RESUMEN
The integration of anoxic filter and aerobic rotating biological contactor shows promise in treating rural domestic sewage. It offers high efficiency, low sludge production, and strong shock resistance. However, further optimization is needed for odor control, pollutant removal, and power consumption. In this study, the investigation on a one-pump-drive lab-scale device of retention anoxic filter (RAF) integrated with hydraulic rotating bio-contactor (HRBC) and its optimal operation mode were conducted. During the 50-day operation, optimal operation parameters were investigated. These parameters included a 175 % reflux ratio (RR), 5-h hydraulic retention time in the RAF (HRTRAF), and 2.5-h hydraulic retention time in the HRBC (HRTHRBC). Those conditions characterized a micro-aerobic environment (DO: 0.6-0.8 mg/L) in RAF, inducing improved deodorization (89.3 % sulfide removal) and denitrification (85.9 % nitrate removal) simultaneously. During the operation period, 84.79 ± 3.87 % COD, 82.71± 2.06 % NH4+-N, 74.83 ± 2.06 % TN, 91.68± 2.12 % S2-, and 89.04 ± 1.68 % TON were removed in RAF-HRBC. Based on large amount of operational data, organic loading rate curves of RAF-HRBC were validated and calibrated as a crucial reference to aid in full-scale designs and applications. The richness of microbial community was improved in both RAF and HRBC. In the RAF, the autotrophic sulfide-oxidizing nitrate-reducing bacteria (a-son) and heterotrophic sulfide-oxidizing nitrate-reducing bacteria (h-son) were selectively enriched, which intensified the sulfide removal and denitrification process. In the two-stage HRBC system, the 1st stage RBC was primarily composed of organics degraders, while the 2nd stage RBC consisted mainly of ammonium oxidizers. Overall, the integrated RAF-HRBC process holds significant potential for simultaneously improving pollutant removal and in-situ odor mitigation in decentralized domestic sewage treatment. This process specifically contributes to enhancing environmental sustainability and operational efficiency.
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The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.
Asunto(s)
Neoplasias Colorrectales , Receptores Quiméricos de Antígenos , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/terapia , Antígeno HLA-A2/genética , Humanos , Pérdida de HeterocigocidadRESUMEN
Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called TmodTM, that contains an activating receptor (activator) and a separate inhibitory receptor (blocker). In proof-of-concept experiments, we use CD19 as the activating antigen and HLA-A*02 as the blocker antigen. This specific Tmod system, which employs a blocker targeting a ubiquitously expressed HLA class I antigen to inhibit CAR activation, has an inherent mechanism for selectivity/safety, designed to activate only when a specific HLA class I antigen is lost. Nonetheless, it is important to test off-target reactivity in functional assays, especially given the disconnect between ligand-binding and function among T cell receptors (TCRs) and chimeric antigen receptors (CARs). We show these cell-based assays yield consistent results with high sensitivity and specificity. The general strategy is likely applicable to more traditional single-receptor CAR- and TCR-T therapeutics.
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Tratamiento Basado en Trasplante de Células y Tejidos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/fisiología , Antígenos CD19/genética , Antígenos CD19/metabolismo , Línea Celular Tumoral , Biología Computacional , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.
Asunto(s)
Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , HumanosRESUMEN
Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.
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Inmunoterapia Adoptiva , Neoplasias/terapia , Infecciones por Papillomavirus/terapia , Receptores Quiméricos de Antígenos/inmunología , Línea Celular , Proteínas Fluorescentes Verdes , Antígeno HLA-A2/inmunología , Humanos , Interferón gamma/inmunología , Luciferasas de Luciérnaga , Neoplasias/inmunología , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/inmunología , Péptidos/inmunología , Proteínas Represoras/inmunología , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Asbestos is a potent carcinogen associated with increased risks of malignant mesothelioma and lung cancer in humans. Although the mechanism of carcinogenesis remains elusive, the physicochemical characteristics of asbestos play a role in the progression of asbestos-induced diseases. Among these characteristics, a high capacity to adsorb and accommodate biomolecules on its abundant surface area has been linked to cellular and genetic toxicity. Several previous studies identified asbestos-interacting proteins. Here, with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry, we systematically identified proteins from various lysates that adsorbed to the surface of commercially used asbestos and classified them into the following groups: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. The surfaces of crocidolite and amosite, two iron-rich types of asbestos, caused more protein scissions and oxidative modifications than that of chrysotile by in situ-generated 4-hydroxy-2-nonenal. In contrast, we confirmed the intense hemolytic activity of chrysotile and found that hemoglobin attached to chrysotile, but not silica, can work as a catalyst to induce oxidative DNA damage. This process generates 8-hydroxy-2'-deoxyguanosine and thus corroborates the involvement of iron in the carcinogenicity of chrysotile. This evidence demonstrates that all three types of asbestos adsorb DNA and specific proteins, providing a niche for oxidative modification via catalytic iron. Therefore, considering the affinity of asbestos for histones/DNA and the internalization of asbestos into mesothelial cells, our results suggest a novel hypothetical mechanism causing genetic alterations during asbestos-induced carcinogenesis.
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Asbesto Amosita/química , Asbesto Crocidolita/química , Asbestos Serpentinas/química , Daño del ADN , Proteínas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Aldehídos/metabolismo , Animales , Asbesto Amosita/metabolismo , Asbesto Amosita/toxicidad , Asbesto Crocidolita/toxicidad , Asbestos Serpentinas/metabolismo , Cromatina/metabolismo , Citoesqueleto/metabolismo , ADN/química , ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biosíntesis , Hemoglobinas/metabolismo , Histonas/metabolismo , Hierro/metabolismo , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/patología , Mesotelioma/etiología , Mesotelioma/patología , Ratones , Oxidación-Reducción , Proteínas/química , Proteínas de Unión al ARN/metabolismo , Ratas , Proteínas Ribosómicas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Propiedades de SuperficieRESUMEN
Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.
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Adenina/análogos & derivados , Transformación Celular Neoplásica/genética , Mapeo Cromosómico/métodos , Genes Relacionados con las Neoplasias/genética , Guanina/análogos & derivados , Neoplasias Renales/genética , Estrés Oxidativo , Acroleína/química , Adenina/análisis , Adenina/química , Animales , Anticuerpos Monoclonales/inmunología , ADN/química , ADN/genética , ADN Glicosilasas/genética , Expresión Génica , Biblioteca de Genes , Genoma/genética , Guanina/análisis , Guanina/inmunología , Inmunoprecipitación , Riñón/química , Masculino , Ratones , Ratones Noqueados , Oxidación-ReducciónRESUMEN
Pre-administration of alpha-tocopherol is protective against oxidative renal tubular damage and subsequent carcinogenesis by ferric nitrilotriacetate (Fe-NTA) in rats. We searched for mechanisms other than the scavenging effect of alpha-tocopherol with microarray analyses, which implicated calnexin, a chaperone for glycoproteins. Renal mRNA levels of calnexin significantly increased 3h after an injection of Fe-NTA in rats fed a standard diet whereas those fed an alpha-tocopherol-supplemented diet showed an increase prior to injection, but after injection showed a decrease in renal calnexin mRNA levels, with unaltered protein levels. In experiments using LLC-PK1 cells, addition of alpha-tocopherol was protective against oxidative stress by H2O2, concomitant with calnexin induction. Knockdown of calnexin by siRNA significantly reduced this protection. Furthermore, COS-7 cells transfected with the calnexin gene were more resistant to H2O2. Together with the fact that alpha-tocopherol induced N-acetylglucosaminyltransferase 3, our data suggest that alpha-tocopherol modifies glycoprotein metabolism partially by conferring mild ER stress. This adds another molecular mechanism of alpha-tocopherol toward cancer prevention.
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Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Estrés Oxidativo/efectos de los fármacos , Lesiones Precancerosas/metabolismo , alfa-Tocoferol/administración & dosificación , Animales , Calnexina , Carcinógenos/toxicidad , Células Cultivadas , Citoprotección/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Compuestos Férricos/toxicidad , Radicales Libres/toxicidad , Neoplasias Renales/prevención & control , Túbulos Renales/efectos de los fármacos , Masculino , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/toxicidad , Lesiones Precancerosas/inducido químicamente , Ratas , Ratas WistarRESUMEN
An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces oxidative renal proximal tubular damage that subsequently leads to a high incidence of renal cell carcinoma in rodents, presenting an intriguing model of free radical-induced carcinogenesis. In the present study, we used gpt delta transgenic mice, which allow efficient detection of point mutations and deletions in vivo, to evaluate the mutation spectra, in association with the formation of 8-oxoguanine and acrolein-modified adenine during the first 3 weeks of carcinogenesis. Immunohistochemical analysis revealed the highest levels of 8-oxoguanine and acrolein-modifed adenine in the renal proximal tubules after 1 week of repeated administration. DNA immunoprecipitation and quantitative polymerase chain reaction analysis showed that the relative abundance of 8-oxoguanine and acrolein-modified adenine at the gpt reporter gene were increased at the first week in the kidney. Similarly, in both 6-thioguanine and Spi(-) selections performed on the renal specimens after Fe-NTA administration, the mutant frequencies were increased in the Fe-NTA-treated mice at the first week. Further analyzes of 79 mutant clones and 93 positive plaques showed a high frequency of G:C pairs as preferred targets for point mutation, notably G:C to C:G transversion-type mutation followed by deletion, and of large-size (>1 kilobase) deletions with short homologous sequences in proximity to repeated sequences at the junctions. The results demonstrate that the iron-based Fenton reaction is mutagenic in vivo in the renal tubular cells and induces characteristic mutations.
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Carcinógenos/toxicidad , Proteínas de Escherichia coli/genética , Compuestos Férricos/toxicidad , Neoplasias Renales/genética , Riñón/efectos de los fármacos , Ácido Nitrilotriacético/análogos & derivados , Pentosiltransferasa/genética , Polimorfismo de Nucleótido Simple , Eliminación de Secuencia , Animales , Secuencia de Bases , Daño del ADN , Análisis Mutacional de ADN , Inmunoprecipitación , Riñón/metabolismo , Neoplasias Renales/inducido químicamente , Neoplasias Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación/genética , Ácido Nitrilotriacético/toxicidad , Reacción en Cadena de la PolimerasaRESUMEN
4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3-5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.
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Actinas/efectos de los fármacos , Aldehídos/farmacología , Actinas/química , Albúminas/química , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Cisteína/química , Compuestos Férricos , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Hemocianinas/inmunología , Histidina/química , Riñón/efectos de los fármacos , Neoplasias Renales/inducido químicamente , Neoplasias Renales/fisiopatología , Lisina/química , Masculino , Metalotioneína/química , Ácido Nitrilotriacético/análogos & derivados , Ratas , Ratas Wistar , Organismos Libres de Patógenos Específicos , Superóxido Dismutasa/químicaRESUMEN
Thioredoxin is a major component of thiol-reducing system. Recently, we identified thioredoxin-binding protein-2 (TBP-2) as a negative regulator of thioredoxin. Here, we report the role of TBP-2 in oxidative renal tubular injury and the subsequent carcinogenesis by ferric nitrilotriacetate. TBP-2 was abundantly expressed in the rat kidney. Immunohistochemical analysis revealed that TBP-2 was present in association with nuclei and mitochondrial intermembrane space in the proximal tubular cells and coimmunoprecipitated with cytochrome c. After acute oxidative tubular damage, TBP-2 protein, but not messenger RNA, markedly decreased, demonstrating shortened half-life of this protein. Most cases of the induced renal cell carcinoma showed undetectable levels of TBP-2 protein, which was associated with the methylation of CpG island in the promoter region. Genome sequence analyses identified the poly-A tract in the 3' untranslated region as a mutation hot spot in this rather nonselective environment. Collectively, the amounts of TBP-2 protein were inversely associated with proliferation of tubular cells, as evaluated by proliferating cell nuclear antigen. These results suggest that loss of TBP-2 is essential for proliferation of not only neoplastic but also non-neoplastic renal tubular cells, and that TBP-2 is a target gene in oxidative stress-induced renal carcinogenesis by ferric nitrilotriacetate.