Biomedical subjects
Publications and source records attributed to Rudi Balling.
The immune system is an important evolutionary invention to battle invaders in young and old organisms. Successful aging in humans who achieve nonagenarian status and beyond depends on how the immune system changes over time. Whether certain immune parameters vary with increased age is influenced by the genotype and lifestyle of the individual.
Experimental and human organ transplant studies suggest an important role for chemokine (C-C-motif) receptor-5 (CCR5) in the development of acute and chronic allograft rejection. Because early transplant damage can predispose allografts to chronic dysfunction, we sought to identify potential pathophysiologic mechanisms leading to allograft damage by using wild-type and Ccr5-deficient mice as recipients of fully MHC-mismatched heart and carotid-artery allografts. Gene expression in rejecting heart allografts was analyzed 2 and 6 days after transplantation using Affymetrix GeneChips. Microarray analysis led to identification of four metalloproteinase genes [matrix metalloproteinase (Mmp)3, Mmp12, Mmp13 and a disintegrin and metalloprotease domain (Adam)8] with significantly diminished intragraft mRNA expression in Ccr5-deficient mice at day 6. Accordingly, allografts from Ccr5-deficient mice showed less tissue remodeling and hence better preservation of the myocardial architecture compared with allografts from wild-type recipients. Moreover, survival of cardiac allografts was significantly increased in Ccr5-deficient mice. Carotid artery allografts from Ccr5-deficient recipients showed better tissue preservation, and significant reduction of neointima formation and CD3+ T cell infiltration. Ccr5 appears to play an important role in transplant-associated arteriosclerosis that may involve metalloproteinase-mediated vessel wall remodeling. We conclude that early tissue remodeling may be a critical feature in the predisposition of allografts to the development of chronic dysfunction.
The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.
It was the aim of this study to further explore the functional role of vitamin D in the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. Pancreatic islets but not exocrine pancreas cells showed strong lacZ reporter gene expression in mutant mice. To rule out possible influences of hypocalcemia on pancreatic endocrine function, a rescue diet enriched with calcium, phosphorus, and lactose was fed to wild-type (WT) and VDR mutant mice. The rescue diet normalized body weight and mineral homeostasis in VDR mutants. In glucose tolerance tests, baseline blood glucose levels were unchanged in fasting VDR mutants. However, blood glucose was elevated after oral or subcutaneous glucose loading, and maximum serum insulin levels were reduced by approximately 60% in VDR mutants vs. WT mice on either diet. In addition, insulin mRNA levels were decreased in VDR mutant mice on both diets, whereas pancreatic beta cell mass, islet architecture, and islet neogenesis were normal. These findings clearly establish a molecular role of the vitamin D-responsive elements in pancreatic insulin synthesis and secretion in vivo.
We urgently need animal models to study infectious disease. Mice are susceptible to a similar range of microbial infections as humans. Marked differences between inbred strains of mice in their response to pathogen infection can be exploited to analyse the genetic basis of infections. In addition, the genetic tools that are available in the laboratory mouse, and new techniques to monitor the expression of bacterial genes in vivo, make it the principal experimental animal model for studying mechanisms of infection and immunity.
The peptide transporter PEPT2 mediates the cellular uptake of di- and tripeptides and selected drugs by proton-substrate cotransport across the plasma membrane. PEPT2 was functionally identified initially in the apical membrane of renal tubular cells but was later shown to be expressed in other tissues also. To investigate the physiological importance of PEPT2 and for a detailed analysis of the protein expression sites, we generated a Pept2 knockout mouse line in which the Pept2 gene was disrupted by insertion of a beta-galactosidase gene under the control of the PEPT2 promoter. The Pept2(-/-) mice showed no obvious phenotypic abnormalities but also no adaptive upregulation in the expression level of related genes in the kidney. The importance of PEPT2 in the reabsorption of filtered dipeptides was demonstrated in knockout animals by significantly reduced renal accumulation of a fluorophore-labeled and a radiolabeled dipeptide after in vivo administration of the tracers. This indicates that PEPT2 is the main system responsible for tubular reabsorption of peptide-bound amino acids, although this does not lead to major changes in renal excretion of protein or free amino acids.
We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.
Despite recent progress in identifying genes underlying deafness, there are still relatively few mouse models of specific forms of human deafness. Here we describe the phenotype of the Beethoven (Bth) mouse mutant and a missense mutation in Tmc1 (transmembrane cochlear-expressed gene 1). Progressive hearing loss (DFNA36) and profound congenital deafness (DFNB7/B11) are caused by dominant and recessive mutations of the human ortholog, TMC1 (ref. 1), for which Bth and deafness (dn) are mouse models, respectively.
The epithelial thymic anlage develops from the third pharyngeal pouch. Pax9 is expressed in the entire pharyngeal endoderm, and its function is required for normal development of organs derived from pharyngeal pouches. Here, we show that in Pax9 null mice, the thymic anlage develops as an ectopic polyp-like structure in the larynx. It expresses Whn/Foxn1, a marker of thymic epithelium, but fails to perform the normal caudo-ventral movement to the upper mediastinum. The thymic rudiment contains mesenchymal cells, blood vessels and is colonized by T cell progenitors. However, from embryonic day 14.5 onwards, the size of the Pax9 mutant thymus is severely reduced. Whereas expression of TCRbeta chain genes is readily detectable in the mutant thymus, no expression of the TCRgamma chain was detectable. Our results identify a new genetically defined control point of thymopoiesis.
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Pax genes encode a family of transcription factors that play key roles in embryonic development. Whereas the functions of Pax genes in the adult organism are largely unknown, upregulated Pax gene expression has been implicated in tumourigenesis. In this study, PAX9-specific monoclonal antibodies have been generated and it has been shown that PAX9 protein is expressed in the normal epithelium of the adult human oesophagus. PAX9 expression was either lost or significantly reduced in the majority of invasive carcinomas and epithelial dysplasias, the latter representing precancerous lesions. Notably, the percentage of PAX9-positive cells within the epithelium decreased with increasing malignancy of the epithelial lesion. These results identify PAX9 as a sensitive marker for deregulated differentiation of oesophageal keratinocytes and indicate a role for PAX9 in the normal differentiation process of internal stratified squamous epithelia. These data suggest that upregulated PAX9 expression is not required for the formation of the majority of squamous cell carcinomas of the human oesophagus.
Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.
During a large-scale ENU mutagenesis screen, a mouse mutant with a dominant cataract was detected and referred to as Aey4. Aim of this study was the morphological description of the mutant, the mapping of the mutation, and the characterization of the underlying molecular lesion. The slit-lamp examination revealed a strong nuclear cataract surrounded by a homogeneous milky opacity in the inner cortex. The histological analysis demonstrated remnants of cell nuclei throughout the entire lens. The mutation was mapped to Chromosome 1 by a genome-wide linkage making the six gamma-crystallin encoding genes and the closely linked betaA2-crystallin encoding gene to relevant candidate genes. Finally, a T-->A exchange in exon 2 of the gammaD-crystallin encoding gene (symbol: Crygd) was demonstrated to be causative for the cataract phenotype; this particular mutation is, therefore, referred to Crygo(Aey4). The alteration in codon 76 leads to an amino acid exchange of Val-->Asp. Val at this position is highly conserved; it is found in all mouse and rat gammaD/E/F-crystallins as well as in the human gammaA- and gammaD-crystallins. It may be replaced solely by Ile, which is present in all bovine gamma-crystallins, in the rat and mouse gammaA/B/C-crystallins, as well as in the human gammaB/C-crystallins. It is predicted that the exchange of a hydrophobic side chain by a polar and acidic one might influence the microenvironment by a dramatic decrease of the isoelectric point by 1.5 pH units in the 10 amino acids surrounding position 76. The Crygd(Aey4) additionally demonstrates the importance of the integrity of the Cryg gene cluster for lens transparency.
We examined the molecular progression of ectopic bone development upon application of recombinant human bone morphogenetic protein-2 (rhBMP2), using a commercial collagen type I carrier, in the hind quarter muscles of mice. We performed a gene expression study using mRNA in situ hybridisation to compare embryonic cartilage and bone formation with BMP2-induced ectopic bone formation. As bone growth can be induced postnatally or in adult animals, we examined the expression of molecules regulating embryonic bone development. We found that the mRNAs of the same molecules, such as Indian hedgehog (IHH), parathyroid hormone (PTH)/PTH-related peptide receptor (PPR) and BMPs, that regulate embryonic cartilage and bone development, are expressed during BMP-induced ectopic bone formation, suggesting parallels in the mechanisms controlling these processes. Our studies support by molecular means the previous findings in rats that BMP2-induced ectopic bone formation in mice undergoes bone development involving both modes, endochondral and intramembranous ossification, simultaneously at different sites of the implant.
The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).