Presence Of Extracellular Matrix Proteins Research Articles

Glioblastoma and cerebral organoids: development and analysis of an in vitro model for glioblastoma migration.

It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.

Piezo1 Response to Shear Stress Is Controlled by the Components of the Extracellular Matrix

Piezo1 is a recently discovered Ca2+ permeable ion channel that has emerged as an integral sensor of hemodynamic forces within the cardiovascular system, contributing to vascular development and blood pressure regulation. However, how the composition of the extracellular matrix (ECM) affects the mechanosensitivity of Piezo1 in response to hemodynamic forces remains poorly understood. Using a combination of microfluidics and calcium imaging techniques, we probe the shear stress sensitivity of single HEK293T cells engineered to stably express Piezo1 in the presence of different ECM proteins. Our experiments show that Piezo1 sensitivity to shear stress is not dependent on the presence of ECM proteins. However, different ECM proteins regulate the sensitivity of Piezo1 depending on the shear stress level. Under high shear stress, fibronectin sensitizes Piezo1 response to shear, while under low shear stress, Piezo1 mechanosensitivity is improved in the presence of collagen types I and IV and laminin. Moreover, we report that α5β1 and αvβ3 integrins are involved in Piezo1 sensitivity at high shear, while αvβ3 and αvβ5 integrins are involved in regulating the Piezo1 response at low shear stress. These results demonstrate that the ECM/integrin interactions influence Piezo1 mechanosensitivity and could represent a mechanism whereby extracellular forces are transmitted to Piezo1 channels, providing new insights into the mechanism by which Piezo1 senses shear stress.

Cell-derived Extracellular Matrix Proteins in Colloidal Microgel as a Self-Assembly Hydrogel for Regenerative Endodontics

IntroductionThis study investigated a colloidal microgel for angiogenic and odontogenic differentiation of cells in the presence of cell-derived extracellular matrix (ECM) proteins using a 3-dimensional culture model. MethodsViscoelastic properties of human dental pulp were determined to understand the native ECM environment. ECM proteins were extracted from dental pulp stem cell (DPSC) cultures, and MaxGel (Millipore Sigma, Burlington, MA) was used as a commercially available ECM protein. DPSCs were incubated in colloidal microgels in the presence of ECM proteins or gelatin methacryloyl (GelMA) as a bulk hydrogel (n = 9/group). The viability and odontogenic differentiation of DPSCs within hydrogels was determined using viability assays, mineralization staining, calcium and alkaline phosphatase assays, and quantitative polymerase chain reaction for odontogenic gene expression. Angiogenic properties of endothelial cells were determined using tubule formation assays and quantitative polymerase chain reaction to detect angiogenic gene expression. ResultsDental pulp had a higher elastic modulus than the viscous modulus, showing a solidlike response similar to hydrogels. DPSC-derived ECM showed higher collagen and GAG than MaxGel (P < .05). The viability of DPSCs was similar in colloidal microgels, whereas higher cell viability, calcium deposition, and alkaline phosphatase activity were observed in GelMA (P < .05). Colloidal microgels allowed tubule-like structures by endothelial cells, whereas no tubular formation was observed in GelMA. DPSC-derived ECM in colloidal microgel up-regulated odontogenic gene expression, whereas MaxGel up-regulated angiogenic gene expression (P < .05). ConclusionsColloidal microgels allowed cellular organization that can improve penetration and nutritional supply in a full-length root canal system. The bioactivity of cell-derived ECM proteins can be modified depending on the external stimulus.

Extracellular matrix extraction techniques and applications in biomedical engineering.

The concept of tissue engineering involves regeneration and repair of damaged tissue and organs using various combinations of cells, growth factorsand scaffolds. The extracellular matrix (ECM) forms the integral part of the scaffold to induce cell proliferation thereby leading to new tissue formation. Decellularization technique provides decellularized ECM (dECM), free of cells while preserving the invivo biomolecules. In this review, we focus on the detailed methodology of diverse decellularization techniques for various organs of different animals, and the biomedical applications employing the dECM. A culmination of different methods of decellularization is optimized, which offers a suitable microenvironment mimicking the native in vivo topography for in vitro organ regeneration. A detailed assessment of the dECM provides information on the microarchitecture, presence of ECM proteins, biocompatibilityand cell proliferation. dECM has also been processed as scaffolds and drug-delivery vehicles, and utilized for regeneration.

Abstract IA04: Stroma-driven resistance to PI3K/mTOR inhibition

Abstract PI3/mTOR pathway, a major regulator of cellular growth and metabolism, is mutated in ~70% of breast cancers and conveys resistance towards estrogen receptor- and HER2-targeted therapies. Unfortunately, targeting the PI3K pathway in the clinic has been hampered by emergence of drug resistance, often through rewiring of their signaling pathways rather than through mutations, a phenomenon called adaptive resistance that is often observed with targeted therapies. Our previous data have shown that adaptive resistance requires input from the tumor microenvironment. Cancer cells require the presence of extracellular matrix proteins to elicit drug resistance; without matrix contact the tumor cells died. Our data also show that fibroblasts secrete matrix proteins that increase cellular survival of epithelial cells under starvation and PI3K and mTOR inhibition. To investigate the identity and role of the secreted proteins that drive resistance, we performed mass-spectrometry and cytokine analysis of fibroblast secretomes treated with PI3K inhibitors. These data have identified several candidate proteins as potential drivers of stroma-mediated drug resistance, and we will discuss how targeting these proteins can increase the efficacy of PI3K- and mTOR-targeted therapies. Citation Format: Taru Muranen. Stroma-driven resistance to PI3K/mTOR inhibition [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA04.

Whole Porcine Pancreatic Extracellular Matrix Protects Isolated Human Islets from Ischemia-induced Damage

Daniel Brandhorst,1,2 Limor Baruch,3 Heide Brandhorst,1,2 Stasia Krishtul,3 Marcelle Machluf,3 Paul R.V. Johnson1,2&#x0D; 1Research Group for Islet Transplantation, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom&#x0D; 2Oxford Consortium for Islet Transplantation, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, University of Oxford, Oxford, United Kingdom&#x0D; 3Laboratory for Cancer Drug Delivery and Cell Based Technologies, Faculty of Biotechnology and Food Engineering, Technion, Israel Institute of Technology, Haifa, Israel&#x0D; Aim&#x0D; Islet isolation essentially requires dissociation of islet basement membranes by collagenolytic enzymes. This is associated with reduced islet function and increased cell death. Previous ex-vivo and in-vivo studies demonstrated that individual extracellular matrix proteins (ECMPs) can increase islet survival. As the natural ECM is a tissue-defined complex network we propose a novel concept for creating a specific islet matrix by using the whole pancreatic ECM (WPM). In contrast to previous studies, ECMPs were dissolved in media rather than coating of culture vessel surfaces.&#x0D; Methods&#x0D; Islets, isolated from pancreases of 6 human DBD donors (52±3 years, 28.5±1.5 BMI, 6.4±0.7 hours CIT), were cultured for 4–5 days in hypoxic atmosphere (2% oxygen). Islets were suspended in CMRL 1066 supplemented with 2% FCS and WPM-gel (200 µg/mL) extracted and purified from porcine pancreases. The WPM-gel was compared with a pre-tested combination of human ECMPs composed of 80 µg/mL collagen-IV, 10 µg/mL laminin-521, and 10 µg/mL nidogen-1. Sham-treated islets (STIs) cultured without ECMPs or WPM-gel served as controls. Post-culture characterisation included IEQ yield or islet number (IN), viability (FDA-PI), early plus late apoptosis (annexin V-PI), glucose stimulation index (SI: 2 vs 20 vs 2 mM) and reactive oxygen species production. All parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Statistical analysis was performed by Friedman test and Dunn’s multiple comparison.&#x0D; Results&#x0D; Post-culture recovery was highest when hypoxic human islets were cultured in WPM-gel and compared with STIs (65±10% vs 38±10%, p&lt;0.01). Although fragmentation (IN/IEQ ratio) increased after all treatments, this increase was lowest in the presence of WPM-gel (0.62±0.05 vs 0.80±0.14 vs 0.93±0.27, NS). Pre-culture viability was nearly completely preserved when human ECMPs (99±10% vs 79±10%, p&lt;0.01) or WPM-gel (92±8%, p&lt;0.05) were administered. Reactive oxygen species production in STIs increased nearly three-fold (127±15 AU/IEQ) but was halved in the presence of ECMPs (61±14 AU/IEQ, p&lt;0.01) or WPM-gel (65±18 AU/IEQ, p&lt;0.05). While initial early apoptosis remained stable when human islets were treated with human ECMPs (90±13% vs 136±14%, p&lt;0.01) or WPM-gel (84±10%, p&lt;0.01), apo-necrosis increased substantially in the presence of human ECMPs (172±33%, NS) or WPM-gel (154±195 vs 214±24%, p&lt;0.05). Glucose-stimulated islets did not respond adequately after sham-treatment (SI 0.85±0.14). In contrast, supplementation with human ECMPs (1.29±0.09, p&lt;0.05) or WPM-gel (1.34±0.09, p&lt;0.01) preserved the physiological insulin response during hypoxia. Overall survival, considering the recovery of viable cells only, was increased by human ECMPs (56±8% vs 34±8%, p&lt;0.01) or WPM-gel (58±8%, p&lt;0.01).&#x0D; Conclusion&#x0D; This initial study presents a new approach to protect human islets from hypoxia-induced damage by supplementing culture media with selected ECMPs or with the whole pancreatic ECM. We assume, that the outcome of our approach will be further improved when the ECM is extracted from human pancreases. These promising findings can be used to develop advanced culture media and innovative encapsulation techniques to protect transplanted islets.

Klotho Deficiency Induces Arteriolar Hyalinosis in a Trade-Off with Vascular Calcification

Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to aging-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency, a state known to induce both renal and vascular phenotypes associated with aging. Histochemistry was used to assess hyalinosis in Klotho-/- kidneys, compared with Klotho+/- and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice, and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho-/- mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, whereas the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells (SMCs). Finally, in Klotho-/- mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and SMC dedifferentiation. In the absence of a calcification-inducing stimulus, SMCs assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing aging-related arteriolar hyalinosis.

Extracellular matrix signaling activates differentiation of adult ovary-derived oogonial stem cells in a species-specific manner

To test if ovarian microenvironmental cues affect oogonial stem cell (OSC) function in a species-specific manner. Animal and human study. Research laboratory. Human ovarian cells obtained from cryopreserved ovarian cortical tissue of reproductive-age women, and ovarian cells and tissues from female C57BL/6 mice. Mouse ovarian tissue, mouse OSCs (mOSCs) and human OSCs (hOSCs) were analyzed for extracellular matrix (ECM) protein expression, and OSCs isolated from adult mouse and human ovaries were cultured in the absence or presence of ECM proteins without or with an integrin signaling inhibitor. Gene expression and invitro derived (IVD) oocyte formation. Culture of mOSCs on a collagen-based ECM significantly elevated the rate of differentiation of the cells into IVD oocytes. Mouse OSCs expressed many integrins, including Arg-Gly-Asp (RGD)-binding subunits, and ECM-mediated increases in mOSC differentiation were blocked by addition of integrin-antagonizing RGD peptides. In comparison, hOSCs expressed a different pattern of integrin subunits compared with mOSCs, and hOSCs were unresponsive to a collagen-based ECM; however, hOSCs exhibited increased differentiation into IVD oocytes when cultured on laminin. These data, along with in silico analysis of ECM protein profiles in human ovaries, indicate that ovarian ECM-based niche components function in a species-specific manner to control OSC differentiation.

Assembly of a functional 3D primary cardiac construct using magnetic levitation

Easily assembled organotypic co-cultures have long been sought in medical research. In vitro tissue constructs with faithful representation of in vivo tissue characteristics are highly desirable for screening and characteristic assessment of a variety of tissue types. Cardiac tissue analogs are particularly sought after due to the phenotypic degradation and difficulty of culture of primary cardiac myocytes. This study utilized magnetic nanoparticles and primary cardiac myocytes in order to levitate and culture multicellular cardiac aggregates (MCAs). Cells were isolated from 2 day old Sprague Dawley rat hearts and subsequently two groups were incubated with either C1: 33 µL nanoshell/million cells or C2: 50 µL nanoshell/million cells. Varying numbers of cells for each concentration were cultured in a magnetic field in a 24 well plate and observed over a period of 12 days. Constructs generally formed spherical structures. Masson’s trichrome staining of a construct shows the presence of extracellular matrix protein, indicating the presence of functional fibroblasts. Many constructs exhibited noticeable contraction after 4 days of culture and continued contracting noticeably past day 9 of culture. Noticeable contractility indicates the presence of functional primary cardiac myocytes in culture. Phenotypic conservation of cardiac cells was ascertained using IHC staining by α-actinin and collagen. CD31 and fibrinogen were probed in order to assess localization of fibroblasts and endothelial cells. The study verifies a protocol for the use of magnetic levitation in order to rapidly assemble 3D cardiac like tissue with phenotypic and functional stability.

The Staphylococcus aureus ArlRS Two-Component System Is a Novel Regulator of Agglutination and Pathogenesis

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

Crosslinked acellular saphenous vein for small-diameter vascular graft

Patients with congenital and acquired heart diseases or arteriopathy require small-diameter vascular grafts for arterial reconstruction. Autologous veins are the most suitable graft, but when absent, an alternative is necessary. This work addresses the issue. Tissue-engineering efforts to create such grafts by modifications of acellular natural scaffolds are considered a promising area. Homologous saphenous veins harvested from cadavers and organ donors were processed by decellularization with detergent and enzymatic digestion, followed by crosslinking by dye-mediated photooxidation. They were validated for acellularity, mechanical strength, and crosslink stability. In-vitro and in-vivo cytotoxicity and hemocompatibility studies were conducted. Collagen conformity was studied by Fourier transform infrared spectroscopy, and heat stability by differential scanning calorimetry. A limited large animal study was performed. The processing method delivered biocompatible, hemocompatible, effectively crosslinked grafts, with high heat stability of 126 , an enthalpy value of 183.5 J·g(-1), and collagen conformity close to that of the native vein. The mechanical strength was 250% better than the native vein. The presence of extracellular matrix proteins allowed the acellular vein to become a triple-layered vascular structure in the sheep venous system. Crosslinking after decellularization by the dye-mediated photooxidation method could be reproduced in any human vein to obtain a small-diameter vascular grafts.

Hepatic differentiation of amniotic epithelial cells.

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, naïve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.

Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement-membrane extracellular matrix proteins

In vitro cultured mesenchymal stromal cells (MSC) are characterized by a short proliferative lifespan, an increasing loss of proliferation capacity and progressive reduction of differentiation potential. Laminin-1, laminin-5, collagen IV and fibronectin are important constituents of the basement membrane extracellular matrix (ECM) that are involved in a variety of cellular activities, including cell attachment and motility. The in vitro proliferation capacity of MSC was significantly improved when the cells were incubated in the presence of basement membrane ECM proteins. For example, a mixture of proteins improved proliferation capacity 250-fold in comparison with standard conditions after five passages. Furthermore, in colony-forming unit-fibroblast (CFU-F) assays colony numbers and size were significantly extended. Blocking specific integrin cell-surface receptors, positive effects on the proliferation capacity of MSC were inhibited. Additionally, when MSC were co-cultivated with ECM proteins, cells maintained their multipotential differentiation capacity throughout many culture passages in comparison with cells cultivated on plastic. However, expansion of MSC on laminin-5 suppressed any subsequent chondrogenic differentiation. Our results suggest that expansion of bone marrow-derived MSC in the presence of ECM proteins is a powerful approach for generating large numbers of MSC, showing a prolonged capacity to differentiate into mesodermal cell lineages, with the exception of the lack of chondrogenesis by using laminin-5 coating.

Microscale adhesion patterns for the precise localization of amoeba

In order to get a better understanding of amoeba-substrate interactions in the processes of cellular adhesion and directional movement, we engineered glass surfaces with defined local adhesion characteristics at a micrometric scale. Amoeba ( Dictyostelium dicoideum) are capable to adhere to various surfaces independently on the presence of extracellular matrix proteins. This paper describes the strategy used to create selective adhesion patterns using an appropriate surface chemistry and shows the first results of locally confined amoeba adhesion. The approach is based on the natural ability of Dictyostelium to adhere to various types of surfaces (hydrophilic and hydrophobic) and on its inability to spread on inert surfaces, such as the block copolymer of polyethylene glycol and polypropylene oxide, named Pluronic. We screened diverse alkylsilanes, such as methoxy, chloro and fluoro silanes for their capacity to anchor Pluronic F127 efficiently on a glass surface. Our results demonstrate that hexylmethyldichlorosilane (HMDCS) was the most appropriate silane for the deposition of Pluronic F127. A complex dependence between the physico-chemistry of the silanes and the polyethylene glycol block copolymer attachment was observed. Using this method, we succeed in scaling down the micro-fabrication of Pluronic-based adhesion patterns to the amoeba cell size (10 μm). This original Pluronic patterning method should prove useful as a tool for controlling cell adhesion and directional movement in amoeba.

Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro

This 3-week protocol produces embryonic-like stem cells from human umbilical cord blood (CBEs) for neural differentiation using a three-step system (cell isolation/expansion/differentiation). The CBE isolation produces a highly purified fraction (CD45-, CD33-, CD7-, CD235a-) of small pluripotent stem cells (2-3 microm in diameter) coexpressing embryonic stem cell markers including Oct4 and Sox2. Initial CBE expansion is performed in high density (5-10 millions per ml) in the presence of extracellular matrix proteins and epidermal growth factor. Subsequent neural differentiation of CBEs requires sequential introduction of morphogenes, retinoic acid, brain-derived neurotrophic factor and cyclic AMP. Described methods emphasize defined media and reagents at all stages of the experiment comparable to protocols described for culturing human embryonic stem cells and cells from other somatic stem cell sources. Neural progenitor and cells generated from CBEs may be used for in vitro drug testing and cell-based assays and potentially for clinical transplantation.

Tumor Necrosis Factor (TNF) Is a Potent Rat Mast Cell Chemoattractant

It is well known that mast cell number increases in local tissues under different pathophysiologic conditions, although the humoral factors that stimulate local mast cell accumulation within tissues are not yet well known. Taking into account that tumor necrosis factor (TNF) influences tissue mast cell activity in various ways, the aim of the present study was to investigate the chemotactic activity of TNF for rat peritoneal mast cells. We have found that TNF induces mast cell migratory response in a dose-dependent manner, even in the absence of extracellular matrix (ECM) proteins. Significant migration was observed at concentrations of TNF as low as approximately 3 fM; higher TNF concentrations caused significant inhibition of spontaneous mast cell migration. In the presence of ECM proteins, TNF induced migration of mast cells in a biphasic manner, with peaks of migration occurring at approximately 0.3 fM and approximately 60 pM (in the presence of fibronectin) and at approximately 0.6 fM and approximately 600 pM (in the presence of laminin). Under the same experimental conditions, RANTES induced dose-dependent mast cell migration, and the optimal concentration of this chemokine for maximal migration was approximately 13 nM. The mast cell migratory response to lower concentrations of TNF was chemotactic and to higher TNF concentrations was due to chemokinesis. TNF-induced mast cell migration was completely blocked by neutralizing anti-TNF and anti-TNFR1 antibodies. The tyrosine kinase inhibitor, genistein, significantly abrogated mast cell migration toward TNF. Additionally, we have documented that TNF does not induce degranulation of rat mast cells. Taken together, our results indicate that TNF serves as an extremely potent chemotactic factor for rat mast cells that would cause accumulation of these cells at the site of diverse pathophysiologic conditions accompanied by inflammation.

Geoffrey Laurence Scott

<h3>Summary</h3> Ovarian cancer metastasizes into the peritoneum through dissemination of transformed epithelia as multicellular spheroids ^{1, 2}. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization...

Glucocorticoid-induced tumour necrosis factor receptor-related protein-mediated macrophage stimulation may induce cellular adhesion and cytokine expression in rheumatoid arthritis

Glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein (GITR) is one of the T cell co-stimulatory molecules and is associated with the pathogenesis of a number of autoimmune diseases. We investigated the expression patterns of GITR in human arthritic synovium and the role of GITR in the pathogenesis of rheumatoid arthritis (RA). Immunohistochemical analyses revealed the expression of GITR and its cognate ligand, GITRL, in macrophages in RA, but not in osteoarthritis (OA), synovium. To investigate the role of GITR in macrophage functions, primary macrophages from RA patients and a human macrophage cell line, THP-1, were analysed. Stimulation of the macrophages with anti-GITR monoclonal antibody induced up-regulation of intercellular adhesion molecule (ICAM)-1 and subsequent aggregation/adhesion, which was enhanced by the presence of extracellular matrix proteins and blocked by anti-ICAM-1 monoclonal antibody. The validity of these in vitro observations was confirmed by immunohistochemical analyses of RA synovium, which showed strong expression of ICAM-1 in GITR-positive macrophages. Additionally, GITR stimulation induced expression of proinflammatory cytokines/chemokines and matrix metalloproteinase-9 in synovial macrophages. These data indicate that GITR, expressed on macrophages in human RA synovium, may enhance inflammatory activation of macrophages by promoting cytokine gene expression and adhesion between cells and to extracellular matrix in RA synovium.

Encapsulation of RIN-m5F cells within Ba2+ cross-linked alginate beads affects proliferation and insulin secretion

The viability, proliferation and insulin production of RIN-m5F cells when loaded into alginate beads to form a 3D culture system has been investigated. The mechanism of alginate cross-linking (calcium ions vs barium ions), the addition of poly(L-lysine) (PLL) and poly(L-ornithine (PLO) and presence of different extra-cellular matrix proteins (ECM) influence the RIN-m5F cell behaviour. Cells in calcium alginate beads (CAB) proliferated and produced more insulin per cell than monolayer culture, but the physical properties of the beads were poor and they ruptured within a few days of culture. Barium alginate beads (BABs) provided a stable encapsulation method. Addition of PLL and PLO at concentrations above 0.1% w/v with the culture medium increased cell proliferation. With the addition of ECMs after bead formation there was a further increase in cell proliferation for certain combinations of ECM and PLO. It was concluded that RIN-m5F-loaded Ba-alginate beads (BABs), when incorporated with varying concentrations of poly (L) lysine (PLL), poly (L) ornithine (PLO) in the presence of extra-cellular matrix proteins (ECMs) were superior to both tissue culture and RIN-m5F-loaded Ca-alginate beads (CABs) in terms of physical stability, cell proliferation and insulin production.

Association of endometriosis in horses with differentiation of periglandular myofibroblasts and changes of extracellular matrix proteins

Periglandular fibrosis and cystic dilation of uterine glands are associated with equine endometriosis. The presence of extracellular matrix proteins (collagen type I, III and IV, laminin and fibronectin) in healthy and endometriotic specimens was demonstrated by immunohistochemistry. The distribution of collagen I, but not collagen III, was dependent on the stage of the oestrous cycle. The arrangement of collagen I and collagen III in endometriotic specimens was similar to that in normal endometrium. In periglandular fibrosis, collagen IV, laminin and fibronectin deposition outside the basement membrane was observed. In these regions, stromal cells were characterized immunohistochemically as myofibroblasts because of their expression of a-smooth muscle actin, and occasionally tropomyosin and desmin. Periglandular differentiation of contractile cells could be interpreted as a reaction to support the extrusion of secretions in cystic dilated glands. Moreover, the changes of extracellular matrix proteins are characteristic for neoplastic lesions, although further development of endometriosis to benign or malignant tumours is not known in horses. Knowledge of the factors responsible for these fibroblastic modulations may be the key to explaining the pathogenesis of endometriosis.