A Non-Isotopic Method for Estimating 11β Hydroxysteroid Dehydrogenase Activity In Vivo

1997 ◽  
Vol 34 (5) ◽  
pp. 521-526 ◽  
Author(s):  
G R Bayly ◽  
W A Bartlett ◽  
A F Jones
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Steroid Production ◽  
Isotopic Method ◽  
Novel Approach ◽  
Large Numbers ◽  
Local Oxidation ◽  
Partial Deficiency ◽  
Dexamethasone Suppression ◽  
Endogenous Steroid

11 β-hydroxysteroid dehydrogenase (11βHSD) has both dehydrogenase (11βDH) and reductase (11βR) activities, which catalyse the interconversion of Cortisol and cortisone, and prednisolone and prednisone. This enzyme confers specificity on the mineralocorticoid receptor by local oxidation of Cortisol to cortisone. Using radiolabeled Cortisol 11βHSD activity has been shown to be lower in some cases of essential hypertension. This study investigated a novel approach to estimating 11βHSD activity in vivo. Plasma steroid kinetics were investigated following oral hydrocortisone (a substrate for 11βDH) and prednisone (a substrate for 11βR) in five normotensive volunteers after dexamethasone suppression of endogenous steroid production. This approach was evaluated by inducing partial deficiency of 11βHSD in the volunteers who took liquorice (to inhibit 11βDH) and then carbenoxolone (to inhibit both 11βDH and 11βR). The ratio of Cortisol to prednisolone (formed from prednisone) provided a measure of the activity of both 11βDH and 11βR. At 75min after the steroid bolus the ratio increased from 1·1 (0·6–1·3) (median, range) under control conditions to 1·2 (0·8–1·7) after liquorice ( P = 0·01, n = 5), and 2·0 (1·3–5·9) after carbenoxolone ( P = 0·02, n = 5). It may therefore be applied to the measurement of 11βHSD activity in vivo in large numbers of hypertensive patients without the use of radioisotopes.

scholarly journals Profiling antibody responses by multiparametric analysis of primary B cells

2008 ◽  
Vol 105 (46) ◽  
pp. 17902-17907 ◽  
Author(s):  
Craig M. Story ◽  
Eliseo Papa ◽  
Chih-Chi Andrew Hu ◽  
Jehnna L. Ronan ◽  
Kara Herlihy ◽  
...  
Keyword(s):  
B Cells ◽  
Neutralizing Antibodies ◽  
Humoral Response ◽  
Cellular Level ◽  
Molecular Characteristics ◽  
Cellular Origin ◽  
Novel Approach ◽  
Large Numbers ◽  
Antibody Secreting Cells

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (≈103-104). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.

scholarly journals 11β-Hydroxysteroid Dehydrogenase Type 1, But Not Type 2, Deficiency Worsens Acute Inflammation and Experimental Arthritis in Mice

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 234-240 ◽  
Author(s):  
Agnes E. Coutinho ◽  
Mohini Gray ◽  
David G. Brownstein ◽  
Donald M. Salter ◽  
Deborah A. Sawatzky ◽  
...  
Keyword(s):  
Acute Inflammation ◽  
Inflammatory Responses ◽  
Inflammatory Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Transfer Model ◽  
Lung Pathology ◽  
Antiinflammatory Therapy ◽  
Novel Approach ◽  
Deficient Mice

Glucocorticoids profoundly influence immune responses, and synthetic glucocorticoids are widely used clinically for their potent antiinflammatory effects. Endogenous glucocorticoid action is modulated by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD). In vivo, 11β-HSD1 catalyzes the reduction of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, thereby increasing intracellular glucocorticoid levels. 11β-HSD2 catalyzes the reverse reaction, inactivating intracellular glucocorticoids. Both enzymes have been postulated to modulate inflammatory responses. In the K/BxN serum transfer model of arthritis, 11β-HSD1-deficient mice showed earlier onset and slower resolution of inflammation than wild-type controls, with greater exostoses in periarticular bone and, uniquely, ganglion cysts, consistent with greater inflammation. In contrast, K/BxN serum arthritis was unaffected by 11β-HSD2 deficiency. In a distinct model of inflammation, thioglycollate-induced sterile peritonitis, 11β-HSD1-deficient mice had more inflammatory cells in the peritoneum, but again 11β-HSD2-deficient mice did not differ from controls. Additionally, compared with control mice, 11β-HSD1-deficient mice showed greater numbers of inflammatory cells in pleural lavages in carrageenan-induced pleurisy with lung pathology consistent with slower resolution. These data suggest that 11β-HSD1 limits acute inflammation. In contrast, 11β-HSD2 plays no role in acute inflammatory responses in mice. Regulation of local 11β-HSD1 expression and/or delivery of substrate may afford a novel approach for antiinflammatory therapy.

scholarly journals HIF1α is a direct regulator of steroidogenesis in the adrenal gland

2021 ◽  
Author(s):  
Deepika Watts ◽  
Johanna Stein ◽  
Ana Meneses ◽  
Nicole Bechmann ◽  
Ales Neuwirth ◽  
...  
Keyword(s):  
Multiple Organ ◽  
Hypoxia Inducible Factors ◽  
Adrenocortical Cells ◽  
Steroid Production ◽  
Hormone Synthesis ◽  
Organ Systems ◽  
Direct Regulator ◽  
Endogenous Steroid ◽  
Endogenous Steroid Hormones

AbstractEndogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.

scholarly journals Marker for the pre-clinical development of bone substitute materials

2017 ◽  
Vol 3 (2) ◽  
pp. 711-715
Author(s):  
Michael de Wild ◽  
Simon Zimmermann ◽  
Marcel Obrecht ◽  
Michel Dard
Keyword(s):  
Bone Graft ◽  
Mechanical Stability ◽  
Clinical Performance ◽  
Ex Vivo ◽  
Region Of Interest ◽  
Defect Site ◽  
Novel Approach ◽  
Bone Substitute Materials ◽  
Clinical Experiments

AbstractThin mechanically stable Ti-cages have been developed for the in-vivo application as X-ray and histology markers for the optimized evaluation of pre-clinical performance of bone graft materials. A metallic frame defines the region of interest during histological investigations and supports the identification of the defect site. This standardization of the procedure enhances the quality of pre-clinical experiments. Different models of thin metallic frameworks were designed and produced out of titanium by additive manufacturing (Selective Laser Melting). The productibility, the mechanical stability, the handling and suitability of several frame geometries were tested during surgery in artificial and in ex-vivo bone before a series of cages was preclinically investigated in the female Göttingen minipigs model. With our novel approach, a flexible process was established that can be adapted to the requirements of any specific animal model and bone graft testing.

Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

1995 ◽  
Vol 23 (1) ◽  
pp. 61-73
Author(s):  
Coenraad Hendriksen ◽  
Johan van der Gun
Keyword(s):  
Quality Control ◽  
Test Methods ◽  
In Vitro Test ◽  
Large Numbers ◽  
Alternative Approach ◽  
Inactivated Vaccines ◽  
Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

scholarly journals Artificial Tumor Microenvironments in Neuroblastoma

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1629
Author(s):  
Colin H. Quinn ◽  
Andee M. Beierle ◽  
Elizabeth A. Beierle
Keyword(s):  
Extracellular Matrix ◽  
Three Dimensional ◽  
Therapeutic Interventions ◽  
3D Bioprinting ◽  
Culture Studies ◽  
In Vivo Models ◽  
Novel Treatments ◽  
Tumor Microenvironments ◽  
Novel Approach

In the quest to advance neuroblastoma therapeutics, there is a need to have a deeper understanding of the tumor microenvironment (TME). From extracellular matrix proteins to tumor associated macrophages, the TME is a robust and diverse network functioning in symbiosis with the solid tumor. Herein, we review the major components of the TME including the extracellular matrix, cytokines, immune cells, and vasculature that support a more aggressive neuroblastoma phenotype and encumber current therapeutic interventions. Contemporary treatments for neuroblastoma are the result of traditional two-dimensional culture studies and in vivo models that have been translated to clinical trials. These pre-clinical studies are costly, time consuming, and neglect the study of cofounding factors such as the contributions of the TME. Three-dimensional (3D) bioprinting has become a novel approach to studying adult cancers and is just now incorporating portions of the TME and advancing to study pediatric solid. We review the methods of 3D bioprinting, how researchers have included TME pieces into the prints, and highlight present studies using neuroblastoma. Ultimately, incorporating the elements of the TME that affect neuroblastoma responses to therapy will improve the development of innovative and novel treatments. The use of 3D bioprinting to achieve this aim will prove useful in developing optimal therapies for children with neuroblastoma.

Activation of the peptidergic neurosecretory system in Diphyllobothrium dendriticum (Cestoda: Pseudophyllidea)

Parasitology ◽  
1981 ◽  
Vol 83 (2) ◽  
pp. 243-247 ◽  
Author(s):  
Margaretha K. S. Gustafsson ◽  
Marianne C. Wikgren
Keyword(s):  
Neurosecretory Cells ◽  
Final Host ◽  
Fish Host ◽  
Neurosecretory System ◽  
Weak Reaction ◽  
Diphyllobothrium Dendriticum ◽  
Large Numbers ◽  
Short Times

SUMMARYThe activation of the peptidergic neurosecretory system in Diphyllobothrium dendriticum was studied following cultivation of plerocercoids for short times in vitro and in vivo. In the plerocercoid the neurosecretory cells gave a very weak reaction with paraldehyde fuchsin (PAF). After cultivation for 1 h large numbers of neurosecretory cells filled with PAF-positive granules were evident. The significance of the activation of the neurosecretory system during the transfer of the worm from the cold-blooded fish host to the warm-blooded final host is discussed.

scholarly journals HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Dan Song ◽  
Ming Guo ◽  
Shuai Xu ◽  
Xiaotian Song ◽  
Bin Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Intellectual Development ◽  
Molecular Mechanisms ◽  
Hsp90 Inhibitor ◽  
Pseudouridine Synthase ◽  
Novel Approach ◽  
Cell Metastasis ◽  
Underlying Mechanisms

Abstract Background Pseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms. Methods We conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry. Results Overexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis. Conclusions The HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.

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