julilili42/papers huggingface dataset:
Retransplantation with Stem Cells from Mismatched Related Donors after Graft Rejection in Pediatric Patients.
2006-11-16

Abstract Graft failure is a rare but life-threatening complication after transplantation of hematopoietic stem cells. Treatment comprises immunoablative reconditioning regimens and a second stem cell donation from the same or from a different donor as soon as possible to minimize the time of pancytopenia and its sequelae. We report a cohort of 11 pediatric patients with leukemias (acute lymphatic n=4, acute or chronic myeloic/MDS n=4) and severe aplastic anemia (n=3) who experienced graft rejection after TBI, busulphan or melphalan based myeloablative transplantation from mismatched related donors (MMRD) (n=6) or after cord blood/matched unrelated donor (MUD) transplantation (n=5) between 2000 and 2006. In the latter the original donor was not available a second time. Thus, all patients were re-transplanted with CD34+ positive selected or CD3/CD19 depleted stem cells from a second, haploidentical parental donor (MACS method, Miltenyi Biotec). Median time span from diagnosis of graft rejection to second donation was 16 days. The reconditioning regimens consisted of total lymphoid irradiation or cyclophosphamide, thiotepa (5mg/kg), fludarabine (120 mg/m2) and ATG/OKT3. A median number of 25×106/kg of body weight stem cells with 60.000/kg residual T cells were infused. Mofetilmycophenolat was given as GvHD prophylaxis, if residual T cells exceeded 25 000/kg bw. Sustained engraftment was achieved in all patients (ANC&gt;500/μl: 9 (11–32) days). No GvHD &gt; grade II was observed. T cell recovery was delayed, however no lethal viral infection occurred. Severe organ toxicity was observed in 2 patients (BOOP, hemorrhagic cystits) and moderate mucositis in 11 patients. 8/11 patients are disease free (median follow up 1.5 (0.3–6.6) years; 1 year EFS=73%). Causes of death were: BOOP (n=1), infection (n=1); only one patient with refractory AML relapsed. Thus, transplantation of stem cells from haploidentical donors represents a realistic option to rescue patients with graft failure within a short time span and for whom a second donation of the original donor is not available. The use of a different donor may help to avoid a second rejection.
Hierarchy of Human B Cell Regeneration after Rituximab Therapy.
2006-11-16

Abstract Constant immunoglobulin levels throughout life-time may be maintained by either long lived plasma cells or continuous formation of immunoglobulin secreting cells (ISC). Rituximab therapy targets CD20+ B cells, thus selectively spares B progenitor cells and plasma cells, providing an in vivo model to investigate human B cell homeostasis. After weekly administration of rituximab in patients with follicular lymphoma, peripheral blood B cells are virtually absent (&amp;lt;0,01%), and no ISC are detected. In contrast, during rituximab maintenance therapy (administered in two months intervals) small numbers of B cells can be detected. These carry the phenotype of new formed plasma blasts (CD27++/CD38++/icIg+++ and Ki67+) and are identified as ISC in ex vivo ELISPOT assay. Surprisingly, ISC detected in treated patients were the same as in healthy donors in absolute numbers, while B cells were still reduced by 100-fold. Only 4 to 6 months after therapy human naive and memory B cells were detected in peripheral blood again. Thus, regeneration of human peripheral blood B lymphocytes follows a hierarchy: Recirculating ISC recover rapidly to normal steady state levels, while B cells with naive or memory phenotype require longer time periods. This hierarchy may represent two elements within the B cells system: a primary system sustaining constant antibody levels and a secondary system providing a diverse repertoire for adaptive immune responses.
Interleukin-10 Severely Impairs TLR Mediated Activation of Antigen Presenting Cells.
2006-11-16

Abstract Danger signals provided by pathogens are sensed by specialized receptors, Toll-like receptors (TLR), on the cell surface of antigen presenting cells (APC) leading to activation of dendritic cells (DC) accompanied by their phenotypic and functional changes. Activation of DC via the TLR is believed to be a critical event regulating the strength and outcome of an immunological response. IL-10 is an immunosuppressive cytokine shown to have inhibitory effects on function and differentiation of APC by interfering with signaling pathways involved in DC activation like the PI3-kinase or NFκB pathway. In our study we analyzed the function of human dendritic cells generated in the presence of IL-10 upon activation by Toll-like receptor ligands (TLR2, TLR3, TLR4, TLR7/8). Exposure of DCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by TLR ligands, a reduced production of cytokines like IL-12, IL-6 or TNF-α and an impaired capacity to stimulate T cell activation. Furthermore, CCR7 upregulation in DC exposed to TLR stimulation as well as migration toward CCL19/MIP-3β were strongly reduced. As a suitable mechanism for these effects, IL-10 was found to downregulate MyD88, an essential adaptor molecule for TLR signaling, and to decrease TLR-induced nuclear expression of the NFκB transcription factor members c-Rel and Rel-B as well as IRF-3 and IRF-8. This was not due to the inhibition of the MAP kinase pathway as phosphorylation of p38 and ERK was not affected but was rather mediated by inhibitory effects on the PI3-kinase pathway. IL-10 treatment of APC resulted in a reduced TLR induced mTOR phosphorylation. In summary, our results demonstrate that IL-10 can inhibit TLR-mediated activation of APC via a MyD88-dependent and independent pathway.
Haploidentical Allogeneic Hematopoietic Cell Transplantation in Adults with Reduced-Intensity Conditioning and CD3/CD19-Depleted Grafts: Low Toxicity and Fast Engraftment.
2006-11-16

Abstract Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with CD34-selected stem cells has been complicated by high regimen related toxicities, slow engraftment and delayed immune reconstitution leading to increased treatment related mortality (TRM). A new regimen using graft CD3/CD19 depletion with anti-CD3 and anti-CD19 coated microbeads on a CliniMACS device and reduced intensity conditioning may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, graft-facilitating- and dendritic-cells. Reduced intensity conditioning was performed with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (5 mg/day, day −5 to +14) and no posttransplant immunosuppression. Twenty two consecutive patients (median age=43 (range, 21–59) years) have been transplanted with this regimen. Diagnosis were AML (n=12), ALL (n=5), NHL (n=2), MM (n=2) and CML (n=1). Patients were “high risk” with refractory disease or relapse after preceding HCT. The CD3/CD19 depleted haploidentical grafts contained a median of 7.6 x 106 (range, 3.4–17x106) CD34+cells/kg, 6.7x107 (range, 0.02–18.2 x107) CD56+cells/kg and 5.5x104(range, 0.006–44x104) CD3+T-cells/kg. Donor-recipient KIR-ligand-mismatch was found in 14 of 22 patients. The regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis. Because of severe neurotoxicity in 4 patients treated with 200 mg/m2 fludarabine, the dose was reduced to 150 mg/m2 with no further cases of neuropathy thereafter. Engraftment was rapid with median time to &gt;500 granulocytes/μL of 12 (range, 10–21) days, &gt;20000 platelets/μL of 11 (range, 7–28) days and full donor chimerism after 2–4 weeks in all patients. Incidence of grade II–IV GVHD was 59% with grade II=9, III=2 and IV=2. One patient, who received the highest T cell dose, developed lethal grade IV GVHD. TRM in the first 100 days was 6/22 (27%) with deaths due to idiopathic pneumonia syndrome (n=1), mucormycosis (n=1), pneumonia (n=3) or GVHD (n=1). Six patients died after day 100, five due to relapse and one with systemic adenoviral infection. Overall survival is 10/22 patients (45%) with a median follow-up of 185 days (range, 17–988). In conclusion, this regimen is promising in high risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing.
Regulation of Dendritic Cell Function by Histone Deacetylase Inhibitors.
2006-11-16

Abstract Histone deacetylases (HDACs) modulate gene transcription and chromatin assembly by modifying histones at the post-transcriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies for malignancies of different histology. Preliminary evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we show that two HDAC inhibitors of clinical use, sodium valproate and MS-275, profoundly affect cytokine-induced differentiation of human monocytes to dendritic cells (DCs), and impair DC phenotypic maturation in response to the Toll-like receptor (TLR) ligand polyinosinic:polycytidylic acid (poly I:C) characterized by a reduced expression of HLA- and costimulatory molecules. HDAC inhibitors reduce cytokine secretion important for T-cell activation like IL-12 and immunostimulatory capacity in DCs and inhibit DC migratory capacity toward the chemoattractant CCL19/MIP-3β. The observed defects in DC function upon exposure to HDAC inhibitors were not due to an increased rate of apoptosis or production of IL-10. We finally investigated the effects of HDAC inhibition on intracellular signalling pathways that play a key role in controlling DC differentiation and function. HDAC inhibitors have been reported to cause a downregulation of the hematopoietic transcription factor PU.1, which contributes to the development of thymic and myeloid dendritic cells and of Langerhans cells in the mouse. We evaluated herein whether PU.1 expression was affected by VPA in monocyte-derived DCs, however, no significant reduction in protein levels could be detected. Therefore, our data rule out PU.1 shortening as a mechanism responsible for the functional defects observed in DCs that were exposed to HDAC inhibitors. Similarly, no significant effect of HDAC inhibitors was detected on Bcl-6, another known target of HDAC inhibitors that, besides its role in lymphomas, has also been linked to the regulation of cytokine and chemokine release in macrophages. In contrast, addition of VPA blocked RelB nuclear relocalization in response to poly I:C in a concentration-dependent manner. Interestingly, IRF-3 and IRF-8, which have been involved in DC differentiation, IL-12 production and DC migration, were also affected by HDAC inhibitors, since their nuclear levels were reduced by VPA in unstimulated as well as in poly I:C-activated DCs. On the other hand, IRAK-1 downregulation in response to poly I:C was not affected by VPA, suggesting that the effects of HDAC inhibition on RelB, IRF-3 and IRF-8 are probably mediated downstream of MyD88 and IRAK-1. In conclusion, HDAC inhibitors exhibit strong immunomodulatory properties on human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders.
Prolonged Survival of Chronic Lymphocytic Leukemia Cells Induced by Sphingosine 1-Phosphate Is Mediated by the G Protein-Coupled Receptor S1P1 and Involves Erk/MAP Kinase, but Not Akt Signaling.
2006-11-16

Abstract Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator which is generated by the hydrolysis of sphingomyelin and released predominantely by activated platelets but also by other cells upon activation. In normal blood plasma it is found in nanomolar to micromolar concentrations. By qualitative and quantitative (TaqMan) RT-PCR, mRNA expression of the S1P receptor S1P1 was detected in CLL cell lines (EHEB, MEC-1) and in all samples of primary chronic lymphocytic leukemia (B-CLL) cells, analyzed after isolation with a CD19 antibody and magnetic cell sorting. mRNA expression of other S1P receptors was less consistently found. When primary CLL cells were cultured in serum-free medium (RPMI 1640 supplemented with fatty acid-free BSA), viability decreased starting from 3 days of culture and was completely lost after 1–2 weeks depending on the individual sample. However, when the cultures were supplemented with S1P (1 nM - 5 μM) the number of viable cells was increased up to 8-fold in a time- and dose-dependent manner as measured using the WST-1 reagent. Moreover, S1P induced a rapid (maximum at 1 min) phosphorylation of the p44/42 Erk/MAP kinase in CLL cell lines and even more prominent in primary CLL cells. The time- and dose dependent phosphorylation was blocked by pretreatment (24h) of the cells with FTY720 (10–100 nM), a compound that binds predominantly to S1P1 and induces loss of cell surface expression by receptor internalization. FTY720 also abrogated the prolonged survival of CLL cells in cultures supplemented with S1P, demonstrating that both the Erk/MAP kinase phosphorylation and the consecutive increased survival were mediated by the S1P1 receptor. Activation of MAP kinase was also inhibited by pertussis toxin, which selectively blocks Gi proteins, the only class of G proteins known to be coupled to S1P1. Phosphorylation of Akt was not observed in CLL cell lines and primary CLL cells after stimulation with S1P. B-CLL cell lines showed a pronounced chemotactic response to S1P at low concentrations. In primary CLL cells however, S1P did neither induce phosphorylation of the focal adhesion kinase (FAK) nor chemotaxis. We conclude that S1P, constitutively present in the blood, prolongs survival of CLL cells involving the Erk/MAP kinase pathway. Since compounds that induce S1P1 receptor internalization like FTY720 are already used as immunosuppressants in humans, our results suggest inclusion of these drugs also in the treatment of B-CLL, where they may potentiate the effect of conventional antiproliferative therapy.
Differential Signaling of G Protein-Coupled Chemotactic Receptors (CXCR4, cysLT1 and S1P1) Expressed in Hematopoietic Progenitor Cells.
2006-11-16

Abstract We have previously demonstrated consistent and high expression of the G protein-coupled receptors (GPCR) CXCR4, cysLT1 and S1P1 in human hematopoetic progenitor and stem cells (HPC). All of these receptors induced chemotactic responses in mobilized CD34+ HPC. As their ligands are present in the hematopoietic microenvironment, these GPCR may be involved in HPC homing and/or proliferation. Ligands for CXCR4 (SDF-1) and cysLT1 (cys-leukotrienes) are both produced by stromal cells, while the S1P1 ligand (sphingosine 1-phosphate, S1P) is released by megakaryocytes and platelets. As GPCR may activate multiple signaling pathways resulting in different functional effects, we compared the signaling capacities of CXCR4, cysLT1 and S1P1 in CD34+ HPC and CD34+ progenitor cell lines. At optimal ligand concentrations, cysLT1 mediated the strongest intracellular calcium fluxes, in contrast to CXCR4 (intermediate response) and S1P1 (almost absent calcium fluxes). Pertussis toxin (PTX) only partially inhibited cysLT1-mediated calcium signaling, indicating coupling of the receptor to both Gi and Gq proteins, while CXCR4-mediated responses were exclusively PTX-sensitive and therefore only Gi-dependent. Both cysLT1 and CXCR4 induced phosphorylation of the proliferation-related p44/42 (Erk) MAP kinase. Again, much stronger responses were mediated by cysLT1, which again were only partially PTX-sensitive. No Erk/MAP kinase activation was mediated by S1P1. We could demonstrate that pyk2, a kinase that links calcium signaling with Erk/MAP kinase activation, was involved in Gq-mediated signaling of cysLT1, as its phosphorylation was not inhibited by PTX. The fact that cysLT1 employs both Gi and Gq-mediated signaling pathways, while CXCR4 signaling was exclusively Gi-dependent, may explain why enhanced proliferation of CD34+ hematopoietic progenitor cells in cytokine-supplemented serum-free liquid cultures was only seen in response to ligands of cysLT1, but not in response to the CXCR4 ligand SDF-1. Activation of CXCR4 resulted in phosphorylation of Akt, which was not observed in response to ligands of cysLT1, while both GPCR induced translocation of NFkappaB to the nucleus. We conclude that in CD34+ HPC, Gi protein-mediated signaling of CXCR4 is mainly involved in cell migration, while Gi and Gq-mediated signaling of cysLT1 also affects cell proliferation predominantly by combined and profound activation of the Erk/MAP kinase pathway. The signaling capacity of S1P1 however was limited compared to the other GPCRs.
Prospective Randomized Comparison of High Dose AraC and AutoPBSCT as Late Consolidation for Patients ≤60 Years with Standard Risk AML: Final Results of the AML SHG-Hannover 01/99 Trial.
2006-11-16

Abstract We treated 520 patients ≤60 years with de novo (n=414) or secondary (n=106) AML. Patients with CBF-leukemias [t(8;21) or inv(16)] or normal karyotype and good response (GR) to induction I (≤5% blasts in d15 BM) were considered standard risk (SR), all others as high risk (HR). Patients with t(15;17) were excluded. Induction I consisted of standard dose araC, idarubicine and etoposide (IVA-I). Patients with GR to IVA-I continued with IVA–II on d21. In patients with bad response (&amp;gt;5% BM blasts on d15), the second cycle consisted of either IVA–II or FlAG/Ida. Induction was followed by early consolidation with intermediate dose araC. As late consolidation, SR patients with normal karyotype and an HLA-matched sibling received matched related donor (MRD) transplantation. The remaining SR patients were randomized between high dose (HD) araC (12 x 3g/m2)/daunorubicine (3 x 45mg/m2) or an autologous peripheral blood stem cell transplantation (autoPBCSCT) with PBSC mobilized after early consolidation. 90% of the 262 SR patients achieved CR in contrast to only 59% of the 249 HR patients (overall CR rate 74%). After 75 months, overall survival (OS) and relapse free survival (RFS) was significantly better for SR than for HR patients. Within the SR group, OS for patients with CBF leukemia (n=62) at 56 months was significantly better than for patients with normal karyotype (n=200). RFS was similar for both groups. 57 SR patients (14 with CBF leukemia) were randomized to receive HD araC and 62 (16 with CBF leukemia) to undergo autoPBSCT. At 70 months, OS and RFS was not different for the patients treated with autoPBSCT and for those receiving HD araC. This was true for patients with normal karyotype as well as CBF leukemias. Median duration of neutropenia (&amp;lt;500/μl) was 9 days for autoPBSCT and 19 days for HD araC (p&amp;lt;0.01) with a significantly higher rate of septicemia (21% vs. 11%) and pneumonia (14% vs. 3%) after HD araC. Duration of thrombocytopenia was 21 days for HD araC and 11 days for autoPBSCT (p&amp;lt;0.01). In SR patients with normal karyotype, OS and RFS after MRD transplantation did not significantly differ from HD araC or autoPBSCT. However, when looking at the FLT3 status in patients with normal karyotype (n=49), chemoconsolidation resulted in an inferior survival (17%) in patients with mutated FLT3 as compared to autotransplantation (67%). In conclusion, outcome is similar in SR AML patients after autoPBSCT, HD araC or MRD transplantation. In SR patients without an HLA-identical sibling donor, autoPBSCT instead of HD araC as used in our study is recommended for late consolidation due to the reduced treatment related toxicity and should be studied prospectively in patients with mutated FLT3.
Secondary Bortezomib-Resistance Is Characterized by Upregulation of Proteasomal Subunits and Their Insufficient Inhibition by Bortezomib, Which Can Be Overcome by Alternative Proteasome Inhibitors Combined with Induction of the ER Stress Response.
2006-11-16

Abstract Resistance towards proteasome inhibition by Bortezomib (Velcade®) represents a challenge for myeloma therapy. Its biology has not yet been characterized in detail. We have demonstrated that Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. We here present the first analysis of proteasome biology and activity, alternative proteolytic pathways, ubiquitin-specific proteases (USP) and the ER stress response (unfolded protein response, UPR) upstream of the proteasome, as well as in vitro cytotoxicity of conventional cytotoxic drugs, alternative proteasome inhibitors and agents that target the UPR in Bortezomib-resistant (BR) cells, compared to wild type (WT) controls. BR cells had higher activities of all subunits of the constitutive and the immunoproteasome, as deferred from turnover of fluorogenic substrates as well as affinity-labelling of active proteasome subunits in intact cells. This was mirrored by increased levels of proteasomal β1 and β2, but especially β5 polypeptides, implicating a homeostatic system that senses and corrects low proteasome activity in cells chronically exposed to Bortezomib. While the vinylsulfone-type proteasome inhibitor NLVS abrogated detectable proteasome activity in both BR and WT cells, Bortezomib at therapeutic concentrations eliminated proteasomal β1 and β5-type activity only in WT cells, while BR cells retained residual activity. These changes in proteasome biology appear to be the molecular hallmark of required Bortezomib resistance, since no changes were observed between WT and BR cells in alternative cytosolic or lysosomal proteolytic pathways, UPR activity as well as the gross activity pattern of USP. As expected, this translated into sensitivity against cytotoxic drugs in vitro: BR cells were less sensitive towards alternative proteasome inhibitors. However, while the IC50 for pan-proteasome inhibitors was only roughly doubled in BR cells, it was nearly tenfold elevated for the β5-preferring vinylsulfone inhibitor NLVS. By contrast, sensitivity towards anthracyclines or cytotoxicity induced by ER stressors as well as the synergy between proteasome inhibitors and UPR-activators remained unaffected in BR cells. Based on our data, proteasome inhibitors with activity profiles different from that of Bortezomib, alone or in combination with induction of the UPR, may represent an appropriate concept to overcome secondary Bortezomib resistance.