Enhancing effect of H-2-linked NZW gene(s) on the autoimmune traits of (NZB X NZW)F1 mice.

To investigate the possible enhancing effect of the H-2z haplotype of the New Zealand White (NZW) strain on the production of autoantibodies and renal disease observed in B/W F1 mice, we developed the ZWD/8 strain, a NZW congenic line carrying the H-2d haplotype, produced (NZB X ZWD/8)F1 (B/WD8 F1) mice, and examined the difference in several immunological abnormalities between the B/W F1 (H-2d/H-2z) and the B/WD8 F1 (H-2d/H-2d) mice. In comparison with B/W F1 mice, the B/WD8 F1 mice showed markedly lower serum levels of the anti-DNA antibodies and the gp70 ICs, and a later onset and a lower incidence of proteinuria with a lower mortality. In contrast, there was no significant difference in the incidences and the amounts of both natural thymocytotoxic autoantibody and anti-erythrocyte autoantibody between these two hybrid strains. Further, the serum levels of IgG and IgM in B/WD8 F1 mice were as high as those in B/W F1 mice. These findings indicate that the gene(s) that is within or closely linked to the H-2 complex of NZW strain specifically acts to intensify the levels of anti-DNA antibodies and gp70 ICs, and to promote the severity of renal disease in B/W F1 mice. This gene may play a role in the class conversion of anti-dsDNA antibodies from IgM to IgG.

Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy.

Studies conducted over the last decade demonstrated variable therapeutic efficacy of angiotensin converting enzyme (ACE) inhibitor on the progression of glomerular diseases, including IgA nephropathy. In this study, among patients with biopsy-proven IgA nephropathy, 53 patients in whom creatinine clearance had been monitored over 5 yr were recruited for study. These patients were classified into two groups according to whether or not renal function had declined as determined by the slope of creatinine clearance against time: group 1 had stable renal function; group 2 had declining renal function (average: -6.7 +/- 1.3 ml/min/yr). 21 of 53 patients were treated with ACE inhibitor and followed for 48 wk. Gene polymorphism consisting of insertion (I) or deletion (D) of a 287-bp DNA fragment (presumed to be a silencer element) of the ACE gene was determined by PCR. 46 age-matched individuals without history of proteinuria were analyzed as controls. The DD genotype was significantly more frequent in group 2 (43%) than in controls (7%) or group 1 patients with stable renal function (16%). 48 wk after ACE inhibitor administration, proteinuria significantly decreased in patients with DD genotype but not in those with ID or II genotypes. The results indicate that deletion polymorphism in the ACE gene, particularly the homozygote DD, is a risk factor for progression to chronic renal failure in IgA nephropathy. Moreover, this deletion polymorphism predicts the therapeutic efficacy of ACE inhibition on proteinuria and, potentially, on progressive deterioration of renal function.

ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4.

To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5'-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5'-ATGACGAGGAGGA-3', restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system.

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells(1).

Interleukin (IL)-4, a pleiotropic cytokine involved in many glomerular diseases, is regulated positively by membrane-bound IL-4R (mIL-4R) and negatively by soluble IL-4R (sIL-4R). Because natural sIL-4R has been documented only in mice, we undertook this study in rats to determine whether they, too, express sIL-4R, particularly in kidney cells. A pair of IL-4R primers was designed for this purpose and used in the polymerase chain reaction. As a result, sIL-4R was found not only in rats spleen cells but also in their glomerular epithelial cells (GEC). Sequence analysis revealed that the mRNA of rat sIL-4R has a 75-bp insert sequence. This insert generated a termination TGA codon upstream from the transmembrane region, resulting in formation of the sIL-4R. Subsequent screening of the kidney cDNA library enabled us to obtain the whole 3605-bp cDNA of sIL-4R; the full-length 3530-bp mIL-4R cDNA was also identified as a much longer sequence than previously published. Among the total 39 clones positive for IL-4R, two were confirmed as sIL-4R, and 37 clones were positive for mIL-4R. Next, the translated portion of sIL-4R cDNA was constructed into an expression vector, enabling us to obtain a recombinant sIL4R-myc fusion protein. By using this recombinant sIL-4R, we proved that sIL-4R can antagonize the IL-4-induced proliferation of spleen cells. Present study demonstrated that sIL-4R is expressed in kidney cells and antagonistically functional.

Biochemical characterization of a T-lymphoma-specific 90,000 molecular weight disulfide linked dimeric glycoprotein.

The biochemical features of a membrane antigen detected by a mouse monoclonal antibody (A1) raised against the murine thymoma cell line EL4 are described. This reagent detected a novel disulfide-linked 90,000 mol. wt dimeric membrane glycoprotein composed of two chains of approx 45,000 mol. wt. Endo-beta-N-acetylglucosaminidase F digestion generated a single 28,000 polypeptide, thus suggesting that the A1 molecule is a homodimer. No structural homology between the A1 molecule and the human T 90/44 protein (9.3 antigen) could be revealed by peptide mapping analysis. In view of the fact that three polypeptides of mol. wts 28,000-30,000, 21,000 and 15,000 respectively co-precipitated with the A1 antigen, the possible relationship of the A1 molecular complex to other known T-cell surface antigens including the antigen receptor is discussed.

The gene encoding mouse lymphocyte antigen Ly-49: structural analysis and the 5'-flanking sequence.

Genomic clones encoding the mouse cell-surface antigen, Ly-49, were isolated, and the gene organization was analyzed. The gene spanned approximately 19 kb, and contained seven exons and six introns. The lengths of introns ranged from 1.3 to 8 kb. A 1067-bp sequence in the 5'-flanking region was determined. Primer extension analysis and S1 nuclease mapping revealed a cap site at 158 bp upstream from the ATG coding the N-terminal Met of Ly-49. The 5'-flanking sequence contained a possible promoter sequence, a potential binding site for the T cell-specific transcription factor (TCF-1 alpha/LEF-1), and three sites for the basic helix-loop-helix-binding basic proteins (bHLH). However, no CAAT box-like sequence was present. These results provide important clues for understanding the mechanism of gene expression of lymphocyte antigens.

Hybridoma autoantibodies to erythrocytes from NZB mice and the induction of hemolytic anemia.

We established two clones of monoclonal hybridoma, from a non-immunized NZB mouse, which produce IgM class hemagglutinating autoantibodies reactive with the exposed murine erythrocyte autoantigens. Absorption studies revealed that one monoclonal antibody exhibits cross-reactivity to chick erythrocytes and mouse liver, and the other antibody to rat erythrocytes and mouse brain. Optimal temperatures for the hemagglutination were 22 degrees C with the former and 4 degrees C with the latter. The specificity and nature of the autoantibodies are apparently distinct from any of the erythrocyte autoantibodies described to date in NZB mice, anti-X, anti-HB, anti-HOL or anti-I antibodies. Implantation of these hybridoma cells in BALB/c mice induced autoimmune hemolytic anemia associated with a marked splenomegaly. These findings provide evidence that the erythrocyte autoantibodies in NZB mice are more heterogenous than generally assumed and suggest that varieties of erythrocyte autoantibodies may be involved in the development of a naturally occurring hemolytic disease in NZB mice.

Specificity of mouse hybridoma antibodies to DNA.

Utilizing the somatic cell hybridization technique, we established 12 clones of mouse hybridoma from MRL/Mp-lpr/lpr(MRL/1) and NZB X NZW(B/W F1) mice, which produce antibodies reacting mainly with ssDNA (anti-ssDNA) and 2 clones from B/W F1 mice, which produce antibodies reacting with both ssDNA and dsDNA (anti-ss/dsDNA). By means of a competitive inhibition radioimmunoassay with synthetic polynucleotides, the anti-ssDNA antibodies were classified into 5 types, in terms of the DNA base specificity. The anti-ss/dsDNA antibodies had polyspecific reactivity to a wide range of polynucleotides, including synthetic RNA.

Detection of antibodies to a recombinant gag protein derived from human endogenous retrovirus clone 4-1 in autoimmune diseases.

To investigate whether human endogenous retroviruses (HERV) contribute to autoimmune diseases, we prepared a recombinant p30gag protein derived from clone 4-1 of the HERV family, using a baculovirus-vector system. This p30gag protein (CA41B) was approximately 30 kDa, as expected, and reacted with antibodies for p30gag purified from both murine and feline leukemia virus. This result suggested that the antigenic determinant for p30gag was well conserved in CA41B. Analysis of serum antibodies to p30gag in patients with autoimmune diseases was done by Western blotting. CA41B detected anti-p30gag antibodies in 48.3% of systemic lupus erythematosus (SLE) patients, 35.0% of Sjögren's syndrome (SS) patients, and 33.3% of mixed connective tissue disease (MCTD) patients, whereas no anti-p30gag antibodies were found in healthy subjects. This suggested that HERV p30gag or other retroviral p30gag proteins possessing the same antigenic determinant as CA41B may play a role in these diseases. Although detection of antibodies to HERV p30gag in autoimmune diseases is indirect evidence that HERV proteins are involved, this study showed that patients with autoimmune diseases have antibodies to HERV p30gag using a recombinant HERV protein rather than synthetic peptides based on HERV or retroviral proteins of other species.

Age-associated changes in renal glomeruli of mice.

To investigate age-associated changes in renal glomeruli of C57BL/6 female mice, we used a single radial immunodiffusion method to measure albumin excretion. Up to 100 mg/dl in urine samples was regarded as microalbuminuria. The mean amount of urinary albumin increased from 14.0 mg/dl at 6 months to 151.1 mg/dl at 24 months of age. Microalbuminuria occurred in 64.6% of tested mice by the time they were 24 months old, and 10% of the mice had marked albuminuria (more than 100 mg/dl) at that time. Parallel morphological study showed that renal mesangial changes were also age-dependent. Mesangial cell proliferation and spike lesions in glomerular capillary walls appeared in aged mice with microalbuminuria, and were then followed by diffuse glomerular sclerosis accompanied by marked albuminuria. Histological scores on damage in the renal mesangium with changes of glomerular basement membrane increased significantly with age from a mean score of 0 at 6 months to 3.24 at 24 months of age. Immunofluorescent study showed a marked deposition of IgG and IgM, but no complement component C3 in enlarged mesangium. Electron microscopic examination of diffuse sclerotic glomeruli in aged mice revealed amyloid substances. These results suggest that assays of albuminuria could be a useful method for early detection of age-associated renal deterioration.

Adrenomedullin amidation enzyme activities in hypertensive patients.

Adrenomedullin (AM) is a potent vasodilating peptide secreted from the vasculature of various organs. It is biologically active when its C-terminus is amidated. Recently, an RIA method was developed for measurement of the active form of AM, or mature AM. We here employed this method to investigate the significance of amidation of AM in controlling cardiovascular function. Thirty-six patients under hemodialysis were recruited and divided into hypertensive (n = 25; 157/86 mmHg) and normotensive (n= 11; 116/66 mmHg) groups. Mature AM, immature AM and blood pressure were monitored during hemodialysis in all patients. There was a significant reduction in blood pressure during hemodialysis in both groups, although after hemodialysis blood pressure was still higher in hypertensives than in normotensives (139 +/-14.8/76 +/- 2.5 mmHg vs. 110 +/- 5.1/66.7 +/- 3.1 mmHg). Mature AM before hemodialysis were lower in hypertensives than normotensives and it decreased in both groups. Although mature AM decreased more in normotensives than in hypertensives (-27 +/- 8% vs. -17 +/- 5%), at the end point, its level was still higher in normotensives. The ratio of mature AM/immature AM decreased only in normotensives (-11.4 8.7%), whereas it remained stable in hypertensives (0.2 +/- 5.6%). Both groups showed similar changes in ANP, endothelin, catecholamines, cGMP, and NOx. The low level in mature AM level in hypertensives may have contributed to the higher blood pressure in this group. The attenuation of AM amidation in normotensives indicates that an unspecified amidative enzyme of AM was regulated in order to normalize blood pressure.

Abnormal calcium metabolism in myotonic dystrophy as shown by the Ellsworth-Howard test and its relation to CTG triplet repeat length.

Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by peculiar clinical features. Its molecular basis is the unstable expansion of a CTG triplet repeat in the gene encoding myotonin protein kinase (Mt-PK), the nucleotide sequence of which has extensive homology to the cyclic AMP (cAMP)-dependent protein kinase gene. Extensive efforts have been made to clarify the signal transduction pathway in which the responsible gene operates, but confirming evidence has yet to be obtained. Because some symptoms in DM are similar to those in hypoparathyroidism, we divided 24 DM patients into two groups on the basis of their serum calcium levels; Group 1, those with normocalcemia (11 patients), and group 2, those with hypocalcemia (13 patients). The highly sensitive parathyroid hormone (HS-PTH) plasma levels in group 1 were within normal limits, whereas those in group 2 were abnormally high. Laboratory findings for the group 2 patients resembled those for pseudohypoparathyroidism (PHP), whereas those for group 1 patients were normal. The Ellsworth-Howard (EH) test was used to determine which type of PHP the group 2 patients belonged to. Both the phosphaturic (delta P) and urinary cAMP (UcAMP) responses were estimated. The delta P responses in group 2 were significantly lower than those in group 1, but their UcAMP responses did not differ. This is evidence that group 2 patients had PHP type II, whereas group 1 patients were normal. We also investigated whether the disease severity differed between the groups. Cataracts, ectopic calcifications, and ossifications, which are associated with PHP, were more frequent in group 2. In addition, the mean IQ in that group was significantly lower. Clinically, the group 2 signs agreed well with those of PHP, whereas for group 1 there was only a slight similarity. These results are additional evidence that the patients in group 2 have abnormal calcium metabolism, the abnormality being in the postadenylate cyclase-cAMP pathway in the renal tubular cells. The degree of (CTG)n expansion, the so-called expanded DNA fragment (EF) size, was determined by standard Southern blot analysis. The allelic EF sizes in both groups were greater than in the healthy controls. Moreover, those in group 2 were significantly longer than those in group 1. We therefore investigated whether EF size is correlated with the serum calcium and plasma PTH levels, the delta P responses in the EH test, and IQ. All these items were significantly correlated with EF size. Our findings show that the expanded DNA fragment size in DM is correlated with the degree of abnormal calcium metabolism.

Genetic variation and interspecific hybridization among natural populations of zoysiagrasses detected by RFLP analyses of chloroplast and nuclear DNA.

Genetic variations among 17 accessions of zoysiagrasses collected from natural populations in Japan were investigated by RFLP analyses of chloroplast DNA (cpDNA) and nuclear DNA. These accessions were classified into five species based on morphological characteristics: Zoysia japonica, Z. matrella, Z. tenuifolia, Z. sinica, and Z. macrostachya. On the basis of eight kinds of RFLPs in cpDNAs detected across accessions, six chloroplast genome types (types A-F) were identified. Although type-A cpDNA was shared by five accessions of japonica and four accessions of matrella, derivative cpDNAs of type A, which each arose by a mutation, were identified in one accession of japonica (type B) and in two accessions of matrella (type C). One accession of japonica which showed spikelets similar to those of shapes macrostachya, contained type-F cpDNA as did sinica and macrostachya. The two accessions of tenuifolia each showed a specific cpDNA type, i.e. types D and E. Genetic relationships among the 17 accessions were investigated by the RFLP analyses of nuclear DNA with 20 genomic and gene probes. A dendrogram constructed with genetic distances calculated from the RFLP patterns indicated four major groups among them. Six accessions of japonica comprised one group, whereas the one accession of japonica possessing the type-F cpDNA was clustered with macrostachya and sinica. Four accessions of matrella with type A cpDNA constituted another group in the dendrogram, showing a closer relationship to the japonica accessions than to the other two accessions of matrella. The remaining two accessions of matrella and tenuifolia accessions were grouped together. These data indicate that zoysiagrasses distributed in Japan harbor highly genetic variations, and that interspecific hybridization has occurred in natural populations.

An autopsy case of light chain deposition disease.

This report describes a case of light chain deposition disease (LCDD) with unusual findings of fibrillar structures in the deposits and marked calcification in several organs. A forty-year-old man was initially diagnosed with LCDD in 1987, and died of sepsis three and one-half-years later. Histological examination of autopsy specimens demonstrated eosinophilic amorphous materials, which differed from amyloid, in vessel walls or around parenchymal cells in almost every organ examined. Ultrastructurally, in addition to granular deposits, fibrillar structures were also seen in the deposits. Marked calcification was present in the myocardium, skeletal muscles, adrenal glands and arteries.

[Studies on toxicity of clobetasone-17-butyrate (II)--chronic toxicity in rats (author's transl)].

Chronic toxicity of clobetasone-17-butyrate, an anti-inflammatory corticosteroid, was investigated in rats. Subcutaneous administration with the drug at dose of 0.003, 0.01 and 0.03 mg/kg/day for three and six months induced no significant changes in the rats. At 0.1 and 0.3 mg/kg/day, however, some dose-dependent symptoms such as suppression of body weight gain, emaciation, regressive changes in adrenals, lymphatic and hematopoietic tissues, decrease in circulating white blood cell and lymphocyte counts, which have been known as toxic effects of synthetic corticosteroids, were induced. The results indicates that the maximum no-toxic dose of clobetasone-17 butyrate was 0.03 mg/kg/day on this experimental condition. In the recovery test for two months no significant differences in the treated rats from controls were found, suggesting that the toxic effects were reversible in the animals treated at 0.3 mg/kg/day and lower than that.

Hyperhomocysteinemia as a possible role for atherosclerosis in CAPD patients.

It has been shown that hyperhomocysteinemia is a risk factor for atherosclerotic vascular disease. In this study, we measured total plasma homocysteine in continuous ambulatory peritoneal dialysis (CAPD) patients and evaluated its correlation with atherosclerosis. Subjects consisted of healthy volunteers, and hemodialysis (HD) and CAPD patients. Fluoro-HPLC was employed to estimate plasma levels of total homocysteine (Hcy). Plasma levels of total Hcy were significantly higher in the CAPD patients compared with the HD patients and controls. Atherosclerotic score (ASS) was calculated, and the correspondence with plasma levels of total Hcy was analyzed. There was a significant correlation between plasma levels of total Hcy and ASS in CAPD patients. However, plasma levels of total Hcy did not correlate with age, plasma vitamin B6 level, residual renal function, protein catabolic rate (PCR), or KT/V. Our present study suggests that elevated concentrations of total plasma Hcy might play a role in the development of atherosclerosis in CAPD patients.

[A case of difficult intubation with tracheobronchial anomaly].

We experienced a case of difficult intubation with tracheobronchial anomaly. A 66-year-old male was scheduled for subtotal esophagectomy. We used a univent bronchial blocker tube (UBT) to separate the lungs because of difficulty with tracheal intubation using a 37 F double lumen tube (DLT). Intraoperatively, we could not separate the lungs due to tracheobronchial anomaly in the right lung, and attempted to change the tube. We could insert a 35 F DLT to the trachea and separate the lungs. In the case of difficult or impossible conventional direct-vision intubation, the use of DLT is a relative contraindication. However, in this case, the separation of the lungs with UBT was difficult because of tracheobronchial anomaly.

[An adult case of glomerulocystic kidney disease].

A 56-year-old female presented with end-stage renal disease. A CT scan of her kidneys demonstrated that the density of the renal parenchyma was quite low as compared with normal kidneys, and that corticomedullary demarcation was obscured. Magnetic resonance imaging (MRI) disclosed low intensity of the kidneys in T1-weighed images, and high intensity in T2-weighed images. In order to elucidate the etiology of her kidney disease, open renal biopsy was performed. The kidney surface was covered with numerous cysts with a diameter of less than 3 mm. Biopsy specimens from the cortical surface showed multiple cystic lesions. Serial sections of more than 200 slices of the biopsy material demonstrated that traces of collapsed glomeruli were present in most of the cysts. On electron microscopy, some epithelial cells lining the cysts were found to be round-shaped and contained a substantial amount of mitochondria, suggesting proximal tubules. These histological findings were compatible with glomerulocystic kidney disease (GCKD). An adult case of GCKD has rarely been reported, but the CT scan as well as MRI of the kidneys appeared to complement the diagnosis of GCKD. Although the cysts of GCKD have been considered to be dilatations of the Bowman's capsules, our observation suggested that part of the cells lining the cysts consisted of proximal tubular epithelium.

An adult case of polycystic kidney disease associated with congenital hepatic fibrosis.

Congenital hepatic fibrosis is often associated with infantile, but not with adult polycystic kidney disease. We report the unusual case of an adult patient with polycystic kidney disease complicated by congenital hepatic fibrosis. A 27-year-old women was admitted to our hospital because of gross hematuria due to hemorrhage from renal cysts. She presented hematemesis from ruptured esophageal varices at the age of 14 years. She was diagnosed as having end-stage renal disease due to polycystic kidney disease at the age of 23 years, and maintenance hemodialysis was initiated the following year. Gross hematuria was managed with supportive therapy. However, the patient developed cholangitis and died of sepsis. Postmortem examinations as well as the patient's clinical course suggested that she had an autosomal dominant type of polycystic kidney disease. Histological findings of the liver were compatible with congenital hepatic fibrosis.

[A case of primary amyloidosis associated with giant cell infiltration within a Bowman's capsule].

A 67-year-old man was hospitalized with a diagnosis of nephrotic syndrome. Physical findings at admission were generalized edema and macroglossia. Urinalysis showed massive proteinuria, + +occult blood, and granular and broad casts. Ig A lambda monoclonal gammopathy was noted in the serum. There was no evidence of myeloma in the bone marrow aspirate, scintigram or X-ray of the bone. A biopsy specimen of the kidney showed massive deposits of structureless material in the glomeruli. Marked cell infiltration was also observed in the interstitium. Multinucleated giant cells were occasionally seen in the Bowman's capsules and the interstitium. There were reactive changes in the Bowman's capsule adjacent to the giant cell. The deposits were proved to be amyloid by positive staining with Congo red and apple-green birefringence by polarized light. In addition, microfibrills seen on electron microscopy displayed deposits. Amyloid depositions were observed in other tissues such as gingiva, skin and tongue. Staining of amyloid with Congo red was resistant to potassium permanganate, and amyloid was positively stained with lambda-light chain of immunoglobulin. These findings indicated that the patient had primary amyloidosis. Infiltration of the multinucleated giant cell has been reported only in patients with familial amyloidosis and secondary amyloidosis associated with rheumatoid arthritis. To our knowledge the present case is a first report of the giant cell infiltration in a Bowman's capsule in primary amyloidosis.