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A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with β1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T

Dysfunction of immunoinhibitory signals and persistent T cell activation reportedly play important roles in the development of vasculitis. The skin is one of the most accessible organs, and it is suitable for the characterization of immune cell signatures. However, the inhibitory checkpoint molecules in the skin and their relevance to vasculitis have not been studied. Here, we investigated the profile of immune checkpoint molecules in the skin and peripheral blood of patients with vasculitis and healthy donors. We found that some of the inhibitory checkpoint molecules, including programmed cell death 1 receptor (PD-1), were elevated in T-cells in the blood of patients with systemic and cutaneous vasculitis. In addition, programmed death-ligand 1 (PD-L1) expression was elevated in the skin of patients with cutaneous vasculitis. Histologically, PD-L1 was highly expressed in the vessels in the skin along with CD4+ and CD8+ T-cell infiltration in patients with cutaneous vasculitis. Notably, plasma soluble PD-L1 levels were increased, and these correlated with C-reactive protein in patients with systemic vasculitis. Our findings suggest that inhibitory checkpoint molecules might be differentially modulated in the skin and peripheral blood of patients with vasculitis, and that the alteration of the PD-L1/PD-1 axis may be associated with the regulation of T-cell activation in vasculitis.

Cold atmospheric pressure plasma (CAP) does not cause thermal damage or generate toxic residues; hence, it is projected as an alternative agent for sterilization in food and pharmaceutical industries. The fungicidal effects of CAP have not yet been investigated as extensively as its bactericidal effects. We herein examined the effects of CAP on yeast proteins using a new CAP system with an improved processing capacity. We demonstrated that protein ubiquitination and the formation of protein aggregates were induced in the cytoplasm of yeast cells by the CAP treatment. GFP-tagged Tsa1 and Ssa1, an H2O2-responsive molecular chaperone and constitutively expressed Hsp70, respectively, formed cytoplasmic foci in CAP-treated cells. Furthermore, Tsa1 was essential for the formation of Ssa1-GFP foci. These results indicate that the denaturation of yeast proteins was caused by CAP, at least partially, in a H2O2-dependent manner. Furthermore, misfolded protein levels in the endoplasmic reticulum (ER) and the oligomerization of Ire1, a key sensor of ER stress, were enhanced by the treatment with CAP, indicating that CAP causes ER stress in yeast cells as a specific phenomenon to eukaryotic cells. The pretreatment of yeast cells at 37 °C significantly alleviated cell death caused by CAP. Our results strongly suggest that the induction of protein denaturation is a primary mechanism of the fungicidal effects of CAP.

Some studies have shown that the estimated glomerular filtration rate (eGFR) at the time of initiating dialysis was associated with mortality. However, the relationship between ratio of blood urea nitrogen to serum creatinine (BUN/Cr) and mortality is unknown. The study was a multicenter, prospective cohort analysis including 1520 patients. Patients were classified into four quartiles based on the BUN/Cr ratio at the dialysis initiation, with Q1 having the lowest ratio and Q4 the highest. All-cause mortality after initiating dialysis was compared using the log-rank test. All-cause mortality of Q1, Q2, and Q3 was compared with that of Q4 using multivariate Cox proportional hazard regression analysis. Moreover, we compared the renal parameters including BUN/Cr ratio, eGFR, and creatinine clearance for sensitivity and specificity using receiver operative characteristic (ROC) curve. Significant differences were observed in all-cause mortality among the four groups (p

Cold atmospheric pressure plasma (CAP) has potential to be utilized as an alternative method for sterilization in food industries without thermal damage or toxic residues. In contrast to the bactericidal effects of CAP, information regarding the efficacy of CAP against eukaryotic microorganisms is very limited. Therefore, herein we investigated the effects of CAP on the budding yeast Saccharomyces cerevisiae, with a focus on the cellular response to CAP. The CAP treatment caused oxidative stress responses including the nuclear accumulation of the oxidative stress responsive transcription factor Yap1, mitochondrial fragmentation, and enhanced intracellular oxidation. Yeast cells also induced the expression of heat shock protein (HSP) genes and formation of Hsp104 aggregates when treated with CAP, suggesting that CAP denatures proteins. As phenomena unique to eukaryotic cells, the formation of cytoplasmic mRNP granules such as processing bodies and stress granules and changes in the intracellular localization of Ire1 were caused by the treatment with CAP, indicating that translational repression and endoplasmic reticulum (ER) stress were induced by the CAP treatment. These results suggest that the fungicidal effects of CAP are attributed to the multiple severe stresses.

1. Definition and background IgA nephropathy (IgAN, also known as Berger’s disease) is a disease characterized by urinary findings suggesting glomerulonephritis; predominantly, IgA is deposited in the glomeruli, with no evidence of other underlying disease. Glomerular hematuria and proteinuria are urinary findings that suggest glomerulonephritis. Renal biopsy findings, which are required for confirming the diagnosis of glomerulonephritis, include IgA deposits mainly in the glomerular mesangium and occasionally in the capillary loops. In many cases, C3 is also co-deposited. The rate of progression to end-stage renal disease (ESRD) is approximately 40 % at 20 years after diagnosis. Treatment may include therapy with renin-angiotensin system (RAS) blockers, antiplatelet agents, oral corticosteroids, fish oil, or non-steroidal immunosuppressive agents; steroid pulse therapy; or tonsillectomy. The therapeutic effects of each have been examined, but an effective treatment regimen is yet to be established.

Renal parenchymal lesions in patients with IgG4-related kidney disease (IgG4-RKD) are characterized by tubulointerstitial nephritis with storiform fibrosis and infiltration by high numbers of IgG4-positive plasma cells. The aim of this study was to evaluate the clinical and pathological effects of corticosteroid therapy in patients with IgG4-RKD. Of six patients who were diagnosed with IgG4-RKD, four patients underwent re-biopsy at approximately 30-50 days after corticosteroid therapy was initiated. Based on the classification of Yamaguchi et al., the degree of tubulointerstitial fibrosis was classified before and after therapy. In addition, tubulointerstitial expression patterns of alpha-smooth muscle actin (alpha-SMA), collagen I, III, and IV protein, and connective tissue growth factor (CTGF) mRNA were examined. Histopathological findings before treatment showed alpha-SMA-positive myofibroblasts in the lesion, and CTGF mRNA-positive cells were found in the cellular infiltrate. Although corticosteroid therapy improved serum creatinine clinically, the stage of fibrosis advanced pathologically as evidenced by increased staining for collagen I and III. However, the number of IgG4-positive plasma cells decreased, and CTGF mRNA expression reduced. In other words, fibrosis had advanced from the time of extensive cell infiltration in patients with IgG4-RKD and inflammation was relieved by corticosteroid. A reduced number of positive CTGF mRNA expression cells in repeat biopsies indicated that the fibrosis process was terminated by corticosteroid therapy. We propose that corticosteroid therapy could terminate the pathway of active fibrosis, thereby inhibiting progression to renal dysfunction

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular (CV) events. Recently, elevated neutrophil gelatinase-associated lipocalin (NGAL) levels have been reported in patients with heart failure, coronary heart disease, or stroke. Our aim was to assess urinary NGAL as a predictor of CV events in patients with CKD. This was a prospective observational cohort study of 404 patients with predialysis CKD. CV events were defined as CV death, acute coronary syndrome, hospitalization for worsening heart failure, stroke and dissection of aorta. During a mean follow-up period of 33 months, 77 CV events (19.1 %) occurred. After adjustment for gender, age, diabetes, previous cardiovascular disease, urinary albumin/creatinine ratio (UACR), estimated glomerular filtration rate, hemoglobin, and high-sensitivity C-reactive protein, patients with the other quartiles of urinary NGAL had significantly higher risk of CV events compared with patients with the lowest quartile (hazard ratio (HR) 2.81, 95 % confidence interval (CI) 1.01–7.81, P = 0.047 for Q2, HR 3.31, 95 % CI 1.22–9.00, P = 0.019 for Q3, and HR 3.27, 95 % CI 1.15–9.29, P = 0.026 for Q4). Regarding the combination of urinary NGAL with UACR, we also stratified patients into four groups according to whether the level of each marker was above or below the median (61.8 μg per gram creatinine (gCr) for NGAL and 351.1 mg/gCr for UACR). Four-year CV event-free survival rates were 89.2, 79.6, 71.8, and 51.5 % in order for the four respective groups (P < 0.0001). Elevated urinary NGAL was able to predict future CV events in CKD patients, and had incremental predictive value with elevated UACR.

In patients on maintenance dialysis, increased serum calcium levels are known to be associated with a poor prognosis. However, it is not known whether serum calcium levels at dialysis initiation have an impact on subsequent prognosis. The subjects were patients who were newly initiated dialysis at the 17 Aichi Cohort Study of Prognosis in Patients Newly Initiated into Dialysis (AICOPP) group centers. The study included 1524 patients who were at least 20 years old, had CKD, and provided written consent. We excluded one patient whose serum adjusted calcium was not assessed and six patients whose outcomes were unknown. Thus, we enrolled 1517 subjects into the study. The patients were divided into the following five groups: (1) G1 with a serum adjusted calcium level

Background To clarify the therapeutic impact of tonsillectomy and combined therapies of tonsillectomy plus steroid on the long-term prognosis of immunoglobulin A nephropathy (IgAN). Methods A retrospective study was conducted on 208 patients with IgAN between 1986 and 2009. According to the strategies for treatments, patients were divided into four groups: tonsillectomy and steroid pulse (TSP, n = 47), tonsillectomy and oral steroid (TOS, n = 33), tonsillectomy alone (T, n = 56), and N group (no particular therapy, n = 72). Multivariate analysis based on the Cox’s regression model was used to assess the relative risk of reaching the outcome of doubling creatinine based on the influence of baseline prognostic factors. Results The mean observation periods were 53.8 months in the TSP group, 122.0 months in the TOS group, 102.9 months in the T group, and 84.6 months in the N group. During an observation period, serum creatinine levels doubled as follows: one in the TSP group (2.1 %), two in the TOS group (6.1 %), five in the T group (8.9 %), histological severity, and 22 in the N group (30.6 %). The Cox’s regression proportional hazard model showed that gender, age, histological activity, dialysis induction risk and therapy were associated with doubling creatinine levels. Hazard ratios (95 % CI) and (P value) in T, TOS, and TSP groups versus N were 0.314 (0.11–0.93, P = 0.037), 0.213 (0.04–1.10, P = 0.065), and 0.032 (0.00–0.28, P = 0.002), respectively. Conclusion A combination therapy of tonsillectomy and steroid pulse had the most significant therapeutic impact compared to other therapies.

Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody-secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.

The KM mouse lacks endogenous genes for immunoglobulins and carries the entire human IgH locus and the IgLk transgene. Therefore, human IgA1 does not provoke a hetero-immune response. We had observed mesangial IgA deposits in KM mice given desialo-degalacto (DeS/DeGal) IgA1. In this study, the mice were immunized with synthetic IgA1 hinge (glyco-)peptide before administration of DeS/DeGal IgA1, and the effects of the pre-immunization were evaluated. Mice were divided into sHP, 5GalNAc-sHP and non-immunization groups. In two pre-immunization groups, KLH-conjugated sHP or KLH-5GalNAc-sHP, which has five GalNAc residues, was subcutaneously given three times every 2 weeks. Two weeks after the final pre-immunization, DeS/DeGal IgA1 was administered daily for 5 weeks. Serial serum levels of anti-sHP and anti-IgA1 antibodies were evaluated by ELISA. On the day of the last administration of IgA1, renal biopsy was performed. Mesangial IgA deposits were observed in all non-immunized mice. In pre-immunized mice, IgA deposition was not detected in 6 of 13 sHP mice and 1 of 4 5GalNAc-sHP mice. The intensities of IgA deposits were significantly different between sHP groups and non-immunized (P = 0.003) groups. There was a significant inverse correlation between the intensities of IgA deposits and the anti-sHP antibody titers (P = 0.016). These results suggest that the anti-IgA1 hinge peptide antibody plays a role in the inhibition of glomerular IgA deposition.

Aims/hypothesis. Visceral adipocytes have different functions than those from the subcutaneous fat area. These differences could contribute to the pathological significance of excessive visceral fat accumulation for accompanying insulin resistance and hyperinsulinaemia. This study addresses this hypothesis and describes a unique method to clarify whether the functional differences between visceral and subcutaneous adipocytes depend on their anatomical location. Methods. 3T3-L1 cells or TNF-α overexpressing CHO cells were implanted into subcutaneous fat area or mesenteric area as visceral fat area in athymic mice of BALB/C strain. Then, serum insulin, glucose, TNF-α, and several markers of lipid metabolism were measured in the fasting condition. OGTT was also analysed. Results. During the course of glucose loading, the mice which had 3T3-L1 cells implanted into mesenteric area but not into subcutaneous fat area showed remarkably increased serum insulin and TMF-α concentrations, compared to the control mice. Moreover, serum insulin concentrations of the mice, implanted with TNF-α overexpressing cells into subcutaneous fat area, were apparently higher than that of control mice. Conclusion/interpretation. This method of implanting adipose cells into subcutaneous or visceral fat area showed high TNF-α concentration and insulin resistance by the adipose cells in visceral area of nude mice. Furthermore, we found that the functional significance of visceral fat accumulation for TNF-α-induced insulin resistance is partly caused by the interaction of adipocytes with surrounding conditions in mesenteric area. [Diabetologia (2002) 45: ▪–▪]

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions1,2,3, and are controlled by intracellular signals such as heterotrimeric G-proteins4, protein kinases5,6 and calmodulin (CaM)7,8. However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM9,10,11, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the β-subunit. The reduced channel activities are due to a decrease in α1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with β-subunits, regulates Ca2+ channel expression at the cell surface.

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.

Though various properties and applications of laser Doppler velocimetry have been extensively studied in the past decade, there is little discussion on the effect of light scattering from the surface of a cell on Doppler beat signals or on methods of reducing it. In this paper, the effect of light scattering from the surface of the cell is treated as a background noise and is studied theoretically and experimentally on the detecting process of Doppler beat signals in off-axis differential-type laser Doppler velocimetry. Laser Doppler velocimetry of an off-axis type is verified to be effective for measurement of the flow velocity in the vicinity of a scattering wall. The effect of the light scattered from the wall surface on the signal-to-noise ratio (SNR) of Doppler beat signals is discussed in detail. The minimum distance, which is close to the wall and at which good Doppler beat signals can be obtained, is defined and determined quantitatively. This minimum distance is found to be strongly affected by the off-axis angle of the detecting optical system.