Frequent somatic mosaicism of NEMO in T cells of patients with X-linked anhidrotic ectodermal dysplasia with immunodeficiency.

Somatic mosaicism has been described in several primary immunodeficiency diseases and causes modified phenotypes in affected patients. X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the NF-κB essential modulator (NEMO) gene and manifests clinically in various ways. We have previously reported a case of XL-EDA-ID with somatic mosaicism caused by a duplication mutation of the NEMO gene, but the frequency of somatic mosaicism of NEMO and its clinical impact on XL-EDA-ID is not fully understood. In this study, somatic mosaicism of NEMO was evaluated in XL-EDA-ID patients in Japan. Cells expressing wild-type NEMO, most of which were derived from the T-cell lineage, were detected in 9 of 10 XL-EDA-ID patients. These data indicate that the frequency of somatic mosaicism of NEMO is high in XL-ED-ID patients and that the presence of somatic mosaicism of NEMO could have an impact on the diagnosis and treatment of XL-ED-ID patients.

Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein.

Familial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4-deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.

Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

Hematopoietic stem cell transplantation with reduced intensity conditioning from a family haploidentical donor in an infant with familial hemophagocytic lymphohistocytosis.

Allogeneic hematopoietic stem cell transplantation is the only curative method for patients with familial hemophagocytic lymphohistiocytosis (FHL). We present a case of a 3-month-old girl with Munc13-4 mutation (FHL3), who underwent bone marrow transplantation (BMT) from her human leukocyte antigen-haploidentical mother following reduced intensity conditioning (RIC) with fludarabine, melphalan, and busulfan. Engraftment after BMT was generally uneventful, with only mild acute graft versus host disease. Munc13-4 protein was restored following BMT, and she is well and free of disease 14 months after BMT. These results suggest that BMT with RIC from a family haploidentical donor may sufficiently restore immune regulation in infants, while lessening treatment-related mortality and long-term sequelae.