RESUMEN
Aplastic anemia (AA) is a rare disorder characterized by the suppression of bone marrow function resulting in progressive pancytopenia. The pathogenesis of AA is complex and involves an abnormal hematopoietic microenvironment, hematopoietic stem cell/progenitor cell deficiencies, and immunity disorders. However, the underlying mechanism of the disease is still not fully uncovered. In this research, we collected both donor and patient samples and found suppressed proliferation, abnormal differentiation as well as increased apoptosis of patient mesenchymal stem cells (MSCs). Considering the close relationship of parathyroid hormone (PTH) and MSCs differentiation, further studies showed that although patients maintained normal serum PTH level, their CD8+ T cells possessed lower PTH receptors. The insensitive to PTH of patients' CD8+ T cells finally lead to reduced expression of key Wnt factors. In all, bone marrow CD8+ T cells may play an important role in inducing MSCs adipogenesis and osteogenesis imbalancement.
Asunto(s)
Anemia Aplásica/genética , Células Madre Mesenquimatosas/metabolismo , Pancitopenia/genética , Hormona Paratiroidea/genética , Adipogénesis/genética , Adolescente , Anemia Aplásica/patología , Apoptosis/genética , Médula Ósea/inmunología , Médula Ósea/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Proliferación Celular/genética , Microambiente Celular/genética , Niño , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/inmunología , Osteogénesis/genética , Pancitopenia/inmunología , Pancitopenia/patología , Hormona Paratiroidea/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Bone loss is a frequent but not universal complication of hyperparathyroidism. Using antibiotic-treated or germ-free mice, we show that parathyroid hormone (PTH) only caused bone loss in mice whose microbiota was enriched by the Th17 cell-inducing taxa segmented filamentous bacteria (SFB). SFB+ microbiota enabled PTH to expand intestinal TNF+ T and Th17 cells and increase their S1P-receptor-1 mediated egress from the intestine and recruitment to the bone marrow (BM) that causes bone loss. CXCR3-mediated TNF+ T cell homing to the BM upregulated the Th17 chemoattractant CCL20, which recruited Th17 cells to the BM. This study reveals mechanisms for microbiota-mediated gut-bone crosstalk in mice models of hyperparathyroidism that may help predict its clinical course. Targeting the gut microbiota or T cell migration may represent therapeutic strategies for hyperparathyroidism.
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Microbioma Gastrointestinal/inmunología , Osteoporosis/etiología , Hormona Paratiroidea/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th17/inmunología , Animales , Trasplante de Microbiota Fecal , Femenino , Vida Libre de Gérmenes , Bacilos Grampositivos Formadores de Endosporas/inmunología , Hiperparatiroidismo Primario/complicaciones , Hiperparatiroidismo Primario/inmunología , Hiperparatiroidismo Primario/microbiología , Intestinos/inmunología , Intestinos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoporosis/inmunología , Osteoporosis/microbiología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Objective: A novel immunochromatographic test strip method was developed to detect tissue parathyroid hormone (PTH) using the immune colloidal gold technique (ICGT). The accuracy and application value of this method for intraoperative parathyroid identification were evaluated. Methods: Serum samples were collected to measure PTH by both ICGT and electrochemiluminescence immunoassay (ECLIA). Patients who underwent unilateral and total thyroidectomy were enrolled to evaluate the feasibility and clinical efficacy of rapid intraoperative identification of parathyroid glands via PTH determination using ICGT. Two sample preparation methods, fine needle aspiration (FNA) and tissue block homogenate (TBH), were used for PTH-ICGT analysis. Results: Bablok analysis showed a linear relationship between the serum PTH measurements obtained by ICGT and ECLIA. Non-parathyroid tissues had much lower PTH concentrations (14.8 ± 2.1 pg/ml, n = 97) detected by ICGT, compared to the parathyroid gland tissues (955.3 ± 16.1 pg/ml, n = 79; P < 0.0001), With biopsy results as the standard, ICGT showed higher diagnosis rates as compared with direct visual inspection, for identifying both parathyroid glands (97.4 vs. 78.2%) and non-parathyroid tissues (100 vs. 68.9%). The cut-off values for parathyroid identification by FNA and TBH methods were 63.99 and 136.30 pg/ml, respectively. The detection time was 2 min by TBH method for in vitro tissue detection and 6 min by FNA method for in situ tissue detection, both of which were faster than traditional intraoperative cryopathological examination (usually >30 min). Intraoperative application of ICGT method was associated with higher postoperative serum calcium and blood PTH levels at 1 and 3 months as well as a lower incidence of postoperative transient hypocalcemia, as compared with direct visual inspection. Conclusion: PTH-ICGT assay shows high potential as a rapid, novel alternative for intraoperative parathyroid identification.
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Oro Coloide/metabolismo , Monitoreo Intraoperatorio/métodos , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/sangre , Glándula Tiroides/metabolismo , Tiroidectomía/métodos , Adulto , Anciano , Cromatografía de Afinidad/métodos , Femenino , Oro Coloide/inmunología , Humanos , Hipoparatiroidismo/sangre , Hipoparatiroidismo/diagnóstico , Masculino , Persona de Mediana Edad , Hormona Paratiroidea/inmunología , Glándula Tiroides/cirugía , Factores de Tiempo , Adulto JovenRESUMEN
Root resorption is usually inflammatory in nature and has a tight link with immune system. Intermittent parathyroid hormone (iPTH) could promote cementum regeneration. The cross-talk of immune cells and cementoblasts may play an important role in the regeneration which stayed to be elucidated. In this study, a CD8+ T cells-OCCM-30 cells coculture system was established in vitro to investigate whether CD8+ T cells could enhance the anabolic effect of iPTH on cementoblasts and to find out the potential link of the effect with Wnt signal pathway. Determined by real-time PCR and Western Blot, we found an amplified cementogenesis in the OCCM-30 cells from coculture system, including increased mRNA and protein expression of Alp, Opn and Runx2, ALP activity and mineralization. We also found iPTH could increase the expression of Wnt10b in CD8+ T cells by ELISA. In addition, Wnt10b would promote the proliferation of OCCM-30 cells, while the effect on differentiation was various in different culture medium. These results demonstrated that the stimulating effect of iPTH on cementoblasts could be mediated through an interaction with CD8+ T cells, and T-cell-induced Wnt10b might be a key mechanism in the mediation.
Asunto(s)
Anabolizantes/inmunología , Linfocitos T CD8-positivos/inmunología , Cemento Dental/inmunología , Hormona Paratiroidea/inmunología , Proteínas Wnt/inmunología , Animales , Línea Celular , Técnicas de Cocultivo , Ratones Endogámicos BALB C , Proteínas Wnt/genéticaRESUMEN
CONTEXT: Immunoassay interference has been most often found with prolactin measurement. However, only few data exist on immunoassay interference for other hormones. CASE DESCRIPTION: A 36-year-old woman with obesity (body mass index, 31 kg/m2) had regularly attended our endocrine unit for type 2 diabetes therapy. When she was included as a control subject in a study for obesity management, detailed laboratory testing was performed, including PTH. In the absence of clinical symptoms, she presented with normal calcium, phosphate, and vitamin D levels. However, the PTH levels were >5000 ng/L. These results were obtained using the Roche Elecsys electrochemiluminescence assay. Repeated measurements with this assay (mouse antibody) led to the same findings. However, using an Euroimmun assay (goat antibody), the exact PTH values were measured at 18.0 ng/L. After pretreatment with a heterophilic antibody blocking reagent, the results of the Roche assay had decreased to a normal level. This phenomenon was explained by the detection of human anti-mouse antibodies in the proband's serum. CONCLUSIONS: In cases of prolactin immunoassay interference, endogenous antibodies will bind to the hormone in vivo, resulting in complexes of a high molecular weight that are less efficiently cleared by the kidneys and, thus, accumulate in the blood. In contrast, the PTH values >5000 ng/L detected in our subject most likely had resulted from the specific interference of the human anti-mouse antibodies present in the proband's serum with the assay antibody, resulting in artificial stimulation of the Roche assay detection system ex vivo.
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Anticuerpos Heterófilos/sangre , Inmunoensayo/métodos , Obesidad/inmunología , Hormona Paratiroidea/inmunología , Adulto , Animales , Anticuerpos Heterófilos/inmunología , Femenino , Humanos , Ratones/inmunología , Obesidad/sangre , Hormona Paratiroidea/sangreRESUMEN
Enzymatically induced silver deposition and subsequent electrochemical oxidation have been widely used in electrochemical biosensors. However, this method is ineffective for producing highly enhanced silver deposition for use in ultrasensitive detection. Herein, we report a fast silver deposition method that simultaneously uses three signal amplification processes: (i) enzymatic amplification, (ii) chemical-chemical (CC) redox cycling, and (iii) chemical-enzymatic (CN) redox cycling. DT-diaphorase (DT-D) is used for enzymatic amplification to convert a nitroso compound, a species incapable of directly reducing Ag+ to an amine compound, which can directly reduce Ag+. NADH acts as a reducing agent for the indirect reduction of Ag+ via the two redox cycling processes. 4-Nitroso-1-naphthol is converted to 4-amino-1-naphthol (NH2-N) in the presence of DT-D. NH2-N initiates two redox cycling processes: NH2-N, along with Ag+ and NADH, are involved in the CC redox cycling, whereas NH2-N, along with Ag+, DT-D, and NADH, are involved in the CN redox cycling. Finally, the deposited silver is electrochemically oxidized to produce a signal. When this triple signal amplification strategy for fast silver deposition is applied to an electrochemical immunosensor for detecting parathyroid hormone (PTH), a detection limit as low as â¼100 fg/mL is obtained. The concentrations of PTH in clinical serum determined using the developed immunosensor are found to agree with those measured using a commercial instrument. Thus, the use of this strategy for fast silver deposition is highly promising for ultrasensitive electrochemical detection and biosensing applications.
Asunto(s)
Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Compuestos Nitrosos/química , Hormona Paratiroidea/sangre , Plata/química , 1-Naftilamina/análogos & derivados , Anticuerpos Inmovilizados/inmunología , Proteínas Bacterianas/química , Geobacillus stearothermophilus/enzimología , Humanos , Límite de Detección , NAD(P)H Deshidrogenasa (Quinona)/química , Oxidación-Reducción , Hormona Paratiroidea/inmunologíaRESUMEN
Immunosuppression may occur for a number of reasons related to an individual's frailty, debility, disease or from therapeutic iatrogenic intervention or misadventure. A large percentage of morbidity and mortality in immunodeficient populations is related to an inadequate response to infectious agents with slow response to antibiotics, enhancements of antibiotic resistance in populations, and markedly increased prevalence of acute inflammatory response, septic and infection related death. Given known relationships between intracellular calcium ion concentrations and cytotoxicity and cellular death, we looked at currently available data linking blockade of calcium ion channels and potential decrease in expression of sepsis among immunosuppressed patients. Notable are relationships between calcium, calcium channel, vitamin D mechanisms associated with sepsis and demonstration of antibiotic-resistant pathogens that may utilize channels sensitive to calcium channel blocker. We note that sepsis shock syndrome represents loss of regulation of inflammatory response to infection and that vitamin D, parathyroid hormone, fibroblast growth factor, and klotho interact with sepsis defense mechanisms in which movement of calcium and phosphorus are part of the process. Given these observations we consider that further investigation of the effect of relatively inexpensive calcium channel blockade agents of infections in immunosuppressed populations might be worthwhile.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio/inmunología , Enfermedades Transmisibles/tratamiento farmacológico , Huésped Inmunocomprometido , Sepsis/tratamiento farmacológico , Calcio/inmunología , Calcio/metabolismo , Canales de Calcio/genética , Enfermedades Transmisibles/genética , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/mortalidad , Farmacorresistencia Microbiana/genética , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/inmunología , Regulación de la Expresión Génica , Glucuronidasa/genética , Glucuronidasa/inmunología , Humanos , Proteínas Klotho , Hormona Paratiroidea/genética , Hormona Paratiroidea/inmunología , Fósforo/inmunología , Fósforo/metabolismo , Riesgo , Sepsis/genética , Sepsis/inmunología , Sepsis/mortalidad , Análisis de Supervivencia , Vitamina D/inmunología , Vitamina D/metabolismoRESUMEN
Enzyme-like nanocatalytic reactions developed for high signal amplification in biosensors are of limited use because of their low reaction rates and/or unwanted side reactions in aqueous electrolyte solutions containing dissolved O2. Herein, we report a nitrosoreductase-like catalytic reaction, employing 4-nitroso-1-naphthol, Pd nanoparticles, and H3N-BH3, which affords a high reaction rate and minimal side reactions, enabling its use in ultrasensitive electrochemical biosensors. 4-Nitroso-1-naphthol was chosen after five hydroxy-nitro(so)arene compounds were compared in terms of high signal and low background levels. Importantly, the nanocatalytic reaction occurs without the self-hydrolysis and induction period observed in the nanocatalytic reduction of nitroarenes by NaBH4. The high signal level results from (i) fast nanocatalytic 4-nitroso-1-naphthol reduction, (ii) fast electrochemical redox cycling, and (iii) the low influence of dissolved O2. The low background level results from (i) slow direct reaction between 4-nitroso-1-naphthol and H3N-BH3, (ii) slow electrode-mediated reaction between 4-nitroso-1-naphthol and H3N-BH3, and (iii) slow electrooxidation of H3N-BH3 at electrode. When applied to the detection of parathyroid hormone, the detection limit of the newly developed biosensor was â¼0.3 pg/mL. The nitrosoreductase-like nanocatalytic reaction is highly promising for ultrasensitive and stable biosensing.
Asunto(s)
Nanopartículas del Metal/química , Naftoles/química , Compuestos Nitrosos/química , Hormona Paratiroidea/análisis , Anticuerpos/inmunología , Técnicas Biosensibles/métodos , Catálisis , Técnicas Electroquímicas/métodos , Humanos , Límite de Detección , Oxidación-Reducción , Paladio/química , Hormona Paratiroidea/inmunologíaRESUMEN
PURPOSE OF REVIEW: This review summarizes studies into the permissive role of T cells in the bone catabolic effects of hyperparathyroidism and parathyroid hormone (PTH). RECENT FINDINGS: Work in animals combined with recent translational studies in humans now highlight the potent amplificatory action of T cells on PTH-induced bone resorption. Mechanistic animal studies reveal a complex pathway by which PTH exploits natural self-renewal functions of CD4+ T cells, to drive TNFα production that promotes formation of IL-17A secreting Th17 T cells. TNFα and IL-17 further amplify osteoblastic receptor activator of NF-κB ligand (RANKL) production and down-modulate osteoprotegerin (OPG), establishing conditions propitious for osteoclastic bone resorption. These findings are consistent with, and add to, the traditional view of PTH-induced bone loss involving only osteoblast-lineage cells. T cells potently amplify traditional pathways and provide permissive costimulatory signals to bone marrow stromal cells, facilitating the development of an increased RANKL/OPG ratio favourable to bone resorption and bone loss.
Asunto(s)
Resorción Ósea/inmunología , Hiperparatiroidismo/inmunología , Hormona Paratiroidea/inmunología , Linfocitos T/inmunología , Resorción Ósea/metabolismo , Linfocitos T CD4-Positivos/inmunología , Humanos , Hiperparatiroidismo/metabolismo , Interleucina-17/inmunología , Osteoclastos , Osteoprotegerina/inmunología , Enfermedades de las Paratiroides/inmunología , Enfermedades de las Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Ligando RANK/inmunología , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
It was the aim of the present investigation to examine whether the stimulating effect of parathyroid hormone (PTH) on human periodontal ligament (hPDL) cell proliferation and differentiation would be enhanced by hPDL/T-cell interaction involving Wnt10b signaling as a mediating pathway. hPDL cells were cultured from healthy premolar tissues of three adolescent orthodontic patients and exposed to PTH(1-34) in monocultures or co-cultures with CD8+ T cells. At harvest, proliferation, alkaline phosphatase-specific activity (ALP), and osteocalcin production were determined by immunofluorescence cytochemistry, real-time PCR, biochemical assay, and ELISA. Wnt10b signaling was analyzed by the use of a specific WNT10b neutralizing antibody. PTH(1-34) stimulation of T cells significantly increased Wnt10b expression and production. Wnt10b exposure of hPDL cells enhanced proliferation and differentiation. PDL cells co-cultured with T cells showed a Wnt10b-dependent regulation of proliferation and differentiation parameters. The addition of a Wnt10b-neutralizing Ab to the co-culture medium resulted in a significant inhibition of the PTH(1-34) effect on proliferation, ALP-specific activity, and osteocalcin protein expression. Our findings provide novel insight into the mechanism of action of PTH on hPDL cells and establish the interplay of T cells and hPDL cells via the Wnt10b pathway as a modulating factor for the anabolic properties of the hormone in periodontal regeneration.
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Linfocitos T CD8-positivos/inmunología , Ligamento Periodontal/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Adolescente , Fosfatasa Alcalina/metabolismo , Anticuerpos Neutralizantes/farmacología , Comunicación Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Niño , Técnicas de Cocultivo , Humanos , Osteocalcina/metabolismo , Hormona Paratiroidea/inmunología , Proteínas Proto-Oncogénicas/inmunología , Regeneración , Transducción de Señal , Proteínas Wnt/inmunologíaRESUMEN
Osteoimmunology is a field of research dedicated to the study of the interactions between the immune system and bone. Among the cells of the immune system that regulate bone turnover and the responsiveness of bone cells to calciothropic hormones are bone marrow T lymphocytes. T cells secrete osteoclastogenic cytokines such as RANKL and TNF-α, as well as factors that stimulate bone formation, one of which is Wnt10b. In addition, T cells regulate the differentiation and life span of stromal cells (SCs) and their responsiveness to parathyroid hormone (PTH) via costimulatory molecules expressed on their surface. The conditioning effect of T cells on SCs is inherited by the osteoblastic and osteocytic progeny of SCs. As a result, osteoblastic cells of T cell-deficient mice have functional characteristics different from corresponding cells of T cell-replete mice. These differences include the ratio of RANKL/OPG produced in response to continuous PTH treatment, and the osteoblastogenic response to intermittent PTH treatment. This article reviews the evidence indicating that the effects of PTH are mediated not only by osteoblasts and osteocytes but also by T cells.
Asunto(s)
Células de la Médula Ósea/metabolismo , Remodelación Ósea , Modelos Biológicos , Osteoblastos/metabolismo , Osteocitos/metabolismo , Hormona Paratiroidea/metabolismo , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Proteínas Morfogenéticas Óseas/metabolismo , Huesos/citología , Huesos/inmunología , Huesos/metabolismo , Comunicación Celular , Linaje de la Célula , Citocinas/metabolismo , Marcadores Genéticos , Humanos , Osteoblastos/citología , Osteoblastos/inmunología , Osteocitos/citología , Osteocitos/inmunología , Hormona Paratiroidea/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Células del Estroma/citología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Proteínas Wnt/metabolismoRESUMEN
Parathyroid hormone (PTH), an 84 amino acid peptide hormone, is an important regulator of calcium homeostasis. Quantitation of PTH in serum is useful for the diagnosis of primary hyperparathyroidism, hypoparathyroidism, and for monitoring osteodystrophy in patients with renal failure. The biological activity of PTH arises from binding of PTH (N terminus) to its target receptor (D'Amour et al., Kidney Int 68: 998-1007, 2005). Several C-terminal and N-terminal fragments circulate in normal subjects. Recent studies have demonstrated that accurate quantitation of PTH fragments may be of clinical value. In this chapter a mass spectrometry based method for quantitation of PTH(1-84) is described. This method involves immunoaffinity capture of PTH followed by trypsinization and quantitation of PTH-specific tryptic peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The N-terminal tryptic peptide, PTH(1-13) as surrogate of 1-84 PTH, is used for quantitation.
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Análisis Químico de la Sangre/métodos , Cromatografía Líquida de Alta Presión/métodos , Hormona Paratiroidea/sangre , Hormona Paratiroidea/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Métodos Analíticos de la Preparación de la Muestra , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Humanos , Microesferas , Hormona Paratiroidea/inmunología , Poliestirenos/química , Estadística como AsuntoRESUMEN
Parathyroid hormone-like hormone (PTHLH) exerts relevant roles in progression and dissemination of several tumors. However, factors influencing its production and secretion have not been fully characterized. The main limitation is the lack of specific, sensitive and widely available techniques to detect and quantify PTHLH. We have developed a lateral flow immunoassay using gold nanoparticles label for the fast and easy detection of PTHLH in lysates and culture media of three human cell lines (HaCaT, LA-N-1, SK-N-AS). Levels in culture media and lysates ranged from 11 to 20 ng/mL and 0.66 to 0.87 µg/mL respectively. Results for HaCaT are in agreement to the previously reported, whereas LA-N-1 and SK-N-AS have been evaluated for the first time. The system also exhibits good performance in human serum samples. This methodology represents a helpful tool for future in vitro and in vivo studies of mechanisms involved in PTHLH production as well as for diagnostics. From the Clinical Editor: Parathyroid Hormone-like Hormone (PTHLH) is known to be secreted by some tumors. However, the detection of this peptide remains difficult. The authors here described their technique of using gold nanoparticles as label for the detection of PTHLH by Lateral-flow immunoassays (LFIAs). The positive results may also point a way to using the same technique for the rapid determination of other relevant cancer proteins.
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Inmunoensayo/instrumentación , Nanopartículas del Metal/química , Técnicas Analíticas Microfluídicas/instrumentación , Neoplasias Experimentales/diagnóstico , Neoplasias Experimentales/inmunología , Hormona Paratiroidea/análisis , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Diseño de Equipo , Análisis de Falla de Equipo , Oro/química , Humanos , Nanopartículas del Metal/ultraestructura , Hormona Paratiroidea/inmunología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Fabrication of a new electrochemical impedance-based biosensor for the analysis of parathyroid hormone (PTH), using self-assembled monolayers (SAMs) of mercaptohexanol and (3-Aminopropyl) triethoxysilane on gold electrodes, was investigated for the first time in the field. Anti-PTH was used as a biorecognition element. To monitor immobilization processes in the biosensor fabrication, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM) techniques were successfully operated. CV and EIS techniques were also used in quantification of PTH. Energy-dispersive X-ray analysis (EDAX) was also applied to identify surface modifications. Fabrication and working parameters of the biosensor were optimized. Moreover, Kramers-Kronig transformations were performed for validation of obtained EIS data in all steps of biosensor fabrication. The linear PTH detection range of the presented biosensor was 10-50 pg/mL PTH. The chrono-impedance technique for real-time monitoring of PTH binding was also implemented. The biosensor has exhibited good repeatability (with a correlation) and reproducibility. Finally, artificial serum samples spiked with known concentrations of PTH were analyzed by the proposed biosensor. To demonstrate the feasibility of the biosensor in practical analysis, real human serum samples and the artificial serum samples were analyzed.
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Anticuerpos Inmovilizados/inmunología , Técnicas Biosensibles , Hexanoles/química , Hormona Paratiroidea/análisis , Propilaminas/química , Silanos/química , Compuestos de Sulfhidrilo/química , Anticuerpos Inmovilizados/química , Espectroscopía Dieléctrica , Electrodos , Oro , Humanos , Hormona Paratiroidea/sangre , Hormona Paratiroidea/química , Hormona Paratiroidea/inmunologíaRESUMEN
CONTEXT: Data on calcium-sensing receptor autoantibodies (CaSRAbs) in hypoparathyroidism are variable. OBJECTIVE: We assessed the prevalence and significance of CaSRAbs in idiopathic hypoparathyroidism. DESIGN: This was a case-control study. SUBJECTS: One hundred forty-seven patients with idiopathic hypoparathyroidism treated during 1998-2011 in a tertiary care setting and 348 controls [healthy, n = 199; type 1 diabetes mellitus (T1DM), n = 99; and chronic lymphocytic thyroiditis (CLT), n = 50] participated in the study. METHODS: CaSRAb assays included Western blot with CaSR protein expressed in Escherichia coli or human embryonic kidney (HEK)-293 cells, immunoprecipitation (IP) using in vitro-transcribed/translated protein, and indirect immunofluorescence on HEK293-CaSR. Functional significance was assessed by ERK1/2 phosphorylation. PTH and CaSR genes were sequenced for mutations. RESULTS: E coli-Western blot assay revealed 16.3% CaSRAb positivity in idiopathic hypoparathyroidism, which was comparable with healthy subjects and CLT but significantly less than the T1DM controls. The prevalence of CaSRAbs on HEK293-Western blot (24.5%) against 150 kDa and/or 168 kDa protein in hypoparathyroidism was significantly higher than the healthy subjects, T1DM, and CLT. IP assay showed CaSRAbs in 12.9% of the hypoparathyroid patients but not in controls. The sensitivity and specificity of CaSRAbs in E coli and HEK-293-CaSR Western blot and IP assays were 16.3% and 83.1%, 24.5% and 88.9%, and 12.9% and 100%, respectively, and 42.1% of the cases detected were common in the IP assay and HEK293-Western blot. Duration of illness and coexistent autoimmunity were similar in patients with and without CaSRAbs. The CaSRAb-positive sera showed no immunofluorescence and phosphorylated ERK1/2 activity. The CaSR gene sequence was normal in all patients. One of the patients showed a novel p.Met1_Asp6del mutation in the signal peptide region of the PTH gene. CONCLUSION: IP performed the best in detecting CaSRAbs in 12.9% of hypoparathyroid patients. Although CaSRAbs were functionally inert, its clinical relevance remains due to 100% specificity. Limited prevalence of CaSRAb suggests heterogeneity in the etiology of idiopathic hypoparathyroidism or the presence of CaSR epitopes other than those measured in the current study.
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Autoanticuerpos/sangre , Hipoparatiroidismo/inmunología , Receptores Sensibles al Calcio/inmunología , Adolescente , Adulto , Anciano , Autoanticuerpos/inmunología , Estudios de Casos y Controles , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Células HEK293 , Enfermedad de Hashimoto/sangre , Enfermedad de Hashimoto/genética , Enfermedad de Hashimoto/inmunología , Humanos , Hipoparatiroidismo/sangre , Hipoparatiroidismo/genética , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Persona de Mediana Edad , Hormona Paratiroidea/genética , Hormona Paratiroidea/inmunología , Fosforilación , Receptores Sensibles al Calcio/genéticaRESUMEN
For uremic patients, Kidney Disease: Improving Global Outcomes suggest that parathyroid hormone (PTH) levels should be maintained within approximately 2-9 times the upper normal limit for the assay. One of reasons for the lack of definite approximate PTH values is the inability to use the Allegro Intact PTH assay from Nicols. We aimed to evaluate whether other parathyroid hormone assays were suitable for these assessments. We compared the parathyroid hormone concentrations measured with five commercial immunoassays by using three serum pools and seven artificially spiked samples of parathyroid hormone; the Total Intact parathyroid hormone assay was used as the reference assay. Although the results of parathyroid hormone assays showed high correlation, the concentrations differed from Yamasa's assay to another. And the third/second-generation assay ratio was approximately 60%. Further, the Immulite assay overestimated the levels of 1-84 PTH. We showed important inter-method variations in the parathyroid hormone assays used in Japan.
Asunto(s)
Enfermedades Renales/complicaciones , Hormona Paratiroidea/sangre , Uremia/complicaciones , Adulto , Enfermedad Crónica , Humanos , Inmunoensayo/métodos , Japón , Laboratorios/normas , Hormona Paratiroidea/inmunología , Juego de Reactivos para Diagnóstico , Diálisis RenalRESUMEN
Patients with unresectable parathyroid carcinoma develop severe hypercalcemia, bone fractures and renal failure, and become unresponsive to conventional treatments. It has been shown that successful induction of anti-parathyroid hormone (PTH) antibodies, using PTH peptide fragments for immunisation, normalized serum levels of calcium as well as improved clinical symptoms. Here, we report our experience of PTH immunization in a Japanese female suffering from refractory hypercalcemia and renal failure caused by unresectable metastatic parathyroid carcinoma. Upon immunization, there were apparent clinical responses including reduction of serum levels of Ca along with anti-PTH antibodies induction. Therefore, we concluded that PTH immunization was an effective treatment against hypercalcemia caused by metastatic parathyroid carcinomas that are unresponsive to conventional treatments.
Asunto(s)
Carcinoma/complicaciones , Hipercalcemia/terapia , Inmunización , Hormona Paratiroidea/inmunología , Neoplasias de las Paratiroides/complicaciones , Adulto , Anticuerpos/sangre , Carcinoma/diagnóstico , Carcinoma/cirugía , Resultado Fatal , Femenino , Insuficiencia Cardíaca , Humanos , Hipercalcemia/etiología , Inmunoterapia Activa , Metástasis de la Neoplasia , Hormona Paratiroidea/sangre , Neoplasias de las Paratiroides/diagnóstico , Neoplasias de las Paratiroides/terapia , Fragmentos de Péptidos/inmunología , Insuficiencia Renal/etiologíaRESUMEN
In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2â»/â» mutants. However, the developmental ontogeny and cellular identity of these "thymic" PTH-expressing cells is unknown. We found that the lethality of aparathyroid Gcm2â»/â» mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2â»/â» mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant "thymic PTH."
Asunto(s)
Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/biosíntesis , Timo/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Organogénesis , Glándulas Paratiroides/embriología , Glándulas Paratiroides/inmunología , Hormona Paratiroidea/sangre , Hormona Paratiroidea/inmunología , Timo/embriología , Timo/inmunología , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Parathyroid hormone (PTH) promotes bone catabolism by targeting bone marrow (BM) stromal cells (SCs) and their osteoblastic progeny. Here we show that a continuous infusion of PTH that mimics hyperparathyroidism fails to induce osteoclast formation, bone resorption, and cortical bone loss in mice lacking T cells. T cells provide proliferative and survival cues to SCs and sensitize SCs to PTH through CD40 ligand (CD40L), a surface molecule of activated T cells that induces CD40 signaling in SCs. As a result, deletion of T cells or T cell-expressed CD40L blunts the bone catabolic activity of PTH by decreasing bone marrow SC number, the receptor activator of nuclear factor-kappaB ligand (RANKL)/OSTEOPROTEGERN (OPG) ratio, and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, life span, and function through CD40L. T cell-SC crosstalk pathways may thus provide pharmacological targets for PTH-induced bone disease.
Asunto(s)
Huesos/metabolismo , Ligando de CD40/metabolismo , Hiperparatiroidismo/metabolismo , Osteoporosis/metabolismo , Hormona Paratiroidea/metabolismo , Linfocitos T/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Huesos/inmunología , Huesos/fisiopatología , Ligando de CD40/inmunología , Proliferación Celular/efectos de los fármacos , Femenino , Hiperparatiroidismo/inducido químicamente , Hiperparatiroidismo/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Osteoclastos/efectos de los fármacos , Osteoclastos/inmunología , Osteoclastos/metabolismo , Osteoporosis/inmunología , Osteoporosis/fisiopatología , Hormona Paratiroidea/inmunología , Hormona Paratiroidea/farmacología , Ligando RANK/inmunología , Ligando RANK/metabolismo , Transducción de Señal/fisiología , Células del Estroma/efectos de los fármacos , Células del Estroma/inmunología , Células del Estroma/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
In this work we explore a new hydrogel stamp material obtained from polymerizing 2-hydroxyethyl acrylate and poly(ethylene glycol) diacrylate in the presence of water for the microcontact printing of proteins directly on gold substrates and by covalent coupling to self-assembled monolayers of alkanethiols. At high cross-link density, the hydrogel is rigid, hydrophilic, and with a high buffer holding capacity to enable the unsupported printing of protein patterns homogeneously and reproducibly, with micrometer-range precision. The stamps were used to print antibodies to human parathyroid hormone, which were shown using immunoassay tests to retain their biological function with binding capacities comparable to those of solution-adsorbed antibodies.