scholarly journals Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems

1999 ◽  
Vol 23 (3) ◽  
pp. 307-313 ◽  
Author(s):  
J Beattie ◽  
V Borromeo ◽  
S Bramani ◽  
C Secchi ◽  
WR Baumbach ◽  
...  
Keyword(s):  
Cell Culture ◽  
Growth Hormone ◽  
Monoclonal Antibodies ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Culture Systems ◽  
Cell Culture Systems ◽  
Responsive Cell

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

scholarly journals Mammary gland 3D cell culture systems in farm animals

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Laurence Finot ◽  
Eric Chanat ◽  
Frederic Dessauge
Keyword(s):  
Cell Culture ◽  
Mammary Gland ◽  
Bacterial Infections ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Farm Animals ◽  
Culture Systems ◽  
Cell Culture Systems

AbstractIn vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D “tissues” called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research.

scholarly journals Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

2014 ◽  
Vol 89 (1) ◽  
pp. 811-823 ◽  
Author(s):  
Yi-Ping Li ◽  
Santseharay Ramirez ◽  
Lotte Mikkelsen ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
Infectious Clones ◽  
Cell Culture Systems ◽  
A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

scholarly journals Alzheimer’s Disease: Current Perspectives and Advances in Physiological Modeling

2021 ◽  
Vol 8 (12) ◽  
pp. 211
Author(s):  
E. Josephine Boder ◽  
Ipsita A. Banerjee
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Culture ◽  
Stem Cell Differentiation ◽  
Experimental Manipulation ◽  
Model Systems ◽  
Culture Systems ◽  
Cell Culture Systems

Though Alzheimer’s disease (AD) is the most common cause of dementia, complete disease-modifying treatments are yet to be fully attained. Until recently, transgenic mice constituted most in vitro model systems of AD used for preclinical drug screening; however, these models have so far failed to adequately replicate the disease’s pathophysiology. However, the generation of humanized APOE4 mouse models has led to key discoveries. Recent advances in stem cell differentiation techniques and the development of induced pluripotent stem cells (iPSCs) have facilitated the development of novel in vitro devices. These “microphysiological” systems—in vitro human cell culture systems designed to replicate in vivo physiology—employ varying levels of biomimicry and engineering control. Spheroid-based organoids, 3D cell culture systems, and microfluidic devices or a combination of these have the potential to replicate AD pathophysiology and pathogenesis in vitro and thus serve as both tools for testing therapeutics and models for experimental manipulation.

Recent Progress in Prediction Systems for Drug-induced Liver Injury Using in vitro Cell Culture

2020 ◽  
Vol 14 ◽  
Author(s):  
Shogo Ozawa ◽  
Toshitaka Miura ◽  
Jun Terashima ◽  
Wataru Habano ◽  
Seiichi Ishida
Keyword(s):  
Cell Culture ◽  
Recent Progress ◽  
Inflammatory Cells ◽  
Protein Adducts ◽  
In Vitro Assays ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Culture Systems ◽  
Cell Culture Systems

Background: In order to avoid drug-induced liver injury (DILI), in vitro assays, which enable the assessment of both metabolic activation and immune reaction processes that ultimately result in DILI, are needed. Objective: In this study, the recent progress in the application of in vitro assays using cell culture systems is reviewed for potential DILI-causing drugs/xenobiotics and a mechanistic study on DILI, as well as for the limitations of in vitro cell culture systems for DILI research. Methods: Information related to DILI was collected through a literature search of the PubMed database. Results: The initial biological event for the onset of DILI is the formation of cellular protein adducts after drugs have been metabolically activated by drug metabolizing enzymes. The damaged peptides derived from protein adducts lead to the activation of CD4+ helper T lymphocytes and recognition by CD8+ cytotoxic T lymphocytes, which destroy hepatocytes through immunological reactions. Because DILI is a major cause of drug attrition and drug withdrawal, numerous in vitro systems consisting of hepatocytes and immune/inflammatory cells, or spheroids of human primary hepatocytes containing non-parenchymal cells have been developed. These cellular-based systems have identified DILIinducing drugs with approximately 50% sensitivity and 90% specificity. Conclusion: Different co-culture systems consisting of human hepatocyte-derived cells and other immune/inflammatory cells have enabled the identification of DILI-causing drugs and of the actual mechanisms of action.

In vitro cytotoxicity of macromolecules in different cell culture systems

1995 ◽  
Vol 34 (3) ◽  
pp. 233-241 ◽  
Author(s):  
Suthummar Choksakulnimitr ◽  
Sada Masuda ◽  
Hideaki Tokuda ◽  
Yoshinobu Takakura ◽  
Mitsuru Hashida
Keyword(s):  
Cell Culture ◽  
In Vitro Cytotoxicity ◽  
Culture Systems ◽  
Cell Culture Systems

scholarly journals Mathematical modelling of fluid flow and solute transport to define operating parameters for in vitro perfusion cell culture systems

2020 ◽  
Vol 10 (2) ◽  
pp. 20190045 ◽  
Author(s):  
Lauren Hyndman ◽  
Sean McKee ◽  
Nigel J. Mottram ◽  
Bhumika Singh ◽  
Steven D. Webb ◽  
...  
Keyword(s):  
Cell Culture ◽  
Fluid Flow ◽  
Mathematical Modelling ◽  
Solute Transport ◽  
Computational Modelling ◽  
Operating Parameters ◽  
Perfusion Cell Culture ◽  
Culture Systems ◽  
Cell Culture Systems

In recent years, there has been a move away from the use of static in vitro two-dimensional cell culture models for testing the chemical safety and efficacy of drugs. Such models are increasingly being replaced by more physiologically relevant cell culture systems featuring dynamic flow and/or three-dimensional structures of cells. While it is acknowledged that such systems provide a more realistic environment within which to test drugs, progress is being hindered by a lack of understanding of the physical and chemical environment that the cells are exposed to. Mathematical and computational modelling may be exploited in this regard to unravel the dependency of the cell response on spatio-temporal differences in chemical and mechanical cues, thereby assisting with the understanding and design of these systems. In this paper, we present a mathematical modelling framework that characterizes the fluid flow and solute transport in perfusion bioreactors featuring an inlet and an outlet. To demonstrate the utility of our model, we simulated the fluid dynamics and solute concentration profiles for a variety of different flow rates, inlet solute concentrations and cell types within a specific commercial bioreactor chamber. Our subsequent analysis has elucidated the basic relationship between inlet flow rate and cell surface flow speed, shear stress and solute concentrations, allowing us to derive simple but useful relationships that enable prediction of the behaviour of the system under a variety of experimental conditions, prior to experimentation. We describe how the model may used by experimentalists to define operating parameters for their particular perfusion cell culture systems and highlight some operating conditions that should be avoided. Finally, we critically comment on the limitations of mathematical and computational modelling in this field, and the challenges associated with the adoption of such methods.

Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1653-1653
Author(s):  
Silvia Locatelli ◽  
Arianna Giacomini ◽  
Anna Guidetti ◽  
Loredana Cleris ◽  
Michele Magni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Drug Combination ◽  
Akt Inhibitor ◽  
Sorafenib Treatment ◽  
Responsive Cell

Abstract Abstract 1653 Introduction: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures: No relevant conflicts of interest to declare.

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