In vitro functional evaluation of isolaureline, dicentrine and glaucine enantiomers at 5-HT2 and α1 receptors

2018 ◽  
Vol 93 (2) ◽  
pp. 132-138 ◽  
Author(s):  
Hui Li Heng ◽  
Chin Fei Chee ◽  
Chun Keng Thy ◽  
Jia Ti Tee ◽  
Sek Peng Chin ◽  
...  
Keyword(s):  
Functional Evaluation ◽  
Α1 Receptors

scholarly journals Cryopreservation of Whole Tumor Biopsies from Rectal Cancer Patients Enable Phenotypic and In Vitro Functional Evaluation of Tumor-Infiltrating T Cells

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2428
Author(s):  
Frank Liang ◽  
Azar Rezapour ◽  
Peter Falk ◽  
Eva Angenete ◽  
Ulf Yrlid
Keyword(s):  
Rectal Cancer ◽  
T Cells ◽  
Γδ T Cells ◽  
T Cell Subsets ◽  
Functional Evaluation ◽  
Mait Cells ◽  
Ifn Γ ◽  
Tumor Biopsies

TILs comprise functionally distinct conventional and unconventional T cell subsets and their role in responses to CRC treatments is poorly understood. We explored recovery of viable TILs from cryopreserved tumor biopsies of (chemo)-radiated patients with rectal cancer to establish a platform for retrospective TIL analyses of frozen tumors from pre-selected study cohorts. Frequencies of TIL subsets and their capacity to mount IFN-γ responses in cell suspensions of fresh vs. cryopreserved portions of the same tumor biopsies were determined for platform validation. The percentages and proportions of CD4+ TILs and CD8+ cytotoxic T lymphocytes (CTLs) among total TILs were not affected by cryopreservation. While recovery of unconventional γδ T cells and mucosal-associated invariant T cells (MAIT cells) was stable after cryopreservation, the regulatory T cells (Tregs) were reduced, but in sufficient yields for quantification. IFN-γ production by in vitro-stimulated CD4+ TILs, CTLs, γδ T cells, and MAIT cells were proportionally similar in fresh and cryopreserved tumor portions, albeit the latter displayed lower levels. Thus, the proposed platform intended for TIL analyses on cryopreserved tumor biobank biopsies holds promises for studies linking the quantity and quality of TIL subsets with specific clinical outcome after CRC treatment.

scholarly journals Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Nayoun Hong ◽  
Seockmo Ku ◽  
Kyungjin Yuk ◽  
Tony V. Johnston ◽  
Geun Eog Ji ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Shuttle Vector ◽  
Sodium Dodecyl ◽  
Interleukin 10 ◽  
Biologically Active ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Evaluation ◽  
Ht 29 ◽  
Cell Free Culture Supernatant

Abstract Background Bifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines. Results The vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473 ± 300 ng/mL (n = 4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n = 6, p < 0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n = 6, p < 0.01) or TNFα-induced HT-29 cells (n = 6, p < 0.001). Conclusion B. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.

scholarly journals Novel MSX1 variants identified in families with nonsyndromic oligodontia

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Jinglei Zheng ◽  
Miao Yu ◽  
Haochen Liu ◽  
Tao Cai ◽  
Hailan Feng ◽  
...  
Keyword(s):  
Current Data ◽  
Phenotypic Characterization ◽  
Functional Evaluation ◽  
Gene Variants ◽  
Chinese Families ◽  
Whole Exome ◽  
Bioinformatics Databases ◽  
Uncertain Significance ◽  
Vitro Analysis

AbstractThe goal of this study was to identify MSX1 gene variants in multiple Chinese families with nonsyndromic oligodontia and analyse the functional influence of these variants. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variants in five families with nonsyndromic oligodontia, and a series of bioinformatics databases were used for variant confirmation and functional prediction. Phenotypic characterization of the members of these families was described, and an in vitro analysis was performed for functional evaluation. Five novel MSX1 heterozygous variants were identified: three missense variants [c.662A>C (p.Q221P), c.670C>T (p.R224C), and c.809C>T (p.S270L)], one nonsense variant [c.364G>T (p.G122*)], and one frameshift variant [c.277delG (p.A93Rfs*67)]. Preliminary in vitro studies demonstrated that the subcellular localization of MSX1 was abnormal with the p.Q221P, p.R224C, p.G122*, and p.A93Rfs*67 variants compared to the wild type. Three variants (p.Q221P, p.G122*, and p.A93Rfs*67) were classified as pathogenic or likely pathogenic, while p.S270L and p.R224C were of uncertain significance in the current data. Moreover, we summarized and analysed the MSX1-related tooth agenesis positions and found that the type and variant locus were not related to the severity of tooth loss. Our results expand the variant spectrum of nonsyndromic oligodontia and provide valuable information for genetic counselling.

Human vascular smooth muscle responses mediated by α2 mechanisms in vivo and in vitro

1985 ◽  
Vol 68 (s10) ◽  
pp. 147s-150s ◽  
Author(s):  
S. Thom ◽  
J. Calvete ◽  
R. Hayes ◽  
G. Martin ◽  
P. Sever
Keyword(s):  
Smooth Muscle ◽  
Physiological Role ◽  
Smooth Muscle Contraction ◽  
Α1 Receptors ◽  
Dose Dependent ◽  
Higher Doses ◽  
Α2 Receptors ◽  
Autoregulatory Mechanism

1. The effects of compounds with α2-agonist and α2-antagonist properties on human forearm blood flow and on isolated human arterial segments have been studied. 2. The findings from these studies in vivo and in vitro did not provide evidence in support of the hypothesis that postsynaptic α2-receptors mediate smooth muscle contraction in the tissues under investigation. 3. The constriction of the forearm vascular bed in response to low intra-arterial doses of idazoxan (RX 781094), an α2-antagonist, provides evidence for a physiological role for a presynaptic α2 autoregulatory mechanism. 4. The variability of the forearm vascular responses to higher doses of idazoxan highlights the pitfalls that may have misled previous authors in their interpretation of the results of similar studies. A U-shaped dose-response curve to compounds with mixed α2-and α1-antagonist properties may be constructed, which emphasizes the importance of the dose-dependent selectivity of these antagonists at α2- and α1-receptors. 5. The effect of idazoxan on the responses of arterial segments in vitro to exogenous catecholamines was dependent on the integrity of the endothelium, and provides evidence that α2-receptors may mediate release of the endothelium-derived relaxing factor.

scholarly journals Interleukin-12 Is the Optimum Cytokine To Expand Human Th17 Cells In Vitro

2009 ◽  
Vol 16 (6) ◽  
pp. 798-805 ◽  
Author(s):  
Soad Nady ◽  
James Ignatz-Hoover ◽  
Mohamed T. Shata
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Interleukin 12 ◽  
Interleukin 17 ◽  
Functional Evaluation ◽  
Optimum Conditions ◽  
T Helper ◽  
Ifn Γ

ABSTRACT Recently, a new lineage of CD4+ T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4+ T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R+ cells from the CD4+ T cells. Functional studies revealed that CD4+ IL-23R+ cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-γ). Furthermore, we expanded the CD4+ IL-23R+ cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4+ IL-23R+ cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN-γ secreted by IL-23R+ CD4+ cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R+ CD4+ cells and the secretion of IFN-γ, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.

scholarly journals (Bio-)Synthesis of the Aquatic Phytotoxin Cyanobacterin – A Paradigm for Furanolide Core Structure Assembly

2021 ◽  
Author(s):  
Paul M. D’Agostino ◽  
Catharina Julia Seel ◽  
Tanja Gulder ◽  
Tobias Gulder
Keyword(s):  
Biosynthetic Gene Cluster ◽  
Core Structure ◽  
Structural Motif ◽  
Functional Evaluation ◽  
One Pot ◽  
Bond Forming ◽  
Amino Acid Precursors ◽  
Structure Assembly ◽  
Gamma Butyrolactone

The gamma-butyrolactone structural motif is commonly found in many natural signaling molecules and other specialized metabolites. A prominent example is the potent aquatic phytotoxin cyanobacterin bearing a highly functionalized gamma-butyrolactone core structure. The enzymatic machinery assembling cyanobacterin and the many structurally related natural products – herein termed furanolides – has remained elusive over decades. Here we discover and characterize the underlying biosynthetic process of furanolide core structure assembly. The cyanobacterin biosynthetic gene cluster (<i>cyb</i>) is identified by targeted bioinformatic screening and validated by heterologous expression in <i>E. coli</i>. Functional evaluation of the recombinant key enzymes provides in-depth mechanistic insights into a streamlined <i>C</i>,<i>C</i>-bond-forming cascade that involves installation of compatible reactivity at seemingly unreactive C-alpha-positions of the amino acid precursors and facilitates development of a one-pot biocatalytic <i>in vitro</i> synthesis. Our work extends the biosynthetic and biocatalytic toolbox for gamma-butyrolactone formation. It thereby provides a general paradigm for the biosynthesis of furanolides and thus sets the stage for their targeted discovery, biosynthetic engineering and enzymatic synthesis.

scholarly journals Structural prope rties, functional evaluation, and in vitro protein digestibility of black and yellow quinoa (Chenopodium petiolare) protein isolates

2019 ◽  
Vol 17 (1) ◽  
pp. 864-872
Author(s):  
Aidee I. Sánchez-Reséndiz ◽  
Anayansi Escalante-Aburto ◽  
Vidalina Andía-Ayme ◽  
Cristina Chuck-Hernández
Keyword(s):  
Protein Digestibility ◽  
Functional Evaluation ◽  
In Vitro Protein Digestibility ◽  
Protein Isolates ◽  
Vitro Protein

Fabrication of liver-derived extracellular matrix nanofibers and functional evaluation in in vitro culture using primary hepatocytes

Materialia ◽  
2018 ◽  
Vol 4 ◽  
pp. 518-528 ◽  
Author(s):  
Ronald Bual ◽  
Haruna Kimura ◽  
Yasuhiro Ikegami ◽  
Nana Shirakigawa ◽  
Hiroyuki Ijima
Keyword(s):  
Extracellular Matrix ◽  
In Vitro Culture ◽  
Functional Evaluation ◽  
Primary Hepatocytes

scholarly journals Functional and Nonclinical Similarity of ABP 980, a Biosimilar of Trastuzumab

2019 ◽  
Vol 36 (12) ◽  
Author(s):  
Shea Jassem ◽  
Wei Wang ◽  
Heather Sweet ◽  
Raffi Manoukian ◽  
Vincent Chow ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Mechanisms Of Action ◽  
Functional Similarity ◽  
Functional Evaluation ◽  
Binding Studies ◽  
Monocyte Cell ◽  
Her2 Signaling ◽  
Primary Assessment

Abstract Purpose The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation. Methods This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters. Results The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action. Conclusions These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.

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