Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice.

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2-/- mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3-/- mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2-/- and TLR3-/- mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2-/- and TLR3-/- mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2-/-, and TLR3-/- mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

MiRNA Regulation of MIF in SLE and Attenuation of Murine Lupus Nephritis With miR-654.

Objective: Macrophage Migration Inhibitory Factor (MIF) is involved in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). MicroRNAs (miRNAs) play important roles in LN but whether specific miRNAs regulate the expression of MIF in LN is unknown. We explore specific miRNAs that can regulate MIF expression, and investigate miR-654 for the treatment of experimentally-induced murine lupus nephritis. Methods: Sera samples from 24 SLE patients and 24 controls were collected to measure the MIF concentration and its correlation with disease activity. A luciferase reporter assay was used to explore the target of miR-654. ELISA was used to detect the downstream cytokines regulated by miR-654 and MIF. Western blot was applied to measure the impact of miR-654 inhibition on downstream MIF signaling. The therapeutic efficacy of miR-654 was tested in the pristine-induced lupus mouse model. We further measured miR-654 expression and analyzed its relationship with MIF expression in SLE patients. Results: The serum MIF level was increased in SLE patients (p < 0.001) and positively correlated with the SLEDAI score (r = 0.5473; p = 0.0056). MiR-654 inhibited MIF and downstream inflammatory cytokine production by selectively inhibiting the phosphorylation of ERK and AKT. Activation of miR-654 reduced IL-1ß, IL-6, IL-8, and TNF-α production, reduced gomerulonephritis, and decreased MIF, IgG, and C3 expression in murine lupus glomeruli. Furthermore, MIF was negatively correlated with miR-654 expression (r = -0.4644; p = 0.0222) in SLE patients. Conclusion: MiR-654 negatively correlated with MIF and disease activity in patients with SLE. MiR-654 inhibits MIF expression via binding to MIF 3'UTR, selectively suppresses the phosphorylation of ERK and AKT, and reduces downstream inflammatory cytokine production. In vivo miR-654 treatment decreases MIF and downstream cytokine production and ameliorates murine lupus nephritis.

Targeting the PI3K pathway in cancer: are we making headway?

The PI3K-AKT-mTOR pathway is one of the most frequently dysregulated pathways in cancer and, consequently, more than 40 compounds that target key components of this signalling network have been tested in clinical trials involving patients with a range of different cancers. The clinical development of many of these agents, however, has not advanced to late-phase randomized trials, and the antitumour activity of those that have been evaluated in comparative prospective studies has typically been limited, or toxicities were found to be prohibitive. Nevertheless, the mTOR inhibitors temsirolimus and everolimus and the PI3K inhibitors idelalisib and copanlisib have been approved by the FDA for clinical use in the treatment of a number of different cancers. Novel compounds with greater potency and selectivity, as well as improved therapeutic indices owing to reduced risks of toxicity, are clearly required. In addition, biomarkers that are predictive of a response, such as PIK3CA mutations for inhibitors of the PI3K catalytic subunit α isoform, must be identified and analytically and clinically validated. Finally, considering that oncogenic activation of the PI3K-AKT-mTOR pathway often occurs alongside pro-tumorigenic aberrations in other signalling networks, rational combinations are also needed to optimize the effectiveness of treatment. Herein, we review the current experience with anticancer therapies that target the PI3K-AKT-mTOR pathway.

Epigallocatechin-3-gallate attenuates acute and chronic psoriatic itch in mice: Involvement of antioxidant, anti-inflammatory effects and suppression of ERK and Akt signaling pathways.

Chronic itch is a distressing symptom of many skin diseases and negatively impacts quality of life. However, there is no medication for most forms of chronic itch, although antihistamines are often used for anti-itch treatment. Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, exhibits anti-oxidative and anti-inflammatory properties. Our previous studies highlighted a key role of oxidative stress and proinflammatory cytokines in acute and chronic itch. Here, we evaluated the effects of green tea polyphenon 60 and EGCG on acute and chronic itch in mouse models and explored its potential mechanisms. The effects of EGCG were determined by behavioral tests in mouse models of acute and chronic itch, which were induced by compound 48/80, chloroquine (CQ), and 5% imiquimod cream treatment, respectively. We found that systemic or local administration of green tea polyphenon 60 or EGCG significantly alleviated compound 48/80- and chloroquine-induced acute itch in a dose-dependent manner in mice. Incubation of EGCG significantly decreased the accumulation of intracellular reactive oxygen species (ROS) directly induced by compound 48/80 and CQ in cultured ND7-23 cells, a dorsal root ganglia derived cell line. EGCG also attenuated imiquimod-induced chronic psoriatic itch behaviors and skin epidermal hyperplasia in mice. In addition, EGCG inhibited the expression of IL-23 mRNA in skin and TRPV1 mRNA in dorsal root ganglia (DRG). Finally, EGCG remarkably inhibited compound 48/80-induced phosphorylation of extracellular signal-regulated kinase (ERK) and imiquimod-induced p-AKT in the spinal cord of mice, respectively. Collectively, these results indicated EGCG could be a promising strategy for anti-itch therapy.

Umbilical cord-derived mesenchymal stem cells alleviated inflammation and inhibited apoptosis in interstitial cystitis via AKT/mTOR signaling pathway.

Interstitial cystitis (IC) is a bladder syndrome characterized by pelvic pain and urinary frequency without infection or other identifiable pathology. There are no effective treatments to cure IC. This study investigated the effects of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) injection on IC rat model. Furthermore, we used a coculture system to find the possible molecular mechanism on the human uroepithelial cells (SV-HUC-1), which was the cell model of IC. A rat model of IC was established via systemic injection with cyclophosphamide (CYP) and a cell model of IC was induced by being exposed to tumor necrosis factor (TNF)-α (10 ng/ml). After one week, UC-MSCs injection significantly ameliorated the bladder voiding function in IC rat model. And the Histo- and immunohistochemical analyses showed that UC-MSCs can repair impaired bladder, reduce mast cell infiltration and inhibit apoptosis of urothelium. ELISA results showed that UC-MSCs can decrease IL-1ß, IL-6 and TNF-α in bladder. In the coculture system, UC-MSCs can promote proliferation of impaired SV-HUC-1 cells, and inhibit apoptosis. However, while knocked down EGF secreted by UC-MSCs with siRNA, the effects would be weaken. Western blot showed that UC-MSCs increase protein expression levels of p-AKT and p-mTOR in SV-HUC-1 cells, and decrease the levels of cleaved caspase-3. Taken together, we provide evidence that UC-MSCs therapy can successfully alleviate IC in a preclinical animal Model and cell model by alleviating inflammation, promoting proliferation and inhibiting apoptosis. In addition, we demonstrate that the AKT/mTOR signaling pathway was activated.

Mechanisms of the action of adenine on anti-allergic effects in mast cells.

INTRODUCTION: Mast cells play an important role in allergic responses. METHODS: We herein demonstrated the mechanisms of inhibitory effect of adenine on IgE/antigen-induced degranulation and TNF-α release in mast cells. RESULTS: We found that these effects were dependent on the amino group of adenine because purine only weakly inhibited degranulation. Adenine also inhibited Ca2+ ionophore- and thapsigargin-induced degranulation, however, this inhibitory effect was weaker than that of the antigen. Therefore, the inhibitory effects of adenine on degranulation may be mediated before as well as after the Ca2+ raise under the antigen stimulus. Adenine inhibited antigen-induced Syk and the subsequent induction of AKT and ERK activation under FcϵRI-mediated signal. Adenine also attenuated antigen-induced increase in Ca2+ . Furthermore, adenine inhibited IgE/antigen-induced IKKα/ß activation, which is involved in degranulation. Finally, adenine protected mice against anaphylactic allergic responses in vivo. CONCLUSIONS: The present study revealed a key role of adenine in the attenuation of allergic responses through the inhibition of Syk-mediated signal transduction and IKK-mediated degranulation.

Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway.

Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

Inhibition of Toll-like receptor 9 attenuates sepsis-induced mortality through suppressing excessive inflammatory response.

Sepsis, a major clinical problem with high morbidity and mortality, is caused by overwhelming systemic host-inflammatory response. Toll-like receptors (TLRs) play a fundamental role in induction of hyperinflammation and tissue damage in sepsis. In this study, we demonstrate a protective role of TLR9 inhibition against the dysregulated inflammatory response and tissue injury in sepsis. TLR9 deficiency decreased the mortality of mice following cecal ligation and puncture (CLP)-induced sepsis. TLR9 knockout mice showed dampened p38 activation and augmented Akt phosphorylation in the spleen, lung and liver. In addition, TLR9 deficiency decreased the levels of inflammatory cytokines and attenuated splenic apoptosis after CLP. These results indicate that TLR9 inhibition might offer a novel therapeutic strategy for the management of sepsis.

Cutting Edge: Differential Regulation of PTEN by TCR, Akt, and FoxO1 Controls CD4+ T Cell Fate Decisions.

Signaling via the Akt/mammalian target of rapamycin pathway influences CD4(+) T cell differentiation; low levels favor regulatory T cell induction and high levels favor Th induction. Although the lipid phosphatase phosphatase and tensin homolog (PTEN) suppresses Akt activity, the control of PTEN activity is poorly studied in T cells. In this study, we identify multiple mechanisms that regulate PTEN expression. During Th induction, PTEN function is suppressed via lower mRNA levels, lower protein levels, and an increase in C-terminal phosphorylation. Conversely, during regulatory T cell induction, PTEN function is maintained through the stabilization of PTEN mRNA transcription and sustained protein levels. We demonstrate that differential Akt/mammalian target of rapamycin signaling regulates PTEN transcription via the FoxO1 transcription factor. A mathematical model that includes multiple modes of PTEN regulation recapitulates our experimental findings and demonstrates how several feedback loops determine differentiation outcomes. Collectively, this work provides novel mechanistic insights into how differential regulation of PTEN controls alternate CD4(+) T cell fate outcomes.

The caffeic acid in aqueous extract of Tournefortia sarmentosa enhances neutrophil phagocytosis of Escherichia coli.

Tournefortia sarmentosa, a Chinese herbal medicine, is considered an antioxidant or a detoxicating agent. Recently T. sarmentosa has received attention for its effects on the immune response. Here we provide evidence that aqueous extract of T. sarmentosa can induce increased phagocytic uptake of Escherichia coli by differentiated HL-60 cells and by neutrophils. Our results also revealed that T. sarmentosa can inhibit E. coli survival within differentiated HL-60 cells. Furthermore, aqueous extract of T. sarmentosa has been shown to enhance cell surface Mac-1 expression and the activated AKT signaling pathway in E. coli-stimulated neutrophils. We also examined the effect of each constituents in aqueous extract of T. sarmentosa on phagocytic uptake of E. coli by differentiated HL-60 cells or neutrophils. Bacterial survival, cell surface Mac-1 expression, and AKT activation of neutrophils were also examined. Our results showed that caffeic acid is an important constituent in mediating aqueous extract of T. sarmentosa-induced phagocytic uptake. Taken together, these results suggest that aqueous extract of T. sarmentosa exerts effects that enhance inflammatory responses by improving phagocytic capability, inhibiting bacterial survival within cells, and increasing Mac-1 expression of neutrophils.

FOXO1 transcription factor: a critical effector of the PI3K-AKT axis in B-cell development.

B-cell development and differentiation are controlled at multiple levels by the complex interplay of specific receptors and a variety of transcription factors. Several receptors involved in regulating this process, such as IL-7R, pre-B cell receptor (pre-BCR), and BCR, share the ability to trigger the signaling via the phosphoinositide 3-kinase (PI3K)-AKT pathway. FOXO1 transcription factor, a major PI3K-AKT downstream effector, regulates the expression of genes critical for progress through consecutive steps of B-cell differentiation. FOXO1 directs or fine-tunes multiple biological functions that are crucial for differentiating cells, including the cell cycle, apoptosis, oxidative stress response or DNA damage repair. Recent studies have highlighted the key role that FOXO1 plays in the maintenance of the hematopoietic stem cell pool, regulation of progenitor commitment, development of early B-cell precursors, induction of B-cell tolerance, peripheral B-cell homeostasis, and terminal differentiation. FOXO1 deficiency impairs B-cell development, due to decreased expression of its critical target genes, that include early B-cell factor (EBF1), IL-7 receptor, recombination activating genes (RAG1 and 2), activation-induced cytidine deaminase (AID), L-selectin, and BLNK. Taken together, FOXO1 is an important node in a dynamic network of transcription factors that orchestrate B-cell differentiation and specialization. Herein, we review molecular mechanisms of the PI3K-AKT-dependent signal transduction and their impact on early B-cell development, peripheral B-cell homeostasis, and terminal differentiation.

[Activation of Kupffer cell and related signal pathway proteins in the liver of high fat and high fructose diet induced NAFLD mice].

OBJECTIVE: To investigate the expression of F4/80, NF-kappaB, p-AKT, AKT in the liver of nonalcoholic fatty liver disease (NAFLD) mice. To determine the role of Kupffer cells (KCs) in the development of NASH (non-alcoholic steatohepatitis), and understand the pathogenic mechanism of NASH. METHODS: Five C3H/HeN mice fed with normal diet were served as controls, while fifteen fed with high fat, high fructose, high fat combined fructose diet respectively for 16 weeks were as NAFLD mice models. The liver inflammation and hepatic damage were examined, and the expression of F4/80, NF-Kb, p-AKT, AKT and the content of lipid in the liver were also detected. RESULTS: Chronic intake of high fat and 30% fructose solution caused a significant increase in hepatic steatosis in animals in comparison to water controls. Liver F4/80 and NF-kappaB were significantly higher in high fat and high fat combined fructose diet fed mice than that in controls (P < 0.01, P < 0.01), F4/80 protein were higher in high fat diet treated mice than those in fructose and high fat combined fructose groups (P < 0.01, P < 0.01). Markers of insulin resistance (e. g, hepatic phospho-AKT, AKT) were only altered in fructose-fed or high fat combined fructose animals (P < 0.01, P < 0.01). CONCLUSION: High fat and fructose diet may induce NAFLD in C3H/HeN mice. Kupffer cells and signal pathway proteins were activated, and they may play key roles in the initiation and progression of NASH.

Inhibitory effects of ZSTK474, a phosphatidylinositol 3-kinase inhibitor, on adjuvant-induced arthritis in rats.

OBJECTIVE: We examined the effects of ZSTK474, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on adjuvant-induced arthritis (AIA). METHODS: AIA was induced in Lewis rats by subcutaneous administration of Freund's complete adjuvant at the base of the tail on day 0. ZSTK474 was orally administered once daily from day 10. The severity of AIA was assessed by measuring the hind paw volume. The number of lymphocytes in inguinal lymph nodes (ILN) was determined by flow cytometry. The in vitro effects of ZSTK474 on the cell proliferation, and the cytokines and prostaglandin E(2) (PGE(2)) production were evaluated by BrdU method, ELISA and cytometric beads array. RESULTS: ZSTK474 ameliorated the progression of AIA. The temporary increases in the number of T cells in ILN, which occurred along with the appearance of arthritis, were inhibited in the ZSTK474-treated groups. In vitro studies revealed that ZSTK474 inhibited the production of IFNγ and IL-17 in concanavalin A-activated T cells. In vitro studies further revealed that ZSTK474 inhibited the proliferation and PGE(2) production by fibroblast-like synovial cells (FLS). CONCLUSION: ZSTK474 demonstrated prophylactic efficacy in a rat model of rheumatoid arthritis (RA) through inhibition of T cell and FLS functions. It was suggested that the inhibitors of PI3K have therapeutic potential for RA.

ESAT-6 induced COX-2 expression involves coordinated interplay between PI3K and MAPK signaling.

Macrophages, as sentinels of robust host immunity, are key regulators of innate immune responses against invading mycobacteria; however, pathogenic mycobacteria survive in the infected host by subverting host innate immunity. Infection dependent expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. As a part of multifaceted immunity to mycobacterial infection, induced expression of cyclooxygenase-2 (COX-2) may act as an important influencing factor towards effective host immunity. In the current investigation, we demonstrate that ESAT-6 triggers COX-2 expression both in vitro and in vivo in a TLR2 dependent manner. Signaling perturbation data suggest that signaling dynamics of PI3K and p38 and JNK1/2 MAPK assume critical importance in ESAT-6 triggered expression of COX-2 in macrophages. Interestingly, ESAT-6 triggered PI3K-MAPK signaling axis holds the capacity to regulate coordinated activation of NF-κB and AP-1. Overall, current investigation provides mechanistic insights into ESAT-6 induced COX-2 expression and unravels TLR2 mediated interplay of PI3K and MAPK signaling axis as a rate-determining step during intricate host immune responses. These findings would serve as a paradigm to understand pathogenesis of mycobacterial infection and clearly pave a way towards development of novel therapeutics.

IL-17A stimulates the production of inflammatory mediators via Erk1/2, p38 MAPK, PI3K/Akt, and NF-κB pathways in ARPE-19 cells.

PURPOSE: To investigate the signaling pathways involved in interleukin (IL)-17A -mediated production of interleukin 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and interleukin 6 (IL-6) by ARPE-19 cells, a spontaneously arisen cell line of retinal pigment epithelium (RPE). METHODS: Flow cytometry analysis and western blot were used to detect the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), p38 mitogen activated protein kinase (MAPK) and protein kinase B (PKB; Akt) in ARPE-19 cells stimulated with IL-17A. These cells were further pretreated with a series of kinase inhibitors and followed by incubation with IL-17A. CXCL8, CCL2, and IL-6 in the supernatant were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Coculture of ARPE-19 cells with IL-17A resulted in significant increases in Erk1/2, p38 MAPK, and Akt phosphorylation. Inhibition of p38MAPK, phosphoinositide 3-kinase (PI3K)-Akt and nuclear factor-kappaB (NF-κB), with the inhibitors SB203580, LY294002 and pyrrolydine dithiocarbamate (PDTC) respectively, reduced IL-17 (100 ng/ml) mediated production of CXCL8, CCL2, and IL-6 in a concentration dependent manner. Inhibition of Erk1/2 with PD98059 decreased the expression of the tested three inflammatory mediators when using low doses of IL-17A (0-10 ng/ml) but not at higher concentrations. CONCLUSIONS: IL-17A-induced production of inflammatory mediators by ARPE-19 cells involves Erk1/2, p38MAPK, PI3K-Akt and NF-κB pathways.

Elevated BCR signaling and decreased survival of Lyn-deficient transitional and follicular B cells.

The Src-family tyrosine kinase Lyn negatively regulates BCR signaling and also myeloid cell activity. Mice deficient in Lyn have substantially decreased numbers of peripheral B cells, despite spontaneously producing IgG anti-DNA antibodies. Here, we examine the mechanism underlying the B-cell depletion in these mice. Lyn-deficient B cells were out-competed by WT B cells in mixed BM chimeras at two steps, at the T1 to T2 transitional maturation stage in the spleen and again between the T2 or T3 stage and the mature follicular B-cell population. Lyn-deficient T2 and follicular B cells expressed elevated levels of the pro-apoptotic factor Bim and deletion of Bim restored splenic B cells of Lyn-deficient mice to close to WT numbers. Lyn-deficient T2 and later stage B cells also had changes in cell surface phenotype consistent with increased in vivo BCR signaling. Similarly, an increased proportion of T2 and follicular B cells had elevated basal intracellular free calcium levels. Overall, these observations suggest that increased BCR signaling is responsible for increased death of weakly self-reactive Lyn-deficient B cells both at the T2 stage and additionally as these cells mature to follicular B cells.

Aberrantly expressed CEACAM6 is involved in the signaling leading to apoptosis of acute lymphoblastic leukemia cells.

OBJECTIVE: The aberrant expression of myeloid antigens on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon. So far, there have been no reports of a functional consequence of this aberrant expression. The granulocytic marker carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c) is a GPI-anchored molecule that is reported to be the most frequently aberrantly expressed myeloid marker in ALL with a strong correlation with genotype. MATERIALS AND METHODS: We mimicked CEACAM6 signaling in ALL cells by cross-linking with anti-CEACAM6 antibody. Next, we measured a response to CEACAM6 signaling by integrin subunits expression, integrin ligand binding, phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt, and p38 mitogen-activated protein kinase (MAPK) and apoptosis by flow cytometry. RESULTS: Following CEACAM6 cross-linking in ALL cells, we detected Erk1/2, Akt, and p38 MAPK phosphorylation and integrin upregulation, as well as enhanced binding of integrin ligands (vascular cell adhesion molecule-1 [VCAM-1] and intercellular cell adhesion molecule-1 [ICAM-1]). However, CEACAM6 signaling resulted in an increase in apoptosis, unlike other GPI-anchored molecules, such as CD24. CONCLUSION: The present study is the first to demonstrate the functional consequences of CEACAM6 cross-linking in B-cell precursor ALL cells.

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Divergent adaptive and innate immunological responses are observed in humans following blunt trauma.

BACKGROUND: The immune response to trauma has traditionally been modeled to consist of the systemic inflammatory response syndrome (SIRS) followed by the compensatory anti-inflammatory response syndrome (CARS). We investigated these responses in a homogenous cohort of male, severe blunt trauma patients admitted to a University Hospital surgical intensive care unit (SICU). After obtaining consent, peripheral blood was drawn up to 96 hours following injury. The enumeration and functionality of both myeloid and lymphocyte cell populations were determined. RESULTS: Neutrophil numbers were observed to be elevated in trauma patients as compared to healthy controls. Further, neutrophils isolated from trauma patients had increased raft formation and phospho-Akt. Consistent with this, the neutrophils had increased oxidative burst compared to healthy controls. In direct contrast, blood from trauma patients contained decreased naïve T cell numbers. Upon activation with a T cell specific mitogen, trauma patient T cells produced less IFN-gamma as compared to those from healthy controls. Consistent with these results, upon activation, trauma patient T cells were observed to have decreased T cell receptor mediated signaling. CONCLUSIONS: These results suggest that following trauma, there are concurrent and divergent immunological responses. These consist of a hyper-inflammatory response by the innate arm of the immune system concurrent with a hypo-inflammatory response by the adaptive arm.

Signaling pathways induced by a tumor-derived vaccine in antigen presenting cells.

We have previously reported on the anti-tumoral potential of a chaperone-rich cell lysate (CRCL) vaccine. Immunization with CRCL generated from tumors elicits specific T and NK cell-dependent immune responses leading to protective immunity in numerous mouse tumor models. CRCL provides both a source of tumor antigens and danger signals leading to dendritic cell activation. In humans, tumor-derived CRCL induces dendritic cell activation and CRCL-loaded dendritic cells promote the generation of cytotoxic T lymphocytes in vitro. The current study was designed to identify the signaling events and modifications triggered by CRCL in antigen presenting cells. Our results indicate that tumor-derived CRCL not only promotes the activation of dendritic cells, but also significantly fosters the function of macrophages that thus appear as major targets of this vaccine. Activation of both cell types is associated with the induction of the MAP kinase pathway, the phosphorylation of STAT1, STAT5 and AKT and with transcription factor NF-kappaB activation in vitro and in vivo. These results thus provide important insights into the mechanisms by which CRCL-based vaccines exert their adjuvant effects on antigen presenting cells.