Higher recipient pre-transplant FOXP3 mRNA expression is associated with acute leukaemia relapse after HSCT.

The effect of higher FOXP3 mRNA expression by recipient pre-transplant CD4+ T cells on leukaemia relapse was analysed in a series of 106 patients who received allogeneic haematopoietic stem cell transplantation after myeloablative conditioning with or without antithymocyte globulin (ATG) due to acute leukaemia in 1st or 2nd complete remission. FOXP3 mRNA was measured by qPCR in purified CD4+ T cells from blood obtained before conditioning. Higher FOXP3 mRNA expression was associated with an increased relapse risk when conditioning included ATG (n = 43, hazard ratio [HR] 11.0 [2.50-48.4], p = 0.00001). No effect was observed in patients not receiving ATG (HR 0.95 [0.53-1.81]).

High preharvest donor Foxp3 mRNA level predicts late relapse of acute lymphoblastic leukaemia after haematopoietic stem cell transplantation.

OBJECTIVES: The curative effect of allogeneic haematopoietic stem cell transplantation (HSCT) for acute leukaemia is due in part to the donor T cell-mediated graft-versus-leukaemia immune reaction (GvL). Several studies have suggested that donor CD25+CD4+Foxp3+regulator T cells (Tregs) may decrease graft-versus-host disease (GvHD) without abrogating GVL. This notion may need modification in acute lymphoblastic leukaemia (ALL). METHODS: Foxp3 mRNA level was measured by qPCR in preharvest donor blood CD4+ T cells. The study comprised 45 patients with ALL in 1st or 2nd CR who received myeloablative HSCT using T-replete bone marrow grafts. RESULTS: Relapse occurred in 17 patients median 363 days after HSCT. The relapse risk was estimated by Cox univariate and multivariate proportional hazard regression. The proportionality assumption was met by analysing the preharvest donor Foxp3 mRNA level as a time-dependent covariate. Early relapse was not modified by the Foxp3 mRNA level. However, a higher Foxp3 mRNA level was associated with a significantly increased relapse risk after day 363 after transplantation, compatible with inhibition of GvL. In contrast, a higher preharvest donor CD4+ T-cell concentration was associated with reduced relapse risk. CONCLUSION: A higher preharvest donor Foxp3 mRNA level may be predictive of late ALL relapse after HSCT.

Only minor stem cell mobilization in head and neck irradiated patients treated with hyperbaric oxygen.

INTRODUCTION: Hyperbaric oxygen, (HBO) is used to treat several conditions including late radiation tissue injury. Previous studies have suggested that HBO mobilizes bone marrow derived stem/progenitor cells (SPC) to the peripheral blood, however possible cumulative effects were highly variable. METHODS: We have investigated a possible HBO-induced mobilization of SPCs by determining CD34+CD45dim cell numbers, as well as SPCs in general. The latter were characterized by high aldehyde dehydrogenase (ALDH) activity by use of the Aldefluor® assay. We included ten patients admitted for HBO treatment of radiation tissue injury. Six patients completed the 29-30 HBO treatment exposures. We also investigated possible HBO-induced effects on platelet activation as measured by flow cytometry and functional analyses. RESULTS: We found a weak and insignificant tendency toward mobilization of CD34+CD45dim cells after a single HBO exposure versus before. Additionally, we found an additive effect of 15 HBO exposures on the increase in CD34+CD45dim cells relative to the pre-1st-HBO values. These changes were significantly more than zero but less than a doubling. We could not demonstrate a significant effect of HBO on the content of Aldefluor® positive SPCs in peripheral blood. There was no significant effect on platelet activation overall. However, in patients with increased expression of activation markers at baseline, we found a decrease after one exposure although this was not reflected in functional tests. CONCLUSION: We found a minor statistically significant mobilizing effect of HBO treatment on the bone marrow derived stem/progenitor cell content in peripheral blood after 15 treatments (n = 10 patients), but no effect after 30 treatments (n = 6 patients). However, because of the low number of patients we cannot confidentially prove or disprove the null hypothesis. The possibility that HBO treatment reduces the number of activated platelets could not be demonstrated nor excluded.

Should the standard dimethyl sulfoxide concentration be reduced? Results of a European Group for Blood and Marrow Transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide.

BACKGROUND: Dimethyl sulfoxide (DMSO) is essential for the preservation of liquid nitrogen-frozen stem cells, but is associated with toxicity in the transplant recipient. STUDY DESIGN AND METHODS: In this prospective noninterventional study, we describe the use of DMSO in 64 European Blood and Marrow Transplant Group centers undertaking autologous transplantation on patients with myeloma and lymphoma and analyze side effects after return of DMSO-preserved stem cells. RESULTS: While the majority of centers continue to use 10% DMSO, a significant proportion either use lower concentrations, mostly 5 or 7.5%, or wash cells before infusion (some for selected patients only). In contrast, the median dose of DMSO given (20 mL) was much less than the upper limit set by the same institutions (70 mL). In an accompanying statistical analysis of side effects noted after return of DMSO-preserved stem cells, we show that patients in the highest quartile receiving DMSO (mL and mL/kg body weight) had significantly more side effects attributed to DMSO, although this effect was not observed if DMSO was calculated as mL/min. Dividing the myeloma and lymphoma patients each into two equal groups by age we were able to confirm this result in all but young myeloma patients in whom an inversion of the odds ratio was seen, possibly related to the higher dose of melphalan received by young myeloma patients. CONCLUSION: We suggest better standardization of preservation method with reduced DMSO concentration and attention to the dose of DMSO received by patients could help reduce the toxicity and morbidity of the transplant procedure.

Addition of plerixafor for CD34+ cell mobilization in six healthy stem cell donors ensured satisfactory grafts for transplantation.

BACKGROUND: In allogeneic hematopoietic stem cell (HSC) transplantation, collection of a sufficient number of HSCs at a fixed time point is crucial. For HSC mobilization into the peripheral blood, the standard regimen, that is, granulocyte-colony-stimulating factor (G-CSF), may be inadequate. Use of plerixafor as adjuvant to G-CSF is so far off-label in healthy donors. STUDY DESIGN AND METHODS: We present six cases in which the "just-in-time" addition of plerixafor ensured proper CD34+ collection from healthy donors with insufficient G-CSF mobilization. In four of these cases a high number of CD34+ cells was needed due to subsequent CD34+ selection or haploidentical transplantation. RESULTS: From all six donors a sufficient number of CD34+ cells was obtained by using plerixafor as an adjuvant to G-CSF. This treatment regimen resulted in only mild side effects for the donor. CONCLUSION: We have presented six cases with different causes leading to insufficient G-CSF mobilization in allogeneic donors and in which the administration of plerixafor just-in-time ensured a proper graft for transplantation.

Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial.

BACKGROUND: Autologous fat grafting is increasingly used in reconstructive surgery. However, resorption rates ranging from 25% to 80% have been reported. Therefore, methods to increase graft viability are needed. Here, we report the results of a triple-blind, placebo-controlled trial to compare the survival of fat grafts enriched with autologous adipose-derived stem cells (ASCs) versus non-enriched fat grafts. METHODS: Healthy participants underwent two liposuctions taken 14 days apart: one for ASC isolation and ex-vivo expansion, and another for the preparation of fat grafts. Two purified fat grafts (30 mL each) taken from the second liposuction were prepared for each participant. One graft was enriched with ASCs (20 × 10(6) cells per mL fat), and another graft without ASC enrichment served as a control. The fat grafts were injected subcutaneously as a bolus to the posterior part of the right and left upper arm according to the randomisation sequence. The volumes of injected fat grafts were measured by MRI immediately after injection and after 121 days before surgical removal. The primary goal was to compare the residual graft volumes of ASC-enriched grafts with those of control grafts. This study is registered at www.clinicaltrialsregister.eu, number 2010-023006-12. FINDINGS: 13 participants were enrolled, three of whom were excluded. Compared with the control grafts, the ASC-enriched fat grafts had significantly higher residual volumes: 23·00 (95% CI 20·57-25·43) cm(3) versus 4·66 (3·16-6·16) cm(3) for the controls, corresponding to 80·9% (76·6-85·2) versus 16·3% (11·1-21·4) of the initial volumes, respectively (p<0·0001). The difference between the groups was 18·34 (95% CI 15·70-20·98) cm(3), equivalent to 64·6% (57·1-72·1; p<0·0001). No serious adverse events were noted. INTERPRETATION: The procedure of ASC-enriched fat grafting had excellent feasibility and safety. These promising results add significantly to the prospect of stem cell use in clinical settings, and indicate that ASC graft enrichment could render lipofilling a reliable alternative to major tissue augmentation, such as breast surgery, with allogeneic material or major flap surgery. FUNDING: Danish Cancer Society, Centre of Head and Orthopaedics Rigshospitalet, and Moalem Weitemeyer Bendtsen.

Direct intramyocardial mesenchymal stromal cell injections in patients with severe refractory angina: one-year follow-up.

In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6-8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up (p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 (p < 0.001), angina attacks per week from 13.8 to 3.2 (p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week (p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life (p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.

Mesenchymal stromal cell derived endothelial progenitor treatment in patients with refractory angina.

AIMS: We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. METHODS AND RESULTS: A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6-8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p < 0.001), reduction in CCS Class (p < 0.001), angina attacks (p < 0.001) and nitroglycerin consumption (p < 0.001), and improved Seattle Angina Questionnaire (SAQ) evaluations (p < 0.001). For all parameters there was a tendency towards improved outcome with increasing numbers of cells injected. In the MRI substudy: ejection fraction (p < 0.001), systolic wall thickness (p = 0.03) and wall thickening (p = 0.03) all improved. CONCLUSIONS: The study demonstrated that it was safe to treat patients with stable CAD with autologous culture expanded MSC. Moreover, MSC treated patients had significant improvement in left ventricular function and exercise capacity, in addition to an improvement in clinical symptoms and SAQ evaluations.

Graft rejection after hematopoietic cell transplantation with nonmyeloablative conditioning.

Graft rejection after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning is a rare but serious clinical problem. Graft rejection and salvage therapy in eight patients in a retrospective analysis of 124 consecutive patients is reported. The patients were conditioned with low-dose fludarabine and total body irradiation (TBI). The association of pretransplantation risk factors with rejection and the effect of chimerism and graft-versus-host disease on rejection were analyzed. Overall survival (OS) and progression free survival (PFS) were compared between patients with and without rejection. Retransplantation was performed with increased TBI conditioning for all patients, and with increased mycophenolate mofetil doses for recipients with HLA-identical sibling donors. No known pretransplantation risk factors were confirmed in this study. Rejection episodes were unevenly distributed over time. The storage temperature of the apheresis products was identified as a risk factor for rejection. Storage of the apheresis products at 5 degrees C diminished the risk of rejection. Low donor T cell chimerism at Day +14 significantly increased the risk of rejection. Seven patients were retransplanted. All but one engrafted successfully, but with decreased OS and PFS. Two patients received pentostatin infusion prior to donor lymphocyte infusions in unsuccessful attempts at reversing rejection. Storage temperature and donor chimerism had a significant effect on rejection. Following rejection, patients are at greater risk of dying from infections and progression/relapse of their malignancy. Retransplantation is feasible and well tolerated after HCT with nonmyeloablative conditioning and should be performed without delay in patients with imminent and manifest graft rejection.

Cytokine gene expression in peripheral blood mononuclear cells and alloreactivity in hematopoietic cell transplantation with nonmyeloablative conditioning.

Cytokines are thought to play an important role in the pathophysiology of graft-versus-host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNCs) from 21 patients with hematologic malignancies and their HLA-identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNCs were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNCs of recipient, donor, or HLA-mismatched third-party origin. The gene expression of interleukin (IL)-2, IL-4, IL-10, IL-18, tumor necrosis factor alpha, and transforming growth factor beta in each culture was then measured by real-time quantitative reverse transcriptase-polymerase chain reaction. The composition of the responder MNCs differed between patients and donors and changed after HCT, with a possible influence on the results. Early after transplantation (day +14), the IL-10 messenger RNA (mRNA) level in response to recipient or donor antigens was higher in patients who did not develop clinically significant acute GVHD when compared with the level in patients who subsequently developed acute GVHD grades II to IV (P = .005 and P = .004, respectively). The IL-10 mRNA level on day +14 was highly correlated with the pretransplantation mRNA level of the recipient MNCs but not with the level of the donor MNCs; this suggests that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNCs obtained before transplantation from recipients whose disease progressed or relapsed after the transplantation when compared with the level in patients whose disease did not progress or relapse (P = .03). In conclusion, a high IL-10 gene expression in the recipient MNCs may be related to a reduced incidence of acute GVHD grades II to IV and a reduced graft-versus-tumor effect after HCT with nonmyeloablative conditioning.

Limiting dilution analysis of interleukin-2 producing helper T-cell frequencies as a tool in allogeneic hematopoietic cell transplantation.

BACKGROUND: A reliable in vitro test that estimates the level of ongoing alloreactivity would be valuable in allogeneic hematopoietic cell transplantation (HCT) as a help to guide clinical interventions such as donor lymphocyte infusions and changes in the immunosuppression. In the present study, the use of limiting dilution analysis of interleukin-2 (IL-2) producing helper T lymphocyte frequencies (HTL assay) as a way to quantify alloreactivity following HCT was investigated. METHODS: Serial HTL assays were performed following allogeneic HCT with myeloablative or nonmyeloablative conditioning in 26 patients with hematologic malignancies. RESULTS: Deviations from single-hit kinetics were frequently observed in the HTL assays and a nonlinear model was therefore used for analysis. The results of this analysis suggested the presence of an inhibitory cell population. Inhibition was observed in the majority of patients and was not restricted to a specific transplant regimen. Inhibition occurred more often with high frequencies of IL-2 producing cells, indicating a physiological role of the putative inhibitory cell population in the regulation of an immune response. Higher frequencies of IL-2 producing cells were observed in patients with acute graft-versus-host disease grades II-IV than in patients with grades 0-I (P = 0.046), indicating that the degree of ongoing alloreactivity is indeed quantified by the HTL assay. CONCLUSIONS: We find that the HTL assay may yield interesting insight into regulation of immune responses following allogeneic HCT, but because of the complexity of the results obtained, its use as a routine procedure to guide immunosuppression cannot be recommended.

Chimerism studies in HLA-identical nonmyeloablative hematopoietic stem cell transplantation point to the donor CD8(+) T-cell count on day + 14 as a predictor of acute graft-versus-host disease.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4(+) and CD8(+) T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4(+) and CD8(+) donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P =.0002 and P =.019, respectively). The rate of disappearance of recipient CD8(+) T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P =.016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8(+) T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P =.003).

Haematopoietic stem cell transplantation with non-myeloablative conditioning in the outpatient setting: results, complications and admission requirements in a single institution.

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft-versus-host disease (GVHD) grades II-IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4-151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP (P = 0.008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year.

Limited protective effect of the CCR5Delta32/CCR5Delta32 genotype on human immunodeficiency virus infection incidence in a cohort of patients with hemophilia and selection for genotypic X4 virus.

The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)-infected patients with hemophilia. One patient (0.6%) had the CCR5Delta32/CCR5Delta32 genotype (which occurs in approximately 2% of the Scandinavian population) and a rapid disease course. His HIV V3 region contained genotypic features attributable to X4 virus and resembled functionally verified X4 virus and virus from patients treated with a CD4 cell-stimulating drug, tucaresol. Age-related differences in disease progression rate and survival time were seen for CCR5/CCR5 patients. Surprisingly, no protective effect of the CCR5/CCR5Delta32 genotype on disease progression or survival was seen for children but was evident for adults. Age group-related immunologic differences might explain this variation, and transmission route and/or viral phenotype variation within donor virus may be related to the limited protection of the CCR5Delta32/CCR5Delta32 genotype. Sequence comparisons indicate that X4 virus can be selected in vivo due to either absence of CCR5 receptors or relative increase of CXCR4 receptors.