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Objectives: With the aim of searching for novel oncofetal tumor biomarkers of lung adenocarcinoma other than carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), we developed a strategy involving monoclonal antibodies generated from embryonic tissue of miniature swine., Materials and Methods: Using immunohistochemistry, we selected suitable hybridoma clones that were reactive against swine fetal lung but not adult lung using tissue microarray loading of human normal lung, lung cancer, and fetal and adult swine tissues., Results: The selected clones included several that were uniquely reactive against both swine fetal lung and human lung adenocarcinoma, and protein microarray revealed that the antigen they recognized was "drebrin" (DBN1). We then examined the association between the pattern of drebrin expression and the clinicopathological characteristics of lung adenocarcinoma using surgically resected samples of human lung adenocarcinoma. Two hundred formalin-fixed and paraffin-embedded tumor samples were immunostained for drebrin using clone B246, one of the clones that were reactive against drebrin. The cases were divided into those with strong (n=85) and weak (n=115) drebrin expression. In terms of disease-free survival, cases showing strong drebrin expression had a significantly poorer prognosis than those with weak drebrin expression (p=0.033)., Conclusion: The present findings indicate that "drebrin" is a unique oncofetal protein that can be applied as a new biomarker of lung adenocarcinoma., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Dimethyl fumarate (DMF), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, has been proven effective for the systemic treatment of multiple sclerosis. The aim of this study is to evaluate the anti-inflammatory effects of Nrf2 activators on human renal mesangial cells (HRMCs) and the development of lupus nephritis (LN) in mice., Methods: To assess Nrf2 activation in vitro, HRMCs were treated with safe doses of Nrf2 activators and prednisolone. The expression levels of Nrf2 and its target genes were measured using quantitative reverse transcription PCR and enzyme-linked immunosorbent assay. The anti-inflammatory effects of these compounds were assessed by measuring tumor necrosis factor alpha-induced cytokine secretion. Experimental LN was induced in female BALB/c mice by a single intraperitoneal injection of pristane. The urine albumin-to-creatinine ratio was measured at 20 weeks after injection. Pathological changes as well as protein and mRNA expression levels were assessed in the kidney obtained at the experimental end point. Oral administration of DMF or prednisolone to these mice was initiated after pristane injection., Results: Nrf2 activators such as sulforaphane and DMF showed anti-inflammatory effects in HRMCs, whereas glucocorticoid (prednisolone) showed partial effects. Moreover, DMF ameliorated the development of kidney diseases in pristane-induced LN mice, whereas glucocorticoid had no effect., Conclusions: Nrf2 activators showed stronger anti-inflammatory and organ-protective effects than glucocorticoid in the kidney. Thus, Nrf2 activators are potential therapeutic targets in glucocorticoid-resistant LN in humans.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Varying degrees of physiological uptake of 18F-fluorodeoxyglucose (FDG) are often noted in the large intestine and can be problematic when interpreting positron emission tomography (PET) images. In relation to colorectal tumor detection with FDG PET, we tentatively classified physiological FDG uptake in the large intestine according to its patterns and intensity. Subjects were 144 asymptomatic individuals (109 men, 35 women; mean age 57.5 ± 10.1 years) in our cancer screening program who underwent total colonoscopy within 24 days of FDG PET study and showed no evidence of colonic lesions on colonoscopy. Distinct FDG uptake on FDG PET images was classified into four types: focal, defined as distinctly nodular and visible on at least 4 axial; localized, 2 to 8 cm with SUVmean ≥ 4; diffuse, > 8 cm with SUVmean ≥ 4; and mixed, of more than one type. SUVmeans were examined by placing multiple circular regions of interest of 1 cm in diameter on the axial images. We found 21 distinct FDG uptakes matching our criteria in 20 of 144 subjects (13.9%): focal (n = 4), localized (n = 1), diffuse (n = 14), and mixed (n = 1; focal and diffuse). With regard to colorectal tumor detection, 6 subjects (4.2%) with focal or localized type of uptake were considered at risk of false-positive tumor identification, and 15 subjects (10.4%) with diffuse type of uptake were considered at risk of their tumors being missed at the site of FDG uptake. To confirm the feasibility of our criteria, this classification should be tested with a larger number of subjects.

Pathogenic effects of reactive oxygen species (ROS) in the salivary glands of patients with Sjögren's syndrome have been demonstrated. Epigallocatechin gallate (EGCG), which is a catechin derivative and exhibits potent antioxidant activity, has been reported to ameliorate autoimmune sialadenitis in a murine model, but the mechanism underlying its protective action remains to be investigated. Herein, we examined the effects of EGCG administration to MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice on disease severity of autoimmune sialadenitis and protein expression levels of 11 sialadenitis-related molecules - heme oxygenase-1 (HO-1) (antioxidant); thymidine glycol (marker of DNA damage); gp91phox/NADPH oxidase 2 (prooxidant); single-stranded DNA (ssDNA) and cleaved caspase 3 (apoptotic cell markers); p53 and Bax (proapoptotic molecules); Bcl-2 (antiapoptotic molecule); SSA/Ro, SSB/La, and Ifi202 (autoantigens). In EGCG-treated mice, the severity of sialadenitis was substantially decreased. Expression levels of thymidine glycol, gp91phox, ssDNA, cleaved caspase 3, p53, Bax, SSA/Ro, SSB/La, and Ifi202 in duct epithelial cells of salivary glands from EGCG-treated mice were reduced, whereas HO-1 and Bcl-2 were overexpressed. Results of correlation analysis among sialadenitis severity and 11 sialadenitis related-molecules, and those of partial correlation analysis between apoptotic related-molecules and sialadenitis severity or HO-1 suggested that the consecutive pathogenic cycle including activated autoimmune reactions, ROS synthesis, DNA damage and p53-dependent apoptosis was associated with the pathogenesis of autoimmune sialadenitis in MRL-Fas(lpr) mice. Overexpression of HO-1 and Bcl-2 mediated by EGCG blocked this pathogenic cycle, subsequently resulting in the inhibition of ROS-mediated DNA damage and apoptosis, and protected salivary gland tissues from oxidative stress. Clinically, green tea catechin may have therapeutic efficacy for Sjögren's syndrome.

Although costimulatory molecules have been shown to play crucial roles in the immune response, their involvement in the pathogenesis of Sjögren's syndrome is incompletely understood. In this study, we evaluated the relationship between the severity of spontaneous Sjögren's syndrome-like autoimmune sialadenitis in MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice and the expression of 6 costimulatory molecules that play important roles in the immune response: CD80, CD86, OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), glucocorticoid-induced TNF receptor-related protein ligand (GITRL), and B cell-activating factor of the tumor necrosis factor family (BAFF). Expression of the costimulatory molecules in the submandibular salivary glands of age-matched autoimmune MRL-Fas(lpr) mice and non-autoimmune MRL/MpJ-+/+(MRL/+) and C3H/HeJ-lpr/lpr (C3H-Fas(lpr)) mice was examined immunohistochemically and scored on a scale of 0 to 3. The severity of sialadenitis was evaluated histologically and scored on a scale of 0 to 3. We found that all of the costimulatory molecules were expressed in duct epithelial cells of salivary glands from MRL-Fas(lpr) mice, whereas immunoreactivity was absent or weak in the MRL/+ mice. The staining intensity for all 6 costimulatory molecules was significantly higher in the MRL-Fas(lpr) than in the MRL/+ mice. Partial correlation analysis was performed to assess the degree of association between costimulatory molecule staining scores and disease scores, which clearly revealed a significant correlation for only GITRL and 4-1BBL. These molecules showed negligible immunoreactivity in the submandibular glands of C3H-Fas(lpr) mice, suggesting that their expression was independent of the Fas(lpr) mutation. In conclusion, the expression of GITRL and 4-1BBL in salivary gland duct epithelial cells is associated with background genes in the MRL strain, but not with the Fas(lpr) mutation itself, and contributes significantly to the pathogenesis of autoimmune sialadenitis in MRL-Fas(lpr) mice. These results suggest that GITRL and 4-1BBL may be effective targets for the development of therapies for Sjögren's syndrome.

Autoantibodies against integral membrane proteins are usually pathogenic. Although anti-endothelial cell antibodies (AECAs) are considered to be critical, especially for vascular lesions in collagen diseases, most molecules identified as autoantigens for AECAs are localized within the cell and not expressed on the cell surface. For identification of autoantigens, proteomics and expression library analyses have been performed for many years with some success. To specifically target cell-surface molecules in identification of autoantigens, we constructed a serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). Here, we present an overview of recent research in AECAs and their target molecules and discuss the principle and the application of SARF. Using SARF, we successfully identified three different membrane proteins: fibronectin leucine-rich transmembrane protein 2 (FLRT2) from patients with systemic lupus erythematosus (SLE), intercellular adhesion molecule 1 (ICAM-1) from a patient with rheumatoid arthritis, and Pk (Gb3/CD77) from an SLE patient with hemolytic anemia, as targets for AECAs. SARF is useful for specific identification of autoantigens expressed on the cell surface, and identification of such interactions of the cell-surface autoantigens and pathogenic autoantibodies may enable the development of more specific intervention strategies in autoimmune diseases.

Glucagon is used as an anti-motility agent during gastrointestinal tract examinations. We experienced subjects with enhanced 18F-fluorodeoxyglucose (FDG) uptake in whole-body skeletal muscle when conducting positron emission tomography (PET). The subjects had been administered glucagon during gastroscopy just prior to PET. This observation prompted us to perform the present retrospective study to determine whether or not glucagon enhances FDG uptake in skeletal muscle. We randomly selected 30 cases, including subjects who had undergone PET and gastroscopy on the same day as cancer screening procedures, and classified them into three groups. In the NO group (n = 10), no medications were used prior to PET. In the SC group (n = 10), scopolamine butylbromide (10 mg) was intravenously administered during endoscopy. In the GL group (n = 10), glucagon (0.5 mg) was intravenously administered during endoscopy. Both drugs were administered 45-60 min prior to FDG administration. The mean standardized uptake value (SUV) for gluteal muscle was 0.7 ± 0.14, 0.69 ± 0.15, and 0.99 ± 0.7 in the NO, SC, and GL groups, respectively. The SUV in the GL group was highest, but the difference was not statistically significant. In the subject with the highest SUV (3.04; GL group), the quality of the oncologic PET image was impaired, perhaps because of a relative decrease of FDG distribution in the chest and abdomen. Because previous literature showed that via hyperglycemia and hyperinsulinemia glucagon has the effect of increasing FDG uptake in skeletal muscle, the use of glucagon should be avoided just prior to FDG PET, although in our subjects, no statistical proof that glucagon enhances FDG uptake in skeletal muscle was obtained.

Background: Pulmonary hypertension (PH) refers to a spectrum of diseases with elevated pulmonary artery pressure. Pulmonary arterial hypertension (PAH) is a disease category that clinically presents with severe PH and that is histopathologically characterized by the occlusion of pulmonary arterioles, medial muscular hypertrophy, and/or intimal fibrosis. PAH occurs with a secondary as well as a primary onset. Secondary PAH is known to be complicated with immunological disorders. The aim of the present study is to histopathologically and genetically characterize a new animal model of PAH and clarify the role of OX40 ligand in the pathogenesis of PAH., Results: Spontaneous onset of PAH was stably identified in mice with immune abnormality because of overexpression of the tumor necrosis factor (TNF) family molecule OX40 ligand (OX40L). Histopathological and physical examinations revealed the onset of PAH-like disorders in the C57BL/6 (B6) strain of OX40L transgenic mice (B6.TgL). Comparative analysis performed using different strains of transgenic mice showed that this onset depends on the presence of OX40L in the B6 genetic background. Genetic analyses demonstrated a susceptibility locus of a B6 allele to this onset on chromosome 5. Immunological analyses revealed that the excessive OX40 signals in TgL mice attenuates expansion of regulatory T cells the B6 genetic background, suggesting an impact of the B6 genetic background on the differentiation of regulatory T cells., Conclusion: Present findings suggest a role for the OX40L-derived immune response and epistatic genetic effect in immune-mediated pathogenesis of PAH.

Recent studies indicate that several transcription units assemble to form a 'transcription factory' where active transcription occurs in the nuclei. Previously, we generated chicken B-lymphocyte-derived DT40 cells lacking six transcriptional regulatory regions scattered in and around the immunoglobulin (Ig)-β gene. The deletions caused a complete shut down of transcription and epigenetic regulation of the Ig-β gene, demonstrating that the scattered regulatory regions cooperated in the transcriptional and epigenetic regulation of the gene. However, the in vivo 3-dimensional spatial relationships between the Ig-β promoter and these six regulatory regions were not investigated. In this study, we used chromosome conformation capture (3C) technology and demonstrated that the Ig-β promoter physically interacted with the scattered regulatory regions. We found that the Ig-β promoter also interacted with two downstream promoters of ubiquitously expressed genes, rad motif 1 (RDM1) and Plekhm1, to form a transcription factory, but not with three ubiquitously expressed genes, BAF60b, p45/SUG, and RRMJ3, located upstream of the Ig-β gene. In this factory, the chromatin from the three promoters and the scattered regulatory regions of the Ig-β gene formed a complex structure with many chromatin loops.

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is an adaptor molecule containing a Src homology 2 (SH2) domain. SAP is expressed in T cells and natural killer (NK) cells and binds to the cytoplasmic domains of SLAM family receptors, resulting in the subsequent recruitment of Fyn. The SAP (SH2D1A) gene is located on the X chromosome and is responsible for X-linked lymphoproliferative disease, characterized by higher susceptibility to Epstein-Barr virus infection. The SAP-mediated signal is not only essential for the development of NKT cells, i.e. unconventional CD1d-restricted T cells with invariant Valpha14 T cell receptors, but also for the regulation of the function of NK cells and conventional T cells. The role of SAP-mediated signaling in the induction of autoimmune diseases has been analyzed using animal models such as lupus, hepatitis, and graft-versus-host disease and is considered important in their pathogenesis in humans. In this review we highlight the current findings on SAP-mediated signaling in hematopoietic cells and discuss its importance in autoimmune diseases and immunological disorders.

Heme oxygenase (HO) catalyzes oxidative breakdown of heme, and constitutes two isozymes, HO-1 and HO-2. Here, we explored the tissue-specific regulation of expression of HO-1 and HO-2 under hypoxemia. There was no significant change in the overall expression levels of HO-1 and HO-2 mRNAs and proteins in the lung during adaptation of C57BL/6 mice to normobaric hypoxia (10% O(2)). However, immunohistochemical analysis revealed the increased expression of HO-1 and HO-2 proteins after 28 days of normobaric hypoxia in the pulmonary venous myocardium that is the extension of the left atrial myocardium into pulmonary venous walls. Moreover, the expression of HO-2 protein was increased in the sub-endocardial myocardium of ventricles under hypoxia, while HO-1 protein level was increased in the full-thickness walls. Thus, hypoxemia induces expression of both HO-1 and HO-2 proteins in the myocardium. Using C57BL/6 mice lacking HO-2 (HO-2(-/-)), which manifest chronic hypoxemia, we also showed that the HO-1 protein level in the lung was similar between HO-2(-/-) mice and wild-type mice. Unexpectedly, HO-1 protein level was lower by 35% in the HO-2(-/-) mouse liver than the wild-type liver. These results indicate that the expression of HO-1 protein is regulated in a tissue-specific manner under hypoxemia.

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)( hage ) mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.

There has been increasing evidence for the involvement of fatty acid-binding proteins (FABPs) in the cytokine production of macrophages and dendritic cells probably through the control of cellular lipid metabolism and signal transduction. Since mast cells (MCs) are recently shown to be involved in immune response through modification of cytokine production, it is possible that some FABPs could also be involved in the immune response of MCs. In this study, we found that epidermal-type FABP (E-FABP) was expressed in murine bone marrow-derived MCs (BMMCs). Using BMMCs from genetically E-FABP-null mutated mice, we demonstrated that E-FABP in BMMCs plays a key role in the production of TNF-alpha following lipopolysaccharide (LPS) stimulation. In the in vivo septic peritonitis model (cecal ligation and puncture model), E-FABP-null mice showed a significantly increased mortality compared to wild-type mice. However, no significant difference in antigen-induced cytokine production was observed between wild-type and E-FABP-null BMMCs, and systemic anaphylaxis was equally induced in vivo in both wild-type and E-FABP-null mice. These results suggest that E-FABP is specifically involved in the LPS-induced cytokine production of MCs, and could play a role in the host-defense against bacterial infection, possibly through regulation of TNF-alpha production.

Genomic recombination events, including VDJ recombination (VDJR) and class-switch recombination (CSR), are indispensable for the adaptation and progression of the acquired immune system. These processes are completed by orderly, temporal onsets of the gene rearrangements along with B-cell differentiation. The presence of various premature transcripts of immunoglobulin heavy chain (IgH) alleles has been demonstrated during B-cell ontogeny. These include D(H)-J(H) (DJ)-mu, J(H)-mu, and sterile transcripts of C(H). Since these transcripts can be detected during the onset of VDJR and CSR, their presence is believed to reflect a structural change in the genome, favoring VDJR and CSR. This report presents evidence of persistent DJ transcription and onset of CSR on an unproductive IgH allele in peripheral tissues. Nucleotide sequence analysis revealed that these transcripts showed DJ-gamma (Dgamma) configuration and that characteristics of the variable region were essentially the same as those of the DJ-mu transcript previously described. It was noted that the small intestine abundantly expresses Dgamma transcripts with gamma2b and gamma1 isotypes of the IgH constant region. The present findings indicate the onset of CSR preceding V(H) to DJ joining in an unproductive IgH allele of the peripheral B cell and the specificity for the gut-associated condition for B-cell differentiation in the small intestine.

Fatty acids have a great influence on the process of lymphocyte apoptosis which is considered as a modulating factor of immune response in both humans and animals. However the mechanism underlying the function of fatty acids in the process of lymphocyte apoptosis is not fully understood. In this study we show that the appearance of adipocyte-type fatty acid binding protein (A-FABP) is induced upon administration of dexamethasone (DEX) in both in vivo and cultured lymphocytes, and its distinct nuclear localization occurs in close relation to the DEX-induced apoptosis process. In immunohistochemistry of mouse spleen, A-FABP-immunoreactivity starts to occur 3 h after DEX stimulation, and it massively localizes in the nucleus 8 h after the treatment, while no A-FABP-immunoreactivity is discerned in the lymphocytes of normal as well as 24 h post-injection spleen. In the murine T-cell leukemia CTLL-2 cells, A-FABP-immunoreactivity is also induced in both of the cytoplasm and nucleus when the apoptosis is induced by IL-2 retrieval together with DEX treatment, while in the presence of IL-2 A-FABP-immunoreactivity is confined to the cytoplasm with DEX treatment. On the other hand, A-FABP-immunoreactivity is not detected by IL-2 retrieval alone. The present findings altogether suggest that A-FABP and its ligands, fatty acids, play an important role in the process of apoptosis and the immune modulation induced by DEX.

Background: The incidence and characteristics of spontaneous ankylosis in the ankle of specific F(1) mice descended from two Fas-deficient strains were reported. Here the coincidence of synovial proliferation and ankylosis in the descendent F(2) mice is reported., Aim: To clarify whether the two distinct manifestations are genetically different., Methods: An arthropathic group of mice (MCF(2)) were bred by intercrossing MRL/Mp.Fas(lpr)-sap(-)/sap(-) and C3H/He.Fas(lpr) mice. All mice were killed by bleeding under anaesthesia when they were 6 months old. Pathological grades for synovial proliferation were determined by microscopical examination. To obtain a linkage locus, the whole genome of male MCF(2) mice was scanned by using 73 microsatellite markers., Results: Synovial proliferation was equally observed in male and female MCF(2) mice. No correlation was observed between the grades of synovial proliferation and the ankylosis occurring in the MCF(2) mice. A suggestive susceptibility locus was shown in the middle of chromosome 11. This locus was an MRL allele with a recessive inheritance mode., Conclusion: The pathogenic mechanisms of synovial proliferation and ankylosis are genetically different. The present locus is overlapped with some loci associated with rheumatoid arthritis and with others associated with experimental arthritides.

Objective: NK026680 is a newly identified type of immunosuppressive agent that inhibits dendritic cell (DC) functions and consequently reduces the mortality of mice with experimental acute graft-versus-host disease. This study was undertaken to evaluate NK026680 suppression of DC functions in preventing development of rapidly progressive glomerulonephritis (RPGN) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) in SCG/Kj mice., Methods: Oral administration of NK026680 to SCG/Kj mice began when mice were 8-10 weeks old, before the onset of disease, and continued for 56 days. The efficacy of NK026680 was evaluated using the mortality of mice, the results of urinalysis, histopathologic evaluation for glomerular injury, and immunofluorescence staining for the detection of immune complex (IC) deposition in glomeruli, and by assessing lymphadenopathy and measuring autoantibody titers., Results: Oral administration of NK026680 at a dosage of 25 mg/kg once daily or 50 mg/kg once daily significantly suppressed 1) spontaneous mortality, 2) proteinuria and hematuria, 3) blood urea nitrogen levels, 4) glomerular damage characterized histopathologically, 5) IC deposition in glomeruli, 6) the development of pANCA and anti-DNA antibodies, and 7) lymphadenopathy., Conclusion: The newly identified DC inhibitor, NK026680, prevented the onset of RPGN, autoantibody production, and lymphadenopathy in SCG/Kj mice, suggesting a crucial role for DC function in these autoimmune phenotypes. NK026680 may be a potent immunosuppressive agent for the treatment of ANCA-associated renovascular disorders.

Objective: Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice., Methods: A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers., Results: Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the delta-granule of platelets., Conclusion: The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-beta locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-beta gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-beta chromatin was resistant to DNase I digestion in these cells. Thus, the collaboration is shown to convert the Ig-beta chromatin from the condensed state to a relaxed state. H3 and H4 acetylation decreased to <8% but H3K4 hypermethylation was observed at the Ig-beta promoter and exon 3. The collaboration of four regions had virtually no effect on CG hypomethylation in the region upstream the transcriptional start site. Accordingly, neither the DNase I general sensitive state in the Ig-beta chromatin nor hyperacetylation of H3 and H4 histones in the promoter proximal region causes H3K4 di-methylation or CG hypomethylation in the promoter. From these analyses, a chromatin situation was found in which both an active state, such as enhanced H3K4 methylation, or CG hypomethylation, and an inactive state, such as DNase I resistance in the Ig-beta chromatin or hypoacetylation of H3 and H4 histones in the Ig-beta locus, coexist.

Autoantibody production and lymphadenopathy are common features of systemic autoimmune disease. Targeted or spontaneous mutations in the mouse germline have generated many autoimmune models with these features. Importantly, the models have provided evidence for the gene function in prevention of autoimmunity, suggesting an indispensable role for the gene in normal immune response and homeostasis. We describe here pathological and genetic characterizations of a new mutant strain of mice, the mutation of which spontaneously occurred in the Fas-deficient strain, MRL/Mp.Faslpr (MRL/lpr). MRL/lpr is known to stably exhibit systemic lupus erythematosus-like diseases. However, the mutant mice barely displayed autoimmune phenotypes, though the original defect in Fas expression was unchanged. Linkage analysis using (mutant MRL/lpr x C3H/lpr)F2 mice demonstrated a nucleotide insertion that caused loss of expression of small adaptor protein, signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). SAP is known to be a downstream molecule of SLAM family receptors and to mediate the activation signal for tyrosine kinase Fyn. Recent studies have shown pleiotropic roles of SAP in T, B, and NK cell activations and NKT cell development. The present study will provide evidence for an essential role for SAP in the development of autoimmune diseases, autoantibodies, and lymphadenopathy in MRL/lpr lupus mice.

Objective: To analyze the influence of the genetic background of an arthritis-prone strain of mice, MRL, on the spontaneous development of arthropathy in DBA/1 mice, which histopathologically resembles enthesopathy in humans, and to clarify the strain-specific gene loci and their interactions that confer susceptibility to arthropathy., Methods: MRL, DBA/1, (MRL x DBA/1)F(1), and (MRL x DBA/1)F(2) intercross mice were prepared, and the severity and onset of arthropathy of the ankle joints in individual mice were quantified (0-3 and 0-5 scale, respectively). A genome-wide scan of 271 male F(2) intercross mice with polymorphic microsatellite markers was performed., Results: Only male DBA/1, (MRL x DBA/1)F(1), and (MRL x DBA/1)F(2) mice developed arthropathy. The macroscopic and histopathologic findings of arthropathy in the F(2) mice were similar to those in the parental DBA/1 mice, but the onset was significantly earlier. In the quantitative trait locus analysis of male F(2) mice, 1 susceptibility locus for both the severity and early onset of the disease in the region of an MRL allele, Amd1, was located at marker D10Mit259 (map position 40.0 cM), which was common to 1 of the sialadenitis susceptibility loci in MRL mice, Asm1. Another susceptibility locus for the severity and early onset of arthropathy in the region of a DBA allele, Amd2, was located at D3Mit46 (29.5 cM). These loci manifested an additive effect on the development of arthropathy., Conclusion: Arthropathy in DBA/1 mice is under the control of an allelic combination of gene loci, one of which is common to the locus for sialadenitis in MRL/MpJ-lpr/lpr mice.

The role of DNase I hypersensitive sites (DHSs) in transcription of the B cell-specific Ig-beta gene and in maintenance of active chromatin state in the Ig-beta locus were examined. A total of 10 DHSs were divided into four regions, and each region was deleted separately in chicken B lymphocyte-derived DT40 cells. Deletion of three DHSs located between the Ig-beta promoter and its upstream Na channelgene, resulted in the absence of Ig-beta mRNA. Three regions except the region in the Na channel gene were involved in the transcription of Ig-beta gene. The enhancing activity of DHSs as determined by transient transfection assays did not always correlate with the effect of DHS deletion on the expression level of Ig-beta mRNA. In each deletion, cells contained the same DHSs as observed in the predeletion cells, indicating that deleted DHSs did not participate in the maintenance of DT40-specific DHSs. Enhanced acetylation of H3 and H4 histones at the Ig-beta promoter and at DT40-specific DHSs was observed in cells in which DHSs between the Na channel gene and Ig-beta promoter were deleted; therefore, these DHSs are prerequisite for transcription of the Ig-beta gene but not required for the maintenance of active chromatin state in the Ig-beta locus. Thus, epigenetic factors required for the maintenance of the active chromatin state are suggested to reside in other regions than those deleted in this study.

Background: To clarify whether vascular endothelial adhesion molecules in glomeruli might contribute to the severity and diversity of glomerular lesions in lupus nephritis, their expression in lupus models was analyzed., Methods: The expression levels of E- and P-selectin and vascular cell adhesion molecule-1 (VCAM-1) in glomeruli of different histopathologic grades of MRL/MpJ-lpr/lpr (MRL/lpr) lupus mice was studied using laser-capture microdissection of the glomeruli, followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The glomerular lesions in SCID mice injected with the 2B11.3 and 7B6.8 clones, which are derived from an MRL/lpr mouse and induce endocapillary proliferative and wire loop type of glomerular lesions, respectively, were analyzed. To investigate the effect of a soluble form of E-selectin (sE-selectin) on the development of glomerular lesions, sE-selectin-producing L cells were prepared by transfection of the cDNA encoding sE-selectin and injected into SCID mice., Results: The glomeruli in MRL/lpr mice showed increased expression of these adhesion molecules, corresponding to the severity of the glomerular lesions. The endocapillary proliferative type lesions in SCID mice induced by the 2B11.3 clone showed significantly increased expression of the adhesion molecules, especially E-selectin and P-selectin, but the wire loop type lesion induced by the 7B6.8 clone expressed only VCAM-1. Formation of the endocapillary proliferative type lesions induced by the 2B11.3 clone was markedly prevented in association with elevation of the serum level of sE-selectin produced by the tansfected L cells., Conclusion: The severity and diversity of the histopathology of lupus nephritis are partially associated with the expression of vascular endothelial adhesion molecules in glomeruli.

A 37-year-old man was admitted to our hospital because of fever, polyarthralgia, and subcutaneous tumors. There was swelling of the bilateral wrists and ankles, and subcutaneous tumors over the bilateral elbow joints. Despite his complaints of multiple symptoms, clinical investigations failed to reveal any abnormality. Although laboratory parameters improved rapidly after steroid therapy, the symptoms remained unchanged, and there was an enormous discrepancy between the laboratory data and his symptoms. A biopsy specimen from one of the subcutaneous tumors revealed foreign-body granuloma associated with a foreign body fragment. Because the nursing staff later discovered that the patient had been carrying out self-injection, a diagnosis of Munchausen syndrome was made. Munchausen syndrome should be included in the differential diagnosis of rheumatic diseases.

Objective: To clarify the mode of inheritance of the tissue distribution of vasculitis in MRL/Mp-lpr/lpr (MRL/lpr) lupus-prone mice and to identify the susceptibility loci., Methods: Vasculitis in individual MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr)F(1), and (MRL/lpr x C3H/lpr)F(2) intercross mice was analyzed by histopathologic grading of main branches of the aorta and of medium-sized arteries in the lower limbs. Genomic DNA samples from F(2) intercross mice were examined by simple sequence-length polymorphism analysis, and the polymorphic microsatellite markers highly associated with vasculitis in each tissue were determined as vasculitis susceptibility loci., Results: A susceptibility locus with significant linkage to vasculitis of main branches of the aorta was mapped on chromosome 4 at D4Mit213 (map position 13.3cM) selectively in males, while vasculitis of medium-sized arteries in the lower limbs was mapped to different chromosomes: at D8Mit31 on chromosome 8 (map position 33.0) selectively in females and at D5Mit36 on chromosome 5 (map position 65.0). All of these were different from the previously defined loci governing susceptibility to vasculitis involving the kidneys., Conclusion: Systemic vasculitis in MRL/lpr mice is genetically controlled with cumulative effects of multiple gene loci, each of which has tissue specificity.

To search for novel regulatory regions, we examined the features of chromatin structure in the rat B29/Ig-beta gene and its flanking regions by determining DNase I hypersensitive sites (DHS) in plasmacytoma-derived Y3 cells. Six Y3 cell-specific DHS were detected at -8.6, promoter, +0.7, +4.4, +6.0, and +8.7 kb. The DHS at +4.4, +6.0, and +8.7 kb were present in the intergenic region between B29/Ig-beta and growth hormone (GH) genes and were mapped inside conserved sequences in rat and humans. In transient transfection into Y3 cells, 2.9-kb DNA containing the +4.4 and +6.0-kb DHS demonstrated six times more enhancing activity than B29/Ig-beta promoter alone. Three intergenic DHS each possessed enhancing activity that was highest in the +4.4-kb region. In the electrophoretic mobility shift assay, a major band shift was demonstrated with Y3 nuclear extract and 0.3-kb DNA containing the +4.4-kb region with a conserved 0.22-kb sequence. By footprint analysis, 20 bases in the middle of the 0.3-kb DNA were protected by Y3 nuclear extract in which the consensus binding site for the OCT family was present. Deletion of the footprinted region reduced enhancing activity to that of the B29/Ig-beta promoter alone. The sequence responsible for the major band shift and transcriptional enhancing activity in the conserved +4.4-kb region thus coincided with the 20-bp footprinted region.