IRAK4 kinase activity is required for Th17 differentiation and Th17-mediated disease.

Both IL-23- and IL-1-mediated signaling pathways play important roles in Th17 cell differentiation, cytokine production, and autoimmune diseases. The IL-1R-associated kinase 4 (IRAK4) is critical for IL-1/TLR signaling. We show here that inactivation of IRAK4 kinase in mice (IRAK4 KI) results in significant resistance to experimental autoimmune encephalomyelitis due to a reduction in infiltrating inflammatory cells into the CNS and reduced Ag-specific CD4(+) T cell-mediated IL-17 production. Adoptive transfer of myelin oligodendrocyte glycoprotein 35-55-specific IRAK4 KI Th17 cells failed to induce experimental autoimmune encephalomyelitis in either wild-type or IRAK4 KI recipient mice, indicating the lack of autoantigen-specific Th17 cell activities in the absence of IRAK4 kinase activity. Furthermore, the absence of IRAK4 kinase activity blocked induction of IL-23R expression, STAT3 activation by IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease.

Study of plasma protein C and inflammatory pathways: biomarkers for dimethylnitrosamine-induced liver fibrosis in rats.

The present investigation was designed to identify potential biomarker(s) and assess the involvement of inflammatory pathway in dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Following DMN-treatment (10 mg/ml/kg, i.p., given three consecutive days each week for 4 weeks) body and liver weights were significantly decreased concurrent with increasing severity of liver damage assessed by bridging fibrosis, a histopathologic assessment and characteristic of human liver disease. Protein C along with albumin, C-reactive-protein (CRP), haptoglobin and total protein were significantly reduced and correlated with changes in liver histopathology. Biochemical markers of liver functions were significantly increased and correlated with changes in liver histopathology and plasma levels of protein C. Soluble intracellular-adhesion-molecule-1 (sICAM-1) levels were increased significantly but were poorly correlated with histopathology and protein C levels. Inflammatory chemokines and other analytes, monocyte-chemoattractant-protein-1 and 3 (MCP-1 and MCP-3), macrophage-colony-stimulating-factor (M-CSF) were significantly increased during the disease progression, whereas macrophage-derived-chemokine (MDC) and CRP were significantly suppressed. Circulating neutrophils and monocytes were also increased along with disease progression. The differential changes in sICAM-1, hyaluronic acid, gamma-glutamyltranspeptidase (GGT), neutrophil and other inflammatory chemokines suggest the involvement of inflammatory pathways in DMN-induced liver fibrosis. In conclusion, the progressive changes in protein C along with other noninvasive biochemical parameters whose levels were significantly correlated with disease progression may serve as biomarkers for pharmacological assessment of targeted therapy for liver fibrosis.

Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models.

The effects of anesthetic agents, commonly used in animal models, on blood glucose levels in fed and fasted rats were investigated. In fed Sprague-Dawley rats, ketamine (100 mg/kg)/xylazine (10 mg/kg) (KX) produced acute hyperglycemia (blood glucose 178.4 +/- 8.0 mg/dl) within 20 min. The baseline blood glucose levels (104.8 +/- 5.7 mg/dl) reached maximum levels (291.7 +/- 23.8 mg/dl) at 120 min. Ketamine alone did not elevate glucose levels in fed rats. Isoflurane also produced acute hyperglycemia similar to KX. Administration of pentobarbital sodium did not produce hyperglycemia in fed rats. In contrast, none of these anesthetic agents produced hyperglycemia in fasted rats. The acute hyperglycemic effect of KX in fed rats was associated with decreased plasma levels of insulin, adrenocorticotropic hormone (ACTH), and corticosterone and increased levels of glucagon and growth hormone (GH). The acute hyperglycemic response to KX was dose-dependently inhibited by the specific alpha2-adrenergic receptor antagonist yohimbine (1-4 mg/kg). KX-induced changes of glucoregulatory hormone levels such as insulin, GH, ACTH, and corticosterone were significantly altered by yohimbine, whereas the glucagon levels remained unaffected. In conclusion, the present study indicates that both KX and isoflurane produce acute hyperglycemia in fed rats. The effect of KX is mediated by modulation of the glucoregulatory hormones through stimulation of alpha2-adrenergic receptors. Pentobarbital sodium did not produce hyperglycemia in either fed or fasted rats. Based on these findings, it is suggested that caution needs to be taken when selecting anesthetic agents, and fed or fasted state of animals in studies of diabetic disease or other models where glucose and/or glucoregulatory hormone levels may influence outcome and thus interpretation. However, fed animals are of value when exploring the hyperglycemic response to anesthetic agents.

LY2875358, a neutralizing and internalizing anti-MET bivalent antibody, inhibits HGF-dependent and HGF-independent MET activation and tumor growth.

PURPOSE: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. EXPERIMENTAL DESIGN/RESULTS: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. CONCLUSIONS: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies.

A model of controlled acute hyperglycemia in rats: Effects of insulin and glucagon-like peptide-1 analog.

A rodent model of controlled acute hyperglycemia that is sensitive to glucose-lowering agents insulin and glucagon-like peptide-1 (GLP-1) analog has been developed. The studies show that anesthesia could be induced in fasted rats with ketamine (100 mg/kg) plus a low dose of xylazine (5 mg/kg) without inducing the acute hyperglycemia typically associated with these agents. Under these conditions, continuous infusion of glucose (10 and 20%) via the jugular vein for 30 to 150 min induced hyperglycemia in a time-dependent fashion. Administration of "loading" boluses of glucose (0.2-0.6 ml of a 20% solution) prior to continuous infusion of 10% glucose produced more immediate and sustained hyperglycemia. Plasma levels of a variety of glucoregulatory and stress hormones such as insulin, growth hormone, glucagon, and corticosterone were determined. Only glucagon levels changed significantly during induction and maintenance of hyperglycemia. The infusion of insulin (0.1 U/kg/h) or GLP-1 analog (10 microg/kg/h) effectively lowered blood glucose from its elevated levels. Insulin produced a significant increase in glucagon levels, and GLP-1 analog produced a significant increase in insulin levels without any change in other glucoregulatory and stress hormone levels. In conclusion, the present studies identified a novel approach for the induction of anesthesia and surgical manipulations without inducing hyperglycemia and further defined an approach for producing acute hyperglycemia in a controlled fashion in rodents. This model will be beneficial to study the influence of hyperglycemia in acute models of critical illness where hyperglycemia develops following the precipitating event. This model was responsive to insulin and GLP-1 analog, both of which were effective in ameliorating hyperglycemia.

Alkaline serine proteinase from Aspergillus fumigatus has synergistic effects on Asp-f-2-induced immune response in mice.

BACKGROUND: Exposure to Aspergillus fumigatus allergens results in the sensitization and the development of allergic bronchopulmonary aspergillosis in susceptible individuals. Aspergillus antigen consists of a number of chemically diverse components and their cumulative or synergistic effect may result in disease. When mice were challenged with individual recombinant allergens, there was only reduced inflammation and immunological responses compared to the whole antigen. Various enzymes identified from A. fumigatus have been thought to cause airway damage. In the present study, we evaluated the effect of exposure to Asp f 13, an alkaline serine proteinase, and Asp f 2 in mice. METHODS: BALB/c mice were challenged intranasally with Asp f 2 and Asp f 13 alone and in combination. The antibody response, pulmonary inflammation, and airway hyperreactivity were studied. RESULTS: Results demonstrated no major difference in antibody response and airway responses among the different groups. The inflammatory responses in the lungs, however, showed marked differences in the various groups. CONCLUSION: In spite of the similar immunological responses in the different groups of mice studied, the results demonstrate enhanced inflammation in the lungs of mice exposed to a combination of both allergens. Allergens with proteinase activity have been found to be involved in airway inflammation and remodeling, which may also apply for Aspergillus-induced allergy.