The graft-versus-leukemia effect of prophylactic donor lymphocyte infusions after allogeneic stem cell transplantation is equally effective in relapse prevention but safer compared to spontaneous graft-versus-host disease.

Therapeutic donor lymphocyte infusions (tDLI) are used to reinforce the graft-versus-leukemia (GvL) effect in relapse after allogeneic stem cell transplantation (alloSCT). In contrast, the role of prophylactic DLI (proDLI) in preventing leukemia relapse has been less clearly established, although supported by retrospective, case-control, and registry analyses. We report a prospective, monocentric, ten year cohort of patients with high risk acute leukemias (AL) or myelodysplasia (MDS) in whom proDLI were applied beyond day +120 post alloSCT to compensate for lack of GvL.272 consecutive allotransplanted AL or MDS patients in complete remission and off immunosuppression at day +120 were stratified according to the prior appearance of relevant GvHD (acute GvHD °II-IV or extensive chronic GvHD) as a clinical indicator for GvL. Escalating doses of unmodified proDLI were applied to 72/272 patients without prior relevant GvHD. Conversely, 157/272 patients with prior spontaneous GvHD did not receive proDLI, nor did 43/272 patients with contraindications (uncontrolled infections, patient refusal, DLI unavailability).By day 160-landmark analysis (median day of first DLI application), proDLI recipients had significantly higher five-year overall (OS) and disease free survival (DFS) (77% and 67%) than patients with spontaneous GvHD (54% and 53%) or with contraindications (46% and 45%) (p=0.003). Relapse incidence for patients with proDLI (30%) or spontaneous GvHD (29%) was significantly lower than in patients with contraindications (39%; p=0.021). With similar GvHD incidence beyond day +160, non-relapse mortality (NRM) was less with proDLI (5%) than without proDLI (18%; p=0.036).In conclusion, proDLI may be able to compensate for lack of GvL in alloSCT recipients with high risk AL or MDS.

Trophectoderm cell failure leads to peri-implantation lethality in Trpm7-deficient mouse embryos.

Early embryogenesis depends on proper control of intracellular homeostasis of ions including Ca2+ and Mg2+. Deletion of the Ca2+ and Mg2+ conducting the TRPM7 channel is embryonically lethal in mice but leaves compaction, blastomere polarization, blastocoel formation, and correct specification of the lineages of the trophectoderm and inner cell mass unaltered despite that free cytoplasmic Ca2+ and Mg2+ is reduced at the two-cell stage. Although Trpm7-/- embryos are able to hatch from the zona pellucida, no expansion of Trpm7-/- trophoblast cells can be observed, and Trpm7-/- embryos are not identifiable in utero at E6.5 or later. Given the proliferation and adhesion defect of Trpm7-/- trophoblast stem cells and the ability of Trpm7-/- ESCs to develop to embryos in tetraploid embryo complementation assays, we postulate a critical role of TRPM7 in trophectoderm cells and their failure during implantation as the most likely explanation of the developmental arrest of Trpm7-deficient mouse embryos.

Neuraminidase Inhibitor Zanamivir Ameliorates Collagen-Induced Arthritis.

Altered sialylation patterns play a role in chronic autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown the pro-inflammatory activities of immunoglobulins (Igs) with desialylated sugar moieties. The role of neuraminidases (NEUs), enzymes which are responsible for the cleavage of terminal sialic acids (SA) from sialoglycoconjugates, is not fully understood in RA. We investigated the impact of zanamivir, an inhibitor of the influenza virus neuraminidase, and mammalian NEU2/3 on clinical outcomes in experimental arthritides studies. The severity of arthritis was monitored and IgG titers were measured by ELISA. (2,6)-linked SA was determined on IgG by ELISA and on cell surfaces by flow cytometry. Zanamivir at a dose of 100 mg/kg (zana-100) significantly ameliorated collagen-induced arthritis (CIA), whereas zana-100 was ineffective in serum transfer-induced arthritis. Systemic zana-100 treatment reduced the number of splenic CD138+/TACI+ plasma cells and CD19+ B cells, which was associated with lower IgG levels and an increased sialylation status of IgG compared to controls. Our data reveal the contribution of NEU2/3 in CIA. Zanamivir down-modulated the T and B cell-dependent humoral immune response and induced an anti-inflammatory milieu by inhibiting sialic acid degradation. We suggest that neuraminidases might represent a promising therapeutic target for RA and possibly also for other antibody-mediated autoimmune diseases.

A Comparative Study on Immobilization of Fructosyltransferase in Biodegradable Polymers by Electrospinning.

Commercial application of biocatalysts depends on the efficiency of the immobilization method and residual enzyme activity. Electrospinning offers a simple and versatile route to immobilize enzymes in submicron-sized fibers and thus improved mass transfer characteristics. Performance of encapsulation of fructosyltransferase from Bacillus subtilis by emulsion, suspension, and coaxial electrospinning was compared. We particularly focused on the effect of hydrophilic properties of a set of biodegradable polymers on support's activity. Bioactivity of electrospun support in aqueous medium increased in order of the matrix hydrophilicity. Additionally, the efficiency of electrospun fibers was compared with Sepabeads®, commercial epoxy-activated resins. In fibers, enzyme loading of 68.1 mg/g and specific enzyme activity of 5.5 U/mg was achieved compared to 49.5 mg/g and 2.2 U/mg on Sepabeads. Fructosyltransferase exhibited high sensitivity towards organic solvents and covalent attachment, respectively. Immobilization of native enzyme in coaxial fibers increased the specific activity to approx. 30 U/mg which corresponds to 24% of that of the free enzyme. Finally, operational stability of fiber supports was examined in a plug-flow reactor and 5% of initial substrate conversion remained after > 2000 cycles. The efficiency of core-shell immobilizates compared to one-dimensional fibers was both in batch and continuous reaction at least 4.4-fold higher.

Imaging Patterns of Rhino-Orbital-Cerebral Mucormycosis in Immunocompromised Patients : When to Suspect Complicated Mucormycosis.

PURPOSE: The aim of this study was to describe radiological imaging findings of a complicated sinusitis, which should raise the suspicion of rhino-orbital-cerebral mucormycosis as being the underlying cause. METHODS: In this retrospective analysis, we describe the cases and imaging findings of 8 patients with proven mucormycosis. These patients presented mostly with new facial or orbital swelling and were referred for imaging to our institution. Magnetic resonance imaging and computed tomography images were classified as abnormal or normal with respect to orbital, paranasal and cerebral signal results. Special emphasis was placed on the distribution of the signal abnormalities regarding involvement of the skull base and the cavernous sinus. RESULTS: Out of a pool of 43 patients with colonization or proven Mucorales infection at different sites of the body, we identified 8 patients with infiltration of the midface and skull base. Unexpectedly seven out of the eight patients with abnormal findings of the paranasal sinuses and the adjacent tissues showed no bony sinus wall destruction. Of the eight patients seven showed inflammatory changes involving the infratemporal fossa and facial/periorbital tissues, three of the eight patients suffered from fungal invasion of the cavernous sinus and the carotid artery and one of the eight patients had a local infection of the hard palate only. CONCLUSION: Imaging findings of inflammatory tissue infiltration adjacent to the paranasal sinuses with possible extension into the pterygopalatine fossa, infratemporal fossa and orbit or the cavernous sinus should raise the suspicion of a mucormycosis, especially in immunocompromised patients.

Oxidative stress predicts progression of peripheral and cardiac autonomic nerve dysfunction over 6 years in diabetic patients.

Oxidative stress is implicated in the pathogenesis of experimental diabetic neuropathy, but prospective studies in diabetic patients are lacking. We aimed to evaluate whether the plasma levels of various biomarkers of oxidative stress predict the progression of diabetic neuropathy and mortality over 6 years. We followed 89 diabetic patients aged 54 ± 14 years (59 % with polyneuropathy), 72 of whom underwent nerve function reassessment after 6.2 ± 0.8 years, whereas 17 died after 4.2 ± 1.0 years. Plasma markers of oxidative stress at baseline included superoxide anion, hypochlorous acid, peroxynitrite, 8-iso-prostaglandin F2α, vitamin E/lipid ratio, and vitamin C. Neuropathy was assessed by symptoms and deficits, motor and sensory nerve conduction velocity (MNCV, SNCV), vibration perception thresholds (VPT), thermal detection thresholds, and heart rate variability (HRV). Despite a reduction in HbA1c by 1.4 ± 1.6 % (p < 0.001), median SNCV, sural SNCV, peroneal MNCV, malleolar VPT, and warm TDT deteriorated after 6 years (all p < 0.05). In multivariate models, increased superoxide generation was associated with a decline in median SNCV (ß = -0.997; p = 0.036) and deterioration in HRV at rest (OR 1.63 [95 % CI 1.09-2.44]; p = 0.017) over 6 years. Low vitamin E/lipid ratio tended to predict a decrease in peroneal MNCV (ß = 0.781; p = 0.057) and an increase in malleolar VPT (ß = -0.725; p = 0.077). Plasma superoxide generation was associated with an increased risk of mortality (HR 23.2 [95 % CI 1.05-513]; p = 0.047). In conclusion, increased plasma superoxide generation predicted the decline in sensory and cardiac autonomic nerve function and mortality over 6 years in diabetic patients, but larger studies are required for confirmation.

Influence of molecular subgroups on outcome of acute myeloid leukemia with normal karyotype in 141 patients undergoing salvage allogeneic stem cell transplantation in primary induction failure or beyond first relapse.

Based on molecular aberrations, in particular the NPM1 mutation (NPM1(mut)) and the FLT3 internal tandem duplication (Flt3-ITD), prognostic subgroups have been defined among patients with acute myeloid leukemia with normal karyotype. Whereas these subgroups are known to play an important role in outcome in first complete remission, and also in the indication for allogeneic stem cell transplantation, data are limited on their role after transplantation in advanced disease. To evaluate the role of molecular subgroups of acute myeloid leukemia with normal karyotype after allogeneic stem cell transplantation beyond first complete remission, we analyzed the data from 141 consecutive adults (median age: 51.0 years, range 18.4-69.3 years) who had received an allogeneic transplant either in primary induction failure or beyond first complete remission. A sequential regimen of cytoreductive chemotherapy (fludarabine, high-dose AraC, amsacrine) followed by reduced intensity conditioning (FLAMSA-RIC), was uniformly used for conditioning. After a median follow up of three years, overall survival from transplantation was 64 ± 4%, 53 ± 4% and 44 ± 5% at one, two and four years, respectively. Forty patients transplanted in primary induction failure achieved an encouraging 2-year survival of 69%. Among 101 patients transplanted beyond first complete remission, 2-year survival was 81% among patients with the NPM1(mut)/FLT3(wt) genotype in contrast to 43% in other genotypes. Higher numbers of transfused CD34(+) cells (hazard ratio 2.155, 95% confidence interval 0.263-0.964, P=0.039) and favorable genotype (hazard ratio 0.142, 95% confidence interval: 0.19-0.898, P=0.048) were associated with superior overall survival in multivariate analysis. In conclusion, patients with acute myeloid leukemia with normal karyotype can frequently be rescued after primary induction failure by allogeneic transplantation following FLAMSA-RIC. The prognostic role of NPM1(mut)/FLT3-ITD based subgroups was carried through after allogeneic stem cell transplantation beyond first complete remission.

Reconstitution and phenotype of Tregs in CMV reactivating patients following allogeneic hematopoietic stem cell transplantation.

In experimental and clinical settings Tregs prevent graft-versus-host disease (GvHD) by inhibiting the proliferation and function of conventional T cells (Tconv). The suppressive potency of Tregs might also lead to the inhibition of protective antiviral T cell responses. As the control of CMV reactivation is important to improve the clinical outcome in allogeneic HSCT, we analyzed the Treg reconstitution in CMV reactivating patients with and without GvHD (n=47) in the first 6 months following transplantation. Most importantly, CMV reactivation does not correlate with the numerical reconstitution of CD4(+)CD25(high)CD127(-) Tregs. During CMV reactivation the proportion of Tregs within the CD4(+) T cell population decreased significantly independent of GvHD manifestation. A comprehensive FACS analysis was performed in order to characterize the phenotype of Tregs and Tconv cells in greater detail for activation, co-stimulation, proliferation, suppressive function and migratory capability. Interestingly, Tregs of patients with CMV reactivation showed a significantly higher CXCR3 expression. CD4(+) Tconv cells expressed significantly higher protein levels of the proliferation marker Ki67 correlating with a numerical increase of CD4(+) T cells. Our results indicate that Tregs are not inhibiting pathogen clearance by Tconv following HSCT, which is of high relevance for future Treg cell-based clinical trials in allogeneic HSCT.

Improving safety of preemptive therapy with oral valganciclovir for cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation.

Valganciclovir (VGC), an oral prodrug of ganciclovir (GCV), has been shown to clear cytomegalovirus (CMV) viremia in preemptive treatment of patients after allogeneic hematopoietic stem cell transplantation (alloHSCT), apparently without significant toxicity. Since VGC obviates hospitalization, it is increasingly being adopted, although not approved, in alloHSCT. When we retrospectively evaluated preemptive treatment with VGC versus GCV, foscarnet or cidofovir, in all 312 consecutive CMV viremias of 169 patients allotransplanted at our institution between 1996 and 2006, we found VGC more efficacious (79%) than non-VGC therapies (69%). The advantage of outpatient VGC, however, was outbalanced by more profound neutropenias (including two cases of agranulocytosis, one with graft loss) requiring subsequent prolonged rehospitalization. Thus, in a second, prospective cohort from 2007 to 2011 (all 202 consecutive CMV viremias of 118 yet older and sicker patients), we implemented twice weekly neutrophil monitoring during outpatient VGC treatment and avoided VGC maintenance therapy. While conserving efficacy (VGC 71%, non-VGC 72%), we could now demonstrate a reduced mean duration of hospitalization with VGC (9 days (0-66)) compared to non-VGC (25 days (0-115)), without any agranulocytosis episodes. We conclude that safe outpatient VGC therapy is possible in alloHSCT recipients, but requires frequent monitoring to prevent severe myelotoxicity.

Reduced-intensity conditioning versus standard conditioning before allogeneic haemopoietic cell transplantation in patients with acute myeloid leukaemia in first complete remission: a prospective, open-label randomised phase 3 trial.

BACKGROUND: Reduced-intensity conditioning regimens have been developed to minimise early toxic effects and deaths after allogeneic haemopoietic cell transplantation. However, the efficacy of these regimens before this procedure has not been investigated in a randomised trial. In this prospective, open-label randomised phase 3 trial we compared a reduced-intensity fludarabine-based conditioning regimen with a standard regimen in patients with acute myeloid leukaemia in first complete remission. METHODS: Patients were aged 18-60 years and had intermediate-risk or high-risk acute myeloid leukaemia (defined by cytogenetics) in first complete remission; an available HLA-matched sibling donor or an unrelated donor with at least nine of ten HLA alleles; and adequate renal, cardiac, pulmonary, and neurological function. Between Nov 15, 2004, and Dec 31, 2009, patients were randomly assigned (1:1, by a computer-based minimisation procedure that balanced patients for age, cytogenetic risk, induction therapy, and donor type) to receive either reduced-intensity conditioning of four doses of 2 Gy of total-body irradiation and 150 mg/m(2) fludarabine or standard conditioning of six doses of 2 Gy of total-body irradiation and 120 mg/kg cyclophosphamide. All patients were given ciclosporin and methotrexate as prophylaxis against graft-versus-host disease. Neither investigators nor patients were blinded to study treatment. Our primary endpoint was the incidence of non-relapse mortality, analysed in the intention-to-treat population. The trial is registered with ClinicalTrials.gov, number NCT00150878. FINDINGS: The trial was stopped early on Dec 31, 2009, because of slow accrual of patients. 99 patients were randomly assigned to receive reduced-intensity conditioning and 96 to receive standard conditioning. The incidence of non-relapse mortality did not differ between the reduced-intensity and standard conditioning groups (cumulative incidence at 3 years 13% [95% CI 6-21] vs 18% [10-26]; HR 0·62 [95% CI 0·30-1·31]). Relapse incidence (cumulative incidence 3 years 28% [95% CI 19-38] vs 26% [17-36]; HR 1·10 [95% CI 0·63-1·90]), disease-free survival (3 year disease-free survival 58% [95% CI 49-70] vs 56% [46-67]; HR 0·85 [95% CI 0·55-1·32]), and overall survival (3 year overall survival 61% [95% CI 50-74] vs 58% [47-70]; HR 0·77 [95% CI 0·48-1·25]) did not differ significantly between groups. Grade 3-4 of oral mucositis was less common in the reduced-intensity group than in the standard conditioning group (50 patients in the reduced-intensity conditioning group vs 73 patients in the standard conditioning group); the frequency of other side-effects such as graft-versus-host disease and increased concentrations of bilirubin and creatinine did not differ significantly between groups. INTERPRETATION: Reduced-intensity conditioning results in a similar incidence of non-relapse mortality and reduced toxic effects compared with standard conditioning without affecting survival outcomes, and thus could be preferentially used in patients younger than 60 years with acute myeloid leukaemia transplanted in first complete remission.

Biomarkers for acute and chronic graft-versus-host disease in regulatory T cells.

Despite improvements in the prevention and treatment of graft-versus-host disease (GvHD) this allogeneic immune response is still one of major complications following allogeneic stem cell transplantation (SCT). Identification of patients at risk for the development of acute and chronic GvHD would facilitate early intervention and thus improve overall survival. Diagnostic biomarkers identified in plasma are largely associated with T cell immune responses. Whereas donor effector T cells promote allogeneic immune responses, regulatory T cells (Tregs) may prevent GvHD by suppression of these alloreactive donor T cells. Therefore, we analyzed molecules associated with Tregs with respect to their potential predictive and prognostic impact on the development of acute and chronic GvHD. For this purpose, the Treg transcriptomes of patients with and without acute/chronic GvHD resulting from dynamical whole genome profiles of CD4(+)CD25(hi)CD127(lo/-) Tregs have been studied for potential GvHD biomarkers. We could identify potential biomarkers for acute/chronic GvHD like the activation marker phosphatidyl-5-kinase-gamma PIP5Kγ, FAS, CD44, CD69, and cell cycle regulators like cyclin A2, B1 and E2. Most importantly, the IKAROS transcription factor Eos, relevant for suppressive Treg function, might be relevant for the prediction of GvHD development. In addition markers like ANK3 (ankyrin), S100A8 and VCAN are indicative for acute GvHD, while IFIT3, IFI44 and IFIT1 are potential biomarkers for chronic GvHD. The identified markers have to be validated prospectively and might help to monitor and guide preventive immune intervention studies, especially adoptive donor Treg cell transfer.

Outcome of high-risk acute myeloid leukemia after allogeneic hematopoietic cell transplantation: negative impact of abnl(17p) and -5/5q-.

The European LeukemiaNet classification combines a heterogeneous group of aberrations as adverse-risk abnormalities. Our goal was to investigate the outcomes associated with distinct high-risk chromosomal abnormalities in acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). We performed a retrospective cohort analysis in patients with high-risk AML who received first, HLA-compatible, allogeneic HSCT between January 2005 and December 2008. Data from 236 patients with a median age of 55 years were included. Because complex karyotype (CK), -5/5q-, and abnl(17p) are overlapping categories, a hierarchical classification system based on the presence or absence of abnl(17p) and -5/5q- was developed. Patients with abnl(17p) had a 2-year event-free survival (EFS) of 11% (95% confidence interval [CI], 0%-25%), patients with -5/5q- but no abnl(17p) a 2-year EFS of 29% (95% CI, 14%-44%), and patients with adverse-risk AML but neither of the 2 marker lesions a 2-year EFS of 49% (95% CI, 39%-59%). Notably, complex and monosomal karyotypes lost their prognostic value when these marker lesions were excluded. In conclusion, hierarchical classification of adverse-risk karyotypes by 2 marker lesions, abnl(17p) and -5/5q-, is effective in prognostication of the outcome of allogeneic HSCT in AML.

Clofarabine-containing conditioning regimen for allo-SCT in AML/ALL patients: a survey from the Acute Leukemia Working Party of EBMT.

Clofarabine (CLO), a second-generation purine analogue, has demonstrated an efficient anti-leukemia activity while showing a favorable toxicity profile. This retrospective multicenter report assessed the outcome of 90 patients who received a CLO-containing conditioning regimen before allo-SCT for AML (n = 69) or ALL (n = 21). Median age was 42 yr at transplant. The majority of cases (n = 66) presented with an active disease at transplant while 38 patients had received previous transplantation(s). A total of 88 and two patients received a reduced-intensity conditioning or a myeloablative regimen, respectively. Engraftment was achieved in 97% of evaluable patients. With a median follow-up of 14 months (range, 1-45), the 2-year OS, LFS, relapse, and NRM rates were 28 ± 5%, 23 ± 5%, 41 ± 6%, and 35 ± 5%, respectively. When comparing AML and ALL patients, OS and LFS were significantly higher for AML (OS, 35 ± 6% vs. 0%, P < 0.0001); LFS: 30 ± 6% vs. 0%, P < 0.0001). In a Cox multivariate analysis, an AML diagnosis was the only factor associated with a better LFS (HR = 0.37; 95%CI, 0.21-0.66; P = 0.001). We conclude that a CLO-containing conditioning regimen prior to allo-SCT might be an effective treatment. Prospective studies are needed to evaluate the potential role of CLO as part of conditioning regimens in acute leukemias.

Cytoreductive treatment with clofarabine/ara-C combined with reduced-intensity conditioning and allogeneic stem cell transplantation in patients with high-risk, relapsed, or refractory acute myeloid leukemia and advanced myelodysplastic syndrome.

The combination of cytoreductive chemotherapy with reduced-intensity conditioning (RIC) is a highly effective antileukemic therapy. Purpose of this retrospective analysis was to evaluate the antileukemic efficacy and toxicity of clofarabine-based chemotherapy followed by RIC and allogeneic stem cell transplantation (SCT) for high-risk, relapsed, or refractory acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). From May 2007 until October 2009, a total of 27 patients underwent allogeneic SCT after treatment with clofarabine and ara-C for 5d and RIC (4Gy TBI/cyclophosphamide/ATG). Prophylaxis of graft-versus-host disease (GvHD) consisted of cyclosporine and mycophenolate mofetil. Unmanipulated G-CSF mobilized PBSC (n=26) or bone marrow cells (n=1) were transplanted from unrelated (n=21) or matched related (n=6) donors. Non-hematological toxicities of this regimen mainly affected liver and skin and were all reversible. Seven patients relapsed within a median time of 5.7 months. The overall survival (OS) and relapse-free survival rates were 56% and 52% at 2 yr, respectively. In this cohort of patients, cytoreduction with clofarabine/ara-C (ClAraC) followed by RIC allogeneic SCT was well tolerated and showed good antileukemic efficacy even in patients with high-risk AML or MDS, with engraftment and GvHD-incidence comparable to other RIC regimens.

P-selectin glycoprotein ligand-1 positive microparticles in allogeneic stem cell transplantation of hematologic malignancies.

P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) mediate adhesion between leukocytes, tumor cells (including leukemias and lymphomas), and platelets, and play an important role in hematopoiesis, T cell activation, and cancer growth and metastasis. As microparticles (MPs) are released from activated or apoptotic cells, there should be significant numbers of circulating PSGL-1-bearing MPs in the blood of patients undergoing allogeneic stem cell transplantation (alloSCT). We enumerated PSGL-1-expressing MPs in plasma samples from 30 consecutive patients with hematologic disorders at different time points during the course of alloSCT by flow cytometry and analyzed their relation to cell counts, patient characteristics, and clinical outcome. Median follow-up time of surviving patients was 1,772 days (range 1272-1981 days). Nineteen patients (63.3%) died, 10 due to progression of disease (33.3%). The PSGL-1 MPs significantly declined during conditioning therapy but increased again after transfusion of donor cells and even more at the time of engraftment. Numbers >250/µL after graft transfusion were associated with a shorter time to engraftment for patients receiving fresh peripheral stem cell grafts (median, 15 vs. 21 days; p = 0.049). Furthermore, low PSGL-1 MP values at those two time points were associated with a higher risk of progress/relapse in univariate analysis (p = 0.008-0.014; hazard ratio [HR] = 15.0-42.0) with cumulative incidences at 5 years of 81.8% versus 28.6% and 85.7% versus 20.0%, respectively. In conclusion, PSGL-1 microparticles show a characteristic course during alloSCT and their possible association with relapse/progress requires further evaluation of the PSGL-1/P-selectin interaction in leukemias and lymphomas.

High lethality of human adenovirus disease in adult allogeneic stem cell transplant recipients with high adenoviral blood load.

BACKGROUND: Human adenoviruses (HAdV) can cause disseminated disease as a severe complication after haematopoietic stem cell transplantation (SCT) and may originate from the reactivation of latent infections. However, data about the clinical relevance of HAdV DNAaemia and disease in adults are scarce. OBJECTIVES: To retrospectively analyse the outcome of adult allogeneic SCT recipients with high HAdV loads in peripheral blood. STUDY DESIGN: Our diagnostic database was screened for allogeneic SCT recipients with peak HAdV DNAaemia above 1.0×10(4)copies/ml (tested by quantitative real-time PCR) and medical records were reviewed retrospectively. RESULTS: From 1674 adult allogeneic SCT recipients 539 (32.2%) received HAdV DNAaemia testing. In twenty-seven of these HAdV blood loads above 1.0×10(4) (range: 1.6×10(4)-1.8×10(9))copies/ml were observed. Seven of these 27 succumbed to HAdV disease and their median peak HAdV DNAaemia was significantly higher than in patients without HAdV-associated death (1.0×10(8) vs. 3×10(5)copies/ml, p<0.001). T-cell depletion was a risk factor for fatal HAdV disease. HAdV of species C predominated (66.7%) and were of high virulence (6 of 7 fatal cases). HAdV of species B were observed more frequently (n=6) in our study than reported for paediatrics, indicating a different pattern of HAdV reactivation in adults. CONCLUSIONS: The presence of several HAdV-associated deaths in adult SCT recipients with high-level HAdV DNAaemia confirmed the clinical relevance of HAdV DNAaemia testing in adults. Quantitative HAdV DNAaemia testing is a promising tool to predict the outcome of HAdV disease.

Human regulatory T cells in allogeneic stem cell transplantation.

GVHD is still one of the major complications after allogeneic stem cell transplantation. Whereas murine data have clearly shown the beneficial effects of regulatory T cells (Tregs) on the prevention of GVHD, data from the human system are rare. Here, we present a comparative dynamic analysis of CD4(+)CD25(hi)CD127(lo/-) Tregs from patients with and without GVHD analyzing the whole genome profile over the first 6 months after stem cell transplantation, representing the most sensitive time window for tolerance induction. The Treg transcriptome showed a high stability. However, the comparison of Treg transcriptomes from patients with and without GVHD uncovered regulated gene transcripts highly relevant for Treg cell function. The confirmative protein analyses demonstrated a significantly higher expression of granzyme A, CXCR3, and CCR5 in Tregs of immune tolerant patients. These results point to a reduced suppressive function of Tregs from GVHD patients with diminished migration capacity to the target organs.

Evaluation of tissue factor bearing microparticles as biomarkers in allogeneic stem-cell transplantation.

BACKGROUND: There is compelling evidence that blood-borne tissue factor that is predominantly found on circulating microparticles (MPs) plays an important role in both, cancer biology and organ and stem-cell transplantation (SCT). Therefore, we hypothesized that numbers of tissue factor bearing MPs might be associated with complications and outcome in allogeneic SCT (allo-SCT). MATERIALS AND METHODS: In a prospective study, we enumerated total, platelet, endothelial, and tissue factor bearing MPs in plasma samples obtained from up to 60 patients with hematologic diseases at different time-points during the course of allo-SCT by flow cytometry. Patient- and transplant-related risk factors were included in statistical analysis. RESULTS: Mean follow-up time was 968 days (0-1981 days). Thirty-four (56.7%) patients died, 17 due to transplant-related mortality (28.3%). High numbers of tissue factor positive MPs more than 500/µL before conditioning were predictive for shorter overall survival (P=0.017, hazard ratio=4.5) in multivariate analysis. This was mainly caused by an increase in transplant-related mortality (P=0.010, hazard ratio=11.0) with cumulative incidences at 1 year of 68.8% compared with patients with lower values (20.1%; P=0.002). CONCLUSIONS: Tissue factor bearing MPs might be useful biomarkers for risk stratification in allo-SCT patients and further studies should investigate their origin, functional properties, and optimal cut-off values.

Acute but not chronic graft-versus-host disease is associated with a reduction of circulating CD4(+)CD25 (high)CD127 (low/-) regulatory T cells.

Defects in central and peripheral tolerance are thought to contribute to life-threatening graft-versus-host disease (GvHD), a severe complication following allogeneic stem cell transplantation (SCT). Recent investigations have demonstrated regulatory T cells (Tregs) to suppress allogeneic immune reactions. Therefore, SCT patients with no or critically low numbers of Tregs may have an increased risk of GvHD. To address this hypothesis, we analyzed the recovery of CD4(+)CD25(high)CD127(low/-) Tregs in the peripheral blood of patients who have never developed GvHD (n = 6), patients who developed acute/chronic GvHD (n = 18), and patients who developed chronic GvHD without an earlier acute manifestation (n = 5) every 30 days for the first 6 months after peripheral blood SCT (PBSCT). The number of Tregs continuously improved in acute/chronic GvHD patients, but always remained lower than Tregs quantified in patients who never developed a GvHD. In contrast, chronic GvHD patients who did not develop acute GvHD earlier displayed significantly increased Treg cell numbers at the timepoint of chronic inflammation. These results indicate that numerically deficient Tregs following PBSCT are associated with the development of acute but not chronic GvHD.

Shedding of the endothelial receptor tyrosine kinase Tie2 correlates with leukemic blast burden and outcome after allogeneic hematopoietic stem cell transplantation for AML.

Angiogenesis plays an important role in the growth and viability of hematologic malignancies. Emerging data suggest a crucial involvement of the endothelial-specific Tie2 receptor and its antagonistic ligand Angiopoietin-2 (Ang-2) in this process. The purpose of this study was to elucidate whether the soluble domain of the Tie2 receptor (sTie2)predicts outcome in patients with acute myeloid leukemia(AML) undergoing allogeneic hematopoietic stem cell transplantation(HSCT). Serum levels of sTie2 and Ang-2 were measured by ELISA in 181 AML patients before conditioning for HSCT. The median follow-up time was 22 months after HSCT. Pre-HSCT sTie2 levels were significantly higher inpatients (median 2.2 (range 1.8-3.0) ng/mL) compared to healthy controls (1.3 (0.9-1.6); p<0.0001). Elevated sTie2 levels were independently associated with active AML but did not relate to cytogenetics/mutational status before transplantation. Logistic regression analysis identified elevated sTie2 (odds ratio (OR) 3.07 (95% confidence interval(CI; 1.56-6.04), p=0.001) as a strong predictor for disease relapse and poor overall survival after HSCT. In a multimarker approach the highest risk for relapse was observed inpatients with both elevated sTie2 and elevated Ang-2 (OR 4.07, (95% CI 1.79-9.25) p<0.0001), as well as patients with both elevated Ang-2 and elevated bone marrow blast count (OR 4.16, (95% CI 1.88-7.36) p<0.0001). Elevated serum sTie2 levels were related to active leukemia,correlated with the percentage of leukemic blasts in the bone marrow, and independently predicted relapse in AML patients after allogeneic HSCT. Furthermore, our data indicate that Tie2 shedding and Ang-2 release seem to reflect overlapping, but nevertheless distinctive features in leukemia-associated neoangiogenesis.