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AIMS/HYPOTHESIS: The Islet Autoantibody Standardization Program (IASP) aims to improve the performance of immunoassays measuring autoantibodies in type 1 diabetes and the concordance of results across laboratories. IASP organises international workshops distributing anonymised serum samples to participating laboratories and centralises the collection and analysis of results. In this report, we describe the results of assays measuring IAA submitted to the IASP 2018 and 2020 workshops. METHODS: The IASP distributed uniquely coded sera from individuals with new-onset type 1 diabetes, multiple islet autoantibody-positive individuals, and diabetes-free blood donors in both 2018 and 2020. Serial dilutions of the anti-insulin mouse monoclonal antibody HUI-018 were also included. Sensitivity, specificity, area under the receiver operating characteristic curve (ROC-AUC), partial ROC-AUC at 95% specificity (pAUC95) and concordance of qualitative/quantitative results were compared across assays. RESULTS: Results from 45 IAA assays of seven different formats and from 37 IAA assays of six different formats were submitted to the IASP in 2018 and 2020, respectively. The median ROC-AUC was 0.736 (IQR 0.617-0.803) and 0.790 (IQR 0.730-0.836), while the median pAUC95 was 0.016 (IQR 0.004-0.021) and 0.023 (IQR 0.014-0.026) in the 2018 and 2020 workshops, respectively. Assays largely differed in AUC (IASP 2018 range 0.232-0.874; IASP 2020 range 0.379-0.924) and pAUC95 (IASP 2018 and IASP 2020 range 0-0.032). CONCLUSIONS/INTERPRETATION: Assay formats submitted to this study showed heterogeneous performance. Despite the high variability across laboratories, the in-house radiobinding assay (RBA) remains the gold standard for IAA measurement. However, novel non-radioactive IAA immunoassays showed a good performance and, if further improved, might be considered valid alternatives to RBAs.
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Autoanticorpos , Diabetes Mellitus Tipo 1 , Animais , Camundongos , Sensibilidade e Especificidade , Curva ROC , Anticorpos Anti-Insulina , Padrões de Referência , Glutamato DescarboxilaseRESUMO
In hematology and coagulation, diligence in the preanalytical phase of testing is of critical importance to obtaining reliable test results. If the sample used for testing is unsuitable, even outstanding analytical procedures and technology cannot produce a clinically-reliable result. Therefore, the intent of this manuscript is to review preanalytical factors intrinsic to the sample that affect the hematology and coagulation testing. Factors intrinsic to the sample (excluding in vivo anomalies) can be controlled, theoretically, by phlebotomists (including nurses) and laboratorians in the preanalytical phase of testing. Furthermore, the management and prevention of such factors is highlighted. Erroneous control of preanalytical factors can produce laboratory errors.
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Coagulação Sanguínea , Hematologia , Humanos , LaboratóriosRESUMO
OBJECTIVES: Dietary glucose is a robust elicitor of central reward responses and ingestion, but the key peripheral sensors triggering these orexigenic mechanisms are not entirely known. The objective of this study was to determine whether glucokinase, a phosphorylating enzyme with known glucosensory roles, is also expressed in taste bud cells and contributes to the immediate hedonic appeal of glucose-containing substances. METHODS AND RESULTS: Glucokinase (GCK) gene transcripts were localized in murine taste bud cells with RNAScope®, and GCK mRNA was found to be upregulated in the circumvallate taste papillae in response to fasting and after a period of dietary access to added simple sugars in mice, as determined with real time-qPCR. Pharmacological activation of glucokinase with Compound A increased primary taste nerve and licking responses for glucose but did not impact responsivity to fructose in naïve mice. Virogenetic silencing of glucokinase in the major taste fields attenuated glucose-stimulated licking, especially in mice that also lacked sweet receptors, but did not disrupt consummatory behaviors for fructose or the low-calorie sweetener, sucralose in sugar naïve mice. Knockdown of lingual glucokinase weakened the acquired preference for glucose over fructose in sugar-experienced mice in brief access taste tests. CONCLUSIONS: Collectively, our data establish that glucokinase contributes to glucose appetition at the very first site of nutrient detection, in the oral cavity. The findings expand our understanding of orosensory inputs underlying nutrition, metabolism, and food reward.
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Glucose , Paladar , Animais , Apetite , Carboidratos , Frutose/metabolismo , Glucoquinase/genética , Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Paladar/fisiologiaRESUMO
In addition to their well-known anxiolytic functions, benzodiazepines produce hyperphagia. Previously, we reported that the benzodiazepine, chlordiazepoxide (CDP), increased consumption of both normally-preferred and normally-avoided taste stimuli during long-term (1 h) tests, primarily through changes in licking microstructure patterns associated with hedonic taste evaluation, whereas there was little effect on licking microstructure measures associated with post-ingestive feedback. In this study, we further examined the hedonic and motivational specificity of CDP effects on ingestive behavior. We tested brief access (15 s) licking responses for tastants spanning all taste qualities after treatment with either CDP (5 or 10 mg/kg) or the non-benzodiazepine anxiolytic, buspirone (1.5 or 3 mg/kg). A between-subjects, counterbalanced design compared the CDP or buspirone effects on licking responses for water and a range of weak to strong concentrations of NaCl, Q-HCl, citric acid, MSG, saccharin, and capsaicin under water-restricted (23 h) conditions; and sucrose, saccharin, and MSG under water-replete conditions. In a dose dependent manner, CDP increased licking for taste stimuli that were normally-avoided after saline treatment, with a notable exception observed for the trigeminal stimulus, capsaicin, which was not affected at any concentration or drug dose, suggesting a taste-specific effect of CDP on orosensory processing. Under water-replete conditions, CDP dose-dependently increased licking to normally-accepted concentrations of sucrose, saccharin, and MSG. There was no effect of either drug on licks for water under either water-restricted or water-replete conditions. Buspirone slowed oromotor coordination by increasing brief interlick intervals, but it did not affect licking for any concentrations of the tastants. Overall, these results indicate that benzodiazepines selectively enhance the hedonic acceptance of gustatory orosensory stimuli, independent of general anxiolytic or oromotor coordination effects, or physiological states such as thirst.
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Ansiolíticos , Benzodiazepinas , Humanos , Ansiolíticos/farmacologia , Benzodiazepinas/farmacologia , Buspirona/farmacologia , Capsaicina/farmacologia , Clordiazepóxido/farmacologia , Hiperfagia/induzido quimicamente , Sacarina/farmacologia , Glutamato de Sódio/farmacologia , Sacarose/farmacologia , Paladar , Água/farmacologiaRESUMO
BACKGROUND: The relative efficacy and safety of allopurinol and febuxostat when used according to current guidelines for the treatment of hyperuricemia are unknown. This double-blind noninferiority trial examined these issues. METHODS: Participants with gout and hyperuricemia (with at least 33% having stage 3 chronic kidney disease) were randomly assigned to allopurinol or febuxostat in this 72-week trial, with doses titrated to target serum urate. The trial had three phases: titration (weeks 0 to 24), maintenance (weeks 25 to 48), and observation (weeks 49 to 72). Allopurinol and febuxostat were initiated at daily doses of 100 and 40 mg, with maximum titration to 800 and 120 mg, respectively. Antiinflammatory prophylaxis was given during phases 1 and 2. The primary end point was the proportion of patients experiencing one or more flares during phase 3, with a prespecified noninferiority margin of less than 8 percentage points between allopurinol and febuxostat. Secondary end points included efficacy in patients with chronic kidney disease, proportion achieving target serum urate levels, and serious adverse events. RESULTS: This study included 940 participants; 20.1% withdrew, with similar proportions in treatment arms. During phase 3, 36.5% of allopurinol-treated participants had one flare or more compared with 43.5% of febuxostat-treated participants (P<0.001 for noninferiority). Overall, 80% of participants achieved mean target urates during phase 2 with no differences by treatment. There were no treatment differences (including cardiovascular events) in serious adverse events. CONCLUSIONS: Allopurinol and febuxostat achieved serum urate goals in patients with gout; allopurinol was noninferior to febuxostat in controlling flares. Similar outcomes were noted in participants with stage 3 chronic kidney disease. (Funded by the Cooperative Studies Program of the Department of Veterans Affairs Office of Research and Development; ClinicalTrials.gov identifier, NCT02579096.).
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BACKGROUND: The distinction between type 1 diabetes (T1D) and type 2 diabetes (T2D) is extremely important for the choice of therapy, body weight and dietary management, screening for coexistent autoimmune diseases and comorbidities, anticipated prognosis, and risk assessment in relatives. Not uncommonly, the presentation of the patient may not allow an unambiguous discrimination between T1D and T2D. To help resolve this challenge, the detection of islet autoantibodies can support the diagnosis of T1D. CONTENT: The presence of islet autoantibodies in a person with diabetes indicates an autoimmune etiology therefore establishing the diagnosis of T1D. Presently 5 islet autoantibodies are available for routine clinical use: islet cell cytoplasmic autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), insulinoma associated-2 autoantibodies (IA-2A), and zinc transporter-8 autoantibodies (ZnT8A). There are caveats to the selection of which islet autoantibodies should be measured. Islet autoantibodies can also predict the development of T1D. Therefore, once safe and effective therapies are available to prevent T1D, islet autoantibody testing is expected to become a routine part of medical practice. A very rare cause of autoimmune diabetes is the type B insulin resistance syndrome resulting from antagonistic autoantibodies to the insulin receptor. Rarely hypoglycemia can result from agonistic insulin receptor autoantibodies, or high-titer IAA causing the autoimmune insulin syndrome (i.e., Hirata disease). SUMMARY: In summary, autoimmune causes of dysglycemia are increasing in clinical importance requiring the scrutiny of laboratorians. The determination of islet autoantibodies can greatly aid in the diagnosis and the prediction of T1D.
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Autoanticorpos , Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Autoanticorpos/análise , Diabetes Mellitus Tipo 1/diagnóstico , Glutamato Descarboxilase , Humanos , Transportador 8 de ZincoRESUMO
There is strong evidence for gut-taste bud interactions that influence taste function, behavior and feeding. However, the effect of gut inflammation on this axis is unknown despite reports of taste changes in gastrointestinal (GI) inflammatory conditions. Lipopolysaccharide (LPS), an inflammatory stimulus derived from gram-negative bacteria, is present in the normal GI tract and levels increase during high-fat feeding and gut infection and inflammation. Recordings from the chorda tympani nerve (CT), which transmits taste information from taste buds on the anterior tongue to the brain, previously revealed a transient decrease in sucrose responses in mice that ingest LPS during a single overnight period. Here we test the effect of acute or chronic, weekly LPS gavage on licking behavior and CT responses. Using brief-access testing, rats treated with acute LPS and mice receiving acute or chronic LPS decreased licking responses to sucrose and saccharin and to NaCl in mice. In long-term (23 h) tests chronic LPS also reduced licking responses to saccharin, sucrose, and NaCl in mice. Neurophysiological recordings from the CT supported behavioral changes, demonstrating reduced responses to sucrose, saccharin, acesulfame potassium, glucose and NaCl in acute and chronic LPS groups compared to controls. Chronic LPS significantly elevated neutrophils in the small intestine and colon, but LPS was not detected in serum and mice did not display sickness behavior or lose weight. These results indicate that sweet and salt taste sensitivity could be reduced even in asymptomatic or mild localized gut inflammatory conditions such as inflammatory bowel disease.
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Comportamento Animal/efeitos dos fármacos , Nervo da Corda do Tímpano/efeitos dos fármacos , Doenças Inflamatórias Intestinais/fisiopatologia , Percepção Gustatória/fisiologia , Paladar/fisiologia , Animais , Comportamento Animal/fisiologia , Nervo da Corda do Tímpano/fisiopatologia , Modelos Animais de Doenças , Feminino , Doenças Inflamatórias Intestinais/induzido quimicamente , Lipopolissacarídeos , Camundongos , Ratos , Ratos Sprague-Dawley , Sacarina/administração & dosagem , Cloreto de Sódio/administração & dosagem , Sacarose/administração & dosagem , Paladar/efeitos dos fármacos , Percepção Gustatória/efeitos dos fármacosRESUMO
BACKGROUND: The Islet Autoantibody Standardization Program (IASP) aims to improve the performance of immunoassays measuring type 1 diabetes (T1D)-associated autoantibodies and the concordance of results among laboratories. IASP organizes international interlaboratory assay comparison studies in which blinded serum samples are distributed to participating laboratories, followed by centralized collection and analysis of results, providing participants with an unbiased comparative assessment. In this report, we describe the results of glutamic acid decarboxylase autoantibody (GADA) assays presented in the IASP 2018 workshop. METHODS: In May 2018, IASP distributed to participants uniquely coded sera from 43 new-onset T1D patients, 7 multiple autoantibody-positive nondiabetic individuals, and 90 blood donors. Results were analyzed for the following metrics: sensitivity, specificity, accuracy, area under the ROC curve (ROC-AUC), partial ROC-AUC at 95% specificity (pAUC95), and concordance of qualitative and quantitative results. RESULTS: Thirty-seven laboratories submitted results from a total of 48 different GADA assays adopting 9 different formats. The median ROC-AUC and pAUC95 of all assays were 0.87 [interquartile range (IQR), 0.83-0.89] and 0.036 (IQR, 0.032-0.039), respectively. Large differences in pAUC95 (range, 0.001-0.0411) were observed across assays. Of formats widely adopted, bridge ELISAs showed the best median pAUC95 (0.039; range, 0.036-0.041). CONCLUSIONS: Several novel assay formats submitted to this study showed heterogeneous performance. In 2018, the majority of the best performing GADA immunoassays consisted of novel or established nonradioactive tests that proved on a par or superior to the radiobinding assay, the previous gold standard assay format for GADA measurement.
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Autoanticorpos/análise , Diabetes Mellitus Tipo 1/imunologia , Adolescente , Adulto , Autoanticorpos/imunologia , Criança , Educação , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Masculino , Pessoa de Meia-Idade , Curva ROC , Padrões de Referência , Adulto JovemRESUMO
Benzodiazepines are one of the most commonly prescribed anxiolytic drugs in America, and between 2006 and 2015 prescription rates increased by an estimated 27.1%. Weight gain is a common side effect of these drugs and it may result from increased feeding caused by drug-enhanced food palatability. We investigated the role of specific GABAA receptor subtypes involved with benzodiazepine-induced food consumption through third ventricle injections of L-838,417, a partial agonist of GABAA α2, α3, and α5 subunits, and a full antagonist of the α1 receptor subunit. A microanalysis of the licking behavior of adult male rats to a sucrose solution was used to isolate drug effects on specific consummatory behaviors that include: hedonic taste evaluation, food approach behavior, and oromotor function. L-838,417 dose-dependently increased intake through increases in the motivation to approach the solution (shorter pause intervals between bouts of licking) and through enhancement of measures associated with hedonic taste evaluation. Oromotor depressant effects previously associated with broad-spectrum benzodiazepine receptor agonists were not observed. These results indicate that nuclei in proximity to the ventricles respond to GABAA α2, α3, or α5 activation to induce motivation to feed, absent of α1 receptor subunit activation. Furthermore, activation of the α1 subunit is not necessary for benzodiazepine hyperphagia and may instead contribute to the oromotor depressant and sedative properties of classic benzodiazepine agonists. Hypothalamic nuclei such as the paraventricular nucleus may be involved in the benzodiazepine-increased motivation to feed, while the parabrachial nucleus of the hindbrain could contribute to benzodiazepine-induced enhancement of taste palatability.
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Ingestão de Alimentos/efeitos dos fármacos , Fluorbenzenos/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Motivação/efeitos dos fármacos , Triazóis/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Ratos , Ratos Sprague-Dawley , SacaroseRESUMO
Previously we have shown that our Healthy Eating Decisions school-based intervention can influence students' selections of the healthiest foods available in their elementary school cafeterias through positive reinforcement techniques. Although effective, we recognized that students were missing fundamental nutrition knowledge necessary to understand why the Healthy Eating Decisions program identified particular beverages and foods as the healthiest in the cafeteria. Therefore, we developed the Boss' Healthy Buddies nutrition education resource as a freely available curriculum matched with South Carolina education standards and designed for elementary school students from kindergarten through fourth grade. The current study implemented Boss' Healthy Buddies and compared its efficacy to a commercially available nutrition program, CATCH. Elementary school students in Spartanburg, South Carolina, received weekly twenty-minute Boss' Healthy Buddies lessons for eight weeks. Results from preassessment and postassessment surveys were compared with a positive control elementary school using the CATCH program and a negative control school receiving no nutrition education. Results show that Boss' Healthy Buddies was equally effective as the CATCH program in improving the nutrition attitudes regarding healthiest beverages and food selections with the advantage of being freely available and minimizing the impact on classroom instruction time. In order to reduce most effectively the high prevalence of childhood overweight and obesity, it is crucial that children are taught nutrition education to support healthy eating habits at an early age. Both the Healthy Eating Decisions school-based intervention and the Boss' Healthy Buddies nutrition education program are available online for use as free resources to aid in reducing childhood overweight and obesity within elementary schools.
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Dieta Saudável , Educação em Saúde , Promoção da Saúde/métodos , Criança , Pré-Escolar , Comportamento Alimentar , Feminino , Preferências Alimentares , Serviços de Alimentação , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Obesidade Infantil/prevenção & controle , Avaliação de Programas e Projetos de Saúde , Instituições Acadêmicas , South Carolina , EstudantesRESUMO
Negative hedonic sensory qualities of HIV antiretroviral drugs often reduce patient adherence particularly in pediatric populations requiring oral consumption. This study examines the palatability of an innovative delivery mechanism utilizing a freeze-drying-in-blister approach to create fast-dissolving tablets (FDTs) containing a fixed-dose combination of lopinavir and ritonavir (LPV/r). Consumption patterns of solutions during brief-access and long-term testing and baby foodstuff consumption were analyzed to evaluate the orosensory detection and avoidance of placebo FDTs containing no LPV/r (FDT-) and FDTs containing LPV/r (FDT+). Rats showed no change in consumption patterns for the placebo FDT- compared with control solutions. Rats can detect but do not avoid FDT+ at body-weight-adjusted dosages in both brief-access (30-s) and long-term (23 h) consumption tests. There is an aversive response to concentrated doses of FDT+ during brief-access tests that cannot be masked by 25% sucrose. However, the strongest FDT+ concentration was not rejected when mixed with 50 g of applesauce, banana sauce, or rice cereal baby foodstuffs. The averseness of the FDT+ was associated with the presence of LPV/r and not the FDT- formulation itself. The novel FDT formulation appears to be a palatable delivery mechanism for oral antiretroviral pharmaceuticals especially when mixed with baby foodstuffs.
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BACKGROUND: Evaluation of repetitive transcranial magnetic stimulation (rTMS) for treatment-resistant major depression (TRMD) in Veterans offers unique clinical trial challenges. Here we describe a randomized, double-blinded, intent-to-treat, two-arm, superiority parallel design, a multicenter study funded by the Cooperative Studies Program (CSP No. 556) of the US Department of Veterans Affairs. METHODS: We recruited medical providers with clinical expertise in treating TRMD at nine Veterans Affairs (VA) medical centers as the trial local investigators. We plan to enroll 360 Veterans diagnosed with TRMD at the nine VA medical centers over a 3-year period. We will randomize participants into a double-blinded clinical trial to left prefrontal rTMS treatment or to sham (control) rTMS treatment (180 participants each group) for up to 30 treatment sessions. All participants will meet Diagnostic and statistical manual of mental disorders, 4 th edition (DSM-IV) criteria for major depression and will have failed at least two prior pharmacological interventions. In contrast with other rTMS clinical trials, we will not exclude Veterans with posttraumatic stress disorder (PTSD) or history of substance abuse and we will obtain detailed history regarding these disorders. Furthermore, we will maintain participants on stable anti-depressant medication throughout the trial. We will evaluate all participants on a wide variety of potential predictors of treatment response including cognitive, psychological and functional parameters. DISCUSSION: The primary dependent measure will be remission rate (Hamilton Rating Scale for Depression (HRSD24) ≤ 10), and secondary analyses will be conducted on other indices. Comparisons between the rTMS and the sham groups will be made at the end of the acute treatment phase to test the primary hypothesis. The unique challenges to performing such a large technically challenging clinical trial with Veterans and potential avenues for improvement of the design in future trials will be described. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01191333 . Registered on 26 August 2010. This report is based on the protocol version 4.6 amended in February 2016. All items from the World Health Organization Trial Registration Data Set are listed in Appendix A.
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Afeto , Transtorno Depressivo Maior/terapia , Transtorno Depressivo Resistente a Tratamento/terapia , Córtex Pré-Frontal/fisiopatologia , Estimulação Transcraniana por Corrente Contínua , Protocolos Clínicos , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/fisiopatologia , Transtorno Depressivo Maior/psicologia , Transtorno Depressivo Resistente a Tratamento/diagnóstico , Transtorno Depressivo Resistente a Tratamento/fisiopatologia , Transtorno Depressivo Resistente a Tratamento/psicologia , Método Duplo-Cego , Humanos , Testes Neuropsicológicos , Indução de Remissão , Projetos de Pesquisa , Inquéritos e Questionários , Fatores de Tempo , Estimulação Transcraniana por Corrente Contínua/efeitos adversos , Resultado do Tratamento , Estados Unidos , United States Department of Veterans AffairsAssuntos
Autoanticorpos/sangue , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Anticorpos Anti-Insulina/sangue , Anticorpos Anti-Insulina/imunologia , Insulina/sangue , Insulina/imunologia , Biomarcadores/sangue , Peptídeo C/sangue , Peptídeo C/imunologia , HumanosRESUMO
Gene-environment interactions play a role in the development of obesity but specific effects of diet on the orosensory detection of fatty acids have yet to be clarified. The objective of this study is to characterize the effect of prolonged (5-week) exposure to a high-fat (60%) diet on the behavioral sensitivity to the fatty acid linoleate following a conditioned taste aversion in obesity-prone and obesity-resistant rats. Exposure to the high-fat diet significantly enhanced the sensitivity of obesity-resistant (S5B/Pl) rats to linoleate while producing no effect on the fatty acid sensitivity for obesity-prone rats. Specifically, high-fat diet fed S5B/Pl rats showed stronger initial avoidance of linoleate and slower extinction rates than their normal diet cohorts. Our study suggests that prolonged dietary fat consumption may alter the behavioral sensitivity to fatty acids particularly in obesity-resistant animals.
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GAD autoantibodies (GADAs) are sensitive markers of islet autoimmunity and type 1 diabetes. They form the basis of robust prediction models and are widely used for the recruitment of subjects at high risk of type 1 diabetes to prevention trials. However, GADAs are also found in many individuals at low risk of diabetes progression. To identify the sources of diabetes-irrelevant GADA reactivity, we analyzed data from the 2009 and 2010 Diabetes Autoantibody Standardization Program GADA workshop and found that binding of healthy control sera varied according to assay type. The characterization of control sera found positive by radiobinding assay (RBA), but negative by ELISA, showed that many of these sera reacted to epitopes in the N-terminal region of the molecule. This finding prompted development of an N-terminally truncated GAD65 radiolabel, (35)S-GAD65(96-585), which improved the performance of most GADA RBAs participating in an Islet Autoantibody Standardization Program GADA substudy. These detailed workshop comparisons have identified a source of disease-irrelevant signals in GADA RBAs and suggest that N-terminally truncated GAD labels will enable more specific measurement of GADAs in type 1 diabetes.
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Autoanticorpos/análise , Diabetes Mellitus Tipo 1/diagnóstico , Ensaio de Imunoadsorção Enzimática , Glutamato Descarboxilase/imunologia , Ensaio Radioligante , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
We have previously identified pancreatic and duodenal homeobox 1 (Pdx1) autoantibodies (PAA) in sera from both non-obese diabetic (NOD) mice and human type 1 diabetic (T1D) patients. A suitable non-radioactive, sensitive and specific assay is needed for large-scale testing to determine the clinical utility of PAA. Here we reported a liquid-phase luciferase immunoprecipitation system (LIPS) assay by generating a renilla luciferase (Rluc)-Pdx1 fusion protein as a sensitive non-radioactive antigen from mammalian cells combined with immunoprecipitation to detect PAA in human sera. Sera from healthy donors and the University of Florida Pathology Laboratories, Endocrine Autoantibody Laboratory were used to validate the LIPS assay for PAA. Antigenic specificity to Pdx1 was confirmed by using a Rluc-only control compared to Rluc-Pdx1 fusion antigen and by competition assays using purified recombinant Pdx1 protein. We then used the LIPS assay to assess the prevalence of triple autoantibodies (GADA, IA-2A, and IA-2ßA), and PAA in non-T1D control sera, recent onset (RO)-T1D sera (mean duration of T1D = 9.5 weeks), and long standing (LS)-T1D sera. Compared to clinical radioimmunoprecipitation assays (RIPA), the LIPS assay showed comparable sensitivity and specificity for detection of GADA and IA-2A. PAA were detectable in human serum samples and higher in triple-positive T1D autoantibodies (21% PAA positive in triple positive sera and 4% PAA positive in triple negative sera). Interestingly, PAA were found to be highest in the non-T1D population, suggesting that PAA might have a clinical utility in screening high-risk population susceptible for developing T1D. In conclusion, we have developed a liquid-phase, non-radioactive, sensitive and specific LIPS assay to detect PAA in human sera, providing a useful tool for evaluating the clinical relevance of PAA.
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Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Proteínas de Homeodomínio/imunologia , Imunoprecipitação/métodos , Luciferases de Renilla , Transativadores/imunologia , Adolescente , Adulto , Ligação Competitiva , Estudos de Casos e Controles , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Feminino , Glutamato Descarboxilase/imunologia , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Extração Líquido-Líquido , Luciferases de Renilla/genética , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Radioimunoensaio , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Proteínas Recombinantes de Fusão , Transativadores/genética , Transfecção , Adulto JovemAssuntos
Serviços de Dietética/organização & administração , Obesidade/prevenção & controle , Serviços de Saúde Escolar/organização & administração , Criança , Fenômenos Fisiológicos da Nutrição Infantil , Comportamento de Escolha , Serviços de Dietética/normas , Humanos , Desenvolvimento de Programas/métodos , Avaliação de Programas e Projetos de Saúde , Recompensa , Serviços de Saúde Escolar/normasRESUMO
A series of brief-access (15s) behavioral assays following the formation of a conditioned taste aversion (CTA) to linoleic acid were performed in order to follow up on observations showing differences in the chemosensory responses to dietary fat in obesity-prone (Osborne-Mendel [O-M]) and obesity-resistant (S5B/Pl) rat strains. Strong aversions to linoleic acid (conditioned stimulus 100 microM) were generated in both O-M and S5B/Pl rats to concentrations as low as 2.5 microM. Observed strain differences were in contrast to expectations based upon electrophysiological studies previously showing greater fatty acid-induced inhibition of delayed rectifying K+ channels in S5B/Pl rats. In the CTA assays, the O-M rats showed aversions at lower fatty acid concentrations with more resistance to extinction in brief-access orosensory tests, suggesting that the obesity-prone strain may be more sensitive in the detection and subsequent avoidance of linoleic acid than the obesity-resistant strain. The independent variable of sex produced even greater differences in the avoidance of linoleic acid following conditioning than the effects of strain. Female rats of both strains were significantly more sensitive to fatty acids, showed greater cross-generalization from linoleic to oleic acid, and showed greater avoidance of linoleic acid than male counterparts. These findings suggest genetic influences on yet to be identified mechanisms potentially within the gustatory system that affect the sensitivity to detect the fatty acid chemicals found in dietary fat during brief-access orosensory testing.
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Ácidos Graxos/farmacologia , Obesidade , Caracteres Sexuais , Paladar/fisiologia , Administração Oral , Animais , Feminino , Cloreto de Lítio/farmacologia , Masculino , Obesidade/genética , Ratos , Ratos Endogâmicos , Cloreto de Sódio/farmacologia , Especificidade da Espécie , Estimulação Química , Paladar/efeitos dos fármacosRESUMO
Sprague-Dawley rats with intact (SHAM) and bilaterally transected chorda tympani nerves (CTX) received conditioned taste aversions (CTAs) to the free fatty acids (FFAs), linoleic and oleic acid, at micromolar quantities. Two-bottle preference tests showed that CTX eliminated avoidance of 88 muM linoleic acid but did not affect CTA avoidance of corn oil or 250 mM sucrose. Short-duration stimulus tests following single-pairing CTAs revealed that 8-s stimulus durations resulted in higher detection thresholds for linoleic acid than 30-s trials. In these short-duration tests, CTX rats showed 2-fold elevations in threshold for linoleic acid compared to the SHAM rats. A single-pairing CTA did not produce avoidance of oleic acid during the short-duration tests; however, 3 consecutive days of CTA pairings did produce avoidance of oleic acid in both male and female rats. Finally, both male and female rats received SHAM or CTX surgery after demonstrating successful CTAs to either 100 microM linoleic or oleic acid. The ability to detect and avoid linoleic and oleic acid was eliminated by CTX for both sexes. Differences in the ability of rats to form CTAs to linoleic and oleic acid suggest that linoleic acid is a more salient stimulus than oleic acid. Our results suggest that FFAs stimulate afferent taste signals in the chorda tympani nerve of male and female rats and that these signals play an important role in the gustatory behavior of accepting or avoiding taste stimuli following a conditioned taste aversion.