Jan Staelens

High-Density Linkage Maps and Sex-Linked Markers for the Black Tiger Shrimp (Penaeus monodon)

We report on the construction of sex-specific high-density linkage maps and identification of sex-linked markers for the black tiger shrimp (Penaeus monodon). Overall, we identified 44 male and 43 female linkage groups (2n = 88) from the analysis of 2306 AFLP markers segregating in three full-sib families, covering 2378 and 2362 cM, respectively. Twenty-one putatively homologous linkage groups, including the sex-linkage groups, were identified between the female and male linkage maps. Six sex-linked AFLP marker alleles were inherited from female parents in the three families, suggesting that the P. monodon adopts a WZ–ZZ sex-determining system. Two sex-linked AFLP markers, one of which we converted into an allele-specific assay, confirmed their association with sex in a panel of 52 genetically unrelated animals.

Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a⁺⁺/⁻ or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.

Hyporesponsiveness of SPRET/Ei mice to lethal shock induced by tumor necrosis factor and implications for a TNF-based antitumor therapy.

Tumor necrosis factor (TNF) is a central mediator in lethal shock and an interesting cytokine for anticancer therapy. To inhibit TNF-induced lethal shock, it is important to identify protective genes. Here we demonstrate that the SPRET/Ei mouse strain, derived from Mus spretus, exhibits an extremely dominant resistance to TNF-induced lethal inflammation. An interspecific backcross experiment revealed that the TNF hyporesponse is linked to loci on chromosomes 2, 6, and 11. Treatment of inoculated tumors with TNF and IFN-γ leads to regression and a highly reduced toxicity in (C57BL/6 × SPRET/Ei)F1 mice.

Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1

TNF is a central actor during inflammation and a well‐recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF‐induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1‐regulating miR‐511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR‐511 down‐regulates TNFR1 and protects against TNF, while anti‐miR‐511 up‐regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR‐511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti‐inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti‐inflammatory effects of glucocorti‐coids are mediated by induction of this miR, resulting in reduced TNFR1 expression.

Breaking the Species Barrier: Derivation of Germline‐Competent Embryonic Stem Cells from Mus spretus × C57BL/6 Hybrids

Embryonic stem (ES) cells, which can differentiate into almost all types of cells, have been derived from the house mouse Mus musculus, rat, rabbit, humans, and other species. Transmission of the genotype to the offspring of chimeras has been achieved only with M. musculus ES cells, limiting targeted mutagenesis using ES cells to this species. Mus spretus, which exhibits many genetic polymorphisms with M. musculus, displays dominant resistance to cancer and inflammation, making derived inbred strains very useful in positional cloning and interspecies mapping. We show here for the first time the derivation of ES cells from hybrid blastocysts, obtained by the mating of two different species, namely Mus musculus and Mus spretus, and their use for the generation of chimeric mice that transmit the Mus spretus genotype and phenotype to the offspring. These hybrid ES cells allow the genetic manipulation of Mus spretus, as an alternative to Mus musculus.

Response of TNF-hyporesponsive SPRET/Ei mice in models of inflammatory disorders.

Most inflammatory disorders are becoming more prevalent, especially in Western countries. The proinflammatory cytokine tumor necrosis factor-α (TNF) plays a prominent role in many of these inflammatory disorders. We have previously shown that SPRET/Ei mice exhibit an extreme and dominant resistance to high doses of TNF. In this report, we investigate the response of heterozygous (C57BL/6xSPRET/Ei)F1 mice in different models of inflammatory diseases. Compared with C57BL/6 mice, (B × S)F1 mice are protected against TNF-induced arthritis and are partially protected against allergic asthma in an ovalbumin-induced model. However, these mice display complete susceptibility to TNF-induced inflammatory bowel disease. These results indicate that the SPRET/Ei genome harbors potent dominant antiinflammatory genes that might be relevant for the treatment of certain chronic inflammatory diseases. It is very well possible that different genes are implicated in the different models.

Description and Mapping of the Resistance of DBA/2 Mice to TNF-Induced Lethal Shock

In our search for genes that inhibit the inflammatory effects of TNF without diminishing its antitumor capacities we found that, compared with C57BL/6 mice, DBA/2 mice exhibit a dominant resistance to TNF-induced lethality. Tumor-bearing (C57BL/6 × DBA/2)(BXD)F1 mice completely survived an otherwise lethal TNF/IFN-γ-antitumor therapy with complete regression of the tumor. This was not the case for C57BL/6 mice. Genetic linkage analysis revealed that TNF resistance is linked to a major locus on distal chromosome 6 and a minor locus on chromosome 17. Compared with littermate controls, chromosome substitution mice carrying a DBA/2 chromosome 6 in a C57BL/6 background were significantly protected against TNF and TNF/IFN-γ, albeit less so than DBA/2 mice. Definition of a critical region of 13 Mb on chromosome 6 was the highest mapping resolution obtained. Further analysis of candidate genes may provide a powerful tool to control TNF-induced pathologies in humans.