Discovery of novel 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives as potent carbonic anhydrase IX inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer.

A novel series of 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives were designed and synthesized as carbonic anhydrase IX (CA IX) inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer (TNBC). The representative compound 9o exhibited more potent inhibitory activity and selective against CA IX over off-target CA II, compared with positive control SLC-0111. Molecular docking study was also performed to gain insights into the binding interactions of 9o in the binding pocket of CAIX. Moreover, compound 9o exhibited superior antitumor activities against breast cancer cells under hypoxia than that of normoxia conditions. Mechanism studies revealed that compound 9o could act as DNA intercalator and effectively suppressed cell migration, arrested the cell cycle at G1/S phase and induced apoptosis in MDA-MB-231 cells, while inducing ferroptosis accompanied by the dissipation of MMP and the elevation intracellular levels of ROS. Notably, in vivo studies demonstrated that 9o effectively inhibited tumor growth and metastasis in a highly metastatic murine breast cancer 4 T1 xenograft model. Taken together, this study suggests that compound 9o represents a potent and selective CA IX inhibitor and ferroptosis inducer for the treatment of TNBC.

Climate and soil stressed elevation patterns of plant species to determine the aboveground biomass distributions in a valley-type Savanna.

Introduction: Extreme environments such as prolonged high temperatures and droughts can cause vulnerability of vegetation ecosystems. The dry-hot valleys of Southwestern China, known for their extremely high annual temperature, lack of water, and unique non-zonal "hot island" habitat in the global temperate zone, provide exceptional sites for studying how plant adapts to the prolonged dry and hot environment. However, the specific local biotic-environment relationships in these regions remain incompletely elucidated. The study aims to evaluate how valley-type Savanna vegetation species and their communities adapt to long-term drought and high-temperature stress environments. Methods: The study investigated the changes in species diversity and communities' aboveground biomass of a valley-type Savanna vegetation along an elevation gradient of Yuanmou dry-hot valley in Jinsha River basin, southwest China. Subsequently, a general linear model was utilized to simulate the distribution pattern of species diversities and their constituent biomass along the elevation gradient. Finally, the RDA and VPH mothed were used to evaluate the impacts and contributions of environmental factors or variables on the patterns. Results and discussion: The field survey reveals an altitudinal gradient effect on the valley-type Savanna, with a dominant species of shrubs and herbs plants distribution below an elevation of 1700m, and a significant positive relationship between the SR, Shannon-Wiener, Simpson, and Pielou indices and altitudes. Relatively, the community aboveground biomass did not increase significantly with elevation, which was mainly due to a decreased biomass of herbaceous plants along the elevation. Different regulators of shrub-herbaceous plant species and their functional groups made different elevation patterns of species diversity and aboveground biomass in valley-type Savannas. Herbaceous plants are responsible for maintaining species diversity and ensuring stability in the aboveground biomass of the vegetation. However, the influence of shrubs on aboveground biomass became more pronounced as environmental conditions varied along the altitudinal gradient. Furthermore, species diversity was mainly influenced by soil and climatic environmental factors, whereas community biomass was mainly regulated by plant species or functional groups. The study demonstrates that the spatial pattern of valley-type Savanna was formed as a result of different environmental responses and the productive capacity of retained plant species or functional groups to climate-soil factors, highlighting the value of the Yuanmou dry-hot Valley as a microcosm for exploring the intricate interactions between vegetation evolution and changes in environmental factors.

Synthesis and Biological Evaluation of Novel 2-Amino-1,4-Naphthoquinone Amide-Oxime Derivatives as Potent IDO1/STAT3 Dual Inhibitors with Prospective Antitumor Effects.

Indoleamine-2,3-dioxygenase 1 (IDO1) and signal transducer and activator of transcription 3 (STAT3) have emerged as significant targets in the tumor microenvironment for cancer therapy. In this study, we synthesized three novel 2-amino-1,4-naphthoquinone amide-oxime derivatives and identified them as dual inhibitors of IDO1 and STAT3. The representative compound NK3 demonstrated effective binding to IDO1 and exhibited good inhibitory activity (hIDO1 IC50 = 0.06 µM), leading to its selection for further investigation. The direct interactions between compound NK3 and IDO1 and STAT3 proteins were confirmed through surface plasmon resonance analysis. A molecular docking study of compound NK3 revealed key interactions between NK3 and IDO1, with the naphthoquinone-oxime moiety coordinating with the heme iron. In the in vitro anticancer assay, compound NK3 displayed potent antitumor activity against selected cancer cell lines and effectively suppressed nuclear translocation of STAT3. Moreover, in vivo assays conducted on CT26 tumor-bearing Balb/c mice and an athymic HepG2 xenograft model revealed that compound NK3 exhibited potent antitumor activity with low toxicity relative to 1-methyl-L-tryptophan (1-MT) and doxorubicin (DOX). Overall, these findings provided evidence that the dual inhibitors of IDO1 and STAT3 may offer a promising avenue for the development of highly effective drug candidates for cancer therapy.

Discovery of novel sulfonamide chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors.

A series of chromone-oxime derivatives containing piperazine sulfonamide moieties were designed, synthesized and evaluated for their inhibitory activities against IDO1. These compounds displayed moderate to good inhibitory activity against IDO1 with IC50 values in low micromolar range. Among them, compound 10m bound effectively to IDO1 with good inhibitory activities (hIDO1 IC50 = 0.64 µM, HeLa IDO1 IC50 = 1.04 µM) and were selected for further investigation. Surface plasmon resonance analysis confirmed the direct interaction between compound 10m and IDO1 protein. Molecular docking study of the most active compound 10m revealed key interactions between 10m and IDO1 in which the chromone-oxime moiety coordinated to the heme iron and formed several hydrogen bonds with the porphyrin ring of heme and ALA264, consistent with the observation by UV-visible spectra that 10m induced a Soret peak shift from 403 to 421 nm. Moreover, compound 10m exhibited no cytotoxicity at its effective concentration in MTT assay. Consistently, in vivo assays results demonstrated that 10m displayed potent antitumor activity with low toxicity in CT26 tumor-bearing Balb/c mice, in comparison with 1-methyl-l-tryptophan (1-MT) and 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L). In brief, the results suggested that chromone-oxime derivatives containing sulfonamide moieties might serve as IDO1 inhibitors for the development of new antitumor agents.

Rapid Screening of Proanthocyanidins from the Roots of Ephedra sinica Stapf and its Preventative Effects on Dextran-Sulfate-Sodium-Induced Ulcerative Colitis.

Proanthocyanidins (PACs) have been proven to exert antioxidant and anti-inflammatory effects. In this study, ultra-performance liquid chromatography (UPLC) coupled with linear ion trap-Orbitrap (LTQ-Orbitrap) high-resolution mass spectrometry was first employed to systematically screen PACs from the roots of Ephedra sinica Stapf, and its ethyl acetate extract (ERE) was found to contain PAC monomers and A-type dimeric proanthocyanidins, which were tentatively identified through characteristic fragmentation patterns. In vitro, the antioxidant activity of ERE was tested through 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. In addition, ERE could inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. In vivo, the preventative effects on dextran-sulfate-sodium-induced ulcerative colitis in mice was investigated. Mice were administered with ERE for 21 days, and during the last 7 days of the treatment period dextran sulfate sodium (DSS) was used to induce experimental colitis. The results showed that ERE treatment alleviated DSS-induced colitis, which was characterized by decreases in disease activity index (DAI) scores, spleen index and colon levels of TNF-α and IL-6, mitigation in pathological damage and oxidative stress and increases in colon length and IL-10 levels. In conclusion, supplementation of PACs derived from ERE may offer a new strategy for the treatment of ulcerative colitis. Moreover, our research will greatly facilitate better utilization of Ephedra plants.

UPLC-LTQ-Orbitrap-Based Cell Metabolomics and Network Pharmacology Analysis to Reveal the Potential Antiarthritic Effects of Pristimerin: In Vitro, In Silico and In Vivo Study.

Rheumatoid arthritis (RA) is characterized by systemic inflammation and synovial hyperplasia. Pristimerin, a natural triterpenoid isolated from plants belonging to the Celastraceae and Hippocrateaceae families, has been reported to exhibit anti-inflammation and anti-proliferation activities. Our study aims to reveal the antiarthritic effects of pristimerin and explore its potential mechanism using in vitro, in silico, and in vivo methods. In the present study, pristimerin treatment led to a dose-dependent decrease in cell viability and migration in TNF-α stimulated human rheumatoid arthritis fibroblast-like synoviocytes MH7A. Moreover, UPLC-LTQ-Orbitrap-based cell metabolomics analysis demonstrated that phospholipid biosynthesis, fatty acid biosynthesis, glutathione metabolism and amino acid metabolic pathways were involved in TNF-α induced MH7A cells after pristimerin treatment. In addition, the adjuvant-induced arthritis (AIA) rat model was employed, and the results exhibited that pristimerin could effectively relieve arthritis symptoms and histopathological damage as well as reduce serum levels of TNF-α, NO and synovial expressions of p-Akt and p-Erk in AIA rats. Furthermore, network pharmacology analysis was performed to visualize crucial protein targets of pristimerin for RA treatment, which showed that the effects were mediated through the MAPK/Erk1/2, PI3K/Akt pathways and directing binding with TNF-α. Taken together, our study not only offered new insights into the biochemical mechanism of natural compounds for RA treatment, but also provided a strategy that integrated in vitro, in silico and in vivo studies to facilitate screening of new anti-RA drugs.

Metabolomics and molecular docking-directed antiarthritic study of the ethyl acetate extract from Celastrus orbiculatus Thunb.

ETHNOPHARMACOLOGICAL RELEVANCE: Celastrus orbiculatus Thunb., an important folk medicine, has long been used for the treatment of rheumatoid arthritis and its ethyl acetate extract (COE) has been reported to possess anticancer, antiinflammation and antiarthritic effects. However, the therapeutic effect and mechanism of COE treatment in rheumatoid arthritis has been rarely studied especially from the perspective of metabolomics. AIM OF STUDY: To reveal the therapeutic effects of COE on adjuvant-induced arthritis (AIA) rats through histopathological analysis, non-targeted metabolomics, and molecular docking study. MATERIALS AND METHODS: Forty-three Wistar rats were randomly divided into normal group, AIA model group, methotrexate group, and COE groups (80 mg/kg, 160 mg/kg and 320 mg/kg of ethyl acetate extract). Paw swelling and arthritis score were monitored through the experiment. Serum levels of tumor necrosis factor α (TNF-α) and nitric oxide were determined and histopathological evaluation was performed. Furthermore, Ultra-high performance liquid chromatography-linear trap quadrupole-Orbitrap-based metabolomics was employed to characterize metabolic changes of AIA rats after COE treatment and molecular docking was performed to predict the potential phytochemicals of COE against TNF-α. RESULTS: COE at three dosages could significantly relieve paw swelling and reduce arthritis scores of AIA rat. Histopathological analysis revealed remarkable decrease in synovial inflammation and bone erosion after COE treatment, especially at middle and high dosage. Additionally, COE down-regulated serum levels of TNF-α and nitric oxide. Serum metabolomics showed that 22 potential biomarkers for the COE treatment of AIA rats were identified, which were closely related to fatty acid metabolism, glycerophospholipid catabolism, and tryptophan metabolism. The molecular docking models predicted that olean-type triterpenes in COE may contribute most to therapeutic effects of rheumatoid arthritis through targeting TNF-α. CONCLUSIONS: COE could significantly relieve the arthritic symptoms in AIA rats and the ultra-high performance liquid chromatography-mass spectrometry based metabolomics proved to be an efficient method to characterize subtle metabolic changes of AIA rats after COE treatment.

StrandAdvantage test for early-line and advanced-stage treatment decisions in solid tumors.

Comprehensive genetic profiling of tumors using next-generation sequencing (NGS) is gaining acceptance for guiding treatment decisions in cancer care. We designed a cancer profiling test combining both deep sequencing and immunohistochemistry (IHC) of relevant cancer targets to aid therapy choices in both standard-of-care (SOC) and advanced-stage treatments for solid tumors. The SOC report is provided in a short turnaround time for four tumors, namely lung, breast, colon, and melanoma, followed by an investigational report. For other tumor types, an investigational report is provided. The NGS assay reports single-nucleotide variants (SNVs), copy number variations (CNVs), and translocations in 152 cancer-related genes. The tissue-specific IHC tests include routine and less common markers associated with drugs used in SOC settings. We describe the standardization, validation, and clinical utility of the StrandAdvantage test (SA test) using more than 250 solid tumor formalin-fixed paraffin-embedded (FFPE) samples and control cell line samples. The NGS test showed high reproducibility and accuracy of >99%. The test provided relevant clinical information for SOC treatment as well as more information related to investigational options and clinical trials for >95% of advanced-stage patients. In conclusion, the SA test comprising a robust and accurate NGS assay combined with clinically relevant IHC tests can detect somatic changes of clinical significance for strategic cancer management in all the stages.

Nuclear translocation of MEK1 triggers a complex T cell response through the corepressor silencing mediator of retinoid and thyroid hormone receptor.

MEK1 phosphorylates ERK1/2 and regulates T cell generation, differentiation, and function. MEK1 has recently been shown to translocate to the nucleus. Its nuclear function is largely unknown. By studying human CD4 T cells, we demonstrate that a low level of MEK1 is present in the nucleus of CD4 T cells under basal conditions. T cell activation further increases the nuclear translocation of MEK1. MEK1 interacts with the nuclear receptor corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT). MEK1 reduces the nuclear level of SMRT in an activation-dependent manner. MEK1 is recruited to the promoter of c-Fos upon TCR stimulation. Conversely, SMRT is bound to the c-Fos promoter under basal conditions and is removed upon TCR stimulation. We examined the role of SMRT in regulation of T cell function. Small interfering RNA-mediated knockdown of SMRT results in a biphasic effect on cytokine production. The production of the cytokines IL-2, IL-4, IL-10, and IFN-γ increases in the early phase (8 h) and then decreases in the late phase (48 h). The late-phase decrease is associated with inhibition of T cell proliferation. The late-phase inhibition of T cell activation is, in part, mediated by IL-10 that is produced in the early phase and, in part, by ß-catenin signaling. Thus, we have identified a novel nuclear function of MEK1. MEK1 triggers a complex pattern of early T cell activation, followed by a late inhibition through its interaction with SMRT. This biphasic dual effect most likely reflects a homeostatic regulation of T cell function by MEK1.

The role of low-level lactate production in airway inflammation in asthma.

Warburg and coworkers (Warburg O, Posener K, Negelein E. Z Biochem 152: 319, 1924) first reported that cancerous cells switch glucose metabolism from oxidative phosphorylation to aerobic glycolysis, and that this switch is important for their proliferation. Nothing is known about aerobic glycolysis in T cells from asthma. The objective was to study aerobic glycolysis in human asthma and the role of this metabolic pathway in airway hyperreactivity and inflammation in a mouse model of asthma. Human peripheral blood and mouse spleen CD4 T cells were isolated by negative selection. T cell proliferation was measured by thymidine incorporation. Cytokines and serum lactate were measured by ELISA. Mouse airway hyperreactivity to inhaled methacholine was measured by a FlexiVent apparatus. The serum lactate concentration was significantly elevated in clinically stable asthmatic subjects compared with healthy and chronic obstructive pulmonary disease controls, and negatively correlated with forced expiratory volume in 1 s. Proliferating CD4 T cells from human asthma and a mouse model of asthma produced higher amounts of lactate upon stimulation, suggesting a heightened glycolytic activity. Lactate stimulated and inhibited T cell proliferation at low and high concentrations, respectively. Dichloroacetate (DCA), an inhibitor of aerobic glycolysis, inhibited lactate production, proliferation of T cells, and production of IL-5, IL-17, and IFN-γ, but it stimulated production of IL-10 and induction of Foxp3. DCA also inhibited airway inflammation and hyperreactivity in a mouse model of asthma. We conclude that aerobic glycolysis is increased in asthma, which promotes T cell activation. Inhibition of aerobic glycolysis blocks T cell activation and development of asthma.

[Comparison study of HBV-P mutation detection by MALDI-TOF Ms and direct PCR sequencing].

OBJECTIVE: To compare the sensitivities of MALDI-TOF MS and direct PCR sequencing on gene mutations detection of hepatitis B virus. METHODS: 100 serum samples from chronic hepatitis B patients were collected, which consisted of 90 serum samples (study group) from 90 chronic hepatitis B patients received nucleoside analogues (NA) therapy for more than 1 year and HBV DNA titer still higher than 500 copies/ml and 10 serum samples (blank group) from 10 chronic hepatitis B patients never treated with antiviral therapy and HBV-DNA titer higher than 1 x 10(5) copies/ml. 9 known mutations associated with HBV P gene in these samples were detected by MALDI-TOF MS and direct PCR sequencing at the same time, TYPE4.0 software and Sequence Navigator software were used to analyze the results separately. RESULTS: (1) In study group, mutations were detected in 53 samples and the total mutation sites were 86 by MALDI-TOF MS with a positive detection rate of 58.89%, whereas only 19 samples were found with mutations and totally 28 mutation sites were detected by direct PCR sequencing, the positive detection rate was 21.11%. The positive detection rate by MALDI-TOF MS was higher than that by direct PCR sequencing and the difference was statistically significant (P < 0.05). In blank group, no mutations were detected by any method. (2) In study group, when the HBV DNA titers were at 500-1000 copies/ml, 10(3)-10(4) copies/ml and 10(4)-10(5) copies/ml, the positive mutation detection rates by MALDI-TOF MS were 50%, 52.08% and 77.27% respectively, higher than that by direct PCR sequencing, which were only 0%, 8.33% and 45.45%. The difference was still statistically significant (P < 0.05). CONCLUSIONS: MALDI-TOF MS had higher detection sensitivity for known mutation sites as compared to direct PCR sequencing method.

Varicella-zoster virus infection of differentiated human neural stem cells.

Primary varicella-zoster virus (VZV) infection in humans produces varicella (chickenpox), after which the virus becomes latent in ganglionic neurons. Analysis of the physical state of viral nucleic acid and virus gene expression during latency requires postmortem acquisition of fresh human ganglia. To provide an additional way to study the VZV-host relationship in neurons, we developed an in vitro model of infected differentiated human neural stem cells (NSCs). NSCs were induced to differentiate in culture dishes coated with poly-l-lysine and mouse laminin in the presence of fibroblast growth factor 2 (FGF-2), nerve growth factor (NGF), brain-derived neurotropic factor (BDNF), dibutyryl cyclic AMP, and retinoic acid. Immunostaining with neuronal (MAP2a and ß-tubulin), astrocyte (GFAP), and oligodendrocyte (CNPase) markers revealed that differentiated neurons constituted approximately 90% of the cell population. These neurons were maintained in culture for up to 8 weeks. No cytopathic effect (CPE) developed in neurons infected with cell-free VZV (Zostavax vaccine) compared to human fetal lung fibroblasts infected with VZV. Weeks later, VZV DNA virus-specific transcripts (open reading frames [ORFs] 21, 29, 62, and 63) were detected in infected neurons, and dual immunofluorescence staining revealed the presence of VZV IE63 and gE exclusively in healthy-appearing neurons, but not in astrocytes. Neither the tissue culture medium nor a homogenate prepared from VZV-infected neurons produced a CPE in fibroblasts. VZV induced apoptosis in fibroblasts, as shown by activation of caspase 3 and by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, but not in neurons. This model provides a unique in vitro system to study the VZV-neuronal relationship.

IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma.

The T cell-driven airway inflammation in chronic asthma is uninhibited and sustained. We examined the resistance of T cells from asthmatic patients against suppression by TGF-ß, IL-10 and glucocorticoids and explored its signaling mechanism. CD4(+)CD25(-) T cells from allergic asthmatic subjects demonstrated increased TCR-stimulated proliferation as compared with healthy and chronic obstructive pulmonary disease controls. This proliferation was resistant to inhibition by TGF-ß, IL-10, and dexamethasone and to anergy induction. CD4 T cells from asthmatic patients, but not chronic obstructive pulmonary disease, allergic rhinitis, and healthy subjects, showed increased expression of MEK1, heightened phosphorylation of ERK1/2, and increased levels of c-Fos. IL-2 and IL-4 stimulated the expression of MEK1 and c-Fos and induced T cell resistance. The inhibition of MEK1 reversed, whereas induced expression of c-Fos and JunB promoted T cell resistance against TGF-ß- and IL-10-mediated suppression. We have uncovered an IL-2- and IL-4-driven MEK1 induction mechanism that results in heightened ERK1/2 activation in asthmatic T cells and make them resistant to certain inhibitory mechanisms.

Uncoordinated 119 preferentially induces Th2 differentiation and promotes the development of asthma.

The Th2 bias is a hallmark of allergic diseases. In this study, we show that the Th1 versus Th2 balance and the development of allergic asthma are strongly affected by the signaling protein uncoordinated 119 (Unc119). The expression of this adaptor protein is significantly increased in Th2 cells. Unc119 activates the Src family and inhibits the Abl family of tyrosine kinases. The activated Src family kinase Lck stimulates the activity of Itk and the expression of the transcription factor JunB. As a result, Unc119 promotes IL-4 production. Through inhibition of Abl kinases, Unc119 dampens IFN-gamma production. Using adoptive transfer of Unc119-knockdown CD4 T cells, we show a critical role for Unc119 in the development of eosinophilic inflammation of airways, mucus production, and bronchial hyperreactivity in a mouse model. Intriguingly, the expression of the Unc119 protein is enhanced in CD4 T cells from patients with asthma. We speculate that the heightened expression of Unc119 promotes Th2, inhibits Th1 differentiation, and contributes to the pathogenesis of asthma in humans.

Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function.

Our objective was to establish an experimental model of a self-sustained and bistable extracellular signal-regulated kinase 1/2 (ERK1/2) signaling process. A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 4 h. Repeated stimulation leads to sustained activation of ERK1/2 but not Jun N-terminal protein kinase (JNK), p38, or STAT6. The ERK1/2 activation lasts for 3 to 7 days and depends upon a positive-feedback mechanism involving Sprouty 2. Overexpression of Sprouty 2 induces, and its genetic deletion abrogates, ERK1/2 bistability. Sprouty 2 directly activates Fyn kinase, which then induces ERK1/2 activation. A genome-wide microarray analysis shows that the bistable phospho-ERK1/2 (pERK1/2) does not induce a high level of gene transcription. This is due to its nuclear exclusion and compartmentalization to Rab5+ endosomes. Cells with sustained endosomal pERK1/2 manifest resistance against growth factor withdrawal-induced cell death. They are primed for heightened cytokine production. Epithelial cells from cases of human asthma and from a mouse model of chronic asthma manifest increased pERK1/2, which is associated with Rab5+ endosomes. The increase in pERK1/2 was associated with a simultaneous increase in Sprouty 2 expression in these tissues. Thus, we have developed a cellular model of sustained ERK1/2 activation, which may provide a mechanistic understanding of self-sustained biological processes in chronic illnesses such as asthma.

Uncoordinated 119 protein controls trafficking of Lck via the Rab11 endosome and is critical for immunological synapse formation.

The activation of T cells through the TCR is essential for development of the adaptive immune response. TCR does not have any enzymatic activity and relies on the plasma membrane-associated lymphocyte-specific protein tyrosine kinase (Lck) for initiation of signaling. Here we uncover a mechanism that is responsible for plasma membrane targeting of Lck. We show that Lck is transported to the membrane via a specific endosomal compartment. The transport depends on the adaptor protein Uncoordinated 119 (Unc119), on the GTPase rat brain 11 (Rab11), and on the actin cytoskeleton. Unc119 regulates the activation of Rab11. Consequently, Unc119 orchestrates the recruitment of the actin-based motor protein, myosin 5B, and the organization of multiprotein complexes on endosomes. The Unc119-regulated pathway is essential for immunological synapse formation and T cell activation.

Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma.

BACKGROUND: Existing asthma models develop tolerance when chronically exposed to the same allergen. OBJECTIVE: We sought to establish a chronic model that sustains features of asthma long after discontinuation of allergen exposure. METHODS: We immunized and exposed mice to a combination of single, double, or triple allergens (dust mite, ragweed, and Aspergillus species) intranasally for 8 weeks. Airway hyperreactivity (AHR) and morphologic features of asthma were studied 3 weeks after allergen exposure. Signaling effects of the allergens were studied on dendritic cells. RESULTS: Sensitization and repeated exposure to a single allergen induced tolerance. Sensitization to double and especially triple allergens broke through tolerance and established AHR, eosinophilic inflammation, mast cell and smooth muscle hyperplasia, mucus production, and airway remodeling that persisted at least 3 weeks after allergen exposure. Mucosal exposure to triple allergens in the absence of an adjuvant was sufficient to induce chronic airway inflammation. Anti-IL-5 and anti-IL-13 antibodies inhibited inflammation and AHR in the acute asthma model but not in the chronic triple-allergen model. Multiple allergens produce a synergy in p38 mitogen-activated protein kinase signaling and maturation of dendritic cells, which provides heightened T-cell costimulation at a level that cannot be achieved with a single allergen. CONCLUSIONS: Sensitivity to multiple allergens leads to chronic asthma in mice. Multiple allergens synergize in dendritic cell signaling and T-cell stimulation that allows escape from the single allergen-associated tolerance development.

Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic airways.

BACKGROUND: Many airway cells manifest signs of chronic activation in asthma. The mechanism of this chronic activation is unknown. OBJECTIVES: We sought to study the activation of the mitogen-activated protein kinase (MAPK) signaling pathway in airway cells. METHODS: Endobronchial biopsy specimens from patients with severe and mild asthma (n = 17 in each group) and healthy control subjects (n = 15) were analyzed for the phosphorylated MAPKs extracellular signal-regulated kinase (ERK) 1/2, p38, and Jun N-terminal kinase (JNK) and their downstream effectors by means of immunofluorescence staining. Airway epithelial activation of ERK1/2 and p38 was studied by using Western blotting. Epithelial function was studied by means of real-time PCR, ELISA, and the thymidine incorporation assay. RESULTS: We detected strong phospho-ERK1/2 staining in airway epithelium and smooth muscle cells in biopsy specimens from asthmatic patients. Fluorescent areas per image, as well as mean fluorescence intensity, were significantly (P < .0001) different among the 3 study groups (patients with severe asthma, patients with mild asthma, and healthy control subjects). Patients with severe asthma also demonstrated strong phospho-p38 staining, mostly in epithelial cells, which was significantly different from that in patients with mild asthma (P = .0001) and healthy control subjects (P = .02). Phospho-JNK primarily stained airway smooth muscle cells. Healthy subjects showed the highest intensity of phospho-JNK staining compared with that seen in patients with severe (P = .004) and mild asthma (P = .003). Inhibition of ERK1/2 and p38 in primary airway epithelial cells blocked their proliferation and expression of select, but not all, chemokines. CONCLUSIONS: Significant phosphorylation of ERK1/2 and p38 and their correlation with disease severity suggests that the foregoing signaling pathways play an important role in asthma. The ERK1/2 and p38 pathways regulate epithelial cell secretory function and proliferation.

A phosphosite screen identifies autocrine TGF-beta-driven activation of protein kinase R as a survival-limiting factor for eosinophils.

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase Cepsilon, and glycogen synthase kinase 3 alpha and beta. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2alpha are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-beta in eosinophils. TGF-beta directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-beta Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phospho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-beta and that this could be modulated by a Th2 microenvironment in vivo.

MK2 controls the level of negative feedback in the NF-kappaB pathway and is essential for vascular permeability and airway inflammation.

We demonstrate that mitogen-activated protein kinase-activated kinase-2 (MK2) is essential for localized Th2-type inflammation and development of experimental asthma. MK2 deficiency does not affect systemic Th2 immunity, but reduces endothelial permeability, as well as adhesion molecule and chemokine expression. NF-kappaB regulates transcription of adhesion molecules and chemokines. We show that MK2 and its substrate HSP27 are essential for sustained NF-kappaB activation. MK2 and HSP27 prevent nuclear retention of p38 by sequestering it in the cytosol. As a result, MK2 precludes excessive phosphorylation of MSK1. By reducing MSK1 activity, MK2 prevents p65 NF-kappaB hyperphosphorylation and excessive IkappaBalpha transcription. IkappaBalpha mediates nuclear export of p65. By reducing IkappaBalpha level, MK2 prevents premature export of NF-kappaB from the nucleus. Thus, the MK2-HSP27 pathway regulates the NF-kappaB transcriptional output by switching the activation pattern from high level, but short lasting, to moderate-level, but long lasting. This pattern of activation is essential for many NF-kappaB-regulated genes and development of inflammation. Thus, the MK2-HSP27 pathway is an excellent target for therapeutic control of localized inflammatory diseases.