scholarly journals Delayed Puberty in Spontaneously Hypertensive Rats Involves a Primary Ovarian Failure Independent of the Hypothalamic KiSS-1/GPR54/GnRH System

Endocrinology ◽  
2009 ◽  
Vol 150 (6) ◽  
pp. 2889-2897 ◽  
Author(s):  
L. Pinilla ◽  
J. M. Castellano ◽  
M. Romero ◽  
M. Tena-Sempere ◽  
F. Gaytán ◽  
...  
Keyword(s):  
Ovarian Function ◽  
Follicular Development ◽  
Experimental Models ◽  
Gnrh Receptor ◽  
Delayed Puberty ◽  
Mrna Levels ◽  
Spontaneously Hypertensive ◽  
And Function

Spontaneously hypertensive (SH) rats, extensively used as experimental models of essential human hypertension, display important alterations in the neuroendocrine reproductive axis, which manifest as markedly delayed puberty onset in females but whose basis remains largely unknown. We analyze herein in female SH rats: 1) possible alterations in the expression and function of KiSS-1/GPR54 and GnRH/GnRH-receptor systems, 2) the integrity of feedback mechanisms governing the hypothalamic-pituitary-ovarian axis, and 3) the control of ovarian function by gonadotropins. Our data demonstrate that, despite overtly delayed puberty, no significant decrease in hypothalamic KiSS-1, GPR54, or GnRH mRNA levels was detected in this strain. Likewise, in vivo gonadotropin responses to ovariectomy and systemic kisspeptin-10 or GnRH administration, as well as in vitro gonadotropin responses to GnRH, were fully preserved in SH rats. Moreover, circulating LH levels were grossly conserved during prepubertal maturation, whereas FSH levels were even enhanced from d 20 postpartum onwards. In striking contrast, ovarian weight and hormone (progesterone and testosterone) responses to human chorionic gonadotropin (CG) in vitro were profoundly decreased in SH rats, with impaired follicular development and delayed ovulation at puberty. Such reduced hormonal responses to human CG could not be attributed to changes in LH/CG or FSH-receptor mRNA expression but might be linked to blunted P450scc, 3β-hydroxy steroid dehydrogenase, and aromatase mRNA levels in ovaries from SH rats. In conclusion, our results indicate that the expression and function of KiSS-1/GPR54 and GnRH/GnRH-receptor systems is normal in SH rats, whereas ovarian development, steroidogenesis, and responsiveness to gonadotropins are strongly compromised.

scholarly journals Astrocytic Connexin43 Channels as Candidate Targets in Epilepsy Treatment

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1578 ◽  
Author(s):  
Laura Walrave ◽  
Mathieu Vinken ◽  
Luc Leybaert ◽  
Ilse Smolders
Keyword(s):  
Transmembrane Protein ◽  
Epileptiform Activity ◽  
Brain Slices ◽  
High Energy ◽  
Experimental Models ◽  
Functional Coupling ◽  
Seizure Outcome ◽  
Mrna Levels

In epilepsy research, emphasis is put on exploring non-neuronal targets such as astrocytic proteins, since many patients remain pharmacoresistant to current treatments, which almost all target neuronal mechanisms. This paper reviews available data on astrocytic connexin43 (Cx43) signaling in seizures and epilepsy. Cx43 is a widely expressed transmembrane protein and the constituent of gap junctions (GJs) and hemichannels (HCs), allowing intercellular and extracellular communication, respectively. A plethora of research papers show altered Cx43 mRNA levels, protein expression, phosphorylation state, distribution and/or functional coupling in human epileptic tissue and experimental models. Human Cx43 mutations are linked to seizures as well, as 30% of patients with oculodentodigital dysplasia (ODDD), a rare genetic condition caused by mutations in the GJA1 gene coding for Cx43 protein, exhibit neurological symptoms including seizures. Cx30/Cx43 double knock-out mice show increased susceptibility to evoked epileptiform events in brain slices due to impaired GJ-mediated redistribution of K+ and glutamate and display a higher frequency of spontaneous generalized chronic seizures in an epilepsy model. Contradictory, Cx30/Cx43 GJs can traffic nutrients to high-energy demanding neurons and initiate astrocytic Ca2+ waves and hyper synchronization, thereby supporting proconvulsant effects. The general connexin channel blocker carbenoxolone and blockers from the fenamate family diminish epileptiform activity in vitro and improve seizure outcome in vivo. In addition, interventions with more selective peptide inhibitors of HCs display anticonvulsant actions. To conclude, further studies aiming to disentangle distinct roles of HCs and GJs are necessary and tools specifically targeting Cx43 HCs may facilitate the search for novel epilepsy treatments.

Silencing of sodium/hydrogen exchanger in the heart by direct injection of naked siRNA

2011 ◽  
Vol 111 (2) ◽  
pp. 566-572 ◽  
Author(s):  
Patricio E. Morgan ◽  
María V. Correa ◽  
Irene L. Ennis ◽  
Ariel A. Diez ◽  
Néstor G. Pérez ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Direct Injection ◽  
Experimental Models ◽  
Left Ventricular ◽  
Maximal Velocity ◽  
Mrna Levels ◽  
Small Interference Rna

Cardiac Na+/H+ exchanger (NHE1) hyperactivity is a central factor in cardiac remodeling following hypertension, myocardial infarction, ischemia-reperfusion injury, and heart failure. Treatment of these pathologies by inhibiting NHE1 is challenging because specific drugs that have been beneficial in experimental models were associated with undesired side effects in clinical practice. In the present work, small interference RNA (siRNA) produced in vitro to specifically silence NHE1 (siRNANHE1) was injected once in vivo into the apex of the left ventricular wall of mouse myocardium. After 48 h, left ventricular NHE1 protein expression was reduced in siRNANHE1-injected mice compared with scrambled siRNA by 33.2 ± 3.4% ( n = 5; P < 0.05). Similarly, NHE1 mRNA levels were reduced by 20 ± 2.0% ( n = 4). At 72 h, siRNANHE1 spreading was evident from the decrease in NHE1 expression in three portions of the myocardium (apex, medium, base). NHE1 function was assessed based on maximal velocity of intracellular pH (pHi) recovery (dpHi/d t) after an ammonium prepulse-induced acidic load. Maximal dpHi/d t was reduced to 14% in siRNANHE1-isolated left ventricular papillary muscles compared with scrambled siRNA. In conclusion, only one injection of naked siRNANHE1 successfully reduced NHE1 expression and activity in the left ventricle. As has been previously suggested, extensive NHE1 expression reduction may indicate myocardial spread of siRNA molecules from the injection site through gap junctions, providing a valid technique not only for further research into NHE1 function, but also for consideration as a potential therapeutic strategy.

Erythropoietin as a Novel Pro-Inflammatory Mediator of Macrophages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3596-3596
Author(s):  
Lilach Lifshitz ◽  
Galit Tabak ◽  
Max Gassman ◽  
Moshe Mittelman ◽  
Drorit Neumann
Keyword(s):  
Mhc Class Ii ◽  
Phagocytic Activity ◽  
Surface Expression ◽  
Class Ii ◽  
Experimental Models ◽  
Splenic Macrophage ◽  
And Function ◽  
Inflammatory Macrophages

Abstract Abstract 3596 Poster Board III-533 The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system were originally described by our group in multiple myeloma patients and have been further elucidated in murine experimental models (Mittelman, 2001; Katz 2005; 2007; Prutchi-Sagiv, 2006). However, the mechanisms of action by which EPO affects lymphocyte number and function are still unknown, particularly since lymphocytes do not carry EPO receptors (EPO-R). We thus set to unravel mechanisms underlying the anti-neoplastic immunomodulatory action of EPO. These studies led us to the novel discovery that dendritic cells (DCs) express EPO-R, and that EPO enhances their survival and function (Prutchi-Sagiv, 2008; Lifshitz, 2009). Here we focus on macrophages as an additional EPO target, since in analogy to DCs, macrophages are also antigen presenting cells, and serve as key effectors of the innate immune response. Using murine models, we first explored the in-vivo effects of EPO using recombinant human EPO (rHuEPO, EPREXR, JC)-injected mice, as well as transgenic mice over-expressing human EPO (termed tg6). EPO treatment was associated with an increased splenic macrophage population, detected by F4/80 expression, and an increased number of macrophages expressing CD11b, CD80 and MHC class II. We further explored the effect of in-vivo EPO administration in an inflammatory model exploiting thioglygollate injection to induce recruitment of peritoneal inflammatory macrophages. The inflammatory macrophages obtained from both EPO injected and from tg6 mice displayed increased expression of F4/80, CD11b, CD80 and MHC class II and augmented phagocytic activity, as compared to the control counterparts. These results are supported by in-vitro studies in bone marrow derived macrophages (BMDMs). We show that BMDMs express EPO-R mRNA, as detected by RT-PCR. In-vitro stimulation of the BMDMs with rHuEPO activated multiple signaling pathways including STAT1, STAT5, MAPK, AKT and NFkB indicating macrophage activation via surface EPO-R. EPO treatment of the BMDMs up-regulated their surface expression of CD11b, F4/80 and CD80, as well as enhanced their phagocytic activity. EPO treatment of LPS-stimulated BMDMs augmented IL-12 secretion, and decreased IL-10 secretion. In conclusion our results show that macrophages are direct targets of EPO and that EPO treatment enhances their pro-inflammatory activity and function. These findings point to the multifunctional role of EPO and may advance its clinical applications as an anti-neoplastic immunomodulator. Disclosures: Mittelman: BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties. Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation).

scholarly journals Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Veedamali S. Subramanian ◽  
Trevor Teafatiller ◽  
Anshu Agrawal ◽  
Masashi Kitazawa ◽  
Jonathan S. Marchant
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Inhibitory Effect ◽  
Acid Uptake ◽  
Mrna Levels ◽  
The Central Nervous System ◽  
Factor Α ◽  
And Function

Vitamin C (ascorbic acid: AA) uptake in neurons occurs via the sodium-dependent vitamin C transporter-2 (SVCT2), which is highly expressed in the central nervous system (CNS). During chronic neuroinflammation or infection, CNS levels of lipopolysaccharide (LPS) and LPS-induced tumor necrosis factor-α (TNFα) are increased. Elevated levels of LPS and TNFα have been associated with neurodegenerative diseases together with reduced levels of AA. However, little is known about the impacts of LPS and TNFα on neuronal AA uptake. The objective of this study was to examine the effect of LPS and TNFα on SVCT2 expression and function using in vitro and in vivo approaches. Treatment of SH-SY5Y cells with either LPS or TNFα inhibited AA uptake. This reduced uptake was associated with a significant decrease in SVCT2 protein and mRNA levels. In vivo exposure to LPS or TNFα also decreased SVCT2 protein and mRNA levels in mouse brains. Both LPS and TNFα decreased SLC23A2 promoter activity. Further, the inhibitory effect of LPS on a minimal SLC23A2 promoter was attenuated when either the binding site for the transcription factor Sp1 was mutated or cells were treated with the NF-κB inhibitor, celastrol. We conclude that inflammatory signals suppress AA uptake by impairing SLC23A2 transcription through opposing regulation of Sp1 and NF-κB factors.

scholarly journals Hyperglycemia downregulates Connexin36 in pancreatic islets via the upregulation of ICER-1/ICER-1γ

2013 ◽  
Vol 51 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Jacques-Antoine Haefliger ◽  
Françoise Rohner-Jeanrenaud ◽  
Dorothée Caille ◽  
Anne Charollais ◽  
Paolo Meda ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Chronic Exposure ◽  
Prolonged Exposure ◽  
Mrna Levels ◽  
Adult Rats ◽  
Junction Protein ◽  
And Function

Channels formed by the gap junction protein Connexin36 (CX36) contribute to the proper control of insulin secretion. We previously demonstrated that chronic exposure to glucose decreases Cx36 levels in insulin-secreting cells in vitro. Here, we investigated whether hyperglycemia also regulates Cx36 in vivo. Using a model of continuous glucose infusion in adult rats, we showed that prolonged (24–48 h) hyperglycemia reduced the Cx36 gene Gjd2 mRNA levels in pancreatic islets. Accordingly, prolonged exposure to high glucose concentrations also reduced the expression and function of Cx36 in the rat insulin-producing INS-1E cell line. The glucose effect was blocked after inhibition of the cAMP/PKA pathway and was associated with an overexpression of the inducible cAMP early repressor ICER-1/ICER-1γ, which binds to a functional cAMP-response element in the promoter of the Cx36 gene Gjd2. The involvement of this repressor was further demonstrated using an antisense strategy of ICER-1 inhibition, which prevented glucose-induced downregulation of Cx36. The data indicate that chronic exposure to glucose alters the in vivo expression of Cx36 by the insulin-producing β-cells through ICER-1/ICER-1γ overexpression. This mechanism may contribute to the reduced glucose sensitivity and altered insulin secretion, which contribute to the pathophysiology of diabetes.

scholarly journals Resistin Regulates Pituitary Lipid Metabolism and InflammationIn VivoandIn Vitro

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
F. Rodriguez-Pacheco ◽  
M. G. Novelle ◽  
M. J. Vazquez ◽  
E. Garcia-Escobar ◽  
F. Soriguer ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Pituitary Gland ◽  
Primary Source ◽  
Chronic Administration ◽  
Metabolic Enzymes ◽  
Experimental Models ◽  
Regulatory Role ◽  
Mrna Levels

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function and also regulates growth hormone (GH) secretion in rat adenopituitary cells cultures with the adipokine. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues, including the pituitary. The aim of this study is to investigate the possible action of resistin on the lipid metabolism in the pituitary glandin vivo(rats in two different nutritional status, fed and fast, treated with resistin on acute and a chronic way) andin vitro(adenopituitary cell cultures treated with the adipokine). Here, by a combination ofin vivoandin vitroexperimental models, we demonstrated that central acute and chronic administration of resistin enhance mRNA levels of the lipid metabolic enzymes which participated on lipolysis and moreover inhibiting mRNA levels of the lipid metabolic enzymes involved in lipogenesis. Taken together, our results demonstrate for the first time that resistin has a regulatory role on lipid metabolism in the pituitary gland providing a novel insight in relation to the mechanism by which this adipokine can participate in the integrated control of lipid metabolism.

scholarly journals Spheres of Influence: Insights into Salmonella Pathogenesis from Intestinal Organoids

2020 ◽  
Vol 8 (4) ◽  
pp. 504 ◽  
Author(s):  
Smriti Verma ◽  
Stefania Senger ◽  
Bobby J. Cherayil ◽  
Christina S. Faherty
Keyword(s):  
Animal Studies ◽  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Primary Cells ◽  
Spheres Of Influence ◽  
Transformed Cell Lines ◽  
And Function

The molecular complexity of host-pathogen interactions remains poorly understood in many infectious diseases, particularly in humans due to the limited availability of reliable and specific experimental models. To bridge the gap between classical two-dimensional culture systems, which often involve transformed cell lines that may not have all the physiologic properties of primary cells, and in vivo animal studies, researchers have developed the organoid model system. Organoids are complex three-dimensional structures that are generated in vitro from primary cells and can recapitulate key in vivo properties of an organ such as structural organization, multicellularity, and function. In this review, we discuss how organoids have been deployed in exploring Salmonella infection in mice and humans. In addition, we summarize the recent advancements that hold promise to elevate our understanding of the interactions and crosstalk between multiple cell types and the microbiota with Salmonella. These models have the potential for improving clinical outcomes and future prophylactic and therapeutic intervention strategies.

scholarly journals Experimental models to study intestinal microbes–mucus interactions in health and disease

2019 ◽  
Vol 43 (5) ◽  
pp. 457-489 ◽  
Author(s):  
Lucie Etienne-Mesmin ◽  
Benoit Chassaing ◽  
Mickaël Desvaux ◽  
Kim De Paepe ◽  
Raphaële Gresse ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Intestinal Bacteria ◽  
Experimental Models ◽  
In Vivo Models ◽  
Intestinal Microbes ◽  
Intestinal Mucus ◽  
Health And Disease ◽  
And Function

ABSTRACTA close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research.

Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of ΔF508-CFTR

2000 ◽  
Vol 278 (2) ◽  
pp. C259-C267 ◽  
Author(s):  
Ronald C. Rubenstein ◽  
Pamela L. Zeitlin
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Ubiquitin Proteasome System ◽  
Common Mutation ◽  
Mrna Levels ◽  
Δf508 Cftr ◽  
Protein Immunoreactivity ◽  
And Function

The most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), ΔF508, is a trafficking mutant that has prolonged associations with molecular chaperones and is rapidly degraded, at least in part by the ubiquitin-proteasome system. Sodium 4-phenylbutyrate (4PBA) improves ΔF508-CFTR trafficking and function in vitro in cystic fibrosis epithelial cells and in vivo. To further understand the mechanism of action of 4PBA, we tested the hypothesis that 4PBA modulates the targeting of ΔF508-CFTR for ubiquitination and degradation by reducing the expression of Hsc70 in cystic fibrosis epithelial cells. IB3-1 cells (genotype ΔF508/W1282X) that were treated with 0.05–5 mM 4PBA for 2 days in culture demonstrated a dose-dependent reduction in Hsc70 protein immunoreactivity and mRNA levels. Immunoprecipitation with Hsc70-specific antiserum demonstrated that Hsc70 and CFTR associated under control conditions and that treatment with 4PBA reduced these complexes. Levels of immunoreactive Hsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBA treatment. These data suggest that 4PBA may improve ΔF508-CFTR trafficking by allowing a greater proportion of mutant CFTR to escape association with Hsc70.

scholarly journals Advanced in vitro Research Models to Study the Role of Endothelial Cells in Solid Organ Transplantation

2021 ◽  
Vol 12 ◽  
Author(s):  
Daphne M. Peelen ◽  
Martin J. Hoogduijn ◽  
Dennis A. Hesselink ◽  
Carla C. Baan
Keyword(s):  
Endothelial Cells ◽  
Allograft Rejection ◽  
Solid Organ Transplantation ◽  
Experimental Models ◽  
Solid Organ ◽  
And Function ◽  
Antigen Presenting

The endothelium plays a key role in acute and chronic rejection of solid organ transplants. During both processes the endothelium is damaged often with major consequences for organ function. Also, endothelial cells (EC) have antigen-presenting properties and can in this manner initiate and enhance alloreactive immune responses. For decades, knowledge about these roles of EC have been obtained by studying both in vitro and in vivo models. These experimental models poorly imitate the immune response in patients and might explain why the discovery and development of agents that control EC responses is hampered. In recent years, various innovative human 3D in vitro models mimicking in vivo organ structure and function have been developed. These models will extend the knowledge about the diverse roles of EC in allograft rejection and will hopefully lead to discoveries of new targets that are involved in the interactions between the donor organ EC and the recipient's immune system. Moreover, these models can be used to gain a better insight in the mode of action of the currently prescribed immunosuppression and will enhance the development of novel therapeutics aiming to reduce allograft rejection and prolong graft survival.

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