Anaphylactic reaction to intravenous corticosteroids in the treatment of ocular toxoplasmosis: a case report.

INTRODUCTION: This case report presents for the first time an acute systemic allergic reaction to corticosteroids in a patient with ocular toxoplasmosis after treatment with intravenous cortisone, and discusses alternative treatments. CASE PRESENTATION: We present the case of a 57-year-old Caucasian woman with an anaphylactic reaction after intravenous injection of prednisolone-21-hydrogensuccinate (Solu-Decortin® H) given for the treatment of toxoplasmosis-associated chorioretinitis. Immediately after the injection, she developed an acute erythema of the legs and abdomen, angioedema, hypotension (blood pressure 80/40mmHg), tachycardia (heart rate 140/minute), hyperthermia (38.8°C), and respiratory distress. Allergological examinations showed a positive skin-prick test to prednisolone and methylprednisolone. In addition, an oral exposure test with dexamethasone (Fortecortin®) and betamethasone (Celestamine®) was conducted to find alternative corticosteroids for future treatments. After oral application, no local or systemic reactions were observed for these two substances. CONCLUSIONS: This case report demonstrates that systemic allergic reactions are possible in patients with uveitis or other inflammatory ophthalmological conditions treated with intravenous corticosteroids. Intravenous administration of cortisone, for example, in the treatment of ocular toxoplasmosis, should always be conducted with caution because of a possible allergic reaction. For patients who react to a particular steroid, it is necessary to undergo allergological testing to confirm that the compound in question is indeed allergenic, and to identify other corticosteroids that are safe for future anti-inflammatory treatments.

Anterior chamber paracentesis after central retinal artery occlusion: a tenable therapy?

BACKGROUND: The goal of this study was to investigate the visual outcome of acute central retinal artery occlusion (CRAO) after current standard therapy with and without paracentesis. In addition, we investigated whether there was a dependence of the resulting visual acuity on the time between first symptoms and implementation of paracentesis. Finally, we analysed risk factors for CRAO. METHODS: We performed a retrospective analysis of data from patients with CRAO who received standard in-patient therapy with and without paracentesis at the Dr. Horst Schmidt Clinics in Wiesbaden, Germany between 2000 and 2012. The primary endpoint was the change of visual acuity 3 days after the initiation of intervention. RESULTS: Data from 74 patients with CRAO were included in the study. Fifteen patients were treated conservatively and 59 patients received additional paracentesis. Clinically significant improvement of BCVA (logMAR ≥ 0.3) after 3 days was observed in 26.7% of patients without paracentesis, 36.4% of patients with paracentesis within 6 hours, 20% of patients with paracentesis within 7-24 hours, and 23.1% of patients with paracentesis more than 24 hours after the onset of symptoms. There was no significant difference in the outcome between patients with (BCVA 1.9 ± 0.31) and without paracentesis (BCVA 1.75 ± 0.32) (p = 0.9), nor among the groups with paracentesis (p = 0.8). One patient suffered a lens injury due to the paracentesis, with subsequent need for cataract surgery. CONCLUSIONS: There was no added gain in visual acuity by performing a paracentesis, independent of the time elapsed between first symptoms and the implementation of paracentesis. In the absence of any tangible effectiveness of paracentesis and the inherent risks of paracentesis such as intraocular infection and injury, paracentesis does not appear to be warranted as a treatment of CRAO.

Phenotypic redifferentiation and cell cluster formation of cultured human articular chondrocytes in a three-dimensional oriented gelatin scaffold in the presence of PGE2--first results of a pilot study.

Modern tissue engineering strategies comprise three elemental parameters: cells, scaffolds and growth factors. Articular cartilage represents a highly specialized tissue which allows frictionless gliding of corresponding articulating surfaces. As the regenerative potential of cartilage is low, tissue engineering-based strategies for cartilage regeneration represent a huge challenge. Prostaglandins function as regulators in cartilage development and metabolism, especially in growth plate chondrocytes. In this study, it was analyzed if prostaglandin E2 (PGE2 ) has an effect on the phenotypic differentiation of human chondrocytes cultured in a three-dimensional (3D) gelatin-based scaffold made by directional freezing and subsequent freeze-drying. As a result, it was clearly demonstrated that low doses of PGE2 revealed beneficial effects on the phenotypic differentiation and collagen II expression of human articular chondrocytes in this 3D cell culture system. In conclusion, PGE2 is an interesting candidate for tissue engineering applications since it represents an already well-studied molecule which is available in pharmaceutical quality.

Human endothelial and osteoblast co-cultures on 3D biomaterials.

Increasingly, in vitro experiments are being used to evaluate the cell compatibility of novel biomaterials. Single cell cultures have been used to determine how well cells attach, grow, and exhibit characteristic functions on these materials and the outcome of such tests is generally accepted as an indicator of biocompatibility. However, organs and tissues are not made up of one cell type and the interaction of cells is known to be an essential factor for physiological cell function. To more accurately examine biomaterials for bone regeneration, we have developed methods to coculture osteoblasts, which are the primary cell type making up bone, and endothelial cells, which form the vasculature supplying cells in the bone with oxygen and nutrients to survive on 2- and 3-D biomaterials.

Anti-angiogenic activity of heparin-like polysulfonated polymeric drugs in 3D human cell culture.

The activity of new anti-angiogenic polymeric drugs was tested in a 3D endothelial cell culture system applied as a model of angiogenesis. The assay was performed in a highly reproducible fibrin matrix that supported endothelial cell attachment, proliferation, migration, and formation of capillary-like structures. Active growth factors (FGF and/or VEGF) were added to the medium to induce the formation of blood vessel-like structures, and the effect of the active polymers was then tested by a semi-quantitative immunostaining protocol and visualized by laser-scanning confocal microscopy. The synthetic heparin-like macromolecules that were tested for their anti-angiogenic efficacy were previously characterized in terms of their anti-proliferative activity in 2D tissue culture. Two different anti-angiogenic monomers, a methacrylic derivative of 5-amino-2-naphthalenesulfonic acid (MANSA) and 2-acrylamido-2-methylpropane sulfonic acid (AMPS), were copolymerized with a hydrophilic monomer (vinyl pyrrolidone, VP) or a hydrophobic monomer (butyl acrylate, BA), giving rise to different copolymeric systems with controlled microstructure and supramolecular organization. Both copolymeric systems have demonstrated a composition-dependent anti-mitogenic effect in "2D in vitro" cell culture experiments using aFGF as pro-angiogenic growth factor and BALB/c 3T3 fibroblast as cell model, as was shown in a previous publication. These 3D experiments provide evidence for the strong and specific modulation of angiogenesis by these systems. The 3D experiments constitute an improvement over 2D in vitro experiments and in vivo experiments with angiogenic drugs and may help to reduce the number of animal experiments.

The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature.

The survival and functioning of a bone biomaterial upon implantation requires a rapidly forming and stably functioning vascularization that connects the implant to the recipient. We have previously shown that human microcapillary endothelial cells (HDMEC) and primary human osteoblast cells (HOS) in coculture on various 3-D bone biomaterial scaffolds rapidly distribute and self-assemble into a morphological structure resembling bone tissue. Endothelial cells form microcapillary-like structures containing a lumen and these were intertwined between the osteoblast cells and the biomaterial. This tissue-like self-assembly occurred in the absence of exogenously added angiogenic stimuli or artificial matrices. The purpose of this study was to determine whether this in vitro pre-formed microvasculature persists and functions in vivo and to determine how the host responds to the cell-containing scaffolds. The scaffolds with cocultures were implanted into immune-deficient mice and compared to scaffolds without cells or with HDMEC alone. Histological evaluation and immunohistochemical staining with human-specific antibodies of materials removed 14 days after implantation demonstrated that the in vitro pre-formed microcapillary structures were present on the silk fibroin scaffolds and showed a perfused lumen that contained red blood cells. This proved anastomosis with the host vasculature. Chimeric vessels in which HDMEC were integrated with the host's ingrowing (murine) capillaries were also observed. No HDMEC-derived microvessel structures or chimeric vessels were observed on implanted silk fibroin when precultured with HDMEC alone. In addition, there was migration of the host (murine) vasculature into the silk fibroin scaffolds implanted with cocultures, whereas silk fibroin alone or silk fibroin precultured only with HDMEC were nearly devoid of ingrowing host microcapillaries. Therefore, not only do the in vitro pre-formed microcapillaries in a coculture survive and anastomose with the host vasculature to become functioning microcapillaries after implantation, the coculture also stimulates the host capillaries to rapidly grow into the scaffold to vascularize the implanted material. Thus, this coculture-based pre-vascularization of a biomaterial implant may have great potential in the clinical setting to treat large bone defects.

Signalling molecules and growth factors for tissue engineering of cartilage-what can we learn from the growth plate?

Modern tissue engineering concepts integrate cells, scaffolds, signalling molecules and growth factors. For the purposes of regenerative medicine, fetal development is of great interest because it is widely accepted that regeneration recapitulates in part developmental processes. In tissue engineering of cartilage the growth plate of the long bone represents an interesting, well-organized developmental structure with a spatial distribution of chondrocytes in different proliferation and differentiation stages, embedded in a scaffold of extracellular matrix components. The proliferation and differentiation of these chondrocytes is regulated by various hormonal and paracrine factors. Thus, members of the TGFbeta superfamily, the parathyroid hormone-related peptide-Indian hedgehog loop and a number of transcription factors, such as Sox and Runx, are involved in the regulation of chondrocyte proliferation and differentiation. Furthermore, adhesion molecules, homeobox genes, metalloproteinases and prostaglandins play a role in the complex regulation mechanisms. The present paper summarizes the morphological organization of the growth plate and provides a short but not exhaustive overview of the regulation of growth plate development, giving interesting insights for tissue engineering of cartilage.

Stability of prostaglandin E(2) (PGE (2)) embedded in poly-D,L: -lactide-co-glycolide microspheres: a pre-conditioning approach for tissue engineering applications.

Prostaglandin E(2) (PGE(2)) is involved in angiogenesis, bone repair and cartilage metabolism. Thus, PGE(2) might represent a suitable signaling molecule in different tissue engineering applications. PGE(2) also has a short half-life time. Its incorporation into poly-D: ,L: -lactide-co-glycolide (PLGA) microspheres was demonstrated in a previous study. However, the stability of bioactive PGE(2) in these microspheres is unknown. With an adjusted mass spectrometry assay we investigated the amount of incorporated PGE(2) and the stability of PGE(2) in conventional cell culture medium and in PLGA microspheres. The stability of PGE(2) was closely pH dependent. Strong acidic or basic environments reduced the half-life from 300 h (pH 2.6-4.0) to below 50 h at pH 2.0 or pH 8.8. The half-life of PGE(2) incorporated into poly-D: ,L: -lactide-co-glycolide increased drastically to 70 days at 37 degrees C and to 300 days at 8 degrees C. Analysis with scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated a distinct nanostructure of the polymeric phase and both nano- and microporosity.

Analysis of the biological response of endothelial and fibroblast cells cultured on synthetic scaffolds with various hydrophilic/hydrophobic ratios: influence of fibronectin adsorption and conformation.

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.

Immobilization and controlled release of prostaglandin E2 from poly-L-lactide-co-glycolide microspheres.

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite involved in physiological homeostasis and numerous pathophysiological conditions. Furthermore, it has been demonstrated that prostaglandins have a stimulating effect not only on angiogenesis in situ and in vitro but also on chondrocyte proliferation in vitro. Thus, PGE(2) represents an interesting signaling molecule for various tissue engineering strategies. However, under physiological conditions, PGE(2) has a half-life time of only 10 min, which limits its use in biomedical applications. In the present study, we investigated if the incorporation of PGE(2) into biodegradable poly-L-lactide-co-glycolide microspheres results in a prolonged release of this molecule in its active form. PGE(2)-modified microspheres were produced by a cosolvent emulsification method using CHCl(3) and HFIP as organic solvents and PVA as emulsifier. Thirteen identical batches were produced; and to each batch 1.0 mL of serum-free medium was added. The medium was removed at defined time points and then analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) to measure the residual PGE(2) content. In this study we demonstrated the prolonged release of PGE(2), showing a linear increase over the first 12 h, followed by a plateau and a slow decrease. The microspheres were further characterized by scanning electron microscopy.

Recombinant protein-co-PEG networks as cell-adhesive and proteolytically degradable hydrogel matrixes. Part II: biofunctional characteristics.

We present here the biological performance in supporting tissue regeneration of hybrid hydrogels consisting of genetically engineered protein polymers that carry specific features of the natural extracellular matrix, cross-linked with reactive poly(ethylene glycol) (PEG). Specifically, the protein polymers contain the cell adhesion motif RGD, which mediates integrin receptor binding, and degradation sites for plasmin and matrix-metalloproteinases, both being proteases implicated in natural matrix remodeling. Biochemical assays as well as in vitro cell culture experiments confirmed the ability of these protein-PEG hydrogels to promote specific cellular adhesion and to exhibit degradability by the target enzymes. Cell culture experiments demonstrated that proteolytic sensitivity and suitable mechanical properties were critical for three-dimensional cell migration inside these synthetic matrixes. In vivo, protein-PEG matrixes were tested as a carrier of bone morphogenetic protein (rhBMP-2) to heal critical-sized defects in a rat calvarial defect model. The results underscore the importance of fine-tuning material properties of provisional therapeutic matrixes to induce cellular responses conducive to tissue repair. In particular, a lack of rhBMP or insufficient degradability of the protein-PEG matrix prevented healing of bone defects or remodeling and replacement of the artificial matrix. This work confirms the feasibility of attaining desired biological responses in vivo by engineering material properties through the design of single components at the molecular level. The combination of polymer science and recombinant DNA technology emerges as a powerful tool for the development of novel biomaterials.

In situ preparation of protein-"smart" polymer conjugates with retention of bioactivity.

Protein-polymer conjugates are widely used in biotechnology and medicine, and new methods to prepare the bioconjugates would be advantageous for these applications. In this report, we demonstrate that bioactive "smart" polymer conjugates can be synthesized by polymerizing from defined initiation sites on proteins, thus preparing the polymer conjugates in situ. In particular, free cysteines, Cys-34 of bovine serum albumin (BSA) and Cys-131 of T4 lysozyme V131C, were modified with initiators for atom transfer radical polymerization (ATRP) either through a reversible disulfide linkage or irreversible bond by reaction with pyridyl disulfide- and maleimide-functionalized initiators, respectively. Initiator conjugation was verified by electrospray-ionization mass spectroscopy (ESI-MS), and the location of the modification was confirmed by muLC-MSMS (tandem mass spectrometry) analysis of the trypsin-digested protein macroinitiators. Polymerization of N-isopropylacrylamide (NIPAAm) from the protein macroinitiators resulted in thermosensitive BSA-polyNIPAAm and lysozyme-polyNIPAAm in greater than 65% yield. The resultant conjugates were characterized by gel electrophoresis and size exclusion chromatography (SEC) and easily purified by preparative SEC. The identity of polymer isolated from the BSA conjugate was confirmed by (1)H NMR, and the polydispersity index was determined by gel permeation chromatography (GPC) to be as low as 1.34. Lytic activities of the lysozyme conjugates were determined by two standard assays and compared to that of the unmodified enzyme prior to polymerization; no statistical differences in bioactivity were observed.

Diurnal rhythm of cone opsin expression in the teleost fish Haplochromis burtoni.

The biochemical and morphological specializations of rod and cone photoreceptors reflect their roles in sight. The apoprotein opsin, which converts photons into chemical signals, functions at one end of these highly polarized cells, in the outer segment. Previous work has shown that the mRNA of rod opsin, the opsin specific to rods, is renewed in the outer segment with a diurnal rhythm in the retina of the teleost fish Haplochromis burtoni. Here we show that in the same species, all three cone opsin mRNAs (blue, green, and red) also have a diurnal rhythm of expression. Quantitative real-time polymerase chain reaction (PCR) with primer pairs specific for the cone photoreceptor opsin subtypes was used to detect opsin mRNA abundance in animals sacrificed at 3-h intervals around the clock. All three cone opsins were expressed with diurnal rhythms similar to each other but out of phase with the rod opsin rhythm. Specifically, cone opsin expression occurs at a higher level near the onset of the dark period, when cones are not used for vision. Finally, we found that the rhythm of cone opsin expression in fish appears to be light dependent, as prolonged darkness changes normal diurnal expression patterns.

Biologically engineered protein-graft-poly(ethylene glycol) hydrogels: a cell adhesive and plasmin-degradable biosynthetic material for tissue repair.

To address the need for bioactive materials toward clinical applications in wound healing and tissue regeneration, an artificial protein was created by recombinant DNA methods and modified by grafting of poly(ethylene glycol) diacrylate. Subsequent photopolymerization of the acrylate-containing precursors yielded protein-graft-poly(ethylene glycol) hydrogels. The artificial protein contained repeating amino acid sequences based on fibrinogen and anti-thrombin III, comprising an RGD integrin-binding motif, two plasmin degradation sites, and a heparin-binding site. Two-dimensional adhesion studies showed that the artificial protein had specific integrin-binding capability based on the RGD motif contained in its fibrinogen-based sequence. Furthermore, heparin bound strongly to the protein's anti-thrombin III-based region. Protein-graft-poly(ethylene glycol) hydrogels were plasmin degradable, had Young's moduli up to 3.5 kPa, and supported three-dimensional outgrowth of human fibroblasts. Cell attachment in three dimensions resulted from specific cell-surface integrin binding to the material's RGD sequence. Hydrogel penetration by cells involved serine-protease mediated matrix degradation in temporal and spatial synchrony with cellular outgrowth. Protein-graft-poly(ethylene glycol) hydrogels represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration.