Analysis of the 5' region of the canine PAX3 gene and exclusion as a candidate for Dalmatian deafness

The causative mutation in a gene related to hearing loss in Dalmatians has been elusive. Because of its role in melanocyte migration and differentiation as integral component of the inner ear, we hypothesized that the canine PAX3 (paired box homeotic gene 3) gene could be a candidate for Dalmatian deafness. Therefore, we isolated the canine PAX3 gene and searched for causative mutations within the coding region of important regulatory domains of PAX3. However, no mutations were identified when comparing the DNA sequences of healthy and affected dogs. These results were confirmed by a two-point linkage analysis in 203 Dalmatians transmitting deafness. Our data clearly show that the canine PAX3 gene can be excluded as candidate for Dalmatian deafness.     

Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.     

Exploitation of herpesvirus immune evasion strategies to modify the immunogenicity of human mesenchymal stem cell transplants

Background

Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected.

Methodology/Principal Findings

We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients'' natural killer (NK) cells were depleted prior to cell transplantation.

Conclusions/Significance

Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable.     

Network analysis of epidermal growth factor signaling using integrated genomic, proteomic and phosphorylation data

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.     

Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates

This study reports the isolation and characterization of a variant of the human CCRF-CEM leukemia cell line that overproduces the carrier protein responsible for the uptake of reduced folates and the folate analogue methotrexate. The variant was obtained by adapting CCRF-CEM cells for prolonged times to stepwise decreasing concentrations of 5-formyltetrahydrofolate as the sole folate source in the cell culture medium. From cells that were grown on less than 1 nM 5-formyl-tetrahydrofolate, a variant (CEM-7A) was isolated exhibiting a 95-fold increased Vmax for [3H]methotrexate influx compared to parental CCRF-CEM cells. The values for influx Km, efflux t0.5, and Ki for inhibition by other folate (analogue) compounds were unchanged. Affinity labeling of the carrier with an N-hydroxysuccinimide ester of [3H]methotrexate demonstrate an approximately 30-fold increased incorporation of [3H] methotrexate in CEM-7A cells. This suggests that the up-regulation of [3H]methotrexate influx is not only due to an increased amount of carrier protein, but also to an increased rate of carrier translocation or an improved cooperativity between carrier protein molecules. Incubation for 1 h at 37 degrees C of CEM-7A cells with a concentration of 5-formyltetrahydrofolate or 5-methyltetrahydrofolate in the physiological range (25 nM) resulted in a 7-fold decline in [3H]methotrexate influx. This down-regulation during incubations with 5-formyltetrahydrofolate or 5-methyltetrahydrofolate could be prevented by either the addition of 10-25 nM of the lipophilic antifolate trimetrexate or by preincubating CEM-7A cells with 25 nM methotrexate. The down-regulatory effect was specifically induced by reduced folates since incubation of CEM-7A cells with 25 nM of either methotrexate, 10-ethyl-10-deazaaminopterin, aminopterin, or folic acid, or a mixture of purines and thymidine, had no effect on [3H]methotrexate influx. Similarly, these down-regulatory effects on [3H]methotrexate transport by 5-formyltetrahydrofolate, and its reversal by trimetrexate or methotrexate, were also observed, though to a lower extent, for parental CCRF-CEM cells grown in folate-depleted medium rather than in standard medium containing high folate concentrations. These results indicate that mediation of reduced folate/methotrexate transport can occur at reduced folate concentrations in the physiological range, and suggest that the intracellular folate content may be a critical determinant in the regulation of methotrexate transport.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.     

Characterization of medium conditioned by irradiated cells using proteome-wide, high-throughput mass spectrometry

Shedding, the release of cell surface proteins by regulated proteolysis, is a general cellular response to injury and is responsible for generating numerous bioactive molecules including growth factors and cytokines. The purpose of our work is to determine whether low doses of low-linear energy transfer (LET) radiation induce shedding of bioactive molecules. Using a mass spectrometry-based global proteomics method, we tested this hypothesis by analyzing for shed proteins in medium from irradiated human mammary epithelial cells (HMEC). Several hundred proteins were identified, including transforming growth factor beta (TGFB); however, no changes in protein abundances attributable to radiation exposure, based on immunoblotting methods, were observed. These results demonstrate that our proteomic-based approach has the sensitivity to identify the kinds of proteins believed to be released after low-dose radiation exposure but that improvements in mass spectrometry-based protein quantification will be required to detect the small changes in abundance associated with this type of insult.     

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.