Mesenchymal stem cells in connective tissue engineering and regenerative medicine: applications in cartilage repair and osteoarthritis therapy.

Defects of load-bearing connective tissues such as articular cartilage, often result from trauma, degenerative or age-related disease. Osteoarthritis (OA) presents a major clinical challenge to clinicians due to the limited inherent repair capacity of articular cartilage. Articular cartilage defects are increasingly common among the elderly population causing pain, reduced joint function and significant disability among affected patients. The poor capacity for self-repair of chondral defects has resulted in the development of a large variety of treatment approaches including Autologous Chondrocyte Transplantation (ACT), microfracture and mosaicplasty methods. In ACT, a cartilage biopsy is taken from the patient and articular chondrocytes are isolated. The cells are then expanded after several passages in vitro and used to fill the cartilage defect. Since its introduction, ACT has become a widely applied surgical method with good to excellent clinical outcomes. More recently, classical ACT has been combined with tissue engineering and implantable scaffolds for improved results. However, there are still major problems associated with the ACT technique which relate mainly to chondrocyte de-differentiation during the expansion phase in monolayer culture and the poor integration of the implants into the surrounding cartilage tissue. Novel approaches using mesenchymal stem cells (MSCs) as an alternative cell source to patient derived chondrocytes are currently on trial. MSCs have shown significant potential for chondrogenesis in animal models. This review article discusses the potential of MSCs in tissue engineering and regenerative medicine and highlights their potential for cartilage repair and cell-based therapies for osteoarthritis and a range of related osteoarticular disorders.

Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation.

Tissue engineering of bone grafts with osteogenic progenitor cells such as adult mesenchymal stem cells (MSC) represents a promising strategy for the treatment of large bone defects. The aim of this experimental study was to evaluate the osteogenic potential of primary osteoblasts on MSCs in co-culture at different ratios. The co-cultures were treated with or without a specific osteogenic induction medium in monolayer and high density cultures. In monolayer co-cultures, MSCs and osteoblasts actively searched for cell-cell contact leading to cell proliferation and only in treated monolayer co-cultures osteogenesis was observed. Ultrastructural evaluation of high density co-cultures using electron microscopy demonstrated osteogenesis with no significant difference between treated or untreated co-cultures. Immunoblotting confirmed expression of collagen type I, beta1-Integrin, the osteogenic-specific transcription factor Cbfa-1 and induction of the MAPKinase pathway (Shc, Erk1/2) in both treated and untreated co-cultures. Although treatment with the induction medium enhanced osteogenesis in the co-cultures compared to untreated co-cultures, the quality of osteogenesis was proportional to the quantity of osteoblasts in the co-cultures. Fifty percent osteoblasts in the co-cultures markedly increased osteogenesis; even the presence of ten percent osteoblasts in the co-culture strongly promoted osteogenesis. This data leads us to conclude that co-culture of MSCs with osteoblasts combined with the three-dimensional environment of the high density culture strongly promotes osteogenesis and stabilizes the osteogenic potential of MSCs. This approach may prove to be of practical benefit in future tissue engineering and regenerative medicine research.

Mesenchymal stem cells as a potential pool for cartilage tissue engineering.

Osteoarthritis (OA) resulting from trauma, degenerative or age-related disease presents a major clinical challenge due to the limited repair capacity of articular cartilage. This poor self-repair capacity of osteochondral defects has resulted in the development of a wide variety of new treatment approaches. Although the use of chondrocytes in applications of cartilage tissue engineering is still prevalent, concerns associated with donor-site morbidity, cell de-differentiation and the limited lifespan of these cells have brought the use of mesenchymal stem cells (MSCs) to the forefront of such applications. Therefore, in the last two decades MSCs have come into the focus of connective tissue engineering and regenerative medicine and have become increasingly sought after as an alternative cell source for improving well-established methods of osteochondrotic cartilage defect repair such as the Autologous Chondrocyte Transplantation method, but are also being tested as an ideal cell source in combination with newly developed implantable scaffolds or as a target/carrier cell in other new concepts of regenerative medicine. However, up to now, although in animal models MSCs have already shown significant potential for cartilage repair and novel approaches using MSCs as an alternative cell source to patient-derived chondrocytes are being tested, much more research is needed before feasible clinical application of MSCs becomes reality.

Diverse roles of integrin receptors in articular cartilage.

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

[Interaction between human chondrocytes and extracellular matrix in vitro: a contribution to autologous chondrocyte transplantation]. / Interaktion zwischen humanen Chondrozyten und extrazellulärer matrix in vitro: ein Beitrag zur autologen Chondrozytentransplantation.

BACKGROUND: Autologous chondrocyte transplantation (ACT) has had reasonable success for repairing small articular cartilage defects. A limiting factor for ACT is, however, the in vitro cultivation of chondrocytes because it leads to dedifferentiation. Therefore, the goal of this work was to optimize the monolayer culture of chondrocytes in vitro. MATERIAL AND METHOD: Human articular chondrocytes were plated on either collagen type II or untreated surfaces. The cells were evaluated morphologically and with immunoblotting. RESULTS: On collagen type II surfaces, a stable chondrogenic phenotype, expression of beta1-integrin, and a significant activation of phosphorylated intracellular proteins and the adaptor protein Shc could be observed up to day 20 in culture. Treatment with beta1 integrin antibody led to a loss of cell adhesion (82%). The results indicate that on collagen type II, beta1-integrin receptors are activated. Through the activation of Shc, these stimulate the Ras-MAPK pathway, which stabilizes the chondrogenic phenotype. CONCLUSION: Our results provide a practical and low-cost solution for improved long-term chondrocyte cultivation, thus providing a new perspective for using ACT on larger or arthrotic cartilage defects.

Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study.

Musculoskeletal diseases with osteochondrotic articular cartilage defects, such as osteoarthritis, are an increasing problem for humans and companion animals which necessitates the development of novel and improved therapeutic strategies. Canine mesenchymal stem cells (cMSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no comprehensive analysis has been undertaken to characterize the ultrastructure of in vitro differentiated cMSCs. The main goal of this paper was to focus on cMSCs and to analyse their differentiation capacity. To achieve this aim, bone marrow cMSCs from three canine patients were isolated, expanded in monolayer culture and characterized with respect to their ability for osteogenic, adipogenic and chondrogenic differentiation capacities. cMSCs showed proliferative potential and were capable of osteogenic, adipogenic and chondrogenic differentiation. cMSCs treated with the osteogenic induction medium differentiated into osteoblasts, produced typical bone matrix components, beta1-integrins and upregulated the osteogenic specific transcription factor Cbfa-1. cMSCs treated with the adipogenic induction medium showed typical adipocyte morphology, produced adiponectin, collagen type I and beta1-integrins, and upregulated the adipogenic specific transcription factor PPAR-gamma. cMSCs treated with the chondrogenic induction medium exhibited a round to oval shape, produced a cartilage-specific extracellular matrix, beta1-integrins and upregulated the chondrogenic specific transcription factor Sox9. These results demonstrate, at the biochemical, morphological and ultrastructural levels, the multipotency of cMSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

Quasilocalization of gravity on a brane by resonant modes.

We examine the behavior of gravity in brane theories with extra dimensions in a nonfactorizable background geometry. We find that for metrics which are asymptotically flat far from the brane there is a resonant graviton mode at zero energy. The presence of this resonance ensures quasilocalization of gravity, whereby at intermediate scales the gravitational laws on the brane are approximately four dimensional. However, for scales larger than the lifetime of the graviton resonance the five-dimensional laws of gravity will be reproduced due to the decay of the four-dimensional graviton. We also give a simple classification of effective gravity theories for general background geometries.

Effect of etoposide on the pharmacokinetics of methotrexate in vivo.

The effect of etoposide on the pharmakokinetics of methotrexate (MTX) was examined in vivo. High-dose (5g/m2/24 h) MTX therapy was combined with two etoposide (100mg/m2/ 1 h) infusions as a part of the medulloblastoma protocol developed in our department. Vepesid therapy was administered in two different schedules. The first group of patients received etoposide immediately before and at the end (24 h) of MTX treatment. The second group was treated with etoposide at 24 and at 48 h after starting MTX infusion. In this latter group both treatment-related grade III and grade IV toxicity developed more frequently than in the first group (58.6 versus 29.2%, for grade 3 toxicity p=0.019, for grade 4 toxic signs p=0.040, respectively). We observed that after the second dose of etoposide given at 48 h (second group) both total and unbound serum MTX levels (determined by high-performance liquid chromatography) were elevated by 53-109 and 26-65%, respectively, by the third hour after completion of Vepesid infusion. This effect was detectable for 6 h. All the liver and kidney functions of the patients were within the normal range. These results suggest the possibility of partial recirculation of extra/intracellular MTX into the blood after etoposide administration. Based on these results, the therapeutic protocol has been modified, and Vepesid is given prior to and at the end (24 h) of high-dose MTX treatment. Under these conditions only a slight decrease of MTX elimination has been detected between 25 and 28 h. These results emphasize the role of possible schedule-dependent interactions of cytostatic drugs.

Effect of L-arginine on adrenal and renal blood flows during hemorrhage in cats.

Our earlier studies have shown development of endothelial dysfunction in the feline renal artery during hemorrhagic hypotension. Because L-arginine (L-Arg), the precursor of nitric oxide (NO), reportedly improves endothelial function in several pathophysiological states including hypotension, we investigated its possible beneficial effect on the adrenal and renal circulations during hemorrhagic hypotension in anesthetized, ventilated cats. Hypotension (mean arterial pressure 50 mm Hg) significantly increased vascular resistance and decreased blood flow (radiolabeled microspheres) in both adrenal and renal cortices. L-Arg (30 mg/kg bolus, 10 mg/kg/min infusion, i.v.) had no significant hemodynamic effects in normotension but prevented the increase of the vascular resistance and improved blood flow in the adrenal cortex during hypotension. In the kidney, L-Arg also prevented hemorrhage-induced vasoconstriction, although its effect on blood flow did not reach significance. The NO synthase inhibitor N(G)-nitro-L-arginine (30 mg/kg bolus, 1 mg/kg/min infusion, i.v.) increased adrenal and renal vascular resistances to a similar extent as that observed during hypotension. It thus seems that an L-Arg-reversible dysfunction of the endothelial NO-synthesizing pathway contributes to hemorrhage-induced adrenal and renal vasoconstriction.

Recombinant human erythropoietin in the prevention of chemotherapy-induced anaemia in children with malignant solid tumours.

This prospective, randomised pilot study was designed to evaluate safety, feasibility and efficacy of recombinant human erythropoietin (rhEPO) in the prevention and treatment of chemotherapy-induced anaemia in children with solid tumours. 20 children (age 4-18 years) undergoing cyclic combination chemotherapy were randomised either to a control group or to receive rhEPO at a dose of 150 U/kg/dose subcutaneously three times/week for a minimum of 12 weeks or three chemotherapy cycles. Of 15 evaluable patients, 8 were randomised to the rhEPO group and 7 to the control group. RhEPO-treated patients showed an increase in the haematocrit over the first 8 weeks of therapy, with a significantly higher mean haematocrit at week 8 (33.2 +/- 2.1% versus 39.3 +/- 4.2% in the control and rhEPO groups, respectively, P < 0.05). Similarly, significantly higher haemoglobin concentrations could be demonstrated in the rhEPO group by week 8 (11.06 +/- 1.35 g/dl versus 13.11 +/- 1.13 g/dl in the control and rhEPO groups, respectively, P < 0.05), with higher precycle haemoglobin before chemotherapy cycles 3 and 4 and higher midcycle haemoglobin between cycles 3 and 4. There was a trend towards a reduction of transfusion requirements during the 3rd month of therapy in rhEPO patients. The results of this pilot study indicate a significant benefit of rhEPO in children treated with intensive combination chemotherapy regimens. Further studies should target issues such as appropriate dosing, timing and duration of rhEPO therapy in children with cancer.

[In vivo effect of etoposide on the pharmacokinetics of methotrexate]. / Az etoposid hatása a metotrexát farmakokinetikájára in vivo.

High dose (5 g/m2/24 h) methotrexate therapy was combined two times with etoposide (100 mg/m2/1h) infusions as a part of the Medulloblastoma protocol developed in our Department Vepesid therapy was administered in two different schedules. The first group of the patients have received etoposide immediately before and at the end (24th h) of methotrexate treatment. The second group was treated with etoposide at 24 h and at 48 hour after starting methotrexate infusion. In this latter group treatment related grade 3-4 toxicity developed more frequently than in the first group (58.6% vs 33.3%). The authors observed that after the second dose of etoposide given at 48 h (second group) both total and unbound serum methotrexate levels (determined by high performance liquid chromatography) were elevated by 53.14-109.19%, and 25.86-64.95%, respectively by the third hour after completion of Vepesid infusion. This effect was detectable for 6 hours. All the liver and kidney functions of the patients were in the normal range. These results suggest the possibility of partial recirculation of extra/intracellular methotrexate into the blood after etoposide administration. Based on these results the therapeutic protocol has been modified and Vepesid is given prior to and at the end (24 h) of high dose methotrexate treatment. Under these conditions only a slight decrease of methotrexate elimination has been detected between the 25-28th h. These results emphasize the role of possible schedule dependent interactions of cytostatic drugs.

Diffuse plasmacytosis in a child with brainstem glioma following multiagent chemotherapy and intensive growth factor support.

The use of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in order to abrogate chemotherapy-induced neutropenia has become a routine part of many cancer treatment regimes. However, there are still very few data available about possible complications related to repeated or prolonged use of these agents in patients with malignant solid tumors. The authors report a child with brainstem glioma who received repeated cycles of multiagent chemotherapy with G- or GM-CSF support. During this period of 10 months, no clinical side effects were observed that could have been attributed to growth factor administration. However, postmortem histological examination revealed the presence of diffuse plasmacytosis, a rare hematological disorder in childhood. Undifferentiated plasma cells of nonmonoclonal origin could be demonstrated infiltrating bone marrow, lungs, and lymph nodes of the patient. Based on previously published in vitro and in vivo evidence on the interleukin-6 (IL-6)-mediated stimulatory effect of G- and GM-CSF on myeloma cell proliferation, the authors suggest a possible link between extensive growth factor support and the development of plasmacytosis in this patient.

Administration of Ethyol (amifostine) to a child with medulloblastoma to ameliorate hematological toxicity of high dose carboplatin.

The first report on the administration of the chemoprotective agent Ethyol (amifostine) in conjunction with high dose carboplatin to a patient in the pediatric/adolescent age group is presented. A 17 year old teenager with recurrent cerebellar medulloblastoma received a total of five courses of high dose carboplatin 2 x 600 mg/m2 (1200 mg/m2 total) in each cycle. A complete response has been observed following the third treatment cycle. However, cumulative grade IV hematological toxicity developed following each of the first four treatments. Therefore, the fifth treatment was administered in conjunction with amifostine, at a dose of 2 x 740 mg/m2. Time to complete hematological recovery (Hb > 100 g/l, granulocytes > 2.0 G/l, platelets > 100 G/l) was 52, 58, 72, 78 and 50 days, respectively, following treatments nos 1, 2,,3, 4 and 5. The duration of grade III-IV neutropenia (< 1.0 G/l) was 3, 7, 8, 10 and 5 days, respectively. The duration of grade II-IV thrombocytopenia (platelets < 75 G/l) was 10, 25, 35, 40 and 32 days, respectively. Grade IV thrombocytopenia (platelets < 25 G/l) lasted for 5, 10, 12, 18 and 3 days, respectively, after each consecutive treatment. The total number of platelet transfusions was 1, 2, 2, 3 and 1, with the transfusion of 6, 9, 11, 11 and 5 units of platelets. The administration of amifostine has not been accompanied by any serious side effect. A short decrease in body temperature and a transient drop of blood pressure have been observed. Although hematological toxicity of high dose carboplatin has not been eliminated by amifostine, we conclude that significant protection was achieved in this situation of progressive bone marrow exhaustion.

Effect of superoxide dismutase on hemorrhagic hypotension and retransfusion-evoked middle cerebral artery endothelial dysfunction.

UNLABELLED: Middle cerebral artery rings (MCA) were prepared from control and hemorrhagic hypotension and retransfusion-subjected (HHR) cats, with or without superoxide dismutase (SOD) treatment. Two-mm-long MCA segments were suspended in organ chambers containing Krebs-Henseleit solution (37 degrees C, gassed with 95% O2-5% CO2) for isometric force measurements. HHR was produced by bleeding to 90, 70, and 50 mmHg MAP and maintained for 15 min at each level, followed by retransfusion. HHR resulted in a marked attenuation of the acetylcholine- and ATP-induced endothelium-dependent relaxations of the MCA in vitro. Relaxations induced by the nitric oxide (NO) donor SIN-1 remained unaltered. In vitro treatment of the vessels with SOD (150 U/ml), facilitated the acetylcholine-induced relaxations both in the control arteries and in the vessels after HHR. In the vessel rings from cats that received in vivo SOD (10 mg/kg initial bolus, followed by 0.1-mg/kg/min infusion) during HHR, cholinergic relaxations were more pronounced than in the HHR untreated cats. The ATP-induced relaxations, however, remained attenuated after SOD treatment, except for the highest dose (10(-5) M) that was applied. CONCLUSION: Superoxide release attenuates the endothelium-dependent relaxation by acetylcholine both in control arteries and after HHR in vitro. The protective effect of in vivo SOD treatment on cerebrovascular endothelium-dependent reactivity in cats suggests that superoxide free radicals contribute to the development of the endothelium dysfunction in MCA rings after HHR.

[Pharmacokinetic study of dibromodulcitol in children with brain tumors]. / A dibrómdulcit farmakokinetikai vizsgálata agytumoros gyermekekben.

Systemic pharmacokinetics of high-dose (500 mg/m2), orally administered dibromodulcitol (Elobromol) were studied in 16 chemotherapeutic courses administered to 5 patients. Cerebrospinal fluid dibromodulcitol levels were also analysed in two patients. Bromoepoxydulcitol, dianhydrodulcitol are cytotoxic, whereas bromoanhydrodulcitol, andhydroepoxydulcitol are inactive metabolites detectable during the biotransformation of dibromodulcitol. The HPLC method, developed by our team, is suitable for the determination of both dibromodulcitol and its main metabolites (dianhydrodulcitol and bromoanhydrodulcitol). Our publication is the first in the literature to describe the pharmacokinetic properties of these three hexite-derivatives in pediatric patients. With the exception of one patient, concentration-time curves were analysed by the one-compartment model. From the 30th minute following administration, dibromodulcitol was detectable in all plasma samples for at least 12 hours, its concentration however was usually undetectable by the 24th hour. Though highly variable in value, dianhydrodulcitol concentrations were detectable during all but one therapeutic courses. The following peak concentrations were observed: dibromodulcitol: 3.46-30.63 microM; dianhydroldulcitol: 1.70-6.17 microM; bromoanhydrodulcitol: 0-5.63 microM. The correlation of area under the curve for bromoanhydrodulcitol and dibromodulcitol was exponential up to 200 microMxh with no additional increase detectable above this limit; the distribution of dianhydrodulcitol values were described by a maximum-curve. The possibility of enterohepatic recirculation could not be excluded for any of the compounds studied. Each of the three hexitol derivatives were detectable in the cerebrospinal fluid even if the concentration of the individual metabolite remained undetectable in plasma. The cerebrospinal fluid concentrations of dibromodulcitol were almost constant in the period from 2.5 to 8 hours following administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetic studies on Elobromol in children with brain tumors.

Systemic pharmacokinetics of high dose (500 mg/m2), orally administered Elobromol (dibromodulcitol, DBD) were studied in 16 chemotherapeutic courses administered to five patients. Cerebrospinal fluid (CSF) DBD levels were also analyzed in two patients. Bromoepoxydulcitol (BED), dianhydrodulcitol (DAD) are cytotoxic, whereas bromoanhydrodulcitol (BAD) and anhydroepoxydulcitol (AED) are inactive metabolites detectable during the biotransformation of DBD. The HPLC method, developed by our team, is suitable for the determination of both DBD and its main metabolites (DAD and BAD). Our publication is the first in the literature to describe the pharmacokinetic properties of these three hexitol derivatives in pediatric patients. With the exception of one patient, concentration time curves were analyzed by the one-compartment model. From 30 min following administration, DBD was detectable in all plasma samples for at least 12 h; its concentration, however, was usually undetectable by 24 h. Though highly variable in value, DAD concentrations were detectable during all but one of the therapeutic courses. The following peak concentrations were observed: DBD = 3.46-30.63 microM, DAD = 1.70-6.17 microM and BAD = 0-5.63 microM. The correlation of AUCBAD and AUCDBD values were exponential up to 200 microM h with no additional increase detectable above this limit: the distribution of AUCBAD and AUCDBD was described by a maximum curve. The possibility of enterohepatic recirculation could not be excluded for any of the compounds studied. Each of the three hexitol derivatives was detectable in CSF even if the concentration of the individual metabolite remained undetectable in plasma. DBD CSF concentrations were almost constant in the period from 2.5 to 8 h following administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Human recombinant granulocyte-macrophage colony-stimulating factor in pediatric oncology practice.

This paper reports preliminary experiences with human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) in children with malignant diseases administered for three indications: (1) chemotherapy-induced neutropenia and sepsis, (2) prolonged neutropenia decreasing dose intensity, and (3) prevention of neutropenia after sublethal doses of chemotherapy. It was concluded that in the daily dose of 5 micrograms/kg subcutaneously, GM-CSF is capable of reducing the duration of chemotherapy-induced neutropenia and may be an effective tool in maintaining dose intensity and achieving dose escalation.

[Poly-electrolyte fractionated porcine factor VIII in the treatment of hemophilia A]. / Polielektrolit-frakcionált porcin VIII. faktor egy esetben való alkalmazásáról gátlótestes haemophiliában.

In approximately 10 to 15 percent of congenital hemophilia A patients circulating antibodies to factor VIII appear in the blood that poses a serious problem in their treatment. A number of methods and preparations are used in the clinical practice to overcome this problem. Authors report their favourable clinical experience with the administration of polyelectrolyte-fractionated porcine factor VIII. concentrate in a hemophiliac child for the first time in Hungary and a brief review of the clinical methods in use in the management of factor VIII. inhibitors.

[First experience with the use of granulocyte-macrophage colony stimulating factor in pediatric oncology]. / Elsö tapasztalataink a granulocita-makrofág kolóniastimuláló faktor alkalmazásáról a gyermekgyógyászati onkológiában.

Authors report their first experiences with the application of granulocyte-macrophage colony stimulating factor in 12 pediatric cancer patients (14 cases). The drug was given in a 5 micrograms/kg single daily dose subcutaneously. Patients were divided into three main indication groups: 1. Severe neutropenia (white blood cell count < 1.0 G/l) and sepsis (6 patients); 2. Prolonged neutropenia (white blood cell count: 1.0-2.0 G/l) and delay in treatment (3 patients); 3. Dose-escalation of chemotherapy in therapy-resistant cases (4 patients). Authors report that in all cases a substantial raise in white blood cell count could be achieved after 5-6 days of granulocyte-macrophage colony stimulating factor treatment. No side effects were detected except of a moderate local pain at the site of the injection. Authors suggest that in the above described dose and way of administration granulocyte-macrophage colony stimulating factor can be an effective agent in the treatment of chemotherapy-induced neutropenia in paediatric oncology.