Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

Reactive oxygen species (ROS) and pro-inflammatory cytokines are crucial in ventricular remodelling, such as inflammation-associated myocarditis. We previously reported that tumour necrosis factor-α (TNF-α)-induced ROS in human aortic smooth muscle cells is mediated by NADPH oxidase subunit Nox4. In this study, we investigated whether TNF-α-induced ventricular remodelling was mediated by Nox2 and/or Nox4. An intravenous injection of murine TNF-α was administered to a group of mice and saline injection was administered to controls. Echocardiography was performed on days 1, 7 and 28 post-injection. Ventricular tissue was used to determine gene and protein expression of Nox2, Nox4, ANP, interleukin (IL)-1β, IL-2, IL-6, TNF-α and to measure ROS. Nox2 and Nox4 siRNA were used to determine whether or not Nox2 and Nox4 mediated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in adult human cardiomyocytes. Echocardiography showed a significant increase in left ventricular end-diastolic and left ventricular end-systolic diameters, and a significant decrease in the ejection fraction and fractional shortening in mice 7 and 28 days after TNF-α injection. These two groups of mice showed a significant increase in ventricular ROS, ANP, IL-1β, IL-2, IL-6 and TNF-α proteins. Nox2 and Nox4 mRNA and protein levels were also sequentially increased. ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems. Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes. Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.     

Efficacy of specific IgY for treatment of lipopolysaccharide-induced endotoxemia using a mouse model

Aims: To estimate the efficacy of specific egg yolk immunoglobulin (IgY) for the treatment of lipopolysaccharide (LPS)‐induced endotoxemia using a mouse model. Methods and Results: Specific IgY was obtained from the yolk of hens immunized with formaldehyde‐killed Escherichia coli O111 and showed a high binding activity to LPS when subjected to an ELISA. Endotoxemia was induced in mice by intraperitoneal injection of LPS at a dose of 20 mg kg^?1 for measuring survival rate and 10 mg kg^?1 for cytokine measurement. The survival rate of mice treated with 200 mg kg^?1 specific IgY or 5 mg kg^?1 dexamethasone was 70% while none of the mice in the normal saline–treated group survived more than 7 days. Specific IgY significantly (P < 0·05) decreased tumour necrosis factor‐α (TNF‐α) level and increased interleukin‐10 (IL‐10) level in the serum of endotoxemia mice. Specific IgY had less of an effect on TNF‐α than dexamthasone, while its effect on increasing IL‐10 was stronger than dexamethasone. Haematoxylin and eosin–stained sections indicated that IgY attenuated the damage to the lung and liver observed in mice with endotoxemia. Conclusions: The specific IgY increased the survival rate of mice with endotoxemia induced by LPS, down‐regulated TNF‐α and up‐regulated IL‐10 in serum and attenuated the extent of damage to the lung and liver. Significance and Impact of the Study: The specific IgY has potential for the treatment of LPS‐induced endotoxemia.     

Malignant potential of H22 hepatocarcinoma cells increases after recovery from IFN-γ-mediated inhibition

IFN-γ (interferon γ) can effectively suppress tumours, but it has also been found to promote tumour progression. However, the underlying mechanisms by which it enhances malignancy have not been fully elucidated. By using a mouse model that expresses IFN-γ locally in muscle, we found that the growth potential of tumours was increased after a quick decrease of IFN-γ. Furthermore, the up-regulation of IRF-2 (IFN regulatory factor 2) and down-regulation of IRF-1 were also found in the tumour cells. Along these lines, IFN-γ led to down-regulated expression of cyclin-D1, Bcl-2 and Bcl-xL and up-regulated expression of p21WAF1 and Bax in tumour cells. Yet, the expression of these genes, as well as activation of ERK (extracellular signal-regulated kinase) and NF-κB (nuclear factor-κB), was also reversed shortly after a decrease in IFN-γ, all of which resulted in increase tumour cell proliferation and apoptosis resistance. These findings indicate that the malignant potential of tumour cells may be suppressed by interfering with IRF-2 signalling pathways during and after decreased IFN-γ in tumour microenvironments.     

Epitope mapping of a chimeric CD137 mAb: a necessary step for assessing the biologic relevance of non‐human primate models

Antibody based manipulation of the CD137 (4‐1BB) co‐signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti‐human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti‐CD137 mAb (4B4‐1), recognized activated PBMCs from both human and non‐human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species (～95% identical), with the extracellular domain differing by only 9–10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co‐stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes. Copyright © 2009 John Wiley & Sons, Ltd.     

LPA-producing enzyme PA-PLA₁α regulates hair follicle development by modulating EGFR signalling

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.