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Background. Calcineurin inhibitors (CNI) have significantly improved patient and graft survival in pediatric liver transplantation (pLT). However, CNI toxicity leads to significant morbidity. Moreover, CNIs cannot prevent long-term allograft injury. Mesenchymal stem (stromal) cells (MSC) have potent immunomodulatory properties, which may promote allograft tolerance and ameliorate toxicity of high-dose CNI. The MYSTEP1 trial aims to investigate safety and feasibility of donor-derived MSCs in pLT. Methods/Design. 7 to 10 children undergoing living-donor pLT will be included in this open-label, prospective pilot trial. A dose of 1 × 106 MSCs/kg body weight will be given at two time points: first by intraportal infusion intraoperatively and second by intravenous infusion on postoperative day 2. In addition, participants will receive standard immunosuppressive treatment. Our primary objective is to assess the safety of intraportal and intravenous MSC infusion in pLT recipients. Our secondary objective is to evaluate efficacy of MSC treatment as measured by the individual need for immunosuppression and the incidence of biopsy-proven acute rejection. We will perform detailed immune monitoring to investigate immunomodulatory effects. Discussion. Our study will provide information on the safety of donor-derived MSCs in pediatric living-donor liver transplantation and their effect on immunomodulation and graft survival. [ABSTRACT FROM AUTHOR]

Purpose: Insufficient production of leukocytes, thrombocytes and erythrocytes after allogeneic peripheral blood stem cell transplantation (PBSCT) represents a life-threatening complication. Methods: In 20 adult patients with poor graft function (PGF defined as transfusion-dependent platelet counts <20,000/µl, or leukocytes <1500/µl, or transfusion-dependent anemia) and variable causes of PGF after allogeneic PBSCT, immunomagnetically selected CD34 stem cell boosts (SCB) from matched unrelated ( n = 8), mismatched unrelated ( n = 11) or haploidentical ( n = 1) donors were applied without prior conditioning. Results: Patients received a median of 4.6 × 10 CD34 cells per kilogram bodyweight (1.9-9.1 × 10) and low T cell numbers (median 0.2 × 10, range 0.04-0.6 × 10). All patients showed responses in at least one hematopoietic lineage. Engraftment for platelets, leukocytes and hemoglobin was 88, 88 and 100 % after a median of 14, 13 and 18 days, respectively. With regard to the complete cohort, 90 % ( n = 18) showed an increase in platelets (median 76,500/µl, range −7000 to 223,000/µl), 95 % ( n = 19) had an increase in leukocytes (median 3110/µl, range 150-13,740/µl) and 90 % ( n = 18) improved with regard to hemoglobin (median 1.9 g/dl, range −0.9 to 5.1 g/dl). Due to effective T cell depletion, only one patient developed graft versus host disease (GvHD, grade III) after SCB. Patients were followed for a median of 7.5 months (1-74 months) with 11 patients being alive and disease free with normalized peripheral blood counts at the end of follow-up. Conclusions: CD34-selected SCB are safe and effective and can durably improve PGF even in patients receiving grafts from unrelated matched or mismatched donors with low incidence of GvHD. [ABSTRACT FROM AUTHOR]

Background aims. The CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαβ+) and CD19 positive (CD19+) cells from apheresis products. Methods. Investigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2-10.4) × 1010 mononuclear cells were used with a median 358 (92-1432) × 106 CD34+ cells. There were 24.8% (6.1-45.7%) median TCRαβ+ cells and 4.4% (1.2-11.7%) median B cells in the apheresis product. Results. After depletion, a median 0.00097% (0.00025-0.0048%) of TCRαβ+ cells could be detected, and B cells, as determined as CD20+ cells, were reduced to 0.0072% (0.0008-0.072%). TCRαβ+ cells were depleted by log 4.7 (3.8-5.5), and B cells were depleted by log 4.1 (3.0-4.7). Recovery of mononuclear cells was 55% (33-77%), and recovery of CD34+ cells was 73% (43-98%). Recovery of CD56+/3− natural killer cells was 80% (35-142%), recovery of TCR gamma/delta positive (TCRγδ+) T cells was 83% (39-173%) and recovery of CD14+ cells was 79% (22-141%). Viability of cells was 98% (93-99%) after separation (all values median). Conclusions. Profound depletion of TCRαβ+ T cells can be achieved with the CliniMACS system. Recovery of CD34+ stem cells is in the same range than after CD34+ enrichment and CD3/CD19 depletion. Transplantation with >4 × 106 CD34+ cells/kg can be performed for every patient with 1-5 × 104 TCRαβ+ cells/kg and about 5-10 × 106 TCRγδ+ cells/kg with two rounds of apheresis. [ABSTRACT FROM AUTHOR]

The impact of human leucocyte antigen (HLA) class I expression intensity and killer-cell immunoglobulin-like receptor (KIR)-mismatch was investigated on natural killer (NK)-cell mediated lysis of B-lineage leukaemic blasts from 21 paediatric patients in vitro. Blast susceptibility to standardised NK-activity and HLA-expression differed widely. A clear association between HLA-molecules/cell (range 442 000–13 000) and specific lysis (mean 59%, range 13–98%) was observed ( r2 = 0·68). A compound model incorporating HLA-expression and KIR-ligand-mismatch provided an even stronger association ( r2 = 0·87), whereas KIR-ligand-mismatch alone was not significant. Assessment of these factors might identify patients who could benefit from NK-mediated graft-versus-leukaemia effects after mismatched stem cell transplantation. [ABSTRACT FROM AUTHOR]

Stick insects were studied while walking on different substrates. The trajectories of swing movements are recorded. The starting position of a swing movement is varied in vertical direction and in the direction parallel to body long axis. The trajectories found cannot be predicted by an ANN (Swingnet1) proposed earlier to describe swing movements. However, a modified network (Swingnet2) allows for a satisfying description of the behavioral results. Walking on a narrow treadwheel leads to different swing trajectories compared to walking on a broad treadwheel. These trajectories cannot be described by Swingnet1, too. The form of the swing trajectory may depend on the direction of the force vector by which the leg acts on the ground in the preceding stance. Based on this assumption, an alternative hypothesis (Swingnet3) is proposed that can quantitatively describe all results of our experiment. When stick insects walk from a wide to a narrow substrate, transition between different swing trajectories does not change gradually over time. Rather, the form of the trajectory is determined by the current sensory input of the leg on a step-to-step basis. Finally, four different avoidance reflexes and their implementation into swing movements are investigated and described by a quantitative simulation. [ABSTRACT FROM AUTHOR]

Transplantation of stem and neural progenitor cells hold great promise in the repair of neuronal tissue lost due to injury or disease. However, survival following transplantation to the adult CNS has been poor, likely due to a lack of neurotrophic factors, such as basic fibroblast growth factor (FGF-2), that are used to maintain and expand these cells in culture. Chromaffin cells produce several neurotrophic agents, including FGF-2, which may aid in both neuroprotection following injury and progenitor cell proliferation and survival. In addition, increased CNS catecholamines have been shown to improve functional recovery following insult. Thus, cotransplants of neural progenitor cells and chromaffin cells may be a useful clinical strategy. To address this, the survival of rat cortical progenitors transplanted to the adult rat striatum with and without bovine chromaffin cell cografts was assessed. Progenitors obtained from E14 embryos were prelabeled with bromodeoxyuridine (BrdU) before transplantation to enable later identification. Transplants were made both unilaterally and bilaterally, where animals received a monograft (progenitor cells alone) on one side and a cograft (progenitors + chromaffin cells) on the other. Histological results after 7, 17, and 30 days posttransplant revealed greatly improved survival of BrdU-labeled cells in the cografts and also less infiltration of presumptive immune cells. In addition, perivascular cuffing was seen in the monografts. In vitro progenitor cohorts stained positive for nestin, GFAP, and β-tubulin III, but in vivo very few cells were found that were double labeled with BrdU and one of these markers. Thus, in contrast to in vitro findings, chromaffin cells did not enhance differentiation of progenitors in vivo during the 30 days posttransplantation. The results of these studies suggest that chromaffin cells may provide neurotrophic support to enhance survival, but not differentiation, of cortical progenitor grafts in the adult CNS. [Copyright &y& Elsevier]

Previous studies showed that neural progenitor cultures could be maintained without exogenously added FGF-2 when co-cultured with chromaffin cells. In addition, progenitor cells displayed dramatically increased neuronal differentiation in the presence of chromaffin cells. These findings suggested an approach to improved neural progenitor transplant outcomes using co-transplantation or administration of chromaffin cell-derived factors. The aim of this study was to determine whether the observed survival and differentiation effects were due to diffusible factors or required direct cell–cell contact (DC). Rat neural progenitors were cultured under six different conditions: (1) Standard N2 media with FGF-2; (2) N2 without FGF-2; (3) N2 with FGF+conditioned media (CM) from chromaffin cultures; (4) N2 without FGF-2+CM; (5) Transwells (TW), progenitor+chromaffin cells grown together but separated by a membrane allowing movement of diffusible agents but preventing direct contact; (6) direct contact co-cultures of progenitors and chromaffin cells. Cultures were evaluated for survival, proliferation, and differentiation. Cultures with FGF-2 proliferated and formed floating neurospheres while those grown in N2 without FGF-2 failed to thrive. Those grown either with CM or in transwells showed significantly improved survival. Survival was comparable to the exogenous FGF groups when progenitors were allowed direct contact with chromaffin cells. Proliferation was low in all cultures except those receiving exogenous FGF-2. Direct contact co-cultures exhibited a marked increase in β-tubulin III+ processes compared to all other groups, indicating differentiation towards a neuronal phenotype. The results of this study suggest that diffusible agents produced by chromaffin cells can sustain viable progenitor cells in vitro even in the absence of added FGF-2 but that the effects on progenitor cell neuronal differentiation require direct cell–cell contact. [Copyright &y& Elsevier]

The transplantation of neural stem cells and progenitors has potential in restoring lost cellular populations following central nervous system (CNS) injury or disease, but survival and neuronal differentiation in the adult CNS may be insufficient in the absence of exogenous trophic support. Adrenal medullary chromaffin cells produce a trophic cocktail including basic fibroblast growth factor (FGF-2) and neurotrophins. The aim of this study was to evaluate whether chromaffin cells can provide a supportive microenvironment for neural progenitor cells. In order to assess this, the growth and differentiation of neural progenitor cell cultures from embryonic rat cortex were compared in standard FGF-2-supplemented neural progenitor growth media, in standard media but lacking FGF-2, or in media lacking FGF-2 but co-cultured with bovine chromaffin cells. Using bromodeoxyuridine (BrdU)-prelabeling, findings indicated poor survival of progenitor cultures in the absence of FGF-2. In contrast, the addition of chromaffin cells in co-culture appeared to ‘rescue’ the progenitor cultures and resulted in robust neurospheres containing numerous BrdU-labeled cells interspersed with and closely apposed to chromaffin cells. As indicated by H3 labeling, cells in co-cultures continued to proliferate, but at a substantially reduced rate compared with standard FGF-2 supplemented growth media. The co-cultures contained more β-tubulin III-positive processes than parallel cultures maintained in FGF-2-supplemented media and these cells displayed a more mature phenotype with numerous varicosities and complex processes. These findings indicate that chromaffin cells can provide a supportive environment for the survival and neuronal differentiation of neural progenitor cells and suggest that their addition may be useful as a sustained source of trophic support to improve outcomes of neural stem cell transplantation. [Copyright &y& Elsevier]

Presents a study that examined whether the compensation of head position is found with respect to the dorso-ventral axis of the body position. Background on the neural system which controls behavior; Comparison of body rotation in animals walking along inclined surfaces and in animals walking on a horizontal plane; Factor that cause the rotation of the body axis.

Disease relapse after one or more allogeneic hematopoietic cell transplantations (HCT) represents a therapeutic challenge with all options bearing a significant morbidity and mortality. Haploidentical HCT may induce more pronounced anti-leukemic effects and was evaluated at our center in 25 consecutive patients with disease relapse after preceding HCT receiving haploidentical grafts after in vitro T cell depletion. Overall survival at 1 and 2 years was 32 and 14%, respectively. Of note, patients with complete remission (CR) before haploidentical HCT had a very favorable overall survival of 41.7% at 2 years. Cumulative incidence of non-relapse mortality was 36 and 40% at 1 and 2 years, respectively. With a cumulative incidence for relapse of 36 and 45.6% at 1 and 2 years, disease-free survival (DFS) was 28 and 14.4%, respectively. Here also, patients with CR before haploidentical HCT had a favorable DFS of 42% at 2 years. Only very limited acute (11 patients (44%) with a median grade 1) and chronic graft versus host disease (GvHD) (5 patients (11%), limited grade only) was observed. The main complications and causes of death comprised-besides relapse-infections and bleeding complications. Hence, haploidentical HCT can achieve long-term survival comparable to second transplantation with matched or mismatched donors for patients with otherwise deleterious prognosis and should be considered as a treatment option for patients experiencing disease relapse after previous allogeneic HCT. [ABSTRACT FROM AUTHOR]

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder primarily affecting the white matter of the nervous system that results from a deficiency of the arylsulfatase A (ARSA). Mesenchymal stem cells (MSCs) are able to secrete ARSA and have shown beneficial effects in MLD patients. In this retrospective analysis, 10 pediatric MLD patients [mesenchymal stem cell group (MSCG)] underwent allogeneic hematopoietic stem cell transplantation (HSCT) and received two applications of 2 × 106 MSCs/kg bodyweight at day +30 and +60 after HSCT between 2007 and 2018. MSC safety, occurrence of graft-versus-host disease (GvHD), blood ARSA levels, chimerism, cell regeneration and engraftment, magnetic resonance imaging (MRI) changes, and the gross motor function were assessed within the first year of HSCT. The long-term data included clinical outcomes and safety aspects of MSCs. Data were compared to a control cohort of seven pediatric MLD patients [control group (CG)] who underwent HSCT only. The application of MSC in pediatric MLD patients after allogeneic HSCT was safe and well tolerated, and long-term potentially MSC-related adverse effects up to 13.5 years after HSCT were not observed. Patients achieved significantly higher ARSA levels (CG: median 1.03 nmol·10−6 and range 0.41–1.73 | MSCG: median 1.58 nmol·10−6 and range 0.44–2.6; P < 0.05), as well as significantly higher leukocyte (P < 0.05) and thrombocyte (P < 0.001) levels within 365 days of MSC application compared to CG patients. Statistically significant effects on acute GvHD, regeneration of immune cells, MRI changes, gross motor function, and clinical outcomes were not detected. In conclusion, the application of MSCs in pediatric MLD patients after allogeneic HSCT was safe and well tolerated. The two applications of 2 × 106/kg allogeneic MSCs were followed by improved engraftment and hematopoiesis within the first year after HSCT. Larger, prospective trials are necessary to evaluate the impact of MSC application on engraftment and hematopoietic recovery. [ABSTRACT FROM AUTHOR]

Investigates the clinical feasibility and safety of CD133-based selection and transplantation in a small number of pediatric patients. Marker of more primitive hematopoietic progenitors; Proportion of CD133 selected cells; Evidence of a rapid platelet recovery; T-cell regeneration.

Steroid-refractory graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation (alloHSCT). Alternative treatment options are often insufficient. Several studies have proven the efficacy of mesenchymal stromal cells (MSCs) in the treatment of therapy-refractory acute GvHD in adult and pediatric patients. Long-term data in pediatric patients are scarce. In this retrospective analysis, a total of 25 patients with a median age of 10.6 years (range 0.6–22.1 years) who received bone marrow-derived MSCs after alloHSCT for the treatment of steroid-refractory III and IV GvHD were analyzed. The median observation period of the surviving patients was 9.3 years (1.3–12.7 years) after HSCT. Among the 25 patients, 10 (40.0%) died [relapse (n = 3), multiorgan failure (n = 6), cardiorespiratory failure (n = 1)] at median 0.5 years (0.2–2.3 years) after HSCT. Partial response and complete remission (PR, CR) of the GvHD were achieved in 76.0% and 24.0% of the patients, respectively. Transplant-related mortality was 0% in the patients who achieved CR after MSC treatment and 26.3% for those with PR. A median improvement by one intestinal or liver GvHD stage (range 1–4) could be achieved after MSC application. No potentially MSC-related long-term adverse effects, for example, secondary malignancy, were identified. In conclusion, the intravenous application of allogeneic MSCs was safe and proved effective for the treatment of steroid-refractory GvHD. However, larger, prospective, and randomized trials are needed to evaluate these findings. [ABSTRACT FROM AUTHOR]

Avascular necrosis (AVN) is a severe complication of immunosuppressant therapy or chemotherapy. A beneficial AVN therapy with core decompression (CD) and intraosseous infusion of mesenchymal stromal cells (MSCs) has been described in adult patients, but there are only few data on MSC applications in pediatric and young adult patients (PYAP). Between 2006 and 2015, 14 AVN lesions of 10 PYAP (6 females) with a median age of 16.9 years (range 8.5–25.8 years) received CD and intraosseous application of autologous MSCs. Data of these patients were analyzed regarding efficacy, safety, and feasibility of this procedure as AVN therapy and compared with a control group of 13 AVN lesions of 11 PYAP (5 females) with a median age of 17.9 years (range 13.5–27.5 years) who received CD only. During the follow-up analysis [MSC group: median 3.1 (1.6–5.8) years after CD; CD group: median 2.0 (1.5–8.5) years after CD], relative lesion sizes (as assessed by magnetic resonance imaging) compared with the initial lesion volume, were significantly lower (P < 0.05) in the MSC group (volume reduction to a median of 18.5%) when compared with the CD group (58.0%). One lesion in the MSC group comprised a complete remission. Size progression was not observed in either group. Clinical improvement (pain, mobility) was not significantly different between the two groups. None of the patients experienced treatment-related adverse effects. CD and additional MSC application was regarded safe, effective, feasible, and superior in reducing the lesion size when compared with CD only. Prospective, randomized clinical trials are needed to further evaluate these findings. [ABSTRACT FROM AUTHOR]

Summary: Donor lymphocyte infusion (DLI) is an effective method to establish full donor chimerism or to prevent and treat relapse after allogeneic haematopoietic cell transplantation (allo‐HCT). Usually, DLIs are collected from naïve donors as steady‐state lymphocytes. When donor lymphocytes are collected during stem cell apheresis, donors are pre‐treated with granulocyte colony‐stimulating factor (G‐CSF). However, the impact of G‐CSF stimulation and the resulting composition of DLIs on beneficial anti‐leukaemic responses and survival remain elusive. Therefore, we performed a retrospective analysis to evaluate the role of G‐CSF‐DLIs: 44 patients received either steady‐state DLIs or G‐CSF‐DLIs to prevent and treat relapse or establish full donor chimerism after allo‐HCT. The G‐CSF‐DLI patient cohort showed an improved conversion to full donor chimerism and a lower cumulative incidence of relapse or disease progression without a significantly increased cumulative incidence of graft‐versus‐host disease (GVHD). CD34+ cells, monocytic myeloid‐derived suppressor cells and monocytes as well as donor age and the subsequent occurrence of chronic GVHD were identified as risk factors that significantly improve overall survival after DLI administration. In conclusion, our data suggest that administration of G‐CSF‐DLIs results in graft‐versus‐leukaemia effects without exacerbating GVHD, therefore, improving survival after DLIs. [ABSTRACT FROM AUTHOR]

Here, we report the case of severe adenoviremia in a 7-year-old boy with highly-resistant, acute leukemia. A combined approach of αβTCR-CD19–depleted stem cell transplantation, enabling immunosuppression-free post-transplant care, and early transfer of adenovirus-specific donor T cells during aplasia resulted in rapid and complete clearance of the treatment-refractory adenoviremia. [ABSTRACT FROM AUTHOR]

Pediatric patients with refractory or relapsed metastatic neuroblastoma (NBL) have a poor prognosis despite autologous stem cell transplantation (SCT). Allogeneic SCT from a haploidentical donor has a remarkable alloreactive effect in patients with leukemia; thus, we evaluated this approach in children with very high-risk NBL. We analyzed data from 2 prospective phase I/II trials. A total of 26 patients with refractory (n = 5), metastatic relapsed (n = 20), or locally relapsed MYCN -positive (n = 1) NBL received a median of 17 × 10 6 /kg T/B cell-depleted CD34 + stem cells with 68 × 10 3 /kg residual T cells and 107 × 10 6 /kg natural killer cells. The conditioning regimen comprised melphalan, fludarabine, thiotepa, OKT3, and a short course of mycophenolate mofetil post-transplantation. Engraftment occurred in 96% of the patients. Event-free survival and overall survival at 5 years were 19% and 23%, respectively. No transplantation-related mortality was observed, and the single death was due to progression/subsequent relapse. The median duration of follow-up was 8.1 years. Patients in complete remission before SCT had a significantly better prognosis than those with residual tumor load ( P  < .01). All patients with progressive disease before SCT relapsed within 1 year. Grade II and grade III acute graft-versus-host disease (GVHD) occurred in 31% and 12% of the patients, respectively. Chronic limited and extensive GVHD occurred in 28% and 10%, respectively. Our data indicate that haploidentical SCT is a feasible treatment option that can induce long-term remission in some patients with NBL with tolerable side effects, and may enable the development of further post-transplantation therapeutic strategies based on the donor-derived immune system. [ABSTRACT FROM AUTHOR]

Poor graft function ( PGF) is a severe complication of haematopoietic stem cell transplantation ( HSCT) and administration of donor stem cell boosts ( SCBs) represents a therapeutic option. We report 50 paediatric patients with PGF who received 61 boosts with CD34+ selected peripheral blood stem cells ( PBSC) after transplantation from matched unrelated ( n = 25) or mismatched related ( n = 25) donors. Within 8 weeks, a significant increase in median neutrophil counts (0·6 vs. 1·516 × 109/l, P < 0·05) and a decrease in red blood cell and platelet transfusion requirement (median frequencies 1 and 7 vs. 0, P < 0·0001 and <0·001), were observed, and 78·8% of patients resolved one or two of their cytopenias. 36·5% had a complete haematological response. Median lymphocyte counts for CD3+, CD3+ CD4+, CD19+ and CD56+ increased 8·3-, 14·2-, 22.- and 1·6-fold. The rate of de novo acute graft-versus-host disease (Gv HD) grade I-III was only 6% and resolved completely. No Gv HD grade IV or chronic Gv HD occurred. Patients who responded to SCB displayed a trend toward better overall survival ( OS) ( P = 0·07). Thus, administration of CD34+ selected SCBs from alternative donors is safe and effective. Further studies are warranted to clarify the impact on immune reconstitution and survival. [ABSTRACT FROM AUTHOR]

Graft failure is a life-threatening complication after allogeneic haematopoietic stem cell transplantation ( HSCT). We report a cohort of 19 consecutive patients (median age: 8·5 years) with acute leukaemias ( n = 14) and non-malignant diseases ( n = 5) who experienced graft failure after previous HSCT from matched ( n = 3) or haploidentical donors ( n = 16) between 2003 and 2012. After total nodal irradiation ( TNI)-based reconditioning combined with fludarabine, thiotepa and anti-T cell serotherapy, all patients received T cell-depleted peripheral blood stem cell grafts from a second, haploidentical donor. Median time between graft failure and retransplantation was 14 d (range 7-40). Sustained engraftment (median: 10 d, range 9-32) and complete donor chimerism was observed in all evaluable patients. 5 patients additionally received donor lymphocyte infusions. Graft-versus-host disease (Gv HD) grade II and III occurred in 1 patient each (22%); no Gv HD grade IV was observed. 2 patients had transient chronic Gv HD. The regimen was well tolerated with transient interstitial pneumonitis in one patient. Treatment-related mortality after one year was 11%. Event-free survival and overall survival 3 years after retransplantation were 63% and 68%. Thus, a TNI-based reconditioning regimen followed by transplantation of haploidentical stem cells is an option to rescue patients with graft failure within a short time span and with low toxicity. [ABSTRACT FROM AUTHOR]

Chronic graft-versus-host disease (cGVHD) is a significant hurdle to long-term hematopoietic stem-cell transplantation success. Insights into the pathogenesis and mechanistical investigations of novel therapeutic strategies are limited as appropriate animal models are missing. The immunodeficient NSG mouse – when humanized with human bone marrow, fetal liver and thymus (BLT NSG) – is prone for cGVHD, yet mainly affects the skin. In contrast, the NSG mouse humanized exclusively with CD34 + -selected, CD3 + -depleted stem cells (CD34 + NSG) has neither been described for acute nor chronic GVHD so far. This is the first report about the development of systemic autoimmune cGVHD ≥24 weeks post stem cell receipt involving lung, liver, skin, gingiva and intestine in two NSG cohorts humanized with CD34 + grafts from different donors. Affected mice presented with sclerodermatous skin, fibrotic lung, severe hepatitis, and massive dental malformation/loss. CD4 + -dominated, T H 2-biased, bulky T-cell infiltrates featured highly skewed T cell receptor (TCR) repertoires, clonal expansions, and autoreactive TCRs. In affected tissues profibrotic IL-13 and -4 dominated over T H 1 cytokines IFN-γ and TNF-α. Thus, the time point of manifestation and the phenotype match human systemic pleiotropic sclerodermatous GVHD. The CD34 + NSG-model's intrinsic deficiency of thymus, thymus-derived regulatory T cells (nTreg) and B cells emphasizes the role of the genetic polymorphism and the cytokines in the pathogenesis of cGVHD. Importantly, the only factor discriminating diseased versus non-diseased CD34 + NSG cohorts were two risk HLA haplotypes that in human mediate susceptibility for autoimmune disease (psoriasis). Thus, the CD34 + NSG model may serve as a platform for addressing issues related to the pathophysiology and treatment of human autoimmunity and chronic GVHD. [ABSTRACT FROM AUTHOR]

Transplantation of T- and B-cell depleted allografts from haploidentical family donors was evaluated within a prospective phase II trial in children with acute lymphoblastic leukaemia, acute myeloid leukaemia and advanced myelodysplastic syndrome ( n = 46). 20 patients had active disease; 19 patients received a second or third stem cell transplantation ( SCT). Toxicity-reduced conditioning regimens consisted of fludarabine or clofarabine (in active disease only), thiotepa, melphalan and serotherapy. Graft manipulation was carried out with immunomagnetic microbeads. Primary engraftment occurred in 88%, with a median time to reach >1·0 × 109/l leucocytes, >20 × 109/l platelets and >0·1 × 109/l T-cells of 10, 11 and 50 days, respectively. After retransplantation, engraftment occurred in 100%. Acute graft-versus-host disease (Gv HD) grade II and III- IV occurred in 20% and 7%, chronic Gv HD occurred in 21%. Both conditioning regimens had comparable toxicity. Transplant-related mortality ( TRM) was 8% at one year and 20% at 5 years. Event-free survival at 3 years was: 25% (whole group), 46% (first, second or third complete remission [ CR], first SCT) vs. 8% (active disease, first SCT) and 20% (second or third SCT, any disease status). This approach allows first or subsequent haploidentical SCTs to be performed with low TRM. Patients in CR may benefit from SCT, whereas the results in patients with active disease were poor. [ABSTRACT FROM AUTHOR]

Haploidentical hematopoietic stem cell transplantation is a curative alternative option for patients without an otherwise suitable stem cell donor. In order to prevent graft-versus-host disease (GvHD), different in vitro and in vivo T cell-depletion strategies have been developed. A delayed immune reconstitution is common to all these strategies, and an impaired immune function after haploidentical transplantation with subsequent infections is a major cause of deaths in these patients. In addition to in vitro and in vivo T cell-depletion methods, posttransplant strategies to rapidly rebuild the immune system have been introduced in order to improve the outcome. Advances in in vitro and in vivo T cell-depletion methods, and adoptive transfer of immune cells of the innate and specific immune system, will contribute to reduce the risk of GvHD, lethal infections, and the risk of relapse of the underlying malignant disease. [ABSTRACT FROM AUTHOR]

Background aims. In the absence of a protective immune response, human cytomegalovirus (HCMV) infection remains a life-threatening complication after allogeneic stem cell transplantation (SCT), especially in recipients of grafts from HCMV-seronegative donors. After allogeneic SCT from a seronegative donor, prolonged and severe immune deficiency often leads to infectious complications. Vaccination with antigen-loaded dendritic cells (DC) has been shown to be a potent approach for the induction of antigen-specific cytotoxic T-cell responses in vivo. For protection from subsequent HCMV reactivation, a sustained immune response is necessary, including antigen-specific CD4+ T cells. Methods. We report the case of an 18-year-old girl with high-risk acute lymphoblastic leukemia that received an allogeneic SCT in CR2. After an HCMV infection, the graft was rejected and she received a second transplant from an HLA-mismatched, HCMV-seronegative family donor. She was treated with pp65-pulsed monocyte-derived DC at day 200 post-SCT, using a recombinant pp65 protein. Until day 200 post-SCT, HCMV reactivated six times with emerging viral resistance to antiviral chemotherapy. Results. After vaccination with protein-pulsed DC, an induction and expansion of HCMV-specific Thelper cells and cytotoxic T lymphocytes was observed, associated with a sustained clearance of the HCMV viremia. Antiviral treatment could be tapered without recurrence of viremia within the first year post-SCT. Conclusions. pp65-pulsed DC could induce antigen-specific T-cell responses even after a SCT from an HCMV-seronegative donor. After vaccination with pp65-pulsed DC, a sustained antigen-specific T-cell response prevented concurrent HCMV viremia. Emergence of antigen-specific Thelper cells may be essential for a sustained, functional T-cell response post-SCT. [ABSTRACT FROM AUTHOR]

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel–Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Abstract: Malaria is a major public health problem in Sub-Saharan Africa. In highly endemic regions infants, children and pregnant women are mostly affected. An effective malaria vaccine would complement existing malaria control strategies because it can be integrated in existing immunization programs easily. Here we present the results of the first phase Ia clinical trial of GMZ2 adjuvanted in aluminium hydroxide. GMZ2 is a malaria vaccine candidate, designed upon the rationale to induce immune responses against asexual blood stages of Plasmodium falciparum similar to those encountered in semi-immune individuals. Ten, 30 and 100μg of GMZ2 were well tolerated in 30 healthy malaria-naïve German volunteers when given three times in monthly intervals. Antigen-specific antibodies as well as memory B-cells were induced and detectable throughout the one year follow-up of the study. We conclude that GMZ2 is a safe and immunogenic malaria vaccine candidate suitable for further clinical development. [Copyright &y& Elsevier]

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO-/-, gp91phox-/-, and NOS-/- mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils. [ABSTRACT FROM AUTHOR]

Abstract: Graft failure is a life-threatening complication after transplantation of hematopoietic stem cells. We report a cohort of 11 pediatric patients with leukemias (n =8) and severe aplastic anemia (n =3) who experienced graft rejection after myeloablative transplantation from mismatched related donors (n =6) or after cord blood or matched unrelated donor transplantation (n =5). In the latter, the original donor was not available anymore. All patients were re-transplanted with CD34+ selected or CD3/CD19 depleted stem cells from a second, haploidentical donor. Median time span from diagnosis of rejection to second transplant was 9 days. Reconditioning regimens comprised total lymphoid irradiation, thiotepa, fludarabine, ATG/OKT3 and were well tolerated. A median number of 23.5×106/kg stem cells with 95,000/kg residual T-cells were infused. Sustained engraftment of neutrophiles/platelets and complete donor chimerism was achieved in all patients (ANC>500/μl: 9 (11–32) days). No GvHD>grade II was observed. 8/11 patients are disease free (median follow up 1.9 years; 1 year-EFS=72%). Causes of death were: pneumonitis, infection, relapse. Thus, haploidentical transplantation represents a realistic option to rescue patients with graft failure within a short time span, for whom a second donation from the original donor is not available. The use of different donors may contribute to avoid a second rejection. [Copyright &y& Elsevier]

Objective: CD3/CD19 depletion may improve engraftment and immune reconstitution after haploidentical hematopoietic cell transplantation (HHCT) as grafts not only contain CD34+ stem cells but also CD34− progenitors and natural killer, dendritic, and facilitating cells. Patients and Methods: Ten consecutive patients received HHCT with CD3/CD19-depleted grafts. Reduced-intensity conditioning was performed with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), and OKT-3 (5 mg/day, day −5 to +14) without additional posttransplant immunosuppression. Diagnoses were AML (n = 4), ALL (n = 3), NHL (n = 2), and multiple myeloma (n = 1). All patients were “high risk” with refractory disease or relapse after preceding HCT. The CD3/CD19-depleted haploidentical grafts contained a median of 7.8 × 106 (range, 5.2–17 × 106) CD34+ cells/kg, 5.5 × 107 (range, 0.02–8.6 × 107) CD56+ cells/kg, and 2.0 × 104 (range, 0.006–44 × 104) CD3+ T cells/kg. Engraftment was rapid with median time to greater than 500 granulocytes/μL of 13 (range, 11–17) days, greater than 20,000 platelets/μL of 11 (range, 8–16) days, and full donor chimerism after 2 weeks in all patients. Six cases of grade II GVHD occurred. One patient, who received the highest T cell dose, developed lethal grade IV GVHD. Treatment-related mortality in the first 100 days was 3/10 (30%) with one death each due to idiopathic pneumonia syndrome, GVHD, and CMV disease. Two patients died after day 100, one due to relapse and one with systemic adenoviral infection. Overall survival is 5/10 patients (50%) with a median follow-up of 435 (range, 229–814) days. Conclusion: This regimen is promising in high-risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing. [Copyright &y& Elsevier]

During periods of immunosuppression, such as postallogeneic stem cell transplantation (SCT), patients are at significant risk for severe viral infections. Human adenovirus (HAdV) infection is a serious complication post-SCT, especially in children. Virus-specific T cells are essential for the clearance of HAdV, as antiviral chemotherapy has revealed limited success. We present feasibility data for a new treatment option using virus-specific donor T cells for adoptive transfer of immunity to patients with HAdV-infection/reactivation. Virus-specific donor T cells were isolated and infused into nine children with systemic HAdV infection after SCT. Isolation was based on γ-interferon (IFN- γ) secretion after short in vitro stimulation with viral antigen, resulting in a combination of CD4+ and CD8+ T cells. 1·2–50 × 103/kg T cells were infused for adoptive transfer. Isolated cells showed high specificity and markedly reduced alloreactivity in vitro. Adoptive transfer of HAdV-specific immunity was successful in five of six evaluable patients, documented by a dose-independent and sustained in vivo expansion of HAdV-specific T cells, associated with a durable clearance/decrease of viral copies. T-cell infusion was well tolerated in all nine patients, except one case with graft- versus-host disease II of the skin. In conclusion, induction of a specific T-cell response through adoptive transfer was feasible and effective. When performed early in the course of infection, adoptive T-cell transfer may protect from HAdV-related complications. [ABSTRACT FROM AUTHOR]

Human adenovirus (ADV) infection is an important complication post allogeneic stem cell transplantation (SCT), especially in children, with significant morbidity and mortality despite new antiviral treatment strategies. Although the control of infection seems to require T cells, characterization of ADV-specific T cells post-SCT has not been reported to date. Therefore, we prospectively studied the occurrence of ADV-specific T cells in children with (n = 21) and without (n = 25) ADV-infection postallogeneic SCT and in healthy donors (n = 53). ADV-associated mortality occurred in seven of 21 children. After stimulationex vivowith ADV-lysate, interferon-γ-secreting T cells were analysed by flow cytometry. All the patients with ADV-associated mortality had no specific T cells, although reconstitution of absolute lymphocyte counts exceeded 0·3 × 109/l within 30 d post-transplant. Patients who cleared ADV infection had significantly higher frequencies (mean ± standard deviation, 0·56 ± 0·5%) of ADV-specific T cells until day 200 post-SCT, than patients without ADV-infection (0·12 ± 0·1%). These data suggest that ADV-specific T-cell reconstitution is protective against life-threatening ADV-infections postallogeneic SCT. [ABSTRACT FROM AUTHOR]

We present an update of our results with transplantation of highly purified stem cells from one to three loci mismatched parental donors. Sixty-three pediatric patients with acute lymphoblastic leukemias (n = 32), acute myeloid, chronic myeloid and myelomonocytic leukemias (n = 13), myelodysplastic syndromes (n = 4), lymphomas (n = 4), and various nonmalignant diseases (n = 10) underwent transplantation. Mobilized peripheral-blood stem cells were selected with either anti-CD34- or anti-CD133-coated microbeads. Patients received a median of 19.5 × 106 purified cells and <25,000 CD3+ T lymphocytes per kilogram, with no regular posttransplant pharmacological immunosuppression. Engraftment occurred in 98% of patients (primary sustained engraftment, 83%; engraftment after reconditioning/stem cell boosts, 15%). Moreover, all survivors but one had a stable three-lineage engraftment with a median follow up of 4.1 years (range 0.6–8 years).Primary acute graft-versus-host disease (GvHD) grade II was seen in only 7% of patients. No severe primary acute GvHD grades III–IV occurred. Thirteen percent of the patients developed transient chronic GvHD. Probability of disease-free survival (DFS) at 3 years was 60% for patients with nonmalignant diseases and 48% for patients with acute lymphatic leukemia (ALL)/non-Hodgkin lymphoma (NHL) in complete remission (CR)1–3. None of the ALL/NHL patients with active disease survived. Children with acute and chronic myeloid leukemias had a poorer outcome (3-year DFS = 18%), whereas two of four patients with myelodysplastic syndrome (MDS) are alive. Relapse probability of the whole group was not significantly increased when compared to a historical control group. The incidence of lethal viral infections was 18% between 1995 and 2002 and has since been reduced to 8% by the introduction of new therapeutic strategies. In summary, the use of stem cells from haploidentical parental donors should be strongly considered in all children who need transplantation but lack an identical donor. [Copyright &y& Elsevier]

Early inductive signals within the embryonic mammalian forebrain establish two major germinal regions along the dorsal–ventral axis. The dorsal germinal zone eventually forms the cerebral cortex while the ventral ganglionic eminence primarily forms the striatum and globus pallidus. The mechanisms leading to patterning of specific forebrain structures from these distinct germinal regions are not fully understood but may involve the adhesive and migratory properties of regionally specified cells and their interactions with the extracellular environments in which they reside. In the present study, we isolated ganglionic eminence neural progenitor cells (geNPC), precursors of the adult striatum, from the ventral forebrain germinal zone and analyzed adhesion, migration, and differentiation of geNPC on various extracellular matrix (ECM) substrates in vitro. Specifically, we evaluated the role of β1 integrins, a family of cell surface receptors important in neural development, in mediating geNPC behavior on ECM molecules expressed in embryonic brain tissue. Adhesion and migration of geNPC were significantly enhanced on laminin (LN) and fibronectin (FN) relative to other ECM substrates. Antibody perturbation experiments revealed that although geNPC express several β1 integrins (α1β1, α2β1, α3β1, α5β1, α6β1, αvβ1), adhesion and migration on LN and FN were primarily mediated by α6β1 and α5β1, respectively, and these interactions were confirmed by biochemical cross-link/extraction procedures. Finally, neuronal differentiation of geNPC was enhanced on LN, indicating a role for LN in geNPC differentiation. β1 integrin–ECM interactions may contribute to basic mechanisms of striatal development and may explain the potent migratory capacity of geNPC transplanted into the adult brain. [Copyright &y& Elsevier]

Transplantation of positively selected, CD34+ peripheral blood stem cells from alternative donors frequently results in delayed immune reconstitution. A shift towards a type 2 cytokine production might be a major contributing factor. We therefore decided to measure IFN-?, IL-2, IL-4, and IL-10 after stimulation of peripheral mononuclear cells with PMA/ionomycin and on a single cell level by intracellular cytokine staining during different stages of immune reconstitution. Immediately after transplantation, secretion of all selected cytokines was substantially diminished, and remained subnormal compared to controls until the end of the first year despite normalizing T-cell levels. IL-2 was predominantly produced by CD4+CD45RA+ naïve, whereas IFN-? originated mainly from CD8+CD45RO+ memory T cells. Secretion of IL-2 was correlated with the numbers of naive CD4+ T cells, whereas IFN-? secretion correlated with total CD3+ T-cell counts. IL-4 and IL-10 were produced by CD4+ and CD8+ memory T cells; secretion of these cytokines was low, however, and did not increase during follow-up. Therefore, a shift towards a preferential production of type 2 cytokines could not be observed. Analysis of CD69 upregulation upon stimulation revealed a deficiency in patient T-cell activation, which unexpectedly comprised both naïve and memory T-cell subpopulations. Therefore, we suggest that a defect in T-cell activation intrinsic to the host and not graft-versus-host disease, post-transplant immunosuppression or a shift towards a type 2 cytokine pattern contributes to the impaired production of cytokines post-transplant. Further studies will focus on the elimination of host factors that may adversely affect T-cell function after transplantation.The Hematology Journal (2004) 5, 329-340. doi:10.1038/sj.thj.6200397 [ABSTRACT FROM AUTHOR]

: ObjectiveIn patients with lymphopenia following allogeneic stem cell transplantation adenovirus (ADV) infection is associated with high morbidity and mortality despite aggressive antiviral drug therapy. Virus-specific T cells seem to be essential for virus elimination. The aim of this study was to isolate and expand donor-derived human ADV-specific T lymphocytes for adoptive transfer of sufficient cell numbers to restore protective immunity after allogeneic stem cell transplantation.: Materials and methodsA clinical-grade strategy to generate ADV-specific T cells using the interferon-γ (IFN-γ) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 (IL-2) and feeder cell stimulation, is described.: ResultsA mean number of 3.4×106 (±3 SD) ADV antigen-specific T lymphocytes were isolated from 0.1 to 2×109 mononuclear cells from peripheral blood (n = 5) or leukapheresis products (n = 6). Characterization of ADV-specific T cells after isolation revealed a mean purity of 85.1% (±12% SD) using antigen-specific intracellular cytokine staining. Isolated cells were expanded ex vivo for a median of 18 days (range 7–29 days; n = 5) to greater than 108 total cells using IL-2 and autologous feeder cell stimulation. ADV-specific response to adenovirus antigen was confirmed in the generated T cell lines, using intracellular cytokine staining, IFN-γ Elispot assay, and 3H-thymidine uptake. Generated T-cell lines showed specific killing of ADV-infected B-LCL (n = 4). Alloreactive proliferation of generated T-cell lines in mixed lymphocyte cultures was significantly reduced when compared to unmanipulated PBMCs.: ConclusionGeneration of adenovirus-specific T cells in a simple and rapid clinical-grade protocol was established, using IFN-γ secretion assay with short expansion times, leading to sufficient numbers of ADV-specific T cells that can be used for adoptive immunotherapy. [Copyright &y& Elsevier]

Studies the locomotor system of slowly walking insects, such as Carausius morosus. Problems with the control system generating animal leg movements; Control of the swing movement.

Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4+, IFNγ-producing THelpertype 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complexin vitroprotocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4+TH1 cellsin vitrofor cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4+T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8+T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4+T cells showed strong specific TH1-responses with IFNγ+, TNFα+, IL-2+and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4+TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients. [ABSTRACT FROM AUTHOR]