bluuebunny/crossref_metadata_2025 huggingface dataset:
Renin as a mediator of pulmonary damage caused by fat embolism and LPS
Authors: Poisner Alan , Herndon Betty , Al Hariri Ahmad , Qin Chang , Quinn Tim , Molteni Agostino
Published: June 2021

Histological lung damage in the model of fat embolism (i.v. triolein) is ameliorated by agents acting on the renin‐angiotensin system (RAS), with pulmonary damage persisting up to 6 wks. Here we report the effect of triolein on the localization of pulmonary renin at various times and its influence on a second hit by a pulmonary toxicant, LPS. Groups of rats were injected i.v. with either saline or triolein and sacrificed at times from 24 hrs to 6 wks. At 6 wks, groups received 3 mg/kg LPS or saline, i.p. 48 hrs later, lungs were studied. Renin was localized with a rat‐specific antiserum (from T. Inagami) and stained with standard immunochemical methods. Following triolein, counts of renin positive cells increased in lungs at 24 hrs and 48 hrs, (p <0.01 and < 0.04) followed by a decline to 3 and 6 wks. LPS (6 wks after triolein) again increased renin staining with cells mostly found in the septa around arteries and the bronchial smooth muscle layer. Since the effects of LPS on the lung are also known to be modified by agents interfering with the RAS, this study suggests that enhanced expression of renin in pulmonary cells relates to triolein; LPS at 6 wk recapitulates the lung damage caused by the initial triolein insult. The data suggest that the sequence of pathological changes after fat embolism includes lipid uptake by lung macrophages, mast cell activation, renin production and angiotensin II generation.

Geldmacher Foundation
Effect of low level laser therapy on lung mechanics and inflammatory response
Authors: Cury Vivian , Lima Thais , Ariga Suely , Barbeiro Denise , Pinheiro Nathalia , Prado Carla Maximo , Moretti Ana Iochabel , Souza Heraldo Possolo
Published: June 2021

Low level laser therapy (LLLT) is currently prescribed as an adjuvant therapy for inflammatory diseases, however its use in internal viscera is controversial. Therefore, we assessed the effect of LLLT on acute lung injury (ALI) induced by intratracheal LPS instillation. C57 black mice (n=10) were exposed to intratracheal LPS (5mg/kg) or PBS. Six hours after instillation, both groups (PBS and LPS) were irradiated with laser at 660 nm, power output 30mW, fluency 10J/cm2 Twenty‐four hours later, bronchoalveolar lavage was collected; lungs were submitted to functional analysis and quantitation of tumor necrosis factor (TNF‐α), interleukin (IL) 1 β and intercellular adhesion molecule 1 (ICAM ‐1).

LPS instillation induced infiltration of large number of inflammatory cells to distal airways, which was reduced by LLLT (2.92±0.34 vs 1.63±051 mononuclear cells; 2.25± 0.84 vs 0.43±0.19 neutrophils; p<0.05). We also observed that LLLT was efficient in reducing secretion of TNF‐α, IL‐1β and ICAM‐1. In spite of reducing inflammation, LLLT treatment did not affect lung mechanical properties, since no change was observed in tissue resistance or elastance.

Therefore, we can conclude that LLLT is able to reduce inflammatory reaction in lungs exposed to LPS, without affecting the pulmonary function and recovery.
Dynamic proteomic analysis of extracellular matrix in the bleomycin‐induced fibrotic lung
Authors: Decaris Martin , Gatmaitan Michelle , FlorCruz Simplicia , Luo Flora , Li Kelvin , Hellerstein Marc , Turner Scott , Emson Claire
Published: June 2021

Idiopathic pulmonary fibrosis (IPF) is characterized by excess deposition of extracellular matrix (ECM), and ultimately organ failure. We have developed a “dynamic proteomics” platform which quantifies the synthesis rates of hundreds of proteins via stable isotope labeling and LC/MS. Here we present the first quantitative analysis of lung ECM protein synthesis in a mouse model of IPF. Mice were dosed with bleomycin and labeled with heavy water. Lung tissue was subjected to sequential protein extraction to enrich for ECM. We identified two kinetically distinct pools of ECM proteins in the lung based on guanidine solubility. Fibrillar collagens, basement membrane proteoglycans and microfibrillar proteins were observed in both pools. The turnover rate of ECM proteins in control lungs was generally higher in the guanidine soluble pool suggesting that the insoluble pool reflects older, more cross‐linked matrix components. In bleomycin treated lungs there was a significant increase in the synthesis of soluble and insoluble ECM proteins. These data show the utility of dynamic proteomics in identifying metabolically distinct protein pools and detecting modulations in protein synthesis associated with disease.
Epoxyeicosanoid regulation of tumor lymphangiogenesis
Authors: Mudge Dayna , Bielenberg Diane R. , Serhan Karolina , Hwang Sung Hee , Hammock Bruce D. , Kieran Mark W. , Panigrahy Dipak
Published: June 2021

Epoxyeicosatrienoic acids (EETs) are lipid autacoids biosynthesized by cytochrome P450 epoxygenases. We recently demonstrated EETs stimulate multiorgan and lymph node metastasis. However, the mechanism of EETs in tumor metastasis is unknown. We hypothesize that EETs stimulate lymph node metastasis via tumor lymphangiogenesis. Using two murine tumor lines, B16F10 melanoma and T241 fibrosarcoma, each transfected with VEGFC we demonstrate EETs stimulate primary tumor growth via lymphangiogenesis. Systemic administration of 14,15‐EET and/or 20‐HETE accelerated growth of both primary tumors. Conversely, systemic administration of EET antagonist (14,15‐EEZE) and/or 20‐HETE antagonist (HET0016) regressed established primary B16F10‐VEGFC tumors. Immunohistochemistry studies revealed an increase in podoplanin‐positive and LYVE‐1‐positive vessels in tumors treated with 14,15‐EET. 14,15‐EET and 11,12‐EET stimulated lymphatic endothelial cell proliferation, viability and migration, as well as increased the production of VEGFC by tumor cells. 14,15‐EEZE and/or siRNA to VEGFR3 inhibited spontaneous lung metastasis and the combination showed additive inhibition. EETs and 20‐HETE stimulate tumor lymphangiogenesis, offering a mechanistic rationale for using EET and 20‐HETE antagonists as novel anticancer therapeutics to inhibit lymph node metastasis and tumor lymphangiogenesis.
Adipocyte morphometric evaluation in a model of DMBA administration
Authors: Costa Eduardo , Dias João , Pereira Paula , Cabrita António Silvério
Published: June 2021

Dimetilbenzantracene is a carcinogenic compound present in several products.

168 Sprague Dawley female rats with 42 days were randomly distributed for seven groups. Besides the group I (control), all the treatment groups were submitted to the 1,12‐dimetilbenzantracene (DMBA). Group I (control) was fed with a standard diet (SD). Group II was submitted to the SD. Group III, was submitted to the SD supplemented with beans. The Group IV was submitted to the SD supplemented with olive oil. The group V was submitted to the SD enriched with fibres. Group VI was submitted to the SD supplemented with fibers, beans and olive oil, and the group VII was submitted to the SD supplemented Mediterranean diet. The experience was 150 days long, and at the end all animal were sacrificed.

The slides of perirenal adipose tissue, stained by HE, were evaluated for morphometric changes, using the software ImageJ.

The adipocytes area were classified according to the following criteria: medium adipocytes, when the value varies between the range [μ−∂; μ+∂], big adipocytes, varies between ]μ+∂; μ+2∂], small adipocytes, varies between ]μ−∂; μ−2∂], very big adipocytes, the value is more than μ+2∂ e very small adipocytes, the value is less than μ−2∂.

In the control group the distribution of each class was significantly different from the observed in test group. This differences suggest that the diet may interfere with the adipocyte volume.
Alterations of arginine in Lysinuric Protein Intolerance (LPI) macrophages
Authors: Barilli Amelia , Rotoli Bianca Maria , Visigalli Rossana , Dall'Asta Valeria
Published: June 2021

Lysinuric Protein Intolerance (LPI) is an inherited transport defect of cationic amino acids (arginine, lysine and ornithine) transport at the basolateral membrane of intestinal and renal tubular cells caused by mutations in SLC7A7 encoding for System y+L‐related y+LAT1 protein. The severe clinical course of this disorder suggests that LPI should be considered a severe multisystem disease with features proper of ‘metabolic’ disorders. In particular, immune dysfunction could be attributed to an altered metabolism of nitric oxide (NO) secondary to abnormal arginine intracellular metabolism. To address this hypothesis we measured arginine transport/metabolism in monocyte‐derived macrophages (MDM), obtained from peripheral blood monocytes isolated from 5 LPI patients. In these cells the efflux of 3H‐arginine through System y+L was markedly lower than in normal MDM, with a decrease that ranged from 60 to 75%. Moreover, SLC7A7 silencing in a human monocytic cell line (THP‐1 cells) caused a significant increase of intracellular arginine content. These results indicate that arginine entrapping takes place in LPI monocytes‐macrophages, supporting the hypothesis that NO overproduction observed in vivo may be due to an abnormal intracellular metabolism of arginine.

Supported by CLIMB (Children living with inherited metabolic diseases, Crewe, UK)
Influence of NADPH oxidase inhibitor apocynin on cardiac structure and function in rats with aortic stenosis
Authors: Bonomo Camila , Martinez Paula Felippe , Lima Aline Regina Ruiz , Damatto Ricardo Luiz , Cezar Marcelo Diarcadia Mariano , Pagan Luana Urbano , Gomes Mariana Janini , Guizoni Daniele Mendes , Okoshi Katashi , Okoshi Marina Politi
Published: June 2021

The aim of this study was evaluate the influence of treatment with the NADPH oxidase inhibitor apocynin on cardiac structures and function in rats with aortic stenosis (AS).

Methods

AS was induced by median thoracotomy by placing a silver clip around ascending aorta. Twenty weeks later, rats were divided into two groups: AS without treatment (n=11), and AS treated with apocynin (AS‐A, 16 mg/kg/day in drinking water for 8 weeks, n=11). Rats subjected to simulated surgery were used as controls (Sham, n=9). Cardiac structures and left ventricular (LV) function were evaluated by transthoracic echocardiogram before and after treatment.

Statistical analysis

ANOVA and Bonferroni.

Results

Echocardiogram parameters did not differ between AS and AS‐A before treatment (p>;0.05). After treatment, both AS groups presented left cardiac chambers dilation. Systolic mitral annular velocity of lateral (Sham 3.10±0.53; AS 2.32±0.44; AS‐A 2.88±0.66; p=0.01) and septal (Sham 3.20±0.76; AS 2.33±0.57; AS‐A 2.54±0.62; p=0.01) wall (tissue Doppler) was decreased in AS compared to Sham and did not differ between AS‐A and Sham or AS groups.

Conclusion

Treatment with the NADPH oxidase inhibitor apocynin attenuates cardiac systolic dysfunction in rats with aortic stenosis. Support: FAPESP, CAPES, CNPq.
Inhibition of Pim1 expression and activity impairs the proliferation of rat bone marrow mesenchymal stem cells
Authors: Jiang Xiaofan , Shen Zhihua , Ding Ranran , Zheng Shaojiang , Li Rujia , Jiang Hanguo , Guo Junli , Jie Wei
Published: June 2021

Bone marrow mesenchymal stem cell (BMMSC) is one of the ideal seed cells in stem cell‐based regenerative medicine such as the myocardial infarction repairing; however, most of the infused BMMSCs would expire within 48h post cell transplantation. To further explore the mechanism involved in regulating the life span of BMMSC itself would be beneficial to understand the stem cell‐based regenerative medicine. Pim1 is one of the serine/threonine kinases involved in cell growth, however, little is known about the role of Pim1 in the proliferation of BMMSC. Here, Immunoblot and quantitative RT‐PCR revealed that Pim1 was decreased in SD rat BMMSCs induced by TGF¦Â1 (5 ng/ml) for 7 days while compared with the cells in the undifferentiated status. Treatment the cells with Pim1 inhibitor, quercetagetin (5.5 ¦ÌM), led to Pim1 changes in its activity but not expression; Transduction the cells with Pim1 shRNA resulted in obvious decrease of Pim1 expression and activity. Treatment with both the quercetagetin and Pim1 shRNA led to significant decrease in cell viability and cell numbers in day 7, and the proportion of G0/G1 in cell cycles also increased after such treatment for 7 days. Thus, our results indicate that the maintenance of Pim1 expression in rat BMMSC, though in a low level, is crucial for cell proliferation. Our results support the rational that Pim1 may be as a potential molecular target in stem cell‐based regenerative medicine.
HIV‐1 gp120 confers a pro‐inflammatory phenotype to macrophages changing the role of the alpha7 acetylcholine receptor
Authors: Delgado Manuel , Baez Carlos , Gerena Yamil , Quesada Orestes , Santiago Laura , Wojna Valerie , Melendez Loyda , Silva Walter , Lasalde‐Dominicci Jose
Published: June 2021

Human immunodeficiency virus (HIV‐1) remains a public health threat. The introduction of combined antiretroviral treatments has markedly increase life expectancy but inflammation has remained unsolved. Recent studies, in control subjects, link macrophage's alpha7 nicotinic acetylcholine receptor (α7‐nAChR) activation with anti‐inflammatory response. This response normally prevents exaggerated immune responses through the cholinergic anti‐inflammatory pathway (CAP) which inhibits the release of pro‐inflammatory cytokines through the activation of the α7‐nAChR. Nonetheless, little is known if this neuroimmune regulation is affected in HIV‐1 infected patients. Here we examined the secretion of 9 cytokines and chemokines from control subjects exposed to HIV gp120 envelope protein to determine if the CAP is disrupted. We found that HIV gp120 induce α7‐nAChR upregulation and a pro‐inflammatory phenotype in macrophages as demonstrated by the inability to control the release of 8 of 9 pro‐inflammatory cytokines. These results suggest that HIV mitigates the natural strategy to control inflammation by promoting the upregulation of α7‐nAChR, a key player in the CAP, contributing to one crucial, unresolved problem of HIV infection despite therapy: inflammation. Sponsored by NIH‐NINDS Specialized Neuroscience Program U54NS0430311
Small Intestine Could be a Major Organ Contributing to Systemic Inflammation and Cardiovascular Complications.
Authors: Navab Kaveh Daniel , Karimi Nika , Farahmand foozhan , Farazmand Maryam , Czerwieniec Gregg
Published: June 2021

Objective

To determine the role of intestine in systemic inflammation.

Introduction

The small intestine receives various ingredients from food and water and the interplay between them, luminal microbiota and the enterocytes appears to play role in many digestive interactions.

Objective

we sought to determine if in a mouse model of inflammatory stress a high fat diet would affect the circulating levels of 12‐ hydroxyeicosatetraenoic acid (12‐HETE) an inflammatory metabolite of arachidonic acid and of serum amyloid A (SAA) a major marker of inflammation.

Methods

Male ApoE deficient mice (n=10 per treatment group) were maintained on the laboratory chow or on a Western Type Diet containing 1.25% cholesterol for two weeks. Blood was removed from fasting mice according to UCLA IRB guidelines, plasma was separated and analyzed using a commercial ELISA system.

Results

12‐HETE an SAA levels were significantly higher in the circulation of the mice on the high fat diet (p=0.012 and p=0.023 respectively. These molecules have systemic effect and their high levels would result in a cascade of interactions producing high levels of other inflammatory molecules.

Conclusion

dietary factors such as lipids can affect intestine and result in elevated levels of inflammatory components. Systemic inflammation brought about by dietary ingredients can in turn produce pathological conditions in various organs and throughout the organism. This emphasizes the importance of the small intestine and the dietary habits in general health and well being.
Effect of a Humanized Anti‐Cocaine Monoclonal Antibody on the Pharmacokinetics of Cocaine in Rats and Mice
Authors: Gooden Felicia CT , Tabet Michael R , Bartley Erin L , Ball William J , Norman Andrew B
Published: June 2021

Monoclonal antibody (mAb) h2E2 is a further humanized version of the high‐affinity anti‐cocaine mAb 2E2. h2E2 is a potential immunotherapeutic for cocaine abuse that retains the same high‐affinity (4 nM Kd) and specificity as 2E2 for cocaine over its inactive metabolites. Infused h2E2 (1.6 μmol/kg i.v.) in both mice and rats produced dramatic increases in the area under the plasma cocaine concentration‐time curve (AUC) and a concomitant decrease of >;90% in the brain AUC following an i.v. injection of an equimolar cocaine dose. Consequently, h2E2 substantially reduced cocaine's volume of distribution in both species by sequestering cocaine in plasma, indicating potential clinical efficacy. Furthermore, h2E2 did not inhibit the clearance of cocaine in either rats or mice. Interestingly, h2E2 restricted cocaine distribution and metabolism from plasma to a single‐compartment model in mice but cocaine disappearance remained multi‐compartmental in rats. This differential effect of h2E2 on cocaine distribution between species may be related to the metabolism of cocaine predominantly to ecgonine methylester (EME) in mice but to benzoylecgonine (BE) in rats. The metabolism of cocaine to BE and EME in humans is between that in mice and rats. Thus, these studies will help determine whether differences in cocaine metabolism are relevant to h2E2's clinical efficacy.

Supported by NIH grant DP1DA031386.
Acetate alters purinergic enzyme and receptor levels in glial cultures
Authors: BHATT DHAVAL , ROSENBERGER THAD ALLEN
Published: June 2021

Acetate supplementation increases brain acetyl‐CoA and histone/non‐histone protein acetylation that attenuates neuroglia activation and pro‐inflammatory cytokine release in rat models of neuroinflammation and neuroborreliosis. Because adenosine (ADO) and ATP are known to modulate neuroglia activation, we postulate that an anti‐inflammatory effect of acetate may involve altering purinergic signaling. To begin to test this hypothesis, we quantified the effect of acetate on purinergic enzymes and receptors in BV‐2 microglial cell cultures treated with lipopolysaccharide (LPS). Cultures were treated with sodium chloride (NaCl), sodium acetate (NaAc), NaCl+LPS, and NaAc+LPS for 12, 24, and 48 hr. The level of enzymes involved in ADO formation [ecto‐5′‐nucleotidase (CD73), ecto‐apyrase (CD39)], removal [ADO deaminase (ADA), ADO kinase (AK)], and ADO receptors (A1 and A2A) were measured using Western blot analysis. We found that at 48 hr, acetate prevented the LPS‐induced 2‐fold increase in CD73, AK, and A2A receptor when compared to controls. Further, ADA and A1 receptor levels were increased to 1.3‐ and 1.5‐fold with LPS which were reduced to 0.5‐ and 0.7‐fold below control levels with acetate. These results suggest that acetate supplementation and an increase in acetyl‐CoA metabolism may modulate purinergic signaling in BV‐2 microglia. This work was supported by a grant from NIH/NIGMS, P30GM103329.
Identification of three distinct calmodulin‐binding domains in the G Protein‐coupled Estrogen Receptor
Authors: TRAN KIM , VerMeer Mark
Published: June 2021

The novel G protein‐coupled estrogen receptor (GPER/GPR30) is being increasingly identified as an important GPCR in many organ system. Calmodulin (CaM) is a ubiquitous Ca2+ transducer that is required for the activities of numerous cellular proteins. Here we show that CaM coimmunoprecipitates with GPER in primary vascular endothelial cells upon stimulation with 17β‐estradiol or Ca2+‐elevating ionophore. To identify the CaM‐binding sites in GPER/GPR30, we have developed a series of novel FRET‐based biosensors whose responses allow identification and highly quantitative characterization of CaM‐binding properties of CaM‐binding domains in GPER/GPR30. Three separate CaM‐binding domains with distinct dissociation constants and Ca2+ sensitivities for complex formation with CaM were found. The functional implications of CaM‐GPER interactions are explored.
Effects of some angiotensin‐converting enzyme inhibitors and angiotensin II receptor blockers on experimental inflammations
Authors: Magyar Ioan , Cret Mihai , Cuparencu Barbu
Published: June 2021

Method

The experiments were carried out in Wistar albino rats, weighing 150–200 g. The inflammations were induced as folows: 1. the hind paw edema evoked by the intraplantar injection of 0.1 mL from a 10% aqueos kaolin suspension; the measurements of the paw volume increases were performed plethismometrically; 2. the insertion of a cotton pellet, weighing 50 mg into the scapulo‐humeral region. The following bood inflammatory markers were measured: fibrinogen and C‐reactive protein (CRP). The tested drugs by intraperitoneally route were: captopril (0.4–4.0–6.0 mg), enalapril (0.2–2.0–4.0 mg), lisinopril (0.2–2.0–4.0 mg), quinapril (0.2–2.0–4.0 mg) and valsartan (4.0–8.0–16.0 mg). The statistical evaluation was carried out by chi square and “size effect” procedures.

Results

All ACEI exhibited in a dose‐dependent and a time‐related manner a proinflammatory activity. The most active compounds were: quinapril and enalapril and lisinopril. Valsartan was ineffective. Fibrinogen and CRP blood levels were increased by all ACEI. However, no clear dose‐response relationships might be seen.

Discussion

Conclusion

From these data it may be inferred that the proinflammatory activity of ACEI was due to an accumulation of bradykinin, a phlogogenic peptide. It is well known that the converting enzyme, besides its splitting effect of angiotensin I, leading to the genesis of angiotensin II has a kininase activity.
Acute nuclear alternation effects of chemiotherapy in buccal mucosa cells in cancer patients
Authors: Alidema Fitim Bexhet , Osmani Behxhet H , Boçari Gezim G , Alidema Majlinda Ilmi , Kryeziu Ilmi Veli , Alidema Mirzade Bexhet
Published: June 2021

The aim of the present study was to evaluate the sensitivity of cancer patients (C 34 Malignant neoplasm of breast) to chemotherapy (Cyclofosfamid, Graviserton and Dexorubicin) by assessing their acute effects on epithelial buccal mucosa cells. In 20 patients – women (control group), and 18 patients with cancer the frequency of binucleated cells, pyknosis, karyolysis and karyorrhexis were compared with the control and with their pretreatment (baseline) values. The chemotherapy employed incuced significant (P <0.001) nuclear alternations closely related to cytotoxicity, such as pyknosis, karyolysis and karyorrhexis in treated patients in comparison with control. The value of karyorrhexis of treated patients was significantly higher (3.9±4.8; 6.4±3.0; P < 0.006) in comparison with pretreatment. A significant positive correlation was established between pyknosis and karyolysis (r = 0.635; P<0.004) and pyknosis and karyorhexis (r = 0.674; P<0.002) in pretreatment cancer patiens and between pyknosis and kariolysis (r = 0.489; P < 0.05) after chemotherapy treatment. These data indicate that acute chemotherapy may not induce chromosomal damage, but is able to promote cytotoxicity.
The Role of Arylamine N‐acetyltransferase 1 in Breast Cancer Progression
Authors: Bourcy Katherine , States J. Christopher , Klinge Carolyn M , Doll Mark A , Hein David W
Published: June 2021

Breast cancer is a leading cause of cancer death among women in the United States. Arylamine N‐acetyltransferase 1 (NAT1) has been linked to breast cancer because of its role in the metabolic activation and detoxification of carcinogens. However, recent studies suggest it may play an important role in cancer biology. Higher NAT1 expression is associated with more aggressive cancers and poorer prognosis. Increased NAT1 levels in primary tumors are significantly associated with increased metastasis to the bone. We have observed NAT1 inhibition decreased proliferation and invasiveness of MDA‐MB‐231 breast cancer cells. To delineate the mechanisms by which NAT1 inhibition decreases cell invasion and proliferation, MDA‐MB‐231 and MCF‐7 breast cancer cell lines were treated with siRNA targeting NAT1, or a non‐targeting siRNA. Cells were collected at 24, 48, 72 and 96 hours after treatment. In vitro assays using a NAT1‐specific substrate, para‐aminobenzoic acid (PABA) were conducted to measure NAT1 activity by HPLC. NAT1 mRNA was measured using qRT‐PCR. Optimal knockdown of NAT1 occurred at 96 and 72 hours in MCF‐7 and MDA‐MB‐231 cells, with 65% (p < 0.005) of NAT1 activity remaining after treatment in both cell lines. NAT1 mRNA decreased 60% (p < 0.0001) and 34% (p < 0.001) in MCF‐7 and MDA‐MB‐231 cells, respectively. These samples will undergo whole genome analysis using a GeneArray to identify potential gene expression signatures which may be altered as a result of NAT1 inhibition. [Partially supported by NIEHS T32‐ESO11564]
Enhancement of the activity of the complement inhibitor CDX‐1135 by a monoclonal antibody
Authors: Pilsmaker Catherine D , Gergel Lauren E. , Forsberg Eric M. , Vitale Laura , Keler Tibor , Marsh Henry C. , Thomas Lawrence J.
Published: June 2021

CDX‐1135 (sCR1) is a soluble recombinant glycoprotein derived from the human complement regulatory protein Complement Receptor Type 1 (CR1, CD35, C3b/C4b receptor). CDX‐1135 inhibits the classical, alternative and lectin pathways of the complement cascade, and is being evaluated for use in certain rare renal diseases involving dysregulated complement. A monoclonal antibody specific for CR1, designated 6B1.H12, appeared to enhance the inhibitory activity of CDX‐1135 in an assay developed to detect anti‐sCR1 antibodies with neutralizing activity in the alternative pathway. The 6B1.H12 antibody has been shown to bind a region of CR1 called long homologous repeat D (LHR‐D). Because LHR‐D does not contain a C3b or C4b binding site, it was expected that 6B1.H12 would have no neutralizing effect on CDX‐1135 in an AP neutralization bioassay. We speculate that the enhancement of CDX‐1135 activity at higher concentrations of 6B1.H12 may be due to the oligomerization of the CDX‐1135, increasing the multivalency of binding to C3b and/or C3b‐dimers, activated components of the alternative complement pathway.
Identification of novel blood brain barrier penetrating peptides using one‐bead‐two‐compound combinatorial library technology
Authors: Lac Diana , Bhardwaj Urvashi , Baek Hyonggee , Jang Allyson , Xiao Wenwu , Le Bach , Fung Gabriel , Sanchez Eduardo , Lam Kit S.
Published: June 2021

Successful development of therapeutics for neurological diseases is limited by the ability of compounds to cross the blood brain barrier (BBB). Brain capillary endothelial cells are sealed by tight junctions forming the BBB which express high levels of efflux transport proteins. This results in molecules like gene based or recombinant protein therapies that cannot cross the BBB. Combinatorial chemistry, namely the one bead two compound (OB2C) method, is a powerful tool to identify brain endothelial cell penetrating peptides. We designed releasable OB2C libraries to discover ligands that specifically penetrate brain endothelial cells (bEnd.3). Screening the libraries, we identified 17 peptides that facilitate the uptake of quantum dot by brain endothelial cells. These peptides were resynthesized in soluble form and its cell penetrating abilities were confirmed by the successful delivery of impenetrable compounds, saporin and imaging agents. One peptide, LBL7, showed uptake specifically in bEnd.3 but not 3T3 or SVEC4–10 cell lines. A different cell entry mechanism is involved since the application of endocytosis inhibitors did not affect uptake. We have successfully used OB2C to identify a series of peptides that can penetrate brain endothelial cells. Our studies indicate LBL7 can carry imaging agents and therapeutics to the BBB thereby showing great promise as a vehicle for CNS delivery. Funding: Department
Development of an assay of neutralizing antibodies to the complement inhibitor CDX‐1135
Authors: Gergel Lauren E. , Pilsmaker Catherine D. , Forsberg Eric M. , Vitale Laura , Keler Tibor , Marsh Henry C. , Thomas Lawrence J.
Published: June 2021

CDX‐1135 (sCR1) is a soluble recombinant glycoprotein derived from the human complement regulatory protein Complement Receptor Type 1 (CR1, CD35, C3b/C4b receptor). CDX‐1135 inhibits the classical, alternative and lectin pathways of the complement cascade, and is being evaluated for use in certain rare renal diseases involving dysregulated complement. A theoretical possibility exists that humans receiving CDX‐1135 may make neutralizing antibodies to this molecule. To monitor for this, we developed a CDX‐1135 neutralizing antibody assay. Briefly, a microtiter plate is coated with S. enteritidis LPS and test samples and controls, heat‐inactivated as needed, are serially diluted in assay buffer that selectively allows alternative complement activation. CDX‐1135 and normal human serum are added to the plate followed by mouse anti‐human C5b‐9 mAb to detect the terminal complement complex (TCC). Peroxidase conjugated F(ab’)2 anti‐mouse IgG in then added. The plate is developed with TMB substrate and endpoint titers are calculated from the baseline patient serum of the same dilution. In conclusion, this assay assesses the neutralizing activity of anti‐CDX‐1135 antibodies on the capacity of CDX‐1135 to inhibit the alternative complement pathway.
FORMULATION AND OPTIMIZATION OF SOLID DISPERSION AS A SUSTAINED RELEASE MATRIX FOR ORAL KETOROLAC TROMETHAMINE
Authors: Abdul Rasool Bazigha K. , Aziz Uday S. , Abu‐Gharbieh Eman , Khan Saeed
Published: June 2021

Objective

In the present study, solid dispersion is employed in the formulation of sustained‐release matrix particles of KT with chitosan (CT) as a carrier.

Methods

The matrix solid dispersion was prepared in three different ratios of KT: CH (1:3, 1:5, and 1:7) by spray‐drying method. The matrix particles were characterized by angle of repose, compressibility, scanning electron microscopic (SEM), FTIR and differential scanning calorimetry. The release profile from the matrix particles was studied at pH 1.2 and 6.8. In vivo biological screening in mice and rats for the analgesics, anti‐inflammatory and ulcerogenicity was also performed.

Results

The formation of composite particles was confirmed by SEM. DSC and FTIR spectroscopy analysis suggested an interaction between the carbonyl group of KT and the amino group of CH formed a hydrogen bond. Optimum sustained in vitro release, retained analgesic and anti‐inflammatory activities with significantly reduced ulcerogenicity was obtained from the matrix particles (1:7) compared to the parent drug.

Conclusion

KT‐CH solid dispersion prepared by spray‐drying is useful as a sustained release preparation.
Matrix Metalloproteinase 2 (MMP‐2) Increases Atherosclerotic Aorta Rings Vasoconstriction.
Authors: Azevedo Aline , do Prado Alejandro Ferraz , Pernomian Laena , Bendhack Lusiane Maria , Gerlach Raquel Fernanda
Published: June 2021

The present study aimed to evaluate the vascular function of atherosclerotic aortic rings after intraluminal injection of MMP‐2. Thoracic aorta isolated from New Zealand rabbits with atherosclerosis (feed 1% cholesterol for 8 weeks) and controls (normal diet) were isolated and tested for vascular reactivity function after intraluminal exposure to Krebs solution (vehicle) or MMP‐2 at 1.2 μg/mL, at 37º C for 30 minutes. Curves for Acetylcholine (ACh) and Phenylephrine (PE) were performed in endothelium‐intact rings. The vascular contraction was normalized by grams of tension by dry tissue (g/g) and the relaxation by % of PE contraction. In the vascular reactivity of atherosclerotic vessels, MMP‐2 increased Emax of PE (1842,08±138,77g/g, n=6) versus vehicle (1432,28±124,13g/g, n=7; p<0.05), but no differences were observed on Emax between vehicle in control vessels (1416,13±120,87g/g, n=7) and MMP‐2 in those vessels (1550,44±110,93g/g, n=7). Atherosclerotic vessels filled with vehicle had an impaired relaxation to ACh (80,98±4,63%, n=7) compared to control vessels filled with vehicle (99,86±3,13%, n=7), and MMP‐2 had no effect on relaxation. Since MMP‐2 was able to increase the effect of PE, it can be speculated that it may worsen the endothelial dysfunction of atherosclerotic arteries.

Supported by the State of Sao Paulo Research Foundation (FAPESP), SP, Brazil.
Conditioning of human embryonic stem cell‐derived endothelial cells with PBMCs confers TLR4 sensing in co‐culture conditions
Authors: Reed Daniel Mark , Foldes Gabor , Gashaw Hime H , Harding Sian E , Mitchell Jane A
Published: June 2021

Human embryonic stem cell‐derived endothelial cells (hESC‐EC) are being investigated as potential therapies for vascular disease. We have previously shown that hESC‐EC do not have functional Toll‐like receptor‐4 (TLR4) responses but do respond avidly to cytokines such as TNFα and IL‐1β1,2, which illustrates that these cells do have fully functional TLR signaling via MyD88 and NF‐κB. In this study we show that, whilst hESC‐EC do not respond to LPS (1μg/ml) to release CXCL8 when treated in mono‐culture, they mediate a synergy when co‐cultured with peripheral blood mononuclear cells (PBMCs). No synergy was found in co‐cultures stimulated with TNFα (10ng/ml) suggesting that the conditioning effects of PBMCs on hESC‐EC is above the level of NF‐κB and downstream pathways leading to CXCL8 release. This data is highly novel but preliminary and more studies need to be performed to establish how this conditioning effect is mediated. These findings will be significant in the use of hESC‐EC in vessel engineering and highlight the importance of cell interactions on stem cell and endothelial cell function.

This work was funded by the Welcome Trust, BHF and an MRC studentship awarded to DMR.
Investigating the catalytic role of Cys150‐Asp29‐Lys117 in maleamate amidohydrolase
Authors: Snider Mark J. , Chiaranunt Pailin , Sullivan Eric D. , Morrison Kelly , Hilmey David
Published: June 2021

Maleamate amidohydrolase (NicF) catalyzes the penultimate reaction in the bacterial oxidation of nicotinate to fumarate. On the basis of sequence homology and structural data, catalysis of maleamate hydrolysis to maleate and ammonia by NicF has been proposed to proceed by a nucleophilic addition‐elimination mechanism involving the thiolate side‐chain of C150. In support of that proposal, NicF activity is shown to be inactivated by iodoacetamide and reversibly inactivated by MMTS. Kinetic analyses of C150S and C150A variants of NicF show a 105–106‐fold drop in kcat, without affecting KM, providing additional functional evidence in support of a critical role in catalysis by C150. Likewise, mutations of D29 or K117, the other two conserved residues that complete a proposed catalytic triad, also produce NicF variants with significant drops in kcat (103–104‐fold). Effects of changing pH (4.0–11.0) on kcat and kcat/KM of wtNicF, and the pH dependence of the rate of wt NicF inhibition by iodoacetamide, both suggest the involvement of two general A/B catalysts. Inhibition of wt NicF by 5‐hydroxyfuran‐2(5H)‐one is competitive and reversible with a Ki of 139 nM, consistent with the formation of a quasi‐stable thiohemiacetal that mimics the structure of the proposed intermediates along the reaction coordinate. This work was supported by a HHMI Undergraduate Science Education Program Award to the College of Wooster.
LMO7 is a MAD1‐interacting protein and has a role in mitosis
Authors: Juang Yue‐Li , Li Dai‐Yu , Tseng Yao‐Wei , Yang Cheng‐Hsiu , Chang Chih‐Yun
Published: June 2021

LMO7, an actin cytoskeleton‐associated protein, was identified to be able to interact with the spindle assembly checkpoint (SAC) protein MAD1 in our yeast two‐hybrid screening. LMO7a and LMO7b are known to be two major isoforms of LMO7 in human and rat cells. LMO7a contains CH, F‐box, PDZ and LIM domains whereas LMO7b does CH, PDZ and LIM domains. We find that LMO7 is a mitotic phosphoprotein and its expression is cell cycle‐regulated. Fluorescence microscopic analysis revealed that MAD1 and LMO7 colocalized to the spindle poles in metaphase. We observed that overexpression but not depletion of LMO7 could cause a defect in the SAC. Furthermore, overexpression of LMO7ΔLIM, a LMO7 mutant protein lacking LIM domain, did not cause a defect in the SAC. Strikingly, the LIM peptide fused to GFP, GFP‐LIM, could localize to the spindle poles, and its expression did not affect kinetochore localization of MAD1 but still could cause a defect in the SAC. These observations suggest that the LIM domain is the determinant for localization of LMO7 to the spindle poles and its role in the SAC. We further identified that YPEL1, a spindle pole‐associated protein, could interact with the LIM domain of LMO7. However, overexpression of YPEL1 could not relieve but instead exacerbate the defect in the SAC caused by overexpression of GFP‐LIM. The significance of LMO7‐YPEL1 interaction in the SAC is under investigation.
Heart rate responses to osteopathic manipulations in healthy young men
Authors: Ioudina Marina , Toder Eric , Boudreaux Brittany
Published: June 2021

Osteopathic manipulative treatment (OMT) is used to treat cardiac patients. Some manipulations are hypothesized to affect the cardiovascular system by altering autonomic tone, although physiological effects of individual techniques have not been well defined. Previous studies from our laboratory have shown that cranial manipulations (CMs) as well as articulatory (A) and inhibitory (I) rib raising techniques decreased cardiac output in healthy individuals. This study measured responses to individual manipulations on heart rate (HR) in healthy men in order to further our understanding of OMT effects on cardiovascular physiology.

25–35‐yr old men participated. HR was measured in response to manipulations thought to stimulate parasympathetic activity (CM), stimulate (A), and inhibit (I) sympathetic activity. ECG recordings were used to measure changes in HR. Manipulations were performed by an osteopathic physician.

CMs caused a significant 6% decrease in HR during manipulations. Both (A) and (I) techniques did not have a significant effect on HR.

These data demonstrate that CMs have a significant effect on HR, likely via an increase in parasympathetic activity. No effects of (A) and (I) rib raising manipulations on HR responses were observed. Future studies should include a larger number of subjects of various age groups.
Calcium‐sensitive potassium channels are not involved in the decreased myogenic tone of posterior cerebral arteries in a genetic model of cerebral ischemic small vessel disease
Authors: Kroigaard Christel , Dabertrand Fabrice , Joutel Anne , Nelson Mark T.
Published: June 2021

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an archetypal monogenetic form of small vessel disease (SVD) of the brain. In a novel transgenic mouse model for CADASIL (R169C), a compromised cerebral blood flow and neurovascular coupling has been shown. This study investigates the consequences of CADASIL on the relationship between intravascular pressure and diameter and the function of potassium (K+) channels in isolated posterior cerebral arteries (PCAs). Elevation of intravascular pressure to 60 mmHg resulted in 38% reduced constriction of isolated CADASIL PCAs. However, vasodilation to NS309, an activator of endothelial small (SK) and intermediate (IK) conductance Ca2+‐sensitive K+ (KCa) channels, was unaffected. In addition, blocking SK, IK and smooth muscle large conductance calcium (Ca2+)‐sensitive (BK) channels constricted CADASIL PCAs to the same extent as control PCAs. In contrast, the vasoconstriction induced by heparin‐binding epidermal growth factor (HB‐EGF), that causes voltage‐dependent K+ (KV) channel endocytosis via activation of EGF, was decreased in CADASIL PCAs. In conclusion, KCa channel function is conserved in PCAs from CADASIL mice, whereas KV channel up‐regulation may play a critical role in the decreased myogenic tone observed. Supported by NIH PO1HL095488, Fondation Leducq and The Lundbeck Foundation.
Effects of training status on circulating angiogenic cell paracrine activity in young men and women
Authors: Landers‐Ramos Rian Q , Jenkins Nathan T , Spangenburg Espen E , Hagberg James M
Published: June 2021

PURPOSE

To determine if a relationship exists between physical fitness and the ability of CD34+ or CD34−/CD31+ circulating angiogenic cells (CAC) to influence angiogenic function through paracrine activity.

METHODS

Peripheral blood was drawn from healthy inactive (n = 7), active (n = 10) and endurance‐trained (n = 7) men and women aged 25–39. Mononuclear cells were isolated by density centrifugation and CD34+ and CD34−/CD31+ fractions were purified by immuno‐magnetic selection. Conditioned media (CM) from cultured CACs was retrieved and used for a HUVEC‐based angiogenesis tube formation assay. Tube length and complexity were imaged and quantified.

RESULTS

CM from CD34+ CACs of endurance‐trained subjects resulted in 21% greater tube length (P<0.05) and 48% greater complexity (P<0.05) than inactive subjects, and 22% greater complexity than active subjects (P<0.05). CD34+ CM from active subjects produced greater length and complexity than inactive subjects (P<0.05, 18% and 33%, respectively). Tube length was 22% greater and complexity was 42% greater (P<0.05 each) with CD34−/CD31+ CAC CM of endurance‐trained subjects compared to inactive individuals.

CONCLUSIONS

These preliminary data provide promising support for a chronic exercise‐induced increase in paracrine‐mediated angiogenic function of CD34+ and CD34−/CD31+ CACs.

Support: NIH AGT3200068 and UMD KNES Graduate Research Initiative Fund
Redox signaling inhibition reverses the age‐related impairment in shear‐mediated collateral growth
Authors: Miller Steven J , Labarrere Carlos A , Wenning Mary Jo , Bills Randall G , Sliva Petr , Unthank Joseph L
Published: June 2021

Previous results demonstrated that antioxidant treatment reversed age‐related collateral growth (CG) impairment. The aim of this study was to determine if impaired CG was due to dysregulation of redox‐mediated signaling and transcription factor activation. CG (% luminal expansion) was assessed with a well‐established mesenteric artery ligation model in Wistar Kyoto retired breeder (10–12 mo‐old) rats. NFkB nuclear translocation indicated by percent p65‐positive intimal nuclei was significantly greater in collateral than control arteries (36.6±3.5 vs. 19.7±2.8, respectively, p<0.01), and apocynin normalized translocation to untreated control values (18.9±2.9). Various inhibitors were used to further assess the potential role of redox‐dependent regulation of transcription factors and signaling pathways, including: NFkB (p65 siRNA), PI3‐kinase (LY294002), mTOR (rapamycin), and renin‐angiotensin type 1 receptor (AT1R; losartan). p65 siRNA stimulated CG compared to control siRNA (27.3±5.9 vs. 1.7±0.8, respectively, P<0.001). CG in LY294002‐ and rapamycin‐treated rats also was significantly increased compared to vehicle (14.5±2.3; P<0.001, and 27.0±2.2; P<0.0001 vs. 0.8±0.8, respectively). In addition, losartan treatment stimulated CG vs. untreated control (25.1±5.2 vs. 5.3±1.4, respectively, P<0.05). The results suggest abnormal redox‐mediated signaling and NFkB activation play a role in impaired CG during aging. Support: HL092012
Development of protein microarray tools for the ex vivo profiling of O‐linked N‐acetylglucosamine transferase (OGT) substrates
Authors: Janetzko John Christopher , Ortiz‐Meoz Rodrigo , Kahne Daniel , Walker Suzanne
Published: June 2021

O‐linked N‐acetylglucosamine transferase, OGT, is an essential mammalian enzyme that transfers a single N‐acetylglucosamine (GlcNAc) moiety to specific serine and threonine residues in numerous cellular proteins. Like protein phosphorylation, protein O‐GlcNAcylation is dynamic. However, unlike protein phosphorylation, which involves many highly specific kinases, OGT is the sole nuclear and cytoplasmic glycosyltransferase. Like protein phosphorylation, glycosylation is believed to play signaling and regulatory roles as aberrant glycosylation has been linked to numerous disease states from cancer to diabetes and neurodegeneration. Previous efforts to identify substrates of OGT have largely been based on the enrichment of in vivo samples and subsequent LC‐MS/MS analysis. This work describes an ex vivo approach that promises to offer unique information about structure‐function relationships within OGT. This approach has several advantages over previous methods including the ability to overcome the challenges of target enrichment and identifying low abundance proteins. The development of chemical and biochemical tools to enable an alternative detection method for O‐GlcNAc modified proteins will be described. Using this tool we have been able to identify previously unknown OGT substrates and our ongoing efforts aim to delineate the substrate profiles of various natural and unnatural OGT constructs as well as examine the roles of glycosylation conditions and accessory proteins on the identified substrates.
Inhibition of the mammalian Target of Rapamycin (mTOR) does not alter nitric oxide or superoxide levels in human skin
Authors: Zhao Joan L , Roman Linda J , Wu Yubo , Johnson John M , Kellogg Dean L
Published: June 2021

In laboratory animals, mTOR inhibition for 7 days causes significant reductions in nitric oxide (NO) levels with concomitant increases in superoxide production. We hypothesized mTOR inhibition by rapamycin would effect similar changes in humans. 8% rapamycin ointment (RAPA) was applied twice daily for 7 days to an 8×8 cm area of skin on the abdomen while a similar second site was treated with the ointment vehicle only. 5 middleaged persons participated. Selective amperometric electrodes placed intradermally within both skin areas monitored NO levels. Superoxide levels were monitored by perfusing the intradermal microdialysis probes at both skin areas with a solution of saline containing oxidized ferricytochrome c and analyzing for reduced cytochrome c in the collected perfusates. Superoxide in the cutaneous interstitial space diffuses across the probe membrane, forming reduced ferrocytochrome c and altering the spectrophotometric absorbance.

Results

NO levels at RAPA treated sites averaged 811±72nM and 526±56nM at vehicle treated sites. These values did not differ between RAPA and vehicle treated sites (p>;0.50 between site). Superoxide levels at RAPA sites averaged 11.73±2.17μM and 11.29±1.90μM at vehicle treated sites. These levels did not differ between sites (p>;0.40). We conclude that chronic RAPA treatment does not alter NO or superoxide levels in human skin. (supported by NIH Grant AG041365)