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Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn disease (CD), is a disorder characterized by diffuse inflammation of the gastrointestinal tract. The immune response and inflammation are mediated by polyunsaturated fatty acids and influenced by dietary fats and lipid metabolism. This study examined the qualitative and quantitative fat intake of IBD patients and healthy controls on plasma phospholipid and erythrocyte membrane phospholipid (EMP) fatty acid content. Measurement of the fatty acid composition of plasma phospholipid and EMP were performed in 29 UC patients, 20 CD patients, and 31 healthy controls. Anthropometric characteristics and data on dietary intake were also collected. We observed significantly lower lipid intake in UC and CD patients vs controls. The UC and CD patients had significantly higher levels of linoleic acid in their EMP than did controls. There were no significant differences in the levels of n-3 polyunsaturated fatty acids, but there were significantly higher levels of the n-6 in the EMP of UC and CD patients compared with controls. The significant differences persisted after the data were adjusted for potential confounders and lipid intake. Higher levels of linoleic acids and n-6 fatty acids, which are involved in production of proinflammatory mediators, were found in IBD patients compared with controls, thereby implicating n-6 fatty acids in the pathophysiology of the disease.

The proliferation and differentiation of hematopoietic stem cells (HSCs) is finely regulated by extrinsic and intrinsic factors via various signaling pathways. Here we have shown that, similar to mice deficient in the lipid phosphatase SHIP, loss of 2 Src family kinases, Lyn and Hck, profoundly affects HSC differentiation, producing hematopoietic progenitors with increased proliferation, reduced apoptosis, growth factor-independent survival, and skewed differentiation toward M2 macrophages. This phenotype culminates in a Stat5-dependent myeloproliferative disease that is accompanied by M2 macrophage infiltration of the lung. Expression of a membrane-bound form of SHIP in HSCs lacking both Lyn and Hck restored normal hematopoiesis and prevented myeloproliferation. In vitro and in vivo studies suggested the involvement of autocrine and/or paracrine production of IL-3 and GM-CSF in the increased proliferation and myeloid differentiation of HSCs. Thus, this study has defined a myeloproliferative transformation-sensitive signaling pathway, composed of Lyn/Hck, SHIP, autocrine/paracrine cytokines, and Stat5, that regulates HSC differentiation and M2 macrophage programming.

We examined the intracellular mechanisms for endothelin-1-induced positive and negative inotropic components that coexist in the mouse ventricular myocardium using isolated ventricular tissue and myocytes from 4-week-old mice. In the presence of SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger, endothelin-1 produced positive inotropy. Endothelin-1, when applied to cardiomyocytes in the presence of SEA0400, did not change the peak amplitude of the Ca2+ transient but increased intracellular pH and Ca2+ sensitivity of contractile proteins. On the other hand, in the presence of dimethylamiloride (DMA), a specific inhibitor of the Na+-H+ exchanger, endothelin-1 produced negative inotropy. In cardiomyocytes, in the presence of DMA, endothelin-1 produced a decrease in peak amplitude of the Ca2+ transient. In the presence of both DMA and SEA0400, endothelin-1 produced neither positive nor negative inotropy. Positive inotropy was blocked by BQ-123 and negative inotropy by BQ-788. These results suggested that endothelin-1-induced positive inotropy is mediated by ET(A) receptors, activation of the Na+-H+ exchanger and an increase in intracellular pH and Ca2+ sensitivity and that the negative inotropy is mediated by ET(B) receptors, activation of the Na+-Ca2+ exchanger and decrease in Ca2+ transient amplitude.

Background: Atopic dermatitis is a chronic or chronically relapsing, pruritic inflammatory skin disease. The incidence of atopic dermatitis has dramatically increased during the past three decades in industrialized countries. We attempted to develop an improved method to induce an animal model of atopic dermatitis and to use it to evaluate the efficacy of a Tec family kinase inhibitor., Methods: We treated dermatitis-prone inbred mice, NC/Nga, by repetitive epicutaneous applications of a house dust mite allergen and staphylococcal enterotoxin B to induce atopic dermatitis-like skin lesions., Results: We established a highly efficient protocol to induce skin lesions in NC/Nga mice, which were histologically and immunologically similar to human atopic dermatitis. Similar to human patients, serum IgE levels were increased in dermatitis-induced mice. Consistent with the proposed roles of infiltrated immune cells in the pathogenesis of human atopic dermatitis, skin lesions were treatable with terreic acid, an inhibitor of Tec family kinases, as well as dexamethasone., Conclusions: We established a highly efficient, highly reproducible protocol to induce skin lesions in NC/Nga mice and successfully applied it to show the efficacy of terreic acid in treating skin lesions. This mouse model of atopic dermatitis will be useful to study the pathogenetic processes of atopic dermatitis and to evaluate the efficacy of drug candidates.

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcepsilonRI). Activated Lyn phosphorylates the FcepsilonRI subunits, beta and gamma, leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcepsilonRI-stimulated hck(-/-) mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck(-/-) cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory events, such as activation of Syk, Btk, JNK, p38, Akt, and NF-kappaB, are substantially reduced in hck(-/-) mast cells. Analysis of lyn(-/-)hck(-/-), lyn(-/-)FcepsilonRIbeta(-/-), and hck(-/-)FcepsilonRIbeta(-/-) cells shows that Hck exerts these functions via both Lyn-dependent and Lyn-independent mechanisms. Thus, this study has revealed a hierarchical regulation among SFK members to fine-tune mast cell activation.

Btk plays crucial roles in the differentiation and activation of B and myeloid cells. Despite drastic reductions of other Ig isotypes, paradoxically high IgE responses have been known in btk mutant mice. Here we show that btk(-/-) dendritic cells exhibit a more mature phenotype and a stronger in vitro and in vivo T cell-stimulatory ability than wild-type cells. Increased IgE responses were induced by adoptive transfer of btk(-/-) dendritic cells into mice. Consistent with the stronger T cell-stimulatory ability of btk(-/-) dendritic cells, btk(-/-) mice exhibited enhanced inflammation in Th2-driven asthma and Th1-driven contact sensitivity experiments. These negative regulatory functions of Btk in dendritic cells appear to be mediated mainly through autocrine secretion of IL-10 and subsequent activation of Stat3.

Mast cells are the crucial effector cells for allergic reactions. They are activated through the aggregation of the high-affinity immunoglobulin E (IgE) receptor (Fc epsilonRI) with allergen and allergen-specific IgE. Tyrosine phosphorylation of Fc epsilonRI subunits and various signaling proteins is an initial triggering event, leading to the activation of several signaling pathways in mast cells. Much has been learned from analysis of mast cells derived from gene-targeted mice. Therefore, in this chapter we will first describe how to generate mast cells from mouse bone marrow cells and how to correct the genetic defect by retroviral transduction. Then, we will describe how to assess early activation events by measuring several protein-tyrosine kinases and serine/threonine kinases such as protein kinase B (= Akt), protein kinase C, and JNK. As signal transduction is highly dependent on protein-protein interactions, we will describe experimental details of co-immunoprecipitation methods that are used to confirm such interactions.

Mast cells are the major effector-cell type for immediate hypersensitivity and other forms of allergic reactions. Expression of 4-1BB, a member of the tumor necrosis factor receptor superfamily, is induced at mRNA and protein levels on stimulation through the high-affinity receptor for immunoglobulin E (IgE; FcepsilonRI). In this study, we present evidence that agonistic anti-4-1BB antibodies can enhance FcepsilonRI-induced cytokine production and secretion. Consistent with this, 4-1BB-deficient mast cells exhibit reduced degranulation and cytokine production on FcepsilonRI stimulation. Analysis of 4-1BB ligand (4-1BBL)-deficient cells supported this notion. As a potential mechanism for these defects, we identified a defect in Ca2+ flux induced by FcepsilonRI stimulation. The defective Ca2+ flux could be accounted for by the reduced activity of Lyn/Btk/phospholipase C-gamma2 pathway and constitutive interactions between 4-1BB and Lyn. Therefore, FcepsilonRI-inducible 4-1BB plays a costimulatory function together with FcepsilonRI stimulation.

Mast cells are the major effector cells for immediate hypersensitivity and chronic allergic reactions. These cells accumulate in mucosal tissues of allergic reactions, where immunoglobulin E (IgE) is produced locally. Here we provide evidence that, in addition to antigen that can attract IgE-bound mast cells, the type of IgE molecules that efficiently activate mast cells can promote the migration of mast cells in the absence of antigen. IgE- and IgE+Ag-mediated migration involves an autocrine/paracrine secretion of soluble factors including adenosine, leukotriene B4, and several chemokines. Their secretion depends on 2 tyrosine kinases, Lyn and Syk, and they are agonists of G-protein-coupled receptors and signal through phosphatidylinositol 3-kinase gamma, leading to mast cell migration. In mouse experiments, naive mast cells are attracted to IgE, and IgE-sensitized mast cells are attracted to antigen. Therefore, IgE and antigen are implicated in mast cell accumulation at allergic tissue sites with local high IgE levels.

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcepsilonRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcepsilonRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcepsilonRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcepsilonRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.

Background/aims: The aims of the present study were to determine the prevalence of patients with cryptogenic liver cirrhosis, and to clarify the clinical characteristics of these patients, with special focus on obesity and type 2 diabetes mellitus., Methodology: For the case-control study, we selected age- and sex-matched control patients with cirrhosis of known etiology., Results: Of 404 patients with liver cirrhosis, 40 (9.9%) were diagnosed as having cryptogenic cirrhosis. Of these 40 patients, 29 (73%) were female. The mean age was 66.1 +/- 8.9 (range 40-87 years old), and was significantly higher than that of either virus, or alcohol-related liver cirrhosis patients. Both obesity and type 2 diabetes mellitus were more common in patients with cryptogenic cirrhosis (53% and 40%, respectively) compared with the case-controls (20%, 18%, respectively)., Conclusions: Cryptogenic liver cirrhosis accounted for approximately 10% of liver cirrhosis, and was associated with both obesity and type 2 diabetes mellitus. Nonalcoholic steatohepatitis sequelae may be the most important etiological factor in patients with cryptogenic liver cirrhosis in Okinawa, Japan, where virus-etiology is more predominant in liver cirrhosis compared with Western countries.

We demonstrate that binding of different IgE molecules (IgEs) to their receptor, FcepsilonRI, induces a spectrum of activation events in the absence of a specific antigen and provide evidence that such activation reflects aggregation of FcepsilonRI. Highly cytokinergic IgEs can efficiently induce production of cytokines and render mast cells resistant to apoptosis in an autocrine fashion, whereas poorly cytokinergic IgEs induce these effects inefficiently. Highly cytokinergic IgEs seem to induce more extensive FcepsilonRI aggregation than do poorly cytokinergic IgEs, which leads to stronger mast cell activation and survival effects. These effects of both types of IgEs require Syk tyrosine kinase and can be inhibited by FcepsilonRI disaggregation with monovalent hapten. In hybridoma-transplanted mice, mucosal mast cell numbers correlate with serum IgE levels. Therefore, survival effects of IgE could contribute to the pathogenesis of allergic disease.

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcepsilonRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk in the membrane compartment of FcepsilonRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcepsilonRI-stimulated mast cells.

Background/aims: The aims of this study were to determine the distribution of serum alanine aminotransferase levels in a normal population and to clarify whether interferon treatment is justified in HCV-infected patients with persistently normal alanine aminotransferase levels., Methodology: The distribution of alanine aminotransferase levels was examined among 949 normal subjects who were negative for hepatitis viruses, denied regular alcohol use. Nineteen patients with chronic hepatitis C and persistently normal alanine aminotransferase levels were treated with alpha interferon (six or ten million units thrice weekly for six months)., Results: Peaks of alanine aminotransferase distribution among the normal subjects were seen at 16-20 IU/L and 11-15 IU/L in males and females, respectively. Fourteen of the 19 patients who received interferon treatment had favorable factors of response to interferon (eight with low pretreatment virus load, four with HCV genotype 2 and two with both). A sustained virological response was achieved in eight (57%) of 14, and alanine aminotransferase levels decreased significantly to within the normal range after interferon treatment in six of eight., Conclusions: Patients with chronic hepatitis C and persistently normal alanine aminotransferase levels should be treated with high doses of interferon if they have favorable factors of response to interferon treatment.

NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.