scholarly journals Heregulin Activity Assays for Residual Testing of Cell Therapy Products

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Paula V. Monje ◽  
Ketty Bacallao ◽  
Gabriela I. Aparicio ◽  
Anil Lalwani
Keyword(s):  
Culture Medium ◽  
Dose Dependency ◽  
Soluble Factors ◽  
Biological Assays ◽  
Tissue Extracts ◽  
Assay Optimization ◽  
Potential Safety ◽  
Kinase Phosphorylation ◽  
Nanomolar Range

Abstract Background Heregulin is a ligand for the protooncogene product ErbB/HER that acts as  a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. Results Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation. Conclusions These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.

scholarly journals Novel Acetylcholinesterase Inhibitors Based on Uracil Moiety for Possible Treatment of Alzheimer Disease

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4191 ◽  
Author(s):  
Vyacheslav E. Semenov ◽  
Irina V. Zueva ◽  
Marat A. Mukhamedyarov ◽  
Sofya V. Lushchekina ◽  
Elena O. Petukhova ◽  
...  
Keyword(s):  
Alzheimer Disease ◽  
Memory Impairment ◽  
Acetylcholinesterase Inhibitors ◽  
Pyrimidine Ring ◽  
Anionic Site ◽  
Biological Assays ◽  
Alkyl Chains ◽  
Nanomolar Range

In this study, novel derivatives based on 6-methyluracil and condensed uracil were synthesized, namely, 2,4-quinazoline-2,4-dione with ω-(ortho-nitrilebenzylethylamino) alkyl chains at the N atoms of the pyrimidine ring. In this series of synthesized compounds, the polymethylene chains were varied from having tetra- to hexamethylene chains, and secondary NH, tertiary ethylamino, and quaternary ammonium groups were introduced into the chains. The molecular modeling of the compounds indicated that they could function as dual binding site acetylcholinesterase inhibitors, binding to both the peripheral anionic site and active site. The data from in vitro experiments show that the most active compounds exhibit affinity toward acetylcholinesterase within a nanomolar range, with selectivity for acetylcholinesterase over butyrylcholinesterase reaching four orders of magnitude. In vivo biological assays demonstrated the potency of these compounds in the treatment of memory impairment using an animal model of Alzheimer disease.

scholarly journals The dialogue between died and viable cells: in vitro and in vivo bystander effects and 1H-NMR-based metabolic profiling of soluble factors

2020 ◽  
Vol 92 (3) ◽  
pp. 399-411
Author(s):  
Elisa Panzarini ◽  
Cristian Vergallo ◽  
Francesco Paolo Fanizzi ◽  
Stefania Mariano ◽  
Ada Maria Tata ◽  
...  
Keyword(s):  
Culture Medium ◽  
Bystander Effect ◽  
Radiation Treatment ◽  
Apoptotic Cell ◽  
Immunohistochemical Analysis ◽  
Inflammatory Cells ◽  
U937 Cells ◽  
Soluble Factors

AbstractThe bystander effect (BE) is an important biological phenomenon that induces damages in distant and not directly affected by a chemical/physical stress cells. This effect, well known in ionizing radiation treatment, relies on reactive signals released by exposed cells and transmitted via cell–cell interaction or culture medium. In this study, cycloheximide (CHX)-induced apoptotic U937 cells and untreated THP-1 cells were chosen to investigate the chemical-induced BE. The effects of apoptotic U937 cells culture medium, Conditioned Medium (CM), on THP-1 cells were evaluated by morphological and immunohistochemical analysis performed by light microscopy; 1D 1H and 2D J-resolved (JRES) NMR metabolomic analysis has been used to characterize the molecules involved in the BE. In summary, this study indicates that: CM of CHX-treated U937 cells induces a time-dependent induction of toxicity, probably apoptotic cell death, and macrophagic differentiation in THP-1 cells; CM contains different metabolites respect fresh culture medium; CM recruits in vivo activated fibroblasts, endothelial cells, macrophages and mononuclear inflammatory cells in rat calf muscles. These data suggest that CHX exposed cells could cause BE through the release, during the apoptotic process, of soluble factors into the medium that could be exploited in anticancer protocols.

Platelet-derived soluble factors induce human extravillous trophoblast migration and differentiation: platelets are a possible regulator of trophoblast infiltration into maternal spiral arteries

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 428-435 ◽  
Author(s):  
Yukiyasu Sato ◽  
Hiroshi Fujiwara ◽  
Bin-Xiang Zeng ◽  
Toshihiro Higuchi ◽  
Shinya Yoshioka ◽  
...  
Keyword(s):  
Culture Medium ◽  
Extravillous Trophoblast ◽  
Soluble Factors ◽  
Explant Cultures ◽  
Endovascular Trophoblast ◽  
Vascular Infiltration ◽  
Extravillous Trophoblasts ◽  
Trophoblast Migration ◽  
Morphologic Changes

Abstract In early pregnancy, human extravillous trophoblasts (EVTs) invade and remodel maternal arteries. We have previously demonstrated that CCR1 is expressed on perivascular/endovascular trophoblasts and that CCR1 ligands promote EVT migration. In this study, we examined the physiologic roles of platelet-derived chemoattractants on EVT invasion. By immunohistochemistry, maternal platelets were localized among endovascular trophoblasts within the lumen of spiral arteries. Extracellular matrices (ECMs) were also detected among endovascular trophoblasts and platelets, suggesting that the platelets in these arteries were activated by ECMs. In vitro, platelets attached to EVTs isolated from human villous explant cultures and expressed P-selectin on the cell surface. Platelets significantly enhanced migration of EVTs without affecting proliferation of EVTs or secretion of MMP-2 or MMP-9. The invasion-enhancing effect of platelet-derived culture medium on EVTs was neutralized by anti-CCR1 antibody. Heat treatment completely abrogated the invasion-promoting effects of platelet-derived culture medium, but charcoal stripping did not. Platelets also induced endovascular trophoblast-like morphologic changes and integrin α1 expression in EVTs during 48-hour culture. These findings suggest that maternal platelets activated in the spiral arteries can regulate trophoblastic vascular infiltration and differentiation by releasing various soluble factors.

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: Modulation by calcium

1991 ◽  
Vol 125 (3) ◽  
pp. 280-285 ◽  
Author(s):  
J. Alan Talbot ◽  
Ann Lambert ◽  
Robert Mitchell ◽  
Marek Grabinski ◽  
David C. Anderson ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Culture Medium ◽  
Follicle Stimulating Hormone ◽  
Dibutyryl Camp ◽  
Immature Rat ◽  
Estradiol Secretion ◽  
Old Rats ◽  
Stimulating Hormone

Abstract We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/1) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091±322 to 1480±84 pmol/l (p<0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360±45 to 1242±133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 μmol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 μmol/l) and A23187 (2 μmol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by >80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 μmol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 μmol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.

Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants

Reproduction ◽  
2000 ◽  
pp. 391-396 ◽  
Author(s):  
AH Duittoz ◽  
M Batailler
Keyword(s):  
Culture Medium ◽  
Primary Cultures ◽  
Pulsatile Secretion ◽  
Olfactory Placode ◽  
Gnrh Secretion ◽  
Individual Culture ◽  
Pulsatile Gnrh

The aim of this study was to investigate the development of pulsatile GnRH secretion by GnRH neurones in primary cultures of olfactory placodes from ovine embryos. Culture medium was collected every 10 min for 8 h to detect pulsatile secretion. In the first experiment, pulsatile secretion was studied in two different sets of cultures after 17 and 24 days in vitro. In the second experiment, a set of cultures was tested after 10, 17 and 24 days in vitro to investigate the development of pulsatile GnRH secretion in each individual culture. This study demonstrated that (i) primary cultures of GnRH neurones from olfactory explants secreted GnRH in a pulsatile manner and that the frequency and mean interpulse duration were similar to those reported in castrated ewes, and (ii) pulsatile secretion was not present at the beginning of the culture but was observed between 17 and 24 days in vitro, indicating the maturation of individual neurones and the development of their synchronization.

Reprogrammed M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 extends lifespan of mice with experimental carcinoma

2017 ◽  
pp. 4-9
Author(s):  
С.В. Калиш ◽  
С.В. Лямина ◽  
А.А. Раецкая ◽  
И.Ю. Малышев
Keyword(s):  
Tumor Growth ◽  
Culture Medium ◽  
Ehrlich Carcinoma ◽  
Macrophage Phenotype ◽  
M1 Macrophages ◽  
M1 Macrophage ◽  
Ec Cells ◽  
Experimental Carcinoma

Цель исследования. Репрограммирование М1 фенотипа макрофагов с ингибированными факторами транскрипции М2 фенотипа STAT3, STAТ6 и SMAD и оценка их влияния на развитие карциномы Эрлиха (КЭ) in vitro и in vivo. Методика. Рост опухоли иницировали in vitro путем добавления клеток КЭ в среду культивирования RPMI-1640 и in vivo путем внутрибрюшинной инъекции клеток КЭ мышам. Результаты. Установлено, что M1макрофаги и in vitro, и in vivo оказывают выраженный противоопухолевый эффект, который превосходит антиопухолевые эффекты М1, M1, M1 макрофагов и цисплатина. Заключение. М1 макрофаги с ингибированными STAT3, STAT6 и/или SMAD3 эффективно ограничивают рост опухоли. Полученные данные обосновывают разработку новой технологии противоопухолевой клеточной терапии. Objective. Reprogramming of M1 macrophage phenotype with inhibited M2 phenotype transcription factors, such as STAT3, STAT6 and SMAD and assess their impact on the development of Ehrlich carcinoma (EC) in vitro and in vivo . Methods. Tumor growth in vitro was initiated by addition of EC cells in RPMI-1640 culture medium and in vivo by intraperitoneal of EC cell injection into mice. Results. It was found that M1 macrophages have a pronounced anti-tumor effect in vitro , and in vivo , which was greater than anti-tumor effects of M1, M1, M1 macrophages and cisplatin. Conclusion. M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 effectively restrict tumor growth. The findings justify the development of new anti-tumor cell therapy technology.

Pertumbuhan Dan Perkembangan Anggrek Dendrobium anosmum Pada Media Kultur In Vitro Dengan Beberapa Konsentrasi Air Kelapa

Agrologia ◽  
2018 ◽  
Vol 1 (1) ◽  
Author(s):  
S. Tuhuteru ◽  
Meity L Hehanussa ◽  
Simon H.T Raharjo
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Water Concentration ◽  
Medium Composition ◽  
Coconut Water ◽  
Orchid Species ◽  
Randomized Design ◽  
Wet Weight ◽  
Completely Randomized Design

Dendrobium anosmum is one of natural orchids in Indonesia. Optimization of medium composition for orchid propagation through in vitro culture is necessary to enhance propagule multiplication capabilities and quality. This study was aimed to study the influence of concentration of coconut water in culture medium on in vitro growth and development of D. anosmum orchid species and to determine the optimal coconut water concentration in culture media.  The experiment were arranged in a Completely Randomized Design with four treatments and eight replications. The treatments consisted of the addition of coconut water with concentrations: 0 ml•l -1 (control), 50 ml•l-1, 100 ml•l-1 and 150 ml•l-1. The results showed that addition of coconut water in culture medium gave different effect on shoot growth and multiplication of D. anosmum orchids.  Coconut water concentration of 100 ml•l-1 was the best concentration for growth and multiplication of D. anosmum orchids, based on both shoots and roots growth, plantlet height and wet weight.

In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions

2020 ◽  
Vol 27 (29) ◽  
pp. 4840-4854 ◽  
Author(s):  
Chrysoula-Evangelia Karachaliou ◽  
Hubert Kalbacher ◽  
Wolfgang Voelter ◽  
Ourania E. Tsitsilonis ◽  
Evangelia Livaniou
Keyword(s):  
Mammalian Cells ◽  
Therapeutic Potential ◽  
Prothymosin Alpha ◽  
Disease States ◽  
Leading Role ◽  
Tissue Extracts ◽  
Biologic Response ◽  
Health And Disease ◽  
Almost All

Prothymosin alpha (ProT&#945;) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProT&#945; is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as “alarmins”. Antibodies and immunochemical techniques for ProT&#945; have played a leading role in the investigation of the biological role of ProT&#945;, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProT&#945; (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

Naphthoquinones and derivatives for chemotherapy: perspectives and limitations of their anti-trypanosomatids activities

2020 ◽  
Vol 26 ◽  
Author(s):  
Luíza Dantas-Pereira ◽  
Edézio F. Cunha-Junior ◽  
Valter V. Andrade-Neto ◽  
John F. Bower ◽  
Guilherme A. M. Jardim ◽  
...  
Keyword(s):  
Large Scale ◽  
Neglected Tropical Diseases ◽  
Folk Medicine ◽  
Leishmania Species ◽  
Parasitic Infections ◽  
Mechanistic Analysis ◽  
Leishmania Spp ◽  
Nanomolar Range

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

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