bluuebunny/crossref_metadata_2025 huggingface dataset:
Leukoreduction System Chambers as a Source of Viable Human Peripheral Blood Mononuclear Cells.
Authors: Kim Sinyoung , Kim Han-Soo , Lee Yangsoon , Song Jaewoo , Kim Hyun Ok , Min Yoo-Hong
Published: October 2019

Many blood banks are using apheresis machines to collect blood components such as platelets (PLTs), RBCs, or plasma. Especially, leukoreduced plateletpheresis using apheresis instrument (Trima Accel, Gambro BCT, Lakewood, CO) provided subsidiary cell products retained in leukoreduction system (LRS) chamber that was originally discarded. The LRS chamber is a conical-shaped chamber that uses saturated, fluidized, particle bed filtration technology to remove WBCs from PLTs. In the current study, a total of 24 LRS chambers from different donors were investigated to determine it would be a valuable source of viable human peripheral blood mononuclear cells (MNCs). The proportions of CD3+, CD19+, CD16+/CD56+, CD14+, CD45+ cells, and absolute CD34+ cell count within the LRS chambers were determined by flow cytometry. Dendritic cells (DCs) were generated from the immunomagnetically purified CD14+ cells from LRS chamber and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the LRS chamber, the total number of WBC count was 1.1 × 109 ± 0.3 × 109 and the mean percentage of MNCs was 80.6 ± 13.1%. The mean proportion of T cells, B cells, NK cells, CD14+ monocytes among CD45+ cells was 54.3 ± 11.5%, 6.4 ± 3.1%, 14.6 ± 3.9%, 12.9 ± 7.5%, respectively. Total absolute CD34+ cell count in LRS chamber was 0.95 × 106 ± 0.65 × 106. Also, we could demonstrate CD14+ cells isolated from LRS chamber was capable of differentiating into functionally mature DCs in vitro. LRS chambers are a valuable and convenient source of viable human peripheral blood mononuclear cell population and could replace standard buffy coat preparations for research applications.
0.1 Grams of Sub-Cutaneous Adipose Tissue (SCAT) as Source of Adult Multipotent Mesenchymal Stromal Cells (MSC) for Cell-Based Therapies.
Authors: Dominici Massimo , Sternieri Rita , De Fazio Domenico , Cafarelli Luigi , Guaraldi Giovanni , Spano Carlotta , Rasini Valeria , Paolucci Paolo , Cilli Michele , Piccardi Federica , Astori Giuseppe , Hofmann Ted J. , Horwitz Edwin M. , Conte Pierfranco
Published: October 2019

The purification of multipotent mesenchymal stromal cell (MSC) from sub-cutaneous adipose tissue (SCAT) represents a promising approach for several clinical applications. The isolation procedures have been applied in humans only to fairly large amount (5–300 g) of harvested SCAT (Zuk PA et al., 2001; Van Harmelen V et al., 2004; Meyerrose TE et al., 2007). Thus, we tested whether it might be possible to isolate MSC from far smaller tissue volumes. SCAT specimens from healthy donors which underwent to plastic surgery operations (n=5; mean weight ± SD=116.3±91.9 mg) were processed with a protocol modified from Zuk et al. 2001. The released cells were filtered, centrifuged and seeded with expansion medium. Solely small, undifferentiated and adherent proliferating fibroblasts were soon evident in the cultures and cells reached the first confluence within 12.6±5.1 days. After 40 days of expansion, the SCAT-MSC displayed a robust proliferative potential, with a cell count of 1.32±0.53x108. FACS analyses showed that SCAT-MSC were positive (>95%) for CD90, CD105, CD73 and negative (<1%) for CD45, CD14, HLA-DR, CD31 and 3G5. CD34 antigen was expressed (<50%) in 2 samples. In vitro differentiation assays successfully drove the cells towards osteogenic, adipogenic and chondrogenic phenotypes. Long-term cryopreservation (2 years) did not affect both phenotype and functional properties. Additionally, SCAT-MSC were gene-modified (57±12%) by retroviruses encoding for GFP and subcutaneously injected into NOD/SCID mice without evidence of uncontrolled proliferation. In 20% of these mice, the transplantation of GFP+ cells was followed by the finding of human derived SCAT three months after the trasplants. We here report a novel and safe approach to isolate multipotent cells from small SCAT specimens to be used as platform for cell based therapies with minimal donors’ discomfort.
CD133 Immunomagnetic Separation: How Effective Is the Method for CD133+ Cell Isolation from Umbilical Cord Blood?.
Authors: Pelagiadis Iordanis , Dimitriou Helen , Kalmanti Maria
Published: October 2019

Umbilical cord blood (UCB) is a rich source of stem cells. Characterisation of stem cell subpopulations residing in UCB requires specific stem cell markers and reliable and reproducible protocols. We assessed the feasibility of CD133 positive cells’ isolation using immunomagnetic cell sorting protocols. The separation was based on the manufacturer’s instructions (Myltenyi Biotec). We compared these results with the corresponding ones of CD34 isolation. Two additional protocols were also applied to increase the efficiency of the CD133+ yield. The first one included an extra-labeling of the selected CD133 subpopulation from the first column with CD133 microbeads before their application to the second column. The second protocol was an indirect labelling procedure, using CD133-PE monoclonal antibody and either anti-PE microbeads or goat anti-mouse IgG microbeads as well as a combination of them. The CD133 isolation was performed in 18 UCB samples, while the CD34 in 17 samples. The volume of the samples used for CD133+ isolation was 38.5 ± 2.1ml and the respective one for the CD34+ protocol was 40.4 ± 2.8ml. The mononuclear cell fraction had 1.29 ± 0.16 x108 cells and 0.53 ± 0.06% of them expressed CD133. The corresponding values for samples used for CD34 isolation were 2.8 ± 0.3 x108 mononuclear cells and 1.64 ± 0.15% CD34. Following the manufacturer’s instructions for CD133 isolation, the number of cells obtained was 21.33 ± 3.3x104 (median 18 x104) and CD133 expression ranged from 10–85% (median=60%). Concerning the CD34 isolation, the number of cells obtained was 54.3 ± 2.1x104 and 93 ± 1.25% expressed CD34. Applying the extra-labeling method, the number of cells obtained was 8±4 x104 and these cells were CD133+ at a percentage of 32.2±4.1%. Applying the indirect immunomagnetic separation the results were as follow: using anti-PE microbeads only, 3 ± 3 x104 cells were retrieved and the percentage of CD133+ was 13.8±9.7. With the use of goat anti-mouse IgG microbeads the cell number obtained was 13±1 x104 of which CD133+ were just 2.0± 2%. In the combination method, the cells initially isolated with anti-PE microbeads and subsequently with goat anti-mouse IgG microbeads were 20 x 104, of which only 18.5% were CD133+. Finally, labeling the cells initially with anti-PE microbeads and consecutively with goat anti-mouse IgG microbeads followed by column isolation yielded 16x104 cells and 19.1% CD133 positive. In conclusion CD34 isolation by the immunomagnetic method results in highly pure CD34+ population confirming the reliability and reproducibility of the method. On the other hand, with the currently available CD133 isolation kit, efficient and reproducible yield of pure CD133 positive population is not feasible.
Stem Cell Recovery Following Implementation of an Automated Cord Blood Processing System in a High Volume Laboratory.
Authors: Harris David T. , Paulson Ingrid E. , Brown Heather , Rosenthal Joseph
Published: October 2019

Background: Many cord blood (CB) processing facilities are transitioning to automated systems to standardize methods and decrease human error. The AutoXpress Platform™ (AXP™) is an automated, functionally closed, sterile CB processing system. When placed within the AXP device and centrifuged, the whole blood product is separated into its composite cell populations and the TNC fraction of the CB unit is separated and automatically delivered into a blow-molded freezing bag at a uniform volume of 21 mL. Objective: This study evaluated the use of the AXP system in a directed donor (DD) family cord blood bank, targeted at producing consistently high TNC and MNC recovery rates regardless of broad variability in collection volume.

Methods: Cord blood units were collected between 12/5/2006 and 2/24/2007 from 1414 consenting mothers who elected to preserve and bank CB at Cord Blood Registry (CBR). Collection kits were provided at the time of enrollment, and after delivery, CB was collected from the umbilical cord and transported to CBR’s processing facility in Tucson, Arizona. During the study period, all units arriving at the CBR laboratory were allocated to either ficoll or AXP processing based on the volume, age, and the degree of clotting in each unit. Units processed using AXP had a volume of 40–130 mL, an age of less than 48 hours since collection, and a clotting score of 0 to 2+ (based on an internal scale). TNC and MNC counts were measured both pre- and post-processing, using the Sysmex analyzer.

Results: The mean age of cord blood units arriving at the laboratory was 23.59 hours, and the mean collection volume was 72.93 mL (±18.10mL). The mean TNC count post-processing was 9.94x108, the TNC percent recovery was 96.19%, and the mean MNC percent recovery was 98.65%.

Volume (mL) Mean Post-Processing TNC (x108) TNC Percent Recovery (%) Mean Post-Processing MNC (x108) MNC Percent Recovery (%) Mean 72.93 9.94 96.19 4.05 98.65 Standard Deviation 18.10 4.43 11.87 1.59 9.16

Conclusions: AXP automated cord blood processing provides consistently high TNC and MNC recovery rates, which has important implications for stem cell dose if the sample is used in transplant. Because limited cell dose is frequently cited as an obstacle to CB transplantation, processing results could impact the usability of each unit. Because DD family CB banks process all units, regardless of collection volume, percent recovery becomes particularly important in evaluating the differences between processing centers. The AXP system yields the highest published cell recovery rate to date and can be easily integrated into a CB processing center such that it decreases the labor and time required for processing while maintaining MNC recovery of greater than 98%.
Characterization of Ex Vivo Expanded Cord Blood Graft Treated with Chromatin Modifying Agents for Potential Clinical Use.
Authors: Yoshinaga Kazumi , Mahmud Dolores , Baluchamy Sudhakar , Parajuli Ritesh , Hiroto Araki , Lindgren Valerie , Rondelli Damiano , Mahmud Nadim
Published: October 2019

The lack of optimal number of repopulating hematopoietic stem cells (HSC) and graft failure following transplantation remain as obstacles to using cord blood (CB) as a true alternative graft for adult patients. Using chromatin modifying agents {5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA)}, we have developed a culture regimen that permits expansion of primitive CB CD34+ CD90+ cells resulting in a 10 fold expansion of in vivo repopulating cells without jeopardizing their multilineage differentiation capacity. Our results also indicate that the chromatin modifying agents likely mediate this function by inducing expression of endogenous genes (HoxB4, Bmi-1, GATA-2, etc.) previously implicated in self-renewal of HSC (Araki H et al. Blood2007:109:3670–3678). In our current studies we have further characterized the allostimulatory function of these expanded CB to assess its potential as grafts to mount an immune response. The percentage of 5azaD/TSA treated expanded CB cells co-expressing HLA-DR and CD86, or CD11c was less than cells expanded in cytokines alone. The expression of HLA-DR/CD40 was not altered following 5azaD/TSA treatment. In a mixed lymphocyte reaction (MLR), the irradiated CB cells (stimulator) were co-cultured with allogeneic peripheral blood T cells (responder) at 1:2 ratios. The MLR results indicate that 5azaD/TSA treated expanded CB grafts possessed diminished allostimulatory response as revealed by their relatively lower stimulation index in comparison to cells expanded in cytokines alone. Interestingly, the stimulation index of 5azaD/TSA treated cells (day 9) was even less than unmanipulated primary CB CD34+ cells (day 0). The reduced allostimulatory capacity of 5azaD/TSA expanded CB cells may favor in the reduction of incidence of graft rejection / graft failure following infusion. Alterations in cell fate regulatory mechanisms raise concerns for tumorigenesis, so the safety of such grafts was further assessed by examining the telomere length and cytogenetic analysis. Both cytogenetic analysis and examination of cell-cycle showed normal karyotype and diploid nature of expanded grafts, respectively. The telomere length of ex vivo expanded grafts as a putative indicator for their proliferative potential did not reveal significant alteration in their length in comparison to unmanipulated CB grafts. Also, in vivo repopulation assays with xenogeneic NOD/SCID mouse model showed no apparent tumor development following transplantation of ex vivo expanded 5azaD/TSA treated CB cells. In summary, our results indicate that the chromatin modifying agents not only result in expansion of in vivo repopulating CB cells but are capable of suppressing immunostimulatory capacity of expanded grafts and these grafts retain their telomere length and normal ploidy and can be expanded from a single unit for potential clinical use.
Reduced Intensity Allogeneic Stem Cell Transplantation in Patients with Myelodysplastic Syndrome: Does the Conditioning Matter?.
Authors: Terriou Louis , Chir Zina , Esperou Helene , Boiron Jean-Michel , Vernant Jean-Paul , Gratecos Nicole , Michalet Mauricette , Milpied Noel , Jouet Jean-Pierre , Yakoub-Agha Ibrahim ,   Societe Francaise de Greffe de Moelle Osseuse et de Therapie Cellulaire
Published: October 2019

Allogeneic transplantation following a reduced-intensity conditioning (RIC) has emerged as an alternative to myeloablative transplantation in pts with myelodysplastic syndrome (MDS). Given the uncertainty regarding the most appropriate conditioning regimen, SFGM-TC conducted a retrospective multicenter study in the attempt to evaluate the impact of conditioning on pts’ outcome. The record of 61 pts (37 males) with MDS who received a RIC were reviewed. The median age was 55 years (range: 18–70). According to the FAB classification, 11 pts had RA at diagnosis, of whom one had progressed to REAB and one to AML before transplantation. Thirty two pts had REAB at diagnosis, of whom 2 had progressed to REAB-T and 7 to AML before transplantation. Twelve pts had REAB-T at diagnosis and 6 CMML, of whom 8 progressed to AML before transplantation. The median time from diagnosis to RIC was 12 months (6–129). Conditioning consisted of Fludarabin (Flu) plus busulfan (FB; n=29), Flu plus 2-Gy TBI (F-TBI; n=20) and idarubicin plus aracytine and Flu (FlagIda; n=12). Donors were HLA-identical siblings (n=52) and HLA-matched unrelated (n=9). All pts received peripheral blood stem cells. The median of CD34+ infused cell dose was 5 x 106/kg (0.5–17.3). The median follow-up was 44.7 months (21–85). Estimated 3-year overall survival (OS), progression free survival (PFS), relapse and transplant-relapse mortality (TRM) were 35%, 27%, 66% and 30%, respectively. Neither of the 3 conditioning regimens used (FB, F-TBI and FlagIda) had impact on patients’ outcome. In multivariable analyses, while acute III/IV grade GVHD development was the only factor found to adversely influencing OS (HR=3.6; 95% CI: 1.1–12.2), chronic GVHD development was the only favourably influencing PFS and relapse ratios (HR=0.3; 95% CI: 0.1–0.7 and HR=0.2; 95% CI: 0.1–0.6, respectively). TRM was adversely influenced by male sex of recipient (HR=9.2; 95% CI: 1.5–66.6). RIC is an effective treatment in MDS patients irrespective of conditioning type. While acute III/IV grade GVHD appeared to be detrimental, the benefit effect of chronic GVHD was to be bound to GVL effect as demonstrated by the improvement of PFS and relapse rates in patients who developed chronic GVHD. This study shows no impact of conditioning in pts’ outcomes. New approaches with focus on immunosuppressive treatment are needed to enhance the GVL effect with an acceptable risk of GVHD.
Complete and Fast Hematologic Reconstitution with Non-Cryopreserved Stem Cell after a 5 Days BEAM Conditioning and Autologous Stem Cell Transplantation.
Authors: Karduss Amado J. , Jimenez Alejo , Gomez Luis R. , Reyes Pedro , De-Ossa N Hilda
Published: October 2019

There are several reports published in the medical literature addressing the roll of non-cryopreserved stem cells for supporting autologous transplant in lymphoma patients. However, in most of them, the conditioning used was a modification of BEAM or CBV with important differences in dose intensity or, in fact with different drugs. We present our experience using non cryopreseved stem cells and BEAM, but without decreasing the dose intensity of it. From Jun 2004 to May 2007, after a signed inform consent, 20 patients were transplanted; 16 men, 4 women, the median age was 37 years (range 15–57), 9 with Hodgkin lymphoma, 10 with large cell lymphoma and 1 with Burkitt. 13 of them were in second complete remission and 7 in third or partial remission. The mobilization was done with filgrastin 5 mg/kg/BID without chemotherapy and in the 5th and 6th day a leukopheresis of 3 or 4 blood volume was performed, the cells obtained were stored at 4oC for 6 days, in the case of the first leukopheresis, and for 5 days in the case of the second leukopheresis The administration of BEAM began in the evening of the last leukopheresis day and it was completed in 5 days as follow: BiCNU 300 mgs/m2 on day −5, etoposide 150–200 mgs/m2 BID for 6 doses on days −5 to −2, citarabine 300–400 mgs/m2 /day from days −4 to −2 and melphalam 140/mg/m2 on day −2. The stem cells were re-infused on day zero after 6 days of refrigeration for the first leokopheresis, and 5 days for the second. All patients received filgrastin from day + 5 until neutrophil recovery or pegfilgrastin one dose The mobilization and recollection were successful in 19 out of 20 patients with a median CD34 of 2.3 x 10(6)/kg (range 1–6) but in one was necessary obtain also bone marrow stem cell. The median viability of the cells after 6 days of refrigeration, measured by trypan blue, was 82% (range 66–90). The median time to achieve an absolute neutrophil count greater than 500 /ml in all patients was 12 days (range 9–20); 18 out of 20 were evaluable for thrombopoesis, they obtained a self sustained platelets count of 20.000 or more at a median of 15 days (range 9–30). The tolerance of the 5 days BEAM was good, one patient died from weak capillary syndrome on day + 18 and another from pneumonia on day + 16 there were no other grade III or IV non hematologic toxicity. With a follow of 3 to 36 months 13 out of 20 patients are alive. We conclude that the use of non-cryopreserved stem cell for supporting autologous transplant after a 5 days BEAM conditioning is feasible, safe, and produce a rapid, complete and sustained hematopoiesis. This simple procedure is a good alternative for saving costs.
Clinical Feature and Prognosis of 60 Patients Presenting Graft Failure (GF) after Allogeneic Stem Cell Transplantation (SCT).
Authors: Hagihara Maki , Kanamori Heiwa , Yoshiba Fumiaki , Aotsuka Nobuyuki , Tanaka Masatsugu , Mori Takehiko , Nakaseko Chiaki , Fujisawa Shin , Sakura Tohru , Uehara Taeko , Kawai Nobutaka , Matsushima Takafumi , Machida Shinichiro , Makita Hisashi , Maruta Atsuo , Sakamaki Hisashi
Published: October 2019

Background: GF after allogeneic SCT is a life-threatening event. To clarify the clinical feature of patients with GF and the feasibility of second SCT for GF, we retrospectively investigated the patients who developed GF after SCT.

Patients and Methods: we surveyed adult patients who received SCT in KSGCT between January 1994 and December 2005. Primary and Secondary GF were defined as a failure of absolute neutrophil count (ANC) ≥ 0.5x109/l for at least 3 consecutive days and a decrease of ANC < 0.5x109/l for at least 7 days consecutive days after initial engraftment, respectively.

Results: Of the 1,963 patients, 60 patients (3.1%) were identified with GF. Their median age was 47 years (range, 16 to 63). There were 52 patients with hematological malignancies and 8 with aplastic anemia. The incidence of GF according to donor source was 9/597 patients (1.5%) in related donor-bone marrow (RBM), 1/359 (0.3%) in related donor-peripheral blood stem cells (RPBSC), 18/839 (2.1%) in unrelated donor-bone marrow (UBM), and 32/168 (19%) in unrelated cord blood (UCB). GF in UCB was significantly higher than that in other donors (P<0.001). Primary GF and secondary GF were 45 patients (2.2%) and 15 (0.7%), respectively. In comparison between patients developing primary GF and those developing secondary GF, UCB transplant (69% vs 7%, P<0.005), malignant disease (98% vs 53%, P<0.005), high risk at transplant (61% vs 13%, P=0.009), and HLA mismatch (76% vs 13%, P<0.005) were significant risk factors. Overall survival (OS) at 3 years was 18% in all patients with GF. Of the 60 patients, 33 patients (55%) underwent second SCT. The median interval between the first and second SCT was 49 days (range, 18 to 1204). Second SCT were performed with RPBSC in 12 patients, UBM in 4, and UCB in 17. Conditioning regimen were non-myeloablative in 29 patients, myeloablative in 2, and no conditioning in 2. Total body irradiation (TBI) was used in 11 patients. Bone marrow engraftment after second SCT was obtained in 22 patients (67%) and one third of the patients again experienced GF. Engraftment in patients with TBI (2–12 Gy) was significantly better than that in those without TBI (100% vs 50%, P=0.004), although there were no difference in donor sources, underlying diseases and use of antithymocyte globulin. OS at 3 years in patients with second SCT and in the others without second SCT was 53% and 9.1%, respectively (P=0.005), Major cause of death in both group was infectious complications.

Conclusions: High frequency of GF in patients with UCB was confirmed. It was not clear which donor source is better for second SCT in patients presenting GF, but conditioning regimen including TBI might contribute to obtain engraftment in this setting.
The Mobilization Effects of G-CSF, GM-CSF and Darbepoetin-α for Allogeneic Peripheral Blood Stem Cell Transplantation.
Authors: Kim Shi Nae , Sohn Sang Kyun , Kim Jong Gwang , Kim Yee Soo , Cho Yoon Young , Moon Joon Ho , Ahn Byung Min , Lee Soo Jung , Kim Yun Jeong
Published: October 2019

Background: Granulocyte colony-stimulating-factor (G-CSF) is widely used for the mobilization of hematopoietic stem cells from normal donors. It has been reported that the addition of granulocyte-macrophage CSF (GM-CSF) to G-CSF mobilized more primitive CD34+ subsets than did G-CSF alone. We investigated the effects of combination regimen (GM-CSF + G-CSF + darbepoetin-α) for mobilization in normal healthy donors and compared its efficacy in mobilizing stem cells with G-CSF alone.

Methods: Thirty donors were randomly assigned to one of the following regimens for mobilization: G-CSF group (G-CSF 5ug/kg/day for 5 to 7 days) and combination group (GM-CSF 10ug/kg/day on 1st and 2nd days, G-CSF 5ug/kg/day for 5 to 7 days and 40mg of dalbepoetin-α on day 1)

Results: The counts of MNC and CD34+ cells/kg were not significantly different between two groups (p=0.129 and p=0.651). The CD3+ cell counts per kilogram were 3.48±1.45×108/kg and 2.54±0.94×108/kg in a G-CSF group and a combination group, respectively (p=0.04). Red blood cell transfusion requirement was not different between two groups (p=0.469). The median time to neutrophil and platelet engraftment was 12.5 vs. 12.0 days (p=0.443) and 11.5 vs. 11.0 days (p=0.752). Red blood cell recovery was 7.5 days and 7 days in G-CSF group and combination group, respectively (p=0.231). Grade II–IV acute graft-versus-host disease (GVHD) was observed in 20% in G-CSF group and 13% in combination group (p=0.50).

Conclusion: The combination regimen was not superior to G-CSF alone in terms of acute GVHD and CD34+ cell dose. But CD3+ cell count was significantly lower in combination regimen group. The role of lower CD3+ cells in the graft need to be elucidated in the future.
Protective Abilities of Interleukin-10 in Blood-Induced Joint Damage.
Authors: Jansen Nathalie W.D. , Van Roon Joel A.G. , Roosendaal Goris , Theobald Matthias , Bijlsma Johannes W.J. , Lafeber Floris P.J.G.
Published: October 2019

PURPOSE: Joint bleeds occur frequently in patients suffering from haemophilia or can occur as a consequence of joint trauma. Even a limited number of bleeds has been demonstrated to lead to severe joint damage in time (1,2). With respect to the mechanisms of blood induced joint damage, we have demonstrated that monocytes/macrophages within the mononuclear cell population together with the red blood cells as present in blood are responsible for the irreversible inhibition of cartilage matrix synthesis. Small amounts of IL-1 that are produced by activated monocytes/macrophages increase the production of hydrogen peroxide (H2O2) by chondrocytes. The hydrogen peroxide reacts with haemoglobin-derived iron from damaged and phagocytosed red blood cells, which results in the formation of hydroxyl radicals in the vicinity of chondrocytes. This leads to chondrocyte apoptosis and with that the irreversible inhibition of cartilage matrix synthesis (2). In search of possible interventions to prevent or limit the cartilage damaging effects of joint bleeds, we tested interleukin-10 (IL-10) as an inflammation controlling cytokine on blood-induced cartilage damage.

METHODS: Healthy human articular cartilage tissue explants were cultured in the presence or absence of 50% v/v blood for 4 days, followed by a recovery period of 12 days. IL-10 was added in 0.1, 1 or 10 ng/ml. Also cartilage and synovium in case of haemophilic arthropathy, obtained at joint replacement surgery, was cultured in the presence of IL-10 (10 ng/ml). The effect on cartilage matrix proteoglycan synthesis, -release, and -content were determined as well as the IL-1β and TNFα production by the synovium.

RESULTS: IL-10 was able to prevent the decrease in proteoglycan synthesis and the increase in proteoglycan release of cartilage exposed to blood dose dependently (p<0.05). As a consequence, the decrease in proteoglycan content after blood exposure could be prevented dose dependently (p<0.05). With 10 ng/ml IL-10, which is still a low dose for local therapeutic treatment, at least 50% reduction in adverse effects was observed (p<0.05). The matrix turnover of haemophilic cartilage improved upon addition of IL-10 and also the production of IL-1β and TNFα by haemophilic synovium decreased in the presence of IL-10 (all p<0.05).

CONCLUSIONS: The present results show that interleukin-10 prevents the direct harmful effects of blood on articular cartilage and has a beneficial effect on the matrix turnover of haemophilic cartilage and pro-inflammatory cytokine production by haemophilic synovium. Therefore, although treatment studies in addition to prevention studies have to be performed; IL-10 might be worthwhile to test in clinical practice for local treatment of joint haemorrhages, to prevent joint damage in time.
Single-Institute Comparable Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation from Unrelated and Related Donor in Patients with Myeloid Malignancies.
Authors: Yano Shingo , Usui Noriko , Dobashi Nobuaki , Takei Yutaka , Yahagi Yuichi , Takahara Shinobu , Otsubo Hiroko , Yamaguchi Yuko , Kasama Kinuyo , Minami Jiro , Kobayashi Tatsunosuke , Hoshi Yasutaka , Kobayashi Tadashi , Asai Osamu , Aiba Keisuke
Published: October 2019

Background: Allogeneic stem-cell transplantation (allo-SCT) from human leukocyte antigen identical related donor (RD) is taken priority over unrelated donor (URD), however, less than 30% of patients do not have a suitable RD. Transplantation from an URD should be considered for those patients. In order to investigate the role of URD, we retrospectively analyzed efficacy and safety of allo-SCT from URD and RD in patients with myeloid malignancies.

Patients and methods: One hundred six patients with myeloid malignancies (acute myeloid leukemia 48, chronic myeloid leukemia 38, and myelodysplastic syndrome 20) who received myeloablative regimens were analyzed. The stem cell source were bone marrow from URD (n=43) and peripheral blood stem cells or bone marrow from RD (n=63). Graft-versus-host disease (GVHD) prophylaxis consisted of taclorimus and short-term methotrexate (sMTX) for URD, or cyclosporine and sMTX for RD.

Results: Median follow-up was 6.3 (0.3–17.2) years, and median age of patients was 37 (15–55) years old. Hematological studies demonstrated donor engraftment in all patients. Incidence of grades II to IV of acute GVHD was 32% in URD and 18% in RD, and extended chronic GVHD was 31% in URD and 28% in RD. Probability of 8-year actuarial survival was 62% in URD and 54% in RD (p=0.4225), and 8-year disease-free survival was 64% in URD and 47% in RD (p=0.1412). There were no apparent differences in transplant-related mortality (TRM) (14% in URD and 24% in RD), relapse (14% in URD and 32% in RD) between both groups.

Conclusion: These results suggested that allo-SCT from URD has comparable safety and effectiveness for adult patients with myeloid malignancies.
Multi-Center Prospective Trial on Conditioning with Treosulfan, Fludarabine and ATG Before Allogeneic HSCT from Unrelated Donors.
Authors: Michallet Mauricette , Le Quoc-Hung , Thiebaut Anne , Ducastelle Sophie , Nicolini Franck E. , Raus Nicole , Tabrizi Reza , Bouabdallah Krimo , Blaise Didier , Mohty Mohamad , Faucher Catherine , Furst Sabine , Bay Jacques-Olivier , Hermet Eric , Milpied Noel
Published: October 2019

This prospective multi-center trial (MiniMud) concerns 27 patients (13 females, 14 males - 56 years[18–65]) who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from unrelated donors. The diagnosis and disease status pre-transplant were AML (n=12; 5 CR1, 7 CR2), ALL (n=1; 1 CR2), MM (n=5; 3 PR2,1 PR3, 1 PR4), NHL (n=4; 1 CR1, 2 PR3, 1 PR4), MPS (n=3; 2 stable disease (SD), 1 Relapse), acute RAEB (n=1; 1 progressive disease (PD) and CLL (n=1; 1 PR2). In total, 14 patients were in CR, 9 in PR, 2 in SD, 2 in PD. The median interval between diagnosis and transplant was 27 months [5;171], 24 patients received PBSC and 3 received bone marrow from unrelated 10/10 antigen HLA-identical (high-resolution level). All patients received Fludarabine 30 mg/m2/dx5d, Treosulfan 12 g/m2/dx3d and ATG 2,5 mg/kg/dx3d. After transplant, we observed 2 graft failures, 7 patients developed an AGVHD ≥ grade II (30%). Only 23 patients were eligibile to be analysed after 3 months of follow-up post transplant, 7 patients developed cGVHD (30%) (5 limited and 2 extensive), 12 patients died (7 from TRM causes and 5 from relapse) and 12 patients are alive in CR with a median follow-up of 14 months. Among the global population we defined a low risk subgroup: 17 patients [12 AML (5 CR1, 7 CR2), 1 ALL (CR2), 3 MM (3 PR2) and 1 NHL (CR1)], 2 patients presented an AGVHD ≥ grade II (13%). With a median follow-up of 18 months, 2 patients had a follow-up of less than 3 months, 3 patients developed a cGVHD (20%), 6 patients died (40%) and 9 patients are alive in CR. At one year, the probability of overall survival (OS) and event-free survival (EFS) were 50.4% [32;79], 40% [23;70] for the total population and 60% [37;96], 51.5% [29;90] for the low risk subgroup respectively. To try to demonstrate if Treosulfan allowed a better transplant outcome we performed a paired match-analysis [center and 4 out of 5 other parameters (age, gender, HSC, pre-transplant status and diagnosis] comparing our Treosulfan series and RIC transplants receiving Fludarabine, Busulfan and ATG (2.5–12.5mg/kg) from the SFGM-TC registry. This paired match-analysis found only RICT from related donors in the SFGM-TC database and showed no difference in term of OS and EFS between our unrelated donor-Treosulfan and related donor-SFGM-TC series.

Figure Figure Figure Figure

In conclusion, these results demonstrate that Treosulfan appears to be a very promising drug that could be included in the conditioning regimen before HSCT from either related or unrelated donors.
Preliminary Results of Phase II Trial of Clofarabine with Parenteral Busulfan Followed by Allogeneic Related or Unrelated Donor Transplantation for the Treatment of Hematologic Malignancies and Diseases.
Authors: Agura Edward D. , Berryman R. Brian , Fay Joseph W. , Pineiro Luis A. , Vance Estil A. , Tadic-Ovcina Mirjana
Published: October 2019

Background: Nonablative transplant regimens are useful to treat patients who are elderly or too ill to undergo a full-intensity ablative allogeneic transplant regimen. However, this approach is limited by a higher relapse rate in some situations. We developed a novel conditioning regimen using the nucleoside analogue clofarabine (CLO) with busulfan (BU) to treat elderly patients with acute leukemia or high risk MDS who are not in remission or are at high risk of relapse.

Methods: Five patients were enrolled on this single institution; phase II, IRB-approved trial, so far. The diagnoses at the time of transplant were two with AML, one with ALL, and two with MDS. All patients received CLO 40mg/m2/day iv on d-7 through d-4 followed by BU 3.2 mg/m2/day iv on d-3 and -2, followed by infusion of HLA matched related or unrelated donor PBSC on day 0. GVHD prophylaxis consisted of oral FK506 and MTX 5mg/m2 iv d + 1, +3 and +6. BU and CLO pharmacokinetic samples were collected on all patients for later PK analysis.

Results: Primary endpoints included toxicity and response to therapy. There were no cases of hepatic, cardiac, or renal toxicity attributed to the conditioning regimen by d+30. Acute toxicities included hand/foot syndrome, n=2 (one grade 1, and one grade 3 by CTCAE v 3.0); fluid retention, n=3 (2 grade 1 and 1 grade 2). All patients had hematologic nadirs lasting 12 to 15 days. GCSF was used in 4 of 5 subjects. 5 of 5 patients achieved allogeneic engraftment with myeloid cells by day +30. Response to therapy was documented in 4 patients by d+30, one is not yet evaluated. One (1) patient relapsed by d+62. 3 of 4 remain in remission with follow up ranging from 60 to 117 days.

Conclusions: CLO + BU is a novel allogeneic conditioning regimen that seems to be well tolerated by the small number of subjects treated so far at our institution. The follow up is not long enough to conclude more at this time. The study continues to accrue patients and will be updated at the meeting.
Intra-Arterial Catheter Directed Immunosuppressive Therapy for Steroid Resistant or Dependent Graft vs. Host Disease (GVHD).
Authors: Shapira Michael Y. , Bloom Allan I. , Or Reuven , Resnick Igor B. , Aker Memet , Slavin Shimon , Verstandig Anthony
Published: October 2019

Introduction: Prolonged treatment of graft vs. host disease (GVHD) is extremely immunosuppressive. Local therapy with intra-arterial (IA) injection of steroids may induce remission with lower extent of systemic immune suppression. Here, we present our experience with IA treatment of gastrointestinal (GI) and/or hepatic steroid resistant/dependent GVHD with 2 consecutive protocols.

Patients and methods: Thirty five patients (37 GVHD events (hepatic, n=15), (GI, n=16), (combined, n=6)) were treated with 53 IA sessions. Most side effects were minor.

Results: We found that IA steroid therapy was associated with partial and complete remission among patients with steroid resistant/dependent hepatic or GI GVHD. Hepatic partial response was observed in 14 (66.6%) patients among whom 7 (33.3%) reached complete response. GI partial response was observed in 19 (86.4%) patients among whom 12 (54.4%) reached complete response. Early administration of the local therapy, female gender, myeloid basic disease, and a non-active status of the basic disease at the day of transplantation were found related for predicting a better response for the intra-arterial treatment. The use of high dose steroids in the hepatic IA protocol from was at least as good as intermediate dose steroids with methotrexate (table 1, figure 1) and may be safer.

Conclusions: Intra-arterial catheter guided steroid therapy is safe and effective in steroid resistant/dependent GVHD. Hepatic artery treatment with methotrexate can be safely substituted with high dose IA methylprednisolone. Further research is warranted characterizing the patients benefit most.

Table 1 - comparison between 1st and 2nd hepatic IA treatment protocols 1st protocol 2nd protocol Significance Median age (range) 25 years (7–42) 32 years (18–59) P=0.09 Sex (M:F) 6:1 8:5 NS family donor vs MUD 6:1 9:4 NS Median time in days SCT-GVHD (range) 27 (13–133) 45 (13–248) NS Median time in days GVHD-IA (range) 15 (6–218) 190 (12–2615) P=0.09 Median peak GVHD grade (range) 3 (3–4) 3 (2–4) NS Highest pre-IA treatment bilirubin level (in mmol/L; normal<17) (range) 186 (138–321) 225.5 (83–672) NS Median time to initial response in days (range) 14.5 (4–100) 8 (1–31) P=0.073 Median time to complete response in days (range) 130.5 (35–226) 49 (17–80) Figure 1A, Figure 1B Figure 1A, Figure 1B.
Increased Interleukin-17 Proportion in Helper T Cell (Th17) in Patients Post Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Lange Andrzej , Dlubek Dorota , Sedzimirska Mariola , Drabczak-Skrzypek Daria , Jaskula Emilia , Lange Janusz
Published: October 2019

Cytokines belonging to interleukin -17 family (IL-17A, B, C, D, E and F) are produced mainly by activated and memory CD4+ cells which are described as Th17. These cells (IL-17+CD4+) promote autoimmune reaction (mouse model), were found in tissues involved in autoimmune process in man and produce IFNgamma. Therefore, Th17 cells constitute a population which may contribute to aGvHD together with Treg cells (FoxP3+). In this study we examined the cytoplasmic expression of IL-17A and FoxP3 in CD4+ lymphocytes in 6 pts post HSCT (3 MUD and 3 SIB, median (mean) age: 35 (33,9) years, 3 AML, 2 ALL-T, 1 SCID). Three of them developed aGvHD. All consecutive pts were included to the study. Peripheral blood lymphocytes were examined or at the very first day of aGvHD manifestation either at the beginning of hematological reconstitution and thereafter in one week intervals. Nine healthy individuals served as controls. PBMC were stimulated with BD Leukocyte Activation Cocktail containing mixture of PMA, Ionomycin and brefeldin A in the presence of BD GolgiStop then stained with CD4, IL-17A and FoxP3 monoclonal antibodies and analyzed in CD4+ and CD4- lymphocytes gates by three colour flow cytometry. Therefore, all results reflect the situation in stimulated cells. FoxP3 mRNA was measured in unstimulated PBMC with the use of qPCR. We found:

Patients post HSCT (during 6 weeks period post transplant) had higher percentages of IL-17A+ cells in CD4+ lymphocytes as compared to healthy controls (2.0%±1.08 vs 0.2%±0.06, p=0.006) independently whether they developed or not aGvHD. In contrast to the lower proportion of total CD4+ cells in pts post HSCT then in controls (18.5%±2.1 vs 34.9%±0.7, p=0.0006) proportions of a subset of CD4+ lymphocytes which produced IL-17A+ were rather higher in pts post HSCT as compared to controls (0.2%±0.06 vs 0.07%±0.02, ns). IL-17A+ cells contributed rather to CD4+ then CD4- lymphocytes in controls (0.2%±0.06 vs 0.05%±0.01, p=0.007) as well as in pts post HSCT (2.0%±1.08 vs 0.05%±0.01, p=0.00004) FoxP3 positive cells in stimulated CD4+ lymphocytes were in lower proportions in pts post HSCT then in healthy individuals (51.6%±3.9 vs 62.2%±2.9, ns). Percentages of FoxP3+CD4+ in stimulated lymphocytes (cytometry) correlated with quantities of FoxP3 mRNA in unstimulated PBMC (qPCR) (R=0,42, p=0,065). Notably, there was a negative correlation between proportions of FoxP3 and IL-17A positive cells in CD4+ lymphocytes in patients post HSCT (R= − 0,55; p=0,012).

In conclusion, HSCT was associated with:

an increase in proportion of Th17 cells, a higher contribution of IL-17A+ cells to the pool of CD4+ cells as compared to healthy individuals and a network in the immune system in which CD4+ FoxP3 + cells and Th17 cells play an opposite role.
Impact of Prophylaxis with Aerosolized Amphotericin-B Deoxycholate (d-AmB) on Respiratory Tract Invasive Fungal Infections (IFIs) after Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Morello Enrico , Cavattoni Irene M. , Fabris Pietro , Coin Silvia , Amato Barbara , Cassibba Vincenzo , Casini Marco , Pagani Leonardo , Cortelazzo Sergio
Published: October 2019

Background and aims IFIs still pose major challenges in HSCT, and effective prophylaxis remains a matter of debate. The aim of this retrospective study was to evaluate the impact of aerosolized d-AmB on respiratory tract IFIs in a homogeneous cohort of allogeneic HSCT patients, transplanted at our institution. Patients and methods Since 1999, 81 consecutive patients were transplanted from matched related (N=61) or unrelated donor (MUD). Analysis was performed on 74 evaluable patients, in order to monitor the prevalence of respiratory tract IFIs within 40 days after HSCT, according to current guidelines (possible, probable, proven IFIs). Conventional antifungal prophylaxis was based on the association of fluconazole (400 mg/d), plus aerosolized d-AmB (15 mg bid) in 54 out of 74 cases (73%). All the patients were screened before transplant and monitored thereafter with CT or x-rays (paranasal sinuses, thorax), surveillance swabs and galactomannan antigenemia. Chi square test was performed to evaluate correlations between variables.

Results Aerosolised d-AmB was administered to 70 patients for a median time of 15 days (range 1–45). Prolonged administration was not associated with increased severe bacterial infections, nor severe adverse events were observed; only a patient developed moderate bronchial spasm. In 13 pts, aerosolized d-AmB was delivered for less than 7 days, due to worsened clinical conditions, or poor compliance. In this group, proven IFIs were diagnosed in 2 patients (1 mucormycosis and 1 fusariosis), possible aspergillosis in one and probable aspergillosis in another one. A shortened administration (<7 days) of aerosolized d-AmB was associated with an increased risk of IFIs (p=0,002). Overall, 95% of patients did not experience IFIs and nobody died due to IFIs. Nine patients had a pre-transplant nasal swab positive for Aspergillus spp., and 8 of them received Aerosolized d-AmB; subsequent surveillance swabs proved negative. On the other hand, the only patient with positive swab who was not able to receive aerosolized d-AmB due to bronchial spasm developed a possible aspergillosis.

Discussion Despite the low number of patients, prolonged aerosolized d-AmB seems to play a role in preventing respiratory tract IFIs, but a randomised controlled trial is recommended to verify the impact of this prophylaxis in the setting of allogeneic HSCT.
Comparison of Cyclosporine A and Mycophenolate Mofetil vs Cyclosporine A and Methotrexate in Reduced Intensity Conditioning HLA Sibling Allogeneic Stem Cell Transplantation. A Case-Match Single-Center Experience.
Authors: Piñana Jose L. , Valcárcel David , Martino Rodrigo , Parody Rocio , Sureda Anna , Briones Javier , Delgado Julio , Snchez Blanca , Brunet Salut , Sierra Jorge
Published: October 2019

BACKGROUND: Graft-versus-host disease (GVHD) is the major obstacle to successful allogeneic stem cell transplantation (SCT) transplantation. Cyclosporine (Csa) in combination with methotrexate (MTX) is the most commonly used prophylactic regimen. The combination of CsA and Mycophenolate Mofetil (MMF) has recently been introduced. In this case-match study we retrospectively analyzed the introduction of MMF as GVHD prophylaxis in comparison with a short course of MTX in the setting of reduced intensity conditioning allogeneic SCT (Alo-RIC).

PATIENTS AND METHODS: We analyzed 59 Alo-RIC recipients who received an Alo-HSCT from an HLA-identical sibling between April 2000 and June 2006. The median follow-up for the whole group was 473 days (8 to 2139 days). The study group included 40 males and 19 females. Median age was 59 years (range 43 to 72). Diagnoses were acute leukemia/myelodisplastic syndrome) (n=19/22), mutiple myeloma (n= 6), chronic myeloid leukemia (n=5) and non- Hodgkin lymphoma (NHL) (n= 4). GVHD prophylaxis consisted of Csa/MTX (MTX group) in 37 patients and Csa/MMF (MMF group) in 22 patients. The conditioning regimen was based on fludarabine in combination with busulfan (42 recipients) or melfalan (17 cases).

RESULTS: The occurrence of mucositis grade 2–4 was higher in the MTX group than in the MMF group (62% vs 28% p= 0.015). No significant differences were found between MMF vs MTX groups in time to neutrophil recovery (16 +/−3 days vs 15 +/− 2days) (p= 0.5). Median time to the achievement of complete T-cell donor chimerism in the MTX and MMF groups was 79 days (range 19–740) and 76 days (range 24–223) respectively (P=0.4). The 1- year non-relapse mortality was similar in MTX and MMF groups 14% (95% C.L. 6–31%) vs 28% (95% C.L. 13–60%) respectively; P=0.2. Cumulate incidence of acute GVHD for MTX and MMF groups was 49% (95% C.L. 35–68%) and 68% (95% C.L. 51–91%) respectively (P= 0.6). Patients in the MTX group showed a trend to a higher incidence of chronic GVHD than the Csa/MMF group, 55% (95% C.L. 40–74%) vs 42% (95% C.L. 23–75%) (P=0.2). No differences were found between MTX vs MMF groups in 1-year overall survival [78%(64–92%) vs 53%(38–76%); P=0.1] nor in 1-year relapse incidence [37% (95%CI 21–64%) vs 19% (95%CI 10–37%) P=0.1) We conclude that the Csa/MMF combination appears to be equivalent to the standard Csa/MTX aGVHD prophylaxis in Alo-RIC. MMF showed significantly less mucositis.