Selective Depletion of αß T Cells and B Cells for Human Leukocyte Antigen-Haploidentical Hematopoietic Stem Cell Transplantation. A Three-Year Follow-Up of Procedure Efficiency.

HLA-haploidentical family donors represent a valuable option for children requiring allogeneic hematopoietic stem cell transplantation (HSCT). Because graft-versus-host diseases (GVHD) is a major complication of HLA-haploidentical HSCT because of alloreactive T cells in the graft, different methods have been used for ex vivo T cell depletion. Removal of donor αß T cells, the subset responsible for GVHD, and of B cells, responsible for post-transplantation lymphoproliferative disorders, have been recently developed for HLA-haploidentical HSCT. This manipulation preserves, in addition to CD34+ progenitors, natural killer, γδ T, and monocytes/dendritic cells, contributing to anti-leukemia activity and protection against infections. We analyzed depletion efficiency and cell yield in 200 procedures performed in the last 3 years at our center. Donors underwent CD34+ hematopoietic stem cell (HSC) peripheral blood mobilization with granulocyte colony-stimulating factor (G-CSF). Poor CD34+ cell mobilizers (48 of 189, 25%) received plerixafor in addition to G-CSF. Aphereses containing a median of 52.5 × 109 nucleated cells and 494 × 106 CD34+ HSC were manipulated using the CliniMACS device. In comparison to the initial product, αß T cell depletion produced a median 4.1-log reduction (range, 3.1 to 5.5) and B cell depletion led to a median 3.4-log reduction (range, 2.0 to 4.7). Graft products contained a median of 18.5 × 106 CD34+ HSC/kg recipient body weight, with median values of residual αß T cells and B cells of 29 × 103/kg and 33 × 103/kg, respectively. Depletion efficiency monitored at 6-month intervals demonstrated steady performance, while improved recovery of CD34+ cells was observed after the first year (P = .0005). These data indicate that αß T cell and B cell depletion of HSC grafts from HLA-haploidentical donors was efficient and reproducible.

Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αß/CD19-depleted haploidentical stem cell grafts.

BACKGROUND: HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αß/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition. METHODS: Ninety healthy donors were evaluated. Single-dose MZ was given to 30 'poor mobilizers' (PM) failing to attain ≥40 CD34+ HSCs/µL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient's body weight. RESULTS: MZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs. CONCLUSIONS: MZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition.

Adoptive immunotherapy with antigen-specific T cells during extracorporeal membrane oxygenation (ECMO) for adenovirus-related respiratory failure in a child given haploidentical stem cell transplantation.

We report on the successful infusion of human adenovirus (HAdV)-specific T cells in a child with congenital amegakaryocytic thrombocytopenia, given T-cell-depleted hematopoietic stem cell transplantation (HSCT) from the HLA-haploidentical mother during extracorporeal membrane oxygenation (ECMO) for severe HAdV-related respiratory failure. Donor-derived, interferon (IFN)-γ-secreting HAdV-specific T cells were enriched using the cytokine capture assay, after in vitro stimulation with overlapping peptides from the immunodominant HAdV5 hexon protein. Two weeks after T-cell transfer, viral load decreased and ECMO was discontinued. T-cell responses to HAdV antigens were documented after four weeks and were associated with viral clearance, immune reconstitution and clinical amelioration.

Extracorporeal photopheresis for paediatric patients experiencing graft-versus-host disease (GVHD).

Extracorporeal photopheresis (ECP) is broadly used in adults with cutaneous T-cell lymphoma, acute or chronic graft-versus-host disease (GVHD), rejection of solid organ transplants, and a variety of autoimmune, cell-mediated diseases. The predominant use of ECP in children and adolescents is for treating GVHD. Children pose specific challenges to ECP, due to their unique physiology and to patient's size. Herein, we will focus on current clinical trials with ECP in children with GVHD, with an emphasis on technical and clinical issues that are peculiar to the paediatric setting.

Soluble Isoform of Suppression of Tumorigenicity 2 (ST2) Biomarker in a Large Cohort of Healthy Pediatric Population: Determination of Reference Intervals.

Introduction: Only little data exists on ST2 reference intervals in healthy pediatric populations despite the high importance of this biomarker in adults with heart failure. The aim of the study was to assess the reference intervals of ST2 in a wide healthy pediatric cohort. Methods: We evaluated the serum concentrations of ST2 biomarker in 415 healthy pediatric subjects referred to our analysis laboratory. Subjects were categorized according to age (i.e., 0−6 (n = 79), 7−11 (n = 142) and 12−18 years (n = 191)) and sex. They were not suffering from any cardiac disorders, metabolic disorders, lung diseases, autoimmune disorders or malignancies. A written consent was obtained for each individual. No duplicate patients were included in the analysis and the presence of outliers was investigated. Reference intervals (Mean and central 95% confidence intervals) were determined. Results: Three outliers have been identified and removed from the analysis (60.0, 64.0 and 150.2 ng/mL). A total of 412 subjects were therefore included. The mean value for the whole population was 15.8 ng/mL (2.4−36.4 ng/mL). Males present a significantly higher mean concentration compared to females (17.2 versus 14.4 ng/mL, p = 0.001). A significant trend toward higher ST2 values with age was also observed, but for males only (r = 0.43, p < 0.0001). If considering age partitions, only males of 12−18 years (mean = 21.7 ng/mL) had significantly higher ST2 values compared to the other groups (ranging from 11.9 for males 0−6 years to 15.2 for females 12−18 years; p < 0.0001). Conclusions: We described age and sex-specific reference intervals for ST2 in a large healthy pediatric population. We found that ST2 values differ between sexes if considering all participants. A significant increase in ST2 with age was also observed, but only for males of 12−18 years.

Reference Intervals for Coagulation Parameters in Developmental Hemostasis from Infancy to Adolescence.

Background: The objective of this study was to establish the age and sex-dependent reference intervals for coagulation assays evaluated in healthy children, ranging from 0 days to 16 years old. Methods: PT, aPTT, Fibrinogen (functional), Antithrombin activity, Protein C anticoagulant activity, Protein S free antigen, Thrombin time, D-Dimer, Von Willebrand Factor antigen, Lupus anticoagulant (screening), extrinsic and intrinsic pathway factors, and activated Protein C resistance were evaluated using STA-R Max2. Results: A total of 1280 subjects (671 males and 609 females) were divided into five groups, according to their age: 0-15 days (n = 280, 174 M and 106 F), 15-30 days (n = 208, 101 M and 107 F), 1-6 months (n = 369, 178 M and 191 F), 6-12 months (n = 214, 110 M and 104 F), and 1-16 years (n = 209, 108 M and 101 F). The 95% reference intervals and the 90% CI were established using the Harrell-Davis bootstrap method and the bootstrap percentile method, respectively. Conclusions: The present study supports the concept that adult and pediatric subjects should be evaluated using different reference intervals, at least for some coagulation tests, to avoid misdiagnosis, which can potentially lead to serious consequences for patients and their families, and ultimately the healthcare system.

Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system.

BACKGROUND: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. STUDY DESIGN AND METHODS: The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. RESULTS: The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. DISCUSSION: Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

Anastomotic ulcers in short bowel syndrome: New suggestions from a multidisciplinary approach.

BACKGROUND AND AIMS: Anastomotic ulceration (AU) is a rare potential life-threatening complication that may occur after intestinal resection. The diagnosis is often delayed after a long-lasting history of refractory anemia. The pathogenesis remains unknown and there are no established therapies. The aim of the study was to analyze the medical history of children with short bowel syndrome (SBS) who were experiencing AU. METHODS: Records of SBS children were retrospectively reviewed. Demographics, baseline characteristics, presentation, diagnosis and treatment of AU cases were analyzed. RESULTS: Eight out of 114 children with SBS were identified as having AU. Mean gestational age was 32.5weeks. Underlying diseases were: 5 necrotising enterocolitis, 2 gastroschisis and 1 multiple intestinal atresia. The mean age at AU diagnosis was 6.5years (diagnosis delay of 35months). All but 2 patients had AU persistency after medical treatment. Endoscopic treatment (2 argon plasma coagulation; 1 platelet-rich fibrin instillation; 2 endoscopic hydrostatic dilations) was effective in 3 out of 5 children. Surgery was required in 3 patients. CONCLUSIONS: Severe bowel ischemic injury, especially in preterm infant, could predispose to AU development. Medical treatment showed discouraging results. We firstly described that different endoscopic treatment could be attempted before resorting to further surgery. LEVEL OF EVIDENCE: IV.

IgE Immunoadsorption Knocks Down the Risk of Food-Related Anaphylaxis.

The effects of an immunoadsorption procedure, specifically designed to remove immunoglobulin E (IgE), on food-induced anaphylaxis have never been evaluated. We evaluate the effects of IgE removal on the allergic thresholds to foods. A 6-year-old boy with anaphylaxis to multiple foods and steroid-resistant unstable allergic asthma displayed serum IgE levels of 2800 to 3500 kU/L. To lower IgE serum concentrations, which could be overridden by a high dose of omalizumab, 1.5 plasma volumes were exchanged in 8 apheresis sessions. During the procedure, serum IgE levels fell to 309 kU/L. After the procedure, the threshold of reactivity to baked milk increased from 0.125 to 5 g of milk protein (full tolerance) after the first session, and the threshold of reactivity to hazelnut increased from 0.037 to 0.142 g of protein after the first session, 0.377 g after the eighth, and 1.067 g (full tolerance) after the first administration of omalizumab. Immediately after the sixth IgE immunoadsorption, we started omalizumab therapy. In the next 40 days, the threshold of reactivity to hazelnut increased to 7.730 (full tolerance). Asthma control was obtained, treatment with montelukast was stopped, and fluticasone was tapered from 500 to 175 µg/day. The boy became partially or fully tolerant to all the tested foods, and quality of life was improved. IgE immunoadsorption, used to establish the starting basis for omalizumab administration, is able to increase the tolerance threshold to foods.