bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in Leukocyte Populations among Children Receiving a Follow‐on Cow's Milk‐Based Formula Containing Lactobacillus rhamnosus GG
Authors: Sarvetnick Nora , Murphy Kevin , Pauley‐Hunter Rosemary , Scalabrin Deolinda 
Published: June 2021

BackgroundLactobacillus rhamnosus GG (LGG) is a probiotic with demonstrated health benefits, and these benefits may be due to LGG‐induced changes in the immune system.ObjectiveWe examined changes in immune cell populations and activation markers produced by the addition of LGG to a children's follow‐on formula.MethodsThis was a double blinded, randomized, controlled, parallel‐designed, prospective study. Data is reported for participants who completed the study. Participants (1–4 years of age) received either a marketed, follow‐on formula (n=8), formula supplemented with 42 mg of β‐Glucan per serving (n=10), or formula with 6 × 109 colony forming units of LGG per serving (n=9), twice a day for a 3 week period. Flow cytometry was used to immunophenotype peripheral blood leukocytes. Thirty serum cytokines were measured via multiplex analysis. Changes from baseline were analyzed by Kruksal‐Wallis. As this was a pilot study, differences with p‐values < 0.10 were considered of interest.ResultsParticipants that received LGG showed reduced levels of IL‐2 and IL‐3. They also had a reduction in the total proportion and general activation of CD4 T cells and a decrease in specific subsets of double negative T cells.ConclusionThese data suggest that LGG has an anti‐inflammatory effect by reducing available T cell populations that can cause inflammation.
Exploring phospho‐signaling pathways in patients with agerelated macular degeneration (AMD), human pigment epithelial cell line ARPE‐19 and porcine ocular tissue
Authors: Fasler‐Kan Elizaveta , Barteneva Natasha , Meyer Peter 
Published: June 2021

The pig has been used extensively in experimental studies of human eye diseases. Pig retina shares many morphological features with the human eye. Retinal pigment epithelial cells constitute an important component of the blood‐retinal barrier and play a pivotal role in the development of AMD. Understanding the underlying molecular mechanisms is a prerequisite for developing therapeutic strategies. We investigated cytokine‐induced changes of the JAK/STAT and NF‐kappa B signaling pathways.MethodsPorcine ocular cells, human ARPE‐19 cell line, eyes with AMD were used to study the nuclear translocation of STAT and NF‐kB proteins by gel‐shift assay, immunohistochemistry, confocal laser microscopy and flow cytometry.ResultsHistological examination of AMD patients revealed a strong formation of choroidal neovascular membranes in the macular region. Strong activation of STAT3 was observed in these membranes. Strong STAT1 activation was demonstrated upon exposure to interferon‐γ in ARPE‐19 and pig ocular cells in vitro. Human recombinant interferon‐α upregulated STAT1, STAT2 and STAT3, while IL‐6 and IL‐4 activated STAT3 and STAT6, respectively. Confocal microscopy identified the nuclear translocation of the STAT proteins.ConclusionOur data demonstrate the upregulation of the members of the JAK‐STAT signaling pathway in AMD patients, porcine ocular cells and ARPE‐19 cell line.
Live kidney slices present a novel method for delineating the mechanisms of calcineurin inhibitor‐mediated nephrotoxicity
Authors: Kelly Mark , Crawford Carol , Loo Ruey‐Leng , Delaney Michael , Farmer Chris , Wildman Scott S.P. , Peppiatt‐Wildman Claire. M. 
Published: June 2021

Cyclosporin A (CsA) and Tacrolimus (FK‐506) are potent immunosuppressant drugs widely used to prevent allograft rejection in organ transplantation. They are calcineurin inhibitors (CNIs) and have limiting side effects including nephrotoxicity that impact upon long‐term renal allograft and patient survival in other organ transplantation. The precise mechanisms of CNI‐induced toxicity are unknown, although ischemic injury resulting from impairment of endothelial cell function, reduced production of endogenous renal vasodilators and concomitant release of vasoconstrictors resulting in net vasoconstriction have been described.We have utilized the live kidney slice model, developed by our group [1], to investigate the mechanisms of CNI‐induced nephrotoxicity in the renal medulla, a region of the kidney that is particularly sensitive to ischemiaData presented here demonstrates both CsA (600 ng/ml) and FK‐506 (800 ng/ml) cause vasoconstriction (9.9% ± 0.88% and 9.2% ± 0.63%, respectively) of vasa recta via their specific action at contractile pericytes. Tubule diameter was unaffected by bath application of both CsA and FK‐506. Data suggest that CNI‐induced constriction of vasa recta by pericytes may exacerbate renal medullary ischemia contributing to observed nephrotoxicity in solid organ transplantation.Research supported by the Medical Research Council and University of Kent.
Altered ZO‐1‐Actin Interaction Induces an Epithelial to Mesenchymal Transition
Authors: Klindworth Kevin O , Lyford Brooks P , Nepomuceno Helene L , King Jonathan M 
Published: June 2021

Zonula occludens‐1 (ZO‐1) is a large scaffold protein necessary for tight junction formation and signaling. The ZO‐1 protein includes a unique C‐terminal structure, including an actin‐binding region (ABR) and ZU‐5 terminal domain, which play a relatively unknown role. Mouse ZO‐1 C‐terminal phrGFP‐fusion constructs (ABR and ABR‐ZU5) were prepared and stably transfected into MDCK II cells. Appropriate localization was observed in all stably cell lines except for the highly expressing GFP‐ABR‐ZO‐1 cells. The GFP‐ABR‐ZO‐1 construct markedly altered cell phenotype into spindle‐like cells deviating from the expected epithelial monolayer. GFP‐ABR‐ZO‐1 appears to interact with bundles of F‐actin as examined by confocal microscopy. These studies highlight the ZO‐1‐ABR region as an important component in the tethering of the actin cytoskeleton to the apical junction complex. Proliferation and migration was enhanced in the in GFP‐ABR‐ZO‐1 cells and wound healing was markedly impaired by 5‐fluorouracil, a cell proliferation inhibitor. qRT‐PCR and Western blotting demonstrated that junctional markers (occludin and claudin‐1) were significantly down regulated by ABR overexpression whereas vimentin, a mesenchymal marker, was elevated. The ZO‐1 C‐terminus mediates critical epithelial cell homeostasis events and in addition to epithelial to mesenchymal transition.Supported by TU‐HHMI program.
Time‐dependent mechanisms of ethanol‐evoked acute hypotensive and cardiac depressant effects in conscious female rats
Authors: Ibrahim Badr Mostafa , Fan Ming , Abdel‐Rahman Abdel A 
Published: June 2021

The molecular mechanisms for the acute hypotensive and cardiac depressant effects of ethanol in proestrus rats are not known. We tested the hypothesis that exacerbated myocardial oxidative stress contribute to the acute ethanol‐evoked myocardial dysfunction and ultimately the hypotensive response in proestrus female rats. Ethanol (1g/kg) or an equal volume of water (control) was administered via nasogastric tube to 48 proestrous rats (divided to 3 ethanol and 3 water groups; n = 6–8), and hemodynamic measurements continued for 30, 60 or 90 min. To elucidate the role of pro‐oxidant and antioxidant mechanisms in the ethanol‐evoked myocardial dysfunction, the catalytic activities of acetaldehyde (ACA) generating [alcohol dehydrogenase (ADH) and catalase] and eliminating (ALDH2) enzymes were measured in the collected cardiac tissues at the specified time points. Further, we measured the levels of markers of oxidative stress (ROS and Nox activity), myocardial kinases (phosphorylated Akt and ERK1/2), and myocardial levels of: (i) the cardiotoxic aldehyde malondialdehyde (MDA), and (ii) 4‐hydroxy‐2‐nonenal (4‐HNE)‐modified proteins. The ex vivo studies revealed: (i) enhanced (ADH), without a change in ALDH2, activity (indicative of elevated ACA levels), (ii) increases in mediators of oxidative stress such as phosphorylated (p) Akt and pERK1/2, Nox activity enhanced along with ROS, (iii), elevated MDA and 4‐HNE‐modified proteins expression. These molecular responses along with the inhibition of myocardial catalase activity coincided with the drastic reductions in cardiac function and BP at 90 min post‐ethanol. The findings implicate local ethanol/ACA metabolizing enzymes in the myocardial oxidative stress, which along with the accumulation of cardiotoxic aldehydes lead to ethanol‐evoked myocardial dysfunction and hypotension in conscious proestrus female rats.Supported by NIAAA Grant 2R01 AA14441–06
Intranasal delivery of pGDNF nanoparticles provides neuroprotection in the rat 6‐hydroxydopamine model of Parkinson's disease
Authors: Harmon Brendan Trevor , Aly Amirah , Cooper Mark J , Dines Karla , Padegimas Linas , Waszczak Barbara L 
Published: June 2021

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to be neuroprotective toward substantia nigra (SN) dopamine neurons in animal models of Parkinson's disease (PD) and in some clinical trials. However, its delivery to brain has required invasive surgical routes that are untenable for many patients with PD. The goal of these experiments was to test whether intranasal delivery of a GDNF expression plasmid could protect dopamine cells in the rat 6‐hydroxydopamine (6‐OHDA) model of PD. If successful, intranasal delivery of pGDNF could provide a renewable source of GDNF within the brain without need for surgical injections or frequent re‐dosing.Rats were given intranasal saline, naked pGDNF or pGDNF in a PEGylated polylysine nanoparticle (NP) preparation 7 days prior to receiving a unilateral 6‐OHDA lesion. Three weeks after 6‐ OHDA, rotational behavior to 5 mg/kg d‐amphetamine was assessed, followed by sacrifice. Tyrosine hydroxylase (TH) immunohistochemistry revealed a significant reduction in lesion severity in the SN of rats given intranasal pGDNF NPs versus those given naked pGDNF or saline. In addition, rotational behavior was reduced in rats treated with pGDNF NPs. These results demonstrate the utility of intranasal delivery of pGDNF NPs as a non‐invasive means of gene therapy for PD and possibly other diseases of the brain. Support provided by: 2011–12 Northeastern University Tier 1 Seed Grant and the M.J. Fox Foundation.
Expression and Function of Hepatic Drug Metabolizing Enzymes and Transport Proteins in Livers of Patients with End‐Stage Renal Disease (ESRD)
Authors: Feere David Alexander , Velenosi Thomas , Gaspar Melisa Lynn , Urquhart Brad 
Published: June 2021

ObjectivesThis study aims to determine the changes in expression and function of hepatic drug metabolizing enzymes (DMEs) and drug transport proteins in liver samples from patients with end‐stage renal disease (ESRD).MethodsLiver samples were collected from nine recently deceased patients with ESRD and six recently deceased controls. Relative gene expression of DMEs and transport proteins were determined by qPCR.ResultsP‐gp and CYP2E1 expression were significantly decreased (P<0.05) in ESRD livers compared to control livers (21% and 17% of control, respectively). There was a trend of decreased expression of CYP2D6, CYP2C9, OATP1B1 MRP2 and MRP3, although this failed to reach significance.ConclusionsOur results demonstrate that DMEs and transport proteins are down‐regulated in ESRD. Current studies are evaluating the enzymatic activity of DMEs in hepatic microsomes from ESRD patients using the specific probe substrates midazolam (CYP3A4), tolbutamide (CYP2C9), chlorzoxazone (CYP2E1) and dextromethorphan (CYP2D6). Our results help explain the large decrease in non‐renal drug clearance observed in patients with ESRD.This research is supported by the Lawson Health Research Institute.
FUNCTIONAL ACTIVITY IN HUMAN HEART EXPRESSING CYP1A1
Authors: Huguet Jade , Gaudette Fleur , Michaud Véronique , Turgeon Jacques 
Published: June 2021

BackgroundDrug metabolism by CYP450s expressed in cardiac myocytes can modulate cardiac drug levels and actions. CYP1A1 and 2C8 are importantly expressed in the human heart. A cocktail approach with 7‐Ethoxyresorufin (7‐ET) and repaglinide (REP) were chosen to study selective metabolism of CYP1A1 and 2C8, respectively.MethodsIncubations were first performed in Supersome™ of each CYP450s founded in the heart. Then, cytosolic (S1) and human heart microsomes (HHM) were isolated by differential centrifugations from one human heart. Resorufin and REP metabolite were quantified by LC‐MS/MS. Western blot analysis was performed using a monoclonal antibody raised against CYP1A1.ResultsREP metabolism was selective towards 2C8 and 7‐ET for 1A1 in Supersome™. For 7‐ET, Km was 0.36 vs. 0.4μM and Vmax was 1640 vs. 1944 pmoles of resorufin/mg of protein/min when incubated alone vs. with REP, respectively. For REP, Km was 3.7 vs. 3.35μM and Vmax was 1283 vs. 1120 pmoles REP metabolite/mg of protein/min when incubated alone vs. with 7‐ET, respectively. No activity was observed when 7‐ET and REP were incubated in HHM but there was a low activity in the S1 fraction (0.3 pmoles of resorufin/mg prot/min). This was confirmed by Western blot analysis where CYP1A1 protein was found in S1 but not in HHM.ConclusionThis cocktail approach allows us to study the CYP450 activities in the heart. Research funded by the CIHR, HSF and FRSQ.
Effects of gender and race on interleukin‐6 production in response to CRP in HUVECs
Authors: Ling Chenyi , Kretzschmar Jan , Grimm Heather , Brown Michael 
Published: June 2021

BackgroundInterleukin‐6 (IL‐6) is a pro‐inflammatory cytokine that can be excreted by endothelial cells. Elevated C‐reactive protein (CRP) level is a risk factor for the development of endothelial dysfunction that can lead to hypertension. The study was to determine whether race or gender affected the IL‐6 response to CRP in HUVECs.MethodsEight HUVEC lines from African American (AA) and Caucasian (CA) donors with gender split evenly were cultured and incubated with CRP for 24‐hrs. Doses of CRP were 0, 25, 50 and 100 μg/mL. IL‐6 was measured in the media that was collected after the incubation and was normalized to protein concentration (pg/mg).ResultsThe female group had higher IL‐6 production than the male group for each CRP condition and there was a significant difference between genders at the 50 μg/mL CRP condition (P=0.03). Though the IL‐6 production tended to be higher in the AA group under 25, 50, and 100 μg/mL CRP conditions, the differences were not significant.

CRP level (μg/mL)
IL‐6 production (pg/mg)

Female
Male

0
66.96 ± 30.88
82.73 ± 28.75

25
144.86 ± 50.15
92.03 ± 40.77

50
177.97 ± 20.73
110.98 ± 25.56

100
179.15 ± 59.41
151.94 ± 103.41

ConclusionIt suggests that females with high CRP levels may have a greater risk of developing endothelial dysfunction due to a greater IL‐6 response. Whether the gender difference is related to differences in CRP receptor function or downstream signaling mechanisms is unknown.
Increased Circulating Microparticles in Diabetic Rats are not only a Result of Cell Activation but Actively Serve as Mediators of Inflammatory Signaling
Authors: Stork Christian , He Pingnian 
Published: June 2021

Diabetes is commonly associated with microvascular dysfunction. Our previous study found that microvessels of diabetic rats had increased basal permeability and enhanced permeability responses to inflammatory mediators. We also found that diabetic rats had increased circulating microparticles (MPs). MPs are small vesicles released by activated or apoptotic cells and carry multiple antigens from activated cells. We hypothesized that MPs serve as vectors to actively disseminate inflammation to the whole vascular system. We used flow cytometry to quantify the numbers of MPs and their cell origins. We tested differences between MPs generated by diabetic rats and MPs from normal rats by directly perfusing them into intact microvessels. The effects of MPs on microvessel walls were examined by perfusing matched numbers of isolated Annexin V positive MPs into normal venules for 30 min. After 10 min resumed blood flow in MP perfused vessel, we found significantly higher number of adherent leukocytes in diabetic MP perfused vessels than in normal MP perfused vessels. Diabetic and normal MP‐ mediated leukocyte adhesion were 17 ± 2.9 and 5.7 ± 0.5 leukocytes per 100 μm of vessel length, respectively. Flow cytometry analysis showed that diabetic MPs had an average 7‐fold increase in phosphatidylserine exposure per MP and a 12‐fold increase in platelet‐leukocyte MP aggregates from those in normal plasma MPs. Our results suggest that the population differences and the larger surface area of PS exposed diabetic MPs may contribute to the greater leukocyte adhesion induced by diabetic MPs. These results support our hypothesis that increased MPs in diabetic rats are more than simply biomarkers of the disease but that they are able to directly mediate leukocyte adhesion and actively disseminate inflammation to remote regions.Supported by: 1F32HL114376, P20GM103434, HL56237, HL084338
Steviol retards cyst growth through inhibition of ERK and wnt/β‐catenin pathways
Authors: Yuajit Chaowalit , Nantavishit Jiriporn , Muanprasat Chatchai , Chatsudthipong Varanuj 
Published: June 2021

Cystogenesis in polycystic kidney disease (PKD) involves multiple pathways. One of them is cAMP‐stimulated epithelial cell proliferation and cyst enlargement. Previously, we found that steviol could inhibit cyst enlargement in part by inhibition of CFTR function. The present study was aimed to investigate the detailed mechanisms of steviol on cyst growth using Madin Darby canine kidney (MDCK) cyst models. We found that steviol (100 μM) reversibly inhibited MDCK cyst growth by 38.3%. Steviol at the dose up to 200 μM had no cytotoxicity on MDCK cells. Western blot analysis of cysts extracted from collagen demonstrated that steviol reduced ERK 1/2 phosphorylation. In addition, treatment with 100 μM of steviol for 24 h significantly abolished ß‐catenin protein expression in MDCK cells by 33.6%. These findings suggest that steviol retards MDCK cyst enlargement, in part, by retarding cell proliferation through inhibition of ERK signaling and wnt/ß‐catenin pathways. Thus, steviol represents a promising natural plant‐based drug candidate for polycystic kidney disease due to its multiple targets.This work was supported by the Thailand Research Fund, the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative and Faculty of Science, Mahidol University, Thailand.
EFFECT OF RESISTANCE TRAINING ON CIRCULATING Th1 CYTOKINES IN HIV+ MEN RECOVERING FROM SUBSTANCE ABUSE
Authors: Vingren Jakob L , Curtis John H , Duplanty Anthony A , Lee Elaine C , Budnar Ronald G , McFarlin Brian K , Hill David W 
Published: June 2021

Human immunodeficiency virus (HIV) and substance abuse (drug and/or alcohol) independently impair the immune system. INF‐γ and IL‐2 are Th1 cytokines involved in modulating immunity relevant to HIV. INF‐γ inhibits HIV replication in latently infected cells and low production of IFN‐γ is correlated to progression to AIDS. IL‐2 slows apoptosis of HIV‐infected cells. Thus, changes in Th1 cytokines could have important implications for HIV+ individuals.PurposeTo examine the effect of resistance training on resting concentrations of circulating Th1 cytokines.MethodsSixteen men (42 ± 11 years, 180 ± 9 cm, 89 ± 21 kg) infected with HIV and enrolled in a 60‐day in‐patient substance abuse treatment program. Participants were assigned to either supervised resistance exercise 3 times per week (RT) or no exercise training (Control) for six weeks. Before (Pre) and after (Post) the 6‐week period, resting and fasted blood samples were obtained and analyzed for serum INF‐γ and IL‐2.ResultsThe 6‐week intervention did not change INF‐γ (RT: Pre: 1.4 ± 2.1 pg·ml−1; Post: 1.2 ± 2.6 pg·ml−1; Control: Pre: 2.3 ± 3.9 pg·ml−1; Post: 2.2 ± 3.9 pg·ml−1) or IL‐2 (RT: Pre: 0.4 ± 0.5 pg·ml−1; Post: 0.2 ± 0.1 pg·ml−1; Control: Pre: 0.1 ± 0.1 pg·ml−1; Post: 0.2 ± 0.1 pg·ml−1)ConclusionA six‐week resistance training program does not elicit changes in the circulating Th1 cytokines INF‐γ and IL‐2 in HIV+ men undergoing substance abuse treatment.
Characterisation of H441 cells as an in vitro model of distal lung epithelial barrier
Authors: Ehrhardt Carsten , Gausterer Julia Clara , Schwagerus Elena , Salomon Johanna Jessica 
Published: June 2021

No continuously growing cell line of human distal lung epithelial phenotype and with the ability to form polarised, electrically tight monolayers has been described to date. Here, we investigated, if the human lung adenocarcinoma cell line H441 has potential to overcome this issue.Epithelial barrier properties were studied by immunocytochemistry (ICC) against ZO‐1 and measurement of RT. The expression of MDR1 and organic cation/carnitine transporters (OCT/Ns) was also investigated by ICC and immunoblot. Uptake of [14C]‐TEA and [3H]‐acetylcarnitine was carried out to assess OCT/N function. Furthermore, the effects of OCT/N modulators on organic cation uptake were studied.H441 cells formed confluent, electrically tight monolayers with peak RT values of 600–1000 Ω·cm2, after 8 days in culture. Presence of P‐gp, OCT1–3, OCTN1 and 2 was confirmed by Western blot and ICC. Uptake of organic cations was time and temperature‐dependent. In the case of [14C]‐TEA, verapamil and MPP+ most efficiently inhibited uptake, whilst amantadine and HC‐3 did not show an effect. D‐carnitine and ergothioneine significantly attenuated [3H]‐acetylcarnitine uptake into H441 cells.H441 cells have the ability to form monolayers with appreciable barrier properties. Moreover, their drug transporter expression and activity is consistent with what has been reported for human alveolar epithelial cells in primary culture.
Expression profiles of three isoforms of phospholipase C Xdomain containing protein (PLCXD) in human and mouse
Authors: Kalujnaia Svetlana , Gellatly Steven , Cramb Gordon 
Published: June 2021

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) enzymes are a family of receptor‐regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. Recently we identified a seventh class of PI‐PLC enzymes (called PLC‐XDs), which contains only the catalytic X domain of the normal multi‐domain‐ containing enzyme family. Three tissue‐specific PLC‐XD isoforms exist in humans, hPLC‐XD1, ‐XD2 and ‐XD3, with ‐XD2 exhibiting three C‐terminal spliceforms. Similar PLC‐XD orthologs have been identified in mice with the mPLC‐XD3 having two C‐terminal spliceforms. PLC‐XD isoforms exhibited their own individual tissue‐specific expression profiles and sub‐cellular localisations with some similarities between species. PLC‐XD1 and ‐XD3 isoforms were expressed in most tissues tested with PLC‐XD2 mainly found in leucocytes and thymus in humans, but predominately in the GI‐tract and stomach of mice. PLC‐XD3, which exhibits the highest sequence homology across species, was mainly expressed in mouse and human heart. The expression profiles suggest that these novel proteins may possess fundamental, and as yet uncharacterised tissue‐specific roles in cell physiology.Funding: School of Medicine, University of St Andrews