bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Mannose Binding Lectin (MBL-2) Deficiency with Pulmonary Infection after Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Chung Dick , Ding Lily , Amigues Isabelle , Kadeishvili Katuna , Lo Theresa , Pamer Eric , Kalambakas Stacey , Buchbinder Aby , Papanicolaou Genovefa
Published: April 2020

Background: Low MBL2 concentration and MBL2 genotype variants have been associated with an increased risk of infection in various clinical settings. Pulmonary infection is a major complication of HSCT. We examined the relationship of MBL genotypes with post-engraftment bacterial (B-PNA) and fungal (F-PNA) pneumonia

Methods: Retrospective review of 236 non-consecutive, non-selected patients who underwent HSCT at MSKCC from 1/1/2000–4/30/2007. Microbiologically confirmed infections and pneumonias were recorded. Antifungal prophylaxis consisted of fluconazole 400 mg daily. Patients at high risk for mold infection received mold-active prophylaxis. After 1/1/2006 voriconazole was the first line anti-mold prophylaxis. Genotype was determined by PR-Melting Curve Analysis on blood or buccal swab specimens. MBL genotype was classified as wild-type: A/A (MBL-sufficient, MBL-S) or variant-type: A/O, O/O (MBL deficient, MBL-D). Patients were followed for up to 2 years. Statistical analysis: Fisher’s Exact test was used to compare the incidence rate between MBL-S and MBL-D patients. Multivariate logistical regression models were used to investigate the relationship between bacterial or fungal pneumonia and MBL genotype, matched related donor (MRD), myeloablative conditioning (MC) peripheral blood as stem cell source (PBSC), acute GVHD grade 2–4 (aGVHD). The results from Maximum Likelihood Estimates were summarized.

Results: Transplant characteristics: 80% MC, 76% PBSC, 48.6% MRD. Incidence of aGVHD: 22.4%. MBL genotypes: One-hundred and forty-two (60%) patients were homozygous for wild-type MBL2 (AA), 85(36%) were heterozygous (A/0) and 9 (3.8%) were homozygous for variant genotypes (OO). Transplant characteristics, rates of GVHD, relapse-free and overall survival were similar between MBL-D and MBL-S. There was higher incidence of overall bacterial infections in MBL-D compared to MBL-S pts (47.87% vs 36.62%, p=0.1049). MBL-D had a higher incidence of B-PNA (12.7% vs 4.9%, p=0.048). In multivariate logistic regression analysis, MBL-D(p=0.04) and aGVHD(p=0.06) were likely associated with B-PNA. Rates of overall fungal infections and F-PNA were similar [among MBL-D and MBL-S pts (12.77% vs 9.86%, p=0.5277) and (8.51% vs 7.75%, p=0.1049) respectively]. In multivariate logistic regression analysis only aGVHD was statistically significantly associated with F-PNA p=0.0002.

Conclusions: 1) MBL-D genotype was likely associated with increased risk of bacterial pneumonia. 2) MBL-D and aGVHD were risk factors for B-PNA. 3) Further analyses are in progress to evaluate the effect of MBL-D on fungal pneumonia in patients who received mold prophylaxis versus patients who did not receive mold prophylaxis. 4) Prospective studies are needed to assess the relative contribution of MBL-D genotype on the risk of pulmonary infection in HSCT.
Improved Overall Survival in Patients Recovering with High Gamma/Delta T Cells after Allogeneic Haematopoietic Stem Cell Transplantation.
Authors: Kordelas Lambros , Junge Matthias , Trenschel Rudolf , Elmaagacli Ahmet H , Beelen Dietrich W
Published: April 2020

T-cells play a crucial role in the Graft-versus-Leukemia (GvL)-effect, since T-cells depleted grafts are associated with a higher relapse risk. Unfortunately, the GvL-effect is often associated with Graft-versus-Host-Disease (GvHD). T-cells can be divided into two phenotypic sub-groups by the expression of specific alpha/beta- and gamma/delta T-cell receptors. Gamma/delta-T-cells might provide a useful source for T-cell-immunotherapy since they may exert a GvL-effect without inducing a GvHD-risk.

Only few studies have been carried out investigating the possible impact of gamma/delta-T-cell recovery following allogeneic hematopoietic stem cell transplantation (HSCT). A recent prospective study (Godder et al., BMT 2007) indicates a survival advantage for patients (pts) recovering with higher gamma/delta-T-cell numbers following HSCT. The data presented here emerge from a single-centre analysis evaluating the possible impact of higher gamma/delta-T-cell numbers following HSCT in pts with hematologic malignancies. We included all patients who had at least three consecutive analyses of alpha/beta- and gamma/delta-T-cell numbers within the first year after HSCT. This cohort of 107 patients includes the following haematological malignancies: AML (n=40), CML (n=19), ALL (n=13), MDS (n=11), OMF (n=9), NHL (n=7), and other diseases (n=8). Median patient age was 41 years (range 16 – 67 years). Median donor age was 38 years (range 18 – 70 years). HSCT was performed with related donors in 37 pts (35%) and with unrelated donors in 70 pts (65%).

We defined the threshold for “high” gamma/delta-T-cell recovery as three ore more absolute gamma/delta-T-cell numbers above the absolute median gamma/delta-T number in the peripheral blood within the first 12 months after HSCT. According to this threshold 29 pts (27%) recovered with “high” gamma/delta-T-cells. These pts achieved a significantly higher overall survival with lower gamma/delta-T-cell numbers (log-rank p .029). This resulted from a lower relapse risk and a lower risk for acute GvHD. In multivariate analysis including other prognostic factors of overall survival (patient age, disease status, donor type, grades of acute GvHD and relapse), the beneficial effect of “high” gamma/delta-T-cell recovery could be confirmed. In contrast, recovery of alpha/beta T-cell numbers in peripheral blood had no significant influence on HSCT endpoints and were further not associated with the recovery of gamma/delta T-cells.

This analysis supports the hypothesis of a beneficial effect of high gamma/delta-T-cells recovery following HSCT regarding overall survival. Further analyses and research are warranted to determine more accurately the importance of increased recovery of gamma/delta-T-cells to possibly develop new therapeutic options in HSCT as e.g. graft engineering.
Rituximab Prophylaxis of EBV Reactivation after Alternative Donor Transplants Following Anti-Thymocyte Globulin-Based Conditioning Regimens.
Authors: Bacigalupo Andrea , Dominietto Alida , Soracco Monica , Raiola Anna Maria , Van Lint Maria Teresa , Lamparelli Teresa , Ibatici Adalberto , Gualandi Francesca , Frassoni Francesco , Tedone Elisabetta
Published: April 2020

Background. Reactivation of Epstein Barr Virus (EBV) frequently occurs after allogeneic stem cell transplantation (ASCT) and EBV-induced lymphoproliferative disease is a potentially fatal complication.

Aim of the study. Prevent EBV reactivation and EBV-induced post-transplant lymphoproliferative disease (LPD) after ASCT from alternative donors following anti-thymocyte globulin (ATG)-based conditioning regimen.

Methods. In 2004, we initiated a pilot study with Rituximab given prophylactically on day +5 after an alternative donor transplant. We treated 68 patients with a fixed dose of 200 mg of Rituximab on day+5 and compared their outcome with a concurrent group of 77 controls who did not receive EBV prophylaxis. The two groups were comparable for patient and disease-associated variables, and all 145 patients received ATG as part of the conditioning regimen.

Results. Rituximab on day +5 was well tolerated without any serious adverse event. Patients receiving rituximab had a significantly lower rate of EBV reactivation 41% vs 78% (p=0.0001), a lower number of EBV copies at reactivation (3 vs 10, p=0.04), a lower viral load expressed as maximum number of EBV copies (79 vs 1321,p=0.001) and a significantly lower proportion of patients with EBV copies exceeding 1000 (10% vs 44% p=0.001). Time to neutrophil and platelet engraftment was not different between the 2 groups. In the group treated with Rituximab, there was a trend for a lower incidence of acute GvHD grade II-IV (21 vs 34%, p=0.07) and grade III-IV (2 vs 8%,p=0.07), and for a reduced transplant related mortality (28 vs 39% p=0.1). Actuarial survival at 1-year was better for the Rituximab group (67 vs 50% p=0.09), but this did reach statistical significance.

Conclusions. The use of Rituximab on day +5 after ASCT from alternative donors was safe and feasible. It did significantly lower the number of EBV copies at reactivation and the number of patients with an EBV load greater than 1000, at higher risk for EBV-related LPD. Thus, this approach may reduce the need of preemptive treatment or adoptive immunotherapy for EBV reactivation after an alternative donor transplant and may warrants further investigation.
Aberrant Intracellular Retention of Mutated Receptor Tyrosine Kinase FLT3 in Human Leukemic Cells as a Potential Mechanism Supporting the Pathogenesis of Acute Myeloid Leukaemia.
Authors: Koch Sina , Ryser Martin , Jacobi Angela , Brenner Sebastian , Bornhaeuser Martin , Ehninger Gerhard , Thiede Christian
Published: April 2020

The exact regulation of FLT3 expression, a type III receptor tyrosine kinase (RTK), contributes to controlled proliferation and differentiation of blood cells at different stages during hematopoiesis. Activating mutations of FLT3 are found in AML, ALL and MDS and contribute to their pathogenesis, but it is unknown how mutant FLT3 deregulates hematopoiesis. It has been reported that murine mutant FLT3 signal transduction, namely with the most frequent Internal Tandem Duplication (ITD), differs from FLT3 wild type signalling. One possible mechanism for a different signalling quality leading to pathogenic effects observed in leukaemia, might be an aberrant intracellular trafficking of mutant FLT3, like shown for the other mutated type III RTKs PDGFR and KIT, which are aberrantly localized in human cancer. The localization of mutant FLT3-ITD as well as wild type FLT3 was investigated in retrovirally transduced COS7 cells, human leukemic cells and cell lines and primary CD34+ hematopoietic stem cells, enriched by MACS technique with an anti-CD34 antibody. Subcellular distribution of FLT3 was determined by confocal laser scanning microscopy. Internalization kinetics was studied by fluorescently labelling the ligand (FL) and time lapse imaging. The effects on signal transduction were investigated by Western blot analysis of protein phosphorylation for FLT3 and downstream targets (ERK, STAT5, PIM1). FLT3 localized to the plasma membrane, whereas mutant FLT3- ITD accumulated in the trans Golgi network (TGN), and not as previously speculated in the Endoplasmic Reticulum. FL was not internalized in FLT3-ITD expressing cells, while internalization in FLT3 expressing cells started at around 4 min. This observation is consistent with the non-surface localization of FLT3-ITD. Inhibition of Golgi specific glycosylation by Swainsonine led to TGN retention of FLT3. The STAT5 pathway was FL-independently activated only by FLT3-ITD. TGN accumulation of human FLT3- ITD, possibly caused by immature glycosylation, and the lack of FL internalization might support FL-independent autonomous signalling. The TGN environment may provide interactions with additional/different substrate(s), altering subsequent signalling quality (as shown here for STAT5) and thereby favouring uncontrolled proliferation of undifferentiated cells.
Newly-Diagnosed Versus Non-Initial Episodes of Thrombotic Thrombocytopenic Purpura: A Comparison of Presenting Clinical Characteristics and Response to Treatment.
Authors: Alvarez-Larrán Alberto , Río-Garma Julio del , Muncunill Josep , de la Rubia Javier , Hernández-Jodrá Manuel , Pujol Misericordia , González-Porras José , Mateo José , Rodríguez-Vicente Pilar , Viejo Aurora , Peña Francisco , Contreras Enric , Pereira Arturo
Published: April 2020

The remission rate with plasma exchange (PE) in thrombotic thrombocytopenic purpura (TTP) exceeds the 80%, but the disease relapses in up to 20–30% of the cases. Clinical characteristics and response to treatment of relapsed TTP is not well defined. The objective of the present study was to analyze the presenting clinical and biological features of relapsed TTP as compared with de novo TTP in a series of 102 TTP episodes (70 de novo and 32 relapses) followed prospectively and treated by daily PE and corticosteroids according to a homogeneous protocol. In comparison with de novo TTP, relapsed TTP episodes showed a higher Hb level (122 g/L versus 91 g/L, p<0.001) and lower serum LDH (2.2 versus 4.5 fold above the upper limit of normality, p<0.001). Neurological symptoms and fever were less frequent in patients with relapsed TTP than in patients with de novo TTP. There were no statistically significant differences in the percentage of patients with a severe deficit of ADAMTS13 activity (72% in de novo TTP versus 81% in relapsed TTP, p not significant) or the presence of inhibitory autoantibodies (87% versus 81%, p not significant). Patients with relapsed TTP needed fewer PE sessions (5 versus 10, p= 0.02) and a smaller volume of plasma (221 ml/kg versus 468 ml/kg, p=0.004) to achieve remission than those with de novo TTP. There were no significant differences in the rate of recrudescence under treatment, the need for complementary treatments (e.g. rituximab or splenectomy) or the frequency of refractoriness to PE therapy. In conclusion, relapsed TTP presents with a milder clinical and biological profile and responds easier to PE than de novo TTP.
Comparison of High-Dose Cyclophosphamide and Growth Factor with Growth Factor Alone for Mobilization of Stem Cells for Transplantation in Patients with Multiple Myeloma.
Authors: Gertz Morie Abraham , Kumar Shaji , Lacy Martha , Dispenzieri Angela , Hayman Suzanne , Buadi Francis , Dingli David , Gastineau Dennis , Winters Jeffery , Litzow Mark
Published: April 2020

Introduction: We retrospectively analyzed outcomes of 716 patients with multiple myeloma who were mobilized using cyclophosphamide and growth factor (n=370) or growth factor alone (n=346) before stem cell transplantation.

Table. Patient Characteristics (N=716)

Variable Cyclophosphamide (n=370) Growth Factor Only (n=346) P Value Abbreviation: IQR, interquartile range. *Or equivalent dosage. Men, No. of patients (%) 224 (61) 202 (58) .50 Age, median (IQR), y 58 (52–64) 60 (53–65) .11 b-2 Microglobulin, median (IQR), mcg/mL 2.7 (1.9–4.0) 2.3 (1.9–3.2) .01 Creatinine, median (IQR), mg/dL 1.1 (0.9–1.3) 1.0 (0.8–1.2) .002 Apheresis, median (IQR) collections, No. 2 (1–3) 4 (3–6) .001 Marrow plasma cells, % 17 (5–34) 5 (1–13) .001 CD34+cells, median (IQR), cells/kg Total collected 10.3x106(7.2x106–14.6x106) 9.9x106(7.6x106–11.9x106) .01 Infused 5.6x106(4.5x106–7.6x106) 4.2x106(3.8x106–5.0x106) <.001 Duration of hospitalization, median (IQR), d 4 (0–10) 4 (0–9) .92 Nonstaphylococcal bacteremia, No. of patients (%) 48 (13) 25 (7) .01 Melphalan dosage, No. of patients (%) .04 200 mg/m2* 337 (91) 298 (86) 140 mg/m2* 33 (9) 48 (14) Exposure before mobilization, No. of patients (%) Melphalan 45 (12) 44 (13) .50 Lenalidomide 14(4) 63(18) .01

Results: Patients receiving cyclophosphamide had higher stem cell yields than the growth factor only group (median number of apheresis sessions needed to achieve stem cell collection goals, 2 vs 4 sessions, respectively [P=.001]). However, patients treated with cyclophosphamide required more time for engraftment of platelets and neutrophils (P<.001 for both).

For patients receiving cyclophosphamide, 75% achieved engraftment (defined as a platelet count of 50x109/L) by day 39, whereas 75% of patients not receiving cyclophosphamide achieved engraftment by day 18. Similar results were observed for neutrophil engraftment. These differences did not affect the duration of hospitalization, but patients treated with cyclophosphamide had a higher incidence of posttransplant nonstaphylococcal bacteremia. For cyclophosphamide-mobilized patients, considerably faster platelet engraftment (5 fewer days) resulted if stem cell reinfusion occurred more than 30 days after the first apheresis session.

Conclusion: Our data suggested that cyclophosphamide damaged the microenvironment and slowed engraftment. By lengthening the period between the completion of apheresis and stem cell reinfusion, the microenvironment may recover and result in faster engraftment. We suggest that the cyclophosphamide used for mobilization may affect the marrow microenvironment. Cyclophosphamide improves yields but increases bacteremia rates after transplantation. Cyclophosphamide increases time required to collect stem cells and slows engraftment.

Figure Figure
The Effect of Physical Conditions on the Transportation of Peripheral Blood Progenitor (PBPC) Products.
Authors: Jansen Jan , Nolan Pamela L , Reeves Margaret I , Akard Luke Paul , Thompson James M. , Dugan Michael J , Hanks Susan G
Published: April 2020

The viability of transported PBPC products has not been studied extensively. Commonly, PBPC products are transported at a concentration of >200 x109/l in containers with −20oC ice packs. Continuous temperature monitoring has shown that the temperatures of these products stays at <10oC for less than 24 hours and reaches room temperature by 48 hours. Samples of freshly collected PBPC from 12 allogeneic donors were studied for various viability parameters during storage for up to 96 hours. The effects of storage time, concentration of cells, temperature, and storage in gas-permeable bags were studied. Trypan-blue exclusion and double fluorescence for 7-AAD and CD34 were used for viability assessment. Over a wide range of temperatures and storage times, the viable CD34+ assay was more sensitive to damage than trypan-blue exclusion (mean Δ 10.7%, p<0.0001 in paired t-test). The viable CD34+ assay was routinely used in parallel with CFU-GM cultures. No difference in survival of viable CD34+ cells or CFU-GM was found whether cells were incubated for 48hr in test-tubes or in gas-permeable bags. When cells at 200 x 109/l were incubated for 48hr at room temperature, the mean viability decreased to 19% and 6% of starting values of viable CD34+ cells and CFU-GM, respectively. Serial dilution to 25 x 109/l improved the survival to 81% and 51% respectively. Similarly, incubation at lower temperatures led to better survival of CD34+ cells and CFU-GM: 67% and 18% at 17oC, 80% and 50% at 13oC, and 95% and 86% at 4oC. At 200 x109/l and 22oC the survivals of CD34+ cells and CFU-GM were 74% and 21% at 24hr, 19% and 7% at 48hr, 7% and 6% at 72hr, and 3% and 13% at 96hr. The effects of concentration, temperature and duration of storage were all significant (p<0.05). Transportation at 4oC leads to the best survival of CD34+ cells and CFU-GM, in particular at a low concentration. If transportation at a slightly higher temperature is necessary, dilution of the PBPC product will enhance the survival of CD34+ cells and CFU-GM. Proliferative assays such as CFU-GM appear the most sensitive parameters of PBPC survival, and should be included in the validation process of PBPC transportation.
Interaction with Human Stromal Cells Enhances CXCR4 Expression and Engraftment of Cord Blood Lin-CD34-Cells Via Wnt Signaling.
Authors: Kobune Masayoshi , Murase Kazuyuki , Iyama Satoshi , Sato Tsutomu , Kikuchi Shohei , Kawano Yutaka , Takada Kohichi , Miyanishi Koji , Takimoto Rishu , Kato Junji
Published: April 2020

Ex vivo manipulation of hematopoietic stem/progenitor cells has been performed in preclinical and clinical settings for expansion of hematopoietic stem cells (HSCs) and enhancement of HSC engraftment. Because the absolute HSC dose in a cord blood (CB) unit is a limiting factor for its use as a graft for adult transplant recipients. Transplantation of hematopoietic stem cells (HSCs) is usually accomplished through intravenous injection, a complex process that requires recognition of bone marrow vasculature and migration to a supportive microenvironment. Hence, some populations of HSCs, including cord blood (CB) Lin-CD34-stem cells, do not engraft well in bone marrow (BM) of NOD/SCID mice. In this study, we examined the effect of human stromal interactions on the properties of CB Lin-CD34-cells. CD34 and CXCR4 expression on fresh CB Lin-CD34-cells and CB Lin-CD34-cells co-cultured with human stromal cells were analyzed. Homing activity and engraftment of these cells were assessed using NOD/SCID mice. Co-culture with human stromal cells induced expression of CD34 and CXCR4 on CB Lin-CD34-cells. Furthermore, these cells acquired homing activity and engrafted in the BM of primary and secondary recipients of NOD/SCID mice after intravenous injection. In an attempt to identify the stromal CXCR4-inducing factor, we conducted comparative experiments using stroma-free, contact culture or non-contact culture. As a result, CXCR4 expression on CB Lin-CD34-cells was induced even in the non-contact culture condition but not stroma-free condition, suggesting that this CXCR4-inducing factor is soluble. Because the function of hematopoietic stem/progenitor cells was modulated by several hematopoietic growth factors, angiogenic factors and morphogens, we next screened for soluble CXCR4 inducing factors using blocking antibodies for hedgehog protein, VEGF receptor (anti-KDR) and angiopoietin-1 receptor (anti-Tie2), BMP inhibitor (noggin), pan Wnt inhibitor (sFRP-1), Wnt/β-catenin signaling inhibitor (DKK1) and Wnt/RhoA signaling inhibitor. We found that DKK1 significantly reduced the expression of CXCR4. This indicated that CXCR4 could be regulated by the Wnt signaling pathway. Subsequently, we analyzed the expression of Wnt family members in human stromal cells by RT-PCR. High expression of Wnt1, Wnt2B, Wnt5A and Wnt11 were observed in human stromal cells. Thus, these Wnts may contribute to the induction of CXCR4 expression on CB Lin-CD34-CXCR4-cells. These findings may be useful for understanding the role of stromal cells in homing and engraftment of HSCs and may provide a new strategy to utilize CB and human stromal cells for HSC transplantation.
Retroviral Gene Therapy for X-Linked Chronic Granulomatous Disease: Results from Phase I/II Trial.
Authors: Kim Joong Gon , Ahn Hyo Seop , Kang Hyoung Jin , Kim Sujeong , Hong Youngtae , Joo Chang-Wan , Yoon Nam-Kyung , Kim Sunyoung
Published: April 2020

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency disease caused by a defect in the gp91phox gene encoding one of the subunits of the NADPH oxidase complex. NADPH oxidase plays an important role in eradicating the pathogen engulfed by the phagocytes. Therefore, CGD patients suffer from recurring life-threatening infection by bacteria or fungi, and die before 30 in most cases. In an effort to treat this life-threatening disease, we initiated a phase I/II gene therapy trial in 2007. Two X-CGD patients were enrolled in the trial. The retroviral vector used for gene delivery was the MLV-based MT vector containing gp91 phox cDNA (Yu et al., Gene Ther2000; 7: 797, Hong et al., J Gene Med2004; 6: 724). Viral vectors have been produced from PG13 packaging cells in compliance with GMP. The clinical protocol was approved by the Korean FDA. G-CSF mobilized peripheral blood CD34+ cells were obtained from patients, and transduced in retronectin-coated gas-permeable bags containing SCGM media supplemented with SCF, FLT3L, TPO, and IL-3. The transduction efficiency was 10.5% for patient #1 and 28.5% for patient #2 when assessed by gp91 FACS analysis. Before receiving transduced cells, patients were treated with a conditioning regimen consisting of busulfan (3.2 mg/kg/day for 2 days) and fludarabine (40 mg/m2/day for 3 days). No adverse effects were observed from the use of busulfan and fludarabine. The percentage of superoxide-producing cells, as determined by DHR assay, was 6.4% and 14.5% at day 17, and decreased to less than 0.1% (after 1 year) and 0.4% (after 7 months). Thus far, abnormal cell expansion has not been observed.
Evaluating the Safety and Efficacy of Anti-HIV Transgenic Cells in a Humanized Mouse Model (NOD/SCIDγc−/−) for HIV Stem Cell Gene Therapy.
Authors: Anderson Joseph S. , Walker Jon , Wirthlin Louisa , Javien John , Nolta Jan A. , Bauer Gerhard
Published: April 2020

In vivo mouse models for safety and efficacy studies of anti-HIV gene therapeutics have been lacking in their ability to evaluate normal systemic hematopoiesis with the development of a functional immune system. These deficiencies in modeling these normal and essential physiologies can now be generated in the recently developed NOD/SCIDγc−/− mouse model. Human CD34+ progenitor cells after intrahepatic injection will undergo multi-organ engraftment generating a functional thymus along with the production of T cells, B cells, and macrophages. Upon injection with various strains of HIV-1, engrafted mice display a disseminated infection due to viral replication in human T cells and macrophages. Infected mice acquire characteristics of increased viremia over time with human CD4+ T cell depletion. Anti-HIV immune responses can also be generated in mice with high viremia. Here we demonstrate the successful engraftment of NOD/SCIDγc−/− mice after injection with human CD34+ progenitor cells isolated from umbilical cord blood. Human cell engraftment was observed in both primary and secondary lymphoid organs including the bone marrow (B cells and macrophages), thymus (T cells), spleen (T cells and B cells), lymph nodes (T cells and B cells), and peripheral blood (T cells and B cells). Human CD34+ cells were found in the liver concluding either organ retention from the initial injection or circulation in the peripheral blood. After displaying a high level of engraftment (>50%), we are now able to proceed with evaluating a number of anti- HIV gene therapeutic constructs for pre-clinical testing. These lentiviral vector constructs include a CCR5 shRNA which has been previously shown to knock down complete expression of CCR5, a human/rhesus macaque chimeric isoform of TRIM5α capable of potently inhibiting HIV-1 infection at the pre-integration step, and combination constructs containing multiple anti-HIV genes. The NOD/SCIDγc−/− mouse model will allow us to evaluate both the safety of these various anti-HIV lentiviral constructs in generating normal functioning transgenic immune system cells and also the efficacy of these anti- HIV genes in inhibiting HIV-1 infection in vivo. With the ability to facilitate multilineage human cell engraftment with the susceptibility for HIV infection, this in vivo model allows for the evaluation of anti-HIV gene therapeutic constructs in a stem cell-based setting. Lentiviral vector transgenic cells can now be tested for their capacity for normal immune system reconstitution.
Systemic Therapy Utilization Patterns among US Dermatologists and Oncologists Treating Cutaneous T-Cell Lymphoma
Authors: Pinter-Brown Lauren , Wisniewski Tami , Katic Bozena , Chandwani Sheenu , Chen Ya-Ting
Published: April 2020

Background: Several systemic therapies are available for patients with cutaneous T-cell lymphoma (CTCL). However, little is known about physicians’ familiarity with and utilization of these therapies. To address this issue, we conducted a web-based survey among physicians treating CTCL with systemic therapies in the United States.

Methods: We surveyed a geographically representative panel of dermatologists and oncologists in September 2007. Physicians had to have treated at least one CTCL patient systemically over the past two years and been practicing medicine for >2 years. Individual systemic therapies were grouped according to NCCN guidelines as Category A (ECP, bexarotene, denileukin diftitox, interferon, vorinostat, and methotrexate) or Category B therapies (gemcitabine, CHOP, and doxorubicin). Chi-square or Fisher’s exact tests assessed statistical differences between specialties.

Results: A total of 250 physicians participated; 128 dermatologists and 122 oncologists. Mean age was 47.5 (std =9.9) years and 20.0% were female. Significantly more (p < 0.0001) dermatologists than oncologists reported being not or somewhat familiar with denileukin diftitox (49.2% vs. 14.8%), gemcitabine (71.1% vs.7.4%), doxorubicin (57.8% vs. 5.7%), CHOP (33.6% vs. 1.6%) and vorinostat (67.2% vs. 36.9%). Overall, use of Category A therapies decreased in later stage disease while use of Category B therapies increased. Dermatologists chose Category A therapies and referred patients more frequently, whereas oncologists chose Category B therapies and referred less frequently.

Conclusions: Familiarity with and utilization of systemic therapies for CTCL differ by physician specialty. Educational efforts aimed at highlighting advances in CTCL treatment and newly implemented treatment guidelines may help optimize patient care.
Economic Analysis of Alemtuzumab in Fludarabine-Refractory and Relapsed Chronic Lymphocytic Leukemia in Canada
Authors: Isogai Pierre K , Cheung Matthew , Mittmann Nicole
Published: April 2020

Background: Alemtuzumab (ALEM), a humanized antibody that recognizes the CD52 antigen, is indicated for the treatment of B-cell chronic lymphocytic leukemia (CLL) in patients who have been treated with alkylating agent and who had failed fludarabine therapy. The objective of this economic analysis was to determine the incremental cost-effectiveness of ALEM in patients with fludarabine-refractory and relapsed CLL from the perspective of a publicly funded health care system in Canada. ALEM was compared to accepted treatments for this patient population.

Methods: A two-year time horizon was used. Effectiveness and adverse event information was obtained from an extensive literature review process. Resource utilization was based on guidelines, literature and expert opinion. Costs were obtained from provincial sources and presented in 2008 Canadian dollars. Only direct medical costs were considered. A number of comparators were used to represent standard treatment: salvage therapy, which included a variety of literature-based treatment options for fludarabine-refractory patients, fludarabine+cyclophosphamide (FC), flud arabine+cyclophosphamide+rituximab (FCR), and best supportive care (BSC). BSC included the palliative costs for caring for individuals previously treated with FC.

Results: Mean survival for ALEM was 20.89 months (1.74 years), salvage therapy 16.32 months (1.36 years), FC 17.44 months (1.45 years), FCR 20.06 months (1.67 years). The incremental cost-effectiveness ratio (ICER) for the base case-ALEM vs. salvage therapy was Canadian $52,021/life year gained (LYG); ALEM vs. FC $68,165/LYG; ALEM vs. FCR $20,886/LYG. For ALEM vs. BSC, there was increased survival and cost savings with ALEM. Base case ICERs were sensitive to four variables: treatment duration of FC; treatment duration of FCR; treatment duration of ALEM; and additional survival due to rituximab for FCR patients compared to FC. Sensitivity analyses of the remaining variables showed minimal change to the base case ICERs. These variables were: ALEM administration (proportion of intravenous to subcutaneous); cytomegalovirus (CMV) complication rate for ALEM; discount rate; neutropenia and thrombocytopenia rates for ALEM; and neutropenia and thrombocytopenia rates for FCR.

Conclusion: The results show that ALEM has a favourable ICER and provided good economic value for patients with fludarabine-refractory and relapsed CLL when compared to salvage therapy, FC and FCR. ALEM was found to be dominant over BSC.
Patient Nonadherence and Treatment Response to Imatinib in Patients with Chronic Myeloid Leukemia: Results from the ADAGIO Study
Authors: Noens Lucien , van Lierde Marie-Anne , De Bock Robrecht , Verhoef Gregor , Zachee Pierre , Berneman Zwi , Martiat Philippe , Mineur Philippe , Van Eygen Koen , MacDonald Karen , De Geest Sabina , Albrecht Tara , Letvak Laurie , Abraham Ivo
Published: April 2020

BACKGROUND. Imatinib therapy for chronic myeloid leukemia (CML) is a long-term treatment potentially compromised by patient nonadherence.

OBJECTIVE. To examine whether patients (pts) at different levels of treatment response differ in adherence to imatinib treatment.

DESIGN & PATIENTS. The ADAGIO study1 is a prospective, 90-day (90d) observational, open-label, multicenter study of pts with chronic myeloid leukemia (CML) and treated with imatinib. 169 evaluable pts who had been on imatinib for a minimum 30 days at enrollment were studied. A sub-analysis included pts with optimal vs. suboptimal response (all patients) and complete vs. incomplete cytogenetic response (CgR; all patients and those treated with imatinib ≥12 months).

MEASUREMENTS. Adherence: imatinib pill count over 90d expressed as % of prescribed imatinib taken. Suboptimal response (SR): incomplete hematologic response at 3 months, and/or less than partial CgR at 6 months, and/or less than major molecular response and, in case of loss of major molecular response, other limitations or chromosomal abnormalities at 18 months (all else: optimal response [OR]). CgR: complete (0% Ph+ metaphases) or incomplete (≥1 Ph+ metaphases).

RESULTS. Pill count percentages ranged from 29%–202% of prescribed dose (M=90.9±20.1). Pts with SR (n=14) had significantly higher %s of imatinib not taken (23.2±23.8) than did those with OR (n=124; 7.3±19.3, P=0.005). Among pts treated with imatinib ≥12 months, those with complete CgR (n=98) had significantly lower mean percentages of imatinib not taken (9.0±18.6) than those with incomplete CgR (n=9; 26.0±24.4, P=0.012). Among all patients regardless of length of treatment, those with complete CgR (n=109) also had significantly lower mean percentages of drug not taken (9.1±18.1) than those with incomplete CgR (n=19; 23.9±19.2, P=0.004).

CONCLUSIONS. Proportions of CML patients with poor treatment response are low (10.1%, 8.4%, and 14.8% resp. for parameters above), underscoring the high efficacy of imatinib in CML. Pts with poor response tended to have higher % of imatinib not taken over 90d, an index of overall adherence behavior. Clinicians should be aware of the association between adherence and imatinib response and should query patients about their adherence behavior. Nonadherence should be ruled out prior to classifying a patient as imatinib-resistant. Enhanced adherence is likely to optimize the effectiveness of imatinib treatment in CML.
Intravital Imaging of Young and Aged Stem Cells in the Marrow of Long Bones: Visualizing Mammalian Stem Cell Behavior in Real-Time in Vivo
Authors: Köhler Anja , Schmithorst Vince , Filippi Marie-Dominique , Ryan Marnie A. , Daria Deidre , Gunzer Matthias , Geiger Hartmut
Published: April 2020

Hematopoiesis, the process in which blood cells are generated from hematopoietic stem and progenitor cells (HSPCs) is primarily confined to the bone cavities. The interactions of hematopoietic cells with stroma cells forming niches inside the bone cavities are central to hematopoiesis, as these regulate cell proliferation, self-renewal and differentiation. Hematopoietic cell/stroma interactions have thus been, in analogy to the immunological synapse, named stem/progenitor cell synapses. So far, visualization of the behavior of somatic stem and progenitor cells in an undisturbed in vivo environment has not been reported for the mammalian system and consequently, the cellular dynamics of stem, progenitor and differentiated cells in vivo are only poorly defined. We developed and performed intravital time-lapse 2-photon microscopy in the marrow of the long bones (tibia) of mice to study the behavior and dynamics of differentiated hematopoietic cells as well as HPCs and HSCs in close vicinity to the endosteum in vivo over time. We demonstrate that HPCs as well as HSCs reside in close vicinity to the endosteum, further supporting the notion of an endosteal stem cell niche, and that they are, in contrast to differentiated macrophages and dendritic cells, solitary and immobile. Both HPCs and HSCs occupy distinct positions relative to the endosteum and show cell protrusion movement consistent with an active stem/progenitor cell synapse. Lastly, we report that aged HSCs show increased protrusion movement and localize more distantly to the endosteum compared to young HSCs. In addition, aged HSCs present with reduced adhesion to stroma as well as reduced polarity upon adhesion in vitro, implying a connection between altered stem cell dynamics in vivo and stem cell aging. The intravital imaging technology developed might establish a basis for further delineating additional important questions in stem cell biology like cellular mechanisms of hematopoietic stem cell self-renewal and differentiation in the context of the stroma/niche in vivo.
Presence of Factor V Leiden or Prothrombin Mutation Influence Inhibitor Development in Children with Severe Hemophilia A: Data of a Multicenter Cohort Study
Authors: Kurnik Karin , Bidlingmaier Christoph , During Christine , Halimeh Susan , Schobess Rosemarie , Nowak-Gottl Ulrike
Published: April 2020

It has been recently suggested that the clinical phenotype of severe hemophilia A (HA) is influenced by co-inheritance with the factor V (FV) G1691A mutation. In addition, we have recently demonstrated that the first symptomatic bleeding onset in children with severe HA carrying prothrombotic risk factors, namely the FV Leiden or the factor II (FII) G20210A variant, is significantly later in life than in non-carriers. The course of bleeding episodes, however, is also controversially discussed with respect to individual therapeutic regimens performed, and recently a protective effect of early use of prophylaxis on inhibitor development in hemophilic children compared with on-demand therapy has been reported. Thus, the present non-concurrent cohort study was performed to investigate the impact of FV and FII (prothrombin) mutations on clinical meaningful inhibitor development (outcome variable) in white children with severe HA. 122 patients with HA consecutively ascertained from four German catchment areas were followed over a median (min-max) period of 15 years (1–24) from HA onset. 19 of 122 children (15.6%) additionally carried either the FV or the FII variant. During the follow-up period 28 of 122 children (22.9%) developed a clinical meaningful inhibitor: 9 out of 19 in carriers of thrombophilia (47.4%) compared with 19 of 103 (18.44%) in the non-thrombophilia affected cohort [p=0.01]. Multivariate analysis (Cox regression), adjusted for age at first bleeding onset, substitution regimen (start of prophylaxis latest at second joint bleed versus more than two joint bleeds), FVIII dosage (≤ 30 IU/kgbw versus > 30 IU/kgbw), and first-line use of plasma-derived versus recombinant FVIII concentrations revealed that the presence of thrombophilia independently increases the risk of clinical meaningful inhibitor development to a hazard of 2.4 [95%CI: 1.1–5.5]. The cumulative high-responder-free survival with respect to FVIII exposure days was significantly reduced in carriers of the factor V or II mutation (logrank p=0.009). None of the confounders adjusted for, showed a statistically significant influence on clinical important inhibitor development. Data presented here support the hypothesis that clinical meaningful inhibitor development is of multifactorial origin and that FV and FII mutations should be included in the aetiology research in future studies.
Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation
Authors: Zhou Lan , Yan Quanjian , Yao David , Li Lebing W , Gerson Stanton L. , Lowe John B
Published: April 2020

Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.
IL7 Counteraction with Notch Signaling Followed by EBF1 Expression Marks the B-Cell Commitment in CLP Stage
Authors: Zandi Sasan , Tsapogas Panagiotis , Månsson Robert , Sigvardsson Mikael
Published: April 2020

Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220−CD19− committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.
In Vivo Cellular Imaging Pinpoints the Role of Reactive Oxygen Species in the Early Steps of Adult Hematopoietic Reconstitution
Authors: Lewandowski Daniel , Barroca Vilma , Ducongé Frédéric , Tavitian Bertrand , Roméo Paul-Henri
Published: April 2020

Progress in regenerative medicine will be greatly helped by the characterization of the in vivo processes that lead to tissue reconstitution by adult somatic stem cells. Many major advances have been achieved in the purification and the ex vivo amplification of the adult somatic stem cells, but very few data are available on the pre-requisites that will enhance their in vivo biological activities. The best studied model of tissue reconstitution is the long-term reconstitution of hematopoiesis after transplantation of adult hematopoietic stem cells (HSCs) following lethal irradiation and imaging strategies are amongst the most promising techniques to reveal the in vivo cellular dynamics of this process. Here, we present a new imaging technique to track labeled cells in living animals and we show that this technique can visualize the hematopoietic reconstitution within the femoral cavity after lethal irradiation. We use a fibered in vivo confocal fluorescence imaging system that can navigate inside the femoral cavity from the knee to the femoral head. Imaging is done in living mice, does not alter their viability, does not interfere with hematopoietic reconstitution and can easily be used to follow the temporal dynamics of tissue reconstitution in the mouse at a cellular level. Using this imaging system, we track the temporal and topological cellular dynamics of in vivo engraftment and proliferation of Lin-Sca+c−Kit+CD34− cells (LSKCD34−), enriched in HSCs, transplanted into lethally irradiated mice. Homing and engraftment of LSKCD34− hematopoietic cells first occur in the two femoral epiphyses where these cells are found associated with vascularized structures as early as one hour after transplantation. Reactive oxygen species (ROS) regulate

the initial homing of transplanted HSCs through the modulation of VCAM-1 expression by the endothelial cells and the proliferation of transplanted HSCs.