RESUMO
With the implementation of the new kidney allocation system (KAS), there is increased reliance on a virtual crossmatch/histocompatibility risk assessment (vXM) for evaluating potential presence, as well as strength, of HLA antibodies against a potential donor. The accuracy of such an assessment depends on the precision in the identification of the recipient's antibody profile and the potential donor's HLA typing. While the development of the single antigen bead (SAB) multiplex assay has improved the sensitivity and specificity of HLA antibody detection, several limitations of the assay (specific to certain sensitized patients) can complicate accurate interpretation of results. In this report, we focus on the "shared-epitope" phenomenon, a condition in which antibody strength can be underrepresented, or its presence completely missed, due to binding of the antibody to competing targets on multiple antigens (beads), effectively "diluting" the resulting MFI readout. Here, we provide a relevant background to understand this phenomenon and present a couple of case studies illustrating how it can be investigated, leading to a more accurate histocompatibility consultation.
Assuntos
Antígenos HLA , Transplante de Rim , Tipagem e Reações Cruzadas Sanguíneas , Epitopos , Teste de Histocompatibilidade , Humanos , IsoanticorposRESUMO
The ubiquitous spread of polybrominated diphenyl ethers (PBDEs) has led to concerns regarding the metabolites of these congeners, in particular hydroxylated PBDEs. There are limited studies regarding the biological interactions of these chemicals, yet there is some concern they may be more toxic than their parent compounds. In this study three hydroxylated PBDEs were assessed for toxicity in embryonic zebrafish: 3-OH-BDE 47, 5-OH-BDE 47, and 6-OH-BDE 47. All three congeners induced developmental arrest in a concentration-dependent manner; however, 6-OH-BDE 47 induced adverse effects at lower concentrations than the other congeners. Furthermore, all three induced cell death; however apoptosis was not observed. In short-term exposures (24-28 hours post fertilization), all hydroxylated PBDEs generated oxidative stress in the region corresponding to the cell death at 5 and 10 ppm. To further investigate the short-term effects that may be responsible for the developmental arrest observed in this study, gene regulation was assessed for embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 hpf. Genes involved in stress response, thyroid hormone regulation, and neurodevelopment were significantly upregulated compared to controls; however, genes related to oxidative stress were either unaffected or downregulated. This study suggests that hydroxylated PBDEs disrupt development, and may induce oxidative stress and potentially disrupt the cholinergic system and thyroid hormone homeostasis.
Assuntos
Morte Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Éteres Difenil Halogenados/administração & dosagem , Éteres Difenil Halogenados/química , Relação Estrutura-Atividade , Hormônios Tireóideos/metabolismo , Fatores de TempoRESUMO
Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.
Assuntos
Técnicas de Genotipagem , Antígenos HLA/genética , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Sondas de Oligonucleotídeos/genética , Transplante de Órgãos , Alelos , HumanosRESUMO
There is a pressing need to develop rapid whole animal-based testing assays to assess the potential toxicity of engineered nanomaterials. To meet this challenge, the embryonic zebrafish model was employed to determine the toxicity of fullerenes. Embryonic zebrafish were exposed to graded concentrations of fullerenes [C(60), C(70), and C(60)(OH)(24)] during early embryogenesis and the resulting morphological and cellular responses were defined. Exposure to 200 µg/L C(60) and C(70) induced a significant increased in malformations, pericardial edema, and mortality; while the response to C(60)(OH)(24) exposure was less pronounced at concentrations an order of magnitude higher. Exposure to C(60) induced both necrotic and apoptotic cellular death throughout the embryo. While C(60)(OH)(24) induced an increase in embryonic cellular death, it did not induce apoptosis. Our findings concur with results obtained in other models indicating that C(60)(OH)(24) is significantly less toxic than C(60). These studies also suggest that that the embryonic zebrafish model is well-suited for the rapid assessment of nanomaterial toxicity.
RESUMO
The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories.
Assuntos
Ensaios de Triagem em Larga Escala , Peixe-Zebra , Animais , Embrião não Mamífero , Estradiol/farmacologia , Etinilestradiol/farmacologia , Tiram/farmacologiaRESUMO
Brominated flame retardants (BFRs) are used to reduce the flammability of plastics, textiles, and electronics. BFRs vary in their chemical properties and structures, and it is expected that these differences alter their biological interactions and toxicity. Zebrafish were used as the model organism for assessing the toxicity of nine structurally-diverse BFRs. In addition to monitoring for overt toxicity, the rate of spontaneous movement, and acetylcholinesterase and glutathione-S-transferase (GST) activities were assessed following exposure. The toxicities of BFRs tested can be ranked by LC50 as tetrabromobisphenol A (TBBPA) < 4,4'-isopropylidenebis[2-(2,6-dibromophenoxyl)ethanol] (TBBPA-OHEE) < Pentabromochlorocyclohexane (PBCH) < 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) < hexabromocyclododecane (HBCD) < hexabromobenzene (HBB) < Tetrabromophthalic anhydride (PHT4). No adverse effect was observed in di(2-ethylhexyl) tetrabromophthalate (TBPH) or dibromoneopentyl glycol (DBNPG)-treated embryos. The rate of spontaneous movement was decreased in a concentration-dependent manner following exposure to four of the nine compounds. GST activity was elevated following treatment with PBCH, TBBPA, HBCD, and HBB. The results indicate that exposure to several BFRs may activate an antioxidant response and alter behavior during early development. Some of the BFRs, such as TBBPA and TBBPA-OHEE, induced adverse effects at concentrations lower than chemicals that are currently banned. These results suggest that zebrafish are sensitive to exposure to BFRs and can be used as a comparative screening model, as well as to determine alterations in behavior following exposure and probe mechanisms of action.
RESUMO
A proposed primary pathway through which polybrominated diphenyl ethers (PBDEs) disrupt normal biological functions is oxidative stress. In the present study, 4 PBDE congeners were evaluated for their potential to initiate oxidative stress in zebrafish during development: BDE 28, BDE 47, BDE 99, and BDE 100. N-acetylcysteine (NAC) was used to increase intracellular glutathione concentrations and only decreased the effects of BDE 28 at 10 ppm and 20 ppm and BDE 47 at 20 ppm. N-acetylcysteine coexposure did not alter the rates of mortality or curved body axis compared with PBDE exposure alone. The activity of glutathione-S-transferase (GST) was not altered at 24 h postfertilization (hpf), but increased following 10 ppm BDE 28 exposure at 120 hpf. Transcription of several genes associated with stress was also evaluated. At 24 hpf, cytochrome c oxidase subunit 6a (COX6a) transcription was up-regulated in embryos exposed to BDE 99, and BDE 28 exposure up-regulated the transcription of Glutathione-S-transferase-pi (GSTpi). At 24 hpf, glutamate-cysteine ligase (GCLC) was slightly down-regulated by all congeners evaluated. At 120 hpf, TNF receptor-associated protein 1 (TRAP1) and COX6A were up-regulated by all congeners, however GSTpi was down-regulated by all congeners. The results of quantitative real-time transcription polymerase chain reaction are mixed and do not strongly support a transcriptional response to oxidative stress. According to the authors' data, PBDEs do not induce oxidative stress. Oxidative stress may occur at high exposure concentrations; however, this does not appear to be a primary mechanism of action for the PBDE congeners tested.
Assuntos
Éteres Difenil Halogenados/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/metabolismo , Acetilcisteína/toxicidade , Animais , Regulação para Baixo/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Glutationa S-Transferase pi/metabolismo , Éteres Difenil Halogenados/química , Bifenil Polibromatos/toxicidade , Fator 1 Associado a Receptor de TNF/metabolismo , Testes de Toxicidade , Regulação para Cima/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Embryonic zebrafish were used to compare the uptake and metabolism of six polybrominated diphenyl ether (PBDE) congeners (BDEs 28, 47, 99, 100, 153, and 183) and identified metabolites from static exposures at 24 and 120 h postfertilization (hpf). An inverse relationship was observed between uptake of PBDEs and their octanol-water partitioning coefficients (uptake of BDEs 28 and 47>99 and 100>153 and 183). Debromination metabolites were identified in all congeners (excluding BDE 28) tested in the 120-hpf tissue samples. Interestingly, BDE 153 underwent meta-debromination, forming BDEs 47 and 99. Gene transcription analysis was conducted at 120 hpf to identify potential metabolic pathways for the PBDEs examined in the present study (gstpi, deiodinases 1 and 2, cyp1a1, cyp1b1, and ugt5g). The greatest induction was of ugt5g for all congeners and deiodinase transcription was also upregulated by BDEs 28, 47, and 183. The cyp1a1 and cyp1b1 were upregulated by BDEs 28, 47, 99, and 183. The least alterations in gene transcription were in the BDE 153-exposed embryos. A clear primary pathway of debromination metabolism was not identified; however, upregulation of these different genes indicated that fish were responding to exposure of PBDEs. Furthermore, the present study demonstrated that the most bioavailable congeners are also those with the highest reported toxicity.
Assuntos
Embrião não Mamífero/metabolismo , Éteres Difenil Halogenados/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Modelos Químicos , Medição de Risco , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Polybrominated diphenyl ethers (PBDEs) have become ubiquitous environmental contaminants with potential for bioaccumulation and maternal-fetal transfer that has led to regulatory bans and/or phasing out of several technical mixtures of PBDEs. In the present study, six PBDE congeners (BDE 28, BDE 47, BDE 99, BDE 100, BDE 153, BDE 183) were evaluated for developmental effects on embryonic zebrafish. These congeners were chosen because they are environmentally relevant and cover a wide range of physical-chemical properties. Alterations in behavior, physical malformations, and mortality were scored daily until 168 h postfertilization (hpf). A concentration-dependent increase in spontaneous movement indicated an early onset of behavioral responses to PBDE exposures. Spontaneous movement was affected the most by BDE 47 and BDE 28, whereas BDE 183 did not alter behavior at any concentration tested. Swimming rates were significantly increased by BDE 28 at 96 and 120 hpf, but decreased swimming activity at 168 hpf. Additionally, BDE 47 significantly decreased the swimming rate at 168 hpf. Other endpoints included malformations and mortality. Congeners with fewer bromines (BDE 28, 47, 99, and 100) also induced a curved body axis starting around 120 hpf, which was followed by mortality. BDEs 153 and 183, however, did not elicit these adverse effects. A relationship was found between log K(OW) and median lethal concentration (LC50) and median effective concentration (EC50). Structure-activity relationships in this study suggest that PBDE acute toxicity results from a receptor-mediated effect and further studies are necessary to determine these pathways.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia , Animais , Comportamento Animal/efeitos dos fármacos , Éteres Difenil Halogenados/química , Relação Estrutura-Atividade , Natação , Poluentes Químicos da Água/química , Peixe-Zebra/fisiologiaRESUMO
Due to its unique physicochemical and optical properties, C60 has raised interest in commercialization for a variety of products. While several reports have determined this nanomaterial to act as a powerful antioxidant, many other studies have demonstrated a strong oxidative potential through photoactivation. To directly address the oxidative potential of C60, the effects of light and chemical supplementation and depletion of glutathione (GSH) on C60-induced toxicity were evaluated. Embryonic zebrafish were used as a model organism to examine the potential of C60 to elicit oxidative stress responses. Reduced light during C60 exposure significantly decreased mortality and the incidence of fin malformations and pericardial edema at 200 and 300 ppb C60. Embryos co-exposed to the glutathione precursor, N-acetylcysteine (NAC), also showed reduced mortality and pericardial edema; however, fin malformations were not reduced. Conversely, co-exposure to the GSH synthesis inhibitors, buthionine sulfoximine (BSO) and diethyl maleate (DEM), increased the sensitivity of zebrafish to C60 exposure. Co-exposure of C60 or its hydroxylated derivative, C60(OH)(24), with H2O2 resulted in increased mortality along the concentration gradient of H2O2 for both materials. Microarrays were used to examine the effects of C60 on the global gene expression at two time points, 36 and 48 h post fertilization (hpf). At both life stages there were alterations in the expression of several key stress response genes including glutathione-S-transferase, glutamate cysteine ligase, ferritin, alpha-tocopherol transport protein and heat shock protein 70. These results support the hypothesis that C60 induces oxidative stress in this model system.
Assuntos
Fulerenos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Butionina Sulfoximina/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Ferritinas/efeitos dos fármacos , Ferritinas/metabolismo , Glutamato-Cisteína Ligase/efeitos dos fármacos , Glutamato-Cisteína Ligase/metabolismo , Glutationa Transferase/efeitos dos fármacos , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Peróxido de Hidrogênio/metabolismo , Luz , Maleatos/farmacologia , Análise em Microsséries , Modelos Animais , Derrame Pericárdico/induzido quimicamente , Fatores de Tempo , Peixe-ZebraRESUMO
With production and use of carbon nanoparticles increasing, it is imperative that the toxicity of these materials be determined; yet such testing requires specific and selective analytical methodologies that do not yet exist. Quantitative liquid-liquid extraction was coupled with liquid chromatography/electrospray ionization mass spectrometry for the quantitative determination of fullerenes from C60 to C98. Isotopically enriched, 13C60, was used as an internal standard. The method was applied to determine the loss of C60 from exposure water solution and uptake of C60 by embryonic zebrafish. The average recovery of C60 from zebrafish embryo extracts and 1% DMSO in aqueous-exposure solutions was 90 and 93%, respectively, and precision, as indicated by the relative standard deviation, was 2 and 7%, respectively. The method quantification limit was 0.40 microg/L and the detection limit was 0.02 microg/L. During the toxicological assay, loss of C60 due to sorption to test vials resulted in the reduction of exposure-solution concentrations over 6 h to less than 50% of the initial concentration. Time-course experiments indicated embryo uptake increased over course of the 12-h exposure. A lethal concentration that caused 50% mortality was determined to be 130 microg/L and was associated with a zebrafish embryo concentration, LD50, of 0.079 microg/g of embryo.