Adsorptive filtration systems for effective removal of blood amyloid ß: a potential therapy for Alzheimer's disease.

Accumulation of amyloid-ß protein (Aß) in the brain causes cognitive impairment in Alzheimer's disease. We hypothesized that an extracorporeal system that rapidly removed Aß from the blood may accelerate Aß drainage from the brain. We previously reported that dialyzers remove blood Aßs effectively, mainly by adsorption on the inner surfaces of the hollow fibers, resulting in lower Aß accumulation in the brains of patients undergoing hemodialysis than the controls without hemodialysis. The aim of the present study was to create a more convenient and effective blood Aß removal system using adsorptive filtration, in which the filtrate returned to the body. Filtration from inside to outside of the fibers may enhance the adsorption of plasma Aßs on the surface of micropores inside the hollow fiber walls. Hence, pool solutions of 4 ng/mL synthetic Aß1-40 and Aß1-42 peptides (300 mL) or human plasma (1000 mL of 250-346 pg/mL Aß1-40 and 30-48 pg/mL Aß1-42) were circulated through polysulfone dialyzers at a flow rate of 50 mL/min to evaluate an adsorptive filtration system. The rates of Aß reduction from the pool solutions significantly increased along with the filtration rates. A filtration rate of > 1 mL/min, preferably 5-10 mL/min resulted in an 80-100% reduction of Aßs within 30 min of circulation. The rates of Aßs passing through the membrane walls were maintained around 0% for plasma Aßs during circulation. Thus, our adsorptive filtration systems may be useful for removing blood Aßs for patients with Alzheimer's disease.

The Level of IgA Antibodies to Endothelial Cells Correlates with Histological Evidence of Disease Activity in Patients with Lupus Nephritis.

Anti-endothelial cell antibodies (AECA) are frequently detected in patients with systemic lupus erythematosus (SLE), but their pathological role remains unclear. We recently developed a solubilized cell surface protein capture enzyme-linked immunosorbent assay (CSP-ELISA) to detect antibodies against membrane proteins involved in autoimmune reactions. In this study, sera from 51 patients with biopsy-proven lupus nephritis (LN), 25 with SLE without renal involvement (non-LN SLE), 42 disease control (DC) subjects, and 80 healthy control (HC) subjects were tested for IgG- and IgA-AECA for human umbilical vein endothelial cells (HUVEC) and human glomerular EC (HGEC) by using CSP-ELISA. IgG- and IgA-AECA titers were significantly higher in LN and non-LN SLE patients than in the DC or HC (P < 0.001) groups. IgG- and IgA-AECA titers for HUVEC corresponded well with those for HGEC. The IgA-AECA level correlated with the SLE disease activity index and with histological evidence of active lesions (cellular proliferations, hyaline thrombi and wire loops, leukocytic infiltration, and fibrinoid necrosis) in LN patients (P < 0.001). The sensitivity of IgA-AECA as a diagnostic test for histological evidence of active lesions in LN patients was 0.92, with a specificity of 0.70. The significant correlation of IgA-AECA with glomerular hypercellularity indicates that IgA-AECA are associated with endothelial damage in LN.

Toward the treatment for Alzheimer's disease: adsorption is primary mechanism of removing amyloid ß protein with hollow-fiber dialyzers of the suitable materials, polysulfone and polymethyl methacrylate.

The accumulation of amyloid ß protein (Aß) in the brain reflects cognitive impairment in Alzheimer's disease. We hypothesized that the rapid removal of Aß from the blood by an extracorporeal system may act as a peripheral Aß sink from the brain. The present study aimed to determine the optimal materials and modality for Aß removal by hemodialyzers. In a batch analysis, hollow-fiber fragments of polysulfone (PSf) and polymethyl methacrylate (PMMA) showed greater removal efficiency of Aß than did other materials, such as cellulose-triacetates and ethylene-vinyl alcohol copolymer (PSf:PMMA at 30 min, 98.6 ± 2.4 %:97.8 ± 0.4 % for Aß1-40 and 96.6 ± 0.3 %:99.0 ± 1.0 % for Aß1-42). In a modality study, the Aß solution was applied to PSf dialyzers and circulated in the dialysis and Air-filled adsorption-mode (i.e., the outer space of the hollow fibers was filled with air) or phosphate-buffered saline (PBS)-filled adsorption modes. The Aß1-40 removal efficiency of the pre/post dialyzer in the Air-filled adsorption-mode was the highest (62.4 ± 12.6 %, p = 0.007). In a flow rate study in the Air-filled adsorption-mode, 200 mL/min showed the highest Aß1-40 reduction rate of pool solution (97.3 ± 0.8 % at 15 min) compared with 20 mL/min (p = 0.00001) and 50 mL/min (p = 0.00382). PMMA dialyzers showed similar high reduction rates. Thus, the optimal modality for Aß removal was the adsorption-mode with PSf or PMMA hollow fibers at around 50 mL/min flow rate, which seems to be suitable for clinical use.

A prospective study on blood Aß levels and the cognitive function of patients with hemodialysis: a potential therapeutic strategy for Alzheimer's disease.

To obtain the proof of concept of a novel therapy for Alzheimer's disease (AD), we conducted two prospective studies with hemodialysis patients who had amyloid ß protein (Aß) removed from their blood three times a week. One major pathological change in the brain associated with AD is Aß deposition, mainly 40 amino acids Aß1-40 and 42 amino acids Aß1-42. Impaired Aß clearance is proposed to be one cause of increased Aß in the AD brain. Thus, we hypothesized that an extracorporeal removal system of Aß from the blood may remove brain Aß and be a useful therapeutic strategy for AD. In the first prospective study, plasma Aß levels and the cognitive function of 30 hemodialysis patients (65-76 years old) were evaluated at baseline as well as 18 or 36 months after. Although plasma Aß1-40 levels either decreased or remained unchanged, levels of Aß1-42 either remained unchanged or increased at the second time point. Mini-Mental State Examination scores of most subjects increased or were maintained at the second time point. Aß1-40 influx into the blood correlated with MMSE at the second time point. In the second prospective study, five patients (51-84 years old) with renal failure were evaluated before and after the initiation of hemodialysis. Plasma Aß levels decreased, while cognitive function improved after initiating blood Aß removal. Therefore, long-term hemodialysis, which effectively removes blood Aß, might alter Aß influx and help maintain cognitive function.

Appropriate nonwoven filters effectively capture human peripheral blood cells and mesenchymal stem cells, which show enhanced production of growth factors.

Scaffolds, growth factors, and cells are three essential components in regenerative medicine. Nonwoven filters, which capture cells, provide a scaffold that localizes and concentrates cells near injured tissues. Further, the cells captured on the filters are expected to serve as a local supply of growth factors. In this study, we investigated the growth factors produced by cells captured on nonwoven filters. Nonwoven filters made of polyethylene terephthalate (PET), biodegradable polylactic acid (PLA), or chitin (1.2-22 µm fiber diameter) were cut out as 13 mm disks and placed into cell-capturing devices. Human mesenchymal stem cells derived from adipose tissues (h-ASCs) and peripheral blood cells (h-PBCs) were captured on the filter and cultured to evaluate growth factor production. The cell-capture rates strongly depended on the fiber diameter and the number of filter disks. Nonwoven filter disks were composed of PET or PLA fibers with fiber diameters of 1.2-1.8 µm captured over 70% of leukocytes or 90% of h-ASCs added. The production of vascular endothelial growth factor (VEGF), transforming growth factor ß1, and platelet-derived growth factor AB were significantly enhanced by the h-PBCs captured on PET or PLA filters. h-ASCs on PLA filters showed significantly enhanced production of VEGF. These enhancements varied with the combination of the nonwoven filter and cells. Because of the enhanced growth factor production, the proliferation of human fibroblasts increased in conditioned medium from h-PBCs on PET filters. This device consisting of nonwoven filters and cells should be investigated further for possible use in the regeneration of impaired tissues.

Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy.

Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.

Global Gene Expression Profiling in PPAR-γ Agonist-Treated Kidneys in an Orthologous Rat Model of Human Autosomal Recessive Polycystic Kidney Disease.

Kidneys are enlarged by aberrant proliferation of tubule epithelial cells leading to the formation of numerous cysts, nephron loss, and interstitial fibrosis in polycystic kidney disease (PKD). Pioglitazone (PIO), a PPAR-γ agonist, decreased cell proliferation, interstitial fibrosis, and inflammation, and ameliorated PKD progression in PCK rats (Am. J. Physiol.-Renal, 2011). To explore genetic mechanisms involved, changes in global gene expression were analyzed. By Gene Set Enrichment Analysis of 30655 genes, 13 of the top 20 downregulated gene ontology biological process gene sets and six of the top 20 curated gene set canonical pathways identified to be downregulated by PIOtreatment were related to cell cycle and proliferation, including EGF, PDGF and JNK pathways. Their relevant pathways were identified using the Kyoto Encyclopedia of Gene and Genomes database. Stearoyl-coenzyme A desaturase 1 is a key enzyme in fatty acid metabolism found in the top 5 genes downregulated by PIO treatment. Immunohistochemical analysis revealed that the gene product of this enzyme was highly expressed in PCK kidneys and decreased by PIO. These data show that PIO alters the expression of genes involved in cell cycle progression, cell proliferation, and fatty acid metabolism.

Abnormalities of glycogenes in tonsillar lymphocytes in IgA nephropathy.

Glycosylation, which represents the most complex post-translational modification, plays a pivotal role during protein maturation, and is orchestrated by numerous glycosyltransferases. Aberrant O-glycosylation of serum and tonsillar IgA1 is presumed to be one of the pathogeneses of IgA nephropathy (IgAN). However, the synthesis of underglycosylated IgA1 in tonsils has not yet been characterized. This study investigated tonsillar B lymphocytes of IgAN using tonsils from patients with chronic tonsillitis and sleep apnea syndrome. Gene expression of ß1,3-galactosyltransferase (ß3GalT), Cosmc, UDP-N-acetyl-α-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase 2, were significantly down regulated in tonsillar CD19-positive B lymphocytes from IgAN patients compared to control as determined by real-time RT-PCR. In contrast, the level of sialyltransferase was not significantly different among the three groups. Tonsillar B cell ß3GalT gene expression significantly correlated with estimated GFR and negatively correlated with proteinuria and glomerular or interstitial injury score. Double immunofluorescent staining showed that some IgA-positive cells in the intrafollicular area were also positive for ß3GalT staining. Western blotting showed the protein expression of ß3GalT in the tonsils to significantly decrease in IgAN in comparison to the controls. These data suggest the downregulation of ß3GalT in tonsillar B lymphocytes to be closely associated with the clinical characteristics of IgAN.

PPAR-gamma agonist ameliorates kidney and liver disease in an orthologous rat model of human autosomal recessive polycystic kidney disease.

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-ß-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Evaluation of blood purification and bortezomib plus dexamethasone therapy for the treatment of acute renal failure due to myeloma cast nephropathy.

Aggressive removal of circulating free light chains (FLC) by blood purification accompanied by chemotherapy is a promising approach for the treatment of acute renal failure due to myeloma cast nephropathy. Plasma exchange has been performed to remove serum FLC; in order to examine an alternative strategy we performed hemodiafiltration using protein-leaking dialyzers for the treatment of dialysis-dependent acute renal failure due to myeloma cast nephropathy. In the first case with κ-light chain cast nephropathy, the pre-treatment serum creatinine was 9.65 mg/dL, and the serum κ-FLC was 27100 mg/L. Plasma exchange or hemodiafiltration was performed from Monday to Friday during the first several weeks. Chemotherapy was started with high-dose dexamethasone and then switched to bortezomib plus dexamethasone. The mean removal rates of κ-FLC were 45.8% (one plasma volume) and 66.9% (one-and-a-half plasma volumes) by plasma exchange. The removal rates of κ-FLC by hemodiafiltration (66.9%, FB210UHß; 71.6%, PES210Dα; 75.2%, FXS220) were comparable to those by plasma exchange. In the second case with λ-light chain cast nephropathy, the pre-treatment serum creatinine was 4.14 mg/dL, and the serum λ-FLC was 4140 mg/L. The mean removal rates of λ-FLC were 60.2% (FXS140) and 64.2% (FB210UHß) by hemodiafiltration. Both cases became dialysis-independent. The combination of an intense blood purification regimen and bortezomib plus dexamethasone therapy appears to be an efficient approach to renal recovery. Hemodiafiltration using protein-leaking dialyzers could become an alternative to plasma exchange as a method of removing FLC.

Differential expression of glycogenes in tonsillar B lymphocytes in association with proteinuria and renal dysfunction in IgA nephropathy.

Aberrant O-glycosylation of serum and tonsillar IgA1 is one of the main pathogeneses of IgA nephropathy (IgAN). However, the synthesis of underglycosylated IgA1 in tonsils has not yet been characterized. This study examined tonsillar B lymphocytes of IgAN (n=34) using tonsils derived from patients with chronic tonsillitis (n=24) and sleep apnea syndrome (n=14) as a control. Gene expression of beta1,3-galactosyltransferase (beta3GalT), and the core 1 beta3GalT-specific molecular chaperone, Cosmc, UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase 2, were significantly decreased in tonsillar CD19-positive B lymphocytes from IgAN patients compared to control tonsillar tissues as determined by real-time RT-PCR. Tonsillar B cell beta3GalT gene expression significantly correlated with estimated GFR and negatively correlated with proteinuria and histological injury score. Western blotting showed the protein expression of beta3GalT in the tonsils to significantly decrease in IgAN in comparison to the controls. These data suggest the downregulation of beta3GalT in tonsillar B lymphocytes to be closely associated with the clinical characteristics of IgAN.

Evaluation of the specific structures of IgA1 hinge glycopeptide in 30 IgA nephropathy patients by mass spectrometry.

BACKGROUND: Although many reports have described the abnormal structures of O-glycan in the IgA1 hinge region in IgA nephropathy (IgAN), the specific glycopeptide that can be used as a diagnostic biomarker for IgAN has not yet been identified. To pursue this diagnostic approach, we used mass spectrometric analysis to search for specific structures in IgA1 hinge glycopeptides in 30 IgAN patients contrasted with 30 healthy controls. METHOD: IgA1 hinge glycopeptides were individually purified from the sera of 30 biopsy-proven IgAN patients and 30 healthy controls. The structure of each glycopeptide was analyzed by ion trap mass spectrometry. Sugar conformations in each glycopeptide were estimated by collision-induced dissociation tandem mass spectrometry. Furthermore, to search for specific O-glycans in IgAN patients with a statistical significance, the identified hinge glycopeptides were analyzed by Fisher exact test. RESULT: A total of 57 hinge glycopeptides were identified from each of the 2 sample groups. Among the structures of the hinge glycopeptides identified, statistical significance was found for 6 glycopeptides (O-glycan compositions were 33-mer hinge core peptides + xGalNAc + yGal + zNeu5Ac; x:y:z = 5:3:3, 5:3:0, 5:2:1, 4:2:2, 3:1:1, 3:1:0) by Fisher exact test (p<0.05). In these 6 O-glycan compositions, 3 compositions (x:y:z = 4:2:2, 3:1:1, 3:1:0) were only observed in IgAN patients and were absent in the 30 controls. CONCLUSION: Statistically specific O-glycans were identified in 30 IgAN patients compared with 30 controls. These results open the possibility for preparation of lectin and/or antibodies binding to specific glycopeptides in IgAN.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Serum levels of 5-s-cysteinyldopa are correlated with skin colors in hemodialysis patients but not in peritoneal dialysis patients.

BACKGROUND: Diffuse hyperpigmentation is common in patients with chronic renal failure undergoing hemodialysis (HD) or peritoneal dialysis (PD). We previously reported that serum levels of 5-S-cysteinyldopa (5SCD, a pheomelanin precursor) and pheomelanin were significantly elevated in HD patients. METHODS: Skin color was assessed using a Mexameter that measures the melanin index (MI) and the erythema index (EI). The upper inner arms (non-sun-exposed site) and the foreheads (sun-exposed site) of HD and PD patients and control subjects were analyzed. RESULTS: MI values on the upper inner arms and on the foreheads of HD and PD patients were significantly higher than in controls. In HD patients, significant correlations were found for serum 5SCD levels with MI and EI on the upper inner arm, and for EI on the forehead. In PD patients, no such correlations were found. CONCLUSIONS: Hyperpigmentation in HD patients results partly from accumulation of pheomelanin in the skin.

Expression of tumor necrosis factor receptors on granulocytes in patients with myeloperoxidase anti-neutrophil cytoplasmic autoantibody-associated vasculitis.

BACKGROUND/AIMS: To clarify the clinical significance of tumor necrosis factor (TNF) receptors in patients with myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, we evaluated the cell surface expression of TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). PATIENTS AND METHODS: 43 patients with MPO-ANCA-associated vasculitis, 16 patients with chronic renal failure, 10 patients with sepsis, 15 patients with systemic lupus erythematosus, and 18 healthy controls were enrolled in this study, and the surface expression levels of TNFR1, TNFR2, CD63, and CD64 on granulocytes were assessed. In 21 patients with MPO-ANCA-associated vasculitis, soluble TNFR1 (sTNFR1), soluble TNFR2(sTNFR2), and TNF-alpha in the serum were also measured. RESULTS: The surface expression levels of TNFR1 and TNFR2 on granulocytes were significantly higher in patients with MPO-ANCA-associated vasculitis than in the healthy controls, and positively correlated with the Birmingham Vasculitis Activity Score (BVAS). The levels of sTNFR1, sTNFR2, and TNF-alpha in the serum were also significantly higher in patients with MPO-ANCA-associated vasculitis than in the healthy controls. Serum levels of sTNFR1 and sTNFR2 correlated with serum creatinine, while the surface expression of TNFR1 and TNFR2 on the granulocytes did not. There was no significant correlation between the BVAS and CD63 or BVAS and CD64. CONCLUSION: The surface expression levels of TNFR1 and TNFR2 on granulocytes were upregulated in patients with MPO-ANCA-associated vasculitis and reflected disease activity.

The HLA-DRB1*1501 allele is prevalent among Japanese patients with anti-glomerular basement membrane antibody-mediated disease.

BACKGROUND: We aimed to clarify the relationship between HLA-DRB1(*)1501 and anti-glomerular basement membrane (GBM) antibody-mediated disease in Japanese patients. MATERIALS: Samples were collected from 16 anti-GBM antibody-positive patients who were admitted to our department or related hospitals from December 1990 to October 2005. We analysed clinical and laboratory data, kidney biopsy findings, and the HLA-DR phenotypes and HLA-DRB1 alleles of the patients. RESULTS: Among the 16 patients, 15 had HLA-DR15 [the phenotype frequency (PF) was 93.8%], 7 were positive for DR4 (the PF was 43.8%) and 5 were positive for DR9 (the PF was 31.3%). The allele frequency of HLA-DRB1(*)1501 was 46.4% (13/28), which was significantly different from Japanese controls (11.6%) (P < 0.001). In contrast, the frequency of HLA-DRB1(*)1502 was not different from controls (0/28). The odds ratio of HLA-DRB1(*)1501 in these patients was 6.4 (95% CI: 2.4-16.5). CONCLUSION: The present study demonstrated that Japanese patients with anti-GBM antibody-mediated disease are very likely to carry the HLA-DRB1(*)1501 but not the HLA-DRB1(*)1502 allele.

Protective role of anti-synthetic hinge peptide antibody for glomerular deposition of hypoglycosylated IgA1.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: These results suggest that the anti-IgA1 hinge peptide antibody plays a role in the inhibition of glomerular IgA deposition.

Severe metabolic acidosis and hypokalemia in a patient with enterovesical fistula.

We report a case of a 59-year-old woman who had severe metabolic acidosis and hypokalemia due to an enterovesical fistula. The patient came to our hospital complaining of systemic weakness and numbness of the fingers. She was found to have hyperchloremic metabolic acidosis (arterial bicarbonate, 2.8 mEq/l) and hypokalemia (serum potassium, 1.9 mEq/l) and was admitted for treatment. Following the correction of metabolic acidosis and hypokalemia, the patient was examined for the underlying cause of these electrolyte and acid-base disorders. She had a history of total hysterectomy followed by radiotherapy due to uterine cancer 30 years previously. After the surgery, she had suffered postoperative neurogenic bladder dysfunction, necessitating intermittent self-catheterization. Two years before admission, she had begun to experience watery diarrhea. A radiographic study after recovery from the acid-base and electrolyte disorders revealed the presence of an enterovesical fistula. The fistula was surgically resected and the metabolic acidosis completely cleared. Unexplained hyperchloremic metabolic acidosis with hypokalemia may suggest the presence of an enterovesical fistula in patients with a surgical history of malignant pelvic tumor and neurogenic bladder dysfunction.

Effects of cytapheresis on tumor necrosis factor receptor and on expression of CD63 in myeloperoxidase--antineutrophil cytoplasmic autoantibody-associated vasculitis.

To evaluate the therapeutic potential of cytapheresis in myeloperoxidase-antineutrophil cytoplasmic autoantibody (MPO-ANCA)-associated vasculitis, plasma levels of soluble tumor necrosis factor receptors (sTNFR1, sTNFR2) and the expression of TNFR1, TNFR2, and CD63 on granulocytes were measured. The levels of sTNFR1 and sTNFR2, and the expression of TNFR1 and TNFR2 were significantly higher in MPO-ANCA-associated vasculitis patients than in normal controls. The levels of sTNFR1 and sTNFR2 increased significantly after cytapheresis (P < 0.001). The expression of TNFR1 showed a tendency to decrease after cytapheresis (P = 0.0535). The expression of CD63 decreased significantly after cytapheresis (P < 0.05). Because sTNFR1 and sTNFR2 act as TNF-antagonists, the increases of sTNFR1 and sTNFR2 after cytapheresis might contribute to inhibit the action of TNF-alpha. The decreased expression of TNFR1, which mediates the signal for polymorphonuclear cell respiratory burst, might also contribute to the reduction of inflammation. From these results, the inhibition of TNF action and removal of degranulated granulocytes appear to be related to the mechanism whereby cytapheresis can exert a beneficial and therapeutic function in the treatment of MPO-ANCA-associated vasculitis.

Downregulation of the beta1,3- galactosyltransferase gene in tonsillar B lymphocytes and aberrant lectin bindings to tonsillar IgA as a pathogenesis of IgA nephropathy.

IgA is a glycoprotein with multiple O-glycans. Under-O-glycosylation of the hinge in IgA in patients with IgA nephropathy (IgAN) is reported. The development of IgAN is frequently preceded by episodes of upper respiratory tract infections such as tonsillitis. Therefore, the tonsils may be related to the pathogenesis of IgAN. However, the mechanism of underglycosylation in tonsillar IgA has not yet been fully elucidated. Since O-glycans in IgA are produced by glycosyltransferases, we hypothesized that dysregulation of the enzymes is associated with underglycosylation.