bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary flaxseed oil decreases PCNA expression in rat lung tissue in a bleomycin‐induced model for pulmonary fibrosis
Authors: Harris Ethan D , Standard Jacob M , Bellfi Emily E , Cook Kelsi M , Sheehy Kathryn G , Gevry Taylor R , Andres Erica L , Pederson Katie L , Woo Anna E , Molteni Agostino , Vanden Heuvel Gregory B , Baybutt Richard C
Published: June 2021

The purpose of the present study was to determine how dietary flaxseed oil altered specific biomarkers in its protection against bleomycin‐induced pulmonary fibrosis. Twenty weanling rats were randomly assigned to one of two groups: (1) a group fed a 15% (w/w) corn oil (control) diet and (2) a group fed a 15% flaxseed oil diet. Rats were fed their respective diets for 4 weeks prior to bleomycin treatment. Half of each group received either bleomycin or saline vehicle via oropharyngeal delivery. All rats were euthanized at day 7 post‐treatment. Formalin‐treated lung tissue were immunohistostained using antibodies for proliferating cell nuclear antigen (PCNA), Cux1 (CUTL1), and cyclin‐dependent kinase inhibitor 1B (p27). We found that PCNA was overly expressed in bleomycin‐induced rat lungs but was notably reduced in rats given the flaxseed oil‐supplemented diet. Expression of p27 was moderately reduced in rats given the flaxseed oil‐supplemented diet as compared with groups given corn oil. No significant differences were found for Cux1. Results indicate that PCNA may be another biomarker for pulmonary fibrosis and its decreased expression suggests that the dietary flaxseed oil inhibits proliferation of bleomycin‐treated lung cells.
Intestinal ROS is reduced in mice after fasting
Authors: Carlsen Harald , Kielland Anders , Blomhoff Rune
Published: June 2021

The intestine contains both red‐ox regulating systems and numerous enzymes producing reactive oxygen species (ROS). It is reasonable to assume that intestinal ROS content is related to the function of the intestine: food digestion. To shed some light on this we have evaluated the effect of fasting and diet change on intestinal ROS. Mice were fasted over night and ROS was evaluated by use of the bioluminescent probe L‐012. This probe emits photons when reacting with ROS, which can be detected by optical imaging. L‐012 was injected i.p. five minutes prior to sacrificing the animals and dissection of the intestine for imaging. Bioluminescence was recorded from duodenum, jejunum, ileum and colon. We found little effect of fasting in duodenum and colon; however, in jejunum and ileum we observed a large reduction in the L‐012 signal. Furthermore, we fed mice a typical unhealthy diet (western diet) for two months. This caused a significant reduction in the L‐012 signal. In conclusion, this suggested that the ROS content in lower part of small intestine is dependent on the presence of specific food components.
Programmed cell death in hyperplasic ovaries of ex‐fissiparous planarian Dugesia arabica
Authors: Harrath Abdul Halim , Ahmed Mukhtar , Mansour Lamjed , Alwasel Saleh
Published: June 2021

Planarians can reproduce exclusively sexually or/and asexually by fissiparity. Among fissiparous populations individuals can sexualize and develop ovaries of remarkable size called hyperplasic ovaries (Gremigni and Banchetti, 1972). Nevertheless, it has been reported that individuals that become sexual are usually sterile because they usually lay infertile cocoons (Grasso and Beanzzi, 1973).

The aim of this study is to demonstrate, through ultrastructural and TdT‐mediated dUTP nick end‐labeling (TUNEL assay) in intact sections of hyperplasic ovaries the cause of infertility of ex‐fissiparous planarians. Our study has been carried out on Dugesia arabica collected from Yemen (Harrath et al., 2013) that reproduce asexually in the field. After having been kept for few months in the laboratory, some of the specimens had been sexualized.

Ultratsructural and TUNEL assay investigations demonstrate that oogonia and young oocytes still develop normally compared to previous studies describing oogenesis in other Dugesia species. At the later stage, very few fertile oocytes of a hyperplasic ovary may succeed in completing their growth whereas the majority at the diplotene stage undergoes a progressive degeneration through programmed cell death. The latter leads to the self‐destruction of oocytes and may represent the most important cause of infertility of ex‐fissiparous planarians. This process should be correlated with polyploidization or genomic variations.
College students’ midpoint sleep time and dietary intake
Authors: Georgakilas Evagelia , Morrell Jesse Stabile , Tagliaferro Tony
Published: June 2021

America is experiencing high rates of obesity, with alarming trends among children and young adults. Research shows there is an association between sleep duration and energy consumption/obesity among young adults (18–24 yrs). The relationship between sleep timing and college students’ dietary intake has not been extensively investigated. Students (n=146, 74.7% female) from the ongoing College Health and Nutritional Assessment Survey (CHANAS) completed a sleep questionnaire and three‐day food record. Macronutrient intake, energy density (g/kg), and empty calories, as defined by MyPlate guidelines, were calculated via Diet Analysis+ 10.0. Midpoint sleep time was calculated using reported onset and rise time of usual weekday sleep; midpoint sleep times were then grouped by quintiles from earliest (1) to latest (5). On average, students reported 8.5±0.1 hours of sleep on weekdays; the average midpoint was 3:56AM. Students in the latest sleep quintile reported the greatest intake of empty calories compared to those in the earliest quintile (776±55 vs 525±60 kcals/day, p ≤ 0.05). The mean energy densities and macronutrient intakes between sleep midpoint quintiles were not different. Findings suggest that later midpoint sleep times may be related to increased consumption of empty calories among college students.
Desmoglein 2 contributes to cell cohesion dependent on the desmosomal cadherin expression pattern
Authors: Hartlieb Eva , Vigh Balázs , Spindler Volker , Waschke Jens
Published: June 2021

Desmoglein (Dsg) 2 is the most widespread isoform of the desmosomal cadherin protein family, of which four desmoglein (Dsg1–4) and three desmocollin (Dsc1–3) isoforms are expressed in keratinocytes. In the blistering skin disease pemphigus vulgaris (PV), Dsg3 is a target of autoantibodies indicating a central role for Dsg3 in keratinocyte cohesion. In contrast, for Dsg2 the role for keratinocyte cohesion has not been defined yet. We demonstrated the importance of Dsg2 for intestinal epithelial cells by incubation of Caco2 cells with a monoclonal Dsg2 antibody (Dsg2 mAb) which reduced cell integrity in a dissociation assay. However, Dsg2 mAb, in contrast to AK23, a pathogenic PV antibody targeting Dsg3, did not reduce cell cohesion in keratinocytes (HaCaT). To confirm that both antibodies are potent to bind their target protein we detected both antibodies’ heavy chains by Western blot analysis. Additionally both antibodies led to a decrease in the number of binding events of their corresponding recombinant Dsg target protein probed by atomic force microscopy (AFM). In contrast to another colorectal adenocarcinoma cell line (HT‐29), siRNA‐mediated silencing of Dsg2 in HaCaT cells reduced cell cohesion only under conditions of increased shear. These results indicate that Dsg2 contributes differently to cell cohesion dependent on the expression pattern of desmosomal cadherin family isoforms. DFG SFB487, SP1300–1
The role of mircoRNA 310/13 in Regulating Cellular Adhesion in Drosophila
Authors: Kapoor Amol Jay
Published: June 2021

We attempted to characterize the mechanism of action of micro RNA 310/13 in healthy Drosophila cells. microRNAs (miRs) are regulators of gene expression. The miR 310/13 complex has been found to act as a potential tumor suppressor due to its suppression of beta‐catenin. Beta‐catenin is a downstream target of the Wnt signaling pathway. Beta‐catenin activates T‐cell factor (TCF) family transcription factors, which causes transcription of proliferation proteins. Beta‐catenin also aggregates around the cell membrane to activate E‐Cadherin, which plays a role in cell‐cell adhesion and maintenance of the epithelial plane. Increased Beta‐catenin production is a common mutation in many cancers. Drosophila miR 310/13 cluster and its human Homologue hsa‐miR‐25 have been shown to bind to beta‐catenin and inhibit the Wnt pathway. p35 is an apoptotic inhibitor. Using transgenic Drosophila and Immunofluorescent staining, we were able to induce miR 310/13 and p35 over‐expression in larval wing discs. Our results confirm the hypothesis that miR 310/13 plays a role in regulating beta‐catenin levels, regulating adhesion, and regulating cell growth. We suggest that miR 310/13 may be a proliferation inhibitor and a cancer therapy. Source of research support from New York University, Langone Medical Center.
New anti‐inflammatory benefits of antibiotics: Tulathromycin promotes apoptosis and efferocytosis, and inhibits pro‐inflammatory CXCL8 production in a model of porcine pleuropneumonia
Authors: Duquette Stephanie , Fischer Carrie D , Muench Gregory P , Lucas Merlyn J , Buret Andre G
Published: June 2021

Neutrophil (PMN) apoptosis and subsequent clearance by surrounding macrophages (MΦ; efferocytosis) is critical to the resolution of inflammation following infection. Abnormal resolution of inflammation is responsible for the severe tissue damage characteristic of pneumonia, as in Actinobacillus pleuropneumoniae porcine pleuropneumonia. The superior clinical efficacy of some antibiotics has been attributed to inherent anti‐inflammatory properties via mechanisms that remain obscure.

Objective

to characterize anti‐inflammatory properties of tulathromycin (TUL) in a model of porcine pleuropneumonia.

Results

In vivo and in vitro, TUL induced porcine PMN apoptosis in a time‐and dose‐dependent manner, as determined by cell death ELISA, TUNEL fluorescent staining, and cleavage (activation) of caspase‐3, detected by western blot. TUL‐treated apoptotic PMN were readily efferocytosed by MΦ. TUL also induced delayed apoptosis and inhibited pro‐inflammatory CXCL8 production in porcine MΦ.

Conclusion

TUL's superior clinical efficacy may be due in part to anti‐inflammatory benefits mediated by PMN apoptosis and inhibition of CXCL8 in MΦ, which together promote the resolution of inflammation. Supported by Pfizer.
Exosomal microRNA modulates pathways of liver fibrosis by regulating connective tissue growth factor (CTGF) expression in fibrogenic cells during chronic injury.
Authors: Chen Li , Brigstock David
Published: June 2021

Aim

To establish the regulation of Connective Tissue Growth Factor (CTGF) expression in hepatic stellate cells (HSC) by delivery of miR‐214 in exosomes.

Methods

Exosomes isolated from conditioned medium of mouse HSC were characterized by Western blot, dynamic light scattering, zeta potential analysis, and cryo‐TEM. MiR‐214 was detected by RT‐PCR. Donor HSC were treated with GW4869 to inhibit exosome production and recipient HSC were transfected with a luciferase reporter vector under the control of a wild type CTGF 3′‐UTR or a mutant CTGF 3′‐UTR lacking the miR‐214 binding site.

Results

HSC exosomes were bi‐membrane vesicles, 80–150nm in diameter, negatively charged (−26mV), and positive for CD9. MiR‐ 214 was present in exosomes and its levels were increased by transfection of HSC with pre‐mir‐214. MiR‐214 levels in exosomes but not in cell lysates were reduced by pre‐treatment of the cells with GW4869. Co‐culture of miR214‐transfected donor HSC with CTGF UTR luciferase reporter‐transfected recipient HSC resulted in miR‐214‐dependent and GW4869‐dependent regulation of activity of the wild type, but not mutated, CTGF 3′‐UTR. CTGF 3‐UTR activity in activated recipient HSC was more greatly inhibited in a GW4869‐dependent manner by co‐culture with freshly isolated donor HSC (high endogenous miR‐214 levels) than with highly activated donor HSC (low endogenous miR‐214 levels).

Conclusions

MiR‐214 is exported exosomally between neighboring HSC, suppressing CTGF production by the cells, After injury, miR‐214 expression is suppressed, resulting in its reduced exosomal delivery and increased CTGF expression in recipient cells. Exosome‐dependent delivery of miRs between neighboring HSC represents a novel mechanism by which fibrotic pathways are regulated during chronic liver injury.
Hypertonic saline acts on septic lung remodeling through nitric oxide‐induced FAK activation pathway
Authors: Petroni Ricardo , Biselli Paolo , Barbeiro Hermes , Kubo Suely , Soriano Francisco
Published: June 2021

Hypertonic saline solution (HS, NaCl 7,5%) has shown to modulates immune function and decrease pulmonary injury triggered by endotoxic shock. Our objective was to investigate the effects of HS on the mechanism involved in pulmonary fibrosis, in an experimental model of endotoxemic shock. Wistar rats received lipopolysaccharide ‐ LPS (10mg/kg i.p.) and volume i.v. after 15 minutes. The animals were assigned in four groups (n=7): control group (not subjected to LPS); LPS group (injected with LPS 10mg/kg i.p); HS group (treated with hypertonic saline, 4 mL/Kg i.v. after LPS) and NS group (treated with normal saline, 34 mL/kg i.v. after LPS). At 24h after treatment, pulmonary mechanics, type I and type III collagen expression, metalloproteinase 9 expression, focal adhesion kinase (FAK) and nitric oxide synthesis were measured. NS increased pulmonary resistance and elastance, compared to other groups. HS inhibited collagen expression compared to LPS and NS groups and prevented pulmonary injury by decreased MMP9 activity in tissue. Expression of FAK was decreased in HS groups compared to LPS and NS groups. NO expression was decreased in HS group, compared to LPS and NS groups. We concluded that treatment of endotoxemic shock with HS solution act on nitric oxide‐induced FAK activation pathway, which could modulate the collagen deposition in pulmonary tissue, and consequently decrease the progression of pulmonary fibrosis
Potential role of pro‐oxidative and pro‐inflammatory mechanisms in blast‐induced neurotrauma
Authors: Cho Hyung Joon , Sajja Sujith V , VandeVord Pamela J , Lee Yong Woo
Published: June 2021

Pro‐oxidative and pro‐inflammatory mechanisms in the brain have been implicated in the onset and progression of neurological disorders. The present study hypothesizes that blast exposure to the brain induces alterations in pro‐oxidative/inflammatory environments with adverse behavioral outcomes. Male Sprague Dawley rats were anesthetized and exposed to calibrated blast overpressure (BOP) of 129.23 ± 3.01 kPa while controls received only anesthesia. Animals underwent novel object recognition (NOR) test following BOP prior to sacrifice. In situ dihydroethidium (DHE) fluorescence staining revealed that BOP significantly increased production of reactive oxygen species (ROS) in brains. In addition, real‐time RT‐PCR, immunofluorescence staining and ELISA demonstrated a significant up‐regulation of mRNA and protein expression levels of pro‐inflammatory mediators, such as interferon‐gamma (IFN‐γ) and monocyte chemoattractant protein‐1 (MCP‐1), in brains collected from BOP‐exposed animals compared with the controls. Furthermore, immunoreactivity of neuronal nuclei (NeuN) indicated that fewer NeuN‐expressing mature neurons were present following BOP. Moreover, NOR paradigm showed a significant impairment in the working memory following BOP. These results suggest that oxidative stress combined with inflammatory responses could play a potential role in blast‐induced neurotrauma.
Investigation of immunological changes in mice subjected to animal model of sepsis
Authors: Jeremias Isabela Casagrande , Victorino Vanessa Jacob , Barbeiro Hermes Vieira , Lima Thais Martins , Soriano Francisco Garcia
Published: June 2021

Sepsis induces multiple organ dysfunction syndrome including septic encephalopathy, that is commonly seen in systemically ill patients. Moreover, knowing that changes in the nervous system are implicated in the induction or amplification of immune system changes, or conversely, and they are not well elucidated in sepsis, we investigate the changes in brain function and immune system of mice in an animal model of sepsis by cecal ligation and puncture (CLP). Balb/c mice underwent CLP and were divided into two groups: CLP or Sham operation. We evaluated brain changes by SHIRPA behavioral test, after 6h of CLP. Mice were sacrificed, spleen were isolated and evaluated by flow cytometry profile of population of lymphocytes. We observe that the neuropsychiatric state in SHIRPA test was significantly decreased in animals with CLP. Regarding lymphocytes, no difference in the number of cells (B lymphocytes, regulatory T cells, helper T lymphocytes and cytotoxic T lymphocytes) compared to Sham and CLP. Thus, we conclude from these preliminary results that the decrease in neuropsychiatric condition is not correlated with cell number of lymphocytes. Support: FAPESP 2012/07898–0
Colorectal cancer cell lines made resistant to SN38‐and Oxaliplatin: Roles of altered ion transporter function in resistance?
Authors: Christensen Sandra , Jensen Niels Frank , Stoeckel Johanne Danmark , Belling Kirstine C , Romer Maria Unni , Gupta Ramneek , Brünner Nils , Pedersen Stine Falsig , Stenvang Jan
Published: June 2021

Colorectal cancer (CRC) is the 3rd most common cancer globally, with 5year survival rates of ~50%. Response rates to standard treatments (irinotecan (SN38) or Oxaliplatin (Oxp)) are 31–56% and drug resistance is a major problem. Thus, we established in vitro CRC models to investigate SN38 and Oxp resistance in HCT‐116, HT‐29 and LoVo cells. Microarray analysis and qPCR validation showed that mRNA expression of glutamate transporters SLC1A1 and SLC1A3 were markedly altered in resistant cells. Remarkably, mRNA levels of SLC1A3 were increased by ~40‐and ~2500‐fold in SN38‐and Oxp‐resistant HT29 cells, respectively. Studies are ongoing to assess glutamate uptake in parental and resistant CRC cells and the effect of inhibition/knockdown of SLC1A1 and ‐3 on SN38‐ and Oxp resistance.

In conclusion, SN38‐and Oxp‐resistance in CRC cells is associated with SLC1A1 and ‐3 dysregulation. As these transporters have not previously been implicated in SN38 or Oxp resistance and are generally restricted to the CNS, they are potential novel biomarkers for resistance and interesting candidates for therapeutic targeting.

Funding: Danish Research Council, Danish Council for Strategic Research, Danish Cancer Society
Transformation of mouse embryonic fibroblasts by dual expression of EBV LMP‐1 and HPV‐16 E6
Authors: Kinjo Takao , Shimabuku Tetsuya , Tamanaha Ayumi , Kitamura Bunta , Tanabe Yasuka , Oshiro Yoshihide
Published: June 2021

Background and Objective

From the high prevalence of Epstein‐Barr virus (EBV) and high‐risk human papillomavirus (HPV) infection among oral cancer patients in Okinawa, southwest islands of Japan, we hypothesize that dual EBV and HPV infection is related to oral carcinogenesis. The purpose of this study is to explore the mechanisms of transformation induced by dual EBV and HPV infection.

Methods

We constructed mouse embryonic fibroblasts expressing singly or dually EBV and HPV genes, and compared transformation properties.

Results

Single expression of EBV latent membrane protein‐1 (LMP‐1) and dual LMP‐1+HPV E6 expression induced increased cell proliferation and resistance to apoptosis, whereas expression of a single viral gene did not exhibit these properties. Dual LMP‐1+E6 expression showed anchorage independence and tumor formation in nude mice, whereas expression of LMP‐1 alone did not. Furthermore, dual LMP‐1+E6‐expressing cells induced increased NF‐κB signaling, and the knockdown of LMP‐1 or E6 expression eliminated the increased cell proliferation, anchorage independence, and NF‐κB activation.

Conclusions

These data suggested that the singular expression of these viral genes is insufficient for inducing transformation and that dual LMP‐1+E6 expression, which is associated with increased NF‐κB activity, is required for transformation.
Expression of miR‐125b and miR‐145 in breast cancer with overexpression of MUC1
Authors: Zanetti Juliana Silva , Darin Patricia R , De Lucca Fernando , Ribeiro‐Silva Alfredo
Published: June 2021

The deregulation of microRNAs (miRNAs) expression is associated with a variety of human diseases including cancer. Recently, it was found that the mucin1 (MUC1) oncoprotein is a direct target of miR‐125b and miR‐145 in breast cancer cells established from invasive ductal carcinoma (IDC). It is known that data obtained using cancer cells are not always validated in intact tumor tissues. The aim of this study was to investigate the expression of miR‐125b and miR‐145 in IDC formalin fixed paraffin embedded (FFPE) tissues with overexpression of MUC1. The FFPE samples of IDC were selected by immunohistochemistry and the expression of miRNAs was evaluated by real time PCR. The normal breast FFPE samples were used as control. Our results indicate that miR‐125b and miR‐145 expression was reduced 40% (P<0.05) and 60% (P<0.05), respectively. These findings suggest that miR‐125b and miR‐145 negatively regulate the expression of MUC1 in tumor tissue from patients with IDC. Thus, this study may contribute to a better understanding of the molecular mechanisms involved in the regulation of MUC1 expression in breast cancer and to develop new therapeutic strategies for IDC.
Morphologic evaluation of dental pulp vascularity during DMBA experimental administration
Authors: Marto Carlos Miguel , Costa Eduardo , Farinha Rodrigo , Cabrita António Silvério
Published: June 2021

7,12 ‐ Dimethylbenz(a)anthracene (DMBA) is a well known carcinogen and immunosuppressor. Oral administrated DMBA circulates in the bloodstream and rich vascularized tissues like dental pulp can be particularity sensible to this compound effect. In this study we evaluate dental pulp morphologic changes during DMBA per os experimental administration.

Control and test groups were constituted by ten male Wistar rats each, 8 weeks old. Control group was maintained without manipulation and test group was submitted to daily administration of 20mg/kg DMBA dissolved in olive oil and administrated by oral gavage, both during 4 weeks. All animals were sacrificed at the end of 4 weeks period and a complete necropsy was performed. Slides of dental pulp were obtained from the lower incisors dental root medium third and hematoxylin‐eosin coloration was performed. Slides were observed in light microscopy and morphometric analysis was performed, evaluating areas of dental pulp vascularity, calcification and presence of inflammatory cells. T‐student test was used in statistical analysis with a significance level of 0,05.

Results show no statistic difference between control and test group and none inflammatory process was observed. We hypothesized that these results may be due the immunosuppressor activity of DMBA, administrated dose or even protective role of olive oil so more studies must be performed.
Dietary modulation of BALT on a rat mammary tumor model
Authors: Lopes Ana Isabel Calado , Pereira Paula , Cabrita António Silvério
Published: June 2021

Murine bronchus associated lymphoid tissue (BALT) is a mirror of systemic immunity since it varies greatly with immune status of the animal and plays a pivotal role in local immune responses at the lung surface. BALT enhanced histopathology (EH) is a semiquantitative screening tool to determine whether there is a potential effect on the immune system of rats under controlled conditions.

168 Sprague Dawley virgin female rats with 42 days were randomly distributed for seven groups of 24 animals each. Besides group I, at 55 days the groups were induced for mammary cancer with 20mg of 1,12‐dimetilbenzantracene (DMBA). Dietary protocols were applied for 150 days as following. Group I: standard diet (SD), 300Kcal/100g; Group II: DMBA +SD; Group III: SD + beans 40g/Kg of SD, 341Kcal/100g; Group IV: SD + olive oil (50ml/Kg of SD); Group V: fiber supplement 4× the SD, 250Kcal/100g; Group VI: fiber supplement 4× the SD + beans 19,95gr + olive oil 25ml on 1Kg of SD; Group VII: SD + mediterranean lyophilized diet (41Kcal). The animals were ethically euthanized and the lungs were routinely fixed and stained with hematoxylin‐eosin. BALT was evaluated by EH.

The BALT morphology of tumoral rats with mediterranean diet and caloric restrain was similar to the BALT of control group. The other tumoral groups had decreased BALT in size and lymphocyte number compared to control group.

These results suggest that diet can interfere on BALT morphology.
Macrophage Phenotypes in Murine Aneurysm
Authors: Morgan Stephanie , Wang Qiwei , Harberg Calvin , Liu Bo
Published: June 2021

A prominent pathological feature of abdominal aortic aneurysm (AAA) is the infiltration of mononuclear phagocytes such as macrophages. Accumulating literature has highlighted the importance of macrophage phenotypes and their role in vascular disease. Inflammatory (M1) macrophages produce proteases and proinflammatory cytokines, while alternatively activated (M2) macrophages produce anti‐inflammatory cytokines and extracellular matrix (ECM) components. Using an elastase‐induced model of murine AAA, we tested the hypothesis that aneurysm induction shifts M1/M2 balance toward inflammation. Leukocytes were isolated from the aortic tissue and analyzed by flow cytometry. Three days after surgery, arteries treated with elastase (aneurysm) or inactivated elastase (control) contained similar levels of total macrophages (7aad‐CD45+CD11b+F4/80+). However, as disease progressed, aneurysmal aorta displayed significantly higher number of macrophages than controls ~2.15 and 1.28 fold higher 7 and 14 days after surgery, respectively. Interestingly, the M1:M2 ratio (CD86+:CD206+) was higher in aneurysm arteries, largely due to a selective suppression of M2 polarization. These data suggest an imbalance between M1 and M2 macrophages may contribute to a chronic inflammatory state during aneurysm progression.

Funding provided by NRSA T32 HL 07936 from the NHLBI.
IL‐17A potentiates TNFα‐induced expression of E‐selectin and cytokine secretion from human endothelial cells thereby controlling neutrophil migration across the capillary wall
Authors: Dissing Steen , Hansen Anker Jon , Bosteen Markus Høybye , Tritsaris Katerina
Published: June 2021

Cytokines and chemotactic peptides are important compounds for inflammatory processes by directing inflammatory cells towards the capillary wall. Interleukin 17A (IL‐17A) and TNF‐α are cytokines that act as potent mediators of inflammation. Activation of human dermal microvascular endothelial cells (HDMEC) by TNFα and IL‐17A causes phosphorylation of IkBα whereby NFkB subsequently becomes phosphorylated resulting in initiation of transcription of adhesion molecules such as E‐selectin. After TNFα and IL‐17A stimulation we observed a strong secretion of IL‐8, GROα and G‐CSF and IL‐17A was able to significantly augment the levels of TNFα induced E‐selectin and ICAM‐1. Stimulation with TNFα and IL17A causes the Src kinase to become deactivated consistent with modifications of the capillary wall structure. We measured chemotactic migration of neutrophils across a HDMEC monolayer and found that IL‐17A on top of TNFα caused an enhanced transmigration across resting and activated endothelial layers. In accordance with this observation, IL‐17A was able to markedly increase TNFα induced neutrophil adherence to HDMEC monolayers in an in vitro adhesion assay. These results show that IL‐17A and TNFα act in cooperation to modify the capillary wall, cause secretion of chemokines and facilitate neutrophil migration across the endothelial cell barrier.
The Borrelia burgdorferi adhesin DbpA is bifunctional, binding to extracellular matrix to foster tissue colonization and to the host complement regulatory protein C4BP to promote bloodstream survival
Authors: Lin Yi‐Pin , Blevins Jon S. , Leong John M.
Published: June 2021

Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, can infect the skin at the site of the tick bite and may subsequently spread via the bloodstream to the heart, nervous system, and joints, leading to multisystemic disease. B. burgdorferi produces two surface lipoproteins, decorin binding protein A (DbpA) and B (DbpB), that promote spirochetal binding to the extracellular matrix (ECM) components decorin and dermatan sulfate. A B. burgdorferi dbpBA deficient mutant is unable to establish mammalian infection. In this study, we found that the expression of wild type DbpA, but not DbpAΔC11, which lacks the C‐terminal 11 amino acids of DbpA and is incapable of binding to either decorin or dermatan sulfate, promoted stable murine colonization by B. burgdorferi dbpBA, indicating that the binding activity of DbpA is critical for colonization. Interestingly, unlike the parental B. burgdorferi dbpBA mutant, derivatives that ectopically produce either DbpA or DbpAΔC11 were detected in the bloodstream at 3 days post infection and were resistant to human serum in vitro. Further, pull‐down assays of human serum and quantitative binding analyses using recombinant DbpA revealed that DbpA recognizes the complement regulatory protein C4BP (C4‐binding protein) with high affinity (KD=0.82 ±0.03μM). These findings suggest that DbpA is a bifunctional protein that binds ECM to promote tissue colonization and C4BP to protect the spirochete from immune clearance at early stages of infection.
DISTINCT EFFECT OF H2O2 IN HEALTH AND UNHEALTHY HEMATOPOIETIC CELLS SURVIVAL
Authors: PEDRO AMANDA NOGUEIRA , MUNHOZ THALYTA AP. CESÁRIO , DIAS CAROLINA CARVALHO , PAREDES‐GAMERO EDGAR JULIAN , ORIGASSA CLARICE C , EÇA LILIAN PIÑERO MARCOLIN , FERREIRA ALICE TEIXEIRA
Published: June 2021

In the last years, many data has suggested the participation of oxidative stress in the regulation of hematopoietic cell homeostasis. Hematopoietic cells are particularly vulnerable to reactive oxygen species (ROS) in a concentration‐dependent manner. It is known that constitutively ROS levels differ between normal and malignant cells. In this respect it is the ideal balance in the redox status for both cell types in order to selectively cause death of cancer but not normal cells. In this work it was induced a pro‐oxidant environment by stimulating murine and fresh human umbilical cord blood (health models) as well as HL‐60 leukemic cells (unhealthy model) with different concentrations (0, 1, 5 and 10 μM) of hydrogen peroxide (H2O2) in vitro. It was observed that H2O2 (5 μM) induced cancer but not normal cells death (28% versus 0,5%). However, HL‐60 hematopoietic stem cells has increased in percentage at the same time progenitors cells diminished. In addition clonogenic capacity was great affected on these cells, enabling to generate myeloid clones. Differentiation was not modulated by the H2O2 in any cell type. HL‐60 loss viability had the participation of phosphor‐AKT which expression has diminished significantly. In summary, we found that at low concentration, H2O2 is able to kill hematopoietic cancer cells without affect normal cells.

Supporting: FAPESP
CD4+ T cells remodel inflammatory ocular surface by disrupting stem cell quiescency and oscillating rhythm
Authors: Chen Ying‐Ting , Chen Feeling , Lazarev Stanislav , Bahrami Ahmad F , Noble Lisa B , Vijmasi Trinka , McNamara Nancy
Published: June 2021

Immune cell‐mediated inflammation and stem cells‐based morphogenesis are intimately integrated during physiological epithelial regeneration, but the pathogenesis underlying the defective squamous metaplastic regeneration (SQM) of chronic inflamed mucosa remains obscure. Using Aire‐KO mouse as an autoimmune ocular SQM model, we employed EdU/BrdU double pulsing‐chasing, and Ki67, p63 and CD4 immunofluorescence to profile the oscillation frequency and quiescence state of ocular stem cells during autoimmune‐deviated ocular surface regeneration. In WT, distinct clusters of EdU+ or BrdU+ basal p63+ progenitors were found uni‐directionally migrating from the limbus to the corneal apex at 40 um/hr. Aire‐KO mice with CD4+ T cells infiltrated limbus demonstrated randomly distributed EdU+ and/or BrdU+ basal progenitors in a faster but non‐directional migratory movement. A 3‐fold increased Ki67 mitotic index in contrast to a 2‐fold increased BrdU‐labeling index in Aire‐KO's progenitors indicates a CD4 T cells‐mediated stem cell priming and a shortened S phase in cell cycle. Label‐retaining cells were significantly reduced in Aire‐KO (8.6 cells/eye vs 25.6 cells/eye in WT), despite the indiscernable colony‐forming efficiency. Collectively, data presented here suggest a rapid cycling nature of SQM‐funding cells without attrition in self‐renewal or clonogeneity of stem cells in autoinflammatory niche.
Effects of PPARα Activation and the Role of HO‐1 in Acute SAH‐Induced Fall in Cerebral Blood Flow in Rat
Authors: Osagie Natalie , Oyekan Adebayo O , Yakubu Momoh A.
Published: June 2021

Traumatic Brain Injury is a serious public health problem; it is a major cause of death and disability globally resulting from secondary subarachnoid hemorrhage (SAH). We have investigated the effects of PPARα activation and the role of HO‐1 in acute SAH‐induced fall in cerebral blood flow (CBF). Rats received PPARα activator clofibrate, HO‐1 activator (CoCl2) and inhibitor (SnMPP) (2.4 mg; 50 μg, and 24 μg/kg or vehicle) 2–4 days before injection (i.c) of 0.2ml of blood (SAH). Changes in CBF were determined by Laser Doppler Scanner (Moor LDI 5152), the data presented as percentage change in CBF. Clofibrate and CoCl2 increased CBF by 3 and 8% while SnMPP and SAH reduced CBF by 30% and 50% at 15 min. Clofibrate and CoCl2 pretreatments prevented SAH‐induce fall in CBF (−11 and −21%), while SnMPP potentiated the fall in CBF {−70 vs. −50% (SAH)}. SnMPP obliterated clofibrate‐induced attenuation of SAH‐induced fall in CBF (−59% vs. −11%). CoCl2 and Clofibrate treatments increased bilirubin levels in the plasma from 189±30 to 202±35 and 257±28 μM with SnMPP reducing it to 33±5 μM. The CoCl2 and Clofibrate‐induced increases in bilirubin were reduced to 40±10 and 159±30 μM in the presence of SnMPP. Thus, PPARα prevented SAH‐induced fall in CBF, an effects which seems to be mediated via activation of HO‐1 as its inhibitor prevented it. PPARα and HO‐1 signaling pathways could be a viable therapeutic target for the treatment of TBI.
Inhibition of prostaglandin E2 or thromboxane A2 promotes human aortic smooth muscle cell differentiation
Authors: Adedoyin Oreoluwa Olufunmilayo , Loftin Charles D
Published: June 2021

INTRODUCTION

Inhibition of cyclooxygenase‐2 (COX‐2) helps maintain vascular SMC differentiation when treating abdominal aortic aneurysms in a disease model. However, mechanisms of COX‐2‐dependent vascular SMC phenotypic modulation have not been identified. We assessed the hypothesis that COX‐2 induces SMC de‐differentiation by the action of prostaglandin E2 (PGE2) or thromboxane A2 (TXA2).

METHODS

Cultured SMCs were treated with enzyme inhibitors or prostanoid receptor agonists or antagonists. Expression of α‐actin as a differentiation marker was examined by real‐time PCR, western blot or immunocytochemistry; and EIA performed to measure inhibitor effects on PGE2 production.

RESULTS

Celecoxib increased α‐actin expression and significantly reduced PGE2 synthesis in stimulated cells (p < 0.0001; n=3; unpaired Students t‐test). Differentiated cells treated with 1μM PGE2 showed significant reduction in differentiation (p < 0.05; n=3; one‐way ANOVA), and treatment with 300nM SQ 29548 (TXA2 receptor antagonist) promoted differentiation. Also, treatment of de‐differentiated cells with 10 μM seratrodast (TXA2 receptor antagonist) increased α‐actin expression by 52%, while 5μM IBOP (TXA2 receptor agonist) reduced differentiation compared with untreated controls.

CONCLUSION

COX‐2 inhibition may increase hASMC differentiation by inhibiting production of PGE2 or TXA2.

Funding: NIH HL083122
Leukemic Relapse Following MMSC Rescue in a Murine Model of GVHD
Authors: O'Kane Barbara Jean , Devetten Marcel , Jackson John , Lacy Jordan , Sharp J Graham
Published: June 2021

Recently, the use of mesenchymal stem cells (MMSC) for the amelioration of acute graft versus host disease (GVHD) has shown promise as a therapeutic intervention. Given that MSC can suppress allogeneic immune responses, there is a concern that the use of these cells may promote leukemic relapse. Here we present a murine model of GVHD in the presence of leukemic cells (L1210). Acute GVHD was induced with or without prior injection of L1210 leukemia cells. The mice were treated with primary MMSC or a mesenchymal cell line (OMA‐AD). The results without L1210 cells, demonstrated that mice treated with primary MMSC had developed moderate GVHD but had increased long‐term survival when compared to controls. The group treated with OMA‐AD cells demonstrated a protective effect against GVHD, and the survival rate was superior to that of animals treated with primary MMSC. However, with L1210 present, the control mice lived outlived the OMA‐AD group. Only the mice receiving primary MMSC survived long term. It appears that although MMSC and OMA‐AD cells can both ameliorate GVHD, the greater immunosuppressive effect of OMA‐AD cells permitted the growth of the leukemic cells. The moderate GVHD that remained after MMSC treatment eliminated the leukemia in the majority of mice. These studies demonstrated that in mice, administration of MMSC can ameliorate GVHD but the complete suppression of GVHD appears to permit leukemic relapse.
Moderate treadmill exercise prevented anxiogenic behavior and impaired basal synaptic transmission in an Alzheimer's disease‐like rat model
Authors: Dao An Thien , Salim Samina , Alkadhi Karim
Published: June 2021

In this study, we investigated the effect of moderate treadmill exercise regimen on non‐cognitive function and basal synaptic transmission in AD‐like pathology model, achieved by i.c.v. infusion of Aβ1–42 (250 pmol/day) for 2 weeks. We have utilized a battery of behavioral tests to assess anxiogenic and exploratory behavior in adult Wistar rats treated with Aβ peptides and/or exercise training. Additionally, in vivo extracellular recording was used to evaluate changes in basal synaptic transmission in the Cornu Ammonis 1 (CA1) area. The animals were randomly assigned into 4 groups: control, exercise, Aβ infusion (Aβ) and Aβ‐infused/exercised (Ex/Aβ). Our data consistently indicated that Aβ rats showed increased anxiety‐like as tested in the open field, elevated plus maze, and light‐dark box paradigms. Interestingly, both Aβ and Ex/Aβ rats showed impaired locomotor activity compared to control and exercise rats. Hence, moderate treadmill exercise exerted an anxiolytic effect but failed to prevent locomotor deficits caused by AD. Additionally, the right shift of the I/O curve of Aβ rats indicated a markedly impaired basal synaptic transmission compared to those of other groups. Aβ rats also required a stronger stimulus to produce the same response at minimum, 30% of maximum, and maximum compared to other groups. Together, these data suggest a protective role of regular exercise in AD.
Derivation and characterization of rat iPS cells using a mouse STEMCCA reprogramming vector
Authors: Rajanahalli Pavan , Weiss Mark L
Published: June 2021

Rats are considered an important animal model system by having many similarities to humans in terms of physiological processes of the cardiovascular, metabolic and neurological systems. Induced pluripotent stem cell (iPSC) technology has great potential to advance in the field of regenerative medicine by generating patient‐specific stem cell for treatment or in vitro disease modeling. Here, we derived rat iPSC's from Fischer 344 embryonic fibroblasts using a mouse STEMCCA cre‐excisable reprogramming vector expressing Oct‐4, Klf4, Sox‐2 and c‐Myc. Once rat ESC‐like colonies were derived, we removed the transgene vector and demonstrated that one of the clones tested showed a normal karyotype by G‐banding. The iPSC colonies demonstrate a morphology that is similar to rat embryonic stem cells (ESCs), are positively stained for pluripotency markers such as alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. The rat iPSCs have been maintained more than 27 passages. Using a custom‐designed microarray, we compared the gene expression profile of rat iPSCs to rat ESCs and there was good similarity between the two with the exception of Cdx2 expression which is expressed in rat ESCs. We plan to further characterize the rat iPSCs to determine whether they can undergo neural differentiation in vitro and whether the micro RNA expression of rat iPSCs is similar to rat ESCs.
Misoprostol, prostaglandin analogue, modulates cytokine activity through cAMP pathway
Authors: Gobejishvili Leila , Barve Shirish , Khan Rehan , Avila Diana , McClain Craig , Hill Daniell
Published: June 2021

Dysregulated cytokine metabolism plays a critical role in the pathogenesis of alcoholic liver disease. We and others have shown that cAMP signaling critically modulates endotoxin‐stimulated expression of pro‐inflammatory cytokine TNF in monocytes and macrophages. The overall aim of this pilot study was to assess the efficacy of Misoprostol, as a potential therapeutic agent, in the treatment of alcoholic hepatitis (AH). Healthy volunteers were given different doses of Misoprostol and baseline and LPS‐inducible cytokine levels were examined ex vivo. Additionally, human peripheral blood mononuclear cells and murine macrophage cell line 264.7 were used to investigate underlying mechanisms of misoprostol effect on cytokine expression. Our results show that lower dose of Misoprostol was well‐tolerated with fewer GI side effects and was effective in modulating cytokine activity. Mechanistically, Misoprostol effect was critically mediated by increase in cAMP levels and consequent changes in activity of transcription factors CRE and NFκB. Further studies are needed to evaluate the effects of Misoprostol on the modulation of cytokine activity, liver function and clinical course in AH patients.
A Single, Ultra‐High Dose Aminoglycoside Therapy in a Rat Model of E. coli Induced Septic Shock
Authors: Pisipati Amarnath , Anand Kumar , Ariano Robert , Blakley Brian , Esfahani Ehsan K
Published: June 2021

Introduction

Septic shock is a life threatening condition often due to Gram negative bacterial infections and is responsible for around 200,000 deaths every year in United States of America alone. Our research focuses on determining the potential of a single, ultrahigh dose aminoglycoside therapy for accelerated bacterial clearance with minimal to manageable side effects in a rat model of E. coli induced septic shock.

Methods: Study in uninfected animals

Gentamicin was administered intravenously at 80 and 160 mg/kg doses to anesthetized and intubated Sprague Dawley rats. Mean arterial pressure, heart rate, respiratory rate and percentage oxygen saturation were determined. The occurrence and time course of neuromuscular paralysis/ventilator dependency, renal injury and auditory toxicity after ultra‐high dosing of gentamicin was determined. Renal injury was determined by Creatinine clearance and histology. Auditory brain stem response studies were performed to assess the auditory toxicity.

Septic shock induced animals

Septic shock was induced in rats by surgical, intra‐peritoneal implantation of a gelatin capsule containing E. coli bort (ATCC 700973) in a fibrinogen and alfa‐cellulose matrix. Gentamicin was intravenously administered at 5, 10, 20, 40, 80 and 160 mg/kg doses.

Results

Ultra high gentamicin dosed animals were found to develop neuromuscular paralysis with ventilator dependency for 20 – 40 minutes and recovered thereafter. Rats did not exhibit any auditory and renal toxicity. Complete eradication of bacterial counts from blood occurred following ultra‐high doses.

Conclusion

Rats were able to tolerate single, ultra‐high doses of gentamicin with complete eradication of bacterial counts from blood.

Research is supported by University of Manitoba
Structure‐Activity Considerations for Heteroaromatic nortropeines and N‐methyltropeines with Nicotinic Acetylcholine Receptor (nAChR) Subtypes and a Serotonin Receptor (5HT3A)
Authors: Serrano Aracely , Nemecz Ákos , Gomez Kimberly , Maksay Gábor , Yamahuchi John George , Taylor Palmer
Published: June 2021

A series of heteroaromatic tropeines have been studied as potentiators of the glycine receptor. We show here a range of potencies for these compounds as agonists and antagonists on excitatory pentameric ligand‐gated ion channels (pLGIC), such as the nAChR and 5HT3A receptor. We have analyzed the interactions of 19 heteroaromatic tropeines, including nor and N‐methyltropeine congeners, with the soluble acetylcholine binding protein (AChBP), a structural extracellulardomain surrogate of pLGICs, and quantified their activities at hα7 and α4β2‐nAChRs and the mouse 5HT3A receptor. Pharmacological studies on hα7‐nAChR reveal the more potent compounds to be agonists with EC50 values ranging between 28–200 nM. By contrast, some of the set of compounds showed antagonist activity on α4β2‐nAChR and 5HT3A receptors with a range of selectivity. In turn, we have compared their pharmacologic spectra of activities with a previously studied series of related azabicyclic heterocycles linked to triazoles by click‐chemistry. Analysis of binding selectivity and pharmacologic activity, when combined with structural analysis, achieved through X‐ray crystallographic structures of tropine‐AChBP complexes, has enabled us to define the molecular determinants of selectivity for the tropine, acylester and linked triazole moieties in this series of pLGIC ligands. Supported by USPHS grant GM18360 and México CONCyT‐18622.
TNF and IL‐6/STAT3 crosstalk revealed in a commercially‐available cell line
Authors: Loring Ralph H , Naiki Pranitha , Wu Ting , Garg Brijesh
Published: June 2021

TNF (Tumor necrosis factor alpha) and IL‐6 (Interleukin 6) are cytokines with important inflammation and cancer roles that exhibit different interactions depending on cell context, e.g. TNF sometimes induces IL‐6 secretion, but TNF also directly alters IL‐6 signaling and vice versa (crosstalk). IL‐6 activates the transcription factor STAT3 in HEK‐Blue® IL‐6 cells (Invivogen) and STAT3 stimulates a reporter gene, secreted alkaline phosphatase (AP), but STAT3 inhibitors block AP secretion. Co‐application of 0.3–10 ng/ml TNF increases IL‐6 induced AP secretion 2–5 fold. However, TNF alone has no effect on basal STAT3 signaling, suggesting that TNF increases IL‐6 sensitivity without inducing IL‐6 secretion. SN50, an inhibitor of the transcription factor NFkB, does not block TNF changes in IL‐6 sensitivity, suggesting a different TNF signaling pathway. TNF effects originally appeared in HEK‐Blue® cells grown in medium giving unusually low responses to IL‐6, and the effects are retained in serum‐free medium. TNF effects on HEK‐Blue® cells grown in chemically‐defined medium may lead to finding serum factors that influence the context of TNF and IL‐6 crosstalk. No external funding.