RESUMO
Activated macrophages contribute prominently to the progression and maintenance of almost all inflammatory and autoimmune diseases. Although non-specific elimination of these phagocytes has been shown to treat animal models of inflammatory disease, the same therapies have been compromised by unacceptable toxicities, because they also kill quiescent macrophages in healthy tissues. In the studies below, we exploit upregulation of folate receptor beta (FRß) on inflammatory (but not resting) macrophages to target a cytotoxic drug selectively to the inflammatory subset of macrophages. Because many of these activated macrophages are nondividing, we also employ verrucarin A as the cytotoxic payload, since it kills both mitotic and nonmitotic cells by blocking protein synthesis. By inserting a redox-sensitive self-immolative linker between the folate and verrucarin A, we further assure that release of unmodified verrucarin A is triggered primarily after internalization by an FRß-positive cell. The resulting folate-verrucarin A conjugate is shown to kill FR-expressing cells in vitro in a manner that can be inhibited by competition with 100-fold excess folic acid. The folate-verrucarin A conjugate is also shown to successfully treat a murine model of inflammatory peritonitis by eliminating inflammatory macrophages without killing other cells in the same peritonitis fluid. Based on this high specificity for inflammatory macrophages, we conclude that folate-verrucarin A warrants continued exploration as a potential therapy for inflammatory and autoimmune diseases in humans.
Assuntos
Modelos Animais de Doenças , Ácido Fólico/farmacologia , Macrófagos/efeitos dos fármacos , Peritonite/tratamento farmacológico , Tricotecenos/farmacologia , Animais , Relação Dose-Resposta a Droga , Receptor 2 de Folato/metabolismo , Ácido Fólico/química , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Peritonite/metabolismo , Relação Estrutura-Atividade , Tricotecenos/químicaRESUMO
The inadvertent severing of a ureter during surgery occurs in as many as 4.5% of colorectal surgeries. To help prevent this issue, several near-infrared (NIR) dyes have been developed to assist surgeons with identifying ureter location. However, the majority of these dyes exhibit at least some issue that precludes their widespread usage such as high levels of uptake in other tissues, overlapping emission wavelengths with other NIR dyes used for other fluorescence-guided surgeries, and/or rapid excretion times through the ureters. To overcome these limitations, we have synthesized and characterized the spectral properties and biodistribution of a new series of PEGylated UreterGlow derivatives. The most promising dye, UreterGlow-11 was shown to almost exclusively excrete through the kidneys/ureters with detectable fluorescence observed for at least 12 h. Additionally, while the excitation wavelength is similar to that of other NIR dyes used for cancer resections, the emission is shifted by ~30 nm allowing for discrimination between the different fluorescence-guided surgery probes. In conclusion, these new UreterGlow dyes show promising optical and biodistribution characteristics and are good candidates for translation into the clinic.
Assuntos
Abdome/cirurgia , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Ureter/cirurgia , Animais , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Rim/cirurgia , Camundongos , Distribuição Tecidual/fisiologia , Ureter/metabolismoRESUMO
Single-particle tracking offers a method to interrogate the organization of transmembrane proteins by measuring their mobilities within a cell's plasma membrane. Using this technique, the diffusion characteristics of the Duffy antigen (DARC), glycophorin A, band 3, and GLUT1 were compared under analogous conditions on intact human erythrocyte membranes. Microscopic diffusion coefficients revealed that the vast majority of all four transmembrane proteins exhibit very restricted movement but are not completely immobile. In fact, only 12% of GLUT1 resolved into a highly mobile subpopulation. Macroscopic diffusion coefficients and compartment sizes were also similar for all four proteins, with movements confined to the approximate dimensions of the "corrals" of the cortical spectrin cytoskeleton. Taken together, these data suggest that almost the entire populations of all four transmembrane proteins are immobilized by either the incorporation within large multiprotein complexes or entrapment within the protein network of the cortical spectrin cytoskeleton.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Glicoforinas , Difusão , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Transportador de Glucose Tipo 1 , Humanos , Espectrina/metabolismoRESUMO
Glucose transporter 1 (GLUT1) is one of 13 members of the human equilibrative glucose transport protein family and the only glucose transporter thought to be expressed in human erythrocyte membranes. Although GLUT1 has been shown to be anchored to adducin at the junctional spectrin-actin complex of the membrane through interactions with multiple proteins, whether other populations of GLUT1 also exist in the human erythrocyte membrane has not been examined. Because GLUT1 plays such a critical role in erythrocyte biology and since it comprises 10% of the total membrane protein, we undertook to evaluate the subpopulations of erythrocyte GLUT1 using single particle tracking. By monitoring the diffusion of individual AlexaFluor 488-labeled GLUT1 molecules on the surfaces of intact erythrocytes, we are able to identify three distinct subpopulations of GLUT1. While the mobility of the major subpopulation is similar to that of the anion transporter, band 3, both a more mobile and more anchored subpopulation also exist. From these studies, we conclude that ~65% of GLUT1 resides in similar or perhaps the same protein complex as band 3, while the remaining 1/3rd are either freely diffusing or interacting with other cytoskeletally anchored membrane protein complexes.
Assuntos
Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Biomarcadores , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Microscopia de Fluorescência , Imagem Molecular , Transporte Proteico , Coloração e RotulagemRESUMO
Activated macrophages play a key role in the development and maintenance of inflammatory diseases such as atherosclerosis, lupus, psoriasis, rheumatoid arthritis, ulcerative colitis, and many others. These activated macrophages, but not resting or quiescent macrophages highly up-regulate folate receptor beta (FR-ß). This differential expression of FR-ß provides a mechanism to selectively deliver imaging and therapeutic agents utilizing folate as a targeting molecule. In an effort to determine whether inflammatory diseases can be targeted utilizing a folate-linked nanosize carrier, a PEG-coated liposome was prepared that incorporated a folate conjugated PEG that also could transport imaging or therapeutic cargo. We demonstrate that these folate-liposomes specifically bind to folate receptor positive cells and accumulate at sites of inflammation in mouse models of colitis and atherosclerosis. These two animal models show that folate-targeted liposomes could be successfully utilized to deliver fluorescent molecules and an anti-inflammatory drug (betamethasone) for diagnostic and therapeutic applications.
Assuntos
Aterosclerose/tratamento farmacológico , Betametasona/farmacologia , Colite/tratamento farmacológico , Modelos Animais de Doenças , Receptor 2 de Folato/metabolismo , Lipossomos/administração & dosagem , Macrófagos Peritoneais/imunologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Betametasona/administração & dosagem , Betametasona/química , Colite/induzido quimicamente , Colite/imunologia , Colite/metabolismo , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Feminino , Ácido Fólico/metabolismo , Regulação da Expressão Gênica , Lipossomos/química , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , NanotecnologiaRESUMO
Folate-receptor-positive activated macrophages are critical for the development and maintenance of many chronic inflammatory and autoimmune diseases. Previously, small-molecule folate-targeted conjugates were found to specifically bind to these activated macrophages in vitro and selectively accumulate at sites of inflammation in vivo. While these small-molecule conjugates have shown promise, the use of a folate-targeted, higher cargo capacity nanovehicle may prove superior in delivering imaging or therapeutic agents in vivo. This nanoparticle strategy has been demonstrated in oncology, where targeted dendrimers have shown superior delivery capabilities; however, little research has been pursued in the area of folate-targeted dendrimers for inflammation and autoimmune diseases. Therefore, we endeavored to create a folate-decorated dendrimer to explore its uptake in mouse models of ulcerative colitis and atherosclerosis. We demonstrate that our final poly(ethylene glycol)-coated, acetic-anhydride-capped, folate-targeted poly(amidoamine) dendrimer exhibits no discernible cytotoxicity in vitro, specifically binds to a folate-receptor-expressing macrophage cell line in vitro, and selectively accumulates in areas of inflammation in vivo.
Assuntos
Aterosclerose/tratamento farmacológico , Colite Ulcerativa/tratamento farmacológico , Dendrímeros/farmacocinética , Ácido Fólico/análogos & derivados , Anidridos Acéticos/química , Animais , Linhagem Celular , Dendrímeros/química , Feminino , Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/farmacocinética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliaminas/química , Polietilenoglicóis/química , Distribuição TecidualRESUMO
As tumors grow, vasculature is often deficient or malformed, resulting in many localized areas of hypoxia. Cells located in these hypoxic regions exhibit an altered gene expression pattern that can significantly alter resistance to conventional anticancer treatments such as ionizing radiation and chemotherapeutic drugs. A priori knowledge of the level of hypoxia within a tumor may better guide clinical care. In an effort to create a hypoxia specific imaging agent, a ligand for the tissue hypoxia marker, carbonic anhydrase IX (CA IX), was synthesized and used as a targeting ligand to deliver an attached (99m)Tc-chelating agent. Binding of the resulting conjugates to hypoxic cancer cells was first characterized in vitro. Whole animal imaging and biodistribution studies then were performed to determine tumor specificity in vivo. Several conjugates were found to bind selectively to CA IX expressing tumors in a receptor-dependent manner. We suggest that such conjugates could prove useful in identifying hypoxic cancers and/or quantitating the level of hypoxia within a tumor.
Assuntos
Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/química , Regulação Neoplásica da Expressão Gênica , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Hipóxia Tumoral , Animais , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/metabolismo , Inibidores da Anidrase Carbônica/farmacocinética , Transformação Celular Neoplásica , Células HT29 , Humanos , Ligantes , Masculino , Camundongos , Polietilenoglicóis/química , Rodaminas/química , Tecnécio/química , Distribuição TecidualRESUMO
Proof-of-principle studies in ovarian, lung, and brain cancer patients have shown that fluorescence-guided surgery can enable removal of otherwise undetectable malignant lesions, decrease the number of cancer-positive margins, and permit identification of disease-containing lymph nodes that would have normally evaded resection. Unfortunately, the current arsenal of tumor-targeted fluorescent dyes does not permit identification of all cancers, raising the need to design new tumor-specific fluorescent dyes to illuminate the currently undetectable cancers. In an effort to design a more universal fluorescent cancer imaging agent, we have undertaken to synthesize a fluorophore that could label all hypoxic regions of tumors. We report here the synthesis, in vitro binding, and in vivo imaging of a near-infrared (NIR) fluorescent dye that is targeted to carbonic anhydrase IX (CA IX), i.e., a widely accepted marker of hypoxic tissues. The low molecular weight NIR probe, named Hypoxyfluor, is shown to bind CA IX with high affinity and accumulate rapidly and selectively in CA IX positive tumors. Because nearly all human cancers contain hypoxic regions that express CA IX abundantly, this NIR probe should facilitate surgical resection of a wide variety of solid tumors.
Assuntos
Anidrase Carbônica IX/metabolismo , Hipóxia Celular/fisiologia , Corantes Fluorescentes/metabolismo , Neoplasias/metabolismo , Neoplasias/cirurgia , Animais , Linhagem Celular Tumoral , Feminino , Fluorescência , Células HT29 , Humanos , Linfonodos/metabolismo , Camundongos , Camundongos Nus , Espectroscopia de Luz Próxima ao Infravermelho/métodosRESUMO
As the delivery of selectively targeted cytotoxic agents via antibodies or small molecule ligands to malignancies has begun to show promise in the clinic, the need to identify and validate additional cellular targets for specific therapeutic delivery is critical. Although a multitude of cancers have been targeted using the folate receptor, PSMA, bombesin receptor, somatostatin receptor, LHRH, and αvß3, there is a notable lack of specific small molecule ligand/receptor pairs to cellular targets found within cancers of the GI tract. Because of the selective GI tract expression of the cholecystokinin 2 receptor (CCK2R), we undertook the creation of conjugates that would deliver microtubule-disrupting drugs to malignancies through the specific targeting of CCK2R via a high affinity small molecule ligand. The cytotoxic activity of these conjugates were shown to be receptor mediated in vitro and in vivo with xenograft mouse models exhibiting delayed growth or regression of tumors that expressed CCK2R. Overall, this work demonstrates that ligands to CCK2R can be used to create selectively targeted therapeutic conjugates.
Assuntos
Oligopeptídeos/farmacologia , Ácidos Pipecólicos/farmacologia , Receptor de Colecistocinina B/metabolismo , Vimblastina/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Células HEK293 , Humanos , Células KB , Ligantes , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.
Assuntos
Corantes Fluorescentes , Fosfatidilinositóis , Fosfolipase C gama , Diester Fosfórico Hidrolases , Cumarínicos/farmacologia , Fosfolipases Tipo CRESUMO
A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various "turn-on" fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Corantes Fluorescentes , Fosfolipase C gama/genética , Ensaios de Triagem em Larga Escala , CumarínicosRESUMO
As humanity begins to venture further into space, approaches to better protect astronauts from the hazards found in space need to be developed. One particular hazard of concern is the complex radiation that is ever present in deep space. Currently, it is unlikely enough spacecraft shielding could be launched that would provide adequate protection to astronauts during long-duration missions such as a journey to Mars and back. In an effort to identify other means of protection, prophylactic radioprotective drugs have been proposed as a potential means to reduce the biological damage caused by this radiation. Unfortunately, few radioprotectors have been approved by the FDA for usage and for those that have been developed, they protect normal cells/tissues from acute, high levels of radiation exposure such as that from oncology radiation treatments. To date, essentially no radioprotectors have been developed that specifically counteract the effects of chronic low-dose rate space radiation. This review highlights how high-throughput screening (HTS) methodologies could be implemented to identify such a radioprotective agent. Several potential target, pathway, and phenotypic assays are discussed along with potential challenges towards screening for radioprotectors. Utilizing HTS strategies such as the ones proposed here have the potential to identify new chemical scaffolds that can be developed into efficacious radioprotectors that are specifically designed to protect astronauts during deep space journeys. The overarching goal of this review is to elicit broader interest in applying drug discovery techniques, specifically HTS towards the identification of radiation countermeasures designed to be efficacious towards the biological insults likely to be encountered by astronauts on long duration voyages.
Assuntos
Radiação Cósmica , Protetores contra Radiação , Voo Espacial , Humanos , Radiação Cósmica/efeitos adversos , Ensaios de Triagem em Larga Escala , Astronautas , Astronave , Protetores contra Radiação/farmacologia , Doses de RadiaçãoRESUMO
Although CAR T cell therapies have proven to be effective in treating hematopoietic cancers, their abilities to regress solid tumors have been less encouraging. Mechanisms to explain these disparities have focused primarily on differences in cancer cell heterogeneity, barriers to CAR T cell penetration of solid tumors, and immunosuppressive microenvironments. To evaluate the contributions of immunosuppressive tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) on CAR T cell efficacies, we have exploited the ability of a folate-targeted Toll-like receptor 7 agonist (FA-TLR7-1A) to specifically reactivate TAMs and MDSCs from an immunosuppressive to pro-inflammatory phenotype without altering the properties of other immune cells. We report here that FA-TLR7-1A significantly augments standard CAR T cell therapies of 4T1 solid tumors in immune competent mice. We further show that co-administration of the FA-TLR7-1A with the CAR T cell therapy not only repolarizes TAMs and MDSCs from an M2-like anti-inflammatory to M1-like pro-inflammatory phenotype, but also enhances both CAR T cell and endogenous T cell accumulation in solid tumors while concurrently increasing their states of activation. Because analogous myeloid cells in healthy tissues ar not altered by administration of FA-TLR7-1A, no systemic activation of the immune system nor accompanying weight loss is observed. These data argue that immunosuppressive myeloid cells contribute prominently to the failure of CAR T cells to eradicate solid tumors and suggest that methods to reprogram tumor associated myeloid cells to a more inflammatory phenotype could significantly augment the potencies of CAR T cell therapies.
Assuntos
Células Supressoras Mieloides , Neoplasias , Animais , Imunoterapia Adotiva , Camundongos , Células Mieloides , Receptor 7 Toll-Like , Microambiente TumoralRESUMO
Aim: Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues. For many ligand-targeted drug conjugates, a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells. Because disulfide bond reduction is frequently exploited to ensure intracellular drug release, an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design. While the redox properties of folate receptor trafficking endosomes have been previously reported, little is known about the trafficking of prostate-specific membrane antigen (PSMA), a receptor that is experiencing increasing use for drug targeting in humans. Methods: To obtain this information, we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green. Results: We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells, and that reduction of its disulfide bond proceeds steadily but incompletely following internalization. The fact that maximal disulfide reduction reaches only ~50%, even after 24 h incubation, suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity. Conclusion: As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates, it also follows that not all internalizing receptors are translocated through similar intracellular compartments. Taken together, these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.
RESUMO
To egress from its erythrocyte host, the malaria parasite, Plasmodium falciparum, must destabilize the erythrocyte membrane by activating an erythrocyte tyrosine kinase. Because imatinib inhibits erythrocyte tyrosine kinases and because imatinib has a good safety profile, we elected to determine whether coadministration of imatinib with standard of care (SOC) might be both well tolerated and therapeutically efficacious in malaria patients. Patients with uncomplicated P. falciparum malaria from a region in Vietnam where one third of patients experience delayed parasite clearance (DPC; continued parasitemia after 3 d of therapy) were treated for 3 d with either the region's SOC (40 mg dihydroartemisinin + 320 mg piperaquine/d) or imatinib (400 mg/d) + SOC. Imatinib + SOC-treated participants exhibited no increase in number or severity of adverse events, a significantly accelerated decline in parasite density and pyrexia, and no DPC. Surprisingly, these improvements were most pronounced in patients with the highest parasite density, where serious complications and death are most frequent. Imatinib therefore appears to improve SOC therapy, with no obvious drug-related toxicities.
Assuntos
Antimaláricos/efeitos adversos , Antimaláricos/uso terapêutico , Mesilato de Imatinib/uso terapêutico , Malária Falciparum/tratamento farmacológico , Adolescente , Adulto , Artemisininas/uso terapêutico , Quimioterapia Combinada , Febre/tratamento farmacológico , Febre/microbiologia , Humanos , Mesilato de Imatinib/efeitos adversos , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Quinolinas/uso terapêutico , Resultado do Tratamento , Vietnã , Adulto JovemRESUMO
Although chimeric antigen receptor (CAR) T cell therapies have demonstrated considerable success in treating hematologic malignancies, they have simultaneously been plagued by a cytokine release syndrome (CRS) that can harm or even kill the cancer patient. We describe a CAR T cell strategy in which CAR T cell activation and cancer cell killing can be sensitively regulated by adjusting the dose of a low molecular weight adapter that must bridge between the CAR T cell and cancer cell to initiate tumor eradication. By controlling the concentration and dosing schedule of adapter administration, we document two methods that can rapidly terminate (<3 h) a pre-existing CRS-like toxicity and two unrelated methods that can pre-emptively prevent a CRS-like toxicity that would have otherwise occurred. Because all four methods concurrently enhance CAR T cell potency, we conclude that proper use of bispecific adapters could potentially avoid a life-threatening CRS while enhancing CAR T cell tumoricidal activity.
Assuntos
Doenças do Sistema Imunitário/prevenção & controle , Imunoterapia Adotiva/efeitos adversos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Engenharia Celular/métodos , Linhagem Celular Tumoral , Citocinas/imunologia , Fluoresceína/metabolismo , Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/metabolismo , Humanos , Doenças do Sistema Imunitário/etiologia , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Síndrome , Linfócitos T/metabolismo , Linfócitos T/transplante , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: Most therapies for autoimmune and inflammatory diseases either neutralize or suppress production of inflammatory cytokines produced by activated macrophages (e.g., TNFα, IL-1, IL-6, IL-17, GM-CSF). However, no approved therapies directly target this activated subset of macrophages. METHODS: First, we undertook to examine whether the folate receptor beta (FR-ß) positive subpopulation of macrophages, which marks the inflammatory subset in animal models of rheumatoid arthritis, might constitute the prominent population of macrophages in inflamed lesions in humans. Next, we utilized anti-FR-ß monoclonal antibodies capable of mediating antibody-dependent cell cytotoxicity (ADCC) to treat animal models of rheumatoid arthritis and peritonitis. RESULTS: Human tissue samples of rheumatoid arthritis, Crohn's disease, ulcerative colitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, chronic obstructive pulmonary disease, systemic lupus erythematosus, psoriasis, and scleroderma are all characterized by dramatic accumulation of macrophages that express FR-ß, a protein not expressed on resting macrophages or any other healthy tissues. A monoclonal antibody to FR-ß accumulates specifically in inflamed lesions of murine inflammatory disease models and successfully treats such models of rheumatoid arthritis and peritonitis. More importantly, elimination of FR-ß-positive macrophages upon treatment with an anti-FR-ß monoclonal antibody promotes the departure of other immune cells, including T cells, B cells, neutrophils, and dendritic cells from the inflamed lesions. CONCLUSIONS: These data suggest that specific elimination of FR-ß-expressing macrophages may constitute a highly specific therapy for multiple autoimmune and inflammatory diseases and that a recently developed human anti-human FR-ß monoclonal antibody (m909) might contribute to suppression of this subpopulation of macrophages.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Reumatoide/imunologia , Receptor 2 de Folato/imunologia , Imunidade Celular , Ativação de Macrófagos , Macrófagos/metabolismo , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Macrófagos/imunologia , Macrófagos/patologia , CamundongosRESUMO
Receptor-targeted delivery of imaging and therapeutic agents has emerged as an attractive strategy to diagnosis and treat many diseases including cancer. One of the most well-studied receptors for targeted therapies is the folate receptor (FR) family. FR-α and FR-ß are present on many cancers with little expression in normal tissues; leading to the testing of at least six folate-targeted drugs in human clinical trials for various cancers. However, the expression of FR in blood cancers has not been fully explored with no reports of FR expression in myelomas. Herein, we report the expression of both FR-α and FR-ß on CD138 + plasma cells isolated from patients with multiple myeloma. In addition, all-trans retinoic acid was shown to increase the levels of FR-α and FR-ß in two myeloma cell lines. Altogether, this data suggests that folate-targeted therapies for the treatment of multiple myeloma warrants further investigation.
Assuntos
Antineoplásicos/farmacologia , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/metabolismo , Mieloma Múltiplo/patologia , Plasmócitos/efeitos dos fármacos , Tretinoína/farmacologia , Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Plasmócitos/metabolismo , Sindecana-1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-ß. Logically, if the FR positive cell population in a tumor mass is dominated by FR-ß positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-ß, no study to date has investigated expression of both FR-α and FR-ß in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-ß (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-ß combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-ß while pancreatic cancers express primarily FR-ß. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-ß should be obtained to predict the potential efficacy of a folate-targeted drug.
RESUMO
AIM OF THE STUDY: The aim of our study was to compare poly(ADP-ribose) polymerase (PARP) activity levels in a porcine model of hemorrhagic shock and resuscitation. MATERIALS AND METHODS: We designed a prospective, comparative randomized survival study of hemorrhagic shock using 20 male Yorkshire-Landrace pigs (15-25 kg). In 16 pigs after splenectomy, we induced hemorrhagic shock to a mean arterial pressure of 50 mm Hg ( approximately 35% bleed). Pigs were randomized to receive normotensive resuscitation (SBP 90 mm Hg), mild hypotensive resuscitation (SBP 80 mm Hg), moderate hypotensive resuscitation (SBP 65 mm Hg), or no resuscitation (n=4 in each group). We also included a group of sham animals that were instrumented and had a splenectomy but not bled (n=4). Muscle and liver biopsies were taken prior to hemorrhage, after 45 min of shock, and 8, 24, and 48 h after resuscitation. PARP activity levels in biopsies were measured using chemical quantitation of NAD+. RESULTS: Irrespective of our resuscitation strategy or outcome, both muscle and liver PARP activity levels rose after 45 min of shock and then returned to baseline. Excluding our control animals, PARP activity levels were significantly higher during shock in non-survivors compared to survivors. CONCLUSIONS: In our model of porcine hemorrhagic shock, PARP activity levels increased during hemorrhagic shock. However, this increase in PARP activity levels was transient as they returned to baseline regardless of resuscitation strategy. Interestingly, PARP activity levels were significantly higher during hemorrhagic shock in non-survivors compared to survivors. These findings suggest that PARP activity may be a part of initial pathways leading from hemorrhagic shock to death.