Reactive oxygen species (ROS) serve several physiological functions; in some settings they act in host defense, while in others they function in cellular signaling or in biosynthetic reactions. We studied the expression and function of a recently described source of ROS, NAD(P)H oxidase 1 or Nox1, which has been associated with cell proliferation. In situ hybridization in mouse colon revealed high Nox1 expression within the lower two-thirds of colon crypts, where epithelial cells undergo proliferation and differentiation. Human multitumor tissue array analysis confirmed colon-specific Nox1 expression, predominantly in differentiated epithelial tumors. Differentiation of Caco2 and HT29 cells with 1α,25-dihydroxyvitamin D3 or IFN-γ enhances Nox1 expression and decreases cell proliferation, suggesting that Nox1 does not function as a mitogenic oxidase in colon epithelial cells. Transduction with retrovirus encoding Nox1 restored activation and differentiation-dependent superoxide production in gp91phox-deficient PLB-985 cells, indicating close functional similarities to the phagocyte oxidase (phox). Furthermore, coexpression of cytosolic components, p47phox and p67phox, augments Nox1 activity in reconstituted K562 cells. Finally, Nox1 partially restores superoxide production in neutrophils differentiating ex vivo from gp91phox-deficient CD34+ peripheral blood-derived stem cells derived from patients with X-linked chronic granulomatous disease. These studies demonstrate a significant functional homology (cofactor-dependent and activation-regulated superoxide production) between Nox1 and its closest homologue, gp91phox, suggesting that targeted up-regulation of Nox1 expression in phagocytic cells could provide a novel approach in the molecular treatment of chronic granulomatous disease.

Key Points• UTX regulates migration and hematopoiesis.• Female UTX-KO mice show key features of myelodysplastic syndrome with chromosomal instability.Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis. (Blood. 2013;121(13):2462-2473

This consensus- based S1 Guideline for perioperative infusion therapy in children is focused on safety and efficacy. The objective is to maintain or re-establish the child's normal physiological state (normovolemia, normal tissue perfusion, normal metabolic function, normal acid- base- electrolyte status). Therefore, the perioperative fasting times should be as short as possible to prevent patient discomfort, dehydration, and ketoacidosis. A physiologically composed balanced isotonic electrolyte solution (BS) with 1-2.5% glucose is recommended for the intraoperative background infusion to maintain normal glucose concentrations and to avoid hyponatremia, hyperchloremia, and lipolysis. Additional BS without glucose can be used in patients with circulatory instability until the desired effect is achieved. The additional use of colloids (albumin, gelatin, hydroxyethyl starch) is recommended to recover normovolemia and to avoid fluid overload when crystalloids alone are not sufficient and blood products are not indicated. Monitoring should be extended in cases with major surgery, and autotransfusion maneuvers should be performed to assess fluid responsiveness.

Hematopoietic stem cells (HSCs) lose marrow reconstitution potential during ex vivo culture. HSC migration to stromal cell-derived factor (SDF)-1 (CXCL12) correlates with CXC chemokine receptor 4 (CXCR4) expression and marrow engraftment. We demonstrate that mobilized human CD34 + peripheral blood stem cells (CD34 + PBSCs) lose CXCR4 expression during prolonged culture. We transduced CD34 + PBSCs with retrovirus vector encoding human CXCR4 and achieved 18-fold more CXCR4 expression in over 87% of CD34 + cells. CXCR4-transduced cells yielded increased

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BackgroundMonogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like receptor family CARD-containing 4 protein (NLRC4).ObjectiveHere we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. Methods: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1β/IL-18 were quantified by using flow cytometry and ELISA, respectively.ResultsThe p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1–dependent cell death, and IL-1β/IL-18 production. ASC contributed to p.W655C NLRC4–mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex.ConclusionThis is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex.

Over the past dozen years, the majority of clinical gene therapy trials for inherited genetic diseases and cancer therapy have been performed using murine onco-retrovirus as the gene delivery vector. The earliest systems used were relatively inefficient in both the rates of transduction and expression of the transgene. Formidable obstacles inherent in the cell biology and/or the immunology of the target cell systems limited the efficacy of gene therapy for many target diseases. Development of novel retrovirus gene transfer systems that are in progress have begun to overcome these obstacles. Evidence of this progress is the recent successful functional correction of the immune T and B lymphocyte deficiency in patients with X-linked severe combined immunodeficiency (X-SCID) and adenosine deaminase (ADA)-deficient SCID following onco-retrovirus vector ex vivo transduction of autologous marrow stem cells [Science 296 (2002) 2410; Science 288 (2000) 669; N. Engl. J. Med. 346 (2002) 1185]. These achievements of prolonged clinical benefit from gene therapy were tempered by the finding of insertional mutageneses in two of the treated X-SCID patients [N. Engl. J. Med. 348 (2003) 255].