R. Willemsen - Academia.edu (original) (raw)

Papers by R. Willemsen

Gene Therapy, 2005

Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes... more Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes has demonstrated its effectiveness in the eradication of cancer and virally infected cells. Clinical trails and in vitro studies have focused on CD8+ cytotoxic T-cell receptor (TCR) alphabeta lymphocytes since these cells directly kill virally infected- and tumor cells after antigen-specific recognition via their TCR alphabeta. However, increasing evidence suggests that induction of sustained immunity against cancer and viral infections depends on the presence of tumor- or virus-specific CD4+ T lymphocytes, which are restricted by MHC class II. Here, we show that these MHC class II-restricted CD4+ T lymphocytes can efficiently be redirected to MHC class I-restricted tumor cells by retroviral introduction of an HLA-A1/MAGE-A1-specific chimeric two-chain TCR ValphaCalphazeta/VbetaCbetazeta (tcTCR/zeta). However, TCR-transduced CD4+ T lymphocytes were only able to specifically bind to HLA-A1/MAGE-A1 complexes and respond to HLA-A1+/MAGE-A1+ melanoma cells when the CD8alpha gene was cointroduced. These CD4+/CD8alpha+/TCR(POS) T lymphocytes produce IFN-gamma, TNFalpha and IL-2 when specifically stimulated via the introduced TCR with immobilized HLA-A1/MAGE-A1 complexes or HLA-A1+/MAGE-A1+ melanoma cells. Furthermore, introduction of the CD8alpha gene into TCR(POS) T lymphocytes rendered these T lymphocytes cytotoxic for HLA-A1+/MAGE-A1+ melanoma cells. These results demonstrate that human CD4+ T lymphocytes when genetically grafted with an HLA-A1/MAGE-A1-specific TCR and CD8alpha are induced to kill and produce cytokines upon specific interaction with the relevant melanoma cells. Hence, CD4+ T lymphocytes, in addition to CD8+ T lymphocytes, may be critical effector cells for adoptive immuno-gene therapy to generate a sustained tumor-specific immune response in cancer patients.

Archives de Pédiatrie, 2003

Molecular and chemical …, 1995

Gaucher mice, created by targeted disruption of the glucocerebrosidase gene, are totally deficien... more Gaucher mice, created by targeted disruption of the glucocerebrosidase gene, are totally deficient in glucocerebrosidase and have a rapidly deteriorating clinical course analogous to the most severely aG fected type 2 human patients. An ultrastructural study of tissues from these mice revealed glucocerebroside accumulation in bone marrow, liver, spleen, and brain. This glycolipid had a characteristic elongated tubular structure and was contained in lysosomes, as demonstrated by colocalization with both ingested carbon particles and cathepsin D. In the central nervous system (CNS), glucocerebroside was diffusely stored in microglia cells and in brainstem and spinal cord neurons, but not in neurons of the cerebellum or cerebral cortex. This rostralcaudal pattern of neuronal lipid storage in these Gaucher mice replicates the pattern seen in type 2 human Gaucher patients and clearly 180 Willemsen et al. demonstrates that glycosphingolipid catabolism and/or accumulation varies within different brain regions. Surprisingly, the cellular pathology of tissue from these Gaucher mice was relatively mild, and suggests that the early and rapid demise of both Gaucher mice and severely affected type 2 human neonates may be the result of both a neurotoxic metabolite, such as glucosylsphingosine, and other factors, such as skin water barrier dysfunction secondary to the absence of glucocerebrosidase activity.

Pediatric Research, 1994

The effect of bone marrow transplantation (BMT) on enzyme and glycosaminoglycan levels of various... more The effect of bone marrow transplantation (BMT) on enzyme and glycosaminoglycan levels of various tissues and isolated parenchymal cells of lethally irradiated gusmPs/ gusmP" mice was studied. These mice have an inherited deficiency of the lysosomal enzyme P-glucuronidase with less than 1% of normal enzyme activity present in all tissues and represent a model of human mucopolysaccharidosis type VII. Tissues were evaluated 200 d after BMT and liver parenchymal cells 300 d after BMT. Normal levels of P-glucuronidase activities were present in spleen and peripheral blood leukocytes of gusmPs/gusmPs mice that underwent transplantations. Intermediate activities were found in lung (73%), kidney (4%), liver (lo%), heart (53%), muscle (55%), brain (6%), and liver parenchymal cells (10% of normal controls). A concomitant decrease in activity of the secondarily increased enzyme P-hexosaminidase was observed. BMT also led to a substantial reduction in storage of glycosaminoglycans in lung (130 to 100%), heart (350 to 106%), kidney (439 to 217%), brain (177 to 91%), liver (613 to 125%), and liver parenchymal cells (443 to 161% of normal controls). These findings were supported by elec-

Neuroscience Research Communications, 2000

Patients with fragile X syndrome show mental retardation, behavioural abnormalities, facial anoma... more Patients with fragile X syndrome show mental retardation, behavioural abnormalities, facial anomalies and macroorchidism due to the lack of the FMRl protein (FMRP). Recently a knockout mouse model for fragile X syndrome has been made through homologous recombination of the murine Fmrl gene by an inactivated Fmrl gene construct in embryonic stem cells. The knockout mouse lacks Fmrp and shows symptoms similar to those found in fragile X patients. To answer the question whether reintroduction of Fmrp can restore the normal phenotype a transgenic mouse was generated expressing human FMRP. The FMRI transgene was under control of a CMV promoter to obtain ubiquitous FMRP expression. Transgenic mice were crossed with knockout mice to obtain a transgenic knockout mouse. The rescue mouse did express FM'RI protein, but did not show a reversal of the phenotype, most likely because the level of FMRP expressed from the transgen.e is inadequate or not time or cell specific.

The Histochemical Journal, 1991

We have used quantitative immunoelectronmicroscopy to compare the in situ localization of acid al... more We have used quantitative immunoelectronmicroscopy to compare the in situ localization of acid alpha-glucosidase, lysosomal acid phosphatase, beta-hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid alpha-glucosidase than it is for beta-hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.

Journal of Medical Genetics, 1997

The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG rep... more The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG repeat in the first exon of the FMRl gene. In patients with an expanded repeat the FMR1 promoter is methylated and, consequently, the gene is silenced and no FMR1 protein (FMRP) is produced, thus leading to the clinical phenotype. Here we describe a prenatal diagnosis performed in a female from a fragile X family carrying a large premutation. In chorionic villus DNA of the male fetus the normal maternal CGG allele and a normal pattern on Southern blot analysis were found in combination with the FRAXAC2 and DXS297 allele of the maternal at risk haplotype. A second chorionic villus sampling was performed giving identical results on DNA analysis and, in addition, expression of FMRP was shown by immunohistochemistry. We concluded that the male fetus was not affected with the fragile X syndrome. Subsequent detailed haplotype analysis showed a complex recombination pattern resembling either gene conversion or a double crossover within a 20 kb genomic region.

Neurobiology of Aging, 2000

Journal of Cell Science, 2010

Cilia length and function are dynamically regulated by modulation of intraflagellar transport (IF... more Cilia length and function are dynamically regulated by modulation of intraflagellar transport (IFT). The cilia of C. elegans amphid channel neurons provide an excellent model to study this process, since they use two different kinesins for anterograde transport: kinesin-II and OSM-3 kinesin together in the cilia middle segments, but only OSM-3 in the distal segments. To address whether sensory signaling modulates the coordination of the kinesins, we studied IFT protein motility in gpa-3 mutant animals, since dominant active mutation of this sensory Galpha protein GPA-3QL) affects cilia length. In addition, we examined animals exposed to dauer pheromone, since dauer formation, which involves gpa-3, induces changes in cilia morphology. Live imaging of fluorescently tagged IFT proteins showed that in gpa-3 mutants and in larvae exposed to dauer pheromone, kinesin-II speed is decreased and OSM-3 speed is increased, whereas structural IFT proteins move at an intermediate speed. These results indicate that mutation of gpa-3 and exposure to dauer pheromone partially uncouple the two kinesins. We propose a model in which GPA-3-regulated docking of kinesin-II and/or OSM-3 determines entry of IFT particles into the cilia subdomains, allowing structural and functional plasticity of cilia in response to environmental cues.