Stereolithography of ceramic scaffolds for bone tissue regeneration: Influence of hydroxyapatite/silica ratio on mechanical properties.

In this paper, the results obtained in the development of ceramic resin feedstock for stereolithography are shown. Hydroxyapatite and silica are used as source of ceramic. Hydroxyapatite is extracted from bovine bone, which enhances bioactivity of ceramic scaffold. The influence of hydroxyapatite amount in polymer-based slurry on the viscosity and printability of feedstock is explored. Hydroxyapatite and silica containing scaffolds are successfully obtained by stereolithography. Influence of hydroxyapatite/silica ratio on the bioactivity, biodegradability and mechanical properties of the scaffolds is also studied. It was observed that higher concentrations of hydroxyapatite led to improved mechanical strength of the scuffolds but increased viscosity of the slurry, affecting printability. Cell viability assays and cell visualization experiments indicated that the scaffolds not cause significant cell toxicity.

Changes in attachment and metabolic activity of rat neonatal cardiomyocytes and nonmyocytes caused by Macrovipera lebetina obtusa venom.

The Caucasian viper Macrovipera lebetina obtusa (MLO) is one of the most prevalent and venomous snakes in the Caucasus and the surrounding regions, yet the effects of MLO venom on cardiac function remain largely unknown. We examined the influence of MLO venom (crude and with inhibited metalloproteinases and phospholipase A2) on attachment and metabolic activity of rat neonatal cardiomyocytes (CM) and nonmyocytes (nCM), assessed at 1 and 24 h. After exposing both CM and nCM to varying concentrations of MLO venom, we observed immediate cytotoxic effects at a concentration of 100 µg/ml, causing detachment from the culture substrate. At lower MLO venom concentrations both cell types detached in a dose-dependent manner. Inhibition of MLO venom metalloproteinases significantly improved CM and nCM attachment after 1-hour exposure. At 24-hour exposure to metalloproteinases inhibited venom statistically significant enhancement was observed only in nCM attachment. However, metabolic activity of CM and nCM did not decrease upon exposure to the lower dose of the venom. Moreover, we demonstrated that metalloproteinases and phospholipases A2 are not the components of the MLO venom that change metabolic activity of both CM and nCM. These results provide a valuable platform to study the impact of MLO venom on prey cardiac function. They also call for further exploration of individual venom components for pharmaceutical purposes.

Antinociceptive, anti-inflammatory, and cytotoxic properties of Origanum vulgare essential oil, rich with ß-caryophyllene and ß-caryophyllene oxide.

Background: Essential oils are of great interest for their analgesic and anti-inflammatory properties. We aimed to study the content of the essential oil of the Origanum vulgare of the Armenian highlands (OVA) in different periods of vegetation and to investigate its antinociceptive and anti-inflammatory effects in mice (in vivo) and cytotoxic action in cultured cells (in vitro). OVA essential oil was extracted from fresh plant material by hydro-distillation. Methods: For OVA essential oil contents determination the gas chromatography-mass spectrometry method was used. Formalin and hot plate tests and analysis of cell viability using the methyl-thiazolyl-tetrazolium (MTT) assay were used. Results: The maximal content of ß-caryophyllene and ß-caryophyllene oxide in OVA essential oil was revealed in the period of blossoming (8.18% and 13.36%, correspondently). In the formalin test, 4% OVA essential oil solution (3.5 mg/mouse) exerts significant antinociceptive and anti-inflammatory effects (P = 0.003). MTT assay shows approximately 60% cytotoxicity in HeLa and Vero cells for 2.0 µL/mL OVA essential oil in media. Conclusions: The wild oregano herb of Armenian highlands, harvested in the blossoming period, may be considered as a valuable source for developing pain-relieving preparations.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

BACKGROUND: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. HYPOTHESIS: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. METHODS AND RESULTS: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. CONCLUSIONS: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.

Downregulation of beta-microglobulin to diminish T-lymphocyte lysis of non-syngeneic cell sources of engineered heart tissue constructs.

The presence of non-autologous major histocompatibility complex class I (MHC-I) molecules on the surface of the grafted cells is one of the main reasons for their rejection in non-syngeneic hosts. We present a straightforward strategy to decrease the presence of MHC-I by shRNA inhibition of beta-2-microglobulin (B2M), a conservative light chain of MHC-I, on the surface of two main cell types that are used to engineer heart tissue constructs. Engineered heart tissue constructs can be generated by combining mouse WT19 fibroblasts and mouse embryonic stem cell-derived cardiac myocytes (mESC-CM). WT19 fibroblasts were stably transduced with an anti-B2M shRNA, which yielded a cell line with dramatically reduced B2M expression levels (16 ± 11% of mock treated control cell line). Interferon gamma treatment increased the levels of B2M expression by >3-fold in both control and transduced fibroblasts; yet, B2M expression levels still remained very low in the transduced cells. When compared with their unmodified counterparts, transduced fibroblasts caused 5.7-fold lesser activation of cognate T-cells. B2M depletion in mESC-CM was achieved by 72 h transduction with anti-B2M shRNA lentiviral particles. Transduced mESC-CM exhibited regular beating and expressed classical cardiac markers. When compared with their unmodified counterparts, transduced mESC-CM caused 2.5-fold lesser activation of cognate T-cells. In vivo assessment of B2M downregulation was performed by analyzing the preferential survival of B2M-downregulated cells in the intraperitoneal cavity of allogeneic mice. Both B2M-downregulated fibroblasts and B2M-downregulated myocytes survived significantly better when compared to their unmodified counterparts (2.01 ± 0.4 and 5.07 ± 1.6 fold increase in survival, respectively). In contrast, when modified WT19 fibroblasts were injected into the intraperitoneal cavity of syngeneic C57Bl/6 mice, no significant survival advantage was observed. Notably, the preferential survival of B2M-downregulated cells persisted in allogeneic hosts with normal levels of natural killer cells, although the effect was lesser in magnitude. Use of shRNA against beta-2-microglobulin offers a simple and effective approach to minimize immunogenicity of the main cellular components of cardiac tissue constructs in non-syngeneic recipients.

Exposure to phthalates affects calcium handling and intercellular connectivity of human stem cell-derived cardiomyocytes.

BACKGROUND: The pervasive nature of plastics has raised concerns about the impact of continuous exposure to plastic additives on human health. Of particular concern is the use of phthalates in the production of flexible polyvinyl chloride (PVC) products. Di-2-ethylhexyl-phthalate (DEHP) is a commonly used phthalate ester plasticizer that imparts flexibility and elasticity to PVC products. Recent epidemiological studies have reported correlations between urinary phthalate concentrations and cardiovascular disease, including an increased risk of high blood pressure and coronary risk. Yet, there is little direct evidence linking phthalate exposure to adverse effects in human cells, including cardiomyocytes. METHODS AND RESULTS: The effect of DEHP on calcium handling was examined using monolayers of gCAMP3 human embryonic stem cell-derived cardiomyocytes, which contain an endogenous calcium sensor. Cardiomyocytes were exposed to DEHP (5 - 50 µg/mL), and calcium transients were recorded using a Zeiss confocal imaging system. DEHP exposure (24 - 72 hr) had a negative chronotropic and inotropic effect on cardiomyocytes, increased the minimum threshold voltage required for external pacing, and modified connexin-43 expression. Application of Wy-14,643 (100 µM), an agonist for the peroxisome proliferator-activated receptor alpha, did not replicate DEHP's effects on calcium transient morphology or spontaneous beating rate. CONCLUSIONS: Phthalates can affect the normal physiology of human cardiomyocytes, including DEHP elicited perturbations in cardiac calcium handling and intercellular connectivity. Our findings call for additional studies to clarify the extent by which phthalate exposure can alter cardiac function, particularly in vulnerable patient populations who are at risk for high phthalate exposure.

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.

Uveitis-associated epitopes of retinal antigens are pathogenic in the humanized mouse model of uveitis and identify autoaggressive T cells.

Noninfectious uveitis is a leading cause of blindness and thought to involve autoimmune T cell responses to retinal proteins (e.g., retinal arrestin [soluble-Ag (S-Ag)]). There are no known biomarkers for the disease. Susceptibility is associated with HLA, but little is known about susceptible class II alleles or the potentially pathogenic epitopes that they present. Using a humanized HLA-transgenic mouse model of S-Ag-induced autoimmune uveitis, we identified several susceptible and resistant alleles of HLA-DR and -DQ genes and defined pathogenic epitopes of S-Ag presented by the susceptible alleles. The sequences of these epitopes overlap with some previously identified peptides of S-Ag ("M" and "N"), known to elicit memory responses in lymphocytes of uveitis patients. HLA-DR-restricted, S-Ag-specific CD4(+) T cells could be detected in blood and draining lymph nodes of uveitic mice with HLA class II tetramers and transferred the disease to healthy mice. Importantly, tetramer-positive cells were detected in peripheral blood of a uveitis patient. To our knowledge, these findings provide the first tangible evidence that an autoimmune response to retina is causally involved in pathogenesis of human uveitis, demonstrating the feasibility of identifying and isolating retinal Ag-specific T cells from uveitis patients and may facilitate their development as biomarkers for the disease.

Immunological barriers to stem-cell based cardiac repair.

Repair of damaged myocardium with pluripotent stem cell derived cardiomyocytes is becoming increasingly more feasible. Developments in stem cell research emphasize the need to address the foreseeable problem of immune rejection following transplantation. Pluripotent stem cell (PSC) derived cardiomyocytes have unique immune characteristics, some of which are not advantageous for transplantation. Here we review the possible mechanisms of PSC-derived cardiomyocytes rejection, summarize the current knowledge pertaining to immunogenicity of such cells and describe the existing controversies. Myocardial graft rejection can be reduced by modifying PSCs prior to their differentiation into cardiomyocytes. Overall, this approach facilitates the development of universal donor stem cells suitable for the regeneration of many different tissue types.

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells.

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.

Clinically relevant concentrations of di (2-ethylhexyl) phthalate (DEHP) uncouple cardiac syncytium.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. The results of studies in experimental animals suggest that DEHP leached from flexible PVC tubing may cause health problems in some patient populations. While the cancerogenic and reproductive effects of DEHP are well recognized, little is known about the potential adverse impact of phthalates on the heart. This study examined the effects of clinically relevant concentrations of DEHP on neonatal rat cardiomyocytes. It was found that application of DEHP to a confluent, synchronously beating cardiac cell network, leads to a marked, concentration-dependent decrease in conduction velocity and asynchronous cell beating. The mechanism behind these changes was a loss of gap junctional connexin-43, documented using Western blot analysis, dye-transfer assay and immunofluorescence. In addition to its effect on electrical coupling, DEHP treatment also affected the mechanical movement of myocyte layers. The latter was linked to the decreased stiffness of the underlying fibroblasts, as the amount of triton-insoluble vimentin was significantly decreased in DEHP-treated samples. The data indicate that DEHP, in clinically relevant concentrations, can impair the electrical and mechanical behavior of a cardiac cell network. Applicability of these findings to human patients remains to be established.

Formation of cardiac fibers in Matrigel matrix.

We report a simple in vitro model of cardiac tissue that mimics three-dimensional (3-D) environment and mechanical load conditions and, as such, may serve as a convenient method to study stem cell engraftment or address developmental questions such as cytoskeleton or intercalated disk maturation. To create in vitro cardiac fibers we used Matrigel, a commercially available basement membrane preparation. A semisolid pillow from concentrated Matrigel was overlaid with a suspension of rat neonatal cardiomyocytes in a diluted Matrigel solution. This created an environment in which the multicellular fibers continuously contracted against a mechanical load. The described approach allows continuous structural and functional monitoring of 20-300-micron-thick cardiac fibers and provides easy access to epitopes for immunostaining purposes.

B-cell delivered gene transfer of human S-Ag-Ig fusion protein protects from experimental autoimmune uveitis.

Uveitis is an important autoimmune disease affecting an estimated 2.3 million Americans. This disease is manifested by inflammation of the retina mediated by the infiltration of T lymphocytes that recognize "S-Antigen" (S-Ag). Current therapies involve the life-long use of immunosuppressive drugs, including steroids. The ability to induce specific tolerance to S-Ag would be desirable and allow patients to be weaned off of steroid therapy. In this study, we determined that S-Ag-Ig retroviral vector was capable of preventing EAU (experimental autoimmune uveoretinitis) in Lewis rats induced by immunization with bovine S-Ag (BoS-Ag). Importantly, B-cell delivered gene therapy with S-Ag-Ig can ameliorate ongoing EAU when the treatment was initiated after rats had been immunized. Furthermore, we have successfully induced tolerance in HLA-DR3 transgenic mice with respect to the T-cell proliferative response. These results demonstrate proof of principle for future efforts to develop this approach for clinical application in patients with uveoretinitis.

Antigen/MHC class II/Ig dimers for study of uveitogenic T cells: IRBP p161-180 presented by both IA and IE molecules.

PURPOSE: Detection and modulation of effector T cells specific to immunodominant epitopes is a central issue in autoimmune diseases. Experimental autoimmune uveitis is a model for human autoimmune uveitis, induced in B10.RIII mice with interphotoreceptor retinoid binding protein or with its immunodominant epitope encoded by residues 161-180. METHODS: The authors generated a dimer composed of p161-180 fused in frame to IA(r) and mouse IgG1, and studied its effects on a CD4(+) uveitogenic T-cell line specific to p161-180 and on a T-cell clone derived from that line. RESULTS: Immunofluorescent staining of the T-cell line with the peptide/IA(r)/Ig dimer revealed that about 90% of the cells bound the reagent, and 10% did not. The T-cell clone failed to bind the reagent. Consistent with this, the line proliferated when stimulated with the reagent plus anti-CD28, and the clone did not. Conversely, after being incubated with the reagent without CD28 cross-linking, the line showed decreased proliferation on subsequent stimulatory exposure to p161-180, whereas the clone was unaffected. Antigen-specific proliferation of splenocytes from B10.RIII mice primed with p161-180 was inhibited by anti-IA as well as anti-IE antibodies; proliferation of the T-cell line was inhibited strongly by anti-IA and poorly by anti-IE, and the clone showed the opposite pattern. Finally, the line, but not the clone, proliferated to p161-180 presented on a B-cell lymphoma expressing IA(r) as its only restriction element. CONCLUSIONS: Uveitogenic T cells can be detected as well as functionally modulated with their cognate peptide-class II reagent, suggesting the potential of such reagents for diagnostic and therapeutic use in uveitic disease; p161-180 can be presented by IA(r) as well as IE(r) major histocompatibility complex (MHC) class II molecules. The possibility that the same immunodominant fragment might be presented by more than one class II molecule should be taken into account when diagnostic or clinical use of peptide-MHC reagents is considered.

Complex formation among murine cytomegalovirus US22 proteins encoded by genes M139, M140, and M141.

The murine cytomegalovirus (MCMV) proteins encoded by US22 genes M139, M140, and M141 function, at least in part, to regulate replication of this virus in macrophages. Mutant MCMV having one or more of these genes deleted replicates poorly in macrophages in culture and in the macrophage-dense environment of the spleen. In this report, we demonstrate the existence of stable complexes formed by the products of all three of these US22 genes, as well as a complex composed of the products of M140 and M141. These complexes form in the absence of other viral proteins; however, the pM140/pM141 complex serves as a requisite binding partner for the M139 gene products. Products from all three genes colocalize to a perinuclear region of the cell juxtaposed to or within the cis-Golgi region but excluded from the trans-Golgi region. Interestingly, expression of pM141 redirects pM140 from its predominantly nuclear residence to the perinuclear, cytoplasmic locale where these US22 proteins apparently exist in complex. Thus, complexing of these nonessential, early MCMV proteins likely confers a function(s) independent of each individual protein and important for optimal replication of MCMV in its natural host.

A humanized model of experimental autoimmune uveitis in HLA class II transgenic mice.

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. Here we report the development of a humanized EAU model in HLA transgenic (TG) mice. HLA-DR3, -DR4, -DQ6, and -DQ8 TG mice were susceptible to IRBP-induced EAU. Importantly, HLA-DR3 TG mice developed severe EAU with S-Ag, to which wild-type mice are highly resistant. Lymphocyte proliferation was blocked by anti-HLA antibodies, confirming that antigen is functionally presented by the human MHC molecules. Disease could be transferred by immune cells with a Th1-like cytokine profile. Antigen-specific T cell repertoire, as manifested by responses to overlapping peptides derived from S-Ag or IRBP, differed from that of wild-type mice. Interestingly, DR3 TG mice, but not wild-type mice, recognized an immunodominant S-Ag epitope between residues 291 and 310 that overlaps with a region of S-Ag recognized by uveitis patients. Thus, EAU in HLA TG mice offers a new model of uveitis that should represent human disease more faithfully than currently existing models.

Identification and characterization of novel murine cytomegalovirus M112-113 (e1) gene products.

The human cytomegalovirus (HCMV) UL112-113 gene products play important roles in viral DNA replication and transcriptional regulation. In this report, we characterize two novel transcripts originating from the homologous M112-113 (e1) region of the murine cytomegalovirus (MCMV) genome. These transcripts of 2.0 and 2.4 kb represent alternatively spliced products of the e1 gene region. Analysis of the e1 proteins demonstrates the presence of a previously unidentified 87-kDa protein that is likely encoded by the 2.4-kb transcript. All four protein products derived from the e1 gene region are expressed with early kinetics, are coordinately regulated, and localize predominantly to the nucleus of MCMV-infected cells. The expression pattern and localization of the e1 proteins show significant similarity to those of the HCMV UL112-113 proteins, signifying that MCMV e1 will serve as a useful model for assessing the role of this early gene region during viral infection.