Robert Pytlik

High-dose immunosuppressive therapy with PBPC support in the treatment of poor risk multiple sclerosis.

High-dose immunoablative chemotherapy with autologous haematopoietic cell support might be beneficial in the treatment of intractable forms of MS. We mobilised PBPC in 11 patients with secondary progressive MS and finally eight patients were grafted after high-dose BEAM chemotherapy with either in vitro or in vivo T cell depletion. Median EDSS and SNRS scores at the time of inclusion were 6.5 (6.5–7.5) and 56 (44–65), respectively. PBPC mobilisation was safe with no serious adverse effects, and without significant aggravation of disability. One patient improved significantly (by 1.0 point on EDSS) after the mobilisation. Two mobilisation failures were observed. No life-threatening events occurred during the transplantation. All grafted patients, except one, at least stabilised their disability status. One patient improved significantly (by 1.5 points on EDSS), two patients improved slightly (by 0.5 points on EDSS), one patient worsened by 1.0 point on the EDSS in 10 months. Improvement occurred with a delay of 2–4 months. Median EDSS and SNRS of grafted patients at the last follow up were 6.5 (5.5–8.5) and 64 (39–73), respectively with median follow-up of 8.5 months. Further follow-up is needed to determine the disease course after complete immune reconstitution. Bone Marrow Transplantation (2000) 25, 525–531.

Vincristine sulfate liposomes injection (Marqibo) in heavily pretreated patients with refractory aggressive non-Hodgkin lymphoma: Report of the pivotal phase 2 study

Marqibo, a sphingosomal/cholesterol encapsulation of vincristine sulfate has targeted, increased, and sustained delivery of vincristine to tumor tissues. A phase 2, open‐label, single‐arm, and multinational study evaluated the efficacy and tolerability of Marqibo as a single agent in patients with multiply relapsed or refractory aggressive non‐Hodgkin lymphoma (NHL).

New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: Biotinylated galectins as tool to detect specific binding sites

Nuclear presence of galectins suggests a role of these endogenous lectins in the regulation of transcription, pre‐mRNA splicing and transport processes. Therefore, detection and localization of nuclear binding sites for galectins by a new methodological step, has potential to further functional analysis. In the first step of our model study we monitored the nuclear expression of galectins‐1 and −3 in cultured stromal cells of human bone marrow and human/porcine keratinocytes. To enable detection and localization of galectin‐specific binding sites, we used purified galectins biotinylated without loss of activity as cytochemical tool. The degree of labeling of the probes was determined by adapting two‐dimensional gel electrophoresis and calculating pI changes in response to stepwise chemical modification of basic and acidic side chains by the biotinylation reagents. Binding studies revealed positivity for galectin‐1, whereas galectins‐3, −5, and −7 were not reactive with nuclear sites under identical conditions in bone marrow stromal cells and keratinocytes prepared from hair follicle enriched for stem cells. Inhibition by lactose indicated an involvement of the carbohydrate recognition domain in nuclear binding of galectin‐1. Colocalization of the galectin‐1‐dependent signal with the SC35 splicing factor and sensitivity toward RNase treatment argued in favor of galectin binding in nuclear speckles, albeit only for a small fraction of the cells. Epidermal cells positive for galectin‐1‐binding sites expressed ΔNp63 known as a potential marker of stem cells. Based on cytokeratin expression cells with nuclear binding of labeled galectin‐1 were basal and not suprabasal cells. Regarding proliferation, no relationship to the expression of a proliferation marker, Ki‐67, was observed. The nucleolar signal colocalized with fibrillarin and nucleophosmin/B23 as representatives of nucleolar proteins in both types of studied cells. In conclusion, the application of labeled galectins to localize accessible binding sites adds a new aspect to the functional analysis of these lectins in the nucleus.

Characterization of dental pulp stem cells from impacted third molars cultured in low serum-containing medium.

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-β1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.

Human dental pulp stem cells--isolation and long term cultivation.

Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2 % of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflicks limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60-90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflicks limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes.

Expression of genes regulating angiogenesis in human circulating hematopoietic cord blood CD34+/CD133+ cells

Objectives: Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors.

Toll-like receptors on B-CLL cells: expression and functional consequences of their stimulation

Toll‐like receptor (TLR) stimulation plays a crucial role in the homeostasis of human B cells. We investigated the expression of TLRs 1–9 on the cells of B‐cell chronic lymphocytic leukemia (B‐CLL) and analyzed the functional consequences of TLR stimulation on leukemic cells. We showed that B‐CLL cells express similar set of TLRs as memory B cells of healthy donors, i.e. TLR‐1, TLR‐2, TLR‐6, TLR‐7 and TLR‐9. However, in contrast to memory B cells, B‐CLL cells lack TLR‐4 expression. Expression of TLRs correlates with their capacity to respond to specific TLR agonists. At the level of phenotype, ODN2006 (TLR‐9 agonist) is the most potent stimulus. B‐CLL cells also respond to the stimulation with loxoribine, Pam3CSK4 and MALP‐2 (TLR‐7, TLR1/TLR2 and TLR2/TLR6 agonists, respectively). TLR‐7 and TLR‐9 stimulation induces production of IL‐6 and TNFα. In 47% of tested patients, treatment with ODN2006, MALP‐2 and Pam3CSK4 reduced leukemic cells survival. Stimulation of B‐CLL cells with TLR‐9 agonists, loxoribine, MALP‐2 and Pam3CSK4 induces significant proliferation. We report that TLR stimulation induces expression of CD38, a negative prognostic marker, on B‐CLL cells. Expression of CD38 is induced by direct stimulation of B‐CLL cells through TLR‐7 and TLR‐9 or CD38 can be induced on B‐CLL cells indirectly by a soluble factor induced in non‐B‐CLL cells after stimulation with TLR‐2, TLR‐3 or TLR‐5 agonists; the nature of this factor remains unknown. Our results argue for cautious evaluation of immunointervention strategies based on the administration of TLR agonists in the treatment of B‐CLL as their effects on B‐CLL cells may be tumor promoting as well as tumor suppressing.

Pre-transplant positron emission tomography in patients with relapsed Hodgkin lymphoma

This retrospective study evaluated the secondary clinical risk score at relapse, the prognostic significance of pre-transplant positron emission tomography (PET), and complete remission (CR) assessed by computed tomography (CT) after salvage chemotherapy before autologous stem cell transplant (ASCT) in 76 patients with relapsed/refractory Hodgkin lymphoma (HL). Median follow-up after ASCT was 23 months. Overall 11/20 PET-positive and 14/56 PET-negative patients relapsed after ASCT. In univariate analysis, only PET negativity before ASCT was significantly associated with better 2-year progression-free survival (PFS) (72.7 ± 6.3% vs. 36.1 ± 11.6%, p = 0.01) and 2-year overall survival (OS) (90.3 ± 4.1% vs. 61.4 ± 11.6%, p = 0.009). Other factors were not significant. In multivariate analysis, none of the evaluated factors were significant for PFS and OS. However, positive pre-transplant PET identified a population with worse PFS and OS at least in univariate analysis.

Circulating Endothelial Progenitor Cells in Patients with ANCA-Associated Vasculitis

Background/Aims: Bone marrow-derived endothelial progenitor cells (EPCs) are believed to contribute to endothelial repair after vascular damage. To investigate the potential for microvascular repair in patients with ANCA-associated vasculitis (AAV), we conducted a cross-sectional study determining the number of circulating EPCs in patients with AAV, chronic uremia, atherosclerosis, and in healthy volunteers. Methods: The number of circulating EPCs was determined by colony-forming assay in 41 patients with AAV, 15 hemodialysis patients (without vasculitis), 13 patients with peripheral arterial occlusive disease (PAOD), and 25 healthy controls. Results: Patients with AAV had a significantly lower number of CFU-Hill than healthy subjects (median 0.3 vs. 19.5 CFU-Hill/ml blood, p < 0.0001), but not than patients on hemodialysis or with PAOD. Neither institution of treatment nor entering remission increased the number of EPCs in AAV patients. The number of EPCs did not correlate with markers of disease activity. AAV patients with glomerular filtration rate <15 ml/min had an even lower number of circulating EPCs than patients with better preserved renal function (median 0.05 vs. 1.2 CFU-Hill/ml, p = 0.015) and patients with anti-MPO positivity had a trend towards a higher number of EPCs than patients with anti-PR3 antibodies (median 3.1 vs. 0.18 CFU-Hill/ml, p = 0.06). Conclusion: Patients with AAV have a significant and persistent deficiency of circulating EPCs. A low number of EPCs could reflect an impaired mechanism of vascular repair and may contribute to repeated relapses in these patients.

In a high-dose melphalan setting, palifermin compared with placebo had no effect on oral mucositis or related patient’s burden

This randomized-controlled trial studied the efficacy of palifermin in a chemotherapy-only, high-dose Melphalan (HDM) transplant setting, to reduce oral mucositis (OM) and its sequelae measured by patient-reported outcomes (PRO) and medical resource use. Palifermin, relative to placebo was given either pre-/post-HDM or pre-HDM in patients with multiple myeloma (MM) undergoing auto-SCT at 39 European centers. Oral cavity assessment (WHO) and PRO questionnaires (oral mucositis daily questionnaire (OMDQ) and EQ 5D) were used in 281 patients (mean age 56, ±s.d.=8 years). 57 patients received placebo. One hundred and fifteen subjects were randomized to pre-/post-HDM receiving palifermin on 3 consecutive days before HDM and after auto-SCT and 109 patients were randomized to pre-HDM, receiving palifermin (60 μg/kg/day) i.v. for 3 consecutive days before HDM. There was no statistically significant difference in maximum OM severity. Severe OM occurred in 37% (placebo), 38% (pre-/post-HDM) and 24% (pre-HDM) of patients. No significant difference was observed with respect to PRO assessments or medical resource use, but more infections and fever during neutropenia were reported in pre-/post-HDM vs placebo (for example, 51 and 26%). To conclude, palifermin was unable to reduce OM or OM-related patient’s burden in MM transplant patients.