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.

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, concentrationdependent 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.

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.The US22 genes of cytomegalovirus (CMV) are members of a multigene family unique to the betaherpesviruses. This gene family is characterized by the presence of one, two, three, or four conserved motifs (4,7,8,12,24,25,27). Consensus sequences for motifs I and II have been identified, and they contain short stretches of hydrophobic and charged residues. The less-well-defined motifs III and IV also have stretches of nonpolar residues. At the left end of betaherpesvirus genomes is a cluster of US22 family genes that exhibit homology among human CMV (HCMV) genes (UL23, UL24, UL28, and UL29), murine CMV (MCMV) genes (M23, M24, m25.1, and m25.2), and human herpesvirus 6 (HHV-6) or HHV-7 genes (U2, U3, U7, and U8). Farther downstream are HCMV US22 genes UL36 and UL43, which are respectively homologous to M36 and M43 in MCMV and to U16/17 and U25 in HHV-6 or HHV-7. At the right end of the MCMV genome is US22 gene M128 (ie2), which is homologous to the HHV-6 or HHV-7 U95 gene. Finally, at the far right end of the HCMV and MCMV genomes lies another cluster of US22 genes not present in HHV-6 or HHV-7. They are HCMV genes US22, US23, US24, US26, and IRS1/TRS1 and homologous MCMV genes M139, M140, M141, m142, and m143.At least two US22 genes within each betaherpesvirus genome appear to function as transcriptional transactivators of heterologous promoters. These include HCMV immediateearly (IE) genes UL36 ...

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.

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Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

BackgroundThe 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 ResultsThe 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.ConclusionsPhthalates 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.

Cytomegaloviruses likely encode numerous gene products involved in regulating virus-host cell interactions and pathogenesis. We previously identified a region of murine cytomegalovirus (MCMV) within HindIII-J and -I that regulates pathogenesis of the virus [open reading frames (ORFs) M139-M141] or is likely required for MCMV replication (ORFs m142 and m143). As a prerequisite for further studies on the structure and function of this gene region, we mapped the transcripts encoded within MCMV HindIII-I. Probes for ORFs M140 and M141 hybridized to 5.4- and 7.0-kb RNA, respectively, which were transcribed with early kinetics and were 3' coterminal with HindIII-J ORF M139. Probes representing ORFs m142, m143, or m144 hybridized to 3' coterminal transcripts of 1.8, 3.8, and 5.1 kb, respectively. ORFs m142 and m143 were transcribed with immediate-early kinetics but were most abundantly expressed at early times. Probes for the rightmost end of HindIII-I hybridized to a 5. 1-kb early/late RNA corresponding to m144 and to a 1.8-kb early RNA transcribed from m145. All of the major transcripts were polyadenylated and therefore are likely coding. Additional minor transcripts of intermediate sizes were also detected. ORFs M139-m143 showed homology to the betaherpesvirus-specific HCMV US22 gene family. Because deletion of these viral genes results in attenuated or helper-dependent phenotypes, this conserved region of US22 family genes may have a role in virus replication as well as in the pathogenesis of betaherpesviruses in their natural hosts.