Clinically pertinent coeliac disease exists despite normal small-bowel mucosal villous architecture. Mucosal transglutaminase 2-specific IgA deposits can be utilized in detecting such patients with genetic gluten intolerance.

The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing.

The new gliadin peptide antibody test proved highly accurate in the diagnostic work-up and follow-up of coeliac disease and can be endorsed in combination with the tissue transglutaminase test.

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze–thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 109 ± 4.74 × 109 cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5–66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.

Some recent reports suggest that cryopreserved and thawed mesenchymal stromal cells (MSCs) may have impaired functional properties as compared to freshly harvested MSCs from continuous cultures. A cryopreservation step in the manufacturing process brings important benefits, since it enables immediate off-the-shelf access to the products and a completion of all quality testing before batch release and administration to the patient. Cryopreservation is also inevitable in MSC banking strategies. In this study, we present the results from the MSC stability testing program of our in-house manufactured clinical-grade allogeneic bone marrow-derived MSC product that is expanded in platelet lysate and frozen in passage 2. The current manufacturing protocol contains only one freezing step and the frozen MSC product is thawed bed-side at the clinic. We can conclude superior viability and cell recovery of the frozen and thawed MSC product utilizing the validated freezing and thawing protocols we have developed. The MSC phenotype and differentiation potential was generally found to be unaltered after thawing, but the thawed cells exhibited a 50% reduced, but not completely abolished, performance in an in vitro immunosuppression assay. The in vitro immunosuppression assay results should, however, be interpreted with caution, since the chosen assay mainly measures one specific immunosuppressive mechanism of MSCs to suppress T-cell proliferation. Since at least two freezing steps are usually necessary in MSC banking strategies, we went on to investigate the impact of repeated freezing on MSC quality attributes. We can conclude that two freezing steps with a preceding cell culture phase of at least one passage before freezing is feasible and does not substantially affect basic cell manufacturing parameters or quality attributes of the final frozen and thawed product. Our results suggest, however, that an exhaustive number of freezing steps (≥4) may induce earlier senescence. In conclusion, our results support the utilization of frozen MSC products and MSC banking strategies, but emphasize the need of always performing detailed studies on also the cryopreserved MSC counterpart and to carefully report the cryopreservation and thawing protocols.

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Delayed and/or insufficient T cell recovery post hematopoietic stem cell transplantation (HSCT) leads to an increased risk of morbidity and mortality. We evaluated thymic function and its association with T cell regeneration post HSCT and identified factors involved in the process among pediatric stem cell transplant recipients. T cell regeneration in 66 pediatric patients was prospectively followed by naive T cell phenotyping, measuring of T cell receptor excision circles (TRECs) and expression of Foxp3 by regulatory T cells for the first 18 months post HSCT. TRECs were lower pre-HSCT in children with a malignant than non-malignant primary disease or immunosuppressed controls (P ¼ 0.001). Naive T lymphocyte reconstitution and thymic recovery were slow in the recipients of allogeneic stem cell grafts post HSCT. Infections caused by herpesviruses had a prognostic impact on mortality. Children with low TRECs had a high mortality (P ¼ 0.05) and low TRECs were also associated with extensive chronic graft-versus-host disease from 6 months onwards. Low amount of Foxp3 pre-HSCT was associated with an increased mortality post HSCT (P ¼ 0.03). Our study indicates an association between impaired T cell regeneration and thymic dysfunction and the clinical post transplant complications in pediatric allogeneic stem cell transplantation.

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