Immunofunctional assay of human growth hormone (hGH) in serum: A possible consensus for quantitative hGH measurement

Confirmation of the diagnosis of GH deficiency in adults and children involves provocative testing for human (h) GH. Different commercially available immunoassays yield largely discrepant results in the measurement of GH levels in human serum. These discrepancies result in doubtful relevance of cut-off levels proposed for GH provocative testing. We have developed an immunofunctional assay method that allows quantitation of only those GH forms in circulation that possess both binding sites of the hormone for its receptor and thus can initiate a biological signal in target cells. An anti-hGH monoclonal antibody recognizing binding site 2 of hGH is immobilized and used to capture hGH from the serum sample. Biotin-labeled recombinant GH-binding protein in a second incubation step forms a complex with those hGH molecular isoforms that have both binding sites for the receptor. The signal is detected after a short third incubation step with labeled streptavidin. The assay is sensitive (detection range, 0.1-100 micrograms/L) and has average inter- and intraassay precisions of 10.3% and 7.3% respectively. Endogenous GH-binding protein does not interfere with the hGH result; placental lactogen slows no detectable cross-reaction in this immunofunctional assay. The degree of immunofunctionally active hGH forms in serum samples, calculated by comparison of immunofunctional assay and RIA results, varied between 52-93%. We propose this immunofunctional assay for GH measurement as a new reference method for hGH quantitation in serum. The immunofunction assay translates only hGH forms into an assay signal that are capable of dimerizing GH receptors and, thus, of initiating a biological effect in target cells

Joint State and Input Estimation of Agent Based on Recursive Kalman Filter Given Prior Knowledge

Modern autonomous systems are purposed for many challenging scenarios, where agents will face unexpected events and complicated tasks. The presence of disturbance noise with control command and unknown inputs can negatively impact robot performance. Previous research of joint input and state estimation separately studied the continuous and discrete cases without any prior information. This paper combines the continuous and discrete input cases into a unified theory based on the Expectation-Maximum (EM) algorithm. By introducing prior knowledge of events as the constraint, inequality optimization problems are formulated to determine a gain matrix or dynamic weights to realize an optimal input estimation with lower variance and more accurate decision-making. Finally, statistical results from experiments show that our algorithm owns 81\% improvement of the variance than KF and 47\% improvement than RKF in continuous space; a remarkable improvement of right decision-making probability of our input estimator in discrete space, identification ability is also analyzed by experiments

T cell immunity rather than antibody mediates cross-protection against Zika virus infection conferred by a live attenuated Japanese encephalitis SA14-14-2 vaccine.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are closely related to mosquito-borne flaviviruses. Japanese encephalitis (JE) vaccine SA14-14-2 has been in the Chinese national Expanded Program on Immunization since 2007. The recent recognition of severe disease syndromes associated with ZIKV, and the identification of ZIKV from mosquitoes in China, prompts an urgent need to investigate the potential interaction between the two. In this study, we showed that SA14-14-2 is protective against ZIKV infection in mice. JE vaccine SA14-14-2 triggered both Th1 and Th2 cross-reactive immune responses to ZIKV; however, it was cellular immunity that predominantly mediated cross-protection against ZIKV infection. Passive transfer of immune sera did not result in significant cross-protection but did mediate antibody-dependent enhancement in vitro, though this did not have an adverse impact on survival. This study suggests that the SA14-14-2 vaccine can protect against ZIKV through a cross-reactive T cell response. This is vital information in terms of ZIKV prevention or precaution in those ZIKV-affected regions where JEV circulates or SA14-14-2 is in widespread use, and opens a promising avenue to develop a novel bivalent vaccine against both ZIKV and JEV. KEY POINTS: • JEV SA14-14-2 vaccine conferred cross-protection against ZIKV challenge in mice. • T cell immunity rather than antibody mediated the cross-protection. • It provides important information in terms of ZIKV prevention or precaution

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6–538.4 µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6–538.4 µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand-receptor fusions A B S T R A C T

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats. Two variants of the LR-fusion, one with a flexible linker (GH-LRv2) and the other without (GH-LRv3), were tested. Comparison was made with native human GH (growth hormone). GH-LRv2 and GHLRv3 demonstrated similar pharmacokinetics in rats, showing reduced clearance compared with native GH and potent agonist activity with respect to body weight gain in a hypophysectomized rat model. In M. fascicularis, a low level of antibodies to GH-LRv2 was found in one sample, but there was no other evidence of any immunogenic response to the other fusion protein. There were no toxic effects and specifically no changes in histology at injection sites after two repeated administrations. The pharmacokinetic profiles in monkeys confirmed long half-lives for both GHLRv2 and GH-LRv3 representing exceptionally delayed clearance over rhGH (recombinant human GH). The results suggest that repeated administration of a GH LR-fusion is safe, non-toxic, and the pharmacokinetic profile suggests that two to three weekly administrations is a potential therapeutic regimen for humans

Structure of the human obesity receptor leptin-binding domain reveals the mechanism of leptin antagonism by a monoclonal antibody

Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling