Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome that is classified into primary and secondary HLH. Primary HLH consists of monogenic disorders that mainly affect the perforinmediated cytotoxicity of cytotoxic T lymphocytes and natural killer cells. Secondary HLH occurs as a complication in various settings such as infection, malignancy, autoimmune disease, and post-allogeneic hematopoietic stem cell transplantation. Both primary and secondary HLH are characterized by uncontrolled hypercytokinemia that results in myelosuppression and vascular endothelium damage. More than 10% of patients with HLH die within 2 months of diagnosis due to bleeding in the visceral organs, opportunistic infection due to neutropenia, or multiple organ failure. The most obvious presentations of HLH are persistent fever refractory to antimicrobial agents and hyperferritinemia due to hypersecretion of various cytokines. The first rule is not to overlook signs of hypercytokinemia and to settle the hyperactivated immunological state as soon as possible. In addition, to improve outcome, it is essential to identify the disorders underlying HLH and provide disorder-appropriate treatment.

The piggyBac transposon system represents a promising non-viral tool for gene delivery and discovery, and may also be of value for clinical gene therapy. PiggyBac is a highly efficient integrating vector that stably transfects (~40%) of primary human T cells for potential adoptive immunotherapy applications. To evaluate the potential genotoxicity of piggyBac, we compared 228 integration sites in primary human T cells to integrations in two other human derived cell lines (HEK293 and HeLa) and randomly simulated integrations into the human genome. Our results revealed distinct differences between cell types. PiggyBac had a non-random integration profile and a preference for transcriptional units (~50% into RefSeq genes in all cell types), CpG islands (18% in T cells and 8% in other human cells), and transcriptional start sites (TSS) (< 5kb, 16–20% in all cell types). PiggyBac also preferred TTAA but not AT rich regions of the human genome. We evaluated the expression of mapped genes into which piggyBac integrated, and found selection of more active genes in primary human T cells compared to other human cell types, possibly due to concomitant T cell activation during transposition. Importantly, we found that in comparison to what has been reported for gammaretroviral and human lenitviral vectors, piggyBac had decreased integration frequency into or within 50kb of the TSS of known proto-oncogenes. Hence the piggyBac non-viral gene delivery system appears to represent a promising gene transfer system for clinical applications using human T lymphocytes.

Of 11 children with juvenile myelomonocytic leukemia (JMML) carrying RAS mutations (8 with NRAS mutations, 3 with KRAS2 mutations), 5 had a profound elevation in either or both the white blood cells and spleen size at diagnosis. Three patients had no or modest hepatosplenomegaly and mild leukocytosis at presentation but subsequently showed a marked increase in spleen size with or without hematologic exacerbation, for which nonintensive chemotherapy was initiated. The other three patients with NRAS or KRAS2 glycine to serine substitution received no chemotherapy, but hematologic improvement has been observed during a 2-to 4-year follow up. In the third group, all hematopoietic cell lineages analyzed had the RAS mutations at the time of hematologic improvement, whereas DNA ob- IntroductionSomatic point mutations of the RAS genes at codons 12, 13, and 61 (NRAS and KRAS2) are found in approximately 20% of patients with juvenile myelomonocytic leukemia (JMML). 1,2 Other patients show inactivation of NF1 or PTPN11 mutations. [3][4][5] Although most patients with JMML die from progressive disease unless treated with hematopoietic stem cell transplantation, there are a few patients who have been reported to spontaneously recover without intervention. 6,7 Some of these children have JMML associated with Noonan syndrome, but others do not. So far, the individual prognosis in JMML-carrying specific genetic aberrations remains unclear. We report the clinical course in 11 patients with RAS mutations. Materials and methodsThis study was approved by the Institutional Review Board of Shinshu University. Informed consent was obtained from the guardians of the patients following institutional guidelines. Cell preparationWe used peripheral blood (PB) or bone marrow (BM) mononuclear cells (MNCs) that had been frozen with liquid nitrogen. CD3-and CD56-positive PB cells were separated immunomagnetically. 8 Ninety-nine percent of the isolated cells were CD3-or CD56-positive according to a flow cytometric analysis. Clonal cell cultureTwenty thousand PB or BM MNCs were plated in a dish containing methylcellulose medium supplemented with or without 0.01 to 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF). 9 To examine the clonal derivation of myeloid and erythroid lineages, 2000 CD34 ϩ PB cells harvested immunomagnetically were cultured in methylcellulose medium supplemented with GM-CSF, stem cell factor, interleukin 3, and erythropoietin. Twelve days after incubation in 5% CO 2 , GM colonies, erythroid colonies, and mixed colonies were individually lifted and prepared as single cell suspensions. Sequence analyses were then performed on individual colonyconstituent cells. Detection of NRAS and KRAS2 mutationsDNA was extracted from PB or BM MNCs and nails. Exon 1 (codons 12 and 13) and exon 2 (codon 61) of NRAS and KRAS2 genes were amplified by polymerase chain reaction (PCR) using primer pairs described previously. 10,11 The PCR products were subjected to direct sequencing from both directions on an automatic DNA se...

Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon= transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19 þ artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

Germline heterozygous IKZF1 mutations cause dysgammaglobulinemia; hematologic abnormalities, including B-cell defect; and autoimmune diseases.