Inhibition Of Downregulation Research Articles

Interventions against hearing loss as an integral component of successful tinnitus therapy.

Tinnitus very often develops from acute or chronic hearing loss, mainly inner ear deafness. The frequency of the tinnitus mostly corresponds to the frequency range of the hearing loss and is enhanced by down-regulation of inhibition in the central auditory pathway for these frequencies, in addition to focused attention and enhanced arousal for the disturbing sound. Therefore, interventions to improve hearing such as mid-ear surgery or-more often-electronic devices including hearing aids or cochlear implants (CI) are important for the treatment of tinnitus. In this review, the current German S3guideline "Chronic tinnitus" and recent literature are discussed.

Interventions against hearing loss are an integral component of successful tinnitus therapy. German version

Tinnitus very often develops from acute or chronic hearing loss, mainly inner ear deafness. The frequency of the tinnitus mostly corresponds to the frequency range of the hearing loss and is enhanced by down-regulation of inhibition in the central auditory pathway for these frequencies, in addition to focused attention and enhanced arousal for the disturbing sound. Therefore, interventions to improve hearing such as mid-ear surgery or-more often-electronic devices including hearing aids or cochlear implants (CI) are important for the treatment of tinnitus. In this review, the current German S3guideline "Chronic tinnitus" and recent literature are discussed.

Primary motor cortex function and motor skill acquisition: insights from threshold-hunting TMS.

Modulation of GABA-mediated inhibition in primary motor cortex (M1) is important for the induction of training-induced plasticity. The downregulation of inhibition during acquisition may promote cortical reorganization, whereas an upregulation once performance has plateaued may promote consolidation of the newly acquired skill. GABA-related inhibition in human M1 is routinely assessed using the paired-pulse transcranial magnetic stimulation (TMS) paradigm of short-interval intracortical inhibition (SICI). However, modulation of SICI with motor skill learning is not a consistent finding and may be influenced by TMS parameters. The aim of this study was to compare the modulation of SICI by motor skill learning between conventional and adaptive threshold-hunting techniques with an anterior-posterior and posterior-anterior induced current. Sixteen participants (21-33years) trained with their dominant (right) hand on a sequential visual isometric pinch task. Electromyographic recordings were obtained from the right first dorsal interosseous muscle. Corticomotor excitability and SICI were examined before and immediately after 12 blocks of training. Skill increased throughout the training, with performance plateauing before completion. Corticomotor excitability increased after motor training for both current directions. The amount of SICI was greater with anterior-posterior stimulation than posterior-anterior for both conventional and adaptive threshold-hunting techniques. SICI increased after motor training, but only for adaptive threshold-hunting with an anterior-posterior-induced current. The increased GABA-mediated inhibition evident after motor skill learning may promote consolidation of the newly acquired skill. The findings also support the notion that adaptive threshold-hunting SICI using an anterior-posterior current provides an effective assessment in interventional studies.

Aging and Central Auditory Disinhibition: Is It a Reflection of Homeostatic Downregulation or Metabolic Vulnerability?

Aging-related changes have been identified at virtually every level of the central auditory system. One of the most common findings across these nuclei is a loss of synaptic inhibition with aging, which has been proposed to be at the heart of several aging-related changes in auditory cognition, including diminished speech perception in complex environments and the presence of tinnitus. Some authors have speculated that downregulation of synaptic inhibition is a consequence of peripheral deafferentation and therefore is a homeostatic mechanism to restore excitatory/inhibitory balance. As such, disinhibition would represent a form of maladaptive plasticity. However, clinical data suggest that deafferentation-related disinhibition tends to occur primarily in the aged brain. Therefore, aging-related disinhibition may, in part, be related to the high metabolic demands of inhibitory neurons relative to their excitatory counterparts. These findings suggest that both deafferentation-related maladaptive plastic changes and aging-related metabolic factors combine to produce changes in central auditory function. Here, we explore the arguments that downregulation of inhibition may be due to homeostatic responses to diminished afferent input vs. metabolic vulnerability of inhibitory neurons in the aged brain. Understanding the relative importance of these mechanisms will be critical for the development of treatments for the underlying causes of aging-related central disinhibition.

Abstract 5878: Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway

Abstract Trichostatin A (TSA), a potent histone deacetylase (HDAC) inhibitor, has been reported to elicit anti-proliferative response in various tumors. Here, we investigated antitumor effect of TSA alone or in combination with conventional chemotherapeutic agents on urothelial carcinoma (UC) cells. We use one high-grade UC cell line (T24) and another UC cells (NTUUC) obtained by primary culture from the surgical specimen of a women with high-grade and metastatic bladder UC. The cytotoxicity and apoptosis induced by TSA alone , chemotherapeutic agents (cisplatin, gemcitabine and doxorubicin) and combined treatment were assessed by MTT assay and fluorescence-activated cell sorting and flow cytometry. The expression of phosphor c-Raf, phosphor-MEK1/2, and phosphor-ERK1/2 were measured by Western blot. urther elucidation on the role of Raf/MEK/ERK pathoway on the TSA-enhanced cytoxicity were exmained by using ERK1 siRNA knockdown and the specific MEK inhibitor (PD98059). Our results showed TSA markedly enhances the cytotoxicity and apoptosis of three chemotherapeutic agents in UC cells with concurrent suppression of Raf/MEK/ERK signaling pathway. Consistently, inhibition of Down-regulation of ERK by MEK inhibitor or by ERK 1 siRNA knockdown potentiated the chemotherapeutic agnet-induced cytotoxicity of TSA in UC cells. We concluded that TSA potentiates the therapeutic efficacy of cisplatin, gemcitabine and doxorubicin in human UC cells through Raf/MEK/ERK signaling pathway. These findings provide a new treatment strategy against UC. Note: This abstract was not presented at the meeting. Citation Format: Kuan-Lin Kuo, Chung-Sheng shi, Ju-Tong Hsieh, Shiang-Peng Chen, Wei-Chou Lin, Shih-Ming Liao, Shing-Hwa Liu, Kuo-How Huang. Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5878. doi:10.1158/1538-7445.AM2017-5878

Effects of sevoflurane preconditioning on zonula occludens-1 during myocardial ischemia-reperfusion in rats in vitro

Objective To evaluate the effects of sevoflurane preconditioning on zonula occludens-1 (ZO-1) during myocardial ischemia-reperfusion (I/R) in rats in vitro.Methods Adult male Wistar rats,weighing 250-300 g,were anesthetized with intraperitoneal pentobarbital 30 mg/kg and heparinized.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 37 ℃.Eighteen isolated rat hearts were randomly assigned into 3 groups (n =6 each) using a random number table:control group (group C),group I/R and sevoflurane preconditioning group (group S).At 10 min of equilibration,the hearts were perfused with K-H solution for 110 min in group C,the hearts were perfused with K-H solution for 20 min,and then subjected to 30 min of ischemia followed by 60 min of reperfusion in group I/R,and the hearts were perfused with K-H solution saturated with 3％ sevoflurane for 15 min followed by 5 min washout,and then subjected to 30 min of ischemia followed by 60 min of reperfusion in group S.At the end of equilibration,immediately before ischemia,and at 15 and 60 min of reperfusion (T1,2),HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP),+ dp/dtmax and-dp/dtmax were recorded.The development of arrhythmias was recorded during reperfusion and scored.At 60 min of reperfusion,myocardial specimens were obtained from the apex of heart for determination of the expression of ZO-1 in myocardial tissues (by Western blot) and for observation of distribution of ZO-1 and connexin43 (Cx43) (by immunofluorescence).Results Compared with group C,HR,LVDP,+ dp/dtmax and-dp/dtmax were significantly decreased and LVEDP was increased at 15 and 60 min of reperfusion,scores of arrhythmia was increased,and ZO-1 expression was down-regulated in I/R group.Compared with group I/R,HR,LVDP,+ dp/dtmax and-dp/dtmax were significantly increased and LVEDP was decreased at 15 and 60 min of reperfusion,arrhythmia was decreased,and ZO-1 expression was up-regulated in group S.ZO-1 and Cx43 were co-localized at the intercalated disk.ZO-1 was redistributed in the lateralization of the membrane and co-localized with Cx43 in group I/R.The incidence of ZO-1 lateralization was significantly decreased in group S.Conclusion The mechanism by which sevoflurane preconditioning decreases reperfusion arrhythmia is related to inhibition of down-regulation of expression and redistribution of ZO-1 and inhibition of redistribution of Cx43 in rats. Key words: Anesthetics, inhalation; Ischemic preconditioning; Myocardial reperfusion injury ; Tight junctions

Immunoregulatory T cells, LFA-3 and HLA-DR in autoimmune thyroid diseases.

Aim:The aim of the present study was to examine the changes in the expression of T-cell activation markers, namely CD4+ CD25+ and CD8+ in patients with AITD, namely Graves’ disease and Hashimoto's thyroiditis as well as colloid nodular goitre. HLA-DR, LFA-3, and peripheral total lymphocytic count are also measured.Materials and Methods:We compared the expression of CD4, CD25, and CD8 surface markers in peripheral blood lymphocyte in Graves’ disease and Hashimoto's thyroiditis as autoimmune thyroid diseases, as well as colloid goitre in comparison with healthy controls. Also, LFA-3 and HLA-DR were measured in the same groups using three-color flow cytometry. Total lymphocytic count in peripheral blood, thyroid function tests, antithyroid antibodies were also included in the laboratory investigations. The total number of participants was 65. All were recruited from endocrine clinics in a tertiary care hospital in the southern region of Saudi Arabia. All participants underwent history taking, clinical examination, laboratory workup, and radiological investigations. Neck ultrasound, technecium pertechnetateψψ thyroid uptake, and fine-needle aspiration and cytology (FNAC) of the thyroid were done when indicated. The study was approved by the Hospital Research Isthics Committee and informed consents were obtained from all participants before enrollment in the study.Results:In comparison with thecontrol group, activation markers CD4, CD25, and CD8 were lower in the autoimmune thyroid diseases. Lymphocyte function antigen-3 (CD58) and total lymphocytic count were higher in the AIT diseases whereas HLA-DR was lower than that in the control group. The CD4/CD8 ratio was lower in the AITD compared with the healthy euthyroid subjects. No difference was found between patients with colloid nodular goitre and the healthy control in any of the study variables except for LFA-3 which was significantly higher in the colloid goitre group.Conclusion:Our findings indicate downregulation of CD4+ CD25+ Treg as well as CD8+ T cells in autoimmune thyroid diseases. Downregulation of suppressor T lymphocytes helps initiation, progression, and maintenance of the autoimmune thyroid diseases. Lower HLA-DR and higher CD58 in AITDs indicate their role in the expression of the autoantigen and its escape from the immune surveillance. High levels of LFA-3 in colloid goitre indicate that the autoimmune process needs interacting factors, and not only the high level of LFA-3.

Down-regulation of suppressor of cytokine signaling 3 expression in hypothalamus attenuates high-fat dietinduced obesity in rats

Objective To investigate whether hypothalamic silencing of suppressor of cytokine signaling 3( SOCS3 ) by RNA interference ( RNAi ) attenuates diet-induced obesity and leptin resistance in rats.Methods Hypothalamic SOCS3-deficient rats were established by means of lentiviral vector ( LV ) mediated RNAi technique,and then were fed with a high-fat diet for 8 weeks.After the rats were sacrificed,the serum leptin and insulin concentrations were measured by RIA, and the expression of SOCS3 in hypothalamus was detected by immunohistochemistry and realtime PCR.Results The immunostaining showed that SOCS3 protein expression was significantly reduced and the expression of SOCS3 mRNA was decreased by 49％ in SOCS3 RNAi group compared to the controls( P＜0.01 ).The rats with hypothalamic SOCS3 knockdown exhibited a significant hampering in gaining body weight with lowered concentrations of leptin[ ( 8.18±2.10 vs 10.85±2.23 ) ng/ml ],insulin[ ( 18.89±4.88 vs 26.78±6.01 )mU/L],glucose [ (4.89±0.91 vs 6.26± 1.41 )mmol/L] and triglyceride [ (0.47 ±0.10 vs 0.62 ±0.16)mmol/L] when exposed to a high-fat diet(P＜0.05 or P＜0.01 ).Conclusion The results provide evidence that rats with hypothalamic SOCS3 silencing by RNAi are resistant to diet-induced obesity,leptin resistance,and metabolic disorder. Key words: Lentiviral ; Suppressor of cytokine signaling 3 ; RNAi ; Hypothalamus ; Obesity

Role of PAI-1 and TAFI in the mediation of fibrinolytic deficit in cancer patients

9727 Background: Cancer associated thrombosis is known to have a multi-factorial pathogenesis involving up regulation of coagulation factors, down regulation of inhibitors and endothelial dysfunction. More recently, cancer patients have been shown to exhibit fibrinolytic deficit with impaired clot lysis. This has been mostly attributed to an increase in the circulating levels of plasminogen activator inhibitor 1 (PAI-1). Thrombin activatable fibrinolytic inhibitor (TAFI) has recently been identified as an additional mediator of fibrinolytic deficit that transforms fibrin to a plamsin resistant variant. Methods: To decipher the role of TAFI in this process, plasma samples obtained from 170 cancer patients with symptomatic thrombosis were analyzed for PAI-1, TAFI functional (F) and TAFI immunologic (I). An ELISA method was used for PAI-1 (Stago, Stamford, CT) and TAFI-I (American Diagnostic, Greenwich, CT), while a functional method was used for TAFI-F (Pentapharm, Ltd., Basel Switerland) Results: Baseline samples exhibited a marked increase in PAI-1 levels (140 ± 21) compared to normal (94 ± 14). The TAFI-I levels ranged from 56 –183 %NHP (123 ± 28 %NHP) which was higher than normal (85.1 ± 14 %NHP; n= 82). Interestingly, TAFI measured by functional levels were disproportionately lower (85 ± 26 %NHP) suggesting the consumption of this protease in cancer patients. In contrast to the TAFI I/F ratio approximately 1 for normal individuals, this ratio for cancer patients was 1.6 ± 0.1 (5.6 –0.7). When the PAI-1 levels were compared with F and I TAFI levels, no correlation was evident. This suggests independent regulation of these two proteins in cancer. The increased PAI-1 and TAFI-I levels were independent of the fibrinogen levels. Conclusions: The results clearly suggest that thrombotic complications in cancer patients may be partly due to the fibrinolytic deficit, which is modulated by an up regulation of both PAI-1 and TAFI. Furthermore, the two regulators of fibrinolysis mediate their effects independently. Since the TAFI-I levels are disproportionately high, this enzyme may complex with endogenous proteins modulating its functionality. No significant financial relationships to disclose.

Down-regulation of inhibition following unilateral deafening

Physiological and neurochemical experiments described here suggest that unilateral deafening causes a reduction in inhibition in the adult gerbil inferior colliculus (IC) contralateral to the deafened ear. Multiple-unit recordings were made from single electrode penetrations in the IC prior to and directly after contralateral cochlear ablation. These recordings showed up to 60% increases in the proportion of sampled loci at which neural activity excited by ipsilateral stimulation was observed after the ablation. Novel excitatory responses were evident within minutes of the ablation. Western blotting for glutamic acid decarboxylase protein levels showed significant decreases in the IC contralateral to cochlear ablation, relative to those in the ipsilateral IC, at 24 h and 7 days survival after the ablation. Four hour or 1 year survival post-ablation did not produce significant contralateral/ipsilateral differences in relation to the control group. Taken together, these results suggest the presence of at least two, short-term mechanisms involved in the central response to cochlear removal, both of which appear to implicate a decreased inhibitory influence. One is a very rapid, stimulus-related, functional unmasking. The other is a more delayed reduction in the capacity of γ-aminobutyric acid synthesis in the IC.

δ-Protein Kinase C Phosphorylation Parallels Inhibition of Nerve Growth Factor– Induced Differentiation Independent of Changes in Trk A and MAP Kinase Signalling in PC12 Cells

We investigated the ability of bryostatin 1 to block nerve growth factor (NGF)–induced differentiation of pheochromocytoma PC12 cells and to effect expression of protein kinase C (PKC) isoforms. Compared with phorbol myristate acetate (PMA), a likewise potent activator of PKC, high doses of bryostatin (> 200 nM) failed to down-regulate δ-PKC, as with ζ-PKC, whereas, α-PKC was completely down-regulated. Two forms of δ-PKC were expressed in PC12 cells, a phosphorylated 78.000 M r species and a de-phosphorylated 76.000 M r form. High-dose bryostatin treatment resulted in a 4.5-fold increase in phosphorylated δ-PKC and a 2.5-fold increase in phosphotyrosine. Inhibition of tyrosine kinase activity, with either herbimycin or genistein, prior to addition of bryostatin abrogated protection from down-regulation and led to simultaneous increases in ubiquitinated 110.000 M r -δ-PKC. Similarly, pre-treatment of cells with N-acetyl- l-leucinyl- l-leucinyl- l-norleucinal, an inhibitor of the proteasome pathway, prior to low-dose treatment with bryostatin resulted in a dose-dependent accumulation of δ-PKC and inhibition of down-regulation. Protection of δ-PKC from down-regulation by high-dose bryostatin requires a counter-balance between protein tyrosine kinase and phosphatase systems. High doses of bryostatin blocked NGF-induced neurite outgrowth without altering Y-490 TrK A phosphorylation or an alteration in pp44/42 mitogen-activated protein kinase. Our findings suggest that the phosphorylation state of δ-PKC may regulate its ability to participate in signal coupling and modulation of cell growth and differentiation pathways. Moreover, these data reveal the existence of a signalling pathway independent of MAP kinase that affects NGF differentiation in a negative fashion.

Inhibition of down regulation by chloroquine in cultured lymphocytes (RPMI-1788 line)

We studied the effect of chronic exposure to insulin on insulin receptors of cultured lymphocytes (RPMI-1788 line). The cells treated with insulin (2 micrograms/ml) at 37 degrees C for 12 h, showed a 36.5% decrease in the number of binding sites. Solubilized extract from the cells treated with insulin showed a 35.9% decrease of binding capacity, suggesting that insulin exposure induced the loss of total (cell surface and intracellular) receptors. Insulin-induced loss of receptors was blocked by chloroquine, suggesting that receptor loss is mediated by a chloroquine sensitive pathway. Bacitracin, which inhibited the insulin degradation, had no effect on insulin-induced receptor loss in this cell line. We also found that vitamin K5, one of the insulin mimickers, induced a 31.5% loss of insulin receptors. Therefore, the post-receptor process appeared to mediate down regulation. In cultured lymphocytes, insulin exposure caused a significant loss of total receptors, suggesting that insulin-induced receptor loss may be due to receptor degradation. Insulin-induced receptor loss is mediated by a chloroquine sensitive pathway, and is related to the post-binding process stimulated by vitamin K5.