RESUMO
Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1-2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIPAM) via reversible addition fragmentation chain transfer (RAFT) polymerization. The resulting thermophobic adjuvants exhibit LCSTs near 37 °C, and self-assembled into nanoparticles with temperature-dependent sizes (90-270 nm). Thermophobic adjuvants activate HEK-mMINCLE and other innate immune cell lines as well as primary mouse bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs). Inflammatory cytokine production is attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvant Rg is observed by DLS, as well as glycolipid-NIPAM shielding interactions are observed by NOESY-NMR. In vivo, thermophobic adjuvants enhance efficacy of a whole inactivated influenza A/California/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+ /44+ /62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity after viral challenge relative to unadjuvanted control vaccine. Together, these results demonstrate the first adjuvants with potency regulated by temperature. This work envisions that with further investigation, this approach can enhance vaccine efficacy while maintaining safety.
Assuntos
Adjuvantes de Vacinas , Vacinas , Animais , Camundongos , Trealose/farmacologia , Trealose/química , Lectinas Tipo C/metabolismo , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/química , Glicolipídeos/farmacologia , Glicolipídeos/química , Anticorpos AntiviraisRESUMO
Multidrug-Resistant (MDR) cancers attenuate chemotherapeutic efficacy through drug efflux, a process that transports drugs from within a cell to the extracellular space via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Conversely, Toll-Like Receptor (TLR) agonist immunotherapies modulate activity of tumor-infiltrating immune cells in local proximity to cancer cells and could, therefore, benefit from the enhanced drug efflux in MDR cancers. However, the effect of acquired drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of TLR 7/8 agonists. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Interestingly, the least potent imidazoquinoline (Imiquimod) was the best P-gp substrate. Next, we compared imidazoquinoline efflux in MDR cancer cell lines with enhanced P-gp expression relative to parent cancer cell lines. Using P-gp competitive substrates and inhibitors, we observed that imidazoquinoline efflux occurs through P-gp and, for Imiquimod, is enhanced as a consequence of acquired drug resistance. This suggests that enhancing efflux susceptibility could be an important consideration in the rational design of next generation immunotherapies that modulate activity of tumor-infiltrating immune cells.
RESUMO
We have recently developed an enzyme-directed immunostimulant (EDI) prodrug motif, which is metabolized to active immunostimulant by cancer cells and, following drug efflux, activates nearby immune cells, resulting in immunogenicity. In this study, we synthesized several EDI prodrugs featuring an imidazoquinoline immunostimulant resiquimod (a Toll-like receptor 7/8 agonist) covalently modified with glycosidase enzyme-directing groups selected from substrates of ß-glucuronidase, α-mannosidase, or ß-galactosidase. We compared the glycosidase-dependent immunogenicity elicited by each EDI in RAW-Blue macrophages following conversion to active immunostimulant by complementary glycosidase. At a cellular level, we examined EDI metabolism across three cancer cell lines (B16 melanoma, TC2 prostate, and 4T1 breast cancer). Comparing the relative immunogenicity elicited by each EDI/cancer cell combination, we found that B16 cells produced the highest EDI prodrug immunogenicity, achieving >95% of that elicited by unmodified resiquimod, followed by TC2 and 4T1 cells (40% and 30%, respectively). Immunogenicity elicited was comparable for a given cell type and independent of the glycosidase substrate in the EDIs or differences in functional glycosidase activity between cell lines. Measuring drug efflux of the immunostimulant payload and efflux protein expression revealed that EDI/cancer cell-mediated immunogenicity was governed by efflux potential of the cancer cells. We determined that, following EDI conversion, immunostimulant efflux occurred through both P-glycoprotein-dependent and P-glycoprotein-independent transport mechanisms. Overall, this study highlights the broad ability of EDIs to couple immunogenicity to the metabolism of many cancers that exhibit drug efflux and suggests that designing future generations of EDIs with immunostimulant payloads that are optimized for drug efflux could be particularly beneficial.
Assuntos
Adjuvantes Imunológicos/metabolismo , Glicosídeo Hidrolases/metabolismo , Imidazóis/metabolismo , Neoplasias/metabolismo , Pró-Fármacos/metabolismo , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Células Cultivadas , Imidazóis/química , Imidazóis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pró-Fármacos/química , Pró-Fármacos/farmacologiaRESUMO
Dysregulated vascular inflammation is the underlying cause of acute lung inflammation/injury (ALI). Bacterial infections and trauma cause ALI that may rapidly lead to acute respiratory distress syndrome (ARDS). There are no pharmacological therapies available to patients with ALI/ARDS, partially as drugs cannot specifically target the lungs. Herein, we developed a stimuli-responsive nanoparticle (NP) to target inflammatory lungs for ALI therapies. The NP is composed of a sharp acid-sensitive segment poly(ß-amino esters) as a core for drug loading and controlled release and a polyethylene glycol-biotin on the particle surface available for bioconjugation, enabling lung targeting and extended circulation. The studies on dissipative particle dynamics simulation and characteristics of NPs suggest that anti-ICAM-1 antibodies can be coated to the particle surface and this coating is required to enhance lung targeting of NPs. A model drug of anti-inflammatory agent TPCA-1 is encapsulated in NPs with a high drug-loading content at 24% (w/w). In the mouse ALI model, our TPCA-1-loaded NPs coated with anti-ICAM-1 can target inflamed lungs after intravenous injection, followed by drug release triggered by the acid environment, thus mitigating lung inflammation and injury. Our studies reveal the rational design of nanotherapeutics for improved therapy of ALI, which may be applied to treating a wide range of vascular inflammation.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Nanopartículas/administração & dosagem , Pneumonia/tratamento farmacológico , Síndrome do Desconforto Respiratório/tratamento farmacológico , Lesão Pulmonar Aguda/complicações , Lesão Pulmonar Aguda/patologia , Amidas/administração & dosagem , Amidas/química , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/química , Infecções Bacterianas/complicações , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Humanos , Concentração de Íons de Hidrogênio , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Nanopartículas/química , Pneumonia/complicações , Pneumonia/patologia , Polietilenoglicóis/química , Ratos , Tiofenos/administração & dosagem , Tiofenos/químicaRESUMO
Drug efflux and enzymatic drug degradation are two cellular mechanisms that contribute to drug resistance in many cancers. Herein, we report the synthesis and in vitro activity of a pro-immunostimulant that exploits both processes in tandem to selectively confer cancer-mediated immunogenicity. We demonstrate that an imidazoquinoline pro-immunostimulant is inactive until it is selectively metabolized to an active immunostimulant by an endogenous α-mannosidase enzyme expressed within multidrug-resistant cancer cells. Following conversion, the immunostimulant is transported to the extracellular space via drug efflux, resulting in the activation of model bystander immune cells. Taken together, these results suggest that enzyme-directed immunostimulants can couple immunogenicity to these mechanisms of drug resistance. We name this process bystander-assisted immunotherapy, and envision that it could be advanced to treat drug-resistant diseases that rely on enzymatic degradation or drug efflux to persist.
Assuntos
Adjuvantes Imunológicos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoterapia , Pró-Fármacos , Neoplasias da Próstata , Quinolinas , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/terapia , Quinolinas/farmacocinética , Quinolinas/farmacologia , Células RAW 264.7RESUMO
The complexity of the immune system creates challenges in exploring its importance and robustness. To date, there have been few techniques developed to manipulate individual components of the immune system in an in vivo environment. Here we show a light-based dendritic cell (DC) activation allowing spatial and temporal control of immune activation in vivo. Additionally, we show time dependent changes in RNA profiles of the draining lymph node, suggesting a change in cell profile following DC migration and indicating that the cells migrating have been activated towards antigen presentation.
Assuntos
Imunidade Inata/imunologia , Ativação Linfocitária/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Animais , Apresentação de Antígeno/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Luz , Linfonodos/imunologia , Camundongos , RNA/imunologiaRESUMO
Synthetic agonists of innate immune cells are of interest to immunologists due to their synthesis from well-defined materials, optimized activity, and monodisperse chemical purity. These molecules are used in both prophylactic and therapeutic contexts from vaccines to cancer immunotherapies. In this review we highlight synthetic agonists that activate innate immune cells through three classes of pattern recognition receptors: NOD-like receptors, RIG-I-like receptors, and C-type lectin receptors. We classify these agonists by the receptor they activate and present them from a chemical perspective, focusing on structural components that define agonist activity. We anticipate this review will be useful to the medicinal chemist as a guide to chemical motifs that activate each receptor, ultimately illuminating a chemical space ripe for exploration.
Assuntos
Inflamação/imunologia , Lectinas Tipo C/agonistas , Oligonucleotídeos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Inflamação/tratamento farmacológico , Lectinas Tipo C/imunologia , Estrutura Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
KEY POINTS: The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. ABSTRACT: The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.
Assuntos
Canal Anal/fisiologia , Canais de Cálcio Tipo L/fisiologia , Canais de Cloreto/fisiologia , Células Intersticiais de Cajal/fisiologia , Canal Anal/metabolismo , Animais , Anoctamina-1 , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Feminino , Expressão Gênica , Técnicas In Vitro , Células Intersticiais de Cajal/metabolismo , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Muscular , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/fisiologiaRESUMO
Herein we report the synthesis and activity of an enzyme-directed immunostimulant with immune cell activation mediated by ß-galactosidase, either exogenously added, or on B16 melanoma cells. Covalent attachment of a ß-galactopyranoside to an imidazoquinoline immunostimulant at a position critical for activity resulted in a pro-immunostimulant that could be selectively converted by ß-galactosidase into an active immunostimulant. The pro-immunostimulant exhibited ß-galactosidase-directed immune cell activation as measured by NF-κB transcription in RAW-Blue macrophages or cytokine production (TNF, IL-6, IL-12) in JAWSII monocytes. Conversion of the pro-immunostimulant into an active immunostimulant was also found to occur using ß-galactosidase-enriched B16 melanoma cells. In co-culture experiments with either immune cell line, ß-galactosidase-enriched B16 cells effected activation of bystander immune cells.
Assuntos
Antineoplásicos/uso terapêutico , Imidazóis/uso terapêutico , Imunoterapia , Melanoma Experimental/terapia , Quinolinas/uso terapêutico , beta-Galactosidase/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Humanos , Imidazóis/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Conformação Molecular , Quinolinas/metabolismoRESUMO
Electron beam (e-beam) lithography was employed to prepare one protein immobilized hydrogel encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 µm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complementary reactivity, and the three-dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the encapsulated Alkyne-PEG core/ AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions.
Assuntos
Elétrons , Peroxidase do Rábano Silvestre/metabolismo , Hidrogéis/química , Polietilenoglicóis/química , Impressão , Alcinos/química , Biotina/química , Cápsulas , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismoRESUMO
Investigating the ecological impacts of contaminants released into the environment requires integration of multiple lines of evidence. Collection and analysis of interstitial water is an often-used line of evidence for developing benthic exposure estimates in aquatic ecosystems. It is a well-established principle that chemical and toxicity data on interstitial water samples should represent in-situ conditions; i.e., sample integrity must be maintained throughout the sample collection process to avoid alteration of the in-situ geochemical conditions. Unfortunately, collection and processing of pore water is not standardized to address possible geochemical transformations introduced by atmospheric exposure. Furthermore, there are no suitable benchmarks (ecological or human health) against which to evaluate adverse effects from chemicals in pore water; i.e., empirical data is lacking on the toxicity of inorganic contaminants in sediment interstitial water. It is clear that pore water data is best evaluated by considering the bioavailability of trace elements and the partitioning of contaminants between the aqueous and solid phases. It is also evident that there is a need for sediment researchers and regulatory agencies to collaborate in developing a standardized approach for sediment/pore water collection and data evaluation. Without such guidelines, the number of different pore water collection and extraction techniques will continue to expand, and investigators will continue to evaluate potentially questionable data by comparison to inappropriate criteria.
Assuntos
Monitoramento Ambiental/normas , Sedimentos Geológicos/análise , Poluentes Químicos da Água/análise , Água/análiseRESUMO
BACKGROUND: Blockade of the vascular endothelial growth factor (VEGF) pathway shows evidence of activity in gastro-oesophageal (GE) and oesophageal cancer. We investigated the efficacy of sunitinib, a multikinase VEGF inhibitor, in patients with relapsed/refractory GE/oesophageal cancer. METHODS: This was a single-stage Fleming phase II study. The primary end point was progression-free survival (PFS) at 24 weeks. If five or more patients out of a total of 25 were free of progressive disease at 24 weeks, sunitinib would be recommended for further study. Patients received sunitinib 37.5 mg orally daily and imaged every 6 weeks. Exploratory correlative analysis included serum growth factors, tumour gene expression and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). RESULTS: Twenty-five evaluable patients participated in the study. Progression-free survival at 24 weeks was 8% (n=2 patients; confidence interval (CI): 95% 1.4-22.5%), and the duration of best response for the patients was 23 and 72 weeks. Ten patients (42%) had stable disease (SD) for >10 weeks. Overall response rate is 13%. Median PFS is 7 weeks (95% CI: 5.6-11.4 weeks) and the median overall survival is 17 weeks (95% CI: 8.9-25.3 weeks). Most common grade 3/4 toxicities included fatigue (24%), anaemia (20%) thrombocytopenia (16%), and leucopenia (16%). No patients discontinued therapy due to toxicity. Serum VEGF-A and -C levels, tumour complement factor B (CFB) gene expression, and DCE-MRI correlated with clinical benefit, defined as SD or better as best response. CONCLUSION: Sunitinib is well tolerated but only a select subgroup of patients benefited. Serum VEGF-A and -C may be early predictors of benefit. On this study, patients with clinical benefit from sunitinib had higher tumour CFB expression, and thus has identified CFB as a potential predictor for efficacy of anti-angiogenic therapy. These findings need validation from future prospective trials.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias Esofágicas/tratamento farmacológico , Junção Esofagogástrica , Indóis/uso terapêutico , Pirróis/uso terapêutico , Adulto , Idoso , Fator B do Complemento/análise , Neoplasias Esofágicas/sangue , Feminino , Humanos , Indóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Pirróis/efeitos adversos , Recidiva , Sunitinibe , Transcriptoma , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/sangue , Fator C de Crescimento do Endotélio Vascular/sangueRESUMO
Inflammatory immune responses are mediated by signaling molecules that are both produced by and recognized across highly heterogeneous cell populations. As such, the study of inflammation using traditional immunostimulants is complicated by paracrine and autocrine signaling, which obscures the origin of a propagating response. To address this challenge, we developed a small-molecule probe that can photosensitize immune cells, thus allowing light-mediated inflammation. This probe was used to control the origin of inflammation using light. Following this motif, inflammation was initiated from fibroblasts or dendritic cells. The contributions of fibroblasts and dendritic cells in initiating inflammation in heterogeneous co-culture are reported, thus providing insights into the future development of vaccines and treatment of inflammation.
Assuntos
Inflamação/imunologia , Lipopeptídeos/imunologia , Sondas Moleculares/imunologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Fibroblastos/imunologia , Humanos , Lipopeptídeos/síntese química , Lipopeptídeos/química , Modelos Moleculares , Sondas Moleculares/síntese química , Sondas Moleculares/química , Estrutura Molecular , Processos FotoquímicosRESUMO
The innate immune response is controlled, in part, by the synergistic interaction of multiple Toll-like receptors (TLRs). This multi-receptor cooperation is responsible for the potent activity of many vaccines, but few tools have been developed to understand the spatio-temporal elements of TLR synergies. In this Communication, we present photo-controlled agonists of TLR7/8. By strategically protecting the active agonist moiety based on an agonist-bound crystal structure, TLR activity is suppressed and then regained upon exposure to light. We confirmed NF-κB production upon light exposure in a model macrophage cell line. Primary cell activity was confirmed by examining cytokine and cell surface marker production in bone-marrow-derived dendritic cells. Finally, we used light to activate dendritic cell sub-populations within a larger population.
Assuntos
Células Dendríticas/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/efeitos da radiação , Luz , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/efeitos da radiação , Imiquimode , Camundongos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
The weathering of coal combustion products (CCPs) in a lotic environment was assessed following the Tennessee Valley Authority (Kingston, TN) fly ash release of 2008 into surrounding rivers. Sampled materials included stockpiled ash and sediment collected from 180 to 880 days following the release. Total recoverable concentrations of heavy metals and metalloids in sediment were measured, and percent ash was estimated visually or quantified by particle counts. Arsenic and selenium in sediment were positively correlated with percent ash. For samples collected 180 days after the release, total concentrations of trace elements downstream of the release were greater than reference levels but less than concentrations measured in stockpiled ash. Total concentrations of trace elements remained elevated in ash-laden sediment after almost 2.5 years. A sequential extraction procedure (SEP) was used to speciate selected fractions of arsenic, copper, lead, nickel, and selenium in decreasing order of bioavailability. Concentrations of trace elements in sequentially extracted fractions were one to two orders of magnitude lower than total recoverable trace elements. The bulk of sequentially extractable trace elements was associated with iron-manganese oxides, the least bioavailable fraction of those measured. By 780 days, trace element concentrations in the SEP fractions approached reference concentrations in the more bioavailable water soluble, ion exchangeable, and carbonate-bound fractions. For each trace element, the percentage composition of the bioavailable fractions relative to the total concentration was calculated. These SEP indices were summed and shown to significantly decrease over time. These results document the natural attenuation of leachable trace elements in CCPs in river sediment as a result of the loss of bioavailable trace elements over time.
Assuntos
Carvão Mineral , Monitoramento Ambiental , Sedimentos Geológicos/química , Rios/química , Poluentes Químicos da Água/análise , Metaloides/análise , Metais Pesados/análise , Modelos Químicos , Centrais Elétricas , Tennessee , Oligoelementos/análiseRESUMO
Agonists of immune cell receptors direct innate and adaptive immunity. These agonists range in size and complexity from small molecules to large macromolecules. Here, agonists of a class of immune cell receptors known as the Toll-like receptors (TLRs) are highlighted focusing on the distinctive molecular moieties that pertain to receptor binding and activation. How the structure and combined chemical signals translate into a variety of immune responses remain major questions in the field. In this structure-focused review, we outline potential areas where the tools of chemical biology could help decipher the emerging molecular codes that direct immune stimulation.
Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Imunidade Inata/efeitos dos fármacos , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacosRESUMO
We report increased stimulation of dendritic cells via heterodimers of immunostimulants formed at a discrete molecular distance. Many vaccines present spatially organized agonists to immune cell receptors. These receptors cluster suggesting that signaling is increased by spatial organization and receptor proximity, but this has not been directly tested for multiple, unique receptors. In this study we probe the spatial aspect of immune cell activation using heterodimers of two covalently attached immunostimulants.
Assuntos
Adjuvantes Imunológicos/química , Polímeros/química , Fosforilação , Transdução de SinaisRESUMO
We present a primary example of a cell surface modified with a synergistic combination of agonists to tune immune stimulation. A model cell line, Lewis Lung Carcinoma, was covalently modified with CpG-oligonucleotides and lipoteichoic acid, both Toll-like receptor (TLR) agonists. The immune-stimulating constructs provided greater stimulation of NF-κB in a model cell line and bone marrow-derived dendritic cells than the components unconjugated in solution.
Assuntos
Carcinoma Pulmonar de Lewis/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Ácidos Teicoicos/farmacologia , Receptores Toll-Like/agonistas , Animais , Carcinoma Pulmonar de Lewis/imunologia , Linhagem Celular Tumoral , Lipopolissacarídeos/química , NF-kappa B/imunologia , Oligodesoxirribonucleotídeos/química , Ácidos Teicoicos/química , Receptores Toll-Like/imunologiaRESUMO
Herein, we report the synthesis of trehalose side chain polymers for stabilization of protein conjugates to environmental stressors. The glycomonomer 4,6-O-(4-vinylbenzylidene)-α,α-trehalose was synthesized in 40% yield over two steps without the use of protecting group chemistry. Polymers containing the trehalose pendent groups were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization using two different thiol-reactive chain transfer agents (CTAs) for subsequent conjugation to proteins through disulfide linkages. The resulting glycopolymers were well-defined, and a range of molecular weights from 4200 to 49 500 Da was obtained. The polymers were conjugated to thiolated hen egg white lysozyme and purified. The glycopolymers when added or covalently attached to protein significantly increased stability toward lyophilization and heat relative to wild-type protein. Up to 100% retention of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated at 90 °C for 1 h; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The glycopolymers were compared to equivalent concentrations of trehalose and poly(ethylene glycol) (PEG) and found to be superior at stabilizing the protein to lyophilization and heat. In addition, the protein-glycopolymer conjugates exhibited significant increases in lyophilization stability when compared to adding the same concentration of unconjugated polymer to the protein.