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Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.
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Doenças Autoimunes/metabolismo , Autoimunidade , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/patologia , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismoRESUMO
Locoregional recurrence negatively impacts both long-term survival and quality of life for several malignancies. For appropriate-risk patients with an isolated, resectable, local recurrence, surgery represents the only potentially curative therapy. However, oncologic outcomes remain inferior for patients with locally recurrent disease even after macroscopically complete resection. Unfortunately, these operations are often extensive, with significant perioperative morbidity and mortality. This review highlights selected malignancies (mesothelioma, sarcoma, lung cancer, breast cancer, rectal cancer, and peritoneal surface malignancies) in which surgical resection is a key treatment modality and local recurrence plays a significant role in overall oncologic outcome with regard to survival and quality of life. For each type of cancer, the current, state-of-the-art treatment strategies and their outcomes are assessed. The need for additional therapeutic options is presented given the limitations of the current standard therapies. New and emerging treatment modalities, including polymer films and nanoparticles, are highlighted as potential future solutions for both prevention and treatment of locally recurrent cancers. Finally, the authors identify additional clinical and research opportunities and propose future research strategies based on the various patterns of local recurrence among the different cancers.
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Oncologia/métodos , Recidiva Local de Neoplasia/terapia , Neoplasias/terapia , Qualidade de Vida , Terapia Combinada/métodos , Terapia Combinada/tendências , Humanos , Oncologia/tendências , Recidiva Local de Neoplasia/complicações , Recidiva Local de Neoplasia/mortalidade , Neoplasias/complicações , Neoplasias/mortalidade , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Resultado do TratamentoRESUMO
OBJECTIVE: The objective was to critically analyze the published literature accounting for sex differences and skeletal age (open vs. closed physis) in preclinical animal models of OA, including the disaggregation of data by sex and skeletal maturity when data is generated from combined sex and/or multi-aged cohorts without proper confounding. METHOD: A scoping literature review of PubMed, Web of Science, EMBASE, and SCOPUS was performed for studies evaluating the effect of sex and age in experimental studies and clinical trials utilizing preclinical large animal models of OA. RESULTS: A total of 9727 papers were identified in large animal (dog, pig, sheep, goat, horse) models for preclinical OA research, of which 238 ex vivo and/or in vivo studies disclosed model type, animal species, sex, and skeletal age sufficient to analyze their effect on outcomes. Dogs, followed by pigs, sheep, and horses, were the most commonly used models. A paucity of preclinical studies evaluated the effect of sex and age in large animal models of naturally occurring or experimentally induced OA: 26 total studies reported some kind of analysis of the effects of sex or age, with 4 studies discussing the effects of sex only, 11 studies discussing the effects of age only, and 11 studies analyzing both the effects of age and sex. CONCLUSION: Fundamental to translational research, OARSI is uniquely positioned to develop recommendations for conducting preclinical studies using large animal models of OA that consider biological mechanisms linked to sex chromosomes, skeletal age, castration, and gonadal hormones affecting OA pathophysiology and treatment response.
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Osteoartrite , Feminino , Masculino , Suínos , Animais , Ovinos , Cavalos , Cães , Modelos Animais de Doenças , Osteoartrite/veterinária , Cabras , Bibliometria , Lâmina de CrescimentoRESUMO
OBJECTIVE: Cationic tantalum oxide nanoparticles (Ta2O5-cNPs), as a newly introduced contrast agent for computed tomography of cartilage, offer quantitative evaluation of proteoglycan (PG) content and biomechanical properties. However, knowledge on the depth-wise impact of cartilage constituents on nanoparticle diffusion, particularly the influence of the collagen network, is lacking. In this study, we aim to establish the depth-dependent relationship between Ta2O5-cNP diffusion and cartilage constituents (PG content, collagen content and network architecture). METHODS: Osteochondral samples (n = 30) were harvested from healthy equine stifle joints (N = 15) and the diffusion of 2.55 nm diameter cationic Ta2O5-cNPs into the cartilage was followed with micro computed tomography (µCT) imaging for up to 96 hours. The diffusion-related parameters, Ta2O5-cNP maximum partition (Pmax) and diffusion time constant, were compared against biomechanical and depth-wise structural properties. Biomechanics were assessed using stress-relaxation and sinusoidal loading protocols, whereas PG content, collagen content and collagen network architecture were determined using digital densitometry, Fourier-transform infrared spectroscopy and polarized light microscopy, respectively. RESULTS: The Pmax correlates with the depth-wise distribution of PGs (bulk Spearman's ρ = 0.87, p < 0.001). More open collagen network architecture at the superficial zone enhances intake of Ta2O5-cNPs, but collagen content overall decreases the intake. The Pmax values correlate with the equilibrium modulus (ρ = 0.80, p < 0.001) of articular cartilage. CONCLUSION: This study establishes the feasibility of Ta2O5-cNPs for the precise and comprehensive identification of biomechanical and structural changes in articular cartilage via contrast-enhanced µCT.
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Cartilagem Articular , Óxidos , Tantálio , Animais , Cavalos , Cartilagem Articular/diagnóstico por imagem , Meios de Contraste , Microtomografia por Raio-X , Proteoglicanas , ColágenoRESUMO
Drug delivery to the esophagus through systemic administration remains challenging, as minimal drug reaches the desired target. Local delivery offers the potential for improved efficacy while minimizing off-target toxicities but necessitates bioadhesive properties for mucosal delivery. Herein, we describe the synthesis of two new mucoadhesive amphiphilic copolymers prepared by sequential ring-opening copolymerization or postpolymerization click conjugation. Both strategies yield block copolymers containing a hydrophilic amine-functionalized poly-amido-saccharide and either a hydrophobic alkyl derivatized poly-amido-saccharide or poly(lactic acid), respectively. The latter resulting copolymers readily self-assemble into spherical, ≈200 nm diameter, positively charged mucoadhesive nanoparticles. The NPs entrap ultrahigh levels of paclitaxel via encapsulation of free paclitaxel and paclitaxel conjugated to a biodegradable, biocompatible poly(1,2-glycerol carbonate). Paclitaxel-loaded NPs rapidly enter cells, release paclitaxel, are cytotoxic to esophageal OE33 and OE19 tumor cells in vitro, and, importantly, demonstrate improved mucoadhesion compared to conventional poly(ethylene glycol)-poly(lactic acid) nanoparticles to ex vivo esophageal tissue.
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With one of the highest mortality rates of all malignancies, the 5-year survival rate for esophageal cancer is under 20%. Depending on the stage and extent of the disease, the current standard of care treatment paradigm includes chemotherapy or chemoradiotherapy followed by surgical esophagogastrectomy, with consideration for adjuvant immunotherapy for residual disease. This regimen has high morbidity, due to anatomic changes inherent in surgery, the acuity of surgical complications, and off-target effects of systemic chemotherapy and immunotherapy. We begin with a review of current treatments, then discuss new and emerging targets for therapies and advanced drug delivery systems. Recent and ongoing preclinical and early clinical studies are evaluating traditional tumor targets (e.g., human epidermal growth factor receptor 2), as well as promising new targets such as Yes-associated protein 1 or mammalian target of rapamycin to develop new treatments for this disease. Due the function and location of the esophagus, opportunities also exist to pair these treatments with a drug delivery strategy to increase tumor targeting, bioavailability, and intratumor concentrations, with the two most common delivery platforms being stents and nanoparticles. Finally, early results with antibody drug conjugates and chimeric antigenic receptor T cells show promise as upcoming therapies. This review discusses these innovations in therapeutics and drug delivery in the context of their successes and failures, with the goal of identifying those solutions that demonstrate the most promise to shift the paradigm in treating this deadly disease.
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Sistemas de Liberação de Medicamentos , Neoplasias Esofágicas , Humanos , Neoplasias Esofágicas/terapia , Sistemas de Liberação de Medicamentos/métodos , Imunoterapia/métodos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Nanopartículas/químicaRESUMO
Breast cancer is among the most prevalent malignancies, accounting for 685,000 deaths worldwide in 2020, largely due to its high metastatic potential. Depending on the stage and tumor characteristics, treatment involves surgery, chemotherapy, targeted biologics, and/or radiation therapy. However, current treatments are insufficient for treating or preventing metastatic disease. Herein, we describe supratherapeutic paclitaxel-loaded nanoparticles (81 wt % paclitaxel) to treat the primary tumor and reduce the risk of subsequent metastatic lesions in the lungs. Primary tumor volume and lung metastasis are reduced by day 30, compared to the paclitaxel clinical standard treatment. The ultrahigh levels of paclitaxel afford an immunotherapeutic effect, increasing natural killer cell activation and decreasing NETosis in the lung, which limits the formation of metastatic lesions.
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Neoplasias da Mama , Glicerol , Neoplasias Pulmonares , Nanopartículas , Polímeros , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Paclitaxel , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Metástase NeoplásicaRESUMO
Pressure sensitive adhesives are components of everyday products found in homes, offices, industries, and hospitals. Serving the general purpose of fissure repair and object fixation, pressure sensitive adhesives indiscriminately bind surfaces, as long as contact pressure is administered at application. With that being said, the chemical and material properties of the adhesive formulation define the strength of a pressure sensitive adhesive to a particular surface. Given our increased understanding of the viscoelastic material requirements as well as the intermolecular interactions at the binding interface required for functional adhesives, pressure sensitive adhesives are now being explored for greater use. New polymer formulations impart functionality and degradability for both internal and external applications. This review highlights the structure-property relationships between polymer architecture and pressure sensitive adhesion, specifically for medicine. We discuss the rational, molecular-level design of synthetic polymers for durable, removable, and biocompatible adhesion to wet surfaces like tissue. Finally, we examine prevalent challenges in biomedical wound closure and the new, innovative strategies being employed to address them. We conclude by summarizing the progress of current research, identifying additional clinical opportunities, and discussing future prospects.
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Polymeric micelles and especially those based on natural diblocks are of particular interest due to their advantageous properties in terms of molecular recognition, biocompatibility, and biodegradability. We herein report a facile and straightforward synthesis of thermoresponsive elastin-like polypeptide (ELP) and oligonucleotide (ON) diblock bioconjugates, ON-b-ELP, through copper-catalyzed azide-alkyne cycloaddition. The resulting thermosensitive diblock copolymer self-assembles above its critical micelle temperature (CMT â¼30 °C) to form colloidally stable micelles of â¼50 nm diameter. The ON-b-ELP micelles hybridize with an ON complementary strand and maintain their size and stability. Next, we describe the capacity of these micelles to bind proteins, creating more complex structures using the classic biotin-streptavidin pairing and the specific recognition between a transcription factor protein and the ON strand. In both instances, the micelles are intact, form larger structures, and retain their sensitivity to temperature.
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Micelas , Fatores de Transcrição , Biomimética , Peptídeos/química , Polímeros/química , TemperaturaRESUMO
Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.
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Neoplasias do Colo/imunologia , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Ceramidase Neutra/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral/transplante , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Ferroptose/efeitos dos fármacos , Ferroptose/imunologia , Humanos , Concentração Inibidora 50 , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Simulação de Acoplamento Molecular , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Ceramidase Neutra/antagonistas & inibidores , Ceramidase Neutra/genética , Estabilidade Proteica/efeitos dos fármacos , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Ring opening polymerization (ROP) of lactams is a highly efficient and versatile method to synthesize polyamides. Within the last ten years, significant advances in polymerization methodology and monomer diversity are ushering in a new era of polyamide chemistry. We begin with a discussion of polymerization techniques including the most widely used anionic ring opening polymerization (AROP), and less prevalent cationic ROP and enzyme-catalyzed ROP. Next, we describe new monomers being explored for ROP with increased functionality and stereochemistry. We emphasize the relationships between composition, structure, and properties, and how chemists can control composition and structure to dictate a desired property or performance. Finally, we discuss biomedical applications of the synthesized polyamides, specifically as biomaterials and pharmaceuticals, with examples to include as antimicrobial agents, cell adhesion substrates, and drug delivery scaffolds.
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Anti-Infecciosos , Nylons , Materiais Biocompatíveis , Caprolactama/análogos & derivados , Lactamas/química , Nylons/química , Preparações Farmacêuticas , Polimerização , PolímerosRESUMO
Polysaccharides are abundant in nature and employed in various biomedical applications ranging from scaffolds for tissue engineering to carriers for drug delivery systems. However, drawbacks such as tedious isolation protocols, contamination, batch-to-batch consistency, and lack of compositional control with regards to stereo- and regioselectivity impede the development and utility of polysaccharides, and thus mimetics are highly sought after. We report a synthetic strategy to regioselectively functionalize poly-amido-saccharides with sulfate or phosphate groups using post-polymerization modification reactions. Orthogonally protected ß-lactam monomers, synthesized from D-glucal, undergo anionic ring-opening polymerization to yield polymers with degrees of polymerization of 12, 25, and 50. Regioselective deprotection followed by functionalization and global deprotection affords the sulfated and phosphorylated poly-amido-saccharides. The resulting anionic polymers are water soluble and non-cytotoxic and adopt helical conformations. This new methodology provides access to otherwise inaccessible functional polysaccharide mimetics for biomedical applications.
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Carboidratos , Sulfatos , Polimerização , Polímeros , PolissacarídeosRESUMO
Arthrofibrosis, or rigid contracture of major articular joints, is a significant morbidity of many neurodegenerative disorders. The pathogenesis depends on the mechanism and severity of the precipitating neuromuscular disorder. Most neuromuscular disorders, whether spastic or hypotonic, culminate in decreased joint range of motion. Limited range of motion precipitates a cascade of pathophysiological changes in the muscle-tendon unit, the joint capsule, and the articular cartilage. Resulting joint contractures limit functional mobility, posing both physical and psychosocial burdens to patients, economic burdens on the healthcare system, and lost productivity to society. This article reviews the pathophysiology of arthrofibrosis in the setting of neuromuscular disorders. We describe current non-surgical and surgical interventions for treating arthrofibrosis of commonly affected joints. In addition, we preview several promising modalities under development to ameliorate arthrofibrosis non-surgically and discuss limitations in the field of arthrofibrosis secondary to neuromuscular disorders.
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Contratura , Artropatias , Contratura/complicações , Contratura/terapia , Fibrose , Humanos , Cápsula Articular/patologia , Artropatias/etiologia , Artropatias/patologia , Artropatias/terapia , Articulações/patologia , Articulação do Joelho/cirurgia , Amplitude de Movimento Articular/fisiologiaRESUMO
Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA- and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis.
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Bursite/tratamento farmacológico , Relaxina/uso terapêutico , Animais , Bursite/patologia , Linhagem Celular , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Injeções Intra-Articulares , Masculino , Camundongos , Amplitude de Movimento Articular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Relaxina/administração & dosagem , Articulação do Ombro/efeitos dos fármacos , Articulação do Ombro/patologiaRESUMO
Carbohydrates are ubiquitous in nature, playing vital roles in all organisms ranging from metabolism to intercellular signaling. Polysaccharides, repeating units of small molecule carbohydrates, are hydrophilic, densely functionalized, stereoregular, and rigid macromolecules, and these characteristics are simultaneously advantageous in biomedical applications while presenting major hurdles for synthetic methodology and development of structure property relationships. While naturally obtained polysaccharides are widely utilized in the biochemical and medical literature, their poor physicochemical definition and the potential for contaminated samples hinders the clinical translation of this work. To address the need for new methods to synthesize carbohydrate polymers, we reported a novel class of biomaterials (Poly-Amido-Saccharides; PAS) in 2012. PASs share many properties with natural polysaccharides, such as hydrophilicity, dense hydroxyl functionality, stereoregularity, and a rigid backbone. PASs are connected by an α-1,2-amide linkage, instead of an ether linkage, that confers resistance to enzymatic and hydrolytic degradation and leads to a unique helical conformation. Importantly, our synthetic methodology affords control over molecular weight distribution resulting in pure, well-defined polymers. This Account provides an overview of the development of PAS, from the factors that initially motivated our research to current efforts to translate functional PAS to biomedical applications. We detail the synthesis of glucose- and galactose-based PAS and their biophysical properties including conformation analysis, lectin interactions, cell internalization, and water solubility. Additionally, we describe postpolymerization modification strategies to afford PASs that act as protein stabilizers. We also highlight our recent efforts toward a mechanistic understanding of monomer synthesis via [2 + 2] cycloaddition reactions in order to develop novel monomers with different stereochemistry and amine or alkyl functionality, thereby accessing functional carbohydrate polymers. Throughout our work, we apply computational and theoretical analysis to explain how properties at the monomer level (e.g., stereochemistry, functionality) significantly impact polymer properties, helical conformation, and bioactivities. Collectively, the results from the theoretical, synthetic, and applied aspects of this research advance us toward our goal of utilizing PASs in key biomedical applications as alternatives to natural polysaccharides. The importance of carbohydrates in nature and the versatility of their functions continue to inspire our investigation of new monomers, polymers, and copolymers, leveraging the advantageous properties of PAS to develop potential therapies.
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Amidas/química , Polissacarídeos/química , Adjuvantes Imunológicos , Reação de Cicloadição , Galactose/química , Glucose/química , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lectinas/química , Lectinas/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , EstereoisomerismoRESUMO
BACKGROUND: Pancreatic peritoneal carcinomatosis (PPC), with the worst median overall-survival (mOS), epitomizes the incurability of metastatic cancer. Cancer stem cells (CSCs) underpin this incurability. However, inhibitors of CSC-stemness fail to increase mOS in cancer patients despite preclinical tumor-reduction. This shortfall reinforces that preclinical efficacy should be defined by increased mOS in the presence of cancer comorbidities, CSC-heterogeneity and plasticity. The primary objectives of this study are: to test the dual endothelin-1/signal peptide receptor, DEspR, as a nodal therapeutic target in PPC, given DEspR induction in anoikis-resistant pancreatic CSCs, and to validate humanized anti-DEspR antibody, hu-6g8, as a potential therapeutic for PPC. METHODS: We used heterogeneous pools of CSCs selected for anoikis resistance from reprogrammed Panc1 and MiaPaCa2 tumor cells (TCs), and adherent TCs reprogrammed from CSCs (cscTCs). We used multiple anti-DEspR blocking antibodies (mAbs) with different epitopes, and a humanized anti-DEspR recombinant mAb cross-reactive in rodents and humans, to test DEspR inhibition effects. We measured DEspR-inhibition efficacy on multiple prometastatic CSC-functions in vitro, and on tumorigenesis and overall survival in a CSC-derived xenograft (CDX) nude rat model of PPC with comorbidities. RESULTS: Here we show that DEspR, a stress-survival receptor, is present on subsets of PDAC Panc1-TCs, TC-derived CSCs, and CSC-differentiated TCs (cscTCs), and that DESpR-inhibition decreases apoptosis-resistance and pro-metastatic mesenchymal functions of CSCs and cscTCs in vitro. We resolve the DNA-sequence/protein-function discordance by confirming ADAR1-RNA editing-dependent DEspR-protein expression in Panc1 and MiaPaCa2 TCs. To advance DEspR-inhibition as a nodal therapeutic approach for PPC, we developed and show improved functionality of a recombinant, humanized anti-DEspR IgG4S228P antibody, hu-6g8, over murine precursor anti-DEspR mabs. Hu-6g8 internalizes and translocates to the nucleus colocalized with cyto-nuclear shuttling galectins-1/3, and induces apoptotic cell changes. DEspR-inhibition blocks transperitoneal dissemination and progression to peritoneal carcinomatosis of heterogeneous DEspR±/CD133 ± Panc1-derived CSCs in xenografted nude rats, improving mOS without chemotherapy-like adverse effects. Lastly, we show DEspR expression in Stage II-IV primary and invasive TCs in the stroma in PDAC-patient tumor arrays. CONCLUSION: Collectively, the data support humanized anti-DEspR hu-6g8 as a potential targeted antibody-therapeutic with promising efficacy, safety and prevalence profiles for PPC patients.
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Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Imunoglobulina G/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/secundário , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/química , Antineoplásicos Imunológicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Imunoglobulina G/química , Imuno-Histoquímica , Imunofenotipagem , Neoplasias Pancreáticas/patologia , Ratos , Receptor de Endotelina A , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic exposure of pancreatic ß cells to high concentrations of free fatty acids leads to lipotoxicity (LT)-mediated suppression of glucose-stimulated insulin secretion. This effect is in part caused by a decline in mitochondrial function as well as by a reduction in lysosomal acidification. Because both mitochondria and lysosomes can alter one another's function, it remains unclear which initiating dysfunction sets off the detrimental cascade of LT, ultimately leading to ß-cell failure. Here, we investigated the effects of restoring lysosomal acidity on mitochondrial function under LT. Our results show that LT induces a dose-dependent lysosomal alkalization accompanied by an increase in mitochondrial mass. This increase is due to a reduction in mitochondrial turnover as analyzed by MitoTimer, a fluorescent protein for which the emission is regulated by mitochondrial clearance rate. Mitochondrial oxygen consumption rate, citrate synthase activity, and ATP content are all reduced by LT. Restoration of lysosomal acidity using lysosome-targeted nanoparticles is accompanied by stimulation of mitochondrial turnover as revealed by mitophagy measurements and the recovery of mitochondrial mass. Remarkably, re-acidification restores citrate synthase activity and ATP content in an insulin secreting ß-cell line (INS-1). Furthermore, nanoparticle-mediated lysosomal reacidification rescues mitochondrial maximal respiratory capacity in both INS-1 cells and primary mouse islets. Therefore, our results indicate that mitochondrial dysfunction is downstream of lysosomal alkalization under lipotoxic conditions and that recovery of lysosomal acidity is sufficient to restore the bioenergetic defects.-Assali, E. A., Shlomo, D., Zeng, J., Taddeo, E. P., Trudeau, K. M., Erion, K. A., Colby, A. H., Grinstaff, M. W., Liesa, M., Las, G., Shirihai, O. S. Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.
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Células Secretoras de Insulina/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Nanopartículas/administração & dosagem , Animais , Células Cultivadas , Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacosRESUMO
Expansile nanoparticles (eNPs) are a promising pH-responsive polymeric drug delivery vehicle, as demonstrated in multiple intraperitoneal cancer models. However, previous delivery routes were limited to intraperitoneal injection and to a single agent, paclitaxel. In this study, we preliminarily evaluate the biodistribution and in vivo toxicity of eNPs in mice after intravenous injection. The eNPs localize predominantly to the liver, without detectable acute toxicity in the liver or other key organs. On the basis of these results, we encapsulated FQI1, a promising lead compound for treatment of hepatocellular carcinoma, in eNPs. eNPs are taken up by cancerous and noncancerous human liver cells in vitro, although at different rates. FQI1-loaded eNPs release FQI1 in a pH-dependent manner and limit proliferation equivalently to unencapsulated FQI1 in immortalized hepatocytes in vitro. eNPs are a versatile platform delivery system for therapeutic compounds and have potential utility in the treatment of liver disease.
Assuntos
Neoplasias Hepáticas , Nanopartículas , Quinolonas , Administração Intravenosa , Animais , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Distribuição TecidualRESUMO
The alkene-isocyanate cycloaddition method affords ß-lactams from glycals with high regio- and stereoselectivity, but the factors that determine substrate reactivity are poorly understood. Thus, we synthesized a library of 17 electron-rich alkenes (glycals) with varied protecting groups to systematically elucidate the factors that influence their reactivity toward the electron-poor trichloroacetyl isocyanate. The experimentally determined reaction rates exponentially correlate with the computationally determined highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) gap and natural bond orbital (NBO) valence energies. The electron-withdrawing ability of the protecting groups, but not bulk, impacts the electron density of the glycal allyloxocarbenium system when oriented pseudo-axially (i.e., stereoelectronics). In this conformation, ring σC-O* orbitals oriented antiperiplanar to the allyloxocarbenium system decrease glycal reactivity via negative hyperconjugation as protecting group electron withdrawal increases. Transition-state calculations reveal that protecting group stereoelectronics direct the reaction to proceed via an asynchronous one-step mechanism through a zwitterionic species. The combined experimental and computational findings, along with experimental validation on an unknown glycal, provide insight on the reaction mechanism and the role of distant protecting groups in glycal reactivity. Together, these studies will aid in the synthesis of new ß-lactam antibiotics, ß-lactamase inhibitors, and bicyclic carbohydrate-ß-lactam monomers prepared by the alkene-isocyanate method.
Assuntos
Elétrons , beta-Lactamas , Reação de Cicloadição , Cinética , Conformação MolecularRESUMO
Sulphated polysaccharides play important roles in a number of physiological and patho-physiological processes including the coagulation cascade, viral transmission, and antioxidation. In nature, sulphated polysaccharides are highly diverse, possessing variations in the carbohydrate backbone, location of the sulphate group(s), and degree of sulphation. These compositional attributes lead to varied sulphated polymers with different negative charge densities and resultant structure-property-activity relationships. Sulphating naturally occurring polysaccharides and their synthetic analogs is challenging, and traditionally requires harsh conditions and long reaction times, often causing non-selective sulphation at different and/or multiple hydroxyl positions. In this Review, we begin with a discussion of both established and novel methods and reagents for sulphation of these polymers, along with the advantages and disadvantages of these various approaches. Next, we describe characterization methods to confirm sulphation. Finally, we provide examples of synthetically sulphated natural polysaccharides and sulphated synthetic polysaccharides, and discuss the utility of these novel polymers in various biomedical applications. This review provides a comprehensive analysis of synthetic sulphated polysaccharides, their current uses, and highlights biomedical opportunities.