ß-Catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation.

The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

Burkitt lymphoma-a retrospective analysis of data from the Registry of the Czech Lymphoma Study Group with external validation of the Burkitt lymphoma International Prognostic Index.

Burkitt lymphoma (BL) is a rare subtype of non-Hodgkin's lymphoma with an aggressive course. To refine the individual patient's prognosis, the International Prognostic Index for BL (BL-IPI) was recently developed and 4 risk factors (RF) were determined as optimal prognostic cut-off by multivariate analysis: age ≥40 years, lactate dehydrogenase >3× upper limit of normal, ECOG performance status ≥2, and central nervous system involvement. The BL-IPI distinguishes 3 prognostic groups, low (without RF), intermediate (1 RF), and high risk (2-4 RF), with significant differences in survival. The aim of the current project was to perform an external validation of the BL-IPI in 101 patients from the Registry of Czech Lymphoma Study Group diagnosed between 1999 and 2016 (median age, 45 years). The median follow-up was 50.4 months. The induction treatment included rituximab plus chemotherapy in 82% and chemotherapy alone in 18%. The overall response rate was 78% and the complete remission rate was 73%. According to BL-IPI, low/intermediate/high risk was present in 21/35/45% of patients, showing high similarity to the training BL-IPI US (United States) dataset (18/36/46%). There were significant differences in progression-free survival (PFS) and overall survival (OS) between patients with high vs. intermediate risk (PFS: hazard ratio 0.16, 95% confidence interval 0.08-0.31, p<0.0001; OS: hazard ratio 0.17, 95% confidence interval 0.09-0.35, p<0.0001) but not between patients with low vs. intermediate risk. The 3-year OS probability according to BL-IPI with low/intermediate/high risk was 96/76/59% in the BL-IPI training dataset vs. 95/85/45% in our external validation cohort; the 3-year PFS probability with low/intermediate/high risk was 92/72/53% in the BL-IPI training dataset vs. 95/85/42% in our cohort. In summary, our external validation of the BL-IPI confirmed a good separation of high-risk patients, who have a poor prognosis and for whom the new therapeutic approaches are needed; patients with low and intermediate risk had favorable clinical outcomes, and differences between these groups were not significant, likely due to a small number of patients.

miR-150 downregulation contributes to the high-grade transformation of follicular lymphoma by upregulating FOXP1 levels.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

The interval between progression and therapy initiation is the key prognostic parameter in relapsing diffuse large B cell lymphoma: analysis from the Czech Lymphoma Study Group database (NIHIL).

Relapsing diffuse large B cell lymphomas (rDLBCL) represent a heterogeneous disease. This heterogeneity should be recognized and reflected, because it can deform the interpretation of clinical trial results. DLBCL patients with the first relapse and without CNS involvement were identified in the Czech Lymphoma Study Group (CLSG) database. Interval-to-therapy (ITT) was defined as the time between the first manifestation of rDLBCL and the start of any treatment. The overall survival (OS) of different ITT cohorts (< 7 vs. 7-21 vs. > 21 days) was compared. In total, 587 rDLBCLs (51.8% males) progressed with a median of 12.8 months (range 1.6 to 152.3) since the initial diagnosis (2000-2017). At the time of relapse, the median age was 67 years (range 22-95). First-line therapy was administered in 99.3% of the patients; CHOP and anti-CD20 were given to 69.2% and 84.7% of the patients, respectively. The salvage immune/chemotherapy was administered in 88.1% of the patients (39.2% platinum-based regimen). The median ITT was 20 days (range 1-851), but 23.2% of patients initiated therapy within 7 days. The 5-year OS was 17.4% (range 10-24.5%) vs. 20.5% (range 13.5-27.4%) vs. 42.2% (range 35.5-48.8%) for ITT < 7 vs. 7-21 vs. > 21 days (p < 0.001). ITT was associated with B symptoms (p 0.004), ECOG (p < 0.001), stage (p 0.002), bulky disease (p 0.005), elevated LDH (p < 0.001), and IPI (p < 0.001). The ITT mirrors the real clinical behavior of rDLBCL. There are patients (ITT < 7 days) with aggressive disease and a poor outcome. Conversely, there are rDLBCLs with ITT ≥ 21 days who survive for a long time.

First-line therapy for T cell lymphomas: a retrospective population-based analysis of 906 T cell lymphoma patients.

Peripheral T cell lymphomas (PTLs) have a globally poor prognosis. The CHOP regimen shows insufficient efficacy; first-line consolidation with autologous stem cell transplantation (auto-SCT) is a promising strategy but has never been confirmed by randomized data. We analyzed retrospectively 906 patients diagnosed with PTL between 1999 and 2015. Chemotherapy was given to 862 patients, and 412 of them were < 60 years. In this subset, we compared induction with CHOP (n = 113) vs. CHOEP (n = 68) and tested auto-SCT (n = 79) vs. no SCT (n = 73) in the intent-to-treat analysis. The median age of the whole cohort at diagnosis was 60 years (range; 18-91); the median follow-up was 4.3 years (range; 0.1-17.8). A shorter overall survival (OS) was associated with the male gender, age ≥ 60 years, stage III/IV, performance status ≥ 2, bulky tumor ≥ 10 cm, and elevated LDH. CHOEP induction showed a better 5-year PFS (25.0% vs. 32.9%; p.001), and 5-year OS (65.6% vs. 47.6%; p.008) than CHOP. Auto-SCT compared to no SCT brought a 5-year OS of 49.2% vs. 59.5% (p.187). Auto-SCT did not influence the OS in low-risk or low-intermediate risk PTLs. The high-intermediate and high-risk IPIs displayed a worse 5-year OS in auto-SCT arm (17.7% vs.46.2%; p.049); however, 73.9% of the patients never received planned auto-SCT. Our population-based analysis showed the superiority of CHOEP over CHOP in first-line treatment. We confirm the 5-year OS of around 50% in PTLs undergoing auto-SCT. However, the intended auto-SCT could not be given in 73.9% of the high-risk PTLs.

Potential loss of prognostic significance of minimal residual disease assessment after R-CHOP-based induction in elderly patients with mantle cell lymphoma in the era of rituximab maintenance.

Rituximab maintenance (RM) prolongs survival of elderly patients with mantle cell lymphoma (MCL). Persistent minimal residual disease (MRD) after induction repeatedly correlated with shorter progression-free survival (PFS). However, none of the published studies analyzed patients treated with RM. The main purpose was to analyze prognostic significance of MRD in the elderly patients with newly diagnosed MCL treated according to the recently published observational trial protocol (alternation of R-CHOP and R-cytarabine, 3 + 3 cycles, GovTrial number NCT03054883) at the centers that implemented RM. Minimal residual disease was evaluated by a EuroMRD standardized real-time PCR approach after 3 and 6 cycles of the induction therapy. Prognostic significance of MRD was analyzed in a subcohort of patients treated at the centers that implemented RM as a standard approach. Bone marrow proved to be a significantly more sensitive source for MRD detection than peripheral blood. In either compartment MRD (positive versus negative) after 3 or 6 cycles of the induction therapy did not correlate with PFS. The observed loss of prognostic significance of MRD after the R-CHOP-based induction appears to be a consequence of RM immune control over the residual lymphoma.

Alternating R-CHOP and R-cytarabine is a safe and effective regimen for transplant-ineligible patients with a newly diagnosed mantle cell lymphoma.

Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first-line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R-CHOP and R-cytarabine for newly diagnosed transplant-ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high-risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography-computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression-free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography-negativity independently correlated with progression-free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R-CHOP and R-cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial (clinicaltrials.gov) NCT03054883.

The incidence of biopsy-proven transformation in follicular lymphoma in the rituximab era. A retrospective analysis from the Czech Lymphoma Study Group (CLSG) database.

The aim of this study is to assess the incidence, risk factors, and outcome of biopsy-proven transformation in follicular lymphoma (FL) patients in the rituximab era. Transformation was analyzed in 1233 patients with initially diagnosed FL grades 1-3A, identified between 2002 and 2012 in the prospectively maintained Czech Lymphoma Study Group database. Only patients with histologically proven transformation (HT) were included. HT occurred in 58 cases at a median of 3.0 years from the initial FL diagnosis; the HT rate was 4% at 5 years. Transformation occurred most frequently at the first relapse (84% patients). Median OS from the HT was 2.5 years (95% CI 0.4-4.6) and 6-year OS with HT was shorter compared to all FLs (60 vs. 83.9%; 95% CI). A bulky tumor (≥ 10 cm), increased lactate dehydrogenase, age ≥ 60 years, and International Prognostic Index (intermediate/high risk), but not Follicular Lymphoma International Prognostic Index, were associated with transformation (p < 0.05). In the first line, 70% of patients received rituximab (including 36% rituximab maintenance), 57% CHOP-like regimens, and 2.6% of patients were treated with fludarabine-based therapy, whereas 11% of patients were watched only. The patients treated with R-CHOP in the first line (n = 591) showed the transformation rate at 5 years of 4.23% (95% CI 2.52-5.93); subsequent rituximab maintenance (n = 276) vs. observation (n = 153) was associated with a lower transformation rate (p.033; HR 3.29; CI 1.10-9.82). The transformation rate seems to be lower than in previous series, which may be influenced by broad use of rituximab, but prognosis of HT developed during therapy continues to be poor.

Can Rituximab Improve the Outcome of Patients with Nodular Lymphocyte-Predominant Hodgkin's Lymphoma?

BACKGROUND: Nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) is a rare subtype of Hodgkin's lymphoma showing strong CD20 expression. The role of rituximab in treating NLPHL still needs clarification. METHODS: We retrospectively reviewed the outcome of 23 patients with NLPHL treated with rituximab alone or in combination with chemotherapy and/or radiotherapy as part of their first- or second-line treatment. RESULTS: The median follow-up of the whole group was 67 months, and all patients remained alive. Twenty-two patients achieved complete remission after rituximab-based therapy, and one of them relapsed 32 months after treatment. One patient treated with rituximab alone achieved partial remission and progressed 22 months after treatment. CONCLUSION: The prognosis of NLPHL is excellent. Rituximab combined with chemotherapy and/or radiotherapy appears to prevent disease progression/relapse.

Complications of MRI-guided stereotactic biopsy of brain lymphoma.

OBJECTIVES: Stereotactic biopsy is a suitable method for sampling intrinsic brain lesions. Although this method is considered to be a safe procedure, some risk of complications still exists. The aim of the study was to retrospectively assess the morbidity and mortality of MRI-guided stereotactic biopsy of lesions which were histologically proven to be brain lymphoma. METHODS: We retrospectively studied all accessible medical records for patients who had undergone MRI-guided stereotactic biopsy of brain lesions with histologically proven brain lymphoma from January 2007 to December 2012. Our cohort included 45 patients, 27 males and 18 females, aged 23-84 (63±14) years. RESULTS: Forty-nine biopsies were carried out on 45 patients; the average number of tissue specimens was 3±1. The diagnostic yield of the stereotactic biopsy was 92%. Overall major morbidity directly related to stereotactic biopsy of brain lymphoma was 6.1% (3 cases) including 4.1% mortality (2 cases). Both deaths after the stereotactic procedure were due to intracranial hemorrhage and subsequent complications and both these patients had a history of treatment of systemic lymphomas. In one patient the stereotactic biopsy was complicated by a brain abscess which was successfully treated. CONCLUSION: Stereotactic biopsy is still a mandatory diagnostic procedure for primary brain lymphomas, with an acceptable risk of complications. However, according to our results, the risk of complications can be higher in patients who have previously been treated for secondary lymphomas.

Characterization of the input material quality for the production of tisagenlecleucel by multiparameter flow cytometry and its relation to the clinical outcome.

Tisagenlecleucel (tisa-cel) is a CD19-specific CAR-T cell product approved for the treatment of relapsed/refractory (r/r) DLBCL or B-ALL. We have followed a group of patients diagnosed with childhood B-ALL (n = 5), adult B-ALL (n = 2), and DLBCL (n = 25) who were treated with tisa-cel under non-clinical trial conditions. The goal was to determine how the intensive pretreatment of patients affects the produced CAR-T cells, their in vivo expansion, and the outcome of the therapy. Multiparametric flow cytometry was used to analyze the material used for manufacturing CAR-T cells (apheresis), the CAR-T cell product itself, and blood samples obtained at three timepoints after administration. We present the analysis of memory phenotype of CD4/CD8 CAR-T lymphocytes (CD45RA, CD62L, CD27, CD28) and the expression of inhibitory receptors (PD-1, TIGIT). In addition, we show its relation to the patients' clinical characteristics, such as tumor burden and sensitivity to prior therapies. Patients who responded to therapy had a higher percentage of CD8+CD45RA+CD27+ T cells in the apheresis, although not in the produced CAR-Ts. Patients with primary refractory aggressive B-cell lymphomas had the poorest outcomes which was characterized by undetectable CAR-T cell expansion in vivo. No clear correlation of the outcome with the immunophenotypes of CAR-Ts was observed. Our results suggest that an important parameter predicting therapy efficacy is CAR-Ts' level of expansion in vivo but not the immunophenotype. After CAR-T cells' administration, measurements at several timepoints accurately detect their proliferation intensity in vivo. The outcome of CAR-T cell therapy largely depends on biological characteristics of the tumors rather than on the immunophenotype of produced CAR-Ts.

Chimeric Antigen Receptor Based Cellular Therapy for Treatment Of T-Cell Malignancies.

T-cell malignancies can be divided into precursor (T-acute lymphoblastic leukemia/lymphoblastic lymphoma, T-ALL/LBL) and mature T-cell neoplasms, which are comprised of 28 different entities. Most of these malignancies are aggressive with rather poor prognosis. Prognosis of relapsed/refractory (R/R) disease is especially dismal, with an expected survival only several months after progression. Targeted therapies, such as antiCD30 immunotoxin brentuximab vedotin, antiCD38 antibody daratumumab, and anti-CCR4 antibody mogamulizumab are effective only in subsets of patients with T-cell neoplasms. T-cells equipped with chimeric antigen receptor (CAR-Ts) are routinely used for treatment of R/R B-cell malignancies, however, there are specific obstacles for their use in T-cell leukemias and lymphomas which are fratricide killing, risk of transfection of malignant cells, and T-cell aplasia. The solution for these problems relies on target antigen selection, CRISPR/Cas9 or TALEN gene editing, posttranslational regulation of CAR-T surface antigen expression, and safety switches. Structural chromosomal changes and global changes in gene expression were observed with gene-edited products. We identified 49 studies of CAR-based therapies registered on www.clinicaltrials.gov. Most of them target CD30 or CD7 antigen. Results are available only for a minority of these studies. In general, clinical responses are above 50% but reported follow-up is very short. Specific toxicities of CAR-based therapies, namely cytokine release syndrome (CRS), seem to be connected with the antigen of interest and source of cells for manufacturing. CRS is more frequent in antiCD7 CAR-T cells than in antiCD30 cells, but it is mild in most patients. More severe CRS was observed after gene-edited allogeneic CAR-T cells. Immune effector cell associated neurotoxicity (ICANS) was mild and infrequent. Graft-versus-host disease (GvHD) after allogeneic CAR-T cells from previous hematopoietic stem cell donor was also observed. Most frequent toxicities, similarly to antiCD19 CAR-T cells, are cytopenias. CAR-based cellular therapy seems feasible and effective for T-cell malignancies, however, the optimal design of CAR-based products is still unknown and long-term follow-up is needed for evaluation of their true potential.

Peripheral T-Cell Lymphomas Involving the Central Nervous System: A Report From the Czech Lymphoma Study Group Registry.

Introduction: We analyzed the incidence, risk factors of central nervous system (CNS) relapse, and outcome of CNS involvement in patients with peripheral T-cell lymphomas (PTCL) from the Czech Lymphoma Study Group Registry NiHiL (Clinical Trial gov. NCT03199066). Materials and Methods: Out of 1,040 patients with PTCL, we identified 29 patients (2.79%) with CNS involvement: 2 patients with primary CNS T cell lymphoma, 11 patients with CNS and systemic disease at diagnosis, and 16 patients (1.54%) at CNS relapse. The most common histology with CNS disease was PTCL, not otherwise specified. Progression-free survival (PFS) was defined as the time interval from diagnosis to progression or death. PFS-2 was defined as the interval from the date of a new relapse until the next relapse. Results: Patients with testicular involvement received intrathecal prophylaxis with methotrexate. High-dose methotrexate-based treatment was administered in 44.8% of patients with CNS disease. Median follow-up was 71.3 months. The difference between the median PFS of 1,027 patients without initial CNS disease (32.6 months) and 11 patients with initial CNS and systemic disease (4.8 months) was significant (p = 0.04). The difference between the median PFS2 in CNS relapses (10.1 months) and 493 relapses outside of CNS (9.1 months) was not significant (p = 0.6). Risk factors for CNS relapses included the following: involvement of more than one extranodal site (p = 0.008), soft tissue involvement (p = 0.003), testicular involvement (p = 0.046), and the presence of B symptoms (p = 0.035). The difference between the median OS of 1,027 patients without initial CNS disease (46.0 months) and 11 patients with initial CNS and systemic disease (18.2 months) was significant (p = 0.02). The median OS2 in CNS relapses was 11.8 months and that in relapses outside of CNS was 21.3 months. CNS involvement was not associated with a significantly worse OS compared to relapsed/refractory patients without CNS involvement (p = 0.1). Conclusions: The incidence of CNS disease at the time of diagnosis and at relapse in PTCL is low and usually associated with other systemic involvement. The prognosis of PTCL with initial CNS involvement is significantly worse when compared to patients without CNS disease at diagnosis. The outcome of CNS relapse is comparable with relapsed PTCL outside of CNS. The optimal treatment is not defined yet.

Circulating microRNAs in Cerebrospinal Fluid and Plasma: Sensitive Tool for Detection of Secondary CNS Involvement, Monitoring of Therapy and Prediction of CNS Relapse in Aggressive B-NHL Lymphomas.

Lymphoma with secondary central nervous system (CNS) involvement represents one of the most aggressive malignancies, with poor prognosis and high mortality. New diagnostic tools for its early detection, response evaluation, and CNS relapse prediction are needed. We analyzed circulating microRNAs in the cerebrospinal fluid (CSF) and plasma of 162 patients with aggressive B-cell non-Hodgkin's lymphomas (B-NHL) and compared their levels in CNS-involving lymphomas versus in systemic lymphomas, at diagnosis and during treatment and CNS relapse. We identified a set of five oncogenic microRNAs (miR-19a, miR-20a, miR-21, miR-92a, and miR-155) in CSF that detect, with high sensitivity, secondary CNS lymphoma involvement in aggressive B-NHL, including DLBCL, MCL, and Burkitt lymphoma. Their combination into an oncomiR index enables the separation of CNS lymphomas from systemic lymphomas or nonmalignant controls with high sensitivity and specificity, and high Receiver Operating Characteristics (DLBCL AUC = 0.96, MCL = 0.93, BL = 1.0). Longitudinal analysis showed that oncomiR levels reflect treatment efficacy and clinical outcomes, allowing their monitoring and prediction. In contrast to conventional methods, CSF oncomiRs enable detection of early and residual CNS involvement, as well as parenchymal involvement. These circulating oncomiRs increase 1-4 months before CNS relapse, allowing its early detection and improving the prediction of CNS relapse risk in DLBCL. Similar effects were detectable, to a lesser extent, in plasma.

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of (3)H-thymidine and differential gating method.

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, (3)H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.

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-ß1/3-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.

The Impact of Various Culture Conditions on Human Mesenchymal Stromal Cells Metabolism.

In vitro and in vivo analyses are closely connected, and the reciprocal relationship between the two comprises a key assumption with concern to the conducting of meaningful research. The primary purpose of in vitro analysis is to provide a solid background for in vivo and clinical study purposes. The fields of cell therapy, tissue engineering, and regenerative medicine depend upon the high quality and appropriate degree of the expansion of mesenchymal stromal cells (MSCs) under low-risk and well-defined conditions. Hence, it is necessary to determine suitable alternatives to fetal bovine serum (FBS-the laboratory gold standard) that comply with all the relevant clinical requirements and that provide the appropriate quantity of high-quality cells while preserving the required properties. Human serum (autologous and allogeneic) and blood platelet lysates and releasates are currently considered to offer promising and relatively well-accessible MSC cultivation alternatives. Our study compared the effect of heat-inactivated FBS on MSC metabolism as compared to its native form (both are used as the standard in laboratory practice) and to potential alternatives with concern to clinical application-human serum (allogeneic and autologous) or platelet releasate (PR-SRGF). The influence of the origin of the serum (fetal versus adult) was also determined. The results revealed the key impact of the heat inactivation of FBS on MSCs and the effectiveness of human sera and platelet releasates with respect to MSC behaviour (metabolic activity, proliferation, morphology, and cytokine production).

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 TNFalpha. 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.

Endothelial cells (EC) and endothelial precursor cells (EPC) kinetics in hematological patients undergoing chemotherapy or autologous stem cell transplantation (ASCT).

Our objective was to study the kinetics of circulating endothelial cells (EC) and endothelial precursor cells (EPC) in hematological patients during chemotherapy and autologous stem cell transplantion (ASCT). Eighteen newly diagnosed patients and 17 patients undergoing ASCT were studied and compared to healthy controls. ECs were evaluated as CD146+CD31+Lin- cells, while EPCs were evaluated as CD34+CD133+Lin-, or CD34+VEGFR2+Lin- cells, or CFU-En colony forming units. Numbers of these cells were evaluated before and after treatment, and, in patients treated with ASCT, during mobilization of hematopoietic progenitors. Both newly diagnosed patients and patients before ASCT had significantly higher number of CD146+CD31+Lin- cells and significantly lower number of CFU-En colonies than healthy controls. These parameters did not return to normal for at least 3 months after chemotherapy or ASCT. Numbers of CFU-En did not correlate either with numbers of CD34+CD133+Lin- cells or with numbers of CD34+VEGFR2+Lin- cells but they did correlate with numbers of CD4+ lymphocytes and NK cells. In conclusion, we have found that hematological patients have higher number of EC and lower numbers of CFU-En than healthy controls and that these parameters do not return to normal after short-term follow-up. Furthermore, our observations support emerging data that CFU-En represent cell population different from flowcytometrically defined EC and endothelial precursors and that their development requires cooperation of monocytes and CD4+ lymphocytes. However, cells forming CFU-En express endothelial surface markers and can contribute to proper endothelial function by NO production.