Abstract Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their ...activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-β production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo.
Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) ...(n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.
We report a prospective multicenter phase II study of haploidentical hematopoietic stem cell transplantation using CD3/CD19-depleted grafts after reduced intensity conditioning with fludarabine, ...thiotepa, melphalan and OKT-3.
Sixty-one adults with a median age of 46 years (range 19-65 years) have been enrolled. Diagnoses were acute myeloid leukemia (n=38), acute lymphoblastic leukemia (n=8), non-Hodgkin's lymphoma (n=6), myeloma (n=4), chronic myeloid leukemia (n=3), chronic lymphatic leukemia (n=1) and myelodysplastic syndrome (n=1). Patients were considered high risk because of refractory disease (n=18), cytogenetics (n=6), complete remission (≥ 2) (n=9), chemosensitive relapse in partial remission (n=4) or relapse after prior hematopoietic stem cell transplantation (n=15 allogeneic, n=8 autologous, n=1 both). At haploidentical hematopoietic stem cell transplantation, 30 patients were in complete remission and 31 in partial remission. Grafts contained a median of 7.0 × 10(6) (range 3.2-22) CD34(+) cells/kg, 4.2 × 10(4) (range 0.6-44) CD3(+) T cells/kg and 2.7 × 10(7) (range 0.00-37.3) CD56(+) cells/kg.
Engraftment was rapid with a median of 12 days to granulocytes more than 0.5 × 10(9)/L (range 9-50 days) and 11 days to platelets more than 20 × 10(9) (range 7-38 days). Incidence of grade IIIV acute graft-versus-host-disease and chronic graft-versus-host-disease was 46% and 18%, respectively. Non-relapse mortality on Day 100 was 23% and 42% at two years. Cumulative incidence of relapse/progression at two years was 31%. Kaplan-Meier estimated 1-year and 2-year overall survival with median follow up of 869 days (range 181-1932) is 41% and 28%, respectively.
This regimen allows successful haploidentical hematopoietic stem cell transplantation with reduced intensity conditioning in high-risk patients lacking a suitable donor. (clinicaltrials.gov identifier:NCT00202917).
Bronchiolitis obliterans (BO) is a late non-infectious pulmonary complication after allogeneic hematopoietic SCT. Among 982 patients after myeloablative hematopoietic SCT between January 2000 and ...October 2010, 68 were diagnosed with BO according to NIH criteria. The median onset of BO was 18 months post transplant, 5-year cumulative incidence was 5.8% and 5-year mortality 41%. BO prevalence rate was 10% among all long-term surviving hematopoietic SCT recipients and 12% among chronic GVHD-patients. Chronic GVHD, peripheral SCT and ABO blood group incompatibility were identified as risk factors associated with BO. IgG levels were significantly decreased at the onset of BO (6.7 g/L±0.7, P=0.001), the mean exhaled NO concentrations were lower in BO-patients than in stem cell recipients without BO (14 p.p.b.±0.9 vs 20 p.p.b.±2.1) or healthy controls (25 p.p.b.±2.4, P<0.001). Hypoxia-inducible factor 1 alpha (HIF-1α) was significantly elevated in BO as compared with healthy controls or GVHD-patients without lung involvement (340±61 vs 127±22 vs 140±32, P=0.02). Calculated 5-year survival was superior in female than in male BO-patients (86 vs 45%, P=0.04). These results emphasize the relevance of BO as serious late complication with substantial mortality and point to essential pathophysiological changes due to regulatory responses to hypoxia.
Introduction
Toll‐like receptors (TLRs) detect invading pathogens through several pattern‐recognition mechanisms and play a central role in the regulation of the immune system. In allogeneic ...hematopoietic stem cell transplantation (HSCT), the frequent opportunistic fungal infections remain an important cause of mortality and morbidity in these highly immunocompromised patients.
Methods
We analyzed 154 patients after allogeneic HSCT for acute leukemia for TLR4 gene variants 1063A/G (D299G) and 1363C/T (T399I) with their respective donors, and correlated the results with the incidence of invasive aspergillosis (IA) infection after transplant.
Results
Probable and proven IA in recipients was significantly increased if either recipients or donors exhibited one of the two TLR4 gene variants. In addition, recipients with TLR gene variants and IA showed a delayed T cell and NKT cell immune reconstitution after transplant. Increased susceptibility for IA was not associated with an increased rate of death‐in‐remission or decreased estimate for overall survival.
Conclusion
These findings reinforce the importance of genetic variants in innate immunity and IA among the recipients of allogeneic HSCT.
Polymorphisms in cytokine genes can influence immune responses and inflammation and thereby affecting the outcome of hematopoietic stem-cell transplantation. We analyzed a single-nucleotide ...polymorphism in the gene for the interleukin-23 receptor (IL-23R) (1142G>A) in a cohort of 221 transplant recipients and their human leukocyte antigen (HLA)-identical sibling donors and in a second cohort of 186 transplant recipients and their HLA-identical unrelated donors. Genotypes were tested for an association with graft-versus-host disease (GVHD) by multivariate analysis. The donor's IL-23R genotype was significantly associated with a reduced risk of acute GVHD in both cohorts for patients after transplant. Analysis of all 407 transplant recipients showed that IL-23R (1142G>A, Arg381Gln) genotype of the donor was associated with a decreased risk of grades 2-4 acute GVHD (31.6 compared to 51.0%, P=0.02) and grades 3-4 severe acute GVHD (3.9 compared to 23.4%, P=0.003). Death in remission was significantly lower in patients transplanted from donors with variant IL23-R (11.7 versus 27.7%, P=0.028), whereas overall survival or relapse rates were not influenced significantly by the IL-23R genotype. Among recipients of hematopoietic cells from HLA-identical donors, the IL-23R (Arg381Gln) gene variant on the donor side has a protective effect on the occurrence of acute GVHD in recipients after transplantation.
An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The safety and efficacy of this new ...conditioning regimen has been investigated prospectively in patients with AML. A total number of 75 patients with AML in CR were treated with 3 × 14 g/m(2) treosulfan and 5 × 30 mg/m(2) fludarabine, followed by matched sibling or unrelated SCT. Patients were evaluated for engraftment, adverse events, GVHD, and for non-relapse mortality, relapse incidence, overall and disease-free survival (DFS). All patients showed primary engraftment of neutrophils after a median of 20 days. Non-hematological adverse events grade III-IV in severity included mainly infections (59%) and gastrointestinal symptoms (7%). Acute GVHD grade II-IV occurred in 21% and extensive chronic GVHD occurred in 16% of the patients. After a median follow-up of 715 days, the 2-year overall and DFS estimates were 61% and 55%, respectively. The 2-year incidences of relapse and non-relapse mortality reached 34% and 11%, respectively. In summary, our data confirm promising safety and efficacy of the treosulfan-based conditioning therapy in AML patients, ClinicalTrials.gov Identifier: NCT01063660.
. Ringdén O, Labopin M, Beelen DW, Volin L, Ehninger G, Finke J, Greinix HT, Kyrcz‐Krzemien S, Bunjes D, Brinch L, Niederwieser D, Arnold R, Mohty M, Rocha V, for the Acute Leukaemia Working Party ...of the European Group for Blood and Marrow Transplantation (EBMT) (Karolinska University Hospital Huddinge, Sweden; CEREST‐TC EBMT, Paris, France; University of Duisburg‐Essen, Germany; Helsinki University Central Hospital, Helsinki, Finland; University Hospital, Dresden, Germany; University Hospital, Freiburg, Germany; Medical University Vienna, Vienna, Austria; Medical University of Silesia, Katowice, Poland; University Hospital, Ulm, Germany; Rikshospitalet, Oslo, Norway; University Hospital, Leipzig, Germany; Charité University Hospital, Berlin, Germany; Université de Nantes, Nantes, France; and Hôpital Saint‐Louis, Paris, France). Bone marrow or peripheral blood stem cell transplantation from unrelated donors in adult patients with acute myeloid leukaemia, an Acute Leukaemia Working Party analysis in 2262 patients. J Intern Med 2012; 272: 472–483.
Background. No survival benefit of using blood stem cells instead of bone marrow (BM) has been shown in matched unrelated donor (MUD) transplantation.
Design and methods. In a retrospective registry analysis, we compared the use of blood stem cells (n = 1502) and BM (n = 760) from unrelated donors in patients aged 18–60 years with acute myeloid leukaemia (AML) undergoing myeloablative conditioning between 1997 and 2008. The blood stem cell recipients were older (P < 0.01), had more advanced disease (P < 0.0001) and received less total body irradiation (P < 0.0001) and more antithymocyte globulin (P = 0.01).
Results. Recovery of neutrophils and platelets was faster with blood stem cells (P < 0.0001). The incidence of acute graft‐versus‐host disease (GVHD) was similar, but there was more chronic GVHD in the blood stem cell group hazard ratio (HR) = 1.29, P = 0.02. There were no significant differences in nonrelapse mortality (NRM), relapse incidence and leukaemia‐free survival (LFS) between the two groups amongst patients with AML in remission. In patients with advanced leukaemia, NRM was lower (HR = 0.61, P = 0.02) and LFS was prolonged (HR = 0.67, P = 0.002) when blood stem cells were used. At 3 years, LFS for all patients, regardless of remission status, was 41% for both treatment groups. The outcome was not affected after multivariable analysis adjusted for confounders.
Conclusion. Blood stem cells compared with BM in MUD transplantation for patients with AML in remission resulted in the same rates of LFS. In patients with advanced leukaemia, the blood stem cell group had reduced NRM and improved LFS.
Vitamin D deficiency is associated with a range of clinical disorders. To study the mechanisms involved and improve treatments, animal models are tremendously useful. Current vitamin D deficient rat ...models have important practical limitations, including time requirements when using, exclusively, a vitamin D deficient diet. More importantly, induction of hypovitaminosis D causes significant fluctuations in parathyroid hormone (PTH) and mineral levels, complicating the interpretation of study results. To overcome these shortcomings, we report the successful induction of vitamin D deficiency within three weeks, with stable serum PTH and minerals levels, in Wistar rats. We incorporated two additional manoeuvres compared to a conventional diet. Firstly, the vitamin D depleted diet is calcium (Ca) enriched, to attenuate the development of secondary hyperparathyroidism. Secondly, six intraperitoneal injections of paricalcitol during the first two weeks are given to induce the rapid degradation of circulating vitamin D metabolites. After three weeks, serum 25-hydroxyvitamin D3 (25D) and 1,25-dihydroxyvitamin D3 (1,25D) levels had dropped below detection limits, with unchanged serum PTH, Ca, and phosphate (P) levels. Therefore, this model provides a useful tool to examine the sole effect of hypovitaminosis D, in a wide range of research settings, without confounding changes in PTH, Ca, and P.
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