Glycosylation, hypogammaglobulinemia, and resistance to viral infections.

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

From bench to bedside: preclinical evaluation of a self-inactivating gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis.

BACKGROUND: Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. OBJECTIVE: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. METHODS: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. RESULTS: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation. CONCLUSION: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.

A novel internalization motif regulates human IFN-γ R1 endocytosis.

This study tested the hypothesis that the IFN-γ R1 287-YVSLI-91 intracellular motif regulates its endocytosis. IFN-γ exerts its biological activities by interacting with a specific cell-surface RC composed of two IFN-γ R1 and two IFN-γ R2 chains. Following IFN-γ binding and along with the initiation of signal transduction, the ligand and IFN-γ R1 are internalized. Two major types of consensus-sorting signals are described in receptors, which are rapidly internalized from the plasma membrane to intracellular compartments: tyrosine-based and dileucine-based internalization motifs. Transfection of HEK 293 cells and IFN-γ R1-deficient fibroblasts with WT and site-directed, mutagenesis-generated mutant IFN-γ R1 expression vectors helped us to identify region IFN-γ R1 287-YVSLI-291 as the critical domain required for IFN-γ-induced IFN-γ R1 internalization and Y287 and LI290-291 as part of a common structure essential for receptor endocytosis and function. This new endocytosis motif, YxxLI, shares characteristics of tyrosine-based and dileucine-based internalization motifs and is highly conserved in IFN-γ Rs across species. The IFN-γ R1 270-LI-271 dileucine motif, previously thought to be involved in this receptor endocytosis, showed to be unnecessary for receptor endocytosis.

Newly diagnosed chronic granulomatous disease in a 53-year-old woman with Crohn disease.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by defective bactericidal activity of white blood cells, specifically, a defect in superoxide production. Patients experience infections, predominantly caused by catalase-positive bacteria and fungal organisms, that may be severe and life-threatening. Most cases of CGD are diagnosed in children; however, it may rarely go undiagnosed until adulthood in individuals with unexplained infections and granulomatous inflammation. OBJECTIVE: To describe an adult with Crohn disease and recurrent infections who was newly diagnosed as having CGD. METHODS: A 53-year-old woman with a history of liver abscesses and Crohn disease presented with Burkholderia cepacia pneumonia and required a right middle lobe resection. Nitroblue tetrazolium test results confirmed the diagnosis of CGD, and Western blot analysis revealed the absence of the 47-phagocyte oxidase protein. Levels of Crohn-associated specific antibodies to Saccharomyces cerevisiae and Escherichia coli outer membrane porin C were elevated. RESULTS: The patient, newly diagnosed as having CGD, was given intravenous trimethoprim-sulfamethoxazole, after which she improved clinically and was discharged from the hospital in stable condition to receive daily oral trimethoprim-sulfamethoxazole treatment. CONCLUSIONS: The concomitant occurrence of Crohn disease and CGD, both characterized by granulomatous inflammation, is noteworthy. This case study demonstrates that CGD should be considered in adults with recurrent infections, especially those caused by catalase-positive organisms, such as B cepacia.

Real-Time PCR: an Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells.

Accurate measurement of gene transfer into hematopoietic progenitor cells is an essential prerequisite for assessing the utility of gene therapy approaches designed to correct hematologic defects. We developed a reliable method to measure transduction efficiency at the level of the progenitor cell with real-time polymerase chain reaction (PCR) analysis of individual progenitor-derived colonies. We hypothesized that this method would demonstrate better sensitivity and specificity than are currently achievable with conventional PCR. An oncoretroviral vector containing the enhanced green fluorescent protein was used to transduce human CD34+ cells derived from bone marrow or granulocyte-colony-stimulating factor-mobilized peripheral blood. Progenitor assays were set up and colonies plucked after visualization by fluorescence microscopy. By analyzing microscopically identified fluorescent samples and nontransduced samples, we calculated an overall sensitivity and specificity of 90.2 and 95.0%, respectively. Real-time PCR had higher specificity and sensitivity than conventional PCR as analyzed by generalized linear models (P = 0.002 and P = 0.019, respectively). In conclusion, we found real-time PCR to have superior sensitivity and specificity compared to conventional PCR in determining transduction efficiency of hematopoietic progenitor cells.

Long-term high-level reconstitution of NADPH oxidase activity in murine X-linked chronic granulomatous disease using a bicistronic vector expressing gp91phox and a Delta LNGFR cell surface marker.

A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91(phox) gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91(phox) and a linked gene for Delta LNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91(phox) expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91(phox) and Delta LNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for Delta LNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for Delta LNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection for Delta LNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed.