Two distinct subsets of LDGs (low density granulocytes) in ANCA-associated vasculitis.

OBJECTIVES AND METHODS: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disorder that causes vasculitis in small blood vessels throughout the body. Low-density granulocytes (LDGs) in autoimmune diseases, such as SLE and AAV, might play a critical role in the pathogenesis of these diseases. Here, we aimed to determine the characteristics of LDGs in patients with AAV. We assessed the number of whole white blood cells, neutrophil extracellular traps (NETs) productivity, proportion of cell surface markers (e.g. CD10), responses to immunosuppressants, and proteomics of LDGs in patients with AAV. RESULTS: We found more LDGs in peripheral blood mononuclear cells (PBMCs) of patients with AAV than PBMCs of healthy controls (HCs) and confirmed that these LDGs in AAV produced more NETs than normal density granulocytes (NDGs) in HCs. We identified CD10-positive LDGs with mature neutrophil features and CD10-negative LDGs with immature granulocyte properties; the proportion of the two LDG types decreased and increased, respectively, in the patients during treatment. Proteomic analysis revealed that the two LDG groups shared protein expression that differed from those of NDGs. CONCLUSION: We identified distinct CD10-positive and CD10-negative LDGs in patients with AAV. The roles of these LDGs in AAV pathology will require further investigation.

Inter- and intra-core laboratory variability in the quantitative coronary angiography analysis for drug-eluting stent treatment and follow up.

AIM: To evaluate inter-core laboratory variability of quantitative coronary angiography (QCA) parameters in comparison with intra-core laboratory variability in a randomized controlled trial evaluating drug-eluting stents. METHODS: A total of 50 patients with 62 coronary lesions were analyzed by four analysis experts belonging to an Angiographic Core Laboratory (ACL: 1 expert) and a Cardiovascular Imaging Core Laboratory (CICL: 3 experts). QCA was based on the same standard operating procedure, but selections of projection and cine frames were at the discretion of each analyst. Inter- and intra-core laboratory variabilities were evaluated by accuracy, precision, Bland Altman analysis, and coefficient of variation. RESULTS: Pre-MLD (minimal lumen diameter) was significantly smaller in results from ACL than those from all CICL experts. Number of analyzed projections did not affect pre-MLD results. Acute gain was larger in ACL than in CICL2. No significant difference was observed in late loss and loss index between inter-core laboratories. Agreement between core labs in the Bland-Altman analysis for each QCA parameter was as follows (mean difference, 95% limits of agreement): pre-MLD (-0.32, -0.74 to 0.10), stent MLD (0.08, -0.28 to 0.44), acute gain (0.22, -0.44 to 0.88), and late loss (-0.07, -0.69 to 0.55). Agreement between analysts in CICL (mean difference, 95% limits of agreement) was: pre MLD (-0.03, -0.37 to 0.31), stent MLD (0.15, -0.15 to 0.45), acute gain (0.05, -0.45 to 0.55), and late loss (0.04, -0.52 to 0.60). The widest limits of agreement among three analyses were shown in both analyses. Width of limited agreement in the intra-core laboratory analysis tended to be smaller than the inter-core laboratory analysis with these parameters. Coefficient of variation tended to be larger in lesion length (LL), acute gain, late loss, and loss index in inter- and in intra- core laboratory comparisons. CONCLUSION: Inter-core laboratory QCA variability in late loss and loss index analysis could be similar to intra-core laboratory variability, but more strict alignment between core laboratories would be necessary for initial procedural data analysis.

Impact of Cine Frame Selection on Quantitative Coronary Angiography Results.

We evaluated intra- and interobserver variability of quantitative coronary angiography (QCA) due to cine frame selection for 9 coronary stenoses. The projection was selected in advance. Cine frames were selected by 2 blinded experts (blind frame QCA) followed by assignment by supervisor (pre-selected frame QCA). Each expert analyzed 18 frames twice with a 3-month interval. A total of 72 measurements by 2 experts were used for intra- and interobserver variability analysis in calibration factor (CF), minimal lumen diameter (MLD), percent diameter stenosis (%DS), interpolated reference diameter (Int R), and lesion length (LL). Accuracy, precision, and coefficient of variation (CV) were calculated based on 2 measurements. For interobserver variability, intraclass correlation coefficient (ICC) was evaluated. Regarding intraobserver variability, precision (CV) was 0.0026 (1.45), 0.220 (25.1), 0.282 (11.0), 7.626 (11.8), and 4.042 (28.7) for blind frame QCA and 0.0044 (2.46), 0.094 (11.2), 0.225 (8.6), 3.924 (5.9), and 1.941 (12.1) for pre-selected frame QCA and regarding interobserver variability, precision (CV) was 0.0037 (2.09), 0.271 (31.8), 0.307 (11.9), 10.10 (15.4), and 5.121 (39.5) for blind frame QCA and 0.0050 (2.82), 0.098 (11.4), 0.246 (9.5), 5.253 (8.0), and 2.857 (19.0) for pre-selected frame QCA in CF, MLD, Int R, %DS, and LL, respectively. Intraclass correlation coefficient of Int R was almost perfect in blind and pre-selected frame QCA. Intraclass correlation coefficient of MLD, %DS, and LL were substantial/lower by blind frame QCA and improved to almost perfect by pre-selected frame QCA. Blind cine film selection might affect intra- and interobserver variability, especially in MLD and LL. In the multiple linear regression analysis, blind frame QCA was selected as an explanatory factor of QCA variability in MLD, %DS, and LL. The error range due to frame selection must be taken into consideration in clinical use.

Streptococcus pneumoniae Meningitis Presenting with Acute Urinary Retention and Emphysematous Cystitis.

A combination of acute urinary retention and aseptic meningitis has occasionally been described, which is referred to as meningitis-retention syndrome. In contrast, acute urinary retention has rarely been reported in bacterial meningitis. We herein report a case of Streptococcus pneumoniae meningitis presenting with acute urinary retention which led to emphysematous cystitis in an elderly woman. She presented with impaired consciousness and a distended lower abdomen. She was diagnosed with pneumococcal meningitis by lumbar puncture. Abdominal computed tomography revealed the presence of emphysematous cystitis. She completely recovered with antibiotic therapy without any complications. Acute urinary retention can occur secondary to pneumococcal meningitis.

Impact of catheter size on reliability of quantitative coronary angiographic measurements (comparison of 4Fr and 6Fr catheters).

To evaluate the feasibility of catheter down sizing for QCA, the reliability of a 4Fr catheter as a calibration device was evaluated. Repeated coronary angiograms of 9 lesions were obtained using 4Fr and 6Fr catheters under otherwise identical conditions. The calibration factor was measured 10 times by 4Fr and 6Fr catheters. QCA measurements including minimal lumen diameter (MLD), interpolated normal reference (Int N), percent diameter stenosis (%DS), and lesion length (LL) were performed by two technicians twice with a 3-month interval. The intraobserver and interobserver variability of each parameter was evaluated using intraclass correlation coefficients (ICCs). Mean of mean SD of calibration factor was significantly larger in 4Fr than in 6Fr in 9 lesions. The mean of mean coefficient of variance was significantly larger in 4Fr catheters vs in 6Fr catheters. A 6Fr catheter showed excellent reliability for both intraobserver and interobserver variability in MLD, Int N, %DS, and LL. In contrast, 4Fr showed that reliability in intraobserver variability depended on the analyst. Although reliability of interobserver variability in Int N measured by the 4Fr catheter was >0.80, the value was less than that by the 6Fr catheter. Taking these results into consideration, 4Fr catheters are less reliable than 6Fr catheters when measuring QCA data especially for follow-up data that need most accurate measurements of MLD and %DS. It would be better to use a 6Fr catheter to evaluate QCA measurements such as acute gain, late loss, restenosis rate, and device size.

Sequential necrotizing fasciitis caused by the monomicrobial pathogens Streptococcus equisimilis and extended-spectrum beta-lactamase-producing Escherichia coli.

Necrotizing fasciitis is a rapidly progressing bacterial infection of the superficial fascia and subcutaneous tissue that is associated with a high mortality rate and is caused by a single species of bacteria or polymicrobial organisms. Escherichia coli is rarely isolated from patients with monomicrobial disease. Further, there are few reports of extended-spectrum beta-lactamase (ESBL)-producing E. coli associated with necrotizing fasciitis. We report here our treatment of an 85-year-old man who was admitted because of necrotizing fasciitis of his right thigh. Streptococcus equisimilis was detected as a monomicrobial pathogen, and the infection was cured by amputation of the patient's right leg and the administration of antibiotics. However, 5 days after discontinuing antibiotic therapy, he developed necrotizing fasciitis on his right upper limb and died. ESBL-producing E. coli was the only bacterial species isolated from blood and skin cultures. This case demonstrates that ESBL-producing E. coli can cause monomicrobial necrotizing fasciitis, particularly during hospitalization and that a different bacterial species can cause disease shortly after a previous episode.

Comparative genome analysis and identification of competitive and cooperative interactions in a polymicrobial disease.

Polymicrobial diseases are caused by combinations of multiple bacteria, which can lead to not only mild but also life-threatening illnesses. Periodontitis represents a polymicrobial disease; Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, called 'the red complex', have been recognized as the causative agents of periodontitis. Although molecular interactions among the three species could be responsible for progression of periodontitis, the relevant genetic mechanisms are unknown. In this study, we uncovered novel interactions in comparative genome analysis among the red complex species. Clustered regularly interspaced short palindromic repeats (CRISPRs) of T. forsythia might attack the restriction modification system of P. gingivalis, and possibly work as a defense system against DNA invasion from P. gingivalis. On the other hand, gene deficiencies were mutually compensated in metabolic pathways when the genes of all the three species were taken into account, suggesting that there are cooperative relationships among the three species. This notion was supported by the observation that each of the three species had its own virulence factors, which might facilitate persistence and manifestations of virulence of the three species. Here, we propose new mechanisms of bacterial symbiosis in periodontitis; these mechanisms consist of competitive and cooperative interactions. Our results might shed light on the pathogenesis of periodontitis and of other polymicrobial diseases.

Significance of small renal artery lesions in patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis.

OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis is a vasculitis affecting the glomerular capillaries and small renal arteries. Although crescent formation has been reported to be characteristic of this condition, the significance of coexisting vasculitis affecting the small renal arteries has not been investigated. METHODS: Fifty patients with ANCA-positive rapidly progressive glomerulonephritis whose renal biopsy specimens contained arterioles and/or interlobular arteries were retrospectively evaluated. Cellular crescents and/or necrotizing glomerulonephritis were noted in all 50 patients. Ten patients had vasculitis of the small renal arteries (group A) and 40 patients were without such vasculitis (group B). The clinical features of these 2 groups were compared. RESULTS: Group A comprised 4 patients who had granulomatosis with polyangiitis (GPA) and 6 with microscopic polyangiitis (MPA), while group B included 1 patient with GPA and 39 with MPA. No patient in either group had eosinophilic granulomatosis with polyangiitis. The C-reactive protein (CRP) level was significantly higher in group A compared with group B (11.58 ± 6.19 vs 2.7 ± 3.55 mg/dl, p < 0.05), and pulmonary involvement was more frequent in group A than group B (80% vs 37.5%, p < 0.05). CONCLUSION: In patients with ANCA-positive glomerulonephritis, vasculitis of small renal arteries may be associated with systemic vasculitis (including pulmonary involvement) because of elevated CRP, a systemic inflammatory marker related to overproduction of interleukin 6.

Inhibition of DNA binding activity of cAMP response element-binding protein by 1,2-naphthoquinone through chemical modification of Cys-286.

1,2-Naphthoquinone (1,2-NQ) is an atmospheric electrophile that reacts covalently with protein thiols. Our previous study revealed that exposure of bovine aortic endothelial cells to 1,2-NQ causes covalent modification of cAMP response element-binding protein (CREB), thereby inhibiting its DNA binding activity and substantial gene expression of B-cell lymphoma-2 (Bcl-2) that is regulated by this transcription factor. In this study, we identified the modification sites of CREB that are associated with the decreased transcriptional activity. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis indicated that three amino acids (Cys-286, Lys-290, and Lys-319) were irreversibly modified by 1,2-NQ. Mutational analysis revealed that electrophilic modification of Cys-286, but not the other two amino acids, at the DNA binding domain is essential for the reduced CREB activity. Substitution of Cys-286 with tryptophan (C286W), which mimics CREB modification by 1,2-NQ, supported this notion. These results suggest that the covalent interaction of CREB with 1,2-NQ through Cys-286 blocks the DNA binding activity of CREB, resulting in the repression of CREB-regulated genes.

Evaluation of serum-free differentiation conditions for C2C12 myoblast cells assessed as to active tension generation capability.

We have compared several serum-free media for the differentiation of C2C12 myoblasts and assessed the extent of differentiation in several ways including as to active tension generation capability. C2C12 cells were allowed to differentiate in Dulbecco's modified Eagle's medium (DMEM) containing Ham's F-12 (F-12), AIM-V (AIM), 0.2% Ultroser-G in DMEM (Ult-G), and 0.1% Sericin in DMEM (Sericin), compared with in DMEM supplemented with 2% horse serum (HS) or 2% calf serum (CS). C2C12 differentiation was assessed as the extent of myotube formation, glucose metabolism, protein expression, sarcomere formation, and active tension generation. All serum-free media examined were capable of inducing myotube formation and the expression of muscle-specific proteins. All serum-free media except for F-12 gave the sarcomere structure. Active tension generation was observed for cells that differentiated in AIM and Ult-G, but the active tension generated by C2C12 cells that differentiated in Ult-G was only ∼25% in the case of myotubes that formed in HS. The addition of Ult-G to the AIM resulted in improvement of the active tension generation capability, the active tension generated being ∼3.4× compared to that in HS. The approach for assessing muscle cell differentiation presented in this study will be suitable for other studies that involve the differentiation of muscle cells.

Catechol estrogens mediated activation of Nrf2 through covalent modification of its quinone metabolite to Keap1.

The catechol metabolites (2-OHE and 4-OHE) of estrogen enter a redox cycle, thereby generating not only reactive oxygen species (ROS) but also electrophilic quinones. It is well recognized that chemicals causing oxidative stress or electrophiles activate a transcription factor Nrf2 that is negatively regulated by Keap1, leading to up-regulation of downstream proteins responsible for detoxification of electrophiles in cells. The purpose of the present study is to explore the roles of oxidative and electrophilic stress in Nrf2 activation caused by redox-active catechol estrogens. Exposure of RAW264.7 cells to 2- and 4-OHE activated Nrf2, resulting in induction of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC). Under these conditions, intracellular oxidants were generated; however, subsequent examinations revealed that quinoid metabolites derived from 2- and 4-OHE mainly participate in the Nrf2 activation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed Keap1 undergoes modification by such quinoid species through multiple reactive thiol groups. These results suggest that Nrf2 activation during redox cycling of catechol estrogens is dominantly attributable to formation of their ortho-quinones that covalently bind to Keap1.

Reduction of arginase I activity and manganese levels in the liver during exposure of rats to methylmercury: a possible mechanism.

The toxicity of methylmercury (MeHg) is, in part, thought to be due to its interaction with thiol groups in a variety of enzymes, but the molecular targets of MeHg are poorly understood. Arginase I, an abundant manganese (Mn)-binding protein in the liver, requires Mn as an essential element to exhibit maximal enzyme activity. In the present study, we examined the effect of MeHg on hepatic arginase I in vivo and in vitro. Subcutaneous administration of MeHg (10 mg/kg) for 8 days to rats resulted in marked suppression of arginase I activity. With purified arginase I, we found that interaction of MeHg with arginase I caused the aggregation of arginase I as evaluated by centrifugation and subsequent precipitation, and then the reduction of catalytic activity. Experiments with organomercury column confirmed that arginase I has reactive thiols that are covalently bound to organomercury. While MeHg inhibited arginase I activity, Mn ions were released from this enzyme. These results suggest that MeHg-mediated suppression of hepatic arginase I activity in vivo is, at least in part, attributable to covalent modification of MeHg or substantial leakage of Mn ions from the active site.

Downregulation of arginase II and renal apoptosis by inorganic mercury: overexpression of arginase II reduces its apoptosis.

Inorganic mercury is a toxic metal that accumulates in the proximal tubules of the kidney, causing apoptosis. Arginase II is known to inhibit apoptosis, but its role in the renal apoptosis caused by inorganic mercury is poorly understood. In the present study, we examined the involvement of arginase II in inorganic mercury-dependent apoptosis. A single exposure to mercuric chloride (HgCl(2), 1 mg/kg) in rats resulted in a dramatic time-dependent reduction in the activity of arginase II in the kidney; for example, the activity at 48 h after exposure was 31% of the control level. The decrease in arginase II activity was due to a decrease in the protein level, not to a reduction in gene expression or to direct inhibition of the activity itself. More interestingly, diminished arginase II activity was well correlated with the induction of apoptosis as evaluated by renal DNA fragmentation (r = 0.99). Overexpression of arginase II in LLC-PK(1) cells blocked cell death during exposure to inorganic mercury. These results suggest that inorganic mercury causes a reduction in protein levels of arginase II, and that impaired arginase II activity is, at least in part, associated with the apoptotic cell damage caused by this heavy metal.

1,2-Naphthoquinone disrupts the function of cAMP response element-binding protein through covalent modification.

1,2-Naphthoquinone (1,2-NQ) is an atmospheric contaminant with electrophilic properties that allow it to react readily with protein thiol groups such as those found on the cAMP response element-binding protein (CREB), a transcription factor with conserved cysteine residues that regulate DNA binding. In the present study, we explored the possibility that the interaction of 1,2-NQ with CREB will affect its activity, resulting in down-regulation of gene expression. With bovine aortic endothelial cells (BAECs) and a cell-free system, 1,2-NQ was found to covalently bind to CREB, and inhibit its DNA binding activity under conditions that were blocked by dithiothreitol. CRE-dependent luciferase activity and the down-regulation of Bcl-2 expression were suppressed by exposure of BAECs to 1,2-NQ. This phenomenon was not seen with the hydrocarbon, naphthalene, which lacks any electrophilic properties. The results indicate that CREB is a molecular target for 1,2-NQ which through irreversible binding, inhibits the function of this transcription factor.

Oxidation of proximal protein sulfhydryls by phenanthraquinone, a component of diesel exhaust particles.

Diesel exhaust particles (DEP) contain quinones that are capable of catalyzing the generation of reactive oxygen species in biological systems, resulting in induction of oxidative stress. In the present study, we explored sulfhydryl oxidation by phenanthraquinone, a component of DEP, using thiol compounds and protein preparations. Phenanthraquinone reacted readily with dithiol compounds such as dithiothreitol (DTT), 2,3-dimercapto-1-propanol (BAL), and 2,3-dimercapto-1-propanesulfonic acid (DMPS), resulting in modification of the thiol groups, whereas minimal reactivities of this quinone with monothiol compounds such as GSH, 2-mercaptoethanol, and N-acetyl-L-cysteine were seen. The modification of DTT dithiol caused by phenanthraquinone proceeded under anaerobic conditions but was accelerated by molecular oxygen. Phenanthraquinone was also capable of modifying thiol groups in pulmonary microsomes from rats and total membrane preparation isolated from bovine aortic endothelial cells (BAEC), but not bovine serum albumin (BSA), which has a Cys34 as a reactive monothiol group. A comparison of the thiol alkylating agent N-ethylmaleimide (NEM) with that of phenanthraquinone indicates that the two mechanisms of thiol modification are distinct. Studies revealed that thiyl radical intermediates and reactive oxygen species were generated during interaction of phenanthraquinone with DTT. From these findings, it is suggested that phenanthraquinone-mediated destruction of protein sulfhydryls appears to involve the oxidation of presumably proximal thiols and the reduction of molecular oxygen.