Toeval en onvermijdelijkheid

Rede, in verkorte vorm, uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht Gentherapie van Hematopoietische Cellen aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam, op 11 april 200

Erythropoietine : enkele aspecten van de humorale regulatie van de erythropoiese

De regulatie van de erythropoiese verloopt in belangrijke mate via een humorale regulator. Deze wordt erythropoietine genoemd, en heeft een specifieke stimulerende werking op de erythropoiese, waarschiinliik in hoofdzaak door de inductie van erythroide differentiatie in een niet geîdentificeerde primitieve hemopoietische cel. Bii de productie van erythropoietine speelt de nier een niet volledig opgehelderde rol. Het hormoon gedraagt zich in serum en urine als een glycoproteïne waarvan de relatieve molecuulmassa ongeveer 30.000 bedraagt. De veronderstelling dat een humorale factor de erythropoiese stimuleert werd in 1906 voor de eerste maal geformuleerd. Enkele jaren na de definiëring van een hormoon door Bayliss en Sterling (1902) concludeerden Carnet en Deflandre (1906a, b) uit een reeks experimenten tot het bestaan van een dergelijke factor, die zij hemopoietine noemden. Ze bestudeerden het effect van intraveneuze toediening van serum van gebloede konijnen bii normale konijnen: " ... Dans un de nos cos, par exemple, un lapin neuf, dont Ie song comprenait, d 1 une façon assez constante, 5 millions et demi d 1 hématies par millimètre cube, après avoir reçu, en injection intraveneuse, 9 cm3 de sérum (recueil I i, chez un autre lap in, 20 heures après une saignée de 30 cm3)r eut une hyperglobulie telle que le nombre des hématies atteignait 8 millions Ie lendema in, plus de 9 millions Ie surlendemain, près de 12 millions Ie trais- Ieme jour, . . . Dit verbazend grote effect kan zeker niet zijn veroorzaakt door erythropoietine, terwijl hun bevinding, dat de serumfactor bij verwarming tot 56°C werd geïnactiveerd, niet tot de thans bekende eigenschappen van erythropoietine behoort. Hun derde conclusie, dat de stimulerende activiteit van het serum 20 uur na de bloeding maximaal is, stemt echter wel ongeveer overeen met latere onderzoekingen

Immune modulation in gene therapy studies

Summary Host immune responses play a major role in clearance of viral infections from the body, and may limit long-term expression and clinical efficacy of viral vectors. Methods to prevent these immune responses may also increase the risk for infections, recombination with wild type virus and affect biodistribution, persistence, shedding and transmission. The study described in this report was initiated to assess possible environmental risks associated with the use of immune modulation in combination with gene therapy and set up as a literature study, by performing PubMed searches for certain keywords, by interviewing experts and by attending selected meetings. Lack of availability of clinical data combining gene therapy and immune modulation and limited animal data warranted additional exploration of relevant non-gene therapy studies from closely related fields such as stem cell and organ transplantation, and vaccination studies with live attenuated vaccines. ...... Finally, we propose the use of a checklist to assess current environmental risks in the use of immune modulation during gene therapy. This report is expected to provide guidance to risk assessors and regulatory officers as well as to applicants for a gene therapy licence

Coexpression of Kit and the receptors for erythropoietin, interleukin 6 and GM-CSF on hemopoietic cells

The detection of functional growth factor (GF) receptors on subpopulations of hemopoietic cells may provide a further dissection of immature cell subsets. Since little information is available about coexpression of different GF receptors at the level of single hemopoietic cells, we studied the feasibility of simultaneous cell staining with a combination of biotin- and digoxigenin-labeled GFs for flow cytometric detection of functional receptors. Using this methodology, coexpression of Kit and receptors for erythropoietin (EPO), interleukin 6 (IL-6), and GM-CSF on hemopoietic cells was studied by triple-staining of rhesus monkey bone marrow (BM) cells with labeled GFs and antibodies against other cell surface markers. Most of the immature, CD34+2 cells were Kit+ but did not display detectable levels of EPO-receptors (EPO-Rs) or GM-CSF-R. Approximately 60% of these CD34+2/Kit+ cells coexpressed the IL-6-R, demonstrating that immature cells are heterogeneous with respect to IL-6-R expression. Maturation of monomyeloid progenitors, as demonstrated by decreasing CD34 and increasing CD11b expression, is accompanied by a decline of Kit and an increase in GM-CSF-R expression in such a way that Kit+/GM-CSF-R+ cells are hardly detectable. IL-6-R expression is maintained or even increased during monomyeloid differentiation. IL-6-R and GM-CSF-R were not identified on most CD71+2 cells, which indicated that these receptors are probably not expressed during erythroid differentiation. Together with previous results, our data show that both Kit and CD71 are upregulated with erythroid commitment of immature progenitors. Upon further differentiation, Kit+/EPO-R-cells lose CD34 and acquire EPO-R. Maturing erythroid cells eventually lose CD71 and Kit expression but retain the EPO-R. In conclusion, this approach enables further characterization of the specificity of GFs for different bone marrow subpopulations. Apart from insight into the differentiation stages on which individual GFs may act, information about receptor coexpression may be used to identify individual cells that can respond to multiple GFs, and allows for further characterization of the regulation of lineage-specific differentiation

The efficacy of recombinant thrombopoietin in murine and nonhuman primate models for radiation-induced myelosuppression and stem cell transplantation

Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys for studying thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the mid-lethal dose of 5 Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow cells, and potentiated the response to growth factors such as GM-CSF and G-CSF. In contrast to the results in the 5 Gy TBI model, TPO was ineffective following transplantation of limited numbers of autologous bone marrow or highly purified stem cells in monkeys conditioned with 8 Gy TBI. In the 5 Gy model, a single dose of TPO augmented by GM-CSF 24 h after TBI was effective in preventing thrombocytopenia. The strong erythropoietic stimulation may result in iron depletion, and TPO treatment should be accompanied by monitoring of iron status. This preclinical evaluation thus identified TPO as a potential major therapeutic agent for counteracting radiation-induced pancytopenia and demonstrated pronounced stimulatory effects on the reconstitution of immature CD34+ hemopoietic cells with multilineage potential. The latter observation explains the potentiation of the hematopoietic responses to G-CSF and GM-CSF when administered concomitantly. It also predicts the effective use of TPO to accelerate reconstitution of immature hematopoietic cells as well as possible synergistic effects in vivo with various other growth factors acting on immature stem cells and their direct lineage-committed progeny. The finding that a single dose of TPO might be sufficient for a clinically significant response emphasizes its potency and is of practical relevance. The heterogeneity of the TPO response encountered in the various models used for evaluation points to multiple mechanisms operating on the TPO response and heterogeneity of its target cells. Mechanistic mouse studies made apparent that the response of multilineage cells shortly after TBI to a single administration of TPO is quantitatively more important for optimal efficacy than the lineage-restricted response obtained at later intervals after TBI and emphasized the importance of a relatively high dose of TPO to overcome initial c-mpl-mediated clearance. Further elucidation of mechanisms determining efficacy might very well result in a further improvement, e.g., following transplantation of limited numbers of stem cells. Adverse effects of TPO administration to myelosuppressed or stem cell transplanted experimental animals were not observed

In Vivo Expansion of Co-Transplanted T Cells Impacts on Tumor Re-Initiating Activity of Human Acute Myeloid Leukemia in NSG Mice

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation

Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.This publication reports that stem cell gene therapy using a codon-optimized gene encoding acid α-glucosidase (GAA) cures the mouse model of Pompe disease, a lysosomal storage disorder

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology