A comparative study of the diagnostic accuracy of ELISA systems for the detection of anti-neutrophil cytoplasm antibodies available in Japan and Europe.

OBJECTIVES: Primary systemic vasculitis associated with anti-neutrophil cytoplasm antibodies (ANCA) differs in its frequency and clinical expression between Japan and Europe. We sought to ascertain whether such differences arise from the performance of enzyme-linked immunosorbent assays (ELISAs) for ANCA. METHODS: Plasma samples from 64 consecutive Japanese patients with a clinical and histological diagnosis of primary systemic vasculitis including microscopic polyangiitis (MPA; n=52), Churg-Strauss syndrome (CSS; n=1), and Wegener's granulomatosis (WG; n=11), or those from disease controls with non-vasculitic glomerulonephritis (n=54) and healthy controls (n=55) were tested for the presence of myeloperoxidase (MPO) by ELISAs available in Japan (Nipro and MBL) and compared with those in Europe (Wieslab). The sensitivity and specificity were calculated for each ELISA, and its diagnostic performance was assessed by receiver operating characteristic curve analysis. RESULTS: The sensitivity and specificity of either MPO-ANCA assays for a diagnosis of MPA were 90.4% and 98.2% (Nipro), 88.2% and 96.3% (MBL), and 86.5% and 99.1% (Wieslab). The overall diagnostic performance, assessed as the area under curve of the MPO-ANCA ELISAs for MPA were 0.946+/-0.022 (Nipro), 0.970+/-0.017 (MBL), and 0.971+/-0.017 (Wieslab), while that of PR3-ANCA ELISAs for WG were 0.986+/-0.025 (Nipro), 0.993+/-0.017 (MBL), and 0.916+/-0.059 (Wieslab). CONCLUSIONS: The MPO-ANCA ELISAs commercially available in Japan exhibited high sensitivity and specificity for the diagnosis of ANCA-associated vasculitides and provided similar diagnostic value to those in Europe. These results facilitate further international comparison of ANCA-associated vasculitides between Japanese and European populations.

Induction of continuous ambulatory peritoneal dialysis after retroperitoneoscopic nephrectomy.

Induction of continuous ambulatory peritoneal dialysis (CAPD) as treatment of end-stage renal disease is difficult for patients requiring nephrectomy with traditional surgery, and usually hemodialysis is selected for these patients. In a 61-year-old woman with end-stage renal failure a left renal tumor was diagnosed by abdominal ultrasonography, enhanced computed tomography and magnetic resonance imaging. Following an urology consultation, we decided to perform left kidney nephrectomy. We estimated that she had to undergo dialysis permanently after nephrectomy. She desired to be treated by CAPD, however, we decided after allowing for a postoperative period for complete healing of the peritoneum to avoid complications. This is why during the interim period between surgery and induction of CAPD she underwent hemodialysis (HD) in a local outpatient HD center and in our hospital. We selected a retroperitoneoscopic approach for nephrectomy. Pathology evaluation revealed a renal cell carcinoma. 4 months after nephrectomy, CAPD catheter implantation was performed by using laparoscopy and CAPD was started. At the present time, the patient is doing well on CAPD. To our knowledge, there are no clear indications regarding initiation of peritoneal dialysis after nephrectomy. The retroperitoneoscopic approach for nephrectomy allows for initiation of peritoneal dialysis after nephrectomy within a relative short postoperative period.

Glomerulonephritis induced in the rabbit by antiendothelial antibodies.

The effects of interaction between endothelial angiotensin converting enzyme (ACE) and goat anti-rabbit ACE (GtARbACE) antibodies were studied in rabbit glomeruli. By immunofluorescence ACE was not detectable in normal glomeruli. However, when kidneys were perfused with GtARbACE antibodies glomerular bound IgG was seven times higher than that of non-immune IgG and granular deposits of goat IgG were found on the endothelium of glomeruli and arteries. Rabbits injected intravenous for 4 d with GtARbACE antibodies showed on day 1 granular deposits of goat IgG on the glomerular endothelium; from day 3 to 24 there was gradual development of subepithelial deposits of goat IgG, rabbit IgG and C3. When GtARbACE antibodies were similarly injected into proteinuric rabbits there was formation of subepithelial granular deposits of goat IgG and ACE. The results document that a glomerular endothelial antigen is redistributed in vivo by a specific ligand, an event associated with formation of immune deposits. Furthermore, if the glomerular permeability is artificially increased, immune complexes shed from nonglomerular endothelia into the circulation can contribute to form subepithelial immune deposits.

Enhanced expression of interleukin-6 in primary human renal cell carcinomas.

We have demonstrated interleukin-6 (IL-6) production by human renal carcinoma cells. The IL-6 gene expression was detected by Northern blot analysis in 22 of 43 primary renal cell carcinoma tissues and in five of seven renal cell carcinoma cell lines. Immunohistochemical analysis confirmed the expression of IL-6 by the tumor cells. Patients with a high-level expression of IL-6 had significantly greater incidences of lymph node metastasis and a larger increase in serum C-reactive protein than those without it. We have also probed for the presence of IL-6 receptor by Northern blot analysis; we detected this receptor in 11 of the 43 primary renal cell carcinoma tissues but in none of the seven renal cell carcinoma cell lines. However, by use of the complementary DNA-polymerase chain reaction, the IL-6 receptor transcript was detected in all specimens, including the seven cell lines. No expression of the interleukin-3 (IL-3) gene was identified in any of the 43 primary renal cell tumors. These data provide evidence that IL-6 and its receptor may play a role in promoting the transformation and/or proliferation of renal cell carcinomas as well as in teh development of symptoms.

LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade.

Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to the neurofibromatosis type 2 (NF2)-Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the whole mechanism of disordered YAP activation in MMs has not yet been well clarified. In the present study, we investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently showed downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor type 2 (LATS2), which is one of the last-step kinases of the NF2-Hippo pathway. Although loss of AJUBA expression was independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We found that AJUBA transduction into MM cells significantly suppressed promoter activities of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of the MM cells that harbored ordinary LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation.

Acute renal failure and degenerative tubular lesions associated with in situ formation of adenovirus immune complexes in a patient with allogeneic bone marrow transplantation.

We describe the development of acute renal failure and degenerative tubular lesions associated with local immune deposits in a patient with allogeneic bone marrow transplantation. A 21-year-old man with an acute myelocytic leukemia received a bone marrow graft from a cousin mismatched for a single HLA-DR locus antigen. Hemorrhagic cystitis due to adenovirus type 11 infection occurred 26 days after transplantation, and 17 days later the patients developed acute renal failure. A study of renal tissue obtained by needle biopsy showed degenerative and necrotic lesions, especially in the distal part of the nephron. By electron microscopy adenovirus type 11 particles were found in the nuclei of tubular cells and in cellular debris in tubular lumina. By immunofluorescence technique, granular immune deposits containing adenovirus type 11 related antigen(s), immunoglobulins, C3, and membrane attack complex (MAC) C5b-9 of the complement system were detected along the tubular basement membranes but not in glomeruli. The patient's IgG did not bind to normal human kidneys. These findings suggest that adenovirus type 11 directly induced acute tubular damage, and that the tubular immune deposits were formed "in situ" by viral antigens and circulating viral antibody.

Increased plasma thrombin-antithrombin III complex levels in non-insulin dependent diabetic patients with albuminuria are reduced by ethyl icosapentatenoate.

Hypercoagulability may increase the risk of cardiovascular disease (CVD) in diabetic patients with albuminuria. Plasma thrombin-anti-thrombin III complex (TAT) levels, representing a functional state of clotting system, were studied in one hundred and fifteen non-insulin-dependent diabetic (NIDDM) patients. The patients were divided into three groups according to the urine albumin index (UAI: mg/g Cr): Group A; UAI < 30, Group B; 30 < UAI < 300, Group C; UAI > 300. The effect of albuminuria on plasma TAT levels was significant (p < 0.02). Ethyl icosapentatenoate (EPA: 1800 mg/day) for 4 weeks significantly (p < 0.0005) decreased plasma TAT levels. These data indicate that the degree of diabetic albuminuria is related to plasma TAT levels in NIDDM patients and that treatment with EPA may reduce TAT levels and possibly therefore the rate of development of CVD in patients with NIDDM.

The glomerular distribution of type IV collagen and laminin in human membranous glomerulonephritis.

The glomerular distribution of type IV collagen and laminin, the major collagenous and noncollagenous components of the glomerular basement membrane, was studied by immunofluorescence microscopy in idiopathic and lupus membranous glomerulonephritis. Affinity-purified antibodies against type IV collagen reacted preferentially with the inner aspect and irregularly with the adjacent outer area of the thickened basement membrane. In contrast, laminin was detected along the inner aspect of the glomerular basement membrane, in subepithelial basement membrane protrusions ("spikes"), and in the newly formed basement membrane layer above the immune deposits. We conclude that type IV collagen and laminin do not codistribute in the newly formed matrix. This aberrant antigenic distribution may reflect a loss of coordinate biosynthesis or degradation of these matrix components by visceral epithelial cells.

Localisation of intrahepatic interleukin 6 in patients with acute and chronic liver disease.

AIM: To evaluate the role of local interleukin 6 (IL-6) in the pathogenesis of acute and chronic liver disease. METHODS: The cellular site of IL-6 in cryostat sections of liver from 31 patients with liver disease was examined using indirect immunofluorescence with a monoclonal antibody. RESULTS: IL-6 staining in sinusoidal endothelial cells was very noticeable and diffusely distributed in the lobules of specimens of acute viral hepatitis. IL-6 expression in endothelial cells, particularly in necrotic areas of hepatocytes, was increased and was accompanied by enhanced expression in Kupffer cells. In contrast, IL-6 staining in infiltrating mononuclear cells was prominent in portal tracts, and the numbers of cytokine positive cells were greater in specimens of chronic active hepatitis compared with chronic persistent hepatitis. In non-specific reactive hepatitis intrahepatic expression of IL-6 was minimal, while in alcoholic liver fibrosis the cytokine distribution in the lobules was similar to that of acute viral hepatitis. CONCLUSIONS: These results indicate that locally produced IL-6 contributes to the inflammatory process and immunological response in acute and chronic liver disease.

Role of transforming growth factor beta 1 on hepatic regeneration and apoptosis in liver diseases.

AIMS: To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on regeneration and induction of apoptosis of liver cell and bile duct in various liver diseases. METHODS: Formalin fixed paraffin wax sections of 18 liver tissue samples were obtained by needle biopsy, surgery, or necropsy; these included six liver cirrhosis, three obstructive jaundice; five fulminant hepatitis, one subacute hepatitis, and three normal liver. Expression of TGF-beta 1, apoptosis related Le(y) antigen, Fas antigen, a receptor for tumour necrosis factor, and biotin nick end labelling with terminal deoxynucleotidyl transferase mediated dUTP (TUNEL) for locating DNA fragmentation, was investigated histochemically. RESULTS: TGF-beta 1 was expressed in areas of atypical bile duct proliferation, where bile duct continuously proliferated from liver cells. In occlusive jaundice and fulminant hepatitis, TUNEL was positive in nuclei and cytoplasm of metaplastic cells which formed incomplete bile ducts, and these cells appeared to extend from TGF-beta 1 expressing liver cells. Fas antigen was found only on the cell membrane of proliferated bile duct in fulminant hepatitis, which differed from TGF-beta 1 and TUNEL positive areas. Le(y) antigen was expressed in liver cell and bile duct at the areas with atypical bile duct proliferation, but its coexpression with TUNEL was rare. CONCLUSIONS: TGF-beta 1 plays a role in the arrest of liver cell regeneration and atypical bile duct proliferation, and in areas of rapidly progressing atypical bile duct proliferation, such as in fulminant hepatitis or bile retention. Apoptosis appears to be induced by TGF-beta 1. This phenomenon may account for the inadequate hepatic regeneration that occurs with liver disease.

Effect of the lipoprotein lipase activator NO-1886 on adriamycin-induced nephrotic syndrome in rats.

Hyperlipidemia associated with nephrotic syndrome may play a role in the deterioration of renal function. Tsutsumi et al have previously reported that the novel compound NO-1886 increases lipoprotein lipase (LPL) activity, resulting in a reduction of plasma triglycerides and an elevation of high-density lipoprotein (HDL) cholesterol in normal rats. The aim of this study was to ascertain whether NO-1886 suppresses the renal injury by treatment of the hyperlipidemia in an Adriamycin (Kyowa Hakko Kogyo, Tokyo, Japan) induced nephrosis rat model fed a high-protein diet that induced renal dysfunction and tubulointerstitial injury. Administration of Adriamycin caused hyperlipidemia, proteinuria, and edema with ascites in rats in 4 weeks. Furthermore, a combination of Adriamycin and a high-protein diet increased plasma creatinine and blood urea nitrogen (BUN) and decreased plasma albumin. Histologically, in Adriamycin-treated rats, marked interstitial cellular infiltration, tubular lumen dilation, and tubular cast formation in the kidney were observed. NO-1886 decreased plasma triglyceride and increased HDL cholesterol in Adriamycin-induced nephrotic rats. NO-1886 treatment reduced plasma creatinine and BUN levels and increased plasma albumin in Adriamycin-treated rats; it also ameliorated the ascites and proteinuria. Histologically, NO-1886-treated rats showed a quantitatively significant preservation of tubulointerstitial lesions. These data suggest that NO-1886 may have a protective effect against Adriamycin-induced nephrosis with tubulointerstitial nephritis in rats by a modification of the plasma lipid disorder.

Central administration of Lys-D-Pro-Thr, an interleukin-1 beta 193-195 analogue, stimulates feeding in rats.

We determined the effect on feeding of Lys-D-Pro-Thr (LDPT), an interleukin-1 beta 193-195 analogue which antagonizes the analgesic effects of interleukin-1 beta. Intracerebroventricular administration of 2 micrograms/rat LDPT increased food consumption at the 0-1 h time period, although food intake was reduced by LDPT at the 1-2 h time period. There was no effect on colonic temperature 1 h later. Subcutaneously injected LDPT (2 micrograms/rat) failed to increase food intake for 1 h. These data suggest that brain interleukin-1 beta may have a physiological role in feeding suppression.

A lipoprotein lipase activator, NO-1886, improves endothelium-dependent relaxation of rat aorta associated with aging.

Endothelial function is closely related to development of atherosclerosis and is impaired with aging. The novel compound NO-1886, 4-diethoxyphosphorylmethyl-N-(4-bromo-2-cyanophenyl)benzamid e, is a lipoprotein lipase activator and its long term administration protects against the development of experimental atherosclerosis in animals. The aim of this study was to ascertain whether NO-1886 ameliorates the impaired endothelium-dependent relaxation of rat aorta associated with aging. NO-1886 (50 mg/kg p.o.) was administered to 7-month old rats for 3 months. Plasma lipid, glucose and insulin levels in old control rats (10 months of age) were significantly higher than those of young rats (2 months of age). NO- 1886 decreased plasma triglyceride levels (old rats, 233+/-10 mg/dl; old rats + NO-1886, 172+/-16 mg/dl, P < 0.01) and increased plasma high density lipoprotein (HDL) cholesterol level (old rats, 72+/-6 mg/dl; old rats + NO-1886, 142+/-6 mg/dl, P < 0.001) in old rats, but had no effects on plasma glucose or insulin. The endothelium-dependent relaxation of the thoracic aorta caused by histamine was significantly impaired in old rats (% relaxation at 10(-5.5) M histamine: young rats 25.4+/-3.1%; old rats 14.1+/-1.9%, P < 0.01), an effect completely prevented by NO-1886 (old rats + NO-1886; 22.8+/-2.8%, P < 0.05 vs. old rats). In contrast, NO-1886 showed no effect on the endothelium-independent relaxation by sodium nitroprusside. These results indicate that NO-1886 improves impaired endothelium-dependent relaxation of rat aorta associated with aging, possibly by correcting lipid metabolism.

50-mile walking race suppresses neutrophil bactericidal function by inducing increases in cortisol and ketone bodies.

To examine the effect of intensive aerobic exercise on the interaction between endocrine and immune systems, we studied in ten normal healthy male subjects the effect of a 50-mile walking race on blood concentration of hormones (insulin, GH, ACTH, cortisol, adrenaline, noradrenaline, and dopamine), ketone bodies, specific immunological functions (IgG, IgM, and PHA/Con A-induced lymphocyte blastformation test), and nonspecific immune (CH50, and neutrophil bactericidal functions). Neutrophil bactericidal activity was measured as chemiluminescences amplified by luciferin analog (CLA-DCL) and luminol (L-DCL). The race increased cortisol and ketone bodies, and decreased insulin, CLA-DCL, and L-DCL (all parameters; P < 0.01). However, other parameters were not significantly changed. There were significant negative correlations between changes of ketone bodies/cortisol and CLA/L-DCL (P < 0.05), however there was no significant correlations between changes of insulin and CLA/L-DCL. These data indicate that extensive aerobic exercise causes impaired neutrophil bactericidal function, probably due to the induced increases in both cortisol and ketone bodies. This impaired neutrophil function may cause the susceptibility to infection after an extensive exercise.

The significance of decreased ambulatory activity during the generation by long-term observation of obesity in ovariectomized rats.

We attempted to determine the significance of ambulatory activity as a cause of overweight in ovariectomized rats. Drinking and ambulation were measured continuously and directly for periods up to 12 months in special apparatus developed at Gunma University. In older rats, ambulatory activity decreased much more in the ovariectomy group than in the control group. There was no difference in food intake between the ovariectomized and the control group. After 2 months, the ovariectomy group increased body weight more than the control group despite no difference in food intake. The decrease in ambulatory activity was consistent in the ovariectomy group, regardless of any differences in age and body weight. These results indicate that decrease of energy expenditure by gradual decrease in ambulatory activity may be an important factor as a cause of overweight in ovariectomized, obese rats.

Surgical treatment of hemodialysis-related shoulder arthropathy.

Thirteen patients with hemodialysis-related shoulder arthropathy were treated either with arthroscopic synovectomy or with open surgery. Arthroscopic synovectomy was performed in eight patients who had shoulder pain, shoulder immobility or both but did not have cystic bone lesions. The therapy was effective for pain relief and improvement of shoulder function for six months but in 12 months the shoulder pain reappeared in most of the patients. Open surgery was done in 5 patients who, in addition to shoulder pain and immobility, had humeral head bone cysts. Resection of the deposited mass on the biceps tendon sheath, of hypertrophied synovium and bursa as well as curettage of cysts and calcium hydroxyapatite ceramic implantation were performed. The therapy was effective for pain relief throughout the follow-up period (12 months). No adverse effects were noted for either procedure. Resected specimens of the synovia contained amyloid as indicated by a positive Congo-red stain by light microscopy and the presence of amyloid fibrils by electron microscopy. Deposition of amyloid in the biceps tendon sheath, synovium and bursa and invasion of the humeral head by amyloid were observed upon open surgery. The results suggest that the resection of deposited material induces the improvement of the shoulder arthropathy.

The kidney disease of Crow-Fukase (POEMS) syndrome: a clinico-pathological study of four cases.

We studied four cases of Crow-Fukase syndrome with renal dysfunction. Kidney specimens obtained by needle biopsy showed glomerular lesions resembling those seen in conditions characterized by microangiopathy. Common glomerular findings by light microscopy were mesangial expansion and narrowing of the capillary lumina. An enlarged subendothelial space and mesangial area with deposition of amorphous material as well as swelling and vacuolization of endothelial cells were observed by electron microscopy. In an active phase, severe mesangial edema and segmental mesangiolysis, and in a late stage, mesangial cell interposition and sclerosis were seen. Tests by immunofluorescence microscopy for the presence of immunoglobulins A, M, G, lambda and kappa light chains, C3, and C4 were negative. Decay accelerating factor was found in glomeruli and in the vascular pole. Other findings included lymph node angiosclerosis, peripheral nerve microangiopathy and hemangioma formation with endothelial cell proliferation. These observations suggest that chronic endothelial injury constitutes the basic pathology of Crow-Fukase syndrome. Hemodialysis was required to manage anasarca in three of the patients although serum creatinine levels were below 5.0 mg/dl. Urinalysis revealed mild abnormalities and did not reflect the severity of the glomerular lesion. Corticosteroids given to three of the patients were effective in controlling fever and the lymphadenopathy; in two cases the corticosteroids induced a recovery of renal function. Thus Crow-Fukase syndrome may be due to chronic endothelial injury; the clinical symptoms and renal involvement respond to corticosteroid therapy.

Urinary levels of IL-6 in patients with active lupus nephritis.

Using the IL-6 dependent hybridoma, MH60.BSF2, we measured urinary levels of interleukin 6 (IL-6) in 29 patients with active lupus nephritis. We detected IL-6 activity in the urine of 24 (83%) of 29 patients before the initiation of therapy. The median value of urinary IL-6 levels in patients with a histologic diagnosis of WHO class IV on renal biopsy was significantly higher than that in patients with other classes (p < 0.01). After treatment, urinary levels of IL-6 decreased significantly (p < 0.001). These data suggest that urinary levels of IL-6 may be a valuable tool for monitoring the progression of lupus nephritis.

Expression of basic helix-loop-helix proteins in the glomeruli.

BACKGROUND: Basic helix loop helix (bHLH) proteins play a critical role in the differentiation of not only striated muscle cells but also adipocytes, neuron cells and smooth muscle cells. Previous studies have established in vitro mouse mesangial cells (MCs) to maintain the differentiated smooth muscle phenotype. MATERIALS AND METHODS: The purpose of the present study was to clone bHLH proteins from these MCs using the primers designed from a homologous sequence specific to bHLH, and to analyze the presence of bHLH proteins in normal kidney in vivo. From the cloning of MCs in vitro, we identified myf5 and herculin mRNA but not myoD. The expression of bHLH proteins in vivo was examined by immunohistochemistry with each specific antibody. RESULTS: The MCs in newborn mice possessed Id but did not express either protein herculin or myoD. On the other hand, mature MCs expressed both myf5 and herculin. The Id protein disappeared in mature glomeruli. CONCLUSION: These results suggest that bHLH proteins are an important factor for mature MCs in vivo.