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Hsu H.-T.,Center for Human Immunobiology | Hsu H.-T.,Baylor College of Medicine | Orange J.S.,Center for Human Immunobiology | Orange J.S.,Baylor College of Medicine
Science Signaling | Year: 2014

Lytic granules in natural killer (NK) cells represent a dangerous cargo that is targeted for secretion to destroy diseased cells. The appropriate management of these organelles enables the mounting of a precise and valuable host defense. The process of NK cell adhesion to a target cell through engagement of the integrin LFA-1 (lymphocyte function-associated antigen 1) promotes lytic granule organization through complex cellular mechanics and a signaling pathway characterized by Zhang et al. in this issue of Science Signaling. A set of signaling molecules was defi ned for their ability to promote the polarization of NK cell lytic granules and the microtubule organizing center (MTOC) toward the interface with a target cell. A subset of these signaling molecules was also required for the convergence of lytic granules on the MTOC.

Stray-Pedersen A.,Baylor College of Medicine | Stray-Pedersen A.,Center for Human Immunobiology | Stray-Pedersen A.,University of Oslo | Backe P.H.,University of Oslo | And 40 more authors.
American Journal of Human Genetics | Year: 2014

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes. © 2014 The American Society of Human Genetics.

Zheng P.,Center for Human Immunobiology | Zheng P.,Baylor College of Medicine | Noroski L.M.,Allergy | Hanson I.C.,Allergy | And 18 more authors.
Journal of Allergy and Clinical Immunology | Year: 2015

Background DiGeorge syndrome affects more than 3.5 million persons worldwide. Partial DiGeorge syndrome (pDGS), which is characterized by a number of gene deletions in chromosome 22, including the chicken tumor virus number 10 regulator of kinase (Crk)-like (CrkL) gene, is one of the most common genetic disorders in human subjects. To date, the role of natural killer (NK) cells in patients with pDGS remains unclear. Objective We sought to define the effect of pDGS-related Crk haploinsufficiency on NK cell activation and cytotoxic immunological synapse (IS) structure and function. Methods Inducible CrkL-silenced NK cells were used to recapitulate the pDGS, CrkL-haploinsufficient phenotype. Findings were validated by using NK cells from patients with actual pDGS. Ultimately, deficits in the function of NK cells from patients with pDGS were restored by lentiviral transduction of CrkL. Results Silencing of CrkL expression inhibits NK cell function. Specifically, pDGS haploinsufficiency of CrkL inhibits accumulation of activating receptors, polarization of cytolytic machinery and key signaling molecules, and activation of β2-integrin at the IS. Reintroduction of CrkL protein restores NK cell cytotoxicity. Conclusion CrkL haploinsufficiency causes functional NK deficits in patients with pDGS by disrupting both β2-integrin activation and activating receptor accumulation at the IS. Our results suggest that NK cell IS quality can directly affect immune status, providing a potential target for diagnosis and therapeutic manipulation in patients with pDGS and in other patients with functional NK cell deficiencies. © 2015 American Academy of Allergy, Asthma & Immunology.

Bonilla F.A.,Harvard University | Khan D.A.,University of Texas Southwestern Medical Center | Ballas Z.K.,University of Iowa | Chinen J.,Lake Houston Allergy and Immunology | And 24 more authors.
Journal of Allergy and Clinical Immunology | Year: 2015

The American Academy of Allergy, Asthma & Immunology (AAAAI) and the American College of Allergy, Asthma & Immunology (ACAAI) have jointly accepted responsibility for establishing the "Practice parameter for the diagnosis and management of primary immunodeficiency." This is a complete and comprehensive document at the current time. The medical environment is a changing environment, and not all recommendations will be appropriate for all patients. Because this document incorporated the efforts of many participants, no single individual, including those who served on the Joint Task Force, is authorized to provide an official AAAAI or ACAAI interpretation of these practice parameters. Any request for information about or an interpretation of these practice parameters by the AAAAI or ACAAI should be directed to the Executive Offices of the AAAAI, the ACAAI, and the Joint Council of Allergy, Asthma & Immunology. These parameters are not designed for use by pharmaceutical companies in drug promotion.

Zheng P.,Center for Human Immunobiology | Zheng P.,Baylor College of Medicine | Bertolet G.,Center for Human Immunobiology | Bertolet G.,Baylor College of Medicine | And 6 more authors.
Journal of Visualized Experiments | Year: 2015

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution. © JoVE 2006-2015. All Rights Reserved.

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