Chipping away at gamma-H2AX foci

The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form gamma-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure gamma-H2AX densities along chromosomal DNA strands broken in G(1) phase mouse lymphocytes. Our experiments revealed that (1) gamma-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous if studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel gamma-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites

Formation of a WIP-, WASp-, actin-, and myosin IIA–containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling

The tumor natural killer (NK) cell line YTS was used to examine the cytoskeletal rearrangements required for cytolysis. A multiprotein complex weighing ∼1.3 mD and consisting of WASp-interacting protein (WIP), Wiskott-Aldrich syndrome protein (WASp), actin, and myosin IIA that formed during NK cell activation was identified. After induction of an inhibitory signal, the recruitment of actin and myosin IIA to a constitutive WIP–WASp complex was greatly decreased. Both actin and myosin IIA were recruited to WIP in the absence of WASp. This recruitment correlated with increased WIP phosphorylation, which was mediated by PKCθ. Furthermore, the disruption of WIP expression by WIP RNA interference prevented the formation of this protein complex and led to almost complete inhibition of cytotoxic activity. Thus, the multiprotein complex is important for NK cell function, killer cell immunoglobulin-like receptor inhibitory signaling affects proteins involved in cytoskeletal rearrangements, and WIP plays a central role in the formation of the complex and in the regulation of NK cell activity

Natural Killer Cell Lytic Granule Secretion Occurs through a Pervasive Actin Network at the Immune Synapse

Super-resolution imaging provides a new look at how the lytic granules in natural killer cells penetrate the filamentous actin network of the immunological synapse

Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network

Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches

Bioavailability of IgG Administered by the subcutaneous route

Purpose US licensing studies of subcutaneous IgG (SCIG) calculate dose adjustments necessary to achieve area under the curve (AUC) of serum IgG vs. time on SCIG that is non-inferior to that on intravenous IgG (IVIG), within the FDA-set limit of ±20 %. The results are interpreted as showing that different SCIGs differ in bioavailability. We used three approaches to determine if the bioavailabilities were actually different. Methods Dose adjustments and AUCs from published licensing studies were used to calculate bioavailabilities using the formula: Bioavailability (% of IVIG) = AUC(SCIG) ÷ AUC(IVIG) x 1/Dose Adjustment. We also compared the increment in serum IgG concentration achieved with varying doses of SCIG in recent meta-analyses with the increment with different doses of IVIG, and determined the serum IgG concentrations when patients switched SCIG products at the same dose. Results The actual bioavailabilities were: Gamunex® 65.0 %, Hizentra® 65.5 %, Gammagard® 67.2 %, Vivaglobin® 69.0 %. Regression analyses of serum IgG vs. dose showed that the mean increase in serum IgG resulting from a 100 mg/kg/month increment in SCIG dosing was 69.4 % of the increase with the same increment in IVIG dosing (84 mg/dL vs. 121 mg/dL). Patients switching SCIG preparations at the same dose had no change in serum IgG levels, confirming that bioavailabilities of the SCIG preparations did not differ. Conclusions Decreased bioavailability appears to be a basic property of SCIG and not a result of any manufacturing process or concentration. Because serum IgG levels do not vary with different SCIG products at the same dose, adjustments are not necessary when switching products

Editorial: NK Cell Subsets in Health and Disease: New Developments

Natural killer (NK) cells were discovered ca 1975, as the first group of lymphoid cells that were neither T cells nor B cells. Since then, the dissection of the biology of NK cells has been growing exponentially with many seminal discoveries from the identification of MHC class I-specific inhibitory receptors to the discovery of receptor\u2013ligand pairs involved in NK cell activation and to the manipulation of NK cells in cancer. In this research topic, we asked a group of thought leaders in NK cell biology to review recent advances in their origins and biology, and their roles in cancer, infection, and inflammation. Together, these 25 articles provide a timely survey of NK cells as critical immunologic components of health and disease. They will hopefully prompt further dialog and developments in basic and translational immunology

Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation

Hypohidrotic Ectodermal Dysplasia and Immunodeficiency with Coincident NEMO and EDA Mutations

Ectodermal dysplasias (ED) are uncommon genetic disorders resulting in abnormalities in ectodermally derived structures. Many ED-associated genes have been described, of which ectodysplasin-A (EDA) is one of the more common. The NF-κB essential modulator (NEMO encoded by the IKBKG gene) is unique in that mutations result in severe humoral and cellular immunologic defects in addition to ED. We describe three unrelated kindreds with defects in both EDA and IKBKG resulting from X-chromosome crossover. This demonstrates the importance of thorough immunologic consideration of patients with ED even when an EDA etiology is confirmed, and raises the possibility of a specific phenotype arising from coincident mutations in EDA and IKBKG

Calculation of a Primary Immunodeficiency “Risk Vital Sign” via Population-Wide Analysis of Claims Data to Aid in Clinical Decision Support

Background: Early diagnosis of primary immunodeficiency disease leads to reductions in illness and decreased healthcare costs. Analysis of electronic health record data may allow for identification of persons at risk of host-defense impairments from within the general population. Our hypothesis was that coded infection history would inform individual risk of disease and ultimately lead to diagnosis.Methods: In this study we assessed individual risk for primary immunodeficiency by analyzing diagnostic codes and pharmacy records from members (n = 185,892) of a large pediatric health network. Relevant infection-associated diagnostic codes were weighted and enumerated for individual members allowing for risk score calculations (“Risk Vital Sign”). At-risk individuals underwent further assessment by chart review and re-analysis of diagnostic codes 12 months later.Results: Of the original cohort, 2188 (1.2%) individuals were identified as medium-high-risk for having a primary immunodeficiency. This group included 41 subjects who were ultimately diagnosed with primary immunodeficiency. An additional 57 medium-high risk patients had coded diagnoses worthy of referral.Conclusions: Population-wide informatics approaches can facilitate disease detection and improve outcomes. Early identification of the 98 patients with confirmed or suspected primary immunodeficiency described here could represent an annual cost savings of up to $7.7 million US Dollars