Idiopathic intracranial hypertension in Asians: a retrospective dual-center study.

BACKGROUND: There have been limited data on idiopathic intracranial hypertension (IIH) in Asians and there remain uncertainties whether a cerebrospinal fluid (CSF) pressure of 250 mm CSF is an optimum diagnostic cutoff. The aims of the present study included (1) characterization of IIH patients in Taiwan, (2) comparisons among different diagnostic criteria for IIH, and (3) comparisons between patients with CSF pressures of > 250 and 200-250 mm CSF. METHODS: This retrospective study involved IIH patients based on the modified Dandy criteria from two tertiary medical centers in Taiwan. Clinical manifestations were retrieved from electronic medical records, and findings on ophthalmologic examination and magnetic resonance images (MRIs) were reviewed. RESULTS: A total of 102 patients (71 F/31 M, mean age 33.4 ± 12.2 years, mean CSF pressure 282.5 ± 74.5 mm CSF) were identified, including 46 (45.1%) with obesity (body-mass index ≥ 27.5), and 57 (62.6%) with papilledema. Overall, 80 (78.4%), 55 (53.9%), 51 (50.0%), and 58 (56.9%) patients met the Second and Third Edition of International Classification of Headache Disorders, Friedman, and Korsbæk criteria, respectively. Patients in the 200-250 mm CSF group (n = 40) were less likely to have papilledema (48.5% vs. 70.7%, p = 0.035), transient visual obscuration (12.5% vs. 33.9%, p = 0.005), and horizontal diplopia (10.0% vs. 30.6%, p = 0.006), and had fewer signs on MRIs (2.2 ± 1.3 vs. 2.8 ± 1.0, p = 0.021) when compared with those with CSF pressures > 250 mm CSF (n = 62). However, the percentages of patients with headache (95.0% vs. 87.1%, p = 0.109) at baseline, chronic migraine at six months (31.6% vs. 25.0%, p = 0.578), and visual field defect (86.7% vs. 90.3%, p = 0.709) were similar. CONCLUSIONS: It was found that obesity and papilledema were less common in Asian IIH patients when compared with Caucasian patients. Although patients with CSF pressures of 200-250 mm CSF had a less severe phenotype, the risks of having headache or visual loss were comparable to those in the > 250 mm CSF group. It is possible that a diagnostic cutoff of > 200 mm CSF could be more suitable for Asians, although further studies are still needed.

Measurement of Lytic Granule Convergence After Formation of an NK Cell Immunological Synapse.

Natural killer (NK) cells contain specialized lysosome-related organelles termed lytic granules allowing them to mediate cytotoxicity against tumorigenic or virally infected target cells. NK cells polarize their lytic granules toward a target cell via the microtubule-organizing center (MTOC). Prior to that, however, lytic granules converge to the MTOC along microtubules utilizing minus-end-directed microtubule motors. Herein we describe how to visualize and quantify lytic granule convergence using confocal microscopy to gain quantitative insight into NK cell cytotoxicity and its regulation.

Quantification of natural killer cell polarization and visualization of synaptic granule externalization by imaging flow cytometry.

Defining immunological mechanisms underlying NK cell biology is crucial for the treatment and prevention of immune deficiency and malignancy. The limited availability of human biological specimens presents a challenge to the study of human immunobiology. The use of high throughput, multi-parametric assays will not only aid in the definition and diagnosis of complex human immune disorders affecting NK cell function but also advance NK cell biology through population-based assessment of molecular signaling. In an effort to garner the most information from limited numbers of human cells, we designed a quantitative method to study NK cell function using imaging flow cytometry (IFC), which combines multiparametric flow cytometry and fluorescence microscopy. Specifically, we developed IFC as a tool to measure polarization and secretion of lytic granules at the immunological synapse formed between an NK cell and a susceptible target. We have further validated our approach through quantitative comparison with high-resolution confocal microscopy. We show that IFC can be used as a quantitative, high throughput measure of NK cell biological function possessing greater dimensionality than standard flow cytometry.

NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing.

Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal-dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector-target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific "bystander" killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.

RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics.

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

PTEN is a negative regulator of NK cell cytolytic function.

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56(dim) NK cell efficiently kills malignant targets at rest, whereas the less mature CD56(bright) NK cells cannot. In this study, we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells.

Follicular helper T cells promote liver pathology in mice during Schistosoma japonicum infection.

Following Schistosoma japonicum (S. japonicum) infection, granulomatous responses are induced by parasite eggs trapped in host organs, particular in the liver, during the acute stage of disease. While excessive liver granulomatous responses can lead to more severe fibrosis and circulatory impairment in chronically infected host. However, the exact mechanism of hepatic granuloma formation has remained obscure. In this study, we for the first time showed that follicular helper T (Tfh) cells are recruited to the liver to upregulate hepatic granuloma formation and liver injury in S. japonicum-infected mice, and identified a novel function of macrophages in Tfh cell induction. In addition, our results showed that the generation of Tfh cells driven by macrophages is dependent on cell-cell contact and the level of inducible costimulator ligand (ICOSL) on macrophages which is regulated by CD40-CD40L signaling. Our findings uncovered a previously unappreciated role for Tfh cells in liver pathology caused by S. japonicum infection in mice.

Endoplasmic reticulum targeting alters regulation of expression and antigen presentation of proinsulin.

Peptide ligands presented by MHC class I (MHC-I) molecules are produced by degradation of cytosolic and nuclear, but also endoplasmic reticulum (ER)-resident, proteins by the proteasome. However, Ag processing of ER proteins remains little characterized. Studying processing and presentation of proinsulin, which plays a pivotal role in autoimmune diabetes, we found that targeting to the ER has profound effects not only on how proinsulin is degraded, but also on regulation of its cellular levels. While proteasome inhibition inhibited degradation and presentation of cytosolic proinsulin, as expected, it reduced the abundance of ER-targeted proinsulin. This targeting and protein modifications modifying protein half-life also had profound effects on MHC-I presentation and proteolytic processing of proinsulin. Thus, presentation of stable luminal forms was inefficient but enhanced by proteasome inhibition, whereas that of unstable luminal forms and of a cytosolic form were more efficient and compromised by proteasome inhibitors. Distinct stability of peptide MHC complexes produced from cytosolic and luminal proinsulin suggests that different proteolytic activities process the two Ag forms. Thus, both structural features and subcellular targeting of Ags can have strong effects on the processing pathways engaged by MHC-I-restricted Ags, and on the efficiency and regulation of their presentation.

No major role for insulin-degrading enzyme in antigen presentation by MHC molecules.

Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.

Cell biological steps and checkpoints in accessing NK cell cytotoxicity.

Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.

Rapid activation receptor- or IL-2-induced lytic granule convergence in human natural killer cells requires Src, but not downstream signaling.

Natural killer (NK) cells participate in host defense by surveying for and ultimately killing virally infected or malignant target cells. NK cell cytotoxicity is a tightly regulated process that proceeds stepwise from adhesion and activation to the secretion of preformed lytic granule contents onto a diseased or stressed cell. We previously characterized rapid dynein-dependent lytic granule convergence to the microtubule-organizing center (MTOC) as an early, prerequisite step in NK cell cytotoxicity. Although multiple activating receptors can trigger granule convergence, the specific signal or signals responsible remained unknown. Using live cell confocal microscopy, NK cell lytic granule movement after NK cell activation was captured and measured. Using inhibitors of common early signaling mediators, we show that Src kinases are required for lytic granule convergence, but downstream signals that promote actin rearrangement, MTOC polarization, and calcium mobilization are not. Exposure to interleukin 2 was also sufficient to induce lytic granule convergence, which required noncanonical Src-dependent signaling. Thus, NK cell lytic granule convergence, prompted by specific receptor-mediated and soluble cytokine signals, depends on a directly downstream early Src kinase-dependent signal and emphasizes the importance of this step in readying NK cells for cytotoxicity.

Isodiospyrin as a novel human DNA topoisomerase I inhibitor.

Isodiospyrin is a natural product from the plant Diospyros morrisiana, which consists of an asymmetrical 1,2-binaphthoquinone chromophore. Isodiospyrin exhibits cytotoxic activity to tumor cell lines but very little is known about its cellular target and mechanism of action. Unlike the prototypic human topoisomerase I (htopo I) poison camptothecin, isodiospyrin does not induce htopo I-DNA covalent complexes. However, isodiospyrin antagonizes camptothecin-induced, htopo I-mediated DNA cleavage. Binding analysis indicated that isodiospyrin binds htopo I but not DNA. These results suggest that isodiospyrin inhibits htopo I by direct binding to htopo I, which limits htopo I access to the DNA substrate. Furthermore, isodiospyrin exhibits strong inhibitory effect on the kinase activity of htopo I toward splicing factor 2/alternate splicing factor in the absence of DNA. Thus, these findings have important implications on naphthoquinone and its derivatives' cellular mode of actions, i.e. these novel DNA topoisomerase I inhibitors can prevent both DNA relaxation and kinase activities of htopo I.