Hypoxia activates SREBP2 through Golgi disassembly in bone marrow-derived monocytes for enhanced tumor growth.

Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.

Reduced antiviral seropositivity among patients with inflammatory bowel disease treated with immunosuppressive agents.

BACKGROUND: Although live-attenuated vaccines are contraindicated under immunosuppression, the immune status of patients with inflammatory bowel disease (IBD) has not been fully assessed prior to immunosuppressive therapy. AIMS: To investigate antiviral serostatus against viruses requiring live vaccines for prevention in IBD patients undergoing immunosuppressive therapy. METHODS: This multicenter study included IBD patients who were aged <40 years and were treated with thiopurine monotherapy, molecular-targeted monotherapy, or combination therapy. Gender- and age-matched healthy subjects (HS) living in the same areas were included as control group. Antibody titers against measles, rubella, mumps, and varicella were measured by enzyme-linked immunosorbent assays. RESULTS: A total of 437 IBD patients (163 ulcerative colitis [UC] and 274 Crohn's disease [CD]) and 225 HS were included in the final analysis. Compared with HS, IBD patients had lower seropositivity rates for measles (IBD vs. HS = 83.91% vs. 85.33%), rubella (77.55% vs. 84.89%), mumps (37.50% vs. 37.78%), and varicella (91.26% vs. 96.44%). Gender- and age-adjusted seropositivity rates were lower in UC patients than in both CD patients and HS for measles (UC, CD, and HS = 81.60%, 85.29%, and 85.33%), rubella (76.40%, 78.23%, and 84.89%), mumps (27.16%, 43.70%, and 37.78%), and varicella (90.80%, 91.54%, and 96.44%); the difference was significant for all viruses except measles. Divided by the degree of immunosuppression, there were no significant differences in seropositivity rates among IBD patients. CONCLUSIONS: IBD patients, especially those with UC, exhibit reduced seropositivity rates and may benefit from screening prior to the initiation of immunosuppressive therapy.

Estimation of population genetic parameters using an EM algorithm and sequence data from experimental evolution populations.

MOTIVATION: Evolve and resequence (E&R) experiments show promise in capturing real-time evolution at genome-wide scales, enabling the assessment of allele frequency changes SNPs in evolving populations and thus the estimation of population genetic parameters in the Wright-Fisher model (WF) that quantify the selection on SNPs. Currently, these analyses face two key difficulties: the numerous SNPs in E&R data and the frequent unreliability of estimates. Hence, a methodology for efficiently estimating WF parameters is needed to understand the evolutionary processes that shape genomes. RESULTS: We developed a novel method for estimating WF parameters (EMWER), by applying an expectation maximization algorithm to the Kolmogorov forward equation associated with the WF model diffusion approximation. EMWER was used to infer the effective population size, selection coefficients and dominance parameters from E&R data. Of the methods examined, EMWER was the most efficient method for selection strength estimation in multi-core computing environments, estimating both selection and dominance with accurate confidence intervals. We applied EMWER to E&R data from experimental Drosophila populations adapting to thermally fluctuating environments and found a common selection affecting allele frequency of many SNPs within the cosmopolitan In(3R)P inversion. Furthermore, this application indicated that many of beneficial alleles in this experiment are dominant. AVAILABILITY AND IMPLEMENTATION: Our C++ implementation of 'EMWER' is available at https://github.com/kojikoji/EMWER. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

IL-9 Blockade Suppresses Silica-induced Lung Inflammation and Fibrosis in Mice.

Recapitulative animal models of idiopathic pulmonary fibrosis (IPF) and related diseases are lacking, which inhibits our ability to fully clarify the pathogenesis of these diseases. Although lung fibrosis in mouse models is often induced by bleomycin, silica-induced lung fibrosis is more sustainable and more progressive. Therefore, in this study, we sought to elucidate the mediator(s) responsible for the pathogenesis of lung fibrosis, through the use of a mouse model of silica-induced lung fibrosis. With a single nasal administration of 16 mg of silica, lung inflammation (assessed by elevated cellular components in the BAL fluids [BALFs]) and lung fibrosis (assessed by lung histology and lung hydroxyproline levels) were induced and sustained for as long as 24 weeks. Of the mediators measured in the BALFs, IL-9 was characteristically elevated gradually, and peaked at 24 weeks after silica administration. Treatment of silica-challenged mice with anti-IL-9-neutralizing antibody inhibited lung fibrosis, as assessed by lung hydroxyproline level, and suppressed the levels of major mediators, including IL-1ß, IL-6, IL-12, CCL2, CXCL1, and TNF-α in BALFs. Moreover, human lung specimens from patients with IPF have shown high expression of IL-9 in alveolar macrophages, CD4-positive cells, and receptors for IL-9 in airway epithelial cells. Collectively, these data suggest that IL-9 plays an important role in the pathogenesis of lung fibrosis in diseases such as IPF.

Measurement of propagation of ultrafast surface plasmon polariton pulses using dual-probe scanning near-field optical microscopy.

We report the construction of diagnostics for ultrafast surface plasmon polariton (SPP) pulses that evolve spatiotemporally in femtosecond and nanometer scales. We constructed two types of scanning near-field optical microscopes (SNOMs) and verified that the temporal waveform of ultrafast SPP pulses can be measured by combining spectral interferometry (SI). In the illumination-collection (I-C) mode SNOM, which uses a single fiber probe to excite samples and collect optical responses, a lock-in detection scheme using a lock-in camera detects SI fringes even for extremely weak signal light pulses. With this I-C SI-SNOM scheme, we measured the complex plasmon response functions of gold (Au) nanorods on Ge2Sb2Te5 thin film, both in the crystal and amorphous phases. For a dual-probe SNOM (DSNOM), a dual-band modulation technique was introduced to independently control the probe-sample and probe-probe distances. With the DSNOM and by employing femtosecond SPP pulse excitation, we successfully measured the temporal waveform of an ultrafast SPP pulse that is propagating on an Au thin layer.

Selective Coherent Anti-Stokes Raman Scattering Microscopy Employing Dual-Wavelength Nanofocused Ultrafast Plasmon Pulses.

Ultrafast surface plasmon polariton (SPP) nanofocusing on a plasmonic metal tapered tip with femtosecond laser pulses enables background-free localized excitation beyond the diffraction limit. We demonstrate simultaneous nanofocusing of ultrafast SPP pulses at 440 and 800 nm, which were coupled with a common diffraction grating structure fabricated on an aluminum (Al) tapered tip, to the tip apex with a radius of ∼35 nm. We achieved selective coherent anti-Stokes Raman scattering (CARS) microscopy that combined an 800 nm (ω) SPP pump pulse, which achieves selective vibrational excitation by spectral focusing, and a 440 nm (2ω) SPP probe pulse. Raman intensity of this novel 2ω-CARS increased by a factor of 3.96 at the G-band and 4.00 at the 2D-band compared with that with ω-CARS for the monolayer graphene. The 2ω-CARS imaging method was applied for imaging a multiwalled carbon nanotube at the D-, G-, and 2D-bands. This dual-wavelength nanofocusing will open up new nanoscale microspectroscopy and optical excitation at the tip apex, such as sum frequency mixing, two-photon excitation.

Leukotriene receptor antagonist attenuated airway inflammation and hyperresponsiveness in a double-stranded RNA-induced asthma exacerbation model.

BACKGROUND: Viral infections are the most common triggers of asthma exacerbation, but the key molecules involved in this process have not been fully identified. Although cysteinyl leukotrienes (cysLTs) have been postulated as the key mediators, their precise roles remain largely unclear. To investigate the roles of cysLTs in virus-induced asthma exacerbation, we developed a murine model using a viral double-stranded RNA analog, polyinosinic-polycytidylic acid (poly I:C), and analyzed the effect of leukotriene receptor antagonist (LTRA) administration. METHODS: A/J mice were immunized with ovalbumin (OVA) + alum (days 0, 28, 42, and 49), followed by intranasal challenge with OVA (phase 1: days 50-52) and poly I:C (phase 2: days 53-55). Montelukast was administered during poly I:C challenge (phase 2) in the reliever model or throughout the OVA and poly I:C challenges (phases 1 and 2) in the controller model. Airway responsiveness to acetylcholine chloride was assessed, and bronchoalveolar lavage (BAL) was performed on day 56. RESULTS: Administration of poly I:C to OVA-sensitized and -challenged mice increased the number of eosinophils and levels of IL-13, IL-9, CCL3, and CXCL1 in BAL fluid (BALF) and tended to increase airway responsiveness. Montelukast significantly attenuated the poly I:C-induced increase in the number of eosinophils and levels of IL-13, IL-9, and CCL3 in BALF and airway hyperresponsiveness in both the reliever and controller models. CONCLUSIONS: This is the first report showing that LTRA functionally suppressed the pathophysiology of a virus-induced asthma exacerbation model, suggesting the importance of cysLTs as a potential treatment target.

[Efficacy and Indication of Fibrinogen Replacement Therapy in Thoracic Aortic Surgery A Retrospective Cohort Study].

BACKGROUND: Fibrinogen replacement therapy con- tributes to effective hemostasis and saving blood trans- fusions in critical hemorrhage. We retrospectively studied the efficacy and indication for cryoprecipitate or fibrinogen concentrate in thoracic aortic surgery. METHODS: In 169 patients undergoing thoracic aortic surgery, 92 (54.4%) patients received dryoprecipitate or fibrinogen concentrate and 77 (45.6%) patients did not We compared them with regard to postoperative bleeding and perioperative blood transfusion. We deter- mined the effective dose of the fibrinogen in cryopre- cipitate or fibrinogen concentrate for increasing the fibrinogen level. RESULTS: The cutoff value of the fibrinogen level at the end of cardiopulmonary bypass between both groups was 100 mg · dl⁻¹. For a fibrinogen level less than 130 mg · dl⁻¹ during cardiopulmonary bypass, the patients who received cryoprecipitate or fibrinogen concentrate had less postoperative bleeding (P<0.01) and fewer transfusions of total blood, fresh frozen plasma, and platelet concentrates (P<0.05). The effec- tive dose 50 of fibrinogen amount was 0.031-0.051 g - kg⁻¹. CONCLUSIONS: The fibrinogen amount of 2-3 g (per 50-70 kg in body weight) in cryoprecipitate or fibrino- gen concentrate effectively reduces postoperative bleeding and perioperative blood transfusions when a fibrinogen level is less than 100-130 mg · dl⁻¹ during cardiopulmonary bypass.

Single-cell colocalization analysis using a deep generative model.

Analyzing colocalization of single cells with heterogeneous molecular phenotypes is essential for understanding cell-cell interactions, and cellular responses to external stimuli and their biological functions in diseases and tissues. However, existing computational methodologies identified the colocalization patterns between predefined cell populations, which can obscure the molecular signatures arising from intercellular communication. Here, we introduce DeepCOLOR, a computational framework based on a deep generative model that recovers intercellular colocalization networks with single-cell resolution by the integration of single-cell and spatial transcriptomes. Along with colocalized population detection accuracy that is superior to existing methods in simulated dataset, DeepCOLOR identified plausible cell-cell interaction candidates between colocalized single cells and segregated cell populations defined by the colocalization relationships in mouse brain tissues, human squamous cell carcinoma samples, and human lung tissues infected with SARS-CoV-2. DeepCOLOR is applicable to studying cell-cell interactions behind various spatial niches. A record of this paper's transparent peer review process is included in the supplemental information.

Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Topological data analysis of protein structure and inter/intra-molecular interaction changes attributable to amino acid mutations.

The presence of some amino acid mutations in the amino acid sequence that determines a protein's structure can significantly affect that 3D structure and its biological function. However, the effects upon structural and functional changes differ for each displaced amino acid, and it is very difficult to predict these changes in advance. Although computer simulations are very effective at predicting conformational changes, they struggle to determine whether the amino acid mutation of interest induces sufficient conformational changes, unless the researcher is a specialist in molecular structure calculations. Therefore, we created a framework that efficiently utilizes molecular dynamics and persistent homology methods to identify amino acid mutations that induce structural changes. We show that this framework can be used not only to predict conformational changes produced by amino acid mutations but also to extract groups of mutations that significantly alter similar molecular interactions, by capturing the resultant protein-protein interaction changes.

Automated time-lapse data segmentation reveals in vivo cell state dynamics.

Embryonic development proceeds as a series of orderly cell state transitions built upon noisy molecular processes. We defined gene expression and cell motion states using single-cell RNA sequencing data and in vivo time-lapse cell tracking data of the zebrafish tailbud. We performed a parallel identification of these states using dimensional reduction methods and a change point detection algorithm. Both types of cell states were quantitatively mapped onto embryos, and we used the cell motion states to study the dynamics of biological state transitions over time. The time average pattern of cell motion states is reproducible among embryos. However, individual embryos exhibit transient deviations from the time average forming left-right asymmetries in collective cell motion. Thus, the reproducible pattern of cell states and bilateral symmetry arise from temporal averaging. In addition, collective cell behavior can be a source of asymmetry rather than a buffer against noisy individual cell behavior.

RENGE infers gene regulatory networks using time-series single-cell RNA-seq data with CRISPR perturbations.

Single-cell RNA-seq analysis coupled with CRISPR-based perturbation has enabled the inference of gene regulatory networks with causal relationships. However, a snapshot of single-cell CRISPR data may not lead to an accurate inference, since a gene knockout can influence multi-layered downstream over time. Here, we developed RENGE, a computational method that infers gene regulatory networks using a time-series single-cell CRISPR dataset. RENGE models the propagation process of the effects elicited by a gene knockout on its regulatory network. It can distinguish between direct and indirect regulations, which allows for the inference of regulations by genes that are not knocked out. RENGE therefore outperforms current methods in the accuracy of inferring gene regulatory networks. When used on a dataset we derived from human-induced pluripotent stem cells, RENGE yielded a network consistent with multiple databases and literature. Accurate inference of gene regulatory networks by RENGE would enable the identification of key factors for various biological systems.

Spatial and single-cell transcriptomics decipher the cellular environment containing HLA-G+ cancer cells and SPP1+ macrophages in colorectal cancer.

The cellular interactions in the tumor microenvironment of colorectal cancer (CRC) are poorly understood, hindering patient treatment. In the current study, we investigate whether events occurring at the invasion front are of particular importance for CRC treatment strategies. To this end, we analyze CRC tissues by combining spatial transcriptomics from patients with a public single-cell transcriptomic atlas to determine cell-cell interactions at the invasion front. We show that CRC cells are localized specifically at the invasion front. These cells induce human leukocyte antigen G (HLA-G) to produce secreted phosphoprotein 1 (SPP1)+ macrophages while conferring CRC cells with anti-tumor immunity, as well as proliferative and invasive properties. Taken together, these findings highlight the signaling between CRC cell populations and stromal cell populations at the cellular level.

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white.

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower ß1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

A bifurcation concept for B-lymphoid/plasmacytoid dendritic cells with largely fluctuating transcriptome dynamics.

Hematopoiesis was considered a hierarchical stepwise process but was revised to a continuous process following single-cell RNA sequencing. However, the uncertainty or fluctuation of single-cell transcriptome dynamics during differentiation was not considered, and the dendritic cell (DC) pathway in the lymphoid context remains unclear. Here, we identify human B-plasmacytoid DC (pDC) bifurcation as large fluctuating transcriptome dynamics in the putative B/NK progenitor region by dry and wet methods. By converting splicing kinetics into diffusion dynamics in a deep generative model, our original computational methodology reveals strong fluctuation at B/pDC bifurcation in IL-7Rα+ regions, and LFA-1 fluctuates positively in the pDC direction at the bifurcation. These expectancies are validated by the presence of B/pDC progenitors in the IL-7Rα+ fraction and preferential expression of LFA-1 in pDC-biased progenitors with a niche-like culture system. We provide a model of fluctuation-based differentiation, which reconciles continuous and discrete models and is applicable to other developmental systems.

Efficacy and Safety of Ultrahigh-Dose Methylcobalamin in Early-Stage Amyotrophic Lateral Sclerosis: A Randomized Clinical Trial.

Importance: The effectiveness of currently approved drugs for amyotrophic lateral sclerosis (ALS) is restricted; there is a need to develop further treatments. Initial studies have shown ultrahigh-dose methylcobalamin to be a promising agent. Objective: To validate the efficacy and safety of ultrahigh-dose methylcobalamin for patients with ALS enrolled within 1 year of onset. Design, Setting, and Participants: This was a multicenter, placebo-controlled, double-blind, randomized phase 3 clinical trial with a 12-week observation and 16-week randomized period, conducted from October 17, 2017, to September 30, 2019. Patients were recruited from 25 neurology centers in Japan; those with ALS diagnosed within 1 year of onset by the updated Awaji criteria were initially enrolled. Of those, patients fulfilling the following criteria after 12-week observation were eligible for randomization: 1- or 2-point decrease in the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) total score, a percent forced vital capacity greater than 60%, no history of noninvasive respiratory support and tracheostomy, and being ambulatory. The target participant number was 64 in both the methylcobalamin and placebo groups. Patients were randomly assigned through an electronic web-response system to methylcobalamin or placebo. Interventions: Intramuscular injection of methylcobalamin (50-mg dose) or placebo twice weekly for 16 weeks. Main Outcomes and Measures: The primary end point was change in ALSFRS-R total score from baseline to week 16 in the full analysis set. Results: A total of 130 patients (mean [SD] age, 61.0 [11.7] years; 74 men [56.9%]) were randomly assigned to methylcobalamin or placebo (65 each). A total of 129 patients were eligible for the full analysis set, and 126 completed the double-blind stage. Of these, 124 patients proceeded to the open-label extended period. The least square means difference in ALSFRS-R total score at week 16 of the randomized period was 1.97 points greater with methylcobalamin than placebo (-2.66 vs -4.63; 95% CI, 0.44-3.50; P = .01). The incidence of adverse events was similar between the 2 groups. Conclusions and Relevance: Results of this randomized clinical trial showed that ultrahigh-dose methylcobalamin was efficacious in slowing functional decline in patients with early-stage ALS and with moderate progression rate and was safe to use during the 16-week treatment period. Trial Registration: ClinicalTrials.gov Identifier: NCT03548311.

Cancer immunotherapy with PI3K and PD-1 dual-blockade via optimal modulation of T cell activation signal.

BACKGROUND: Immune checkpoint blockade (ICB) induces durable clinical responses in patients with various types of cancer. However, its limited clinical efficacy requires the development of better approaches. In addition to immune checkpoint molecules, tumor-infiltrating immunosuppressive cells including regulatory T cells (Tregs) play crucial roles in the immune suppressive tumor microenvironment. While phosphatidylinositol 3-kinase (PI3K) inhibition as a Treg-targeted treatment has been implicated in animal models, its effects on human Tregs and on the potential impairment of effector T cells are required to be clarified for successful cancer immunotherapy. METHODS: The impact of a selective-PI3K inhibitor ZSTK474 with or without anti-programmed cell death 1 (PD-1) monoclonal antibody on Tregs and CD8+ T cells were examined with in vivo animal models and in vitro experiments with antigen specific and non-specific fashions using peripheral blood from healthy individuals and cancer patients. Phenotypes and functions of Tregs and effector T cells were examined with comprehensive gene and protein expression assays. RESULTS: Improved antitumor effects by the PI3K inhibitor in combination with ICB, particularly PD-1 blockade, were observed in mice and humans. Although administration of the PI3K inhibitor at higher doses impaired activation of CD8+ T cells as well as Tregs, the optimization (doses and timing) of this combination treatment selectively decreased intratumoral Tregs, resulting in increased tumor antigen-specific CD8+ T cells in the treated mice. Moreover, on the administration of the PI3K inhibitor with the optimal dose for selectively deleting Tregs, PI3K signaling was inhibited not only in Tregs but also in activated CD8+ T cells, leading to the enhanced generation of tumor antigen-specific memory CD8+ T cells which contributed to durable antitumor immunity. These opposing outcomes between Tregs and CD8+ T cells were attributed to the high degree of dependence on T cell signaling in the former but not in the latter. CONCLUSIONS: PI3K inhibitor in the combination with ICB with the optimized protocol fine-tuned T cell activation signaling for antitumor immunity via decreasing Tregs and optimizing memory CD8+ T cell responses, illustrating a promising combination therapy.

[A case of Guillain-Barré syndrome accompanied by syndrome of inappropriate secretion of antidiuretic hormone].

A 73-year-old Japanese male was admitted because of difficulty in standing up after acute upper respiratory inflammation with mild fever followed by watery diarrhea. Neurological examination revealed moderate proximal muscle weakness and loss of tendon reflexes in all extremities. The blood sodium level was 106 mEq/l on admission. The blood level of antidiuretic hormone (ADH), renin and aldsterone was 11.3 pg/ml (normal value 0.3-4.2), 0.2 ng/ml/h (0.2-2.7) and less than 10.0 pg/ml (38.9-307.0), respectively. The plasma osmolarity was 221 mOsm/kg (270-295), and the urine osmolarity was 416 mOsm/kg (50-1400). EMG and nerve conduction studies suggested acute demyelination in the motor and sensory nerves. CSF revealed 10 cells/mm3 and elevated protein to 98 mg/dl. The clinical course, laboratory data and electrophysiological findings suggested coexistence of the syndrome of inappropriate secretion of ADH (SIADH) and Guillain-Barré syndrome (GBS) from the very early clinical stage of the diseases. The clinical and laboratory findings improved after intravenous administration of saline over three weeks. When GBS is associated with SIADH, hyponatremia is commonly seen at the peak of motor paralysis, often accompanied by autonomic or respiratory failure requiring mechanical ventilation. This was not the case in the present patient. It is postulated that SIADH, like GBS, might be caused by an autoimmune mechanism.

Intracellular protein phosphorylation in eosinophils and the functional relevance in cytokine production.

BACKGROUND: Eosinophils play a pivotal role in the pathogenesis of asthma. Thus, it is of paramount importance to investigate the mechanism of eosinophil activation. Although a number of factors including cytokines/chemokines activate eosinophils, the potency of each stimulus to phosphorylate intracellular molecules and activate eosinophils remains to be elucidated. In the present study, we performed inclusive analyses of protein phosphorylation in eosinophils and studied the functional relevance of such phosphorylation in cytokine production. METHODS: Blood eosinophils were purified using Percoll and anti-CD16 antibody-coated magnetic beads. Purified eosinophils were stimulated with various stimuli. The eosinophil lysates were subjected to phosphoprotein analysis using the Luminex system. In some of these experiments, we studied the effect of a few signaling inhibitors on cytokine production from eosinophils. RESULTS: We found that several factors such as IL-5, eotaxin, platelet-activating factor (PAF), and PGD2 phosphorylated Akt, ERK1/2, p38 MAPK, and glycogen synthase kinase-3 (GSK-3) in the eosinophils. Because eotaxin most potently induced the production of various cytokines, we performed the inhibition study using eotaxin-stimulated eosinophils. Eotaxin-induced production of IL-1beta, IL-6, and MIP-1beta was significantly reduced by the MEK inhibitor PD98059, p38 MAPK inhibitor SB203580, or PI3K inhibitor LY294002. In contrast, the GSK-3 inhibitor SB216763 blocked only IL-1beta production from the eosinophils. CONCLUSIONS: In terms of the phosphorylation of intracellular signaling molecules, we could quantify the potency of various stimuli that activate eosinophils. We are the first to demonstrate the role of GSK-3 in cytokine production from eosinophils. The Luminex system aids in examining the mechanism of eosinophil activation.