bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I/II Study of Bortezomib in Combination with Liposomal Doxorubicin and Melphalan in Relapsed or Refractory Multiple Myeloma.
Authors: Chari Ajai , Kaplan Lawrence , Linker Charles , Damon Lloyd E. , Navarro Willis H. , Martin Thomas
Published: April 2020

Phase I/II studies using either liposomal doxorubicin (D) or oral melphalan (M) in combination with the proteasome inhibitor bortezomib (V) have found favorable efficacy and tolerance in patients with relapsed and refractory MM. Since these drugs have different mechanisms of action and toxicity profiles, we initiated a phase I/II study to examine the safety and efficacy of combining all three agents (DMV) in relapsed or refractory myeloma.

Study Aims: Starting at 25–50% of the doses used in the two drug combination studies, the objective of this dose escalation study was to evaluate the safety/tolerability of DMV until the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) could be identified. Efficacy was also evaluated.

Methods: Patients with relapsed/refractory MM and progressive disease requiring treatment were enrolled in the study. Dose level 1 consisted of D 10 mg/m2 and M 5 mg/m2 given IV on day 1 with V given by IV push on days 1, 4, 8, and 11. Cycles were repeated every 28 days up to a maximum of 6 cycles. Dose levels of D, M, V (mg/m2) in the subsequent three cohorts were (10, 10, 0.7), (20, 10, 0.7), and (20, 10, 1.0) respectively. Once the MTD has been identified, an additional 17 patients will be enrolled at this dose level in the phase II portion of the study.

Results: 5 patients (2 males, median age 65, range 33–79) have been enrolled in the study thus far. The myeloma subtypes include 2 IgG, 1 IgA, and 2 with light chains only. In this heavily pretreated population (range 2–7 prior therapies), 4 patients received prior autologous stem cell transplantation, 2 had a prior nonmyeloablative allogenic transplant, 4 prior VAD, 1 prior bortezomib, 2 prior oral melphalan, 3 prior thalidomide, 2 prior CC-5013, and 2 prior spinal radiation.

The first dose cohort has been completed without any DLT. One patient had 3 days of Grade 3 neutropenia. All other toxicities were Grade 1–2 including asthenia, vomiting, thrombocytopenia, and headache. Of note, a patient with baseline Grade 2 peripheral neuropathy and on hemodialysis remained stable. A patient with Grade 2 chronic graft versus host disease also remained stable.

Four patients have been enrolled at dose level one. One patient, who had progressive disease after an autologous and a nonmyeloablative allogenic transplant as well as CC5013, had a near CR (IFE+) after the second cycle of DMV. Two patients have had stable disease. One had disease progression after one cycle and subsequently died. The patient on dose level two is too early to evaluate.

Conclusion: Thus far, DMV appears to be well tolerated and no DLT has been observed even in elderly patients with significant comorbidities. The finding of a near CR and SD at only dose level 1 is encouraging particularly since the patients were significantly pretreated. Dose escalation continues to determine the MTD.
Hematopoietic Development of Multipotent Germline Stem Cells Derived from Neonatal Mouse Testis In Vitro and In Vivo.
Authors: Yoshimoto Momoko , Heike Toshio , Chang Hsi , Baba Shiro , Fujino Hisanori , Shinohara Mito K. , Shinohara Takashi , Nakahata Tatsutoshi
Published: April 2020

Recently multipotent stem cells have been established from neonatal mouse testis (Shinohara et al, Cell 2004). This multipotent germline stem cell (mGS) is very similar to embryonic stem cells. It differentiates into various types of somatic cells in vitro and produces teratomas after inoculation into mice. Here we show the characteristics of hematopoietic development from mGS cells.

mGS cells were maintained on mitomycin C treated mouse embryonic fibroblasts. In undifferentiated state, surface markers were almost the same among mGS, ES and EG cell; E-cadherin+, β1 integrin+, CD31+ and c-kit+. For induction, undifferentiated mGS cells were cultured on OP9 stromal cell line. Four days after induction, we detected and sorted FLK1+ cells as much as ES cells. Flk1+ cells were further cultured on OP9 with various cytokines. Erythroid, myeloid, lymphoid, megakaryocyte and mast cells as well as endothelial cells and beating cells were obtained in the same manner as the induction of ES cell and EG cells. mGS cells had colony forming abilities including mix and magakaryocyte colonies. The number of mixed colony was dependent on the combination of cytokines. In erythroid differentiation, mGS showed the two waves of embryonic and definitive erythroid production, which was proved by RT-PCR and immunochemistry. When hematopoietic cells derived from GFP+ mGS cells that were cultured on OP9 for 6 days were transplanted into BM of NODγ mice directly, GFP+ cells were detected in the BM and Spleen by FACS analysis and PCR 4 months after transplantation. FACS analysis showed that GFP+ cells were detected in the side population when BM cells of transplanted mice were stained with Hoechst33324. Immunostaining of the slice of BM showed some GFP+ cells were attached to the endosteal region that is thought to be the niche for hematopoietic stem cells.

Thus, mGS cells have as much capabilities of blood cell production as ES cells and were showed having hematopoietic reconstitution ability. Considering mGS cells can be obtained from postnatal mice, they have strong advantages in research for clinical application.
Immune Dysfunctions of Cultured T Lymphocytes Are Due to a Preferential Expansion of CD4+CD25+ Suppressor Cells.
Authors: Mesel-Lemoine Mariana , Cherai Mustapha , Le Gouvello Sabine , Guillot Maude , Leclercq Virginie , Klatzmann David , Thomas-Vaslin Véronique , Lemoine Francois M.
Published: April 2020

We investigated the causes of the altered functionality of T-cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T-cells cultured for 6-days with anti-CD3 ± anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T-cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft versus host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amount of cultured T-cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and human. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T-cells (Treg) and thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T-cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T-cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.
Stable Mixed Hematopoietic Chimerism in Dogs Conditioned with Donor Antigen, Anti-CD154 Antibody and 100cGy TBI.
Authors: Jochum Christoph , Beste Mechthild , Zellmer Eustacia , Harkey Michael A. , Graves Scott S. , Storb Rainer F.
Published: April 2020

Stable mixed chimerism can be established in dogs given a sublethal dose of 200cGy total body irradiation (TBI) before and immunosupression with mycophenolate mofetil (MMF) and Cyclosporine (CSP). When the TBI dose is reduced to 100 cGy, only transient engraftment is observed. Here we asked whether stable engraftment after 100 cGy TBI could be accomplished by first reducing the intensity of host immune responsiveness with the help of an anti-CD154 antibody which blocks the CD40-CD154 pathway.

Accordingly, recipients were given a single i.v. injection of 5 mg/kg anti-CD154 antibody (day -5) followed one day later by combined i.v./sc. injections of 107 peripheral white blood cells/kg from their intended DLA-identical littermate marrow donors. TBI, 100cGy (delivered at 7 cGy/min), was given on day 0 followed by infusion of a median of 4.22x108 marrow cells/kg. Postgrafting immunosuppression consisted of MMF (10mg/kg, BID, days 0 to 28) and CSP (15 mg/kg BID, days -1 to 35). Six dogs have been transplanted. Two dogs are too early after transplant for evaluation, while four dogs have been followed for >9, 12, >18 and >26 weeks. All four dogs had initial engraftment. Three of four dogs have continued to show stable mixed donor/host hematopoietic chimerism among lymphocytes and granulocytes from the peripheral blood. One dog rejected the graft 12 weeks after transplantation. All six transplanted dogs are alive and well. Besides mild allergic reactions to the antibody in five of the six dogs (redness of the eyes, skin rashes and dry mucous membranes), no additional side effects attributed to the pretransplant treatment were observed.

Data were consistent with previous findings in dogs, in which the costimulatory signal between B7 and CD28 was blocked, and support the hypothesis, that stable marrow allografts would be established by combining non-myeloablative pretransplant host immunosuppression and posttransplant immunosupression of both host and donor cells using MMF and CSP.
Fluorescence In Situ Hybridization Detected MRD and Chimerism in Patients with Hematologic Malignancies after allo-HSCT.
Authors: Qiu Huiying , Xue Yongquan , Jin Jinlan , Wu De Pei
Published: April 2020

Objective Monitoring of minimal residual disease (MRD) and cellular chimerism in patients with hematopoietic malignancies after allogeneic hematopoietic stem cell transplantation(allo-HSCT).

Methods From May 2001 to June 2005, seventy four patients were received allo-HSCT. Including 50 Males and 24 females. 41 patients received sibling HLA-matched BMT, 7 patients received un-related BMT, 9 patients received Nonmyeloablative stem cell transplantation (NST), 14 patients received related haploidentical transplantation and 3 patients received allo-PBSCT. Among them, 45 patients were diagnosed with CML, 13 patients with AML, 14 patients with ALL, one patient with Multiple myeloma and one patient with malignant lymphoma. Chimerism and MRD were monitored using X and Y specific centromeric probes or gene probes for BCR/ABL, MLL and AML1/ETO by fluorescence in situ hybridization (FISH),1000 cells were analysised.

Results Among 18 patients, received sex-matched transplant, we did not found the former chromosome rearrangements in 14 patients after transplantation, MRD were detected in 17% and 10% of cells in two patients, MRD were decreased from 10% to 1% of cells after the reduction of the dose of immunotherapy in one patient four month later, the patient was still in remission one year after transplantation. Another patient died of sever GVHD after the reduction of immunotherapy. 2 patients were found to have the former chromosomal rearrangement 1 and 4 month after transplantation respectively who did not achieve remission after chemotherapy and died 3 and 5 months respectively after transplantation. Over 99% donor chimerisms were found in 44 patients on day 25, donor cells were at a low level (96.2%~98.7) in 7 patients on day 25, and increased over 99% on day 180, they were in remission without relapse. The donor chimerisms decreased gradually in 6 patients, host cells were found over 20 cells, 3 patients showed cytogenetic or hematologic bone marrow relapse, two patients died of sever GVHD after the reduction of cyclosporine A, Over 99% donor chimerisms was achieved in one patient. Conclusion FISH could play a pivotal role in the detection of MRD and chimerism. It is helpful to the evaluation of graft and relapse, to the guide of implement of early immunotherapy.
Kinetics of Hematopoietic Progenitor Cell Mobilization with Cyclophosphamide or Cyclophosphamide Plus AMD3100 Using a Mouse Model.
Authors: Gai Bill Q. , Holt Matthew S. , Nervi Bruno , Rettig Michael P. , Ritchey Julie K. , Bridger Gary , DiPersio John F.
Published: April 2020

Hematopoietic progenitor cells (HPC) mobilized from bone marrow into peripheral blood (PB) are used to reconstitute hematopoiesis following myeloablative therapy. To date, a variety of agents including cytokines, chemotherapy and the combination have been used to mobilize HPC for autologous-HST. Cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF) are two of the most commonly used agents for HPC mobilization. Recently, AMD3100 (AMD), a bicyclam molecule that specifically and reversibly blocks SDF-1 binding to CXCR4, has been shown to have a unique HPC mobilizing capability characterized by a rapid mobilization within 1 to 2 hours after subcutaneous (sc) administration, compared with several days for G-CSF or CY. In contrast to AMD-induced mobilization, G-CSF and CY-induced HPC mobilization require functional neutrophils and an intact G-CSF receptor. We hypothesize that AMD will increase CY mobilization, and early intervention with AMD after CY treatment will increase the migration of HPC into PB in advance of neutrophil recovery. Wild type C57/BL6 mice were divided into 7 groups (n=3). One group was an untreated control and each of the other six groups received CY (200mg/kg) intraperitoneally on day 0, followed by single dose AMD (5 mg/kg sc) on days 3, 4, 5, 6, 8, and 12 after CY, respectively. WBC recovery and HPC mobilization into PB was evaluated with white blood cell (WBC) counts and standard colony-forming unit (CFU) assay, before and 2 hours after AMD administration. Our results showed that AMD can mobilize WBC within 30 minutes after treatment, reaching its peak effect in 2–3 hours with a 3–5 fold WBC increase, and a > 20 fold increase of PB neutrophils compared to baseline. We also demonstrated that AMD, CY, and CY + AMD increased the CFU/ml by 7.1 + 0.4, 22.6 + 8.4, and 42.9 + 16 fold over the baseline, respectively, reaching the peak mobilization for CY on day 6. The CFU/ml returned to baseline 12 days after treatment with CY or CY + AMD. Although the kinetics of mobilization of HPC after CY + AMD were similar to those seen with CY alone (Figure 1), significant mobilization of murine HPC after CY + A occurred 1–2 days (days 4–5) in advance of that seen with CY alone (days 6–8), 1–2 days in advance of neutrophil recovery (days 6–8) and coincident with period of maximal cytoxan-induced neutropenia. The addition of AMD to CY results in synergistic mobilization of HPC which occurs throughout the entire period of CY-induced mobilization. These data suggest that the addition of A to CY for PBSC mobilization will not significantly alter the kinetics of cytoxan-induced mobilization but may permit earlier collections in patients even before significant WBC recovery occurs.

Figure Figure
Clinical Scale Preparation of B Lymphoblastoid Cell Line-Tumor Cell Hybridoma Cells for Vaccination in Patients with Relapsed Ewing’s Sarcoma and Neuroblastoma.
Authors: Lucas Kenneth G. , Mierski Joseph , Kullen Jennifer , Sun Qi
Published: April 2020

Despite improvements in survival for pediatric patients with cancer, for those with recurrent, high risk Ewing’s sarcoma (ES) or neuroblastoma (NB) treatment options are limited. Tumor-specific antigens in these malignancies are poorly characterized, and expression of human leukocyte antigen (HLA) and co-stimulatory molecules may be down-regulated, imparing a cellular immune response. Several groups have studied the use of tumor cell-dendritic cell (DC) hybridoma vaccines in murine tumor models as well as in phase I clinical trials in human cancer. One problem in implementing these vaccine strategies on a clinical scale is the requirement for repeat blood collections or leukapheresis to expand a limited number of DC. B lymphoblastoid cell lines (BLCL) are effective APC expressing HLA as well as key co-stimulatory molecules, and can be rapidly expanded from relatively few peripheral blood mononuclear cells. We have studied the ability of BLCL to be fused to a series of NB and ES cell lines using polyethylene glycol, and the ability to propagate these hybrid cells long term in culture. Using monoclonal antibodies specific for tumor markers (anti-GD2 and CD56 for NB), BLCL (CD19), and by labeling ES tumor cells with fluorescent dye prior to fusion, 3–11% of the resulting cell products represented true fusion cells. This was confirmed in studies in which BLCL transduced to express green fluorescent protein (GFP) were fused with adherent tumor cells, resulting in a population of large green adherent cells with abnormal morphology. Dual fluorescent staining and flow cytometry of green fluorescent dye labeled tumor cells and unlabelled BLCL consistently revealed a mixed population of unfused BLCL, unfused tumor cells, and cells with irregular morphology expressing both CD19 and green fluorescent dye. Despite these findings, these hybrid cells could not be expanded in long term culture, and adherent hybrid cells did not survive in cell culture over 2 weeks. In addition, these hybrid cells could not be selected when tumor cells and BLCL were transduced with different antibiotic resistance genes and then subjected to selection media. Despite the inability to selectively expand tumor cell-BLCL hybridomas for long-term culture, the choice of BLCL as an APC for vaccine permits the preparation of relatively large numbers of fusion cells for serial vaccination or for in vitro stimulation of antigen specific CTL.
Multipotent Adult Progenitor Cells (MAPC) Are Immunoprivileged and Demonstrate Immunosuppressive Properties on Activated T Cell Population.
Authors: Kovacsovics-Bankowski Magdalena , Streeter Philip R. , Hewett Erin , Van’t Hof Wouter J. , Deans Robert J. , Maziarz Richard T.
Published: April 2020

Multipotent Adult Progenitor cells (MAPC) are rare progenitor cells present within the bone marrow that have the capacity to differentiate into mesodermal, endodermal and ectodermal lineage cells. The capacity to differentiate into these diverse cell types distinguishes MAPC from Mesenchymal Stem Cells (MSC), and suggests that MAPC may provide biological solutions for tissue repair or regeneration in multiple organ systems (Jiang Y et al. Nature.2002; 418:41–9). We have developed the technology for the large-scale expansion of MAPC with stable phenotype and biological properties. Cell surface marker analysis of MAPC revealed that these cells express low levels of MHC class I, CD44, CD90 and CD49c and are negative for MHC class II, CD45 and CD106 suggesting that MAPC do not express markers associated with the hematopoietic lineage. However, to optimize in vivo utilization, the identification of MAPC immunological properties is necessary. The objectives of the current studies were 1) to determine the immunogenicity of MAPC and 2) to assess the immunomodulatory properties of MAPC. First, MAPC from two different rat strains did not stimulate allogeneic T cells proliferation, while splenocytes of the same rat strains elicited strong proliferative responses in a mixed lymphocytic culture. Second, the immunosuppressive properties of MAPC were investigated using alloactivated T cells. Addition of MAPC at the initiation of a mixed lymphocyte reactions (MLR) suppressed T cell proliferation in a dose dependent manner. The inhibition was detectable at as low as 3,000 MAPC/well. Lymphocyte proliferation in MAPC-containing cultures was inhibited by up to 80% when compared to cultures without MAPC. The ability to inhibit T cell alloresponses was independent of the MHC, allowing the use of “third party” MAPC as inhibitory cells. MAPC also inhibited proliferative responses to the T cell mitogen Concanavalin A (50%), although inhibition required higher MAPC:responder cell ratios. Taken together, these data suggest that MAPC are progenitor cells that do not express markers of the hematopoietic lineage; they are non-immunogenic, suggesting that universal MAPC donors may be used for tissue repair or regeneration; and MAPC exhibit potent immunosuppressive properties, a result which suggests that these calls may be useful in the management and/or prevention of GVHD.
Multi-Tissue Homing of Repopulating Hematopoietic Cells after Bone Marrow Transplantation in Mice.
Authors: Bistrian Roxana , Martin Hans , Seifried Erhard , Mueller Albrecht , Henschler Reinhard
Published: April 2020

We wished to assess the tissue distribution of repopulating hematopoietic stem cells after bone marrow transplantation (BMT), since so far only sub-fractions of repopulating cells have been found to home to hematopoietic organs and tissues in BMT models. Two x 105 lineage depleted (lin−) bone marrow cells from C57BL/6 Ly5.1 mice were transplanted into 2 x 5.5 Gy irradiated Ly5.2 congenic C57BL/6 recipient mice. On day 1, 7 or 28 post BMT, cell suspensions were prepared from bone marrow, spleen, muscle, brain, lung and liver, and CD45+ cells were enriched by magnetic beads separation. Enriched CD45+ cells from brain, quadriceps muscle, or other organs and tissues were transplanted into Ly5.2 secondary recipients in groups of three (mean transplanted cell number per mouse: 0.68 x 105; range: 0.26–2.9 x 105) along with 1 x 106 freshly isolated Ly5.2 competitor whole bone marrow cells. A total of 39 primary and 273 secondary recipient mice were analyzed. Here we show that whereas bone marrow displayed substantial donor-derived repopulation potential in non-irradiated and non-transplanted controls (mean contribution of Ly5.1 cells to bone marrow of secondary recipients after 4–7 weeks: 23.5%), muscle, brain, lungs and liver showed little competitive repopulating activity (<2%). In transplanted recipients, however, high donor-derived repopulating activity was detected in bone marrow (38–72%), spleen (7–15%) and, surprisingly, in liver (25–36%), muscle (24–37%), brain (7–14%) and lung (4–12%) at 1, 7 and 28 days post BMT. In contrast, hematopoietic colony-forming cells localized exclusively to hematopoietic tissues and were not found in muscle, brain, or lung. Repopulation potential was present in blood in all groups including non-irradiated, non-transplanted controls (donor contribution, 6–20%). Our data indicate that, following BMT, bone marrow, spleen and additional tissues take up substantial numbers of repopulating cells. This provides an explanation for the published low homing efficiency of repopulating cells to hematopoietic organs.
Molecular Mechanisms of the AICD Paradoxon in Human T Cells Revealed by an Apototic Role for CD25.
Authors: Richter Guenther , Hanewinkel Karin , Mollweide Andreas , Burdach Stefan
Published: April 2020

The IL2R is a heterotrimeric receptor consisting of the alpha-chain (CD25) and the two signal transducing beta-, gamma-chains. CD25 monoclonal antibody (e.g. daclizumab) binding to the α-chain, blocks high affinity IL2 binding thereby preventing complete T cell activation. This opportunity to hinder T cell triggering is of ample importance in transplantation medicine and the treatment of autoimmune disease; e.g. for the prevention of an acute graft versus host reaction during allogeneic hematopoietic cell transplantation. However, gene-targeting experiments revealed, that CD25 has an important role in mediating activation induced cell death (AICD) thereby maintaining T cell homeostasis. Thus, CD25 antibodies may not only block T cell activation but may also prevent AICD attributing a dual function to IL2, which has been described by the term AICD paradoxon. The molecular mechanisms of AICD remain to be elucidated. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of CD25 mAb was investigated with the aim to identify genes that are involved in T cell activation or in AICD. PBMC were stimulated with OKT3 together with recombinant IL2 in the absence or presence of 30 microgram/ml Daclizumab. Cells were incubated for 16 hrs, RNA extracted and subjected to microarray analysis on U133A gene chips (Affymetrix). Gene chip profile revealed up-regulation of 60 genes and down regulation of 36 genes respectively, by Daclizumab. Anti-CD25 treatment inhibitied cytokine genes typically expressed during T cell activation including CD40L, IL9, TNF-alpha and IFN-gamma as previously shown (e.g. Burdach et al., JCI). Surprisingly, daclizumab also blocked expression of several genes important for susceptibility to apoptosis, such as DR6. In addition, daclizumab reversed IL2-mediated repression of anti-apoptotic genes, such as TOSO. Microarray analysis of these apoptosis related genes was confirmed by RT-PCR and functional assays. In conclusion, CD25-mediated induction of pro-apoptotic as well as repression of anti-apoptotic gene clusters should be considered for future drug development of CD25-antibodies in the clinical arena: these apoptosis related gene products may represent new pharmacologic targets in hematopoietic cell transplantation as well as in the treatment of autoimmune diseases.
Effective Ex Vivo-Generation and Quantification of Leukemia-Derived DC Has To Precede a Specific T-Cell Stimulation for Adoptive Immunotherapy in AML and MDS.
Authors: Schmetzer Helga Maria , Kremser Andreas , Loibl Julia , Hentschel Nina , Steiner Karin , Schmid Christoph , Kroell Tanja , Kufner Stefanie , Salih Helmut , Valle Francesco del , Doehner Constanze , Schweiger Claudia , Schwartz Christian , Hiddemann Wolfgang , Kolb Hans-Jochen
Published: April 2020

The presentation of leukemic antigens can be improved in AML and MDS by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). In preliminary analyses we could already define optimal serum-free culture conditions, generate and characterize DC from 55 MDS- and 99 AML-cases and characterize their T-cell activating function (Kufner S. 2005 I-III). DC/DCleu were quantified according to their surface DC /blast -marker profiles. However in 6/30% of cases with AML/MDS less than 10% of DC could be generated and only 50–60% of leukemic cells were convertible to DCleu. Therefore we tested 5 alternative methods to improve the harvest of DC/DCleu in 99 AML and 55 MDS-patients (1) ‘MCM-mimic’ (Lee, 2003), 2) ‘Ca-Ionophore’ (Houtenbos, 2003), 3) ‘Picibanil’ (Sato, 2003), 4) ‘Cytokines’ (Westers, 2003) and 5) ‘Poly I:C’ (Rouas, 2004)). 1. Although the percentual harvest of DC/DCleu was comparable in all of the methods (28–40% DC, with 45–65% of those DC being DCleu and about 47% of blasts being convertible to DCleu) not every method was successful in individual patients. 2. However we could demonstrate that DC can be generated from every sample with at least one of the following methods: MCM-mimic, Ca-Ionophore and Picibanil. 3. Highest DC/DCleu yield was seen in monocytoid differentiated FAB-types and was independent of the cytogenetic risk. 4. Many ‘DC-surface markers’ can be aready expressed on naive blasts in AML/MDS-patients before there conversion to DCleu. Moreover the ‘DC-marker expression’ is variable in individual patients and culture methods.

In summary our data show 1. that the generation of DC/DCleu is possible independent from the karyotype in every patient under serum-free conditions with at least one of the 3 methods MCM-mimic, Ca-Ionophore and Picibanil. 2. The overall percentual harvest of DCleu from MNC-fractions after culture in the 5 methods compared is low. Possibly a combination of the optimal method with optimal ‘DC- and blast markers’ in every single case could improve the detectability and quantification of DC/DCleu and residual blasts. This contributes to improve the generability and quality of DCleu to stimulate and expand anti-leukemia-directed T-cells for the immunotherapy of AML and MDS.
IL10-Transduced Mesenchymal Stem Cells Attenuate Experimental Acute Graft-Versus-Host Disease.
Authors: Min Chang-Ki , Kim Bo-Kyung , Park Gyeongsin , Cho Bin , Oh Il-Hoan
Published: April 2020

Recent data suggest that, due to their immunosuppressive nature, adult mesenchymal stem cells (MSCs) may be of interest to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD).

Using a murine model of acute GVHD, this study investigated whether the immunosuppressive properties of MSCs could reduce the severity of experimental GVHD. In a MHC-mismatched C57BL/6→B6D2F1 model, recipient animals were transplanted with bone marrow (BM) cells (10x106) plus 20x106 splenocytes after 1100 cGy on day 0. Various doses (1~3x106) of donor BM-derived MSCs were given intravenously from day 1 to day 5 and subsequently evaluated the clinical and immunologic parameters.

MSCs did not attenuate the severity of acute GVHD. Using GFP expressing MSCs, we were unable to detect labeled cells in liver, spleen, lymph nodes and lung day 7 post-BMT. Because MSCs fail to display any immunosuppression in this experimental GVHD model and induce an immunosuppressive microenvironment through the production of IL10, we investigated whether the use of genetically modified MSCs expressing IL10 (IL10-transduced MSCs, IL10 MSCs) could improve the GVHD protection. Lethally irradiated recipients were transplanted and injected with 2x106 IL10 MSCs, MSC expressing MIG as a vector (MIG MSCs), or diluent on day 1. Compared with MIG MSCs or controls, recipients of IL10 MSCs demonstrated significantly reduced mortality at day 50 after BMT (percent survival, 0% or 10% vs 70%, P<0.001). The reduction in mortality was confirmed by the semi-quantitative GVHD score (P<0.01). It was associated with decreased serum levels of pro-inflammatory cytokines, IFNγ on day 7 (IL10 MSCs vs MIG MSCs; 297±116 pg/ml vs 681±87 pg/ml, P=0.015). Serum levels of TNFα or splenic donor CD3+ cell numbers were not different between the groups. Thus, benefical effects on GVHD could be observed when MSCs were engineered to express an anti-inflammatory cytokine, IL10. The mechanisms for the GVHD protection effect of IL10 MSCs in this murine model are currently under investigation.
Human T Lymphocyte Activation Kinetics for Identifying and Targeting Alloreactive T Cells.
Authors: Bajwa Rajinder P.S. , McCarthy Philip L. , Wallace Paul K. , Wallace Stephen , Wu Yiyuan , Battiwalla Minoo
Published: April 2020

Selective depletion of alloreactive T cells from a stem cell graft has the potential of reducing graft versus host disease (GvHD) while preserving graft versus malignancy and third party responses. For this purpose several techniques generate and deplete alloreactive cells, which are donor-derived T cells activated by recipient tissue. The kinetics of T cell activation in donor-recipient co-culture systems is critical in optimizing the timing of depletion of alloreactive T cells. We present the T cell activation kinetics in our preclinical system. Peripheral blood mononuclear cells (PBMCs) were derived from several pairs of unrelated healthy human volunteers. 2500 cGy irradiated cells (stimulators) were co-cultured with PBMCs (responders) in a 1:1 ratio and a concentration of 5 x 106/ml in serum free medium. Stimulator cells were labeled with PKH67 and the co-cultures were, analyzed for CD3, CD4, and CD25, expression by flow cytometry on days 0 through 7, using Topro-3 to exclude dead cells. We also added low dose Interleukin-2 (IL2) for augmenting the generation of alloreactive cells, to see if this made a difference. Our results show that alloreactive CD8 cells (CD3+, CD4−, CD25+) increased from ≤1 % on day 0, to 6.5 percent by day 5; the addition of IL2 amplified this to nearly 10% by day 5. CD4 + activated T cells (CD3+, CD4+, CD25+dim or total) did not appreciably increase over time and ranged between 2 to 5%; the addition of IL2 did not have any effect. T regulatory cells (CD3+, CD4+, CD25+bright) increased from ≤1 percent at baseline to 5% by day 5 and these were unaffected by the addition of IL2. The proportion of non-activated T lymphocytes, decreased with time of co-culture progression. Our results show that T lymphocyte activation, defined by CD25 expression, progressively increases through the first week of co-culture. This important observation will help in establishing the timing of allograft manipulation, for the selective depletion of alloreactive T cells in clinical hematopoietic stem cell transplantation.

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Therapeutic Anti-Tumor Immunity Mediated by Transiently Engrafting Allogeneic Lymphocytes: The “Allogeneic Effect” Revisited.
Authors: Symons Heather J. , Levy M. Yair , Wang Jie , Zhou Xiaotao , Fuchs Ephraim J.
Published: April 2020

The “allogeneic effect” refers to the induction of host B cell antibody synthesis or host T cell cytotoxicity, including tumoricidal activity, by an infusion of allogeneic lymphocytes. We have previously shown that treatment of mice with cyclophosphamide (Cy) followed by infusion of CD8+ T cell-depleted allogeneic spleen cells (Cy + CD8− DLI) induces anti-tumor activity in a model of minimal residual leukemia, even though the donor cells are eventually rejected by the host immune system. The purpose of the current investigation was to test the activity of Cy + CD8− DLI in the treatment of well-established cancer, and to characterize the mechanisms of the anti-tumor effect. BALB/c mice were inoculated intravenously (IV) with the syngeneic A20 lymphoma/leukemia or the RENCA renal cell carcinoma on day 0 and were then treated with nothing, Cy alone on day 14, or Cy + CD8− DLI from MHC-mismatched C57BL/6 donors on day 15. In both tumor models, the combination of Cy + CD8− DLI significantly prolonged survival compared to mice treated with nothing or with Cy alone. While depletion of CD4+ T cells from the DLI significantly diminished the beneficial effect of CD8− DLI, purified CD4+ T cells alone were inactive, demonstrating that donor CD4+ T cells and another population of cells were required for optimal anti-tumor activity. Several observations pointed to an active role for the host immune system in the anti-tumor activity of Cy + CD8− DLI. First, host T cells participated in the anti-tumor effect of treatment with Cy alone, since the drug’s activity was diminished in tumor-bearing scid mice or in normal BALB/c mice depleted of T cells. Second, while Cy + CD8− DLI caused no GVHD in tumor-bearing but immunocompetent BALB/c recipients, it caused fatal acute GVHD in either tumor-bearing scid or T-cell depleted BALB/c mice. Finally, the anti-tumor effect of Cy + CD8- DLI was also significantly inhibited in BALB/c mice that were depleted of CD8+ T cells. These results demonstrate that transiently engrafting T cells administered after Cy can induce significant anti-tumor effects against both solid and liquid tumors. We propose that upon recognition of alloantigen on host antigen-presenting cells (APCs), allogeneic donor CD4+ T cells deliver activating ligands to the APCs, thereby generating effective “help” to break tolerance in tumor-specific host CD8+ T cells. This mechanism may correspond to the “allogeneic effect” in the anti-tumor response described over three decades ago.
Comparing Platelet Loss in 2 Common Apheresis Machines for Collecting Autologous Peripheral Blood Stem Cells (PBSC).
Authors: Law Ping , Yasir Fathalha , Mah Joanna K.Y. , Soh Teck Guan , Lim Susan T.T. , Tan Lip Kun
Published: April 2020

Chemotherapy together with G-CSF is commonly used to mobilize patients prior to leukapheresis (LP) of PBSC for autologous transplantation. For many patients, peripheral blood platelet counts (PLT) were below 75,000/μl when LP was initiated. Minimizing PLT loss is an important consideration, especially for these patients. The aim of this retrospective analysis is to compare PLT loss during LP for autologous PBSC in two common apheresis machines (Baxter Amicus and Cobe Spectra). Between August 2004 and May 2005, 57 autologous PBSC products were collected from 28 adult patients (weight > 40 kg) with results (mean ± SD) summarized in Table 1. LP were collected using automated procedures and 3 times blood volume was processed. Collection efficiencies (EFF) were calculated as total cells (CD34+ or PLT) in product divided by the circulating count prior to LP times processed volume. Statistical significance was determined by unpaired Student’s t-test. There were no significant differences (p > 0.1) (NS) in patient age, weight, CD34+ cell count (data not shown) and PLT count prior to LP. There were also no differences in the proportion and the total CD34+ cells in the PBSC products collected by either machines. While both devices collected CD34+ cells with similar EFF (data not shown), the Amicus PBSC products have a trend (p = 0.08 and 0.07 when all LP or only those initiated with PLT count below 75,000/μl, respectively)towards a smaller volume (VOL) and contained significantly (p < 0.05) fewer PLT than those collected by Spectra. The PLT EFF was also significantly lower with Amicus, indicating a smaller PLT loss. The same findings were observed whether all LP or only those LP initiated with low PLT counts (< 75, 000/μl) were considered. Our results showed that lower PLT loss is achieved with Amicus during autologous PBSC collection. The machine would be more appropriate for collecting patients with low PLT counts.

Table 1: PLT Loss during Autologous PBSC Collection in Amicus or Spectra N Pre LP PLT (x103/μ l) LP VOL (ml) LP Total PLT (x1010) PLT EFF (%) ALL LP Spectra 17 49.5 ± 20.2 178.0 ± 41.7 8.7 ± 3.5 17.6 ± 16.9 Amicus 40 57.0 ± 32.9 164.5 ± 16.5 6.8 ± 2.6 8.4 ± 4.1 p value NS ≤ 0.08 ≤0.05 ≤0.05 Low PLT Spectra 17 49.5 ± 20.2 178.0 ± 41.7 8.7 ± 3.5 17.6 ± 16.9 Amicus 35 46.8 ± 15.4 162.9 ± 16.8 6.5 ± 2.4 9.0 ± 3.9 p value NS 0.07 ≤ 0.05 ≤ 0.05
Highly Efficient Peripheral Blood Progenitor Cell Mobilization in Myeloma Patients by Single-Dose Application of 12 mg Pegylated G-CSF.
Authors: Fruehauf Stefan , Seeger Timon , Topaly Julian , Ho Anthony D. , Goldschmidt Hartmut
Published: April 2020

Autologous peripheral blood stem cell (PBSC) transplantation leads to significant prolongation of survival in patients with different malignancies. For PBSC mobilization a combination of myelosuppressive chemotherapy and granulocyte colony stimulating factor (G-CSF) are administered. The optimal application of G-CSF is twice daily. New G-CSF formulations with prolonged half-life carry the promise of reduced patient strain, increased compliance and via continuously high G-CSF serum levels possibly improved PBSC mobilization. We initiated a study with pegfilgrastim-supported mobilization chemotherapy in stage II and III myeloma patients. Patients received up-front treatment with three cycles of vincristin, doxorubicin, dexamethason (VAD) or thalidomide, doxorubicin, dexamethason (TAD).

The mobilization regime consisted of four days chemotherapy with cyclophosphamide 1g/m2 day1, doxorubicin 15mg/m2 day 1–4, dexamethason 40 mg d 1–4 p.o. (CAD) and a single administration of 12 mg pegfilgrastim subcutaneously on day five. 25 patients (median age 57, 12 female, 13 male) received pegfilgrastim and leukapheresis was started 6–17 days (mean 10 days) after treatment with a range of maximum CD34 cell count between 7,2 and 842 (mean: 119) CD34+ cells/μl peripheral blood. After leukapheresis 6.2 * 10e6 to 40.5 * 10e6 (mean 13.8 *10e6) CD34+ cells per kilogram body weight were collected. 11 patients achieved the target number of 7.5 * 10e6 CD34+ cells per kilogram body weight during a single apheresis, while 5 patients needed two, 7 patients needed three and 2 patients needed four apheresies on consecutive days. 3 patients required additional administration of filgrastim. 2 patients received 6 mg pegfilgrastim off study. Both achieved sufficient numbers of CD34+ cells (12.7 and 20.8 * 10e6 CD34+ per kg BW). There were only minimal adverse effects. Four patients reported of bone pain or nausea. Mobilization failures did not occur in this patient population. To date, 16 patients have been transplanted with a mean of 6,03* 10e6 CD34+ per kg BW (range 2,4 to 13,5). Reconstitution (reaching a leukocyte count of >1,0/nl) was reached within a mean of 13 days (range 11–21). On the basis of these first results we conclude that a single dose application of 12 mg pegfilgrastim after CAD treatment allows collection of more than 3 autografts in two or less apheresis session in the majority of myeloma patients. Reduction of apheresis sessions is a significant clinical cost-effectiveness end point in PBPC mobilization.
Hyaluronic Acid Enhances To Differentiate into Myelo-Megakaryocytic Lineages during Ex-Vivo Expansion of Human Cord Blood(HCB) Using TPO, SCF and FL.
Authors: Mun Yeung-Chul , Ahn Jee-Young , Lee Kyung-Eun , Yoo Eun-Sun , Lee Sang min , Bae Yun-Kyung , Lee Seung-Eun , Kim Min Kyoung , Hyun Myung Soo , Seong Chu-Myong
Published: April 2020

Hyaluronic acid(HA) is the major glycosaminoglycan component of extracellular matrices. CD44 is a ligand for HA and expresses on a wide variety of cell types. CD44 has been implicated in many cell-cell and cell-matrix interactions. In the present study, we studied the effects of HA on proliferation, differentiation and apoptosis of CD34+ cells during HCB ex vivo expansion using TPO, SCF and FL. CD34+ cells from cord blood MNC were cultured for 4 weeks in the combination of TPO, SCF and FL on HA-coated well and CD34+cells were cultured in identical conditions except HA-coated well. At day 4, 7, 14, 21, 18, we counted the number of expanded cells and analyzed the cell surface markers (CD3, CD14, CD19, CD34, CD44, CD61, CD66b) and apoptosis using 7-AAD staining. To analyze the effects of HA on CD34+ cell differentiation, anti-CD44 antibodies (clone A3D8 and 515) were used. The morphology on the expanded cells was examined using wright’s staining. In results, cell counts were increased in HA-coated well compared with the control group until D7, but proliferation rate was decreased after D7. Apoptosis portion of CD34+ using 7-AAD staining was increased in HA-coated well and % of CD34+ cells was decreased in HA-coated wells compared with that of the control groups. The expanded cells in HA-coated well were negative for CD3 (pan T cell), CD19 (B cell) but CD14 (monocyte), CD61 (megakaryocyte) and CD66b (neutrophil) were highly expressed than those of control groups. Apoptotic portion was also increased in the HA-coated group. CD66b (neutrophil) expression in HA-coated well was significantly higher than those of control groups at D14 and above findings were confirmed by wright’s stain. CD66 expression was reduced by the anti-CD44 antibody clone, A3D8 and however CD66 expression was increased by the clone, 515. Therefore, it might be possible that A3D8 and clone 515 recognize each different epitopes of CD44. In conclusion, CD34+ cell differentiation from HCB into CD66, CD14 and CD61 positive cells was enhanced by the contact of HA during ex vivo expansion using TPO, SCF and FL. This result suggests that the expanded human cord blood graft with HA might be useful as the “post-progenitor” to shorten the severe cytopenia period after myeloablative conditioning for HCB transplantation.
Platelet Count Is a Sensitive Predictor of Bone Marrow Reserve and Autologous Peripheral Blood Progenitor Cell Mobilization.
Authors: Zubair Abba , Grant Rhonda , Tun Han , Rivera Candido , Moreno-aspitia Alvaro , Roy Vivek , Colon-Otero Gerry , Solberg Lawrence
Published: April 2020

Background: Previous studies have proposed combination of various factors that predict poor mobilization after GCSF stimulation. However, there is no single clinical or laboratory test that reliably correlates with bone marrow reserve and PBPC mobilization. Peripheral blood CD34+ cell count (pCD34) reliably correlates with PBPC mobilization and yield but only after GCSF administration.

Methods: We have prospectively followed 36 autologous PBPC transplant candidates, from the time of initial evaluation to transplant, to evaluate factors the correlate with bone marrow reserve and autologous PBPC mobilization.

Results: The patients median age was 63 (range 26 – 76). 21 of the 36 patients were diagnosed with dysproteinemia, primarily multiple myeloma, 10 patients had non-Hodgkin’s lymphoma while the remaining 5 had other form of hematological malignancies. Our evaluation shows baseline platelet count prior to growth factor administration significantly correlate with total CD34+ cell yield (spearman: r = 0.64, p <0.001, Figure 1). In addition, daily platelet count during PBPC harvest correlate with CD34+ cell yield for the day (spearman: r = 0.6, p <0.001). Using multiple linear regression model (F = 19.1, p <0.001, R2 = 0.64), we have determined patients’ age (p = 0.03) and type of disease (p <0.001), in addition to platelet count (p<0.001), significantly correlate with total CD34+ cell yield. Further analysis showed low baseline platelet count predicts poor PBPC mobilization. Using Receiver Operating Curve (ROC), we have determined that platelet count of 174,000/ul was the best cut-off for collecting at least 2 million/Kg CD34+ cells with sensitivity of 81% and specificity of 78% and estimated correct classification of 81%. Even though pCD34 counts were slightly more sensitive and were better at identifying poor mobilizers in our cohort (best cut-off 10/ul, sensitivity 96%, specificity 67%, correct classification 89%), their clinical utility were diminished since the values were only known after GCSF administration.

Conclusion: Baseline platelet count is a sensitive predictor of bone marrow reserve and can be used prior to growth factor administration to predict poor mobilization.

Figure 1: Correlation of baseline platelet count and total CD34+ cell yield Figure 1:. Correlation of baseline platelet count and total CD34+ cell yield
Multivariate Analysis of Risk Factors for Clinically Overt Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Zhu Kanger , Ma Chunhui , Zhang Tao , Zhong Juan
Published: April 2020

Objective: To analyze the risk factors of clinically overt hemorrhagic cystitis (HC) (grade ≥II) in 114 patients undergoing allo-HSCT to predict the occurrence of HC.

Methods: We retrospectively analyzed 29 cases of clinically overt HC from a series of 114 patients given allo-HSCT from April 1997 to December 2004. The time of follow-up began from the day of initiating conditioning to day 180 post-transplant. The 11 clinical parameters were selected for univariate analysis using a Cox regression: age, sex, underlying disease, conditioning regimen, disease status at transplant, aGVHD, donor type, use of ATG, GVHD prophylaxis, platelet and neutrophil engraftment. Factors that were significant at the 0.1 level on univariate analysis were evaluated by multivariate analysis using a Cox regression. The cumulative incidence of grade ≥ II HC within the day 180 after transplantation was calculated by the method of Kaplan and Meier.

Results: 29 out of 114 patients (26%) developed HC with grade II in 12/29 cases (41.4%), grade III in 11/29 cases (37.9%) and grade IV in 6/29 cases (20.7%). The following factors were associated with an increased risk of HC by univariate analysis: male gender (RR=2.885, P=0.021),younger than 26 years (RR=3.265, P=0.007),grade III~IV aGVHD (RR=4.039, P=0.002),unrelated doner (RR=4.347, P=0),intense GVHD prophylaxis (RR=2.218, P=0.045),advanced disease (RR=2.668, P=0.009). These risk factors were entered into a multivariate model. Only male gender (RR=2.993, 95% CI 1.218–7.358; P=0.017) and unrelated donor (RR=4.478, 95% CI 2.049–9.786; P=0.000) were identified as being significantly associated with the occurrence of hemorrhagic cystitis.

Conclusion: We found that in multivariate analysis, patients were at increased risk of HC if they were male or had received graft from unrelated donors.