Biomedical subjects
Publications and source records attributed to R Mertelsmann.
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Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.
New strategies based on gene transfer technology are employed in cancer therapy. Cytokines are polypeptides involved in immunity and inflammation, and essentially control the magnitude of the immune response. Genetically modified tumor cells releasing various cytokines have been shown to enhance tumor immunogenicity and to induce the regression of preexisting tumors. In some instances, immunological memory has been generated to resist the subsequent challenge with unmodified, parental tumor cells. Cytokine gene transfer into antitumor effector cells, as well as antigen presenting cells, is also being investigated to augment antitumor immune responses.
A 46-year-old female presented with acute myeloid leukemia during complete remission of multiple myeloma after extensive treatment with alkylating agents. Leukemic blasts expressed CD34, platelet esterase and gp IIIa. RT-PCR analyses of peripheral blood cells detected a p190 type BCR-ABL rearrangement and high levels of MDR1. The patient expired during neutropenia shortly after induction chemotherapy. Autopsy revealed persistent blasts in the bone marrow, spleen and liver. 'Secondary' acute myeloid leukemia with megakaryoblastic features and p190-type BCR-ABL rearrangement has not previously been reported. The possibility that the combination of a BCR-ABL rearrangement with overexpression of MDR1 may have contributed to the treatment-refractory course is discussed.
Growth factors have had an increasingly strong impact in breast cancer management in recent years. The main role of growth factors, particularly hematopoietic growth factors, in this setting has been to promote better tolerance of standard-doses, and for the implementation of high-dose chemotherapy in innovative protocols. After a brief overview of growth factor biology, the current clinical guidelines for their use in cancer management is reviewed. Finally, the role growth factors in palliative and curative chemotherapy of breast cancer and methods to reduce tumor cell contamination of peripheral stem cell harvests will be discussed.
The increasing use of high-dose chemotherapy with autologous hematopoietic transplantation for the treatment of solid malignancies has raised concern about the role of tumor cells contaminating the grafts. Minimal residual disease (MRD) in autologous grafts has became a dynamic and intensively studied field in oncology. This review discusses the current status of MRD in breast cancer autografts and presents existing data on detection methodology, clinical relevance, biologic characteristics and purging techniques.
Thrombocytosis is the cause of various complications in myeloproliferative disorders. We present the case of a 54-year-old woman with chronic myelogenous leukaemia who developed large ulcers on both lower legs that were refractory to standard treatment. As concomitant thrombocytosis persisted despite treatment with hydroxyurea, the new megakaryocyte inhibitor anagrelide (Agrelin) was administered and led to normalization of the platelet count within 11 days. The leg ulcers started to heal after 2 weeks and disappeared over a period of 5 months. Our findings argue for a pathogenic role of platelets in the development of leg ulcers in patients with thrombocytosis due to a myeloproliferative disorder.
The possibility of maintaining, manipulating, and expanding human hematopoietic stem cells in ex vivo culture could help to provide patients with autologous and allogeneic stem cell transplants improved in purity and performance and could offer access to gene therapy of the hematopoietic system. Recent advances in the ex vivo culture of immature human hematopoietic progenitor cells and human hematopoietic stem cells have led to experimental evidence for the qualitative and quantitative maintenance and possible numerical expansion of hematopoietic stem cells in ex vivo culture, making ex vivo graft engineering a realistic possibility. This review summarizes recent developments in the field, their regulatory implications and their application in hematopoietic gene therapy.
Supportive care in hematologic malignancies includes a wide range of topics. We have selected the following issues for a review of recently published developments: new insights into the benefits and risks of established hematopoietic growth factors (HGF), such as granulocyte- or granulocyte-macrophage colony-stimulating factor (G-CSF, GM-CSF); the emerging role of newly introduced HGFs such as keratinocyte-growth factor (KGF) and thrombopoietin; the prophylactic and therapeutic use of amifostine, a cytoprotective agent; the role of hematopoietic growth factors and the demethylating agent decitabine in myelodysplastic syndromes (MDS); infectious complications of anticancer therapy; and, recent improvements in and complications of transfusional therapy, including the renewed interest in granulocyte transfusions.
STAT proteins become activated upon tyrosine and serine phosphorylation, are subsequently translocated from the cytosol to the nucleus where they exert DNA-binding activity. Several STAT binding consensus motifs have been identified in the promoters of distinct genes. These consensus elements mediate STAT recruitment and influence the kind of STAT proteins that are bound at a specific promoter site. Recent structure function analyses have revealed conserved amino terminal sequences to be crucial for phosphatase dependent deactivation of the STAT proteins. To date an increasing amount of data is available concerning the on- and off-regulation of STAT activity. Considerable convergence as well as crosstalk has been shown between the JAK-STAT pathway and the MAPK, RAS, PI3K, PKC, and PKA involving pathways. Moreover, the nature of the genes that are regulated by STAT proteins as well as the cell functions that result from STAT activation are of great current interest. Understanding the critical functional role of STAT mediated signalling events as well as their regulation by interfering pathways provides new insights into the mechanisms involved in malignant cell proliferation.
Combined-modality treatment for limited-disease small cell lung cancer using conventional chemotherapy and chest irradiation achieves high response rates, but most patients relapse over a period of 12 to 16 months. To improve current results, we performed a phase II trial including high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) as part of an early intensification strategy after two cycles of induction therapy. Moreover, to reduce the risk of local recurrence, the protocol included surgical resection in stages I to IIIA patients as well as chest irradiation. Between January 1991 and July 1994, 16 consecutive patients (median age, 50 years; age range, 30 to 59 years) were treated in this single-center trial. The patients received two cycles of conventional chemotherapy consisting of etoposide 500 mg/m2, ifosfamide 4 g/m2, cisplatin 50 mg/m2, and epirubicin 50 mg/m2 plus granulocyte colony-stimulating factor 5 microg/kg at a 3-week interval, followed by PBPC collection and subsequent high-dose etoposide 1,500 mg/m2, ifosfamide 12 g/m2, carboplatin 750 mg/m2, and epirubicin 150 mg/m2 with PBPCT. The duration of the entire chemotherapy program was 9 weeks. Six of 10 patients in stages I to IIIA and one of six patients in stage IIIB received neoadjuvant or adjuvant surgery before high-dose chemotherapy, followed by thoracic (50 Gy) and prophylactic (30 Gy) cranial irradiation. Hematopoietic reconstitution after high-dose chemotherapy occurred within 11 days (range, 9 to 17 days) for both neutrophils (>0.5 x 10(9)/L) and platelets (>20 x 10(9)/L). Oral mucositis (World Health Organization grade 2 to 4) was the predominant nonhematologic toxicity, which was observed in 12 of 16 patients. One patient developed neutropenic septicemia with fatal multiorgan failure. At a median follow-up of 44 months (range, 32 to 77 months) after PBPCT, nine patients are alive and well, resulting in a disease-free and overall survival rate of 56.3% +/- 12.4%. The median overall survival has not yet been achieved. None of the patients who had surgery relapsed or died after therapy. All relapses occurred within the first 12 months after PBPCT. Patients in stages I to IIIA (10 patients) had a 70% +/- 14% overall survival rate at 4 years, while patients in stage IIIB (six patients) had a 33% +/- 19% survival rate at 4 years, with a median survival of 17 months posttransplant. These data demonstrate that a multimodality treatment including early high-dose chemotherapy with PBPCT may lead to a prolonged disease-free survival in the majority of patients. A randomized phase III study has now been initiated to prospectively investigate the role of high-dose chemotherapy, surgery, and chest irradiation in the multidisciplinary approach to limited-disease small cell lung cancer.
Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clinical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38lo Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primitive progenitor cells was successful. Equal to fresh CD34+/CD38lo cells, >90% of reselected CD34+/CD38lo cells were in G0/G1. CD34+/CD38lo reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38lo cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38lo population) and recovery (62% of initial LTC-IC number in CD34+/CD38lo cells were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38lo cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.
Loss of physical performance is a universal problem of cancer patients undergoing chemotherapy. We postulated that this impairment can be partially prevented by aerobic exercise. In a randomized study, 33 cancer patients receiving high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (training group, T) performed an exercise program consisting of biking on an ergometer in the supine position after an interval-training pattern for 30 minutes daily during hospitalization. Patients in the control group (C, n = 37) did not train. Maximal physical performance was assessed with a treadmill test by admission and discharge. Physical performance of the two groups was not different on admission. The decrement in performance during hospitalization was 27% greater in the control group than in the training group (P = .05); this resulted in a significantly higher maximal physical performance at discharge in the trained patients (P = .04). Duration of neutropenia (P = .01) and thrombopenia (P = .06), severity of diarrhea (P = .04), severity of pain (P = .01), and duration of hospitalization (P = . 03) were reduced in the training group. We conclude that aerobic exercise can be safely carried out immediately after high-dose chemotherapy and can partially prevent loss of physical performance. Based on the potential significance of the observed outcomes, further studies are warranted to confirm our results.
The conditions that control the migratory status of hematopoietic progenitor cells on extracellular matrix (ECM) and that decide whether a cell migrates or adheres are incompletely understood. We analyzed the migratory behavior of murine hematopoietic progenitor cells factor-dependent-cell-paterson (FDCP)-mix and purified lin-Sca1+ bone marrow cells on ECM. We found that migration on fibronectin (Fn) or laminin (Lam) becomes dependent on beta1-integrins if a surface restraint force is introduced by tilting the ECM-coated culture vessels. Under these conditions, migration specifically occured on Fn and Lam, and was not detected on collagen IV-, hyaluronate-, or bovine serum albumin- coated surfaces. Migration depended on the continuous presence of hematopoietic cytokines interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), or stem cell factor (SCF), whereas other cytokines, such as IL-8, macrophage inflammatory protein-1alpha, macrophage-chemotactic and activating factor, and erythropoietin resulted in very little or no migratory response. IL-3 induced migration was synergistically enhanced by other CSFs, but was completely inhibited by addition of transforming growth factor-beta1. In contrast to firm local adhesion of previously cytokine depleted progenitors that was rapidly inducible within 1 hour after exposure to cytokines, preincubation on Fn matrix for 4 to 6 hours was required before cytokines could induce migration. A sudden increase of cytokine concentration reversibly inhibited migration and induced a fully adhesive state; this effect could be prolonged by consecutive stimulation with heterologous cytokines. Whereas cytokines activated resting progenitor cells to migrate on ECM, cell migration speed was regulated by Fn concentration. These results indicate that beta1-integrin-mediated progenitor cell adhesion and migration are differentially regulated by external stimuli and suggest that this regulation corresponds to different activation states of beta1-integrins in hematopoietic progenitor cells.
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Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Recombinant G-CSF (rG-CSF) is routinely used in the prevention of chemotherapy-induced neutropenia and in the setting of bone marrow transplantation. Chronic idiopathic and congenital neutropenic disorders also show improvement after rG-CSF injections. Applications of either rG-CSF or G-CSF gene transfected cells into mice give rise to leukocytosis, which can be measured easily. This makes G-CSF a versatile tool for studying systemic effects of therapeutic proteins delivered by genetically modified cells in vivo. Although the biological activity of G-CSF is not species-specific, studies on long-term expression would require the use of species-identical proteins in order to avoid host immune reactions against the foreign gene product. Because of the physiological and immunological similarity of pigs and human, the pig has become an important large-animal model for biomedical research. We have therefore cloned porcine G-CSF cDNA from RNA isolated from pig PBLs. Pig G-CSF is a 195-amino-acid polypeptide that shares a high degree of homology to human (78%), murine (71%) as well as rat (68%) G-CSF. In contrast to human and murine, but not to rat G-CSF, a different ATG translation start codon is used, resulting in a shorter, but still functional signal sequence.
Interfering with the endotoxin-mediated cytokine cascade is thought to be a promising approach to prevent septic complications in gram-negative infections. The synthetic lipid A analog SDZ MRL 953 has been shown to be protective against endotoxic shock and bacterial infection in preclinical in vivo models. As part of a trial of unspecific immunostimulation in cancer patients, we conducted a double-blind, randomized, vehicle-controlled phase I trial of SDZ MRL 953 to investigate, first, its biologic effects and safety of administration in humans and, second, its influence on reactions to a subsequent challenge of endotoxin (Salmonella abortus equi). Twenty patients were treated intravenously with escalating doses of SDZ MRL 953 or vehicle control, followed by an intravenous application of endotoxin (2 ng/kg of body weight [BW]). Administration of SDZ MRL 953 was safe and well-tolerated. SDZ MRL 953 itself increased granulocyte counts and serum levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), but not of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), IL-1beta, and IL-8. Compared with vehicle control, pretreatment with SDZ MRL 953 markedly reduced the release of TNF-alpha, IL-1beta, IL-8, IL-6, and G-CSF, but augmented the increase in granulocyte counts to endotoxin. Induction of tolerance to the endotoxin-mediated cascade of proinflammatory cytokines by pretreatment with SDZ MRL 953 in patients at risk may help to prevent complications of gram-negative sepsis.
Cleidocranial dysplasia (CCD) is an autosomal-dominant condition characterized by hypoplasia/aplasia of clavicles, patent fontanelles, supernumerary teeth, short stature, and other changes in skeletal patterning and growth. In some families, the phenotype segregates with deletions resulting in heterozygous loss of CBFA1, a member of the runt family of transcription factors. In other families, insertion, deletion, and missense mutations lead to translational stop codons in the DNA binding domain or in the C-terminal transactivating region. In-frame expansion of a polyalanine stretch segregates in an affected family with brachydactyly and minor clinical findings of CCD. We conclude that CBFA1 mutations cause CCD and that heterozygous loss of function is sufficient to produce the disorder.