scholarly journals A Central Role for the Armadillo Protein Plakoglobin in the Autoimmune Disease Pemphigus Vulgaris

2001 ◽  
Vol 153 (4) ◽  
pp. 823-834 ◽  
Author(s):  
Reto Caldelari ◽  
Alain de Bruin ◽  
Dominique Baumann ◽  
Maja M. Suter ◽  
Christiane Bierkamp ◽  
...  
Keyword(s):  
Steric Hindrance ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Pemphigus Vulgaris ◽  
Cell Types ◽  
Linear Distribution ◽  
Igg Binding ◽  
Vitro Model ◽  
First Time

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG−/−) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG−/− cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG−/− keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.

scholarly journals Autologous human immunocompetent white adipose tissue-on-chip

2021 ◽  
Author(s):  
Julia Rogal ◽  
Raylin Xu ◽  
Julia Roosz ◽  
Claudia Teufel ◽  
Madalena Cipriano ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
White Adipose Tissue ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Associated Diseases ◽  
Chip Technology ◽  
Vitro Model ◽  
On Chip

Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In the era of 'diabesity' and due to its central role for metabolic and endocrine processes, adipose tissue (specifically white adipose tissue; WAT) has become a target of high interest for therapeutic strategies. To gain insights in cellular and molecular mechanisms of adipose (patho-)physiology, researchers traditionally relied on animal models since in vitro studies on human WAT are challenging due to the large size, buoyancy, and fragility of mature white adipocytes. Leveraging the Organ-on-Chip technology, we introduce a next-generation microphysiological in vitro model of human WAT based on a tailored microfluidic platform featuring vasculature-like perfusion. The platform integrates a 3D tissue comprising all major WAT-associated cellular components in an autologous manner, including not only mature adipocytes but also organotypic endothelial barriers and stromovascular cells featuring tissue-resident innate immune cells, specifically adipose tissue macrophages. This microphysiological tissue model recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. The combination of all individual cell types with extra cellular matrix-like hydrogels in a precisely controllable bottom-up approach enables the generation of a multitude of replicates from the same donors circumventing issues of inter-donor variability and paving the way for personalized medicine. Moreover, it allows to adjust the model's degree of complexity to fit a specific purpose via a flexible mix-and-match approach with different cell component modules. This novel WAT-on-chip system constitutes a human- based, autologous and immunocompetent in vitro model of adipose tissue that recapitulates almost full tissue heterogeneity. In the future, the new WAT-on-chip model can become a powerful tool for human-relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized- and precision medicine applications.

scholarly journals Crocetin Mitigates Irradiation Injury in an In Vitro Model of the Pubertal Testis: Focus on Biological Effects and Molecular Mechanisms

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1676
Author(s):  
Giulia Rossi ◽  
Martina Placidi ◽  
Chiara Castellini ◽  
Francesco Rea ◽  
Settimio D'Andrea ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Biological Effects ◽  
Cellular Damage ◽  
X Rays ◽  
Adolescent Cancer ◽  
Radiation Induced ◽  
Vitro Model ◽  
Underlying Mechanisms

Infertility is a potential side effect of radiotherapy and significantly affects the quality of life for adolescent cancer survivors. Very few studies have addressed in pubertal models the mechanistic events that could be targeted to provide protection from gonadotoxicity and data on potential radioprotective treatments in this peculiar period of life are elusive. In this study, we utilized an in vitro model of the mouse pubertal testis to investigate the efficacy of crocetin to counteract ionizing radiation (IR)-induced injury and potential underlying mechanisms. Present experiments provide evidence that exposure of testis fragments from pubertal mice to 2 Gy X-rays induced extensive structural and cellular damage associated with overexpression of PARP1, PCNA, SOD2 and HuR and decreased levels of SIRT1 and catalase. A twenty-four hr exposure to 50 μM crocetin pre- and post-IR significantly reduced testis injury and modulated the response to DNA damage and oxidative stress. Nevertheless, crocetin treatment did not counteract the radiation-induced changes in the expression of SIRT1, p62 and LC3II. These results increase the knowledge of mechanisms underlying radiation damage in pubertal testis and establish the use of crocetin as a fertoprotective agent against IR deleterious effects in pubertal period.

Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

2008 ◽  
Vol 294 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Ellen Steward Pentz ◽  
Maria Luisa S. Sequeira Lopez ◽  
Magali Cordaillat ◽  
R. Ariel Gomez
Keyword(s):  
Chromatin Remodeling ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Histone H4 Acetylation ◽  
Renin Gene ◽  
Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

scholarly journals SPINK1-induced amelioration of impaired autophagy contributes to suppression of trypsinogen activation in a model of acute pancreatitis

2020 ◽  
Author(s):  
Yuxiao Zhao ◽  
Jianlong Jia ◽  
Abdullah Shopit ◽  
Yang Liu ◽  
Jun Wang
Keyword(s):  
Acute Pancreatitis ◽  
In Vitro Model ◽  
Autophagic Flux ◽  
Trypsin Activity ◽  
Autophagy Flux ◽  
Trypsinogen Activation ◽  
Vitro Model ◽  
First Time ◽  
Induced Amelioration

AbstractSPINK1 has been regarded as a reversible trypsinogen inhibitor for the inappropriate activation of trypsin, a key step in the initiation of acute pancreatitis (AP). However, the mechanisms of its action remains largely unclear and controversial. Here, we reported an unexpected effects of SPINK1 on inhibiting trypsinogen activation through the regulation of impaired autophagy in cerulein-stimulated AR42J cells, a well-established in vitro model of acute pancreatitis. Firstly, we found that the impaired autophagic flux was induced and trypsinogen activity enhanced in the above setting. Then, we showed that SPINK1 overexpression could inhibit the level of increased autophagic activity, improving the hindered autophagy flux, and significantly decreased the trypsinogen activity, whereas shRNA-caused downregulation of SPINK1 exacerbated the impairment of autophagic flux and trypsin activity, in the same cerulein-processed cells. More importantly, the trypsinogen activation in this model could be ameliorated by 3-Methyladenine(3-MA), an autophagy inhibitor. Thus, this study revealed, possibly for the first time, that SPINK1 greatly blocked the trypsinogen activation possibly through the modulation of impaired autophagy in cerulein-induced in vitro model of acute pancreatitis.

Iron-related transcriptomic variations in CaCo-2 cells, an in vitro model of intestinal absorptive cells

2006 ◽  
Vol 26 (1) ◽  
pp. 55-67 ◽  
Author(s):  
Céline Chicault ◽  
Bertrand Toutain ◽  
Annabelle Monnier ◽  
Marc Aubry ◽  
Patricia Fergelot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Annotation ◽  
In Vitro Model ◽  
Iron Absorption ◽  
Intracellular Iron ◽  
Absorptive Cells ◽  
Number Of Genes ◽  
Vitro Model ◽  
First Time

Regulation of iron absorption by duodenal enterocytes is essential for the maintenance of homeostasis by preventing iron deficiency or overload. Despite the identification of a number of genes implicated in iron absorption and its regulation, it is likely that further factors remain to be identified. For that purpose, we used a global transcriptomic approach, using the CaCo-2 cell line as an in vitro model of intestinal absorptive cells. Pangenomic screening for variations in gene expression correlating with intracellular iron content allowed us to identify 171 genes. One hundred nine of these genes are clustered into five types of expression profile. This is the first time that most of these genes have been associated with iron metabolism. Functional annotation of these five clusters indicates potential links between the immune response, proteolysis processes, and iron depletion. In contrast, iron overload is associated with cellular metabolism, especially that of lipids and glutathione involving redox function and electron transfer.

scholarly journals CELLULAR IMMUNITY IN VITRO

1971 ◽  
Vol 133 (6) ◽  
pp. 1377-1389 ◽  
Author(s):  
Harvey B. Simon ◽  
John N. Sheagren
Keyword(s):  
Cellular Immunity ◽  
In Vitro Model ◽  
Specific Antigen ◽  
Cell Types ◽  
Intracellular Bacteria ◽  
Migration Inhibition ◽  
Vitro Model ◽  
And Control ◽  
Macrophage Migration Inhibition

An in vitro model of cellular immunity in the guinea pig was established. Animals were immunized with tubercle bacilli, bovine gamma globulin, or picrylated human serum albumin in complete Freund's adjuvant. Oil-induced peritoneal exudates from immune and control animals were cultured overnight with and without specific antigen. The cultures were washed and the macrophage monolayers were infected with Listeria monocytogenes. At intervals the monolayers were lysed and the numbers of viable intracellular bacteria were quantitated by pour plate cultures. Random monolayers were also evaluated in sequence by visually counting the intracellular bacteria on Gram-stained plates. Both methods demonstrated that the macrophages from immune animals had markedly enhanced listericidal activity when the peritoneal exudates were cultured with antigen before infection. Macrophage migration inhibition was also demonstrated under these conditions. The experiments reported here describe an in vitro model of cellular immunity which will allow separation and recombination of cell types and direct assay of cell products in efforts to elucidate further the mechanisms of the immunologically mediated enhancement of macrophage bactericidal capacity.

scholarly journals Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells

2003 ◽  
Vol 372 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Nathalie NAUD ◽  
Aminata TOURÉ ◽  
Jianfeng LIU ◽  
Charles PINEAU ◽  
Laurence MORIN ◽  
...  
Keyword(s):  
Germ Cells ◽  
In Vitro Model ◽  
Cell Types ◽  
Glutathione S Transferase ◽  
Rac Gtpase ◽  
Male Germ Cells ◽  
Rho Family ◽  
Vitro Model ◽  
Null Mutant Mice

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP-null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2–MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.

scholarly journals Comparative analysis of neuroinvasion by Japanese encephalitis virulent and vaccine viral strains in an in vitro model of human blood-brain barrier

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252595
Author(s):  
Cécile Khou ◽  
Marco Aurelio Díaz-Salinas ◽  
Anaelle da Costa ◽  
Christophe Préhaud ◽  
Patricia Jeannin ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Japanese Encephalitis ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Brain Barrier ◽  
Blood Brain ◽  
The Central Nervous System ◽  
Vitro Model ◽  
Viral Determinants

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.

208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

2006 ◽  
Vol 18 (2) ◽  
pp. 212
Author(s):  
J. Y. Won ◽  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. H. Kang ◽  
H. Shim
Keyword(s):  
Human Serum ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
In Vitro Model ◽  
Regulatory Protein ◽  
Cell Types ◽  
Human Complement ◽  
Transgenic Pigs ◽  
Vitro Model

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

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