Sanja Mazić

Expression of Bone Morphogenetic Proteins in Stromal Cells from Human Bone Marrow Long-term Culture

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.

Large volume leukapheresis: Efficacy and safety of processing patient's total blood volume six times

Large-volume leukapheresis (LVL) differs from standard leukapheresis by increased blood flow and an altered anticoagulation regimen. An open issue is to what degree a further increase in processed blood volume is reasonable in terms of higher yields and safety. In 30 LVL performed in patients with hematologic malignancies, 6 total blood volumes were processed. LVL resulted in a higher CD34+ cell yield without a change in graft quality. Although a marked platelet decrease can be expected, LVL is safe and can be recommended as the standard procedure for patients who mobilize low numbers of CD34+ cells and when high number of CD34+ cells are required.

Toxicity related to autologous peripheral blood haematopoietic progenitor cell infusion is associated with number of granulocytes in graft, gender and diagnosis of multiple myeloma

Background and Objectives  We prospectively evaluated the infusion‐related toxicity of autologous peripheral blood progenitor cells (PBPC) in 215 patients with haematologic malignancies or solid tumours.

Large volume leukapheresis is efficient and safe even in small children up to 15 kg body weight.

BACKGROUND The collection of peripheral blood stem cells, although now a routine procedure, is still a challenge in low body weight children because of specific technical and clinical issues. For paediatric patients it is crucial to obtain an adequate number of CD34+ cells with the minimum number of procedures: this can be done using large volume leukapheresis (LVL). MATERIALS AND METHODS We analysed the efficacy and safety of 54 autologous LVL performed in 50 children (33 [66%] males and 17 [34%] females), median age 2 years (range, 1-5) and median body weight 12 kg (range, 6-15). The procedures were performed with a COBE Spectra previously primed with red blood cells; ACD-A solution and heparin were used as anticoagulants. RESULTS The target CD34+ cell dose (≥5×10/kg body weight) were collected with one LVL in 46 (92%) patients, while four (8%) patients needed another procedure. All our LVL were well tolerated. Side effects were observed in five (9.2%) patients and one procedure had to be discontinued because of catheter-related haemorrhage. The platelet count decreased significantly (p<0.001) after each procedure but without bleeding or need for transfusion support. DISCUSSION Our experience confirms that LVL is efficient and safe even in small children, if the procedure is adjusted considering the weight and age of child. The most important factors are good venous access, adequate preparation of the childs electrolyte status, and surroundings in which the small child as well as parents feel comfortable, and can tolerate the procedure better. Although a median platelet loss of 50% can be expected, LVL is safe and reduces the overall number of procedures required. It can be recommended for peripheral blood stem cell collection even in small body weight children with malignant diseases, particularly those who mobilise low numbers of CD34+ cells.

Treatment of chronic GVHD with extracorporeal photochemotherapy

We analyzed 351 ECP procedures performed in 6 patients with cGVHD; median ECP per patient was 52 (range 13–127). Patients’ median age was 29 years (range, 12–76 years). The patients suffered from generalized sclerodermatou s skin changes, impaired join mobility and one patient had symptoms of oral disease. In all patients concomitan t immunosupp ressive treatments for cGVHD were used as necessary. ECP procedures were performed for two consecut ive days: in initial phase weekly, followed every two weeks and then monthly accordin g to clinical response. ECP was performed using the ‘‘off line’’ technique. MNCs were collected using COBE Spectra cell separator (Caridian BCT). In all leukapheresi s 2 patient’s total blood volumes were processed. Collected MNC concentrate was transferred to Extracorporeal UV-A Bag Set (Cell Max GmbH) and diluted with saline solution. 8-MOP (Gerot) was injected into the UV-A Bag Set and bag was irradiated by PUVA Combi-Ligh t UVA Illuminator at wave length of 350 nm with the irradiation dose of 2 J/cm . Irradiated cells were reinfused back to the patient. During apheresis and reinfusion of irradiated cells patients were monitored for adverse reactions . Number of T-lymphocyte subsets (CD3+, CD3 + 4+, CD3 + 8+, CD4 + CD8 + ratio) and B-lymphocytes (CD 19+), in patient’s peripheral blood were tested monthly.