Christina Hart

Expression and Function of Homing‐Essential Molecules and Enhanced In Vivo Homing Ability of Human Peripheral Blood‐Derived Hematopoietic Progenitor Cells after Stimulation with Stem Cell Factor

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the α4β1 and α5β1 integrins very‐late antigen (VLA)‐4 and VLA‐5 on peripheral blood‐derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA‐4 and VLA‐5 on CD34+/c‐kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient‐derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long‐term culture‐initiating cell. The improvement of the homing abilities of SCF‐stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic‐scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.

Genotype and Laboratory and Clinical Phenotypes of Protein S Deficiency

The diagnosis of thrombophilia caused by protein S deficiency remains difficult. From 2005 to 2010, we documented 135 patients with suspected hereditary protein S deficiency for whom mutational analysis of the PROS1 gene had been performed by direct double-stranded sequencing of the amplified 15 exons including splice sites. Multiplex ligation-dependent probe amplification was performed on 12 of 15 exons in cases with no mutation found but a large deletion in the PROS1 gene was suspected. Mutations were identified in 49 patients, 9 by familial screening. Altogether, 17 new and 11 previously described mutations of PROS1 were identified among the 49 patients. After the exclusion of acquired protein S deficiency due to pregnancy or hormonal contraceptives, there remained only 1 case with protein S activity levels less than 40% that could not be explained by sequence variations or deletions in the examined regions of the PROS1 gene. After the exclusion of conditions associated with acquired protein S deficiency, persistently low protein S activity levels are highly indicative of a genetic alteration in PROS1. We observed a clear correlation between the laboratory phenotype and the type of mutation.

Safety and feasibility of long term administration of recombinant human granulocyte-colony stimulating factor in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuronal disease resulting in a loss of the upper and lower motor neurons and subsequent death within three to four years after diagnosis. Mouse models and preliminary human exposure data suggest that the treatment with granulocyte-colony stimulating factor (G-CSF) has neuro-protective effects and may delay ALS progression. As data on long-term administration of G-CSF in patients with normal bone marrow (BM) function are scarce, we initiated a compassionate use program including 6 ALS patients with monthly G-CSF treatment cycles. Here we demonstrate that G-CSF injection was safe and feasible throughout our observation period up to three years. Significant decrease of mobilization efficiency occurred in one patient and a loss of immature erythroid progenitors was observed in all six patients. These data imply that follow-up studies analyzing BM function during long-term G-CSF stimulation are required.

Ifosfamide, epirubicin, and etoposide (IEV) mobilize peripheral blood stem cells more efficiently than cyclophosphamide/etoposide

High-dose chemotherapy with autologous stem cell support is an effective treatment in advanced multiple myeloma. In this study, we compare chemotherapy with ifosfamide, epirubicin, and etoposide (IEV) or cyclophosphamide and etoposide (CE) in 47 patients with multiple myeloma with regard to stem cell mobilization, toxicity, and tumor response. The proportion of patients reaching the threshold of >6 × 106 CD34+ cells/kg body weight was significantly higher in the IEV group (97% vs 71%), and more CD34+ cells (10 × 106 vs 3.5 × 106 cells/kg; p = 0.002) could be collected by the first leukapheresis associated with less leukaphereses needed. Non-hematopoietic side effects were mild with nausea being more frequent after IEV treatment (30% vs 7%). Grade 3/4 neutropenia (thrombocytopenia) occurred in 89 and 100% (55 and 44%) of the patients. There was one treatment-related death due to septic shock in the IEV group. Grade 3/4 anemia was more frequent in the IEV group (19% vs 0%). Forty-two percent (IEV) and 50% (CE) received inpatient treatment for neutropenic fever. In 20 and 7% of the patients, a partial response was observed after IEV and CE. However, the overall response rate (complete response and partial tumor response) after mobilization and tandem high-dose chemotherapy was 75% after IEV and 78% after CE and, thus, independent of the mobilization. In summary, both treatment protocols can readily be used for the mobilization of peripheral blood stem cells with comparable major toxicities and similar tumor response rates. However, the efficiency of the stem cell mobilization was significantly higher after IEV treatment.

Splenic pooling and loss of VCAM-1 causes an engraftment defect in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation

Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n=31) and acute myeloid leukemia (n=31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34+ were significantly reduced at early time points in myelofibrosis patients, whereas CD34+CD38− and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34+ hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation.

A standardized treatment regimen for patients with severe haemophilia A undergoing orthopaedic or trauma surgery: a single centre experience.

Recommendations on the administration of clotting factor concentrates in patients with severe haemophilia undergoing surgery are usually determined by monitoring target clotting factor levels. In this retrospective cohort study, we enrolled patients with severe haemophilia A who underwent major orthopaedic or trauma surgery. We wanted to evaluate the feasibility and the safety of a standardized medical treatment procedure. Further on, we wanted to assess whether our standardized treatment regimen enables surgical procedures in certain situations in which measuring clotting factor VIII (FVIII) activity is not available. We created a standardized medical treatment procedure that included a medical protocol and close cooperation with the Haemophilic Treatment Centre. Thirteen surgical procedures in nine patients were examined. The feasibility and safety of this standardized treatment concept were assessed by identifying perioperative complications and by means of a questionnaire. Depending on the surgery, the amount of FVIII administered within the first 10 days ranged between 653 and 1027 units/kg body weight. No allogeneic blood transfusion was required. The measurement of FVIII activity was performed repeatedly in five patients. In all patients activated partial thromboplastin time monitoring was performed during the hospital stays. The surgeons and patients were satisfied with our treatment concept and adhered to the medical regimen protocol. By means of a detailed, standardized medical protocol and by ensuring close cooperation between the patient, the surgeons and the Haemophilic Treatment Centre, we could show that elective and emergency operations can be safely performed even in situations in which FVIII activity could not be monitored.

Detection and quantification of functionally defined hematopoietic progenitor cells and tissue specific mRNA within the peripheral blood of myeloma patients after administration of granulocyte colony-stimulating factor and erythropoietin.

Objective:  Hematopoietic progenitor cells (HPC) as well as tissue committed stem cells expressing mRNA specific to various somatic tissues are thought to be part of the CD34+ bone marrow compartment. In this study, we explore and quantify their mobilization in patients with multiple myeloma undergoing chemotherapy upon administration of granulocyte colony‐stimulating factor (G‐CSF) plus/minus erythropoietin (EPO).

Pre- and post-diagnosis physical activity, television viewing, and mortality among hematologic cancer survivors

Purpose The associations of physical activity and television (TV) viewing with mortality risk among individuals with hematologic malignancies remain unclear. Methods We examined the relations of physical activity and TV viewing time before and after diagnosis with mortality among 5182 U.S. adults aged 50–71 years from the NIH-AARP Diet and Health Study cohort who survived a first primary hematologic cancer between 1995–1996 and 2011. Results For the pre- and post-diagnosis analyses, we confirmed 2606 and 613 deaths respectively. In multivariable-adjusted Cox proportional hazard regression models, comparing high (≥4 hrs/wk) versus low (<1 hr/wk) activity levels, pre-diagnosis physical activity was associated with 18%-22% reduced risks of all-cause mortality among all hematologic cancer survivors, and survivors of non-Hodgkin lymphoma, myeloma, and leukemia, respectively. Additional control for BMI had little impact on the results, expect for myeloma survivors, for whom the association was no longer significant. Post-diagnosis physical activity was related to risk reductions in mortality ranging from 36%-47%. The associations for TV viewing did not show a clear pattern. Conclusion Our study suggests that pre- and post-diagnosis physical activity is associated with lower risk of all-cause mortality among hematologic cancer survivors. Further research is required to confirm this observation.

Warum blutet Ihr Patient

ZusammenfassungEine hämorrhagische Diathese kann sich anamnestisch, klinisch und laborchemisch auf unterschiedliche Art und Weise zeigen. Das Erkennen und die weitere Abklärung ist für Betroffene nicht nur vor operativen Eingriffen essenziell. In diesem Beitrag geben wir einen Überblick über die wichtigsten Gerinnungsstörungen und stellen dar, wie der Verdacht auf eine hämorrhagische Diathese erhärtet und erste diagnostische Schritte eingeleitet werden können.

TGF-β1 and CXCL12 modulate proliferation and chemotherapy sensitivity of acute myeloid leukemia cells co-cultured with multipotent mesenchymal stromal cells

ABSTRACT Objectives: Multipotent mesenchymal stromal cells (MSCs) play a central role within the bone marrow (BM) niche, supporting hematopoiesis via soluble factors like cytokines and chemokines. In our study, we sought to investigate the effect of blocking transforming growth factor beta 1 (TGF-β1) and C-X-C motif chemokine 12 (CXCL12) receptor CXCR4 on acute myeloid leukemia (AML) cells in an MSC co-culture system. Methods: Human MSCs were obtained by BM aspirates and their phenotype and functional properties were confirmed in vitro. Co-cultures of AML cells on MSCs were initiated and compared to those on mouse fibroblasts (MS-5) and liquid cultures. Additionally, the effect of blocking CXCR4 and TGF-β1 on AML cells was tested with and without the addition of cytarabine. Results: MSCs from BM showed a typical phenotype and differentiation pattern. Co-culture of AML cells on MSCs resulted in a significantly higher proliferation capacity than on MS-5 or liquid culture. Blockade of TGF-β1 increased AML cell proliferation and chemosensibility, while the CXCR4 antagonist plerixafor showed anti-proliferative effects and did not change cytarabine-induced cell death compared to control. Discussion: Human MSCs are potent feeder cells, able to maintain AML cells in long-term culture. This favorable co-existence seems to be due in part to molecules important for communication within the niche. Blockade of TGF-β1 and CXCL12 was associated with different effects on AML cell proliferation and chemotherapy resistance. Conclusion: These findings suggest a strong supporting affinity between MSCs and AML cells within the leukemic niche, where TGF-β1 and CXCL12 pathways play an important role.