Repair of Annulus fibrosus with Genipin-enhanced Fibrin Hydrogel and silk membrane- fleece

Introduction Low back pain is often caused by a trauma causing disc herniation and /or disc degeneration. Although there are some promising approaches for nucleus pulposus repair, the inner tissue of the intervertebral disc (IVD) so far no treatment or repair is available for annulus fibrosus (AF) injuries. Here we aimed to develop a new method to seal and repair AF injuries by using a silk fleece composite and a genipin enhanced hydrogel. Methods Bovine (b) IVDs were harvested under aseptic conditions and kept in free swelling conditions for 24h in high-glucose DMEM containing 5% bovine serum for equilibration (1). A circular 2mm biopsy punch (Polymed Medical Center, Switzerland) was used to form a reproducible defect in the AF. For filling the defect and keeping the silk composite in place a human-derived fibrin gel (Baxter Tisseel, Switzerland) enhanced with 4.2mg/ml of the cross linker genipin (Wako Chemicals GmbH, Germany) was used. The silk composite consists of a mesh- and a membrane side (Spintec Engineering GmbH, Germany); the membrane is facing outwards to form a seal. bIVDs were cultured in vitro for 14 days either under dynamic load in a custom-built bioreactor under physiological conditions (0.2MPa load and ±2° torsion at 0.2Hz for 8h/day) or static diurnal load of 0.2MPa (2). At the end of culture discs were checked for seal failure, disc height, metabolic activity, cell death by necrosis (LDH assay), DNA content and glycosaminoglycan content. Results Silk composite maintained its position throughout the 14 days of culture under loaded conditions. Although repaired discs performed slightly lower in cell activity, DNA and GAG content were in the range of the control. Also LDH resulted in similar values compared to control discs (Fig 1). Height loss in repaired discs was in the same range as for static diurnal loaded control samples. For dynamically loaded samples the decrease was comparable to the injured, unrepaired discs. Fig 1 LDH of repaired discs compared to control disc after 24h in free swelling conditions for equilibration and first three loading cycles. Conclusions Silk-genipin-fibrin reinforced hydrogel is a promising approach to close AF defects as tested by two degree of freedom loading. In further experiments cytocompatibility of genipin has to be investigated. References 1. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. 2. Walser J, Ferguson SJ, Gantenbein-Ritter B. Design of a mechanical loading device to culture intact bovine caudal motional segments of the spine under twisting motion. In: Davies J, editors. Replacing animal models: a practical guide to creating and using biomimetic alternatives. Chichester, UK: John Wiley & Sons, Ltd.; 2012. p. 89-105. Acknowledgements This project is funded by the Gerbert Rüf Stiftung project # GRS-028/13 and the Swiss National Science Project SNF #310030_153411.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke

Each year about 650,000 Europeans die from stroke and a similar number lives with the sequelae of multiple sclerosis (MS). Stroke and MS differ in their etiology. Although cause and likewise clinical presentation set the two diseases apart, they share common downstream mechanisms that lead to damage and recovery. Demyelination and axonal injury are characteristics of MS but are also observed in stroke. Conversely, hallmarks of stroke, such as vascular impairment and neurodegeneration, are found in MS. However, the most conspicuous common feature is the marked neuroinflammatory response, marked by glia cell activation and immune cell influx. In MS and stroke the blood-brain barrier is disrupted allowing bone marrow-derived macrophages to invade the brain in support of the resident microglia. In addition, there is a massive invasion of auto-reactive T-cells into the brain of patients with MS. Though less pronounced a similar phenomenon is also found in ischemic lesions. Not surprisingly, the two diseases also resemble each other at the level of gene expression and the biosynthesis of other proinflammatory mediators. While MS has traditionally been considered to be an autoimmune neuroinflammatory disorder, the role of inflammation for cerebral ischemia has only been recognized later. In the case of MS the long track record as neuroinflammatory disease has paid off with respect to treatment options. There are now about a dozen of approved drugs for the treatment of MS that specifically target neuroinflammation by modulating the immune system. Interestingly, experimental work demonstrated that drugs that are in routine use to mitigate neuroinflammation in MS may also work in stroke models. Examples include Fingolimod, glatiramer acetate, and antibodies blocking the leukocyte integrin VLA-4. Moreover, therapeutic strategies that were discovered in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, turned out to be also effective in experimental stroke models. This suggests that previous achievements in MS research may be relevant for stroke. Interestingly, the converse is equally true. Concepts on the neurovascular unit that were developed in a stroke context turned out to be applicable to neuroinflammatory research in MS. Examples include work on the important role of the vascular basement membrane and the BBB for the invasion of immune cells into the brain. Furthermore, tissue plasminogen activator (tPA), the only established drug treatment in acute stroke, modulates the pathogenesis of MS. Endogenous tPA is released from endothelium and astroglia and acts on the BBB, microglia and other neuroinflammatory cells. Thus, the vascular perspective of stroke research provides important input into the mechanisms on how endothelial cells and the BBB regulate inflammation in MS, particularly the invasion of immune cells into the CNS. In the current review we will first discuss pathogenesis of both diseases and current treatment regimens and will provide a detailed overview on pathways of immune cell migration across the barriers of the CNS and the role of activated astrocytes in this process. This article is part of a Special Issue entitled: Neuro inflammation: A common denominator for stroke, multiple sclerosis and Alzheimer's disease, guest edited by Helga de Vries and Markus Swaninger.

Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12–24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 μmol g−1 fresh weight h−1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Limits of the power of Tissue P systems with cell division

Tissue P systems generalize the membrane structure tree usual in original models of P systems to an arbitrary graph. Basic opera- tions in these systems are communication rules, enriched in some variants with cell division or cell separation. Several variants of tissue P systems were recently studied, together with the concept of uniform families of these systems. Their computational power was shown to range between P and NP ? co-NP , thus characterizing some interesting borderlines between tractability and intractability. In this paper we show that com- putational power of these uniform families in polynomial time is limited by the class PSPACE . This class characterizes the power of many clas- sical parallel computing models

Building intuition of iron evolution during solar cell processing through analysis of different process models

An important aspect of Process Simulators for photovoltaics is prediction of defect evolution during device fabrication. Over the last twenty years, these tools have accelerated process optimization, and several Process Simulators for iron, a ubiquitous and deleterious impurity in silicon, have been developed. The diversity of these tools can make it difficult to build intuition about the physics governing iron behavior during processing. Thus, in one unified software environment and using self-consistent terminology, we combine and describe three of these Simulators. We vary structural defect distribution and iron precipitation equations to create eight distinct Models, which we then use to simulate different stages of processing. We find that the structural defect distribution influences the final interstitial iron concentration ([Fe-i]) more strongly than the iron precipitation equations. We identify two regimes of iron behavior: (1) diffusivity-limited, in which iron evolution is kinetically limited and bulk [Fe-i] predictions can vary by an order of magnitude or more, and (2) solubility-limited, in which iron evolution is near thermodynamic equilibrium and the Models yield similar results. This rigorous analysis provides new intuition that can inform Process Simulation, material, and process development, and it enables scientists and engineers to choose an appropriate level of Model complexity based on wafer type and quality, processing conditions, and available computation time.

A synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496–13505]. Here, we show that a synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance–fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides’ chirality is not necessarily required for peptide–peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1+, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1− cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1+ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.

Rho-dependent Regulation of Cell Spreading by the Tetraspan Membrane Protein Gas3/PMP22

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid–induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Membrane-anchored Plakoglobins Have Multiple Mechanisms of Action in Wnt Signaling

In Wnt signaling, β-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic β-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize β-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of β-catenin turnover. Expression of cnxPg increases levels of cytosolic β-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize β-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with β-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both β-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on β-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

GERp95, a Membrane-associated Protein that Belongs to a Family of Proteins Involved in Stem Cell Differentiation

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: Possible role of membrane TNF

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic β cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in β cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LTα) locus (whose sole source of TNF are the β cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LTα mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.