bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of multivitamin, multimineral and phytonutrient supplementation on selected nutrient levels in blood and heart health risk factors in Russian population (831.4)
Authors: Isakov Vasily , Golubeva Alexandra , Selezneva Ksenia , Bessonov Vladimir , Sentsova Tatiana , Tutelyan Victor , Lin Yumei , Kazlova Valentina
Published: June 2021

The primary objective of this exploratory study was to evaluate the effect of a multivitamin multimineral with phytonutrient supplement on heart health risk factors. One hundred twenty healthy adults (40‐70 y) were recruited in this 8‐week randomized double blind placebo control study. B‐carotene, a‐tocopherol, vitamin C, red blood cell (RBC) folate, B6, B12, Zn, Se content in blood were measured on Day 1, 28 and 56 and quercetin was measured on Day 1 and 56. Heart health risk factors such as homocysteine (Hcy), high‐sensitivity C‐reactive protein (hs‐CRP), oxidized LDL (ox‐LDL), gamma‐glutamyl transferase (GGT), uric acid and blood lipid profile were monitored on Day 1 and 56. Results showed that supplementation significantly improved quercetin (p=0.0047), vitamin C (p=0.0011), RBC folate (p=0.0027) and B6 (p=0.0012) status after 8 weeks of consumption. A greater decrease of Hcy (p=0.0314), GGT (p=0.0545) and uric acid (p=0.1683) was also observed. However, a lesser degree of increase of hs‐CRP (p=0.2511) was observed, but not ox‐LDL (p=0.5444). Overall, supplementation may contribute nutrient status improvement and reduction of Hcy, which may play a role in supporting healthy heart in this Russian population.

Grant Funding Source: Access Business Group, LLC and OOO Amway
Endogenous VEGF deficiency causes intestinal microcirculatory dysfunction in a model of neonatal necrotizing enterocolitis (832.1)
Authors: Yan Xiaocai , Sabnis Animesh , Liu Shirley , Marek Catherine , Tan Xiaodi , De Plaen Isabelle
Published: June 2021

Ischemic necrosis is a feature of necrotizing enterocolitis (NEC). We have previously found that VEGF is decreased in a neonatal mouse model of NEC. Here, we hypothesized that, in NEC, VEGF deficiency causes altered neonatal intestinal microvasculature and impaired microcirculation leading to NEC development. To test this hypothesis, neonatal pups were injected intraperitoneally with SU5416, a VEGF receptor kinase inhibitor prior to exposure to a NEC protocol. The intestinal mucosal microvasculature morphology was assessed by imaging after intracardiac dye injection. Endothelial (CD31+) cells were quantified by flow cytometry analysis following collagenase digestion of intestinal tissues. We found that: 1) Intestinal vascular networks were decreased in pups exposed to the NEC protocol for 48 hours compared to controls, and further decreased in pups treated with VEGF inhibitor; 2) The percentage of CD31+ cells in the CD45‐ Epcam‐ population was decreased in NEC small intestines (1.20%) vs. dam‐fed controls (4.64%), and further decreased when VEGF pathway was blocked (0.37%, p<0.05 vs dam‐fed). Pups exposed to the NEC protocol treated with VEGF inhibitor had increased incidence of severe NEC (score蠅2), compared to controls (18/29 vs 8/32, χ2=7.1, p<0.01) and had increased mortality. In conclusion, we found that inhibition of endogenous VEGF causes intestinal microcirculatory dysfunction and facilitates the development of neonatal NEC..

Grant Funding Source: Supported by a Thrasher Foundation Grant
Insulin‐like growth factor I (IGF‐1) suppressed foam cells via downregulation of 12/15‐lipoxygenase: potential mechanism responsible for IGF‐1‐induced atheroprotective effect (832.14)
Authors: Sukhanov Sergiy , Snarski Patricia , Vaughn Charlotte , Kim Catherine , Delafontaine Patrick
Published: June 2021

We have shown that IGF‐1 reduced foam cells and suppressed atherosclerosis, however mechanism mediating these effects was not identified. Macrophage (MΦ) 12/15‐lipoxygenase (LOX) known to mediate oxidation of low density lipoprotein (LDL) and promote formation of foam cells contributing to atherogenesis. We hypothesized that IGF‐1 downregulates LOX and via this mechanism reduces foam cells and plaque size.

IGF‐1 infusion into Apoe‐null mice (1.5 mg/kg/d, 12 weeks on a Western diet) reduced LOX plaque immunopositivity (74±9% decrease), suppressed plaque oxidative stress (64±7% decrease, immunohistochemistry with 8‐oxodG a/b), reduced plaque lipids (35%±6 decrease, Oil Red staining) and decreased atherosclerotic lesion area (IGF‐1: 0.259 ± 0.018 mm2, saline: 0.356 ± 0.031 mm2).

To further define mechanisms THP‐1‐derived MΦ were exposed to IGF‐1. IGF‐1 (50 ng/ml, 16h) reduced LOX mRNA (49±6% decrease), decreased LOX protein (42±6% decrease, immunoblotting) and also IGF‐1 suppressed STAT6 transcription factor binding to LOX gene promoter region (CHIP assay with STAT6 a/b) suggesting that IGF‐1 downregulates LOX via STAT6‐dependent transcriptional mechanism. IGF‐1 reduced oxidation of LDL mediated by THP‐1 or mouse peritoneal MΦ (85±6% and 67±5% decrease, respectively; MDA assay) and also IGF‐1 decreased cellular LDL uptake (i.e. formation of foam cells) by THP‐1 or WT peritoneal MΦ (45±8% and 51±3% decrease, respectively), however both lipid uptake and IGF‐1 effect were blocked in LOX‐deficient peritoneal MΦ. These data indicate that LOX downregulation mediates IGF‐1‐induced suppression of LDL oxidation and foam cell formation.

In summary, IGF‐1 reduced LOX expression in atherosclerotic plaque and in vitro in cultured MΦ. We demonstrated that LOX downregulation mediates IGF‐1‐induced reduction in foam cells representing potential mechanism mediating IGF‐1‐induced atheroprotective effect.

Grant Funding Source: Supported by NIH/NHLBI
Regulation of TGF‐β1‐inducible angiotensinogen gene transcription in lung cells by core promoter SNPs (834.6)
Authors: Dang My‐Trang , Abdul‐Hafez Amal , Uhal Bruce
Published: June 2021

Previous work from our laboratory demonstrated that JunD and hypoxia inducible factor (HIF‐1α) mediate transforming growth factor beta (TGF‐β1) inducible angiotensinogen (AGT) transcription in human pulmonary fibroblasts (FASEB, 2009:23(6)). The core promoter of AGT contains binding sites for JunD and HIF‐1α that overlap with single nucleotide polymorphisms. In non‐pulmonary cells, AGT promoter sequence variants affect the basal transcription rate of AGT. Increase in the basal transcription rate is seen with the presence of the C allele at ‐20 and ‐18 or the presence of the A allele at ‐6. Furthermore, these same SNPs have been associated with the severity and/or progression of various diseases. Recent work from our lab demonstrated that AGT promoter sequence variants at the ‐20 and ‐6 positions predicted low diffusing capacity in cohorts from the United States (p = 0.0028) and Spain (p = 0.017). From this, we hypothesize that AGT promoter sequence variants can influence the rate of TGF‐β1‐inducible AGT transcription. AGT promoter‐luciferase reporter studies demonstrated that AGT transcription in human pulmonary fibroblasts was significantly increase in cells transfected with the CA haplotype (at ‐20 and ‐6 respectively) compared to the AG haplotype (p = 0.05). Preliminary data suggest that these variants alter the binding affinity of HIF‐1α and JunD to the core promoter ‐ with increase binding of HIF‐1α to the CCA haplotype (at ‐20, ‐18, and ‐6 respectively) and JunD to the ATG haplotype. We hypothesize that this effect may be due to the competition between JunD and HIF‐1α for similar binding sites at the core promoter.

Grant Funding Source: Supported by PHS‐HL 45136
Knocking out RAGE disrupts natural repair of condylar cartilage during osteoarthritis (835.14)
Authors: Chavez Matias Elizabeth , Mecham David , Wilhelm Spencer , Black Collin , Mitchell Jacob , Andersen Karl , Graf Justin , Macdonald James , Reynolds Paul , Kooyman David
Published: June 2021

We have previously shown that knockout (KO) mice lacking the Receptor for Advanced Glycation End Products (RAGE) demonstrate attenuated osteoarthritis (OA) in articular cartilage of the knee. The objective of this research is to discover if knocking out RAGE has a similar protective effect in the condylar cartilage of the Temporomandibular joint (TMJ). Misalignment of the TMJ of Wild‐type (WT) and RAGE KO mice was performed, and severity of OA was assessed along with immunohistochemical staining of several known biomarkers of OA. An inverse relationship is known to exist between the expression of Htra1 and TGF‐B 1 in association with OA. We observed a reciprocal expression of these two proteins in WT but not RAGE KO mice. Condylar cartilage, unlike articular cartilage, has been shown to self‐repair when stress is applied. We hypothesize that the WT mice expressed higher levels of TGF‐B 1 as a result of natural repair processes. In contrast, RAGE KO mice lacked this pattern, suggesting that this receptor may play a role in the natural repair of condylar cartilage of the TMJ.
Improving the diagnostic approach to type I diabetes: the introduction of anti‐zinc transporter protein autoantibodies (ZnT8A) (835.15)
Authors: Zago Silvia , Fabris Martina , Liguori Marco , Trevisan Mariateresa , Zanatta Manuela , Comici Alberto , Zanette Giorgio , Tonutti Elio , Curcio Francesco
Published: June 2021

Aim of the study. To evaluate the prevalence and persistence of ZnT8A, in a cohort of Pediatric diabetic patients, to assess reliability and relationship with other T1DM diagnostic markers. Methods. Sera from 97 T1DM pediatric patients (53.6% M/46.4% F, age 11.1 ± 3.9 yrs), and 112 controls (51.8% M/48.2% F, age 9.6 ± 4.9 yrs; with no T1D or family history of autoimmune diseases) were analyzed for IgG ZnT8, GADA, IA2A and AIA by ELISA, and for IgG Islet Cell Antibodies (ICA) by an indirect immunofluorescence test on cryostat primate’s pancreas.

Results. ZnT8A (cut‐off 蠅 15 UA/ml) were present in 54.6% (53/97) T1DM patients (in two cases as the unique autoantibody) vs 97.3% (3/112) controls (p<0.0001). ZnT8A prevalence decreases among onset (25/40, 62.5%) and follow‐up patients with 5 or more years of disease (12/29, 41.3%). The prevalence of ICA at disease onset was 33.3% compared to 66.7% for GADA, 73.3% for IA2A, 73.3% for ZnT8A and 6% for AIA. Conclusions. The sensitivity (54.6%) and specificity (97.3%) of the ZnT8A in T1DM were in line with previous literature and comparable to those of the other major known diabetes autoantibodies. Even if ZnT8A do not persist as long as GADA after diagnosis, it represent a valid independent diagnostic marker for T1DM and its introduction in the Lab analysis is strongly recommended. In contrast, the sensibility of ICA appears unsatisfactory and its use as a screening test of limited value.
Identifying cytokines associated with acute and chronic Lyme arthritis (835.6)
Authors: Sridharan Ramya , Maduskie Victoria , Fawcett Paul
Published: June 2021

Symptoms of untreated infection with Borrelia burgdorferi, which is the causative agent of Lyme disease, frequently include arthritis, particularly of large joints. In pediatric patients, symptoms of arthritis typically resolve following treatment with an adequate course of antibiotics. However, in 10 to 20 percent of pediatric patients with Lyme arthritis, symptoms persist despite patients having received adequate treatment with antibiotics. Patients whose symptoms resolve in less than 6 months after initiation of antibiotic therapy are classified as having acute Lyme arthritis. Patients whose symptoms do not resolve within 6 months are classified as having chronic Lyme arthritis.

Cells were obtained from the synovial fluid from pediatric patients with both chronic and acute Lyme arthritis. These were analyzed to determine if cytokines produced by synoviocytes could be used to predict the outcome of treatment with antibiotics. Primary cell cultures were cultured in transformation media to change fibroblasts into adipocytes and osteocytes. Culture supernatants were then tested for cytokine concentration changes.

Results showed that transformation of synoviocytes caused significant variation of inflammatory cytokine concentrations associated with arthritis inflammation: MCP‐1 and IL‐6. IL‐6 rapidly declined in both adipogenic and osteogenic transformations. MCP‐1 also declined. Results confirm that transformation of synoviocytes from synovial fluid samples caused significant variation of inflammatory cytokine concentrations associated with arthritis inflammation.
Cytokines in asthma: effects on human pulmonary fibroblasts (835.8)
Authors: Lankala Shreya , Quinn Tim , Herndon Betty
Published: June 2021

In the asthmatic patient, CD4+ T lymphocytes elevate chemokines in airway fluids and blood. This study assayed effects of serum from asthmatic adults on the metabolism of healthy human lung fibroblasts in culture, comparing effects to those seen with sera from lung‐healthy volunteers. WI38 human lung fibroblasts (ATCC) were cultured in MEM + 15% fetal calf serum. At assay, 1500 cells in 100 µL were added to 96‐well plates, and after attachment, media was replaced with serum‐free MEM with 15 µL asthma or control sera. After 24 hr incubation, MTT formazan cell proliferation was assayed at 570 nm. Tests were repeated 3x.

MTT of 24 asthmatics averaged 1.37 ± 0.12 vs 1.28 ± 0.11 for 16 controls, p=0.03 with 3 replications. Sera with high IgE (ELISA) were compared to low by MTT values and were not different. Serum and breath condensate have recently been assayed for multiple chemokines but not for effects of these effluents on healthy lung cells. Our tests, while not identifying components demonstrate the expression of substances in asthma that raise the inflammatory response of healthy cells (measured by MTT upregulation). We showed the upregulation was significantly greater by asthmatic sera than by control sera. The use of asthma serum‐sensitized cells in culture offers high possibility of clarifying the disease of asthma and its humoral processes beyond cell‐related triggers.

Grant Funding Source: Sarah Morrison Student Grant to SL
Persistence of Streptococcus pyogenes in the lysosomal environment (836.9)
Authors: Snipper Joshua , No Hyewon , Till Andreas , Okumura Cheryl
Published: June 2021

Streptococcus pyogenes is a bacterial pathogen that can cause a range of infections from pharyngitis to necrotizing fasciitis. During an infection, the phagocytes of the host engulf the bacteria and fuse the phagosome with a lysosome to degrade the bacteria and prevent further infection. Previous experiments using a pH‐sensitive GFP construct demonstrated that while bacteria are localized to the lysosomal compartment of THP‐1 monocytes, the construct was not degraded. Because we determined the GFP construct was indeed pH‐sensitive, one possible explanation is that S. pyogenes is able to survive the low pH conditions of a lysosome. We tested the growth of S. pyogenes in different pH medias, ranging from pH = 4 ‐ 7. Our results indicated that within the pH range of a lysosome, bacteria persisted, but did not proliferate. Upon more detailed examination of the bacteria in a narrower pH range (pH = 4 ‐ 5), we found that bacteria were able to survive at pH = 4.5 ‐ 4.7. These results were corroborated with fluorescence microscopy of pH‐sensitive GFP‐expressing bacteria incubated in low pH media. Thus, S. pyogenes can survive the low pH‐environment of the lysosome. Our data do not however exclude the possibility that S. pyogenes can also prevent the acidification of the lysosome and future experiments will be aimed at measuring the pH of the lysosome in an infected cell as well as elucidating bacterial factors involved in pH resistance.
Gravin mutant mice show resistance to the development and progression of cardiac hypertrophy (837.12)
Authors: Singh Sonal , Diaz Diaz Andrea , Wijaya Cori , Suryavanshi Santosh , Fan Qiying , McConnell Bradley
Published: June 2021

Gravin, an A kinase anchoring protein is involved in the desensitization cycle of the β2‐adrenergic receptors (β2‐ARs) upon agonist stimulation via localized scaffolding of important β2‐AR pathway signaling components. Gravin scaffolds key proteins including protein kinase A (PKA), protein kinase C and other signaling molecules to the β2‐AR. The goal of this study was to evaluate whether disruption of PKA/gravin interaction restricts cardiac hypertrophy induced by chronic β‐AR stimulation with isoproterenol (ISO) (60mg/kg/day for 14 days) in gravin knockout (t/t) mice. Ascorbic acid (AA) was used as control. The heart to body weight (HW/BW) ratio, an indicator of hypertrophy, showed that WT‐ISO had a significantly higher ratio compared to WT‐AA whereas there was no significant difference between t/t‐ISO and t/t‐AA groups. Left ventricular contractility was significantly lower in the WT‐ISO compared to the WT‐AA as indicated by the decreased dp/dtmax values in the WT‐ISO group with no significant difference between the t/t‐ISO and t/t‐AA groups. Also no significant difference was found in the basal PKA activity between the WT‐AA and t/t‐AA groups. However there was a significant reduction in the PKA activity in the ISO treated groups compared to their respective controls. Thus, disruption of gravin signaling renders the t/t animals resistant to the development of cardiac hypertrophy.

Grant Funding Source: Source of research support was RO1HL085487
The interplay between heme oxygenase and nitric oxide synthase and downstream PI3K/sGC/ERK signaling ameliorates the estrogen‐dependent depressant effect of chronic nicotine on reflex bradycardia (837.2)
Authors: El‐Mas Mahmoud , El‐Gowelli Hanan , Fouda Mohamed , El‐Gowilly Sahar
Published: June 2021

Chronic nicotine impairs reflex bradycardia in estrogen (E2)‐replaced, but not depleted, female rats. We tested the hypothesis that nitric oxide synthase (NOS) and/or heme oxygenase (HO) and their downstream mitogen‐activated protein kinases mediate the nicotine‐baroreflex interaction. The depressant effect of nicotine (2 mg/kg/day i.p., 2 weeks) on reflex bradycardic responsiveness to phenylephrine in E2‐replaced ovariectomized (OVXE2) rats was abolished after treatment with hemin (HO inducer) or L‐arginine (NOS substrate), denoting the importance of reduced availability of their respective gaseous products, CO and NO, in the nicotine effect. The roles of CO and NO appear to be mutually facilitated because the hemin and L‐arginine effect was abolished after NOS (L‐NAME) and HO inhibition (ZnPP), respectively. Estrogen receptor blockade (ICI 182,780) reduced baroreflexes in OVXE2 rats but failed to abolish the improved baroreflex gain caused by hemin or L‐arginine in OVXE2/nicotine rats. Baroreflex facilitation by hemin was eliminated after inhibition of PI3K (wortmannin), sGC (ODQ) or ERK (PD98059) in central neurons, in contrast to no effect for the inhibition of p38 (SB203580) or JNK (SP600125). Together, the upregulation of NOS/HO‐coupled PI3K/sGC/ERK signaling downstream of E2 receptors rectifies the E2‐dependent, nicotine‐evoked impairment of reflex bradycardia in female rats.

Grant Funding Source: Supported by Grant STDF ID 502, Egypt
Resveratrol partly protect aged heart from reperfusion injury in rat (837.4)
Authors: Li Jing , Zhang Jincai , Tian Wencong , Yang Lei , Yang Liang , Qi Zhi , Tang Xiangdong
Published: June 2021

Objective: We aimed to compare the anti‐aging effects of 2‐deoxy‐D‐glucose (2‐DG) and resveratrol (RSV) using conventional dietary restriction (DR) as control.

Methods: Forty‐eight male SD rats were divided into 6 groups control group (Ctrl), 2‐DG group, RSV group, DR group, 2‐DG + DR group, and RSV + DR group. Parameters such as body weight (BW), left ventricular development of pressure (LVDP), and selected genes were determined and compared among different groups.

Results: Compared to the Ctrl animals, all DR groups experienced a sustained weight loss paralleled by a decrease in wet weight of liver, kidneys, and epididymal fat. By comparison, animals that fed on 2‐DG or RSV alone only dropped their body weight very briefly. DR, 2‐DG, and RSV all increased mRNA expression of MnSOD and catalase, accompanied by an increase in mRNA of FOXO1, Sirt2, and Sirt1. DR or RSV protected aged animals from reperfusion injury while 2‐DG worsened them as demonstrated by Langendorff measurements and cardiac histology. At cellular level, both 2‐DG and RSV increased mRNA expression of MnSOD and catalase and protected H9c2 cells from ischemia‐reperfusion injury.

Conclusions: DR and RSV could protect aged hearts from reperfusion injury, which is associated with mRNA up‐regulation of MnSOD and catalase accompanied by increased FOXO1, Sirt2 and Sirt1. 2‐DG does not seem to have any beneficial role in aged rats.

Grant Funding Source: supported by NSFC 81072629; Tianjin NSF 10JCYBJC14800; Project “973” grant 2010CB945001
Inhibition of endocannabinoid‐inactivating enzymes and cannabinoid (CB1) discrimination (838.1)
Authors: Bergman Jack , Kangas Brian , Leonard Michael , Nikas Spyros , Shukla Vidya , Alapafuja Shakiru , Makriyannis Alexandros
Published: June 2021

The discriminative‐stimulus (Sd) and other behavioral effects of CB1 ligands can be reproduced in mice by combined inhibition of the anandamide‐ (AEA‐) and 2‐arachidinoyl glycerol‐ (2‐AG‐) inactivating enzymes fatty‐acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL; Long et al. 2009). The present studies using selective and non‐selective ligands [FAAH (URB 597;AM 2018), MGL (AM 2036); mixed (AM 2129), and FAAH /CB1 (AM 3506) were conducted to further explore effects of mixed FAAH and MGL inhibition in squirrel monkeys that discriminate CB1 agonists (AM 4054;THC) from vehicle (n=4). Results indicate that: 1) mixed (but not FAAH‐ or MGL‐selective) inhibition or the combination of FAAH inhibition with some CB1 activity reproduced CB1‐related Sd effects, and 2) anandamide produced these effects after treatment with relatively low doses of non‐selective and FAAH‐selective enzyme inhibitors. Schild analysis of data from antagonism studies suggest some differences in the interaction of the several types of agonist effect with the CB1 receptor. The present data strengthen the idea that complementary, but not individual, actions of the endocannabinoids AEA and 2‐AG produce CB1‐related Sd, and perhaps subjective, effects in primate species.

Grant Funding Source: Supported by NIH/NIDA RO1‐31020
Effects of chronic Δ9‐tetrahydrocannabinol and ovariectomy on cannabinoid type‐1 and type‐2 receptors and receptor trafficking in the hippocampus of female rats (838.3)
Authors: Weed Peter , Filipeanu Catalin , Winsauer Peter
Published: June 2021

Previous research from this laboratory has shown that 40 days of chronic Δ9‐tetrahydrocannabinol (THC) administration in female rats during adolescence increased their adult sensitivity to the acute error‐increasing effects of THC on a learning and performance task. These error‐increasing effects of THC were correlated with increased cannabinoid type‐1 receptor (CB1R) levels in the hippocampus, a region of the brain associated with learning. To determine the direct effects of long‐term chronic administration of THC on cannabinoid receptors, we administered THC daily to rats for five months, beginning in adolescence. Female rats underwent either ovariectomy or sham surgery on post‐natal day 30, which was then followed by either saline or 5.6 mg/kg of THC daily. This yielded four treatment groups with respect to hormone status and THC administration (intact/saline, intact/THC, OVX/saline, OVX/THC). Hippocampal tissue was collected and analyzed by western blotting. Dependent measures included chaperone proteins responsible for maturation and trafficking of CB1R, such as heat shock protein 90 (HSP90) and its co‐chaperone activator of heat‐shock 90kDa protein ATPase homolog 1 (AHA1). The results indicated that OVX rats, irrespective of chronic treatment, had decreased total CB1R, CB2R, and AHA1 levels. In addition, brain‐derived neurotrophic factor (BDNF) and HSP90β levels were increased in OVX rats. Together, these results show that ovarian hormones directly modulate cannabinoid receptors and specific chaperone proteins in the hippocampus. These findings may help explain the many sexually dimorphic effects of the cannabinoids.
Spontaneous and rimonabant‐precipitated cannabinoid withdrawal in Δ9‐tetrahydrocannabinol and AM2389‐treated mice (838.5)
Authors: Chopda Girish , Nikas Spyros , Shukla Vidyanand , Makriyannis Alexandros , Paronis Carol
Published: June 2021

Cannabinoid (CB) withdrawal following exposure to THC has been described for human and nonhuman primates. Yet, despite several reports of antagonist‐precipitated CB withdrawal, spontaneous CB withdrawal has not been characterized in mice. Experiments compared the effects of rimonabant or vehicle in mice injected daily with THC or AM2389 (high efficacy CB agonist) using two indices of drug effect: increased paw tremors and decreased operant responding. For paw tremor studies, mice received 10‐20 mg/kg/day THC or 0.03‐1.0 mg/kg/day AM2389 for 5‐28 days and effects of vehicle or 1‐10 mg/kg rimonabant on observable behaviors (e.g. paw tremors) were evaluated at 4‐48 hr after the last CB agonist injection. Other mice, trained to respond (nosepoke) for 0.02 ml of sweetened milk, received 0.1 mg/kg/day AM2389 and the effects of AM2389, rimonabant, or vehicle on response rate were recorded. Results show increased paw tremors following vehicle injection at 24hr after AM2389 whereas rimonabant increased paw tremors at 4‐24 hr after AM2389 or THC injection, with some evidence that contextual cues may influence the expression of withdrawal. Similarly, both rimonabant and interruption of the daily dosing regimen decreased operant responding in AM2389‐treated mice. These results indicate that daily treatment with a high efficacy CB agonist will result in dependence that can be measured via spontaneous withdrawal effects.
Blockade of serotonin uptake by decynium‐22 enhances sociability (839.3)
Authors: Smolik Corey , Zhang Wynne , Vitella Melissa , Sanchez Jesus , Javors Martin , Koek Wouter , Daws Lynette , Gould Georgianna
Published: June 2021

Impaired social behavior occurs in several psychiatric disorders including autism, schizophrenia and depression. This symptom has proven difficult to treat with available pharmaceuticals. Based on clinical findings and experiments in rodents, serotonin (5‐HT) transmission is often disrupted in the socially‐impaired brain, and limbic regions and frontal cortex appear to be involved. Two drugs are commonly used to treat autism; risperidone curbs aggression but blunts social interaction, and fluoxetine improves social behavior somewhat, but its efficacy is diminished with reduced 5‐HT transporter (SERT) function. Our goal was to characterize the effects of blocking ancillary transporters of 5‐HT, with lower affinity but greater capacity than SERT to remove 5‐HT from extracellular fluid. These auxillary transporters, known as 'uptake 2' include organic cation and plasma membrane monoamine transporters. We utilized two models of impaired social behavior, inbred BTBR T+tf/J and SERT knock‐out (‐/‐) mice, to examine uptake 2 blockade effects on 5‐HT uptake and social behavior. The pseudoisocyanine decinium‐22 (D‐22) blocked 5‐HT uptake (Km = 92 +/‐ 12 nM) but had negligible affinity for SERT (Ki > 3000 nM) in hippocampal synaptosomes. In SERT ‐/‐ and BTBR mice, social behavior improved with acute D‐22 treatment at doses of 0.01 ‐ 0.1 mg/kg. This strategy may be an effective treatment for inpaired social behavior, and warrants further study.

Grant Funding Source: Supported by CDMRP Autism Idea Award AR110109, NIH MH086708 and NIH GM097632.
Nerves and immunity: reflexes in immunological homeostasis (84.2)
Authors: Tracey Kevin
Published: June 2021

The nervous system and the immune system co‐evolved to sense infection and injury, and to maintain physiological and metabolic homeostasis. Molecular products of infection, injury, inflammation, or metabolic stress activate sensory neurons to initiate neural reflexes. The activity of the descending (also termed “efferent” or “motor”) reflex arc modulates organ function to maintain homeostasis. The motor arc that suppresses inflammation is termed “the cholinergic anti‐inflammmatory pathway.” Recent advances have elucidated a prototypical reflex that regulates immunity, termed “the inflammatory reflex.” Efferent vagus nerve signaling controls immune function through molecular mechanisms that converge on the alpha 7 nicotinic acetylcholine receptor subunit expressed on mitochondria, which suppresses release of mitochondrial DNA, and inhibits inflammasome activity. Insufficient signaling or dysfunction of the inflammatory reflex results in excessive inflammatory responses to infection and injury. Clinical trials of stimulating the inflammatory reflex to treat rheumatoid arthritis have shown promise.
Cortricotropin‐releasing factor (CRF) injected into the brain or the periphery induces protection against indomethacin‐caused gastric injury through glucocorticoids and the CRF receptors types 1 and 2 (840.10)
Authors: Filaretova Ludmila , Mayzina Marina , Bagaeva Taniana , Morozova Olga
Published: June 2021

In the present study we investigated whether CRF injected into the brain or the periphery induces protection against indomethacin‐caused gastric injury and the role of glucocorticoids, the CRF receptor types 1 and 2 (CRF1 and CRF2 receptors) in the protection. Gastric injury was caused by indomethacin (35 mg/kg, subcutaneously) in preliminary fasted rats and CRF was injected 30 min before indomethacin administration. The participation of glucocorticoids was studied by inhibitor of glucocorticoids’ synthesis metyrapone, and glucocorticoid receptor antagonist RU‐38486. The role of the CRF1 and CRF2 receptors was investigated by the nonselective CRF1/CRF2 receptor antagonist astressin, the selective CRF1 receptor antagonist NBI 27914, and the selective CRF2 receptor antagonist astressin2‐B. Intracerebroventricular or intraperitoneal injection of CRF at the doses, which markedly increased plasma corticosterone levels, significantly suppressed the occurrence of gastric erosion induced by indomethacin. The protective effect of CRF on the gastric mucosa against indomethacin‐produced injury was significatly attenuated by metyrapone and RU‐38486 as well as by astressin, NBI 27914 and also astressin2‐B. The results suggest that CRF injected into the brain or the periphery may induce protection against indomethacin‐caused gastric injury through glucocorticoids, CRF1 and CRF2 receptors.

Grant Funding Source: Supported by grants from RFBR N13‐04‐01680‐a and Programs of Presidium RAS 7P
Enhanced host adaptive response prerequisite to prevent relapse during maintenance therapy of IBD (840.11)
Authors: Hahm Ki Baik , Park Jong Min , Kim Eun Hee
Published: June 2021

Oral administration of DSS in drinking water can induce acute and chronic colitis. Mice that developed colitis showed signs of diarrhea and bleeding within 6‐10 days after 3%‐10% DSS, but pathology of colitis and permeability change was not seen up to 4 days of administration, leading to question why intact colon was preserved in spite of damaging DSS. To answer question, we measured sequential changes of host adaptive phase 2 enzyme as well as damaging genes at 2, 6, 12, 24, 48, 72, 120, and 168hr of DSS. COX‐2 expressions were significantly increased after 120hr accordant with NF‐κB activations/IκBα inhibition. HO‐1 increased between 2‐6hr, NQO1 between 12‐24hr, γ‐GCS between 72‐168hr, of which were all associated with significant activations of Nrf2. Next, we challenged COX‐2‐/‐ and Nrf2‐/‐ mice with DSS to compare pathologic conditions. COX‐2‐/‐ mice showed milder colitis than wild‐type littermates, whereas Nrf2‐/‐ mice showed aggravated colitis followed with increased expression of COX‐2, but no expression of NQO1. Since phase 2 enzyme induction to cope with DSS challenge was essential, we administered oligonol, low molecular polyphenol, in recovered mice from previous DSS challenge and found significantly lower rates of relapse than control group (p<0.001). Our experiments signified the potentiating host adaptive response to prevent relapse of IBD under maintenance therapy.
Anti‐infammatory effect of Cucurbita maxima sweet seed extract on foregut induced injury: role of oxidative stress (840.2)
Authors: Pshyk‐Titko Irena , Zayachkivska Oksana
Published: June 2021

The established therapy of foregut disorders has resulted in incomplete efficacy, in long term use it is risky by unexpected effects, as altered microbial profile which elicits inflammation, increased oesophageal sphincters, hypokinetic gastric dysfunctions, inhibition of activity of dimethylarginine dimethylaminohydrolase (DDAH), which increase the risk of vascular inflammation (Ghebremariam YT, 2013) etc. In this study, to investigate effect of Cucurbita Maxima sweet seed extract (CMSE) on foregut mucosal integrity during blocking gastric acid by histamine H‐2‐receptor antagonist ranitidine (R), antagonist of the muscarinic acetylcholine receptors, atropine (A) given separately or in combination with the non‐selective blocker NOS L‐NAME.

Rats were used with without/with CMSE treatment (200 mg/kg) during 1 to 3 days A (3 mg/kg), R (100 mg/kg), L‐NAME (10 mg/kg) and their combination applying; oral, esophageal, gastric lesions by histology score index (HIS); NO by biochemical study, IL‐1ß, GRO/CINC‐1 via ELISA.

In rats R, A and L‐NAME pre‐treatment a significant accelerated production of IL‐1ß (14,3%) GRO/CINC‐1 ‐ 191,4%, rise of HIS vs control. NO content showed increasing tendency with maximum rise after 3 days during combination of triple blocking cholinergic, histaminergic and NOS via L‐NAME administration. CMSE decrease foregut lesions and signs of inflammation and IL‐1ß and GRO/CINC‐1 secretion.

Histamine and NO synthesis exert biphasic biological effect in foregut when in physiological dose are essential for natural functioning but in the high or low application seems to be a reason of cell and tissue damage. CMSE has ability to maintain foregut mucosal integrity normalizing redox system activity and inflammatory mediators.

Grant Funding Source: supported by prof. S. Szabo
Palmitoylethanolamide ameliorates development of colitis caused by injection of dinitrobenzene sulfonic acid in mice (840.3)
Authors: Cuzzocrea Salvatore , Campolo Michela , Impellizzeri Daniela , Paterniti Irene , Bruschetta Giuseppe , Cordaro Marika , Esposito Emanuela
Published: June 2021

Dinitrobenzene sulfonic acid (DNBS) induced colitis has proven to be a useful experimental model of intestinal bowel disease (IBD). N‐palmitoylethanolamide (PEA) is an endogenous fatty acid amide with important analgesic, anti‐inflammatory and neuroprotective effects.

The aim of the study was to examine the effects of a new formulation of PEA (PEA ultramicronized) in mice subjected to experimental colitis. PEA was administered daily i.p, (10 mg/kg, 10% ethanol). Four days after DNBS, colon nuclear factor NF‐ kB was increased as well as TNF‐a and IL‐1b production, neutrophil infiltration and adhesion molecules expression. Immunohistochemistry for inducible nitric oxide synthase (iNOS), nitrotyrosine and poly (ADP‐ribose) polymerase (PARP) showed an intense staining in the inflamed colon. PEA significantly reduced the appearance of diarrhea and body weight loss. This was associated with a significant reduction of MPO activity, NF‐kB, pro‐inflammatory cytokines release, iNOS, nitrotyrosine and PARP and reduced up‐regulation of ICAM‐1, P‐selectin and metalloproteinases. Thus, the results of this study suggested that administration of PEA may be beneficial for treatment of intestinal inflammatory diseases.
Involvement of reactive nitrogen species production in diazoxide‐induced preconditioning in cultured cortical neurons of rats (841.1)
Authors: Dutta Somhrita , Rutkai Ibolya , Katakam Prasad , Busija David
Published: June 2021

Diazoxide (DZ), a mitochondrial ATP‐sensitive potassium (mito K‐ATP) channel opener, has been shown to be protective in models of brain ischemia. We have previously shown that reactive oxygen species production and mitochondrial depolarization are major initiating steps in DZ preconditioning, but the signaling pathway is incomplete. We hypothesized that other signaling pathways, like those involved in RNS production, may participate in the protective effects of DZ. Confluent rat primary neuronal cultures were pre‐treated with DZ (500µM) alone or co‐treated with DZ+ Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME, 100µM or 150µM) for 3 days to block nitric oxide (NO) synthase. Additionally, NO was exogenously supplied by co‐applying SNP (5µM) with DZ and L‐NAME to potentially rescue protection by DZ. After the 3 day treatment period, neurons were subjected to 3 h oxygen glucose deprivation (OGD) followed by re‐oxygenation, an in vitro model of stroke. Neuronal viability was assessed using the MTS colorimetric assay 24 h after OGD. OGD reduced neuronal viability 24 h from 100% (time control) to 54.1±1.4% (P<0.05; n=32 wells). DZ protected cells and increased viability after OGD (67±2.4%, P<0.05; n=32 wells). Blocking RNS using L‐NAME 100µM and 150µM decreased viability to the untreated control level (53.2±3.9% and 50.6±3.9% respectively, NS compared to time control; n=32 wells). Replacing NO using SNP rescued the effect of L‐NAME treatment at both doses (63.5±6.3% and 60.8±6.5%, respectively) (NS compared to time control; n=32 wells). This study shows for the first time that RNS production is a crucial step to preconditioning using DZ in neurons.

Grant Funding Source: HL‐077731, HL‐030260, HL‐065380, and HL‐093554
A calcium‐independent PKC isoform mediates cerebral vasoconstriction through actin cytoskeleton reorganization (841.2)
Authors: El‐Yazbi Ahmed
Published: June 2021

The role of PKC in force production and vasoconstriction in resistance arterioles is hypothesized to be through several mechanisms including: increased calcium influx, calcium sensitization, and thin filament regulation. PKC involvement in these pathways was proposed based on pharmacological experiments without direct biochemical measurement of the phosphoproteins. We studied the effect of phorbol dibutyrate (PDBu, 1 microM) on rat middle cerebral artery in a pressure myography setup. A slow profound constriction was obtained as a result of PDBu treatment. The constriction was associated with increased myosin light chain (LC20) phosphorylation that was similar to that evoked by a maximal dose of 5‐HT producing a smaller constriction. PDBu constrictions were abolished by GF109302X, but neither with a calcium‐dependent PKC inhibitor, nor with calcium‐free Krebs’, though the latter reduced LC20 phosphorylation. There was no evidence of increased phosphorylation of caldesmon and calponin, ruling out a role for thin filament regulation. G‐actin content was greatly reduced in both regular and calcium‐free Krebs’ solution together with an increase in paxillin phosphorylation in both conditions implicating actin cytoskeleton reorganization as a potential mechanism contributing to force generation independent of LC20 phosphorylation following PKC activation.

Grant Funding Source: Supported by CIHR MOP‐97988
Preserved mitochondrial function in cerebral arteries following ischemia‐reperfusion in the rat: the role of endothelium (841.7)
Authors: Rutkai Ibolya , Dutta Somhrita , Walter Korey , Katakam Prasad , Busija David
Published: June 2021

Mitochondrial function following IR in the cerebral circulation has not been examined. We examined the effects of IR on the mitochondrial‐endothelial axis in middle cerebral arteries (MCAs) exposed to 10‐100 μM of diazoxide (DZ), a mitoKATP opener, and 10 μM of bradykinin (BK) from Sprague‐Dawley rats exposed to 90 min of ischemia by the occlusion of the MCA followed by 4 and 48 h of reperfusion. At both 4 and 48 h, responses to BK were reduced on the ischemic (IPSI) compared with the non‐ischemic (CONTRA) sides whereas DZ responses at 4 h were normal on both sides compared with Control. At 48 h, DZ responses were substantially reduced on the CONTRA compared with IPSI (5.4±2 μm vs 22.6±3 μm at 50 μM; n= 22) and Control (21.1±2 μm). At 48 h, eNOS phosphorylation and mitochondrial mass, determined by western blot for voltage dependent anion channel, Complex V, and mitoDNA were greater on the IPSI compared with CONTRA. Endothelium denudation completely eliminated the response to DZ in IPSI but not in Control, indicating that endothelial mechanisms predominate in previously ischemic arteries. Our findings suggest that targeting the mitochondria in endothelium in cerebral arteries may be a new therapeutic target in stroke treatment for improving blood flow. Our data also revealed for the first time that non‐ischemic cerebral arteries are compromised by IR occurring at a distant site in the brain.

Grant Funding Source: NIH grants HL‐077731, HL‐030260, HL‐065380, and HL‐093554.
Enhanced in vitro cancer cell cytotoxicity of dual ligand‐targeted liposomes (842.10)
Authors: Sriraman Shravan Kumar , Torchilin Vladimir
Published: June 2021

A broad variety of cancer cells over‐express different types of cell surface receptors like transferrin (TfR) and folate (FR) receptors compared to normal cells. This receptor expression increases as the tumor progresses and grows aggressively. With this in mind, polyethylene glycol (PEG)‐coated long‐circulating liposomes targeted with varying densities of transferrin and folic acid were loaded with doxorubicin (Dox), a frequently used chemotherapeutic drug.

Purpose: To enhance the uptake of drug‐loaded liposomes by cancer cells using the combination of these two tumor‐specific ligands resulting in increased cytotoxic effects even at low drug concentrations.

Methods: Transferrin and folic acid were conjugated to the distal ends of the PEG chain and post‐inserted into liposomes made of eggphosphatidylcholine (ePC), cholesterol and dioleoylphosphatidylethanolamine (DOPE). For cell association studies, the liposomes were fluorescently labeled with rhodamine. The optimized formulations were loaded with Dox, and their in vitro cytotoxic potential was evaluated with human cervical cancer (HeLa) cells.

Results: Flow cytometry demonstrated a 7‐fold increase in cell association of the dual ligand‐targeted liposomes (DL) compared to any single ligand‐targeted liposomes (SL). The association and subsequent internalization of the DL was further confirmed using the confocal microscopy. The DL also significantly increased cytotoxicity of Dox when compared to SL and non‐targeted Dox‐liposomes in vitro. The IC50 for DL was also decreased by almost 7‐fold compared to SL.

Conclusions: Simultaneous targeting of TfR and FR has been shown to be an effective strategy to deliver drugs and enhance their cytotoxic effects on cancer cells in vitro. The decrease in IC50 of DL allows us to work with lower drug concentrations thereby reducing the off‐target drug toxicity.

Grant Funding Source: 86R01CA128486
Stable knockdown of LDH‐A in MDA‐MB‐231 breast cancer cells does not affect cell survival or lactate production (842.14)
Authors: Mack Nzinga , Mazzio Elizabeth , Soliman Karam
Published: June 2021

A well‐known characteristic of tumor cells involves the propensity to produce lactate under normoxia and hyperoxia, also referred to as the Warburg effect. High levels of lactate are believed to be associated with elevated expression of lactate dehydrogenase A (LDHA), also up‐regulated in diverse aggressive malignancies. In this study, ShRNAs were used to permanently silence the expression of LDHA in human breast cancer MDA‐MB 231 cells using five different strains of lentaviral vectors. Cells were cloned,selected and evaluated for LDHA gene and protein expression using diverse methodologies. The data suggest that [1] LDHA is the predominant form of LDH in the transcriptome of MDA‐MB 231 cells [2], cells can survive with stable silenced transduction and [3] silencing LDHA does not correlate to halted production of lactic acid. These findings show a lack of correlation between LDHA and lactate suggesting there is likely another dominant pathway involved with maintaining lactate production in tumor cells.

Grant Funding Source: Supported by NIMHD G12 MD007582 and P20 MD006738
Involvement of PI3K/AKT and beta‐catenin signaling in anti‐cancer activity of 7‐O‐succinyl macrolactin A (842.4)
Authors: Park Su‐Young , Kim Mi‐Young , Kim Dong‐Hee , Kim Jung‐Ae
Published: June 2021

Colorectal cancer is the 4th most common cancer and the second most common cause of cancer‐related death in the world. One of the most commonly involved pathways in cancer cell survival, growth and proliferation is the PI3K/AKT/mTOR pathway. In addition, Wnt/beta‐catenin signaling pathway has been implicated in colorectal tumorigenesis by upregulating genes related to cell proliferation such as c‐Myc and cyclin D1. In the present study, we investigated anti‐cancer activities of macrolactin A (MA) and its derivative, 7‐O‐succinyl macrolactin A (SMA) was mediated through PI3K/AKT/mTOR and beta‐catenin signaling. In the growth‐arrested HT‐29 cells by SMA, which was revealed by proliferation and colony formation assays, phosphorylation of PI3K/AKT/mTOR was suppressed. In addition, SMA inhibited Wnt‐induced beta‐catenin nuclear translocation and TCF/LEF transcriptional activity. Western blot analyses showed that SMA inhibited serum‐induced c‐Myc, cyclin D1, and survivin expressions in colon cancer cells. Taken together, the results suggest that anti‐cancer activity of SMA was mediated through both PI3K/AKT signaling and beta‐catenin pathway in colon cancer cells.
Effects of selective inhibitors of the isoprenoid biosynthetic pathway on androgen‐dependent and independent prostate cancer cells (842.6)
Authors: Reilly Jacqueline , Zhou Xiang , Tong Huaxiang , Wiemer David , Hohl Raymond
Published: June 2021

The isoprenoid biosynthetic pathway (IBP) provides substrates, such as geranylgeranyl diphosphate (GGDP), which are used to post‐translationally modify proteins that are key regulators of malignant cell properties such as proliferation, migration, and invasion. Clinically relevant IBP inhibitors, including statins and nitrogenous bisphosphonates, affect these malignant cell properties. However, they broadly deplete all mevalonate derivatives, thus affecting farnesylated proteins and cholesterol. XZ‐1‐269 and XZ‐4‐37 were developed as selective inhibitors of GGDP synthase to reduce protein GGation. In the androgen‐dependent prostate cancer (PCa) cell line LNCaP and the highly metastatic androgen‐independent PCa cell line PC3, lovastatin and XZ‐1‐269 more potently reduce protein GGation and cell viability than digeranyl bisphosphonate, XZ‐4‐37, and zoledronate. In the androgen‐independent PCa cell line 22Rv1, all compounds are less potent in reducing protein GGation and cell viability than in LNCaP and PC3 cells. Our findings detail novel compounds for selectively reducing protein GGation in three prostate cancer cell lines. Further work defining the isoprenoid proteins involved in advancing PCa will identify new targets for therapeutic intervention.

Grant Funding Source: Supported by NIH Pharmacological Sciences Training Grant T32 GM067795