Mesenchymal stem cell therapy in proteoglycan induced arthritis.

OBJECTIVES: To explore the immunosuppressive effect and mechanism of action of intraperitoneal (ip) and intra-articular (ia) mesenchymal stem cell (MSC) injection in proteoglycan induced arthritis (PGIA). METHODS: MSC were administered ip or ia after establishment of arthritis. We used serial bioluminescence imaging (BLI) to trace luciferase-transfected MSC. Mice were sacrificed at different time points to examine immunomodulatory changes in blood and secondary lymphoid organs. RESULTS: Both ip and local ia MSC injection resulted in a beneficial clinical and histological effect on established PGIA. BLI showed that MSC ip and ia in arthritic mice are largely retained for several weeks in the peritoneal cavity or injected joint respectively, without signs of migration. Following MSC treatment pathogenic PG-specific IgG2a antibodies in serum decreased. The Th2 cytokine IL-4 was only upregulated in PG-stimulated lymphocytes from spleens in ip treated mice and in lymphocytes from draining lymph nodes in ia treated mice. An increase in production of IL-10 was seen with equal distribution. Although IFN-γ was also elevated, the IFN-γ/IL-4 ratio in MSC treated mice was opposite to the ratio in (untreated) active PGIA. CONCLUSIONS: MSC treatment, both ip and ia, suppresses PGIA, a non-collagen induced arthritis model. MSC are largely retained for weeks in the injection region. MSC treatment induced at the region of injection a deviation of PG-specific immune responses, suggesting a more regulatory phenotype with production of IL-4 and IL-10, but also of IFN-γ, and a systemic decrease of pathogenic PG-specific IgG2a antibodies. These findings underpin the potential of MSC treatment in resistant arthritis.

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder which maps to chromosome 4qter, distal to the D4S139 locus. The cosmid clone 13E, isolated in a search for homeobox genes, was subsequently mapped to 4q35, also distal to D4S139. A subclone, p13E-11, detects in normal individuals a polymorphic EcoRI fragment usually larger than 28 kilobases (kb). Surprisingly, using the same probe we detected de novo DNA rearrangements, characterized by shorter EcoRI fragments (14-28 kb), in 5 out of 6 new FSHD cases. In 10 Dutch families analysed, a specific shorter fragment between 14-28 kb cosegregates with FSHD. Both observations indicate that FSHD is caused by independent de novo DNA rearrangements in the EcoRI fragment detected by p13E-11.

Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1.

Mutations in APC or beta-catenin inappropriately activate the transcription factor Tcf4, thereby transforming intestinal epithelial cells. Here it is shown that one of the target genes of Tcf4 in epithelial cells is Tcf1. The most abundant Tcf1 isoforms lack a beta-catenin interaction domain. Tcf1(-/-) mice develop adenomas in the gut and mammary glands. Introduction of a mutant APC allele into these mice substantially increases the number of these adenomas. Tcf1 may act as a feedback repressor of beta-catenin-Tcf4 target genes and thus may cooperate with APC to suppress malignant transformation of epithelial cells.

Uncoupling of S phase and mitosis in cardiomyocytes and hepatocytes lacking the winged-helix transcription factor Trident.

In order to maintain a stable karyotype, the eukaryotic cell cycle is coordinated such that only one round of S phase precedes each mitosis, and mitosis is not initiated until DNA replication is completed. Several checkpoints and regulatory proteins have been defined in lower eukaryotes that govern this coordination, but little is known about the proteins that are involved in mammalian cells. Previously, we have shown that the winged-helix transcription factor Trident - also known as HFH-11, FKL16 and WIN [1] [2] [3] - is exclusively expressed in cycling cells and is phosphorylated during mitosis [1] [4]. The cellular function of Trident has yet to be described, however. Here, we have shown that disruption of the Trident gene in mice resulted in postnatal death, most probably because of circulatory failure. Histological analysis of Trident -/- embryos from embryonic day 10 (E10) onwards revealed a specific, characteristic defect in the developing myocardium. The orientation of the myocytes was highly irregular and the nuclei of these disorganized cardiomyocytes were clearly polyploid with up to a 50-fold increase in DNA content. Polyploidy was also observed in embryonic hepatocytes. Our results indicate that expression of Trident is required to prevent multiple rounds of S phase in the heart and the liver. Trident therefore appears to have a role in preventing DNA re-replication during the G2 and M phases.

Sox-4 facilitates thymocyte differentiation.

The mouse Sry-like transcription factor Sox-4 is expressed in thymus, bone marrow, and gonads of adult mice. Sox-4-deficient mice die at embryonic day E14 due to cardiac malformation. In transfer experiments to irradiated recipients, B cell development was shown to be severely impaired in Sox-4-deficient progenitor cells. However, no drastic effects on T lymphocyte development were noted, despite the high level expression of the Sox-4 gene in the thymus of normal mice. Here, we report a detailed analysis of T cell development from Sox-4-deficient progenitors. Explanted fetal thymic organ cultures (FTOC) of Sox-4-deficient thymi yielded 10-50-fold fewer CD4 CD8 double-positive and single-positive cells than FTOC of littermates. This effect was T cell-autonomous, since similar observations were made when FTOC were performed by culturing of Sox-4-deficient progenitors in wild-type thymus lobes. When Sox-4-deficient fetal liver cells were injected together with normal cells intrathymically, they did not compete efficiently for reconstitution. It is concluded that Sox-4 facilitates thymocyte development.

Isolation and characterization of the rat gene encoding glutamate dehydrogenase.

The concentration of glutamate dehydrogenase (GDH) varies strongly between different organs and between different regions within organs. To permit further studies on the regulation of GDH expression, we isolated and characterized the rat gene encoding the GDH protein. This gene contains 13 exons and spans approximately 34 kbp. The GDH gene is present as a single, autosomally located copy in the Wistar rat genome, but shows an extensive restriction-fragment-length polymorphism for several enzymes. Promoter activity of the 5'-flanking sequence is shown by transient transfection experiments. The 5'-flanking sequence contains a TTAAAA sequence at position -29, instead of a consensus TATA box and, like many other TATA-less promoters, is characterized by a very high G + C content. In addition, consensus sequences for the binding sites of the transcription factors Sp1 and Zif268 are present in the G + C-rich upstream region.

Critical involvement of Tcf-1 in expansion of thymocytes.

T cell maturation in Tcf-1(-/-) mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4- 8- thymocytes are observed at two distinct stages (CD44+ 25+ and CD44- 25-), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1(-/-) progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1(-/-) mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Furthermore, their peripheral T cells have an activated phenotype (increased CD44 and decreased CD62L expression) and proliferate normally in response to Ag or mitogen, suggesting that these cells may have arisen from the early wave of development during embryogenesis and are either long lived or have subsequently been maintained by peripheral expansion. As Tcf-1 is a critical component in the Wnt/beta-catenin signaling pathway, these data suggest that Wnt-like factors play a role in the expansion of double-negative thymocytes.

Location of facioscapulohumeral muscular dystrophy gene on chromosome 4.

The autosomal dominant disorder facioscapulohumeral muscular dystrophy (FSHD) is the last of the major progressive muscular dystrophies in which the gene had not been located. In linkage analysis on ten Dutch families with this disorder a lod score of 6.34 at a recombination fraction of 0.13 was obtained with the microsatellite marker Mfd 22 (D4S171). This maps the FSHD gene to chromosome 4. Only one family was uninformative for this marker. We found no evidence of genetic heterogeneity.

The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors.

Tcf/Lef transcription factors mediate signalling from Wingless/Wnt proteins by recruiting Armadillo/beta-catenin as a transcriptional co-activator. However, studies of Drosophila, Xenopus and Caenorhabditis elegans have indicated that Tcf factors may also be transcriptional repressors. Here we show that Tcf factors physically interact with members of the Groucho family of transcriptional repressors. In transient transfection assays, the Xenopus Groucho homologue XGrg-4 inhibited activation of transcription of synthetic Tcf reporter genes. In contrast, the naturally truncated Groucho-family member XGrg-5 enhanced transcriptional activation. Injection of XGrg-4 into Xenopus embryos repressed transcription of Siamois and Xnr-3, endogenous targets of beta-catenin-Tcf. Dorsal injection of XGrg-4 had a ventralizing effect on Xenopus embryos. Secondary-axis formation induced by a dominant-positive Armadillo-Tcf fusion protein was inhibited by XGrg-4 and enhanced by XGrg-5. These data indicate that expression of Tcf target genes is regulated by a balance between Armadillo and Groucho.

Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription.

T cell factor / lymphocyte enhancer factor (Tcf/Lef) transcription factors complex with the transcriptional co-activator beta-catenin to transduce Wnt signals in a variety of developmental systems. The prototypic family member Tcf-1 is highly expressed in T lineage cells. Tcf1-/- mice are defective in cell cycling of early thymocyte stages. Here, we show that the interaction of beta-catenin with Tcf-1 is required for full thymocyte development. This interaction may be established by signals mediated by Wnt1 and Wnt4, leading to increased Tcf-dependent transcriptional activity in thymocytes, as demonstrated in Tcf-LacZ reporter mice. Transduction of fetal thymocytes with Wnt1 and Wnt4 results in increased survival in an in vitro cell culture system. Retroviral expression of soluble Wnt receptor mutants that block Wnt signaling inhibits thymocyte development. These results imply an important role for the Wnt cascade in thymocyte development.

Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization.

We have recently assigned the facioscapulohumeral muscular dystrophy (FSHD) gene to chromome 4 by linkage to the microsatellite marker Mfd 22 (locus D4S171). We now report that D4S139, a VNTR locus, is much more closely linked to FSHD. Two-point linkage analysis between FSHD and D4S139 in nine informative families showed a maximum combined lod score (Zmax) of 17.28 at a recombination fraction theta of 0.027. Multipoint linkage analysis between FSHD and the loci D4S139 and D4S171 resulted in a peak lod score of 20.21 at 2.7 cM from D4S139. Due to the small number of recombinants found with D4S139, the position of the FSHD gene relative to that of D4S139 could not be established with certainty. D4S139 was mapped to chromosome 4q35-qter by in situ hybridization, thus firmly establishing the location of the FSHD gene in the subtelomeric region of chromosome 4q. One small family yielded a negative lod score for D4S139. In the other families no significant evidence for genetic heterogeneity was obtained. Studies of additional markers and new families will improve the map of the FSHD region, reveal possible genetic heterogeneity, and allow better diagnostic reliability.

Genetic linkage map of facioscapulohumeral muscular dystrophy and five polymorphic loci on chromosome 4q35-qter.

A genetic map of five polymorphic markers in the area of the facioscapulohumeral muscular dystrophy (FSHD) gene on chromosome 4q35-qter has been constructed. With these five markers, a number of recombinants have been identified that allow ordering of the marker and the disease loci. The most likely locus order and the relative position of the FSHD gene supported by the recombinants is centromere-D4S171-F11-D4S187-D4S163-D4S139-FS HD-telomere. However, at least one recombination event appears to be inconsistent with this order and suggests a location of FSHD proximal to D4S139.

Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4.

A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro.