bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of CD38 gene on TLR4 and inflammatory cytokines and its mechanisms (835.10)
Authors: Li Rong , Chen Cheng , Li Ling , Li Li , Kong Yuan , Yang Xiao , Liao Jie
Published: June 2021

Protective mechanisms of the host will be initiated by the activation of Toll‐like receptor (TLR), including the chemotaxis and activation of immune cells. TLR4 defection always accompanied with inflammation. CD38 gene is associated with myocardial infarction. However, impact of CD38 gene on TLR4 is not well defined. Objective To study the influences of CD38 gene to the distribution of TLR4 and it's effects and mechanisms on expression of TLR4 and inflamatory cytokines. Methods Distribution of TLR4 in organs (such as heart, liver spleen, lung, kidney, cerebrum, thymus and lymph node) of C57/B6 mice and CD38‐/‐ C57/B6 mice were elucidated by polymerase chain reaction (PCR), Different expression of TLR4 in spleen of C57/B6 mice and CD38‐/‐ C57/B6 mice was analyzed by realtime PCR and westernblot (WB). Expression of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α), monocyte chemotactic protein 1 (MCP‐1), Interleukin‐1 (IL‐6) and Interleukin‐1β (IL‐1β) in spleen were analyzed by realtime PCR individually. Different expression of NF‐ĸB in spleen was analyzed by WB. Results TLR4 distribute broadly in all of the above organs in both C57/B6 mice and CD38‐/‐ C57/B6 mice. But comparing to C57/B6 mice, CD38‐/‐ C57/B6 mice showed an significantly decrease in the expression of TLR4 on both mRNA and protein level in spleen. Expression of inflammatory cytokines such as TNF‐α, MCP‐1 and IL‐6 were found to be significantly decreased accompanied by significantly increased expression of IL‐1β in spleen. Meanwhile, the expression of NF‐ĸB in spleen was greatly enhanced. Conclusions Overall, these data suggest that CD38 has a profound influence on TLR4, which may contribute to the dysregulated expression of inflammatory factors under physiological and such pathological conditions as myocardial infarction through NF‐ĸB pathway.

Grant Funding Source: Supported by GJJ81302600 and 30960357
Increase of splenic lymphocyte apoptosis in septic encephalopathy (835.12)
Authors: Jeremias Isabela , Victorino Vanessa , Lima Thais , Soriano Francisco
Published: June 2021

Sepsis is a systemic response to infection that can affect any organ system, including the central and peripheral nervous systems. Authors reported that changes in the nervous system are implicated in the induction or amplification of immune cell changes. We investigated the changes in brain function and late immune response in an animal model of sepsis by cecal ligation and puncture (CLP). Balb/c mice were divided into two groups: Sham operation or CLP. We evaluated brain changes by SHIRPA behavioral test, after 6h of CLP. 15 days after surgery, mice were sacrificed, the spleen was isolated and lymphocytic apoptosis was evaluated by flow cytometry. We observed that the neuropsychiatric state in SHIRPA test was significantly decreased in animals with CLP. Regarding apoptosis of lymphocytes, there was also a significant difference, in which the CLP apoptotic lymphocytes were greater than in the sham group. Correlating (r=‐0.96) the two factors we observed that the higher the neurological damage greater the number of apoptotic lymphocytes. Thus, we conclude from these preliminary results that neurological status observed 6 hours after CLP may determine delayed immune response impairment.Support: FAPESP 2012/07898‐0

Grant Funding Source: Support by: FAPESP 2012/07898‐0
Blocking the receptor for advanced glycation end‐products but not Toll‐like receptor 4 attenuates the progression of OA (835.13)
Authors: Wilhelm Spencer , Mecham David , Chavez Matias Elizabeth , Kartchner Jeff , Crepeau Philip , Andersen Karl , Vandruff James , Robinson MarLeice , Christensen Ian , Taylor Kara , Reynolds Paul , Kooyman David
Published: June 2021

Receptors for Advanced Glycation End Products (RAGE) and Toll Like Receptor 4 (TLR‐4) have been shown to play a role in the development of Osteoarthritis (OA). We have previously shown that knocking out RAGE in mice slows the disease progression in articular cartilage of the knee. The objective of this study was to determine if application of the compound TAK‐242, a TLR‐4 specific inhibitor, in conjunction with knocking out RAGE could further attenuate the disease. Destabilization of the medial meniscus of RAGE KO and Wild Type (WT mice) was performed, and severity of OA was qualitatively analyzed through two standardized scoring systems (Mankin and OARSI). We also performed immunohistochemistry to analyze levels of HtrA1 and TGF‐β1, known biomarkers for the disease. Surprisingly, addition of the TLR blocker disrupted the protection afforded by knocking out RAGE, and its application to WT mice had no protective effect. We conclude that while blockage of the RAGE pathway alone is beneficial to the attenuation of OA, hampering the TLR‐4 pathway offers no protective benefits. We hypothesize that blocking TLR‐4 receptor mitigates the beneficial effects of knocking out RAGE on OA.
Differential regulation of chemokine (C‐X‐C Motif) ligand 12 by lipopolysacchrides in differentiated and un‐differentiated human THP‐1 cells (835.4)
Authors: Yu Lu , Yu Liangli , Wang Thomas
Published: June 2021

Chemokine (C‐X‐C Motif) ligand (CXCL) 12 is a pleiotropic chemokine that act through its receptor CXCR4/7. CXCL12 plays crucial roles in the regulation of autoimmune, inflammation, angiogenesis and cancer development. However, tissues expression, physiological role and regulation of CXCL12 and CXCR4/7 remain largely unclear. Using RT‐PCR, we found in both phorbol 12‐myristate 13‐acetate‐differentiated (d‐) and undifferentiated (u‐) human THP‐1 cells CXCL12 mRNA levels were similar. However, CXCL12 mRNA levels were induced by Toll like receptor (TLR) 4 activator lipopolysaccharides (LPS, 10 ng/mL, 2 h) 14‐fold in d‐THP‐1 but only ~3‐fold in u‐THP‐1. Treatments with TLR2 ligand Pam3CSK also induced CXCL12 mRNA but only by ~2‐fold. Inhibition of the binding of LPS to TLR4 by anti‐CD14 antibody resulted in a significant inhibition of LPS induction of CXCL12 mRNA level. Increase in CXCL12 protein secretion by d‐THP‐1 was confirmed using ELISA. Condition‐media from LPS‐treated macrophages led to increased migration of u‐THP‐1 by 2.57‐fold as compared to control media. In conclusion, our study indicated that CXCL12 is differentially expressed and regulated in monocyte and macrophage by LPS. In human d‐THP‐1 cells, TLR4 and TLR2 pathway appeared to be the regulatory pathways. Secretion of CXCL12 by macrophages could lead to increased attraction of additional monocytes.
Novel role of cathepsin V in TLRs‐MyD88 mediated signaling revealed in HMGB1‐TLR4 inhibitor screening (835.9)
Authors: Xu Hongbo , Montenegro Marcelo , Pribis John , Billiar Timothy
Published: June 2021

Toll‐like receptors (TLRs) recognize pathogen‐derived molecular patterns (PAMP) and danger‐associated molecular patterns (DAMP), and induce NF‐κB‐dependent pro‐inflammatory cytokines release such as TNF‐α and IL‐6. High Mobility Group Box 1 (HMGB1) is a prototypical DAMP that drives inflammation in many sterile and infectious diseases signaling through TLR4. A cell‐based high‐throughput screening identified a FDA‐approved HIV protease inhibitor Saquinavir (SQV) as a potent inhibitor of HMGB1‐TLR4 mediated signaling. We have previously identified human cathepsin V (CTSV) as a mammalian target of SQV and confirmed CTSV involves in HMGB1‐TLR4 signaling using genetic knockdown and pharmacological inhibition. In this study, we expanded these previous evidence for TLR4 and tested the effects of SQV and SID266 (a known CTSV inhibitor) on TNF‐α production induced by TLR 1/2, 4 and 9, using the TLR ligands Pam3CSK4, LPS and ODN2006, respectively. Similar to HMGB1, SQV and SID266 diminish TNF‐α production in a concentration‐dependent manner. SQV also acts upstream in the TLR‐NF‐κB pathway by attenuating phosphorylation of IRAK4 and IKKα/β. The activities of SQV are shared by SID266. Hinted from the mechanistic properties of SQV and SID266, we found that CTSV physically interacts with MyD88 in PMA‐differentiated THP‐1 cells stimulated with HMGB1, LPS or Pam3CSK4, which could be blocked by SQV administration. Our study uncovers a previously unknown mechanism of cathepsin V in TLRs‐MyD88 mediated signaling and suggests a potential novel therapeutic target in related inflammatory diseases, and paves the way for re‐purposing a drug already safely in humans.

Grant Funding Source: Department of Surgery, University of Pittsburgh
Antibodyome development post‐immunization with hepatatis B virus vaccine (836.1)
Authors: Zarhoud Josef
Published: June 2021

Medical students at Sahlgrenska Academy receive Hepatatis B surface antigen (HBsAg) vaccination prior to their studies in infectious medicine. Peripheral blood samples are collected prior to and post‐vaccination for detection of antibodies developed for core and surface antigen. A small population never develop the required anti‐HBsAg titers required for a full protection despite multiple vaccination doses. We intend to utilize FACS to select the mature B‐cell population of the blood samples. RNA will be extracted and the variable region of immunoglobulin will be amplified and subsequently sequenced using NGS (next generation sequencing). The newly developed antibodies against HBsAg will be detected through subtraction of antibodyome post‐ and pre‐immunization.
Group A Streptococcus evades lysosomal degradation in macrophages (836.3)
Authors: No Hyewon , Snipper Joshua , Till Andreas , Okumura Cheryl
Published: June 2021

Group A Streptococcus (GAS) has evolved multiple immune evasion strategies in order to successfully establish infections in otherwise healthy individuals. In this study, we sought to create a molecular tool to study intracellular trafficking and degradation of GAS by host phagocytic cells. We created a construct using a tandem red fluorescent protein linked to a pH‐sensitive green fluorescent protein (mWASABI), which degrades upon contact with the low pH environment of the lysosome. We confirmed successful cloning and expression of the construct into GAS and degradation of the green fluorescent protein under low pH conditions. However, to our surprise, infection of THP‐1 macrophage cells with the RFP‐mWASABI expressing bacteria in combination with immunofluorescent assays indicates the failure of the GFP to degrade in host cells despite bacterial colocalization with lysosomes. Our data suggests GAS either disables lysosomal acidification, thus preventing the activation of lysosomal degradative enzymes, or withstands the low pH environment and proteolytic enzymes present in the lysosomes. Current work focuses on elucidating specific bacterial factors involved in lysosomal resistance mechanisms. These studies contribute to our understanding of how GAS has evolved to circumvent cellular defense mechanisms and will aid in improving treatment options for GAS infections.
Cleavage of DAP5 by coxsackievirus B3 protease 2A causes its nuclear translocation and inhibition of IRES‐containing gene transcription (836.5)
Authors: Hanson Paul , Qiu Ye , Ye Xin , Zhang Huifang Mary , Hemida Maged , Lim Travis , Gu Ada , Cho Brian , Kim Hyein , Yang Decheng
Published: June 2021

Coxsackievirus B3 (CVB3) is a primary causative agent of viral myocarditis which may progress to dilated cardiomyopathy (DCM), a leading cause of unexpected death in children and young adults. Upon infection, CVB3 hijacks host cellular translation machinery, generating an environment that favors viral replication. Death associated protein 5 (DAP5) is an internal ribosomal entry site (IRES) specific translation initiation factor, utilized by the cell in conditions of stress, differentiation, cell‐cycle and apoptosis. We previously showed that DAP5 is cleaved during CVB3 infection. In this study we aimed to identify the responsible protease and site of cleavage, as well as the functional impact. Our results found that DAP5 was cleaved by viral protease 2A but not by cellular proteases. We further identified the cleavage site at amino acid G434. Upon cleavage, the N‐terminal product could translocate to the nucleus and inhibited transcription of IRES‐containing pro‐survival genes such as Bcl‐2, GRP‐78 and VEGF. This is the first report to reveal the function and mechanism of DAP5 cleavage in regulating viral and host gene expression during CVB3 infection. Moreover, this study may identify key components necessary for facilitating viral pathogenesis and thus provide novel pharmaceutical targets for viral myocarditis.

Grant Funding Source: Canadian Institutes of Health Research
Serum‐derived bovine immunoglobulin/protein isolate binds to pathogen‐associated molecular patterns (836.6)
Authors: Horgan Alan , Maas Kenneth , Henderson Abigail , Detzel Christopher , Weaver Eric
Published: June 2021

A key feature of the innate immune system is the panoply of pattern recognition receptors which bind a wide array of highly conserved pathogen‐associated molecular patterns (PAMPs). Under normal conditions, PAMP binding initiates downstream signaling cascades culminating in pro‐inflammatory cytokine production, inflammation, and subsequent antigen elimination. Continual PAMP binding leads to a persistent state of inflammation associated with numerous chronic inflammatory disorders such as IBS, IBD, and HIV enteropathy. We hypothesize that immune exclusion of PAMPs through antigen/antibody interactions can reduce inflammatory signaling and lessen the gastrointestinal manifestations of these disorders.

Serum‐derived bovine immunoglobulin/protein isolate (SBI) is comprised primarily of immunoglobulins (Ig) and is indicated for the clinical dietary management of gastrointestinal enteropathy under physician supervision. We investigated the binding of SBI’s Ig components (IgG, IgA, and IgM) to PAMPs associated with intestinal inflammatory disorders. Using a modified ELISA technique we have shown that IgG, IgA, and IgM contained in SBI bind to a wide array of bacterial, viral, and fungal PAMPs.Oral administration of SBI has been shown clinically to manage GI symptoms in patients with IBS‐D and HIV enteropathy; however, the mechanism of action is poorly understood. Here we show that SBI binds a wide variety of PAMPs. We propose that Ig‐PAMP binding may inhibit immune recognition and the chronic inflammation associated with persistent immune activation.
Improved diastolic function in rats with volume overload heart failure may be due to phosphodiesterase (PDE) 3 inhibition (837.1)
Authors: Lewis Kristin , Zhang Xiaojin , Lucchesi Pamela
Published: June 2021

This study investigated whether drugs with (milrinone (Mil), levosimendan (Levo)) and without (omecamtiv mecarbil (OM)) demonstrated phosphodiesterase (PDE) 3 inhibitory activity improve diastolic function in rats with aortocaval fistula (ACF)‐induced volume overload (VO). ACF or sham surgery was performed in male Sprague‐Dawley rats (200‐240g) 8 weeks prior to acute IV infusion with either Levo (2.8 µg/kg/min), Mil (9.9 µg/kg/min), OM (11.7 µg/kg/min) or vehicle (Veh; 0.4% DMSO) for 30 min. Levo and Mil improved diastolic function compared with Veh (tau; 9.0 and 9.8, respectively vs. 10.7 msec, p<0.05), while OM worsened diastolic function compared with Veh, Levo, and Mil (tau and dP/dtmin; 12.4 msec (p<0.05 vs. ACF‐Veh and p<0.001 vs. ACF‐Levo and ACF‐Mil) and ‐5967 mmHg/sec (p<0.05 vs. ACF‐Veh and p<0.01 vs. ACF‐Levo and ACF‐Mil)). Following infusion, %FS improved in ACF‐Levo, ACF‐Mil and ACF‐OM (39.8, 37.9 and 36.8%, respectively) compared with ACF‐Veh (32.1%; p<0.01), and there was no significant decrease in mean arterial pressure and no increase in arrhythmias. These results suggest that PDE3 inhibition improves diastolic function and that Levo’s improved diastolic function may be due to PDE3 inhibition independent of changes in blood pressure.

Grant Funding Source: Supported by: R01 HL‐056046 (to PAL), ACVP/STP Coalition (to KL)
Characterization of erectile dysfunction and overactive bladder in heart failure rats: model of volume overload (837.6)
Authors: Claudino Mário , Silva Fabio , Ramos‐Filho Celso , Rojas‐Moscovo Julio , Priviero Fernanda , Nucci Gilberto , Antunes Edson
Published: June 2021

In chronic heart failure (CHF) 85% of patients have been reported some episode of erectile dysfunction and 34% reported overactive bladder. We evaluate the muscle reactivity of isolated corpus cavernosum (CC) and detrusor muscle (DM) of CHF rats (CHFR). Concentration responses curves to phenilephrine (PE) in CC (4wks); and carbachol (CCh), KCl in DSM were obtained from CHFR (4‐12wks). Nitrergic and neurogenic responses induced by electrical field stimulation were obtained in CC and DM (respect). Intracavernosal pressure (ICP) and cystometry (in vivo, respect.) also were evaluated. PE contracted CC with a potency (pEC50) of 5.44 ± 0.04. The pEC50 of PE did not change in CHFR (5.36 ± 0.06), but, the maximal response (Emax) was increased (2.5 ± 0.1mN/mg; P<0.05) compared with sham rats (SR; 1.9 ± 0.1 mN/mg). Nitrergic relaxation in CC was significantly decreased at higher frequencies in CHFR. ICP was reduced in CHFR vs SR (P<0.05). CCh and KCl contracted DM with a pEC50 of 5.42 ± 0.07 and 0.90 ± 0.12, respectively. The pEC50 of CCh and KCl did not change in CHFR (5.68 ± 0.07; 1.34 ± 0.11, respect.), but, the Emax were increased (1.09 ± 0.07; 0.88 ± 0.05mN/mg, respect; P<0.05) vs SR (1.45 ± 0.10; 1.29 ± 0.11 mN/mg, respect.). In CHFR neurogenic contractions in DM were significantly increased in all frequencies vs SR. Cystometry were increase in CHFR vs SR (P<0.05). In conclusion, CHFR exhibit an impaired nitrergic response (CC) and erectile function, and an increase of contractile‐mechanism in DSM and cystometry featuring the genitourinary dysfunctions in CHFR.

Grant Funding Source: FAPESP/CNPq
Behavioral effects of the cannabinoid CB1 receptor negative modulator ORG27569 in rats (838.2)
Authors: Li Jun‐Xu , Ding Yuanyuan , Qiu Yanyan , Zhang Yanan
Published: June 2021

Orthosteric cannabinoid CB1 receptor antagonists are effective for treating obesity but also have serious side effects (e.g., depression) which limit their clinical utility. Negative allosteric modulators might possess different therapeutic and side effect profiles. This study examined the behavioral effects of a CB1 receptor negative allosteric modulator ORG27569 in rats. CB1 receptor agonists CP55940 (0.1‐1 mg/kg) and anandamide (3.2‐32 mg/kg) dose‐dependently decreased the body temperature in rats. ORG27569 alone had no effect on the body temperature but markedly antagonized CP55940‐ and anandamide‐induced hypothermic effects. CP55940 (0.032‐1 mg/kg) produced marked cataleptic effect. Although the CB1 receptor orthosteric antagonist rimonabant markedly antagonized the cataleptic effect of CP55940, ORG27569 did not alter such an effect. Rimonabant dose‐dependently increased the frequency of grooming and scratching behaviors in rats. ORG27569 alone did not increase the frequency of these observational behaviors and did not alter the potency of rimonabant in this assay. These data suggest that ORG27569 has a different antagonism profile from the orthosteric CB1 receptor antagonist rimonabant in vivo. A better understanding of the antagonism profile of ORG27569 is warranted which may eventually lead to useful therapeutic actions via biased CB1 receptor blockade.(DA034806 and DA033426)
Functional consequences of synthetic cannabinoid metabolites and CYP2C9 polymorphisms (838.4)
Authors: Allen Catherine , Patton Amy , Seely Kathryn , Fantegrossi William , Radominska‐Pandya Anna , Prather Paul , Ford Benjamin , James Laura , Moran Jeffery
Published: June 2021

Synthetic cannabinoid (SC) abuse is a growing problem. Previous studies using recombinant cytochrome P450 (CYP) enzymes identified CYP2C9 as an isozyme responsible for SC oxidation, but the role of genetic variability in SC metabolism has not been studied. To examine the relationship of CYP2C9 polymorphisms and SC effects, mice were treated with JWH‐018 or the chiral (ω‐1)‐hydroxyl metabolite ((ω‐1)‐OH) that is formed via CYP2C9 action. Administration of JWH‐018 (i.p. 3 mg/kg) produced Δ9‐THC‐like effects on core temperature and locomotor activity in mice that corresponded to brain concentrations of 10 ng/g for JWH‐018 and 2 ng/g for (R/S)‐(ω‐1)‐OH. Administration of purified (R)‐ or (S)‐(ω‐1)‐OH enantiomers (i.p. 3 mg/kg) equally reduced locomotor activity and core temperature in the mice. Pre‐treatment with the cannabinoid receptor‐1 antagonist rimonabant attenuated the physiologic effects associated with administration of the chiral (ω‐1)‐OH metabolites. Both enantiomers also retained nanomolar affinity and intrinsic activity at cannabinoid receptors. In vitro enzyme kinetic assays performed using human recombinant CYP2C9 variants (*1, *2, and *3) showed reduced metabolite formation with the CYP2C9*3 variant. Thus, data demonstrate comparable biologic activity for the chiral (ω‐1)‐OH metabolites of CYP2C9 and suggest a potential role for CYP2C9 polymorphisms as a modulator of drug toxicity.

Grant Funding Source: Supported by NIH grants RR020146 and RR029884 and an institutional ASPET SURF award.
Differential contributions of the two cell surface G‐protein coupled neurotensin (NT) receptors (NTr1 and NTr2) in open field accommodation (839.7)
Authors: Smith Misty , Nielsen Shannon , Wada Etsuko , Wada K , Fleckenstein Annette , Hanson Glen
Published: June 2021

Neurotensin (NT) is an endogenous neuropeptide with diverse central and peripheral effects. Open field accommodation is a dopamine (DA) ‐related behavioral model that appears to be sensitive to NT systems. Although NT has previously been shown to have modulatory effects on CNS DA activity and to be involved in motivation, the relative contribution of the two cell surface G‐protein coupled NT receptors (NTr1 and NTr2) to exploratory drive in a novel open field is unknown: to investigate their roles in this model, C57BL/6J mice deficient in either NTr1 (‐/‐) or NTr2 (‐/‐) were compared to wild‐type, littermate C57BL/6J (WT +/+) mice. Open field locomotor activity (40 cm L × 40 cm W × 30 cm H) was quantified using the automated Accuscan activity monitoring system (OmniTech Instruments Inc., Columbus, OH) in 10 min epochs for up to 4 hours. The total distance travelled, horizontal activity, and vertical activity were compared between groups and statistically analyzed using Two way ANOVA with repeated measures (GraphPad Prism, La Jolla, CA). Compared to WT (+/+) mice, mice deficient in the NTr1 receptor accommodated to the open field significantly less, whereas, mice deficient in the NTrs2 receptor accommodated faster, than WT (+/+) mice in all three parameters of locomotor activity quantified. These results suggest that the NTr1 subtype of the NT system facilitates, whereas NTr2 subtype inhibits, locomotor accommodation in a novel environment. The significance of these opposing effects of the NTr1 and 2 receptors will be discussed.

Grant Funding Source: Supported by NIDA DA031883 (GRH), NIDA DA000378 (GRH), & NIDA DA019447 (AEF)
The impact of hydrogen sulfide in oesophagoprotection (840.15)
Authors: Hrytsevych Nazar , Zayachkivska Oksana , Yaschenko Antonina
Published: June 2021

Various studies have shown that novel cytoprotective gasotransmitter hydrogen sulfide (H2S) has cytoprotective and preventive effects in the gastrointestinal system (Wallace J., 2010‐2012). However, the mechanism of H2S on oesophageal barrier remains unclear. Therefore, the aim of present study was to examine the protective role of endogenous H2S in oeosphageal mucosal (OM) lesions related to stress‐associated injury and postprandial hyperglycemia (PHG) which we recently recognized as a serious trigger in the oesophageal injury genesis (Hrytcevych N., 2011‐2013).

Aim: To determine whether or not H2S attenuates oesophageal mucosal cytoprotection in rats with nonerosive oeosphagitis and long term PHG, and explored the underlying mechanisms may be involved.

Methods: Rats without and with 3,5 h water immersion and restraint stress (WIRS) and 28 days of PHG received i.p. of vehicle, cystathionine gama‐lyase (CSE) inhibitor, DL‐propargylglycine (PAG, 25 mg/kg i.p.), CBS inhibitor, beta‐cyano‐alanine (BCA, 50 mg/kg i.p.), or H2S donor NaHS (100 mlmol/kg) prior to WIRS. Scoring lesion index (LI), vascular index (VI) by OM histomorphology, IL‐10, IL‐17 by ELISA.

Results: WIRS & PHG induced non‐erosive injury which characterized by rise LI and VI vs control. PAG or BCA pretreatment enhanced EM lesions escalating LI and VI in twice, drop IL‐10 and rise IL‐17. NaHS mediated decrease of OM lesions via decreased LI, VI and increase IL‐10 and decrease IL‐17 scores vs control.

Conclusion: These results indicate that endogenous H2S plays oesophagoprotective role by inhibiting inflammation, oedeme and vasodilator effect, highlighting the therapeutic potential for H2S donor in postprandial hyperglycemic stress‐associated conditions.
Anti‐inflammatory effect of miR‐17‐3p in human umbilical vein endothelial cells (840.16)
Authors: Zhang Yu , Leung Susan , Man Ricky
Published: June 2021

Anti‐inflammatory effect of miR‐17‐3p in human umbilical vein endothelial cells Yu ZHANG, Susan WS LEUNG and Ricky YK MAN Department of Pharmacology & Pharmacy, the University of Hong Kong, Hong Kong SAR, China Aim: MicroRNAs (miRNAs) are a class of small, noncoding RNAs of ≍22 nucleotides that negatively regulate gene expression. They appear to play a role in the development/progression of many disorders, including inflammation, cardiovascular diseases and endothelial dysfunction. This study examines the role of miR‐17‐3p in vascular inflammation.

Methods: Human umbilical vein endothelial cells (HUVECs) were transfected with miR‐17‐3p agomir (miR‐17‐3p mimic) or its negative control using lipofectamine 2000. They were incubated with lipopolysaccharide (LPS, 10 ng/ml) for 16 hours to induce inflammatory reactions. The level of miR‐17‐3p and the expressions of IκBα, p65 and phosphorylated p65 (p‐p65) were measured by qPCR and Western blotting. The amount of interleukin‐8 (IL‐8) and tumor necrosis factor (TNF)‐α released in the culture medium was detected with ELISA kit.

Results: The levels of miR‐17‐3p, IL‐8, TNF‐α and p‐p65 were increased following LPS stimulation. While the expression of p65 was not changed, the expression of IκBα was reduced. LPS‐induced increase in IL‐8 level and decrease in IκBα expression were smaller in HUVECs transfected with miR‐17‐3p agomir than in those transfected with the negative control.

Conclusion: MiR‐17‐3p is up‐regulated in LPS‐induced inflammation. It may produce an anti‐inflammatory effect by inhibiting NF‐κB pathway and reducing IL‐8 production.
Emetic and defecatory action of the prostanoid EP receptor agonist, sulprostone, in ferrets (840.9)
Authors: Kan Kelvin , Lu Zhengbing , Wai Man , Wong Yuen , Jones Robert , Rudd John
Published: June 2021

Prostanoid EP receptor agonists are used extensively for gynecological purposes but may be associated with gastric disturbance. Endogenously produced prostaglandins may also be associated with gastric dysfunction. In the present studies, we investigate the mechanism of the EP1/3 receptor agonist, sulprostone (30 µg/kg, i.p.), to induce emesis and defecation in ferrets. Sulprostone induced tachygastria (dominant frequency increased from 9.7±0.1 to 11.8±0.6 cpm; P<0.05) and hypertension (mean arterial pressure increased from 117.9±4.1 to 165.9±7.3 mmHg; P<0.05). Promethazine (3 mg/kg) potentiated sulprostone‐induced emesis (P<0.05). The emetic response was not antagonized significantly by metoclopramide (0.3 and 3 mg/kg), ondansetron (0.1 and 1 mg/kg), or scopolamine (3 mg/kg). Only scopolamine reduced significantly the defecatory response (P<0.05). Bilateral abdominal vagotomy was ineffective to reduce sulprostone‐induced emesis and defection. Sulprostone (10 µg) administered into the fourth ventricle was emetic (P>0.05). These data suggests that mechanisms controlling sulprostone‐induced emesis and defecation are distinct.
Activation of central nicotinic acid receptor GPR109A increases blood pressure in conscious rats (841.11)
Authors: Rezq Samar , Abdel‐Rahman Abdel
Published: June 2021

The hypolipidemic drug nicotinic acid (NA) elicits prostaglandins (PGs)‐dependent flushing reaction via GPR109A activation. There are no reports on GPR109A expression or function in the rostral ventrolateral medulla (RVLM), despite evidence that NA reaches the brain. We present the first evidence for GPR109A expression in the RVLM (Western blot). Next, we showed in conscious rats that intra‐RVLM NA (estimated doses to reach the RVLM following systemic administration) increased BP and reduced HR, which resembled responses elicited by intra‐RVLM L‐glutamate. The pressor effect of NA was abolished by selective NMDAR blockade (2‐amino‐5‐phosphonopentanoic acid; AP5) and enhanced by L‐glutamate uptake inhibition (L‐trans‐Pyrrolidine‐2,4‐dicarboxylic acid; PDC). Further NA elevated L‐glutamate level in culture medium of PC12 cells, which exhibit neuronal phenotype and express GPR109A. These novel findings suggest that L‐glutamate release is involved in RVLM GPR109A‐mediated brief pressor response. Ongoing integrative and molecular studies will elucidate the role of PGs and oxidative stress in the delayed pressor response caused by intra‐RVLM NA. The findings are clinically relevant because they elucidate the mechanisms of a centrally mediated deleterious effect of NA on BP, and yield new insight into the potential use of concurrent therapeutics to circumvent such deleterious effect.
Contribution of Rho kinase to TRPM4‐mediated myogenic tone in cerebral parenchymal arterioles (841.5)
Authors: Li Yao , Baylie Rachael , Tavares Matthew , Brayden Joseph
Published: June 2021

Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to cerebral pial arteries, as defined by their critical roles in neurovascular coupling and distinct sensitivities to mechanical and chemical stimulants. Elevation in intraluminal pressure, a physiologic vasomotor stimulant, causes smooth muscle membrane depolarization and contraction (myogenic tone) in cerebral arteries. However, the mechanisms by which this myogenic tone is regulated in the cerebral microcirculation remain unclear. Prior research in our laboratory indicates that myogenic tone of PAs is mediated primarily through stimulation of mechano‐sensitive P2Y purinergic receptors. The objective of the present study was to determine the roles of TRPM4 channels and Rho kinase signaling in PA myogenic tone. We show that suppression of TRPM4 channel expression in vivo using antisense oligonucleotides reduced (by ~40%) both myogenic and P2Y‐receptor agonist‐induced tone in PAs. Further, we found that the Rho kinase inhibitor H1152 reversibly and nearly completely inhibited myogenic tone in these brain arterioles (IC50:0.2 µM). H1152 (1 µM) also attenuated P2Y4 and P2Y6 receptor‐mediated vasoconstriction by 85% and 87%, respectively. Finally, H1152 (1 μM) reduced constitutive and UTPγS (0.5 μM)‐activated TRPM4 currents in parenchymal myocytes by 61% and 75%, respectively. These results indicate an important role for Rho kinase in regulation of TRPM4 channel activity and myogenic tone in the cerebral microcirculation.

Grant Funding Source: Supported by NIH HL095488
Glyoxalase‐1 gene transfer prevents cerebral deficits in rats with type 1 diabetes (841.9)
Authors: Alomar Fadhel , Shao Chun , Xia Jianxun , Xiong Huangui , Mayhan William , Bidasee Keshore
Published: June 2021

Background: Individuals with type 1 diabetes (T1D) confront daily challenges including reduction in sustained attention, decreased decision‐making abilities and increased risks for accidents. Studies attribute these insufficiencies in part to reduced psychomotor efficiency, slowing in information processing speed, impaired visuo‐construction and cerebral vascular dysfunction. To date, the molecular cues responsible for these cerebral deficits in T1D remain poorly defined. Here we investigated whether the reactive carbonyl species methylglyoxal (MGO), whose production is upregulated in T1D, is an underlying cause.

Methods and results: Brain levels of MGO were 6 times higher in T1D compared to non‐diabetic rats. Chronic administration of MGO to control rats impaired their nest building capabilities, an activity that requires decision‐making, visual and motor functions. MGO treatment also impaired endothelial‐mediated vasodilatation of cerebral microvessels, induced localized microvascular leakages, astrocytes activation and impaired synaptic transmission, akin to that seen in T1D. Increasing expression of the MGO‐degrading enzyme glyoxalase‐1 (Glo‐1) in rats after the onset of diabetes using an adeno‐associated viral construct, decreased brain MGO level and blunted cerebral impairments induced by T1D.

Conclusion: From these data we conclude that upregulation of MGO is an underlying cause for cerebral deficits in T1D and MGO detoxification is sufficient to prevent these cerebral dysfunctions.
Inhibitory effect of AHCC and GCP on tumorigenesis of C6 glioma cells in culture medium and rats mediated by downregulating CD133 and Nestin expressions (842.13)
Authors: Zhang Xiao‐Hong , Nishioka Hiroshi , Fujii Hajime
Published: June 2021

The CD133 and Nestin proteins are described as cancer stem cell markers. Norepinephrine (NE) is a sympathetic neurotransmitter, studies have indicated the level of NE or the active degree of adrenoceptors in certain bodily tissues correlates with the degree of tumorigenesis in those tissues. To explore the relation between the activation of adrenoceptors and the expression level of CD133 and Nestin proteins, we investigated whether NE might regulate the expression of CD133 and Nestin and its signaling pathway; the effect of AHCC (Active Hexose Correlated Compound) &and GCP (Genistein Concentrated Polysaccharide) on the expression of CD133 and Nestin and the sphere‐colony formation of C6 cells in medium and the tumorigenesis in rats inoculated subcutaneously with C6 cells.

We measured the expression level of CD133 and Nestin in C6 cells with western blot analysis, examined the sphere‐colony formation of the cells by sphere assay and measured the tumor volume in rats calculated as length × width × depth × 0.5236.

We found out the expressions of CD133 and Nestin in C6 cells were significantly upregulated by NE stimulation. Further analysis showed prazosin completely blocked the NE‐induced upregulation of CD133 and Nestin expressions, while propranolol had no influence to them. Pretreatment with LY294002, blocked the NE‐induced upregulation of CD133 expression, but showed no influence to Nestin expression. Pretreatment with B3806, blocked the NE‐induced upregulation of Nestin expression, but had no significant influence to CD133 expression in the cells. Co‐culture with AHCC or GCP, the upregulated effects on the expressions of CD133 and Nestin induced by NE in the cells were weaken and the sphere size of the cells became smaller. The animal experiment in rats showed that the tumor size was markedly smaller in AHCC or GCP group than in control group.

Based on these findings, we thus assume that AHCC & GCP can ameliorate some kinds of tumorigenesis in cancer stem cells expressing CD133 and Nestin proteins.
Investigating sulforaphane effects on gene regulation and protein expression in HL‐60 cells (842.15)
Authors: Lehner Kristen , Sanger Julia , O'Donnell Robert
Published: June 2021

Sulforaphane (SFN), a naturally occurring isothiocyanate, has shown promising chemopreventive and anti‐cancer potential. Our preliminary studies demonstrate that SFN induces apoptosis in HL‐60 leukemia cells. Our data show a significant increase in SFN‐induced apoptotic and dead cells, as well as slowed cellular division over 72 hours. However, the mechanism by which SFN affects HL‐60 cells to induce apoptosis and block cellular division is not well understood. To this end, microarray experiments were used to compare gene expression profiles of untreated and SFN‐treated cells. Of particular interest, there was a 3.52‐fold upregulation of Bcl‐2 as well as upregulation of other Bcl‐2 related genes in the presence of SFN and a 100‐fold downregulation of Myc after treatment with SFN. The Myc gene is constitutively active in most cancers and this downregulation is of particular interest for potential anti‐cancer effects of the mechanism of SFN. Using FITC labeled monoclonal antibodies and flow cytometry, we are currently investigating SFN’s effects on protein expression of Bcl‐2 and Myc to determine the consistency or inconsistency with microarray data. Initial experiments suggest a difference in Myc expression between SFN treated and untreated HL‐60 cells while no difference was observed in Bcl‐2 levels. Subsequent experiments will utilize different doses of SFN and exposure times.
Silencing of regulator of G‐protein signaling 10 (RGS10) increases phosphorylation of eukaryotic initiation factor 4E binding protein (4E‐BP1), and enhances growth and survival signaling pathways in ovarian cancer (843.11)
Authors: Altman Molly , Patel Mihir , Hooks Shelley , Murph Mandi
Published: June 2021

Ovarian cancer is a devastating disease that is the 5th leading cause of cancer related deaths in women in the US. It is difficult to treat in that more than 70% of patients that are initially responsive to first‐line chemotherapeutics become refractory in less than 2 years. The problem of drug‐resistance or chemoresistance has plagued cancer researchers and hindered the success of cancer therapy development. In our previous studies examining computational bioinformatics, we demonstrated that reduced RGS gene expression develops with the occurrence of drug resistance in ovarian cancer cell lines. Specifically, we have shown that silencing of RGS10 protein increases viability of ovarian cancer cells in the presence of cycle arrest compounds such as paclitaxel. In our present study, we have shown that silencing RGS10 in ovarian cancer cell lines increases phosphorylation of Eukaryotic initiation factor (eLF) 4E, the mRNA 5’ cap‐binding protein (phosphorylated‐4E‐BP1). Phosphorylation of 4E‐BP1 has been shown to be a hallmark of more aggressive cancer phenotypes in prostate cancer and ovarian cancer. HeyA8 Parental and SKOV3 ovarian cancer cell lines were transiently transfected with siRNA non‐targeted control and human RGS10. Approximately 36 hrs post‐transfection cells were serum‐starved and treated with INK128 a selective mTOR inhibitor overnight. Cells were treated the following day with lysophosphatidic acid (LPA) and collected for western blot analysis. The same treatment conditions were also analyzed by immunofluorescence assays and imaged using a Cellomics ArrayScan VTI reader and HCS software. We found that when RGS10 was silenced in these ovarian cancer cell lines we observed changes in cell growth and size. Based on the results from these assays, we believe that we have elucidated a novel role of RGS10 in the growth and survival signaling pathways involved in the development of chemoresistance in ovarian cancer.

Grant Funding Source: NIH
Intragenic suppressors of McCune‐Albright syndrome R201H mutations also suppress other constitutively active Gs alleles (843.5)
Authors: Rylaarsdam Robin , Turcic K , Dafour A , Carroll J
Published: June 2021

McCune‐Albright Syndrome (MAS) is a genetic disorder caused by a mutation in Gs alpha at Arg201 that inhibits GTP hydrolysis, constitutively activating the protein. We identified three sites that, when mutated, suppressed the constitutive activity caused by R201H: F142, R231, and L266. None of the three mutations by themselves caused constitutive activity of the protein or significantly changed cellular responsiveness to hormone, as measured by the ability of a luteinizing hormone receptor agonist to activate cAMP production. We have now examined the ability of mutations at these three sites to suppress other activating mutations of Gs alpha. Other McCune‐Albright associated mutations include R201C and R201S. Both F142S and R231C were able to suppress the constitutive activity of both of previously untested R201 substitutions. L266N was able to suppress R201S, and experiments investigating its effects on R201C are ongoing. Gs alpha can also be constitutively activated by the Q227L substitution, associated with a variety of adenomas. The mutations identified as suppressors of R201H were all unable to suppress the constitutive activity of Q227L, suggesting that the action of the suppressors is specific to the R201 site and not generalizable to other activating perturbations of Gs alpha.

Grant Funding Source: Supported by NIH grant 1R15ED020190‐01
Acrolein exacerbates anti‐retroviral therapy‐associated hepatotoxicity by increasing ER stress, apoptotic gene expression and cell death (844.14)
Authors: Ghare Smita , Donde Hridgandh , Joshi‐Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

In HIV patients prolonged ART usage accompanied by other life style components (e.g. alcohol consumption, smoking) has been associated with adverse hepatic events caused by abnormal lipid accumulation and peroxidation. However, role of cofactors that can significantly alter the hepatic interaction of ART leading to pathogenic outcomes is only poorly understood. In this regard, ART mediated hepatic steatosis can give rise to highly reactive hepatotoxic aldehydes, like acrolein, via lipid peroxidation. Hence the present study examined the role of acrolein as a cofactor in ART drug, azidothymidine (AZT)‐induced hepatotoxicity.

Cytotoxic effects of acrolein and AZT were investigated using human hepatoma cells (HepG2) and rat‐primary cells. Cells were treated with various concentrations of acrolein (ACR) and AZT alone or in combination (AZT+ACR). Our results showed that acrolein or AZT alone had minimal effect on hepatocyte survival. However combinatorial treatment of AZT+ACR significantly increased apoptotic death. Further, the hepatotoxic effects of AZT+ACR were observed to involve ER stress and mitochondrial dysfunction which was documented by Ca 2+ release, up‐regulation in ER stress genes and mitochondrial membrane depolarization and ATP depletion. Additionally, the combinatorial treatment increased apoptotic gene expression of FasL, a known contributor to liver damage. Notably, acute acrolein exposure enhanced AZT responsive epigenetic modifications at the FasL promoter contributing to transcriptional activation. Overall, the data suggest that presence of acrolein plays a significant pathogenic role in the development of ART‐induced hepatotoxicity.

Grant Funding Source: Supported by NIH and Veterans Administration
Study the effect of drug resident time on in vivo efficacy (844.6)
Authors: Lee Kin Sing Stephen , Yang Jun , Ng Connie , Niu Jun , Morisseau Christophe , Hammock Bruce
Published: June 2021

When developing a new drug, it is difficult to predict from in vitro parameters to in vivo efficacy. It is generally accepted that drugs with the strong interactions with their target are more likely to show better efficacy. Therefore, thermodynamic parameter: dissociation constant or inhibition constant (Ki) are often considered as the most important factor to be determined as the first screening to prioritize the drug candidates. However, the kinetic parameter: the dissociation rate constant (koff) of the drugs is often neglected although several reports suggested that koff is as important as or more than Ki to estimate in vivo efficacy.

Soluble epoxide hydrolase (sEH) has been proven to play an important role on regulating important lipid mediators, epoxy‐polyunsaturated fatty acid. Inhibition of sEH has been showed to be beneficial to human health in several aspects, anti‐inflammatory, analgesic and promoting tissue regeneration. We recently synthesized a series of new sEH inhibitors with different Kis and koffs. These inhibitors will be used as tools to test if koff can predict in vivo efficacy as equal as or better than the Ki.

Here, a series of inhibitors with improved in vitro efficacy will be reported. Our in vivo results suggested that koff is crucial parameters to affect drug efficacy in an alternate way.

Grant Funding Source: National Institute of Environ‐ mental Health Sciences (NIEHS) grant ES002710
A new therapeutic target for Alzheimer’s disease: effects of palmitoylethanolamide with luteolin association on neuroinflammation pathway (845.10)
Authors: Paterniti Irene , Campolo Michela , Bruschetta Giuseppe , Siracusa Rosalba , Esposito Emanuela , Cuzzocrea Salvatore
Published: June 2021

Activation of glial cells and the consequent neuroinflammatory response is recognized as a prominent neuropathological feature of Alzheimer’s disease (AD). To counteract the neuroinflammation of AD, in our study we used the association of palmitoylethanolamide (PEA) with the antioxidant luteolin (Lut), in a ratio of 10:1 respectively, in in vitro and in ex vivo organotypic model of AD. For in vitro model we used a human neuroblastoma cell line SHSY‐5Y, that were pre‐treated with PEALut (0.1‐1‐10 μM) for 2h. The damage was induced by Aβ1‐42 stimulation (1µM) and 24hrs after damage we performed cell death assay and western blot analysis. Moreover, we carried out an ex vivo organotypic model of AD; to this purpose organotypic hippocampal slice cultures were prepared from P6 mice, and placed onto semiporous inserts. On day 21 of culturing, organotypic hippocampal slides were pre‐treated with PEALut (0.1‐1‐10 μM) for 2h, and the damage was induced by Aβ1‐42. 24hrs after damage, sections were gently removed from inserts and processed for MTT assay, NO assay and western blot analysis. Our data indicate that PEALut compound is able to blunt a Aβ1‐42 ‐induced neurotoxicity and to exert a marked protective effect on glial cells, providing an effective strategy for AD.
The effects of alpha1A adrenergic receptor knockout on learning, memory, and seizure incidence (845.2)
Authors: Collette Katie , Thorsen Arthur , Goldenstein Brianna , Lyons Mariaha , Wood Sarah , Cooper Albertine , Simpson Paul , Perez Dianne , Doze Van
Published: June 2021

The role of the α1 adrenergic receptor (α1ARs) subtypes, α1A‐ and α1BAR, in the central nervous system is not well understood. Specifically, there are conflicting data regarding the role of the α1AR in learning and memory, possibly because agonists and antagonists highly selective for each subtype are not available. Our lab has used transgenic and chronic pharmacologic models of activation to examine the role of the α1AAR subtype in modulating seizure threshold, learning, and memory. Previously, we found that stimulation of the α1AAR subtype has positive effects. When exposed to flurothyl, transgenic animals overexpressing the α1AAR (CAM‐α1AAR mice) have an increased seizure threshold when compared with wild type (WT) mice. CAM‐α1AAR and mice treated with the α1AAR agonist cirazoline have enhanced spatial learning when compared with WT animals. Stimulation of the α1AAR also increases proliferation in the adult mouse dentate gyrus, which has been linked to enhanced learning in other studies. In the current study, α1AAR knock‐out (A‐KO) mice were used to determine the effects on learning and memory in the Barnes maze, Morris water maze, and novel object recognition task. An assessment of general health and neurological reflexes did not show any abnormalities in the A‐KO mice. The A‐KO mice did not spend more time with the novel object than chance would dictate, suggesting impaired recognition memory. In the Barnes maze, A‐KO mice took significantly longer to solve the maze than WT mice. The Morris water maze is ongoing but, thus far, no differences in learning have been observed. A subset of A‐KO mice experienced novelty‐induced seizures during the testing period; the data will be examined for effects of seizures on learning ability.