos , Animais , Glicoproteína Mielina-Oligodendrócito/química , Glicoproteína Mielina-Oligodendrócito/metabolismo , Epitopos , Camundongos Endogâmicos C57BL , Peptídeos/uso terapêutico , Fragmentos de PeptídeosRESUMO
BACKGROUND: ICOS and its ligand ICOSL are immune receptors whose interaction triggers bidirectional signals that modulate the immune response and tissue repair. AIM: The aim of this study was to assess the in vivo effects of ICOSL triggering by ICOS-Fc, a recombinant soluble form of ICOS, on skin wound healing. METHODS: The effect of human ICOS-Fc on wound healing was assessed, in vitro, and, in vivo, by skin wound healing assay using ICOS-/- and ICOSL-/- knockout (KO) mice and NOD-SCID-IL2R null (NSG) mice. RESULTS: We show that, in wild type mice, treatment with ICOS-Fc improves wound healing, promotes angiogenesis, preceded by upregulation of IL-6 and VEGF expression; increases the number of fibroblasts and T cells, whereas it reduces that of neutrophils; and increases the number of M2 vs. M1 macrophages. Fittingly, ICOS-Fc enhanced M2 macrophage migration, while it hampered that of M1 macrophages. ICOS-/- and ICOSL-/- KO, and NSG mice showed delayed wound healing, and treatment with ICOS-Fc improved wound closure in ICOS-/- and NSG mice. CONCLUSION: These data show that the ICOS/ICOSL network cooperates in tissue repair, and that triggering of ICOSL by ICOS-Fc improves cutaneous wound healing by increasing angiogenesis and recruitment of reparative macrophages.
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Fragmentos Fc das Imunoglobulinas , Ligante Coestimulador de Linfócitos T Induzíveis , Proteína Coestimuladora de Linfócitos T Induzíveis , Cicatrização , Animais , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas Recombinantes/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Store-operated Ca2+-entry is a cellular mechanism that governs the replenishment of intracellular stores of Ca2+ upon depletion caused by the opening of intracellular Ca2+-channels. Gain-of-function mutations of the 2 key proteins of store-operated Ca2+-entry, STIM1 and ORAI1, are associated with several ultra-rare diseases clustered as tubular aggregate myopathies. Our group has previously demonstrated that a mouse model bearing the STIM1 p.I115F mutation recapitulates the main features of the STIM1 gain-of-function disorders: muscle weakness and thrombocytopenia. Similar findings have been found in other mice bearing different mutations on STIM1. At present, no valid treatment is available for these patients. In the present contribution, we report that CIC-39Na, a store-operated Ca2+-entry inhibitor, restores platelet number and counteracts the abnormal bleeding that characterizes these mice. Subtle differences in thrombopoiesis were observed in STIM1 p.I115F mice, but the main difference between wild-type and STIM1 p.I115F mice was in platelet clearance and in the levels of platelet cytosolic basal Ca2+. Both were restored on treatment of animals with CIC-39Na. This finding paves the way to a pharmacological treatment strategy for thrombocytopenia in tubular aggregate myopathy patients.
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Miopatias Congênitas Estruturais , Trombocitopenia , Animais , Cálcio/metabolismo , Camundongos , Mutação , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Trombocitopenia/genéticaRESUMO
The inducible T-cell co-stimulator (ICOS) is a T-cell receptor that, once bound to ICOS ligand (ICOSL) expressed on several cell types including the B-cell lineage, plays a decisive role in adaptive immunity by regulating the interplay between B and T cells. In addition to its immunomodulatory functions, we have shown that ICOS/ICOSL signalling can inhibit the activity of osteoclasts, unveiling a novel mechanism of lymphocyte-bone cells interactions. ICOS and ICOSL can also be found as soluble forms, namely sICOS and sICOSL. Here we show that: (i) levels of sICOS and sICOSL are increased in multiple myeloma (MM) compared to monoclonal gammopathy of undetermined significance and smouldering MM; (ii) levels of sICOS and sICOSL variably correlate with several markers of tumour burden; and (iii) sICOS levels tend to be higher in Durie-Salmon stage II/III versus stage I MM and correlate with overall survival as an independent variable. Moreover, surface ICOS and ICOSL are expressed in both myeloma cells and normal plasma cells, where they probably regulate different functional stages. Finally, ICOSL triggering inhibits the migration of myeloma cell lines in vitro and the growth of ICOSL+ MOPC-21 myeloma cells in vivo. These results suggest that ICOS and ICOSL represent novel markers and therapeutic targets for MM.
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Mieloma Múltiplo , Humanos , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Ligantes , Mieloma Múltiplo/metabolismo , Linfócitos T , Microambiente TumoralRESUMO
Photodynamic therapy (PDT) has been pointed out as a candidate for improving melanoma treatment. Nanotechnology application in PDT has increased its efficacy by reducing side effects. Herein, mesoporous silica nanoparticles (MSNs) conjugated with verteporfin (Ver-MSNs), in use with PDT, were administered in mice to evaluate their efficacy on lymphoangiogenesis and micrometastasis in melanoma. Melanoma was induced in mice by the subcutaneous injection of B16-F10 cells. The mice were transcutaneously treated with MSNs, Ver-MSNs, or glycerol and exposed to red light. The treatment was carried out four times until day 20. Lymphangiogenesis and micrometastasis were identified by the immunohistochemical method. Lymphoangiogenesis was halved by MSN treatment compared with the control animals, whereas the Ver-MSN treatment almost abolished it. A similar reduction was also observed in lung micrometastasis. PDT with topically administrated Ver-MSNs reduced melanoma lymphoangiogenesis and lung micrometastasis, as well as tumor mass and angiogenesis, and therefore their use could be an innovative and useful tool in melanoma clinical therapy.
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Linfangiogênese/efeitos dos fármacos , Melanoma Experimental , Nanopartículas , Dióxido de Silício , Verteporfina , Administração Tópica , Animais , Feminino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Nanopartículas/química , Nanopartículas/uso terapêutico , Metástase Neoplásica , Porosidade , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Verteporfina/química , Verteporfina/farmacologiaRESUMO
Hepatic ischemia/reperfusion injury (IRI) is aggravated by steatosis and is a main risk factor in fatty liver transplantation. Adenosine receptors (ARs) are emerging as therapeutic targets in liver diseases. By using cellular and in vivo systems of hepatic steatosis and IRI, here we evaluated the effects of pharmacological A2AR and A1R activation. The A2AR agonist CGS21680 protected the primary steatotic murine hepatocyte from IR damage and the activation of ASK1 and JNK. Such an effect was attributed to a phosphatidylinositol-3-kinase (PI3K)/Akt-dependent inhibition of ASK1. By contrast, the A1R agonist CCPA enhanced IR damage, intracellular steatosis and oxidative species (OS) production, thereby further increasing the lipid/OS-dependent ASK1-JNK stimulation. The CGS2680 and CCPA effects were nullified by a genetic ASK1 downregulation in steatotic hepatoma C1C7 cells. In steatotic mice livers, CGS21680 protected against hepatic IRI and ASK1/JNK activation whereas CCPA aggravated hepatic steatosis and IRI, and enhanced ASK1 and JNK stimulation. These results evidence a novel mechanism of CGS21680-mediated hepatoprotection, i.e., the PI3K/AKT-dependent inhibition of ASK1, and they show that CGS21680 and CCPA reduces and enhances the IRI of fatty liver, respectively, by preventing or increasing the activation of the cytotoxic ASK1/JNK axis. They also indicate the selective employment of A2AR agonists as an effective therapeutic strategy to prevent IRI in human fatty liver surgery.
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Progressão da Doença , Fígado Gorduroso/complicações , MAP Quinase Quinase Quinase 5/metabolismo , Substâncias Protetoras/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Traumatismo por Reperfusão/complicações , Agonistas do Receptor A1 de Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inativação Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipídeos/análise , Masculino , Camundongos Endogâmicos BALB C , OxirreduçãoRESUMO
BACKGROUND: Glaucoma is currently the leading cause of irreversible blindness; it is a neuropathy characterized by structural alterations of the optic nerve, leading to visual impairments. The aim of this work is to develop a new oral formulation able to counteract the early changes connected to glaucomatous degeneration. The composition is based on gastrodin and vitamin D3 combined with vitamin C, blackcurrant, and lycopene. METHODS: Cells and tissues of the retina were used to study biological mechanisms involved in glaucoma, to slow down the progression of the disease. Experiments mimicking the conditions of glaucoma were carried out to examine the etiology of retinal degeneration. RESULTS: Our results show a significant ability to restore glaucoma-induced damage, by counteracting ROS production and promoting cell survival by inhibiting apoptosis. These effects were confirmed by the intracellular mechanism that was activated following administration of the compound, either before or after the glaucoma induction. In particular, the main results were obtained as a preventive action of glaucoma, showing a beneficial action on all selected markers, both on cells and on eyecup preparations. It is therefore possible to hypothesize both the preventive and therapeutic use of this formulation, in the presence of risk factors, and due to its ability to inhibit the apoptotic cycle and to stimulate cell survival mechanisms, respectively. CONCLUSION: This formulation has exhibited an active role in the prevention or restoration of glaucoma damage for the first time.
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Inflammatory bowel disease (IBD) is a complex, chronic, and dysregulated inflammatory condition which etiology is still largely unknown. Its prognosis and disease progression are highly variable and unpredictable. IBD comprises several heterogeneous inflammatory conditions ranging from Ulcerative Colitis (UC) to Crohn's Disease (CD). Importantly, a definite, well-established, and effective clinical treatment for these pathologies is still lacking. The urgent need for treatment is further supported by the notion that patients affected by UC or CD are also at risk of developing cancer. Therefore, a deeper understanding of the molecular mechanisms at the basis of IBD development and progression is strictly required to design new and efficient therapeutic regimens. Although the development of animal models has undoubtedly facilitated the study of IBD, such in vivo approaches are often expensive and time-consuming. Here we propose an organ ex vivo culture (Gut-Ex-Vivo system, GEVS) based on colon from Balb/c mice cultivated in a dynamic condition, able to model the biochemical and morphological features of the mouse models exposed to DNBS (5-12 days), in 5 h. Indeed, upon DNBS exposure, we observed a dose-dependent: (i) up-regulation of the stress-related protein transglutaminase 2 (TG2); (ii) increased intestinal permeability associated with deregulated tight junction protein expression; (iii) increased expression of pro-inflammatory cytokines, such as TNFα, IFNγ, IL1ß, IL6, IL17A, and IL15; (iv) down-regulation of the anti-inflammatory IL10; and (v) induction of Endoplasmic Reticulum stress (ER stress), all markers of IBD. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of IBD, in a time- and cost-effective manner.
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Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.
RESUMO
Ischemia-reperfusion injury (IRI) consequent to major liver surgery is a still unmet clinical problem. The activation of endogenous systems of hepatoprotection can prevent the damaging effects of ischemia-reperfusion (IR) as shown by the phenomenon known as 'ischemic preconditioning'. The identification of endogenous signal mediators of hepatoprotection is of main interest since they could be targeted in future therapeutic interventions. Qiu et al. recently reported in Clin. Sci. (Lond.) (2020) 134(17), 2279-2294, the discovery of a novel protective molecule against hepatic IR damage: dual-specificity phosphatase 12 (DUSP12). IR significantly decreased DUSP12 expression in liver whereas DUSP12 overexpression in hepatocytes protected IRI and DUSP12 deletion in DUSP12 KO mice exacerbated IRI. The protective effects of DUSP12 depended on apoptosis signal-regulating kinase 1 (ASK1) and acted through the inhibition of the ASK1-dependent kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). These results enlighten DUSP12 as a novel intermediate negative regulator of the pro-inflammatory and pro-apoptotic ASK1/JNK-p38 MAPK pathway activated during hepatic IR and identify DUSP12 as potential therapeutic target for IRI.