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

Regulation of haemopoietic stem‐cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone‐marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU‐s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU‐e and CFU‐G/M) kinetics after TBI and +/+ bone‐marrow transplantation in Slj/+ and +/+ mice. the Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU‐E and CFU‐G/M as colonies from +/+ spleens, while their CFU‐s content was increased. On day 10 post‐transplantation, the macroscopic ‘missing’ colonies could be detected at the microscopic level. These small colonies contained far fewer CFU‐s than the macroscopic detectable colonies. Analysis of CFU‐s proliferation‐inducing activities in control and post‐LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem‐cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal ‘niches’ for colony formation. However, 35% of these niches is defective in its proliferative support. Copyrigh

Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of Cl²MDP liposomes

Transplantation of normal and malignant human hematopoietic cells into severe combined immunodeficient (SCID) mice allows for evaluation of long-term growth abilities of these cells and provides a preclinical model for therapeutic interventions. However, large numbers of cells are required for successful engraftment in preirradiated mice due to residual graft resistance, that may be mediated by cells from the mononuclear phagocytic system. Intravenous (i.v.) injection of liposomes containing dichloromethylene diphosphonate (Cl2MDP) may eliminate mouse macrophages in spleen and liver. In this study outgrowth of acute myeloid leukemia (AML) cells and umbilical cord blood (UCB) cells in SCID mice conditioned with a single i.v. injection of Cl2MDP liposomes in addition to sublethal total body irradiation (TBI) was compared to outgrowth of these cells in SCID mice that had received TBI alone. A two- to 10-fold increase in outgrowth of AML cells was observed in four cases of AML. Administration of 107 UCB cells reproducibly engrafted SCID mice that had been conditioned with Cl2MDP liposomes and TBI, whereas human cells were not detected in mice conditioned with TBI alone. As few as 2 x 104 purified CD34+ UCB cells engrafted in all mice treated with Cl2MDP liposomes. In SCID mice treated with macrophage depletion unexpected graft failures were not observed. Histological examination of the spleen showed that TBI and Cl2MDP liposomes i.v. resulted in a transient elimination of all macrophage subsets in the spleen, whereas TBI had a minor effect. Cl2MDP liposomes were easy to use and their application was not associated with appreciable side-effects. Cl2MDP liposome pretreatment in combination with TBI allows for reproducible outgrowth of high numbers of human hematopoietic cells in SCID mice

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

Benefits of a dermocosmetic formulation with vitamins B3 and a B6 derivative combined with zinc-PCA for mild inflammatory acne and acne-prone skin

Acne is a chronic inflammatory disorder of the pilosebaceous follicles that affects 80% of the population. As topical agents for acneic skin treatment are often irritants, dermocosmetics, may improve therapy. Thus, we developed cosmetic formulations with nicotinamide (vitamin B3), pyridoxine tris-hexyldecanoate (a vitamin B6 derivative) and zinc- pyrrolidone carboxylic acid (PCA) in association, and evaluated their clinical efficacy, skin compatibility, and sensory properties. The formulation (vehicle) added with vitamin B3, the vitamin B6 derivative and zinc-PCA in combination was applied twice daily for six weeks on the forehead, malar and chin skin regions of sixteen subjects. Before (pre-treatment) and after treatment, these regions were evaluated using biophysical and skin imaging techniques. Inflammatory acne lesions were reduced by 60% after application of the complete formulation. Porphyrine reduction was shown in the majority of volunteers. The results shown an improvement of inflammatory acne lesions based on porphyrine reduction, lesion counts, skin compatibility and comedogenicity testing. The skin barrier function was not impaired by the experimental formulation, which demonstrates its efficacy in acne treatment without undesirable effects. The combination of Zn-PCA and vitamins B3 and B6 vehiculated in an adequate topical formulation can be considered as a safe and effective alternative treatment for mild inflammatory acneic skin