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It remains unclear whether Shiga toxin-2 (Stx-2)-induced acute encephalopathy contributes to an inappropriate activation of the renal sympathetic outflow. This investigation set out to examine the impact of Stx-2 administered into the brain on the neural control of the kidney.Using acutely anaesthetised male Wistar rats (300-350 g), saline, Stx-2 (10 μg/kg) or lipopolysaccharide (LPS 50 μg/kg) was administered intracerebroventricularly (icv) and measurements of renal haemodynamic and excretory function or renal nerve activity were made over the following 4 h.There were minimal changes in renal blood flow, glomerular filtration rate, urine flow or sodium excretion, irrespective of whether saline, Stx-2 or LPS was administered into the brain. The renal nerve recordings showed that whereas saline and LPS caused small inconsistent changes in renal nerve activity over the 4-h period, there was a significant (P0.05) doubling of renal nerve activity in the rats which were administered Stx-2 icv. Immunocytochemical examination demonstrated that Stx-2 induced globotriaosylceramide receptors, the proposed functional receptors for Stx-2, on the blood vessel walls around the hypothalamus and hippocampus, and histological evaluations showed that changes in the kidney were beginning to occur to the renal tubular epithelial cells, consistent with developing lesions.Stx-2 crosses either the blood-brain barrier or the blood-cerebrospinal fluid barrier where it can alter neuronal function and trigger neuronal derangements. These structural changes could contribute, at least in part, to the raised renal sympathetic nerve activity.

Hepatopulmonary syndrome (HPS) (hypoxaemia due to intrapulmonary vasodilation and a right-to-left shunt associated with liver disease) resolves after liver transplantation. The authors describe a case of spontaneous resolution of HPS prior to liver transplantation. This patient was diagnosed with HPS associated with extra-hepatic biliary atresia when she was 10 years old. She exhibited digital clubbing, facial vascular dilation, cyanosis, and suffered from dyspnoea during exercise. The patient's PaO(2) at rest was 53.8 mm Hg in room air and a Technetium-99m macro-aggregated albumin lung perfusion scan demonstrated a right-to-left shunt. Although her symptoms and laboratory data supported a diagnosis of HPS, she nevertheless showed spontaneous resolution within 2 years. When she was 14 years old, pulmonary hypertension was evident upon examination of her echocardiogram. HPS may be improved or masked by an accidental overlap with pulmonary hypertension in the terminal stage of liver disease.

Successful gene therapy requires gene delivery that is efficient, has an optimal route of administration and has biosafety. The aims of the present study were to evaluate the safety and applicability of the subcutaneous delivery route for adenoviral transgenes containing the human beta(2)-adrenoceptor (adeno-beta(2)-AR) and to investigate whether this approach prevented renal dysfunction in a rat model of endotoxaemic shock induced by LPS (lipopolysaccharide). Subcutaneous administration of adeno-beta(2)-AR (a total of 10(10) viral particles) significantly increased beta-AR density in the kidney, lung and liver, but was without effect on physiological and plasma biochemical parameters. Moreover, this dose of virus did not cause any of the potential toxic responses of viral administration, such as inflammation and tissue TNF (tumour necrosis factor)-alpha expression. Although the LPS challenge caused a decrease in glomerular filtration rate, fractional excretion of sodium and renal beta-AR density in all groups, the reduction in renal function was significantly less in the rats given adeno-beta(2)-AR compared with non-treated rats. Thus, although further evaluation will be required, this initial study demonstrated that the subcutaneous injection of adeno-beta(2)-AR was efficient, comparatively non-pathogenic and potentially therapeutic to deal with acute renal failure associated with sepsis.

Bacterial superantigens have been implicated in the pathogenesis of several human diseases. Among them, toxic shock syndrome (TSS) is a prototypic acute intoxication caused by the pyrogenic exotoxin family of superantigens. In this study, we investigated the pathophysiological mechanism of TSS using the Yersinia pseudotuberculosis-derived mitogen (YPM) and its point mutants. The results indicated that YPM could induce toxic shock in BALB/c mice but not in T cell-deficient SCID mice. We found that Vbeta8(+) T cells activated by YPM migrated from peripheral blood to liver as early as 1 h after injection of YPM and that serum level of IFN-gamma was significantly elevated 4 h after YPM injection. Co-administration of anti-IFN-gamma antibody or anti-YPM monoclonal antibody alleviated the liver injury and protected mice from YPM-induced death. Moreover, anti-YPM antibody also suppressed the early migration of Vbeta8(+) T cells from the peripheral circulation and the elevation of serum IFN-gamma level, indicating a pivotal role of T cells in inducing shock in our mouse model.

Mutations of human jagged 1 (JAG1) gene are responsible for Alagille Syndrome (AGS), whose 2 main symptoms are intrahepatic bile duct hypoplasia and pulmonary stenosis. We examined the JAG1 mutation in extrahepatic biliary atresia (EHBA), which is similar in phenotype to AGS, although a different pathogenesis is suggested. In 102 cases of EHBA, 9 missense mutations were detected, including 2 intrafamilial expressions in the propositus and an aunt of one family. These mutations were all missense and sporadic except for those of this particular family. The JAG1 gene mutations were generally found in severely ill patients subjected to liver transplantation at less than 5 years of age. None of the 9 cases of EHBA revealed any of the 5 major symptoms of AGS nor any identical pathological findings after 3 years of follow-up. Our cases were clearly separated from AGS by pathological findings and clinical features, and could be diagnosed as EHBA and not as atypical AGS. The increase of interleukin 8 (IL-8) production induced by tumor necrosis factor alpha (TNF-alpha) in Huh 7 cells was suppressed by the coexistence of JAG1 protein. We examined the different influences between wild-type cells and the 3 kinds of mutants detected in EHBA on Huh 7 cells and found that 2 of 3 mutants showed about half of the repressed activity compared with that of wild type. In conclusion, these results suggest that the JAG1 gene abnormality may be an aggravating factor in EHBA.

Previous findings indicated that cAMP had an inhibitory effect of on tumour necrosis factor (TNF)-alpha production. Angiotensin II (Ang II) may activate the cAMP-protein kinase A (PKA) pathway in renal mesangial cells through synthesis of prostaglandins (PGs) and the possibility arises that inhibition of Ang II-induced cAMP formation might result in the overproduction of TNF-alpha in the cell and this hypothesis was tested in the present study. Rat mesangial cells were exposed to Ang II in the presence or absence of cyclooxygenase inhibitor (indomethacin) or cAMP-PKA inhibitor (H-89). Exposure of mesangial cell to Ang II (10 −6 M–10 −8 M) significantly increased intracellular cAMP level through type 1 Ang II receptor but had no effect on TNF-alpha protein release, transcriptional activity, or mRNA. However, following the addition of indomethacin or H-89, Ang II significantly increased TNF-alpha release, transcriptional activity, and mRNA level. These data suggested that in mesangial cells after blockade of cAMP-PKA by PG inhibition, Ang II was capable of stimulating TNF-alpha transcription which subsequently increased TNF-alpha mRNA accumulation and protein release.

Alagille syndrome (AGS) is a congenital multi-system anomaly mainly characterized by paucity of intrahepatic bile ducts caused by haploinsufficiency of the Jagged 1 gene (JAG1). To explore the relationship between genotype and phenotype, we analyzed the JAG1 gene in 25 Japanese AGS families at the genomic DNA level and identified 15 point mutations and one large deletion. Analysis of the genotype and phenotype strongly indicated that the Delta/Serrate/Lag-2 (DSL) domain in JAG1 protein played an essential role in determining the severity of the liver disorder. In four sporadic cases, missing an entire DSL domain in mutant JAG1 resulted in progressive liver failure and all 4 patients needed a liver transplant at a very young age. This correlation was further confirmed by statistical analysis (chi2=9.143, p

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

CD28 engagement by specific monoclonal antibody (mAb) or binding of the natural ligands, CD80 and CD86, induces tyrosine phosphorylation of CD28, which in turn recruits and activates the signal transducer and activator of transcription 6 (Stat6). The Stat6 association with CD28 is specifically induced by CD80 or CD86 ligand binding and is not dependent upon the secretion of IL-4 or IL-13. Activated Stat6 translocates to the nucleus and binds to a Stat6-responsive element on the human IL-4 promoter. CD28 ligation induces Stat6-dependent transcriptional activation of a reporter gene under the control of a multimerized Stat6-responsive element fused to an essential part of the IL-4 promoter. Primary stimulation of naive CD4(+) T cells with anti-CD28 mAb in the presence of IL-2, but in the absence of anti-CD3 mAb induces preferential production of IL-4 and expression of CCR4 mRNA after secondary stimulation with anti-CD3, indicating the preferential differentiation of Th2 cells. These findings suggest that initial IL-4 production required for commitment of naive T cells toward Th2 cells may be provided in response to signals delivered via CD28 by antigen-presenting cells.

The bacterial superantigens, staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, are grouped in a family by the conservation of amino acid sequence and polypeptide folding patterns. In the case of Yersinia pseudotuberculosis-derived mitogen (YPM), however, there is no noticeable homology with this family, although many of the in vitro functional features conform to the criteria for a superantigen. To study the mode of action of YPM at the molecular level, we first generated a number of YPM point mutants with reduced T-cell proliferative activity using random mutagenesis and localized the amino acid positions involved in either major histocompatibility complex class II or T-cell receptor Vbeta-interaction. Plotting the elucidated positions on the hydrophilicity profile suggested that they reside mostly on the outer portion of the molecule. We also report that the two cysteines positioned almost at opposing ends of the YPM molecule are connected by an S-S bond the destruction of which causes fatal damage. Finally, we obtained evidence that YPM partially competes with staphylococcal enterotoxin E for human leukocyte antigen-DR binding. This raises the question of whether these different types of superantigens have acquired the same function by genetic convergence or originated from a common ancestral gene.

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are rare diseases caused by the mutations of Wilms tumor gene, WT1. The common denominator in these syndromes is a nephropathy which is manifested by early-onset proteinuria, nephrotic syndrome and end stage renal failure. Although these syndromes are genetic models of nephropathy and the mutations of WT1 gene are characterized in these patients the mechanism how mutations of WT1 gene affect the embryonic kidney adversely has not been elucidated. Recently, there was a report that FS is caused by mutations in the donor splice site of WT1. These mutations predicted loss of +KTS isoform, which is one of the four splicing variants of WT1. In this study, two +KTS deletion mutants of WT1 were made as well as a WT1 mutant mimicking a mutation found in a patient who had diffuse mesangial sclerosis, end stage renal failure and Wilms tumor. Mutant embryonic kidney cell lines were established by transfection of 293 embryonic kidney cells with WT1 mutants. We investigated the transcription regulation of mutant WT1 among these cell lines using the reporter vectors containing PDGF-A and TGF-beta promoter sequence. Our results showed that the promoter activity of PDGF-A and TGF-beta, which are related to the progression of glomerular diseases, was modestly increased in the mutant cell mimicking the patent, while those activities were markedly increased in other two deletion mutant cell lines. This study demonstrated that +KTS WT1 mutation found in DDS affected the cytokine expression adversely in vitro. From these results, we suggest that the alteration of +KTS WT1 expression be responsible for the rapid progression of renal diseases in DDS and FS.

The present study was designed to clarify the role of beta2-adrenoceptors in modulating the level of TNF and IL-6 gene transcription and their respective mRNA accumulations. Astrocytes were transfected with the 5'-flanking region of the TNF and IL-6 genes linked to a luciferase coding sequence as a reporter. The addition of isoproterenol had an inhibitory effect on the TNF and IL-6 promoter activity induced by LPS. The inhibitory effect was blocked in the presence of a beta2-adrenoceptor antagonist but not in the presence of a beta1-adrenoceptor antagonist. TNF and IL-6 mRNA and protein levels in the astrocytes were depressed by beta2-adrenoceptor activation which corresponded to changes in TNF and IL-6 promoter activity. These studies demonstrated that isoproterenol, via beta2-adrenoceptors, suppressed LPS-induced TNF and IL-6 promoter activities, mRNA accumulations, and protein levels in the astrocytes. Beta2-adrenoceptor activation may be an important mechanism for regulating TNF and IL-6 expression in brain diseases.

We have shown previously in the spontaneously hypertensive rat (SHR) kidney that interleukin-6 (IL-6) and tumor necrosis factor (TNF) mRNA levels were low under conditions of acute anaesthesia and surgical stress. The reasons for the suppression of IL-6 and TNF gene expression in the SHR were investigated by examining the influence of enhanced beta-adrenergic stimulation, high blood pressure, and renal function (renal blood flow, glomerular filtration rate, plasma creatinine levels) on renal IL-6 and TNF mRNAs. The experiments were performed by means of the following three studies; (1) SHR and Wistar rats at 4, 7, 9 week old were injected with lipopolysaccaride (LPS), and then a relationship between blood pressure levels and IL-6 and TNF mRNA levels were estimated, (2) isoproterenol and propranolol were administered into SHR and WKY rats, and the levels of IL-6 and TNF mRNA were compared, (3) under condition of anaesthesia and surgical stress, blood pressure and renal functions in SHR were measured, and then the relationships between these factors and IL-6 or TNF mRNA levels were analyzed. Renal IL-6 and TNF mRNAs in SHR remained low even though blood pressure increased with age and there was no significant correlation between IL-6 or TNF mRNA levels and values of blood pressure or renal function under anaesthesia and surgical stress. However, the inhibition of the IL-6 and TNF mRNAs in SHR was prevented by propranolol treatment. These results suggested that suppression of IL-6 and TNF mRNAs in the SHR kidney could be due to overactivity of beta-adrenergic influences which may importantly contribute to the development of hypertension.

Alagille syndrome (AGS) is a genetic disease and the responsible gene has already been mapped at 20p12. To more accurately detect the region of the AGS gene on the linkage map of chromosome 20p, 14 yeast artificial chromosome (YAC) clones were screened to construct a YAC contig in the candidate region and 13 locus markers and 2 sequence-tagged sites (STS) were ordered. Combining all of the analyses, a 1.3 Mb critical region from D20S507 to D20S61 for the AGS gene was identified. As the human Jagged 1 gene (JAG1) lies just in this region and is responsible for the AGS disease, the genomic DNA in an AGS family without a visible deletion were analyzed by single-strand conformational polymorphism (SSCP) and direct DNA sequencing, and a 2-bp (CT) deletion mutation at exon 26 of the JAG1 was identified.

In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC). After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed. The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction. The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma. The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells. These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration.

OBJECTIVE: To assess the influence of elevated renal sympathetic nerve activity on renal interleukin-6 (IL-6) and interleukin-1 alpha (IL-1 alpha) production and to compare the specificity of the constitutive production of these cytokines in the spleen and kidney of the spontaneously hypertensive rat (SHR). DESIGN: In acute studies, using anaesthetized and surgically stressed rats, the renal nerves either were not stimulated or were stimulated electrically to reduce renal blood flow by 15 or 30% for 1 h. The kidneys were then extracted for analysis. For the chronic study, Wistar rats and SHR aged 9 weeks were briefly anaesthetized and kidneys and spleen extracted for cytokine measurement. METHODS: Tissue samples were subjected to reverse transcriptase and polymerase chain reaction amplification to assess levels of IL-6 and IL-1 alpha. RESULTS: Renal nerve stimulation decreased levels of IL-6 messenger RNA in the SHR kidney, from a control value of 0.677 +/- 0.043 units to 0.624 +/- 0.049 and 0.383 +/- 0.078 units during the 15 and 30% reductions in renal blood flow induced neurally. The chronic experiments showed that, although there was no difference in constitutive production of IL-1 alpha between the kidney and spleen of Wistar rats and SHR, spleen levels of IL-6 were higher in the SHR and IL-6 was expressed constitutively neither in the Wistar rat nor in the SHR kidney. CONCLUSION: These data demonstrate that renal IL-6 gene expression is low in the SHR kidney under conditions of anaesthesia and surgical stress and that it is further decreased when the kidney is subjected to adrenergic influences. These studies help to define the deficiencies and abnormalities of the SHR Immune system.

膜性増殖性腎炎 (MPGN) において低補体血漿を伴うことはよく知られている。また低補体血漿を伴うpartial lipodystrophyに膜性増殖性腎炎が発症することより低補体血漿がその発症の一要因として重要視されている。 母から子に遺伝する低C3血症については，Westらが2家系を報告したのに始まり1)2)H因子に対して結合を異にするC3異常症と，腎生検組織所見によってMPGN typeIIの所見をあわせて示すことより，腎炎の発症機序と低補体の関連について興味がもたれた。我々はC3の機能異常を伴う例について，遺伝子学的検索を行い，C3のI因子の作用部位に異常を示す症例について報告した3)。現在まで家族性の低C3血症を示すその他の例についてもI因子，H因子，F，Sの移動度を決定する部位についての遺伝子学的検討を行った。しかしながらI因子，H因子の部位の遺伝子配列に関しては異常は認められず，またF，Sの部位についてはすべてSであり，特にcontrolとの遺伝子学的な差異を見い出しえなかった。これらの患者にしばしばC3NeF活性や，補体のbreakdown productsの存在が認められることより，補体の活性化による低下という面からの検討を行った。 Spitzerらは，C3NeFについてはidiotype検体の出現があり，これがC3NeFは産生亢進，抑制を認節している可能性について報告4)5)している。このような観点から家族性低C3血症について検討し，idiotype検体の問題点について患者および母親について検索したのでその結果について報告する。

To date, no detectable biotinidase activity has been reported in the human urine. In the present study, a simple and convenient HPLC-fluorimetric assay method was applied to biotinidase activity measurement of urine samples. Biotinyl-6-aminoquinoline was utilized as the enzyme substrate, and the liberated product (6-aminoquinoline) was monitored with a fluorimetric detector. Biotinidase activity was assessed in urine specimens from: twenty-five patients with various renal disorders associated with proteinuria, and forty age- and sex-matched healthy control subjects. The examined samples from the patients with renal diseases exhibited biotinidase activity with the exception of six of the patients (enzyme activity, median: 49.9 pmol · min−1 · ml−1 of urine; range: 0–2498 pmol · min−1 · ml−1); specific activity, median: 19.4 pmol · min−1 · mg−1 of protein; range: 0–176 pmol · min−1 · mg−1) while none of the urine specimens from the control subjects showed detectable enzyme activity. Thus, this metho...

In this study the complement breakdown products C3d, C4d, Bb and membrane attack complex were measured in plasma of patients with Kawasaki syndrome. The results suggested strong activation of the classical activation pathway. However, there was no significant decrease in hemolytic titer or in the concentrations of the intact proteins C3, C4, and B. The relationship between the serum concentrations of cytokines and complement components was examined; increased interleukin-6 concentration on the fifth day after the onset of fever was found to correlate well with the C3 and B concentrations in serum obtained 5 days later. We conclude that complement activation occurred in Kawasaki syndrome via the classical pathway but that the inflammatory reaction was accompanied by increased production of complement components. As a result, there was increased formation of activation products without changes in the serum complement levels.

OBJECTIVE Leukocytapheresis (LCAP) is a nonpharmacologic therapy that has recently been used to treat ulcerative colitis (UC). This multicenter open-label study prospectively assessed the efficacy and safety of LCAP in pediatric patients with UC. PATIENTS AND METHODS Twenty-three patients ages 8 to 16 years with moderate (n = 19) to severe (n = 4) steroid-resistant UC were enrolled. One of 2 LCAP columns with different volumes (model EX and the half-volume model EI) was selected, according to body weight. LCAP was performed once per week for 5 consecutive weeks. Clinical and laboratory data were collected at predetermined time points. The primary endpoint was decreased stool frequency/hematochezia score, and secondary endpoints were clinical, laboratory, and endoscopic improvements. RESULTS The stool frequency/hematochezia score decreased significantly from 4.5 ± 1.2 before treatment to 1.6 ± 1.9 after the fifth treatment. Clinical parameters, including stool frequency, presence of visible blood, abdominal pain, and body temperature, were significantly improved. Fecal calprotectin decreased significantly. Endoscopic findings evaluated using Matts score also improved (P < 0.01). The steroid dose decreased from 1.1 ± 0.4 mg/kg before treatment to 0.8 ± 0.5 mg/kg after treatment. There were no significant differences in changes between the EX and EI columns. The incidence of adverse effects was 61%, although none was serious. The most common adverse effects were decreased hematocrit and hemoglobin concentration. CONCLUSIONS The present study showed that LCAP was well tolerated in children with UC, mostly moderate, and was as effective as in adults. The types of pediatric patients best suited to LCAP remain to be determined.

OBJECTIVE The aim of the study was to evaluate the influence of renal sympathetic nerves on the production of interleukin (IL)-6 in the kidney of normotensive Wistar rats and spontaneously hypertensive rats (SHR). DESIGN In acute studies, the left kidney was exposed and the renal nerves were electrically stimulated to decrease renal blood flow by either 15 or 30% for 1 h. The changes in renal function induced by nerve stimulation were measured and IL-6 production estimated at the end of the experiment. METHOD Pentobarbitone anaesthetized rats were prepared for the measurement of renal blood flow, glomerular filtration rate and water and sodium excretion. IL-6 production was estimated by the level of IL-6 messenger (m)RNA present in the kidney tissue. RESULTS At the lower level of nerve stimulation there were reductions in the glomerular filtration rate of 12 and 24% in Wistar and SHR, respectively, and a decrease in sodium excretion of approximately 30% in both rat strains. At higher rates of stimulation these haemodynamic and tubular responses were proportionately larger. The mRNA for IL-6 and beta-actin were measured by densitometric analysis of Northern blot gels following hybridization. Renal IL-6 mRNA levels in the Wistar rat demonstrated that the gene was actively expressed and was increased some threefold by renal nerve stimulation. By contrast, IL-6 mRNA was extremely low in SHR compared with that found in the kidneys of Wistar rats and did not appear to be changed by renal nerve stimulation. CONCLUSIONS These findings suggest that the renal sympathetic nerves are an important regulatory mechanism for IL-6 production under normal conditions. However, in the SHR, production of IL-6 in the kidney appears to be suppressed.

CAPD療法は, blood accessが不必要であること, 経済性に優れていること, 手技上の容易さなどの利点から, 小児期における慢性腎不全の主流の治療法となりつつある. しかしながら, 本法は優れた方法であるが, 欠点として長期間の施行により, 除水不全, 透析不全という腹膜透過性の変化を生ずることがあげられる. 我々は, ラットCAPDモデルを作成し, 糖濃度が腹膜透過性に及ぼす影響について検討した. 種々の糖濃度の溶液を腹腔に留置したチューブを介しラットに投与し, 腹膜機能を評価した. その結果, 7.0%の糖濃度溶液はリンパ系吸収の変化なしに腹膜の透過性の変化をもたらした. 一般に高浸透圧溶液の反復投与によっては腹膜透過性を増加させ, 限外濾過量を減少させるという結論を得た.

Increased frequency of C4 gene deletions in IgA nephropathy and Henoch-Schönlein purpura nephritis. To determine the frequency of complement 4 (C4) deficiency among the patients with IgA nephropathy (IgAN) and Henoch-Schönlein purpura nephritis (HSPN), C4 and factor B protein allotypes and the DNA restriction fragment length polymorphism (RFLP) of C4, steroid 21-hydroxylase (21OHase), HLA DQ beta and DR beta chain genes were studied. Genomic DNA from 32 patients with IgAN, 24 patients with HSPN and 143 controls was digested with restriction enzyme TaqI or BamHI and subjected to Southern analysis. The frequency of C4 gene deletions was significantly increased in the patients (16.1 vs. 2.8%, p = 0.002). The serum C4 concentration in patients with C4 gene deletion was significantly lower than in patients without gene deletion (11.4 +/- 2.4 vs. 19.7 +/- 5.2 mg/dl, p = 0.001). DNA-RFLP typing of DQB/BamHI and DRB/TaqI showed increased frequency of 10.26 kb of D-DQw4/8/9 (87.5 vs. 64.5%, p = 0.004) and 5.22 kb of D-DR4 (66.1 vs. 41.7%, p = 0.03) among the patients. Segregation analysis showed that the association of the D-DQw4/8/9 and D-DR4 with the diseases was not responsible for the increased frequency of C4 gene deletion in the patients. The decreased concentration of C4 in sera in C4 gene-deleted patients might be directly involved in the pathogenesis of IgAN and HSPN.

Wilson disease is an autosomal recessive disorder with copper metabolism. In Japan, the standard treatment is the administration of copper chelating agents, such as D-penicillamine and trientine. In this study, the authors used zinc acetate to treat Japanese patients with Wilson disease and investigated its efficacy. The 37 patients that comprise this study were found to have Wilson disease using clinical and biochemical tests and were administrated zinc acetate for 48 weeks. The authors followed the clinical symptoms and laboratory findings of the patients by assessing their complete blood counts, biochemical findings, as well as the results of urinalysis and special laboratory tests for copper and zinc metabolism. We also examined side effects of the treatment. Zinc acetate did not aggravate the hepatic or neurological symptoms of any of the patients. Blood biochemical analysis also did not reveal elevation of alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltranspeptidase levels. Zinc treatment did not aggravate the patients' clinical signs and/or laboratory findings. However, it did improve some clinical symptoms of the Wilson disease patients. Although this agent had some side effects, none of them were severe. The authors measured spot urinary copper excretion, which gave an indication of the efficacy of treatment and of the sufficient dosage of zinc. We recommend maintaining a spot urinary copper excretion less than 0.075-μg/mg creatinine. The authors conclude that zinc acetate is an effective and safe treatment for Japanese patients with Wilson disease.

DNA restriction fragment length polymorphism (RFLP) typing of HLA-DR and DQ alleles of 60 Japanese type 1 (insulin dependent) diabetic patients and 115 controls was performed. RFLP typing of DRB1 showed increased frequency of DR9 and decreased frequencies of DR2 and DRW6 among patients compared to controls. In the RFLP typing of BamHI- digested DNA to DQ β probe (BamHI-DQB1), the incidence of the 10.26 kb fragment, which represents either DQW4, DQW8 or DQW9, was markedly elevated in the patients, whereas the incidence of DQW6 was reduced. The predicted DR-DQ haplotype study revealed that DR4-DQW4 or DQW8, DRW8-DQW4 or DQW8 and DR9-DQW9 may contribute to susceptibility to type 1 diabetes. When serological typing of the 13 DRW8 patients was performed, all the 11 DRW8 patients carrying DQW4 or DQW8 (BamHI-10.26 kb) were positive for DQW3. These results indicated that the HLA-DQ locus may play an important role in the development of type 1 diabetes in the Japanese as well as other ethnic groups and that the DRW8- DQW8 haplotype may predispose to the disease in Japan.

Among the various systemic manifestations in Y. pseudotuberculosis infection, renal complication such as acute renal failure, and an erythematous skin rash were more strongly related to the elevation of anti-YPM titer. Acute renal failure might be caused by the T cells that was stimulated and matured under the Th1 type inflammatory cytokine milieu produced by YPM during the acute phase of the disease. Patients with erythematous skin rash had an elevated level of serum IL-12p40 during the acute phase. The in vitro experiments indicated that YPM could contribute to the pathogenesis of skin rash through the enhanced production of IL-12 and the expansion of the skin-homing receptor CLA+ T cells in patients.

Alagille syndrome (AGS, MIM 118450) is an autosomal dominant inherited disease. Paucity of interlobular bile ducts is one of the major abnormalities. To explore the molecular mechanism by which mutation in the human Jagged 1 gene (JAG1, MIM 601920) causes liver defects, we investigated the gene regulation of JAG1 to hepatocyte growth factor gene (HGF). By transfecting wild-type and mutant JAG1 into COS-7 cells in vitro, we found that HGF is a target gene of JAG1 downstream. Wild-type JAG1 is inhibitory for HGF expression and mutant JAG1s relieve the inhibition. Several domain disruptions in mutant JAG1 protein reveal a reduced inhibition to HGF expression at different levels. JAG1 mutations actually result in HGF overexpression. Furthermore, JAG1 controls HGF expression by a dosage-dependent regulation and Notch2 signaling seems to mediate JAG1 function. Given that HGF plays a critical role in differentiation of hepatic stem cells, overexpression of HGF acts on off-balanced cell fate determination in AGS patients. Hepatic stem cells may differentiate towards more hepatocytes but less biliary cells, thus causing the paucity of interlobular bile ducts in liver development of AGS. Our novel findings demonstrated that dosage-dependent regulation by mutations of JAG1 is a fundamental mechanism for liver abnormality in AGS.

Abnormalities in the beta(2)-adrenergic control of organ function have been implicated in the pathogenesis of several disease states, such as septic shock. The objectives of the present study were to define the contribution of beta(2)-adrenoceptors (beta(2)-AR) to normal renal physiology and to investigate whether overexpression of renal beta(2)-AR might be potentially beneficial in preventing progressive renal damage associated with endotoxemia. Adenoviral transgenes containing the human beta(2)-AR (Adeno-beta(2)-AR) were constructed and delivered into the rat kidney by means of intraparenchymal injections. Administration of 10(9) total viral particles of Adeno-beta(2)-AR induced an approximately threefold increase in beta(2)-AR density in the renal tissue, which 2 wk after delivery, enhanced GFR and sodium reabsorption compared with control rats. The enhanced GFR was abolished by the addition of the beta(2)-AR antagonist, ICI 118,551. Administration of lipopolysaccharide (LPS) caused a reduction in GFR, beta(2)-AR density, and cAMP together with enhanced TNF-alpha mRNA in the kidney. In rats overexpressing beta(2)-AR, the reduction in baseline GFR and elevation of TNF-alpha mRNA and leukocyte infiltration into the kidney associated with the endotoxin were blocked. These findings suggested the possibility that a renal-specific overexpression of beta(2)-AR preserves basal renal function in response to a ligand-independent beta(2)-AR activation and that the delivery of Adeno-beta(2)-AR gene is a potential novel therapeutic strategy for treatment of acute renal failure associated with sepsis.

YPM had an acute toxic effect on BALB/c mice, which was dependent on the presence of T cells and correlated with the elevation of serum IFN-gamma. One monoclonal antibody (YSA8E3) rescued BALB/c mice from development of toxic shock after YPM injection.

Yersinia pseudotuberculosis-derived mitogen (YPM), a superantigen with no amino-acid sequence similarity to other known superantigens, has been crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 138.67, b = 78.66, c = 32.91 A, beta = 91.97 degrees. A native data set has been collected to a resolution of 1.8 A using synchrotron radiation. Self-rotation function calculations suggest the presence of three molecules in the asymmetric unit, corresponding to a solvent content of 45%.

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children worldwide. Shiga toxin (Stx) associated HUS, the most common type, is now known to be caused by Escherichia coli O157:7, which produces Stxl or the more potent, Stx2. Since the renal tubule is the major tissue affected in the course of HUS and Stx2 is known to be toxic to the renal tubular cells (RTC), we attempted to elucidate the mechanism of renal injury in HUS by studying the alteration of cytokines in the RTC evoked by Stx2. cDNA-array is a powerful tool for evaluating changes in the expression of a group of critical genes and also gives insights on the overview of the gene activation. In this study, we purified Stx2 from the E. coli O157:7, which was isolated from a typical diarrhea-associated HUS patient and then tried to compare the cytokine gene expression between the stimulated RTC and un-stimulated RTC using cDNA-array. Our results showed that one third of the examined cytokine genes were up regulated at least twice by the addition of Vtx2. These up-regulated genes represented the chemokines (macrophage related cytokines), fibrosis-related cytokine (TNF, PDGF) and leukemia inhibitory factors. However, the expression of IL-6, one of the pleiotropic cytokines, was significantly decreased and this finding was confirmed by northern analysis. Our results suggest that VT2 up-regulates the pro-inflammatory cytokines and fibrosis prone growth factors in RTC and that the inhibition of the activation of these cytokines may ameliorate the renal tubular injury in the HUS caused by E. coli O157:7.

Mutations of human jagged 1 (JAG1) gene are responsible for Alagille Syndrome (AGS), whose 2 main symptoms are intrahepatic bile duct hypoplasia and pulmonary stenosis. We examined the JAG1 mutation in extrahepatic biliary atresia (EHBA), which is similar in phenotype to AGS, although a different pathogenesis is suggested. In 102 cases of EHBA, 9 missense mutations were detected, including 2 intrafamilial expressions in the propositus and an aunt of one family. These mutations were all missense and sporadic except for those of this particular family. The JAG1 gene mutations were generally found in severely ill patients subjected to liver transplantation at less than 5 years of age. None of the 9 cases of EHBA revealed any of the 5 major symptoms of AGS nor any identical pathological findings after 3 years of follow-up. Our cases were clearly separated from AGS by pathological findings and clinical features, and could be diagnosed as EHBA and not as atypical AGS. The increase of interleukin 8 (IL-8) production induced by tumor necrosis factor alpha (TNF-alpha) in Huh 7 cells was suppressed by the coexistence of JAG1 protein. We examined the different influences between wild-type cells and the 3 kinds of mutants detected in EHBA on Huh 7 cells and found that 2 of 3 mutants showed about half of the repressed activity compared with that of wild type. In conclusion, these results suggest that the JAG1 gene abnormality may be an aggravating factor in EHBA.

It is well known genetic predisposition may play an important role in the pathogenesis of renal diseases. Recently, there has been some controversy about the possible role of the polymorphism of the immunoglobulin heavy chain switch region gene. We have studied this gene by SstI restriction fragment length polymorphisms using DNA from 41 children with IgA nephropathy, 44 with Henoch-Schönlein nephritis and 60 with idiopathic nephrotic syndrome. There was no association of specific genotype with these diseases, in contrast to previous reports. These results are probably due to the differing genetic background of Japanese and Caucasoid patients as far as the switch region is concerned; no switch region genotype constitutes a genetic risk factor for the Japanese in these diseases.

Kidney biopsy is an indispensible procedure for making a pathologic diagnosis of renal diseases by fixing and staining the biopsy specimen. However, it is not a routine procedure to culture the cells from a renal biopsy specimen directly, or to utilize the cultured cells for any kind of diagnostic or functional evaluation. In this study, primary culture of the renal tubular epithelial cells was tried from a piece of percutaneous kidney biopsy specimen. Successive passages of the cells were possible until fourth passage. With these cells, morphologic characteristics of the cultured cells and integrin expression profiles were investigated. On light and electron microscopy, these cells were characterized by the cobblestone-like growth, presence of microvilli and tight junction, and the preservation of polarity. Immunohistochemical studies demonstrated the epithelial nature of these cells and particularly their differentiation from renal tubular epithelial cells, of either proximal or distal nephronic segment. The integrin profile confirms the epithelial nature of the cell. We hope that our results facilitate the understanding of pathophysiology of renal tubular cells from the patient directly.

Background: Mutations in the FBN1 gene, encoding fibrillin-1, result in Marfan syndrome (MFS). According to previous reports, the mutations in FBN1 share certain characteristics in each family with variable penetrance and overlapping symptoms, even in the same genotype. In the present study, we report six novel mutations and evaluate the clinical significance of these nucleotide changes. Methods: To screen for nucleotide changes in all 65 exons of the FBN1 gene in 38 unrelated Korean patients, we performed polymerase chain reaction, single-strand conformational polymorphism (SSCP) and sequencing for the shift of the band in SSCP. Results: We identified six mutations: a 2253 del 7 b.p., N1043S, C1254S, L1421F, C1895R and S2662P. Conclusions: These results suggest that many different mutations are responsible for MFS in the Korean population.

This study aimed to investigate the time-course of the effect of beta2-adrenoceptor stimulation with terbutaline on lipopolysaccharide (LPS)-induced tumour necrosis factor(TNF)-alpha production in rat mesangial cells. Cells were cultured from 0-24 h in the presence of LPS (1 microg/ml) and/or terbutaline (10(-7)-10(-8) mol/l). After 1 h of incubation, terbutaline inhibited TNF-alpha protein release as well as transcription and translation of TNF-alpha and mitogen activated protein kinase (MAPK, p42/p44) activity. At 3 h, terbutaline enhanced intracellular cAMP but suppressed TNF-alpha release and transcription. By 24 h, whereas terbutaline was no longer influencing transcription or translation, TNF-alpha release remained depressed which correlated with an increase in supernatant interleukin (IL)-6. Terbutaline did not affect the LPS-induced IL-10 produced in the cell. These findings indicate that beta2-adrenoceptor stimulation during an LPS challenge prevented TNF-alpha production as a consequence of MAPK inhibition and enhanced cAMP generation, which at a later stage was associated with an anti-inflammatory effect of IL-6.

Staphylococcal enterotoxins (SEs) are a family of structurally related pyrogenic exotoxins consisting of the five prototypic SEs (types A to E) and three newly characterized SEs (types G to I) produced by Staphylococcus aureus (S. aureus). They also work as superantigens and cause food poisoning and shock symptoms in humans. In this study, we cloned a new variant gene of the seg and characterized its superantigenic properties and distribution among the clinical isolates of S. aureus. The gene encodes a 233 amino acid protein which is highly homologous to SEG (97.7%). The variant SEG (SEGv) expressed by the cloned gene exerted mitogenic activity on human peripheral blood mononuclear cells at the concentration of 100 pg/ml. T cells bearing Vbeta3, 12, 13.1, 13.2, 14 and 15 were preferentially expanded after stimulation with the recombinant protein. The mRNA of the variant seg gene was detected in the total RNA of the organisms bearing this gene. By PCR, 27 out of 48 clinical isolates of S. aureus (56%) possessed either the seg or variant seg gene. These findings suggest that SEG, or SEGv, is one of the most frequently produced superantigen exotoxins by S. aureus and may participate in the inflammatory process of the host by activating a distinct set of Vbeta families of T cells.

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 × BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6–9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-γ expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.

We report new mutations in exon 9 of the WT1 gene that did not alter the ratio of +/– KTS splice isoforms in two unrelated patients with Frasier syndrome (FS). The mutation of intron 9 inducing defective alternative splicing was reported to be responsible for this syndrome. The mutations found in our cases occurred in the same exon of the WT1 gene as detected in Denys-Drash syndrome (DDS) and could not be explained by the previously proposed mechanism. The results suggest that the two syndromes originate from the same WT1 gene abnormality. From a molecular biological point of view, we concluded that the two diseases were not separable, and that FS should be included as an atypical form of DDS. Hum Mutat 14:466–470, 1999. © 1999 Wiley-Liss, Inc.

This study aimed to examine whether lipopolysaccharide (LPS)-induced increase in tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) gene transcription was regulated by beta-adrenoceptor activation and whether TNF-alpha and IL-6 gene transcription was regulated by angiotensin II in rat renal resident macrophage cells. The cells were transfected with a fusion gene with the 5'-flanking region of rat TNF-alpha or IL-6 genes linked to a luciferase coding sequence as a reporter. The stimulatory effect of LPS on TNF-alpha transcription was suppressed by isoproterenol (10(-8)-10(-5)M) in a dose-dependent manner, whereas IL-6 transcription was only decreased by the high concentration (10(-5)M) of isoproterenol. The addition of beta(2)-adrenoceptor antagonist (ICI118,551), but not a beta(1)-adrenoceptor antagonist (atenolol), blocked the inhibitory effect of isoproterenol. By contrast, angiotensin II (10(-8)-10(-5)M) enhanced IL-6 gene transcription in the cells in a dose-dependent manner which was inhibited by type 1 angiotensin II receptor antagonist (CV11,974). TNF-alpha and IL-6 secretion from the cells was altered with beta(2)-adrenoceptor agonists (terbutaline, formoterol) and angiotensin II corresponding to changes of TNF-alpha and IL-6 gene transcription. Angiotensin II had no effect on TNF-alpha secretion and gene transcription. These findings suggested that beta(2)-adrenoceptor agonist and angiotensin II potentially could influence renal immune function through the regulation of TNF-alpha and IL-6 gene transcription by the renal resident macrophage cells.

Modulation of interleukin-6 by β2-adrenoceptor in endotoxin-stimulated renal macrophage cells.BackgroundActivation of the cAMP signaling pathway by means of β2-adrenoceptor agonists has been shown to up-regulate interleukin-6 (IL-6) gene expression and to stimulate IL-6 production in macrophage cells. However, whether β2-adrenoceptor activation can also modify the rate of IL-6 production in macrophage cells activated by the bacterial endotoxins has not yet been determined. Using renal resident macrophage cells treated with endotoxin, lipopolysaccharide (LPS), and β2-adrenoceptor agonist, terbutaline, we investigated the role of cAMP pathway, tumor necrosis factor (TNF)-α and mitogen-activated protein kinase (MAPK) pathway (p42/p44) in regulating IL-6 production.MethodsIL-6 protein, mRNA, and promoter activity were measured in these cells exposed to LPS (1 μg/ml) and/or terbutaline (10-9 to 10-6M). Furthermore, the time course effects of terbutaline on cAMP, MAPK (p42/p44), and TNF-α release were evaluated in the cells.ResultsTerbutaline at high concentrations (10-6M) significantly up-regulated IL-6 by approximately 25% (P < 0.05), whereas at a lower concentration (10-8M), it down-regulated IL-6 production by 42% (P < 0.05). Terbutaline (10-8 and 10-6M) caused a concentration- and time-dependent stimulation of cAMP (P < 0.05) and TNF production (P < 0.05) and a time-dependent decrease in MAPK activity (P < 0.05). Following the addition of a cAMP inhibitor, IL-6 promoter activity was correlated with TNF-α levels and MAPK activity.ConclusionsA biphasic effect of β2-adrenoceptor agonist on IL-6 production in renal resident macrophage cells became apparent when LPS was exposed to the cells. The terbutaline-induced down-regulation of IL-6 gene production was mediated by an inhibitory effect of terbutaline on TNF-α, which was exerted through the MAPK and cAMP pathways, whereas the up-regulation appeared to be due to a direct action of intracellular cAMP.

CTLA-4 (CD152) is transiently induced on the cell surface of activated T cells and expression is limited at a low level. In this study, we investigated the possibility that phosphatidylinositol 3 kinase (PI 3-K) and other related PI kinases associated with the cytoplasmic domain of CTLA-4 are involved in intracellular trafficking and sorting of CTLA-4 protein. Treatment with micromolar concentrations of wortmannin (WN) for >4 h enhanced both cell-surface and intracellular CTLA-4 without affecting its transcriptional activities in a murine mastocytoma cell line transfected with the human CTLA-4 gene and normal activated CD4(+) T cells. However, a more specific PI 3-K inhibitor, LY294002, failed to affect CTLA-4 expression, indicating that the action of WN is independent of conventional PI 3-K activities. WN down-regulated specific association of CTLA-4 with adaptor proteins and its endocytosis. The fact that lysosomotropic agents, ammonium chloride and monensin, enhanced CTLA-4 expression suggests that WN may also block lysosomal sorting and consequent degradation of CTLA-4. Co-localization of CTLA-4 and lysosome-associated membrane protein-1 detected by immunofluorescence microscopy indicates the actual lysosomal sorting of CTLA-4. Our data suggest the existence of WN-sensitive enzymes, which promote lysosomal sorting of CTLA-4. In addition to rapid endocytosis by clathrin-associated adaptor complex, a prompt sorting of CTLA-4 to lysosomes may be one of the regulatory mechanisms for managing CTLA-4 signals in intracellular trafficking pathways.

The superantigenic Yersinia pseudotuberculosis-derived mitogen (YPM) may contribute to severe complications in Y. pseudotuberculosis infections. Since the pathogenic mechanism of a superantigen (SAg) is based on its capability for T-cell overstimulation, by introducing point mutations into YPM an attempt was made to abrogate this effect. Six mutants studied exhibited a variety of T-cell proliferating responses. Two had activity reduced by 80-90%, three had activity reduced by approximately 50%, and one mutant showed almost no attenuation. The SAg-associated in vitro pathogenic functions, cytotoxic activation and the production of proinflammatory cytokines, were also diminished, in parallel. Since these mutants were confirmed to be defective in TCR Vbeta binding, it was possible to compare them with native YPM. Our results suggested that the intensity of TCR Vbeta binding is a crucial factor determining the severity of pathogenesis and that single amino acid alterations might be useful for producing immunotherapeautical agents from native YPM.