Critical early events in hematopoietic cell seeding and engraftment.

Durable hematopoietic stem cell engraftment requires efficient homing to and seeding in the recipient bone marrow. Dissection of cellular and molecular mechanisms by retrospective analysis of functional engraftment studies imposes severe limitations on the understanding of the early stages of this process. We have established an experimental approach for in vivo functional imaging of labeled cells at the level of recipient bone marrow in real time. The adhesive interaction of hematopoietic cells with the bone marrow stroma evolves as the most important early event. Adhesion to the marrow, rather than the vascular endothelium, determines the efficiency of both homing and seeding, and is absolutely essential to maintain cell viability in the marrow. Seeding and engraftment may be improved either by bypassing homing or by localized transplant of a large number of cells in a relatively small marrow space. There is functional redundancy in the molecular pathways that mediate the cell-stroma interaction, such that blockage of a single pathway has only minor effect on homing and seeding. We hypothesize that successfully seeding-engrafting cells undergo extensive phenotypic changes as a consequence of interaction with the stroma, without engaging in rapid proliferation. Surprisingly, Fas-ligand appears to promote hematopoietic cell engraftment by immunomodulatory and trophic effects

Characterization and Comparison of the 10-2 SITA-Standard and Fast Algorithms

Purpose: To compare the 10-2 SITA-standard and SITA-fast visual field programs in patients with glaucoma. Methods: We enrolled 26 patients with open angle glaucoma with involvement of at least one paracentral location on 24-2 SITA-standard field test. Each subject performed 10-2 SITA-standard and SITA-fast tests. Within 2 months this sequence of tests was repeated. Results: SITA-fast was 30% shorter than SITA-standard (5.5 ± 1.1 vs 7.9 ± 1.1 minutes, P < 0.001). Mean MD was statistically significantly higher for SITA-standard compared with SITA-fast at first visit (Δ = 0.3 dB, P = 0.017) but not second visit. Inter-visit difference in MD or in number of depressed points was not significant for both programs. Bland-Altman analysis showed that clinically significant variations can exist in individual instances between the 2 programs and between repeat tests with the same program. Conclusions: The 10-2 SITA-fast algorithm is significantly shorter than SITA-standard. The two programs have similar long-term variability. Average same-visit between-program and same-program between-visit sensitivity results were similar for the study population, but clinically significant variability was observed for some individual test pairs. Group inter- and intra-program test results may be comparable, but in the management of the individual patient field change should be verified by repeat testing

Outcome of Children Developing Grade III-IV Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Acute graft versus host disease (aGvHD) remains one of the major causes of procedure-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Information on the outcome of pediatric patients experiencing this complication is limited. We conducted a retrospective registry-based analysis on children who developed grade III-IV acute GVHD and were reported to the European Blood and Marrow Transplantation (EBMT) registry. Included in the study were children below age of 18 years who were transplanted between 2004 and 2016 (n=28109). Of these children, 1968 experienced grade III-IV acute GvHD: 1370 were had malignancies, while 598 were affected by a non-malignant disorder (NMD). Median year at HSCT was 2009 for patients with malignancies and 2010 for patients with NMD. In this latter group, as expected, the median age at HSCT was lower (5.8 years), in comparison with those affected by malignancies (9 years). The donor was an HLA-identical sibling in 576 cases and an unrelated donor in 895 cases. Umbilical cord blood (UCB) was employed in 282 cases, while a relative other than a compatible sibling in 215 cases. Overall, 1075 patients were given bone marrow (BM), while 598 received peripheral blood stem cells (PBSC). A fully myeloablative conditioning regimen has been employed in 94% of patients with malignancies in comparison with 75% of children with NMD. As a post-transplant pharmacological GvHD prophylaxis, a different strategy of immune suppressive treatment have been used: it consisted in the association of Cyclosporine-A (CSA) and Methotrexate in 40%, CSA alone in 30% and CSA plus Mycophenolate mofetil in 10% of patients. Grade III aGvHD occurred in 1383 patients (70%), while grade IV aGvHD was diagnosed in 585 (30%). Chronic GvHD occurred in 48.2% and 49.3% of patients with malignant and NMD, respectively. It was extensive in 262 (26.8%) patients with malignancies and in 111 (28%) children affected by NMD. Within patients with malignancies, the 2-year Kaplan-Meyer probability of overall survival (OS) was 65.7% (confidence interval 95, 63 - 68.4). In this group, the cumulative incidence of non-relapse mortality (NRM) was 23.1%. Notably, the occurrence of GvHD was responsible of death in 228 patients (CI 14.5%). In the NMD cohort, the 2-year Kaplan-Meyer probability of overall survival (OS) was 67.8% (confidence interval 95, 63.8 - 71.9). Sixty-one patients died to GvHD, being the 2-year cumulative incidence of GvHD-related mortality 19%. These data indicate that the occurrence of grade III-IV aGVHD is associated with a dismal outcome also in pediatric patients. The main cause of fatality is represented by NRM, while leukemia recurrence affected outcome of a lower number of children. Thus, strategies aimed at preventing this immune-mediated complication and at optimizing its treatment are desirable

The role of anion gap normalization time in the management of pediatric diabetic ketoacidosis

IntroductionOur aims were to determine whether anion gap normalization time (AGNT) correlates with risk factors related to the severity of diabetic ketoacidosis (DKA) in children, and to characterize AGNT as a criterion for DKA resolution in children admitted with moderate or severe disease.MethodsA ten-year retrospective cohort study of children admitted to the intensive care unit with DKA. We used a survival analysis approach to determine changes in serum glucose, bicarbonate, pH, and anion gap following admission. Using multivariate analysis, we examined associations between patients' demographic and laboratory characteristics with delayed normalization of the anion gap.ResultsA total of 95 patients were analyzed. The median AGNT was 8 h. Delayed AGNT (&gt;8 h) correlated with pH &lt; 7.1 and serum glucose &gt;500 mg/dL. In multivariate analysis, glucose &gt;500 mg/dL was associated with an increased risk for delayed AGNT, by 3.41 fold. Each 25 mg/dL elevation in glucose was associated with a 10% increment in risk for delayed AGNT. Median AGNT preceded median PICU discharge by 15 h (8 vs. 23 h).DiscussionAGNT represents a return to normal glucose-based physiology and an improvement in dehydration. The correlation observed between delayed AGNT and markers of DKA severity supports the usefulness of AGNT for assessing DKA recovery

Allogeneic Stem Cell Transplantation From HLA-Mismatched Donors for Pediatric Patients with Acute Lymphoblastic Leukemia Treated According to the 2003 BFM and 2007 International-BFM Studies: Impact of Disease Risk on Outcomes.

Summary Rational Allogeneic HSCT is beneficial for pediatric patients with relapsed or (very) high-risk ALL in remission. A total of 1115 consecutive patients were included in the ALL SCT 2003 BFM study and the ALL SCT 2007-International study and were stratified according to relapse risk (Standard vs. High vs. Very High Risk of Relapse) and donor type (Matched Sibling vs. Matched Donor vs. Mismatched Donor). Patients and methods A total of 148 patients (60% male, median age 8.7 years; B-cell precursor ALL: 75%) were transplanted from MMD, which was defined as either less than 9/10 HLA-compatible donors or less than 5/6 unrelated cord blood after myelo-ablative conditioning regimen (TBI-based: 67%) for HRR (n=42) or VHRR disease (n=106). The stem cell source was either BM (n=31), unmanipulated PBSCs (n=28), T-cell ex vivo depleted PBSCs (n=59) or cord blood (n=25). The median follow-up was 5.1 years. Results The 4-year OS and EFS was 56±4% and 52±4%, respectively, for the entire cohort. Patients transplanted from MMD for HRR disease obtained remarkable 4-y OS and EFS values of 82±6% and 80±6%, respectively, while VHRR patients obtained values of 45±5% and 42±5% (p Conclusion HSCT with a mismatched donor is feasible in pediatric ALL patients but leads to inferior results compared to HSCT with better matched donors, at least for patients transplanted for VHRR. The results are strongly affected by disease status. The main cause of treatment failure is still relapse, highlighting the urgent need for interventional strategies after HSCT for patients with residual leukemia before and/or after transplantatio

The impact of donor type on the outcome of pediatric patients with very high risk acute lymphoblastic leukemia. A study of the ALL SCT 2003 BFM-SG and 2007-BFM-International SG

Allogeneic HSCT represents the only potentially curative treatment for very high risk (VHR) ALL. Two consecutive international prospective studies, ALL-SCT-(I)BFM 2003 and 2007 were conducted in 1150 pediatric patients. 569 presented with VHR disease leading to any kind of HSCT. All patients >2 year old were transplanted after TBI-based MAC. The median follow-up was 5 years. 463 patients were transplanted from matched donor (MD) and 106 from mismatched donor (MMD). 214 were in CR1. Stem cell source was unmanipulated BM for 330 patients, unmanipulated PBSC for 135, ex vivo T-cell depleted PBSC for 62 and cord-blood for 26. There were more advanced disease, more ex vivo T-cell depletion, and more chemotherapy based conditioning regimen for patients transplanted from MMD as compared to those transplanted from MSD or MD. Median follow up (reversed Kaplan Meier estimator) was 4.99 years, median follow up of survivals was 4.88, range (0.01–11.72) years. The 4-year CI of extensive cGvHD was 13 ± 2% and 17 ± 4% (p = NS) for the patients transplanted from MD and MMD, respectively. 4-year EFS was statistically better for patients transplanted from MD (60 ± 2% vs. 42 ± 5%, p < 0.001) for the whole cohort. This difference does not exist if considering separately patients treated in the most recent study. There was no difference in 4-year CI of relapse. The 4-year NRM was lower for patients transplanted from MD (9 ± 1% vs. 23 ± 4%, p < 0.001). In multivariate analysis, donor-type appears as a negative risk-factor for OS, EFS, and NRM. This paper demonstrates the impact of donor type on overall results of allogeneic stem cell transplantation for very-high risk pediatric acute lymphoblastic leukemia with worse results when using MMD stem cell source

Effector and Naturally Occurring Regulatory T Cells Display No Abnormalities in Activation Induced Cell Death in NOD Mice

BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-), FoxP3(-)) and suppressor (CD25(+), FoxP3(+)) CD4(+) T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4(+) subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+)CD25(-) T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis

Supportive Care During Pediatric Hematopoietic Stem Cell Transplantation : Prevention of Infections. A Report From Workshops on Supportive Care of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT)

Specific protocols define eligibility, conditioning, donor selection, graft composition and prophylaxis of graft vs. host disease for children and young adults undergoing hematopoietic stem cell transplant (HSCT). However, international protocols rarely, if ever, detail supportive care, including pharmaceutical infection prophylaxis, physical protection with face masks and cohort isolation or food restrictions. Supportive care suffers from a lack of scientific evidence and implementation of practices in the transplant centers brings extensive restrictions to the child's and family's daily life after HSCT. Therefore, the Board of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) held a series of dedicated workshops since 2017 with the aim of initiating the production of a set of minimal recommendations. The present paper describes the consensus reached within the field of infection prophylaxis.Peer reviewe

Apoptosis of Purified CD4+ T Cell Subsets Is Dominated by Cytokine Deprivation and Absence of Other Cells in New Onset Diabetic NOD Mice

BACKGROUND: Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures. PRINCIPAL FINDINGS: Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25(+) T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25(-) T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25(+) T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays. CONCLUSIONS: The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world