Production of IL-10 and IL-12 in CD40 and interleukin 4-activated mononuclear cells from patients with Graves' disease

We investigated the effect of T cell-dependent B cell activation on the production of IL-10 and IL-12 by peripheral blood mononuclear cells (PBMCs) obtained from patients with Graves' disease vs Hashimoto's thyroiditis, type 1 diabetes or normal controls. Incubation of PBMCs, from each of the subject groups, with a combination of anti-CD40 monoclonal antibodies and interleukin 4 (IL-4)-activated B cells, as shown by an increased level of soluble CD23. There was also a notable increase in the number of CD23(+)cells in PBMCs from patients with Graves' disease as compared to the other subject groups. This combination of B cell stimulants increased production of IL-10 in PBMCs obtained from patients with Graves' disease relative to those patients with Hashimoto's thyroiditis, type 1 diabetes, or the control subjects. The production of IL-12 showed wide variation that depended on the basal IL-12 level. In subjects with a low basal IL-12 level there was a positive correlation between the production of IL-12 and that of IL-10 from PBMCs stimulated with anti-CD40 antibodies plus IL-4. On the contrary, in the patients with a high basal IL-12 level, no change or a decrease of IL-12 production was observed after the stimulation. Thus, T cell-dependent B cell activation via a CD40 pathway triggers the overproduction of IL-10 and overcome the effect of IL-12 to shift the Th(1)/Th(2)balance to Th(2)dominance in patients with Graves' disease but not in Hashimoto's thyroiditis or type 1 diabetes.     

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Interferon-gamma: a key contributor to hyperoxia-induced lung injury in mice

Hyperoxia-induced lung injury complicates the care of many critically ill patients who receive supplemental oxygen therapy. Hyperoxic injury to lung tissues is mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines. IFN-gamma is known to be induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. To determine whether IFN-gamma contributes to hyperoxia-induced lung injury, we first used anti-mouse IFN-gamma antibody to blockade IFN-gamma activity. Administration of anti-mouse IFN-gamma antibody inhibited hyperoxia-induced increases in pulmonary alveolar permeability and neutrophil migration into lung air spaces. To confirm that IFN-gamma contributes to hyperoxic lung injury, we then simultaneously exposed IFN-gamma-deficient (IFN-gamma-/-) mice and wild-type mice to hyperoxia. In the early phase of hyperoxia, permeability changes and neutrophil migration were significantly reduced in IFN-gamma-/- mice compared with wild-type mice, although the differences in permeability changes and neutrophil migration between IFN-gamma-/- mice and wild-type mice were not significant in the late phase of hyperoxia. The concentrations of IL-12 and IL-18, two cytokines that play a role in IFN-gamma induction, significantly increased in bronchoalveolar lavage fluid after exposure to hyperoxia in both IFN-gamma-/- mice and wild-type mice, suggesting that hyperoxia initiates upstream events that result in IFN-gamma production. Although there was no significant difference in overall survival, IFN-gamma-/- mice had a better early survival rate than did the wild-type mice. Therefore, these data strongly suggest that IFN-gamma is a key molecular contributor to hyperoxia-induced lung injury.     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

ASP4058, a Novel Agonist for Sphingosine 1-Phosphate Receptors 1 and 5, Ameliorates Rodent Experimental Autoimmune Encephalomyelitis with a Favorable Safety Profile

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P₁–S1P₅). S1P₁ is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P₁. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P₁ and S1P₅. ASP4058 preferentially activates S1P₁ and S1P₅ compared with S1P_{2, 3, 4} in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.     

Soluble guanylate cyclase-α1 is required for the cardioprotective effects of inhaled nitric oxide

Reperfusion injury limits the benefits of revascularization in the treatment of myocardial infarction (MI). Breathing nitric oxide (NO) reduces cardiac ischemia-reperfusion injury in animal models; however, the signaling pathways by which inhaled NO confers cardioprotection remain uncertain. The objective of this study was to learn whether inhaled NO reduces cardiac ischemia-reperfusion injury by activating the cGMP-generating enzyme, soluble guanylate cyclase (sGC), and to investigate whether bone marrow (BM)-derived cells participate in the sGC-mediated cardioprotective effects of inhaled NO. Wild-type (WT) mice and mice deficient in the sGC α(1)-subunit (sGCα(1)(-/-) mice) were subjected to cardiac ischemia for 1 h, followed by 24 h of reperfusion. During ischemia and for the first 10 min of reperfusion, mice were ventilated with oxygen or with oxygen supplemented with NO (80 parts per million). The ratio of MI size to area at risk (MI/AAR) did not differ in WT and sGCα(1)(-/-) mice that did not breathe NO. Breathing NO decreased MI/AAR in WT mice (41%, P = 0.002) but not in sGCα(1)(-/-) mice (7%, P = not significant). BM transplantation was performed to restore WT BM-derived cells to sGCα(1)(-/-) mice. Breathing NO decreased MI/AAR in sGCα(1)(-/-) mice carrying WT BM (39%, P = 0.031). In conclusion, these results demonstrate that a global deficiency of sGCα(1) does not alter the degree of cardiac ischemia-reperfusion injury in mice. The cardioprotective effects of inhaled NO require the presence of sGCα(1). Moreover, our studies suggest that BM-derived cells are key mediators of the ability of NO to reduce cardiac ischemia-reperfusion injury.     

Secretion of inflammatory factors from chondrocytes by layilin signaling

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA).