scholarly journals Ex vivo Pretreatment of Islets with Mitomycin C

2017 ◽  
Vol 26 (8) ◽  
pp. 1392-1404 ◽  
Author(s):  
Naoya Sato ◽  
Junichiro Haga ◽  
Takayuki Anazawa ◽  
Akira Kenjo ◽  
Takashi Kimura ◽  
...  
Keyword(s):  
Mitomycin C ◽  
Validation Studies ◽  
Ex Vivo ◽  
Chemotactic Activity ◽  
Vitro Assay ◽  
Proinflammatory Mediators ◽  
Genes Encoding ◽  
Matrix Metallopeptidase

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

scholarly journals An in-vitro assay using human spermatozoa to detect toxicity of biologically active substances

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tino Vollmer ◽  
Börje Ljungberg ◽  
Vera Jankowski ◽  
Joachim Jankowski ◽  
Griet Glorieux ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Toxicity Testing ◽  
Biologically Active ◽  
Human Spermatozoa ◽  
Vitro Assay ◽  
Cellular Behavior ◽  
Novel Method ◽  
Set Up

Abstract Identifying the key toxic players within an in-vivo toxic syndrome is crucial to develop targeted therapies. Here, we established a novel method that characterizes the effect of single substances by means of an ex-vivo incubation set-up. We found that primary human spermatozoa elicit a distinct motile response on a (uremic) toxic milieu. Specifically, this approach describes the influence of a bulk toxic environment (uremia) as well as single substances (uremic toxins) by real-time analyzing motile cellular behavior. We established the human spermatozoa-based toxicity testing (HSTT) for detecting single substance-induced toxicity to be used as a screening tool to identify in-vivo toxins. Further, we propose an application of the HSTT as a method of clinical use to evaluate toxin-removing interventions (hemodialysis).

scholarly journals Generation of strong casein kinase 1 inhibitor of Arabidopsis thaliana

2019 ◽  
Author(s):  
Ami N Saito ◽  
Hiromi Matsuo ◽  
Keiko Kuwata ◽  
Azusa Ono ◽  
Toshinori Kinoshita ◽  
...  
Keyword(s):  
Bromine Atom ◽  
Casein Kinase ◽  
In Planta ◽  
Vitro Assay ◽  
Casein Kinase 1 ◽  
Yeast Fungi ◽  
Genes Encoding ◽  
Period Lengthening

AbstractCasein kinase 1 (CK1) is an evolutionarily conserved protein kinase among eukaryotes. Studies on yeast, fungi, and animals have revealed that CK1 plays roles in divergent biological processes. By contrast, the collective knowledge regarding the biological roles of plant CK1 lags was behind those of animal CK1. One of reasons for this is that plants have more multiple genes encoding CK1 than animals. To accelerate the research for plant CK1, a strong CK1 inhibitor that efficiently inhibits multiple members of CK1 proteins in vivo (in planta) is required. Here, we report a novel strong CK1 inhibitor of Arabidopsis (AMI-331). Using a circadian period-lengthening activity as estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship (SAR) study of PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride), a potent CK1 inhibitor of Arabidopsis, and found that PHA767491 analogues bearing a propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) enhance the period-lengthening activity. The period lengthening activity of a hybrid molecule of AMI-212 and AMI-23 (AMI-331) is about 100-fold stronger than that of PHA767491. An in vitro assay indicated a strong inhibitory activity of CK1 kinase by AMI-331. Also, affinity proteomics using an AMI-331 probe showed that targets of AMI-331 are mostly CK1 proteins. As such, AMI-331 is a strong potent CK1 inhibitor that shows promise in the research of CK1 in plants.

scholarly journals Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

2019 ◽  
Author(s):  
Takehito Sugasawa ◽  
Tome Yoshiya ◽  
Yoshinori Takeuchi ◽  
Naoya Yahagi ◽  
Rahul Sharma ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Binding Protein ◽  
Cold Stimulation ◽  
Vitro Assay ◽  
Mitochondrial Dna Copy Number ◽  
Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injury. However, its molecular mechanisms are unknown. To clarify these mechanisms, in this study, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cAMP response element-binding protein 1 (CREB1) was markedly phosphorylated (as pCREB1) and CREB-binding protein (CBP) was recruited to pCREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes such as Pgc-1α involved in mitochondrial biogenesis. The foregoing results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes in a manner dependent on cold stimulation frequency and duration. This information may serve as an impetus for further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

scholarly journals Impact of Bicarbonate on PBP2a Production, Maturation, and Functionality in Methicillin-Resistant Staphylococcus aureus (MRSA)

2021 ◽  
Author(s):  
Selvi C. Ersoy ◽  
Henry F. Chambers ◽  
Richard A. Proctor ◽  
Adriana E. Rosato ◽  
Nagendra N. Mishra ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Carotenoid Content ◽  
Methicillin Resistant ◽  
Genes Encoding ◽  
Regulatory Impact ◽  
Mrsa Strains ◽  
The Impact

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam-susceptibility in vitro, ex vivo and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Herein, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on: expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA); membrane PBP2a and PrsA protein content; and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (vs - nonresponsive) MRSA, there was significantly reduced expression of: i) mecA and blaZ; ii) the vraSR-prsA gene axis; and iii) pbp4. Carotenoid production was reduced, while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for maintenance of the MRSA phenotype through PBP2a functionality and maturation.

The High Potential of Ozone Gas to Inactivate Echinococcus granulosus Protoscoleces During Hydatid Cyst Surgery

2020 ◽  
Vol 20 (5) ◽  
pp. 708-712
Author(s):  
Hossein Mahmoudvand ◽  
Majid Fasihi Harandi ◽  
Massumeh Niazi ◽  
Abdolreza Rouientan ◽  
Fazel Mohammadi-Moghadam ◽  
...  
Keyword(s):  
Hydatid Cyst ◽  
Ex Vivo ◽  
Human Subjects ◽  
Oral Bacteria ◽  
Vitro Assay ◽  
Low Concentrations ◽  
Wide Range ◽  
Resistant Strains

Background: In medicine, ozone therapy is effectively used in a broad spectrum of diseases. Reviews have shown that ozone gas demonstrates potent antimicrobial effects against a wide range of pathogenic microorganisms, such as oral bacteria, fungi, viruses, and parasite even in resistant strains. The present investigation was designed to assess the protoscolicidal effects of ozone gas on hydatid cysts protoscoleces in vitro and in vivo. Methods: Hydatid cyst protoscoleces were acquired from sheep livers that were slaughtered at Kerman slaughterhouse, Iran. The viability of protoscoleces was assessed by the eosin exclusion examination after exposure with ozone gas for 1 to 14 min in vitro and ex vivo. Results: In this study, in vitro assay showed that ozone gas at the concentration of 20 mg/L killed 85 and 100% of hydatid cyst protoscoleces after 4 and 6 min of treatment, respectively. However, in the ex vivo analysis, a longer time was needed to confirm a potent protoscolicidal activity such that ozone gas after an exposure time of 12 min, 100% of the protoscoleces were killed within the hydatid cyst. Conclusion: : In conclusion, the findings of the present study showed that ozone gas at low concentrations (20 mg/L) and short times (4-6 min) might be used as a novel protoscolicidal drug for use in hydatid cyst surgery. However, more clinical surveys are required to discover the precise biological activity of ozone gas in animal and human subjects.

scholarly journals Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

2020 ◽  
Vol 21 (13) ◽  
pp. 4588
Author(s):  
Takehito Sugasawa ◽  
Yoshiya Tome ◽  
Yoshinori Takeuchi ◽  
Yasuko Yoshida ◽  
Naoya Yahagi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Binding Protein ◽  
Cold Stimulation ◽  
Vitro Assay ◽  
Mitochondrial Dna Copy Number ◽  
Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

scholarly journals Cysteinyl leukotriene 2 receptor promotes endothelial permeability, tumor angiogenesis, and metastasis

2018 ◽  
Vol 116 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Ernest Duah ◽  
Lakshminarayan Reddy Teegala ◽  
Vinay Kondeti ◽  
Ravi K. Adapala ◽  
Venkateshwar G. Keshamouni ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Ex Vivo ◽  
Endothelial Permeability ◽  
Tumor Vasculature ◽  
In Vivo Model ◽  
Mouse Tumor ◽  
Proinflammatory Mediators ◽  
Myosin Light Chain 2

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLT2R, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R-null mice (Cysltr2−/−) compared with WT and CysLT1R-null mice (Cysltr1−/−). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared with CysLT1R. Further, the tumor vasculature in Cysltr2−/− recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLT2R as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLT2R as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLT2R as a possible target for intervention.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

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