Zaruhi Karabekian

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.

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.

Products of US22 Genes M140 and M141 Confer Efficient Replication of Murine Cytomegalovirus in Macrophages and Spleen

ABSTRACT Efficient replication of murine cytomegalovirus (MCMV) in macrophages is a prerequisite for optimal growth and spread of the virus in its natural host. Simultaneous deletion of US22 gene family members M139, M140, and M141 results in impaired replication of MCMV in macrophages and mice. In this study, we characterized the proteins derived from these three genes and examined the impact of individual gene deletions on viral pathogenesis. The M139, M140, and M141 gene products were identified as early proteins that localize to both the nucleus and cytoplasm in infected cells. Gene M139 encodes two proteins, of 72 and 61 kDa, while M140 and M141 each encode a single protein of 56 (pM140) and 52 (pM141) kDa, respectively. No role for the M139 proteins in MCMV replication in macrophages or mice was determined in these studies. In contrast, deletion of either M140 or M141 resulted in impaired MCMV replication in macrophages and spleen tissue. Replication of the M140 deletion mutant was significantly more impaired than that of the virus lacking M141. Further analyses revealed that the absence of the pM140 adversely affected pM141 levels by rendering the latter protein unstable. Since the replication defect due to deletion of M140 was more profound than could be explained by the reduced half-life of pM141, pM140 must exert an additional, independent function in mediating efficient replication of MCMV in macrophages and spleen tissue. These data indicate that the US22 genes M140 and M141 function both cooperatively and independently to regulate MCMV replication in a cell type-specific manner and, thus, to influence viral pathogenesis.

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.

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.

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.

Complex Formation among Murine Cytomegalovirus US22 Proteins Encoded by Genes M139, M140, and M141

ABSTRACT 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.

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.

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.

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.