Biomedical subjects
Publications and source records attributed to N Yoshikawa.
The clinical, histological, and electron microscopic features of a case of malignant amelanotic melanoma of the esophagus are described. Amelanotic melanoma is difficult to distinguish from other malignant lesions, but in our case electron microscopy was helpful in the diagnosis.
The clinical features, renal biopsy findings, and subsequent course in 53 children with asymptomatic constant isolated proteinuria were studied retrospectively (1) to determine the prevalence of renal pathologic abnormalities among these children, (2) to identify those clinical characteristics that may enable recognition of children with an increased likelihood of having renal pathologic abnormalities, and (3) to assess the clinical course. All biopsy specimens were examined by light, immunofluorescence, and electron microscopy. Twenty-five patients (47%) had significant glomerular changes (15 had focal segmental glomerulosclerosis, 4 IgA nephropathy, 3 diffuse mesangial proliferative glomerulonephritis without IgA deposition, and 3 membranous glomerulonephritis), and 28 had minimal glomerular changes. There were no differences between the patients with significant glomerular changes and those with minimal glomerular changes with regard to clinical and laboratory findings except for a predominance of boys in the former group. At the latest follow-up, seven patients with significant glomerular changes, but none with minimal glomerular changes, had chronic renal impairment. Because of the high incidence of significant glomerular changes and the high rate of progression to chronic renal impairment, we believe that a renal biopsy is indicated for a child with asymptomatic constant isolated proteinuria.
In order to examine T lymphocyte function in childhood IgA nephropathy, 13 patients and 10 age-matched control subjects were studied. T lymphocyte function was examined in terms of in vitro immunoglobulin synthesis by peripheral blood mononuclear cells (PBMC) and CD4-depleted (suppressor-rich) and CD8-depleted (helper-rich) PBMC in both unstimulated and pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SAC) stimulated cultures. T lymphocyte subpopulations were examined by two-color immunofluorescence analysis using Fluorescein-Activated Cell Sorter (FACS). Children with IgA nephropathy showed (1) a significant increase in IgA synthesis by PBMC with or without mitogen stimulation, (2) a significant increase in IgG and IgA synthesis by CD4-depleted (suppressor-rich) PBMC, (3) a significant increase in IgG and IgA synthesis by CD8-depleted (helper-rich) PBMC, and (4) a significant decrease in suppressor-inducer T cells (Leu3a+Leu8+). These results suggest that a decrease in suppressor-inducer T cells, impaired suppressor T cell function and hyperactivity of helper T cell function are responsible for the increase in IgA production in children with IgA nephropathy.
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To elucidate the mechanism of thyrotropic activity of human chorionic gonadotropin in sera of normal pregnant women, we examined the effect of blockade of the TSH receptor on the serum-induced cAMP accumulation and the effect of hCG on the TSH binding to FRTL-5 cells. In the presence of crude immunoglobulin fractions in sera from patients with primary hypothyroidism, cAMP accumulation induced by both crude and purified hCG, and normal pregnant women serum were significantly inhibited compared with that in the presence of normal IgGs. The mode of inhibition of these IgGs on the cAMP accumulation was similar for TSH and hCG when analysed by Lineweaver-Burk plots. Moreover, binding of [125I]bTSH to the TSH receptor in porcine thyroid cell membrane was apparently inhibited by adding 4 x 10(6) IU/l of purified hCG. Binding studies of TSH in FRTL-5 cells also indicated the dose-dependent displacements of [125I]TSH by hCG. Although half-maximal inhibitory concentration of hCG was about 20 times as high as that of TSH on a molar basis, displacement of [125I]TSH was observed at a concentration of hCG of 10(5)IU/l or more, which could be a physiological concentration of hCG in sera of normal pregnant women. These results suggest that thyrotropic activity of hCG in sera of normal pregnant women is, at least in a part, mediated by TSH receptors.
Quantitative analysis of the mesangial matrix and cells was performed on serial renal biopsies from 41 children with IgA nephropathy. In the repeat renal biopsy, nine patients showed a significant increase of mesangial matrix, 29 showed no change and in three there was a significant decrease. Eight of the nine patients (89 per cent) with a matrix increase had persistent proteinuria at the second biopsy, whereas only 14 of the 32 (44 per cent) without a matrix increase had persistent proteinuria (P less than 0.05). Although the mesangial matrix increased in patients with persistent proteinuria, there was no decrease in patients with clinical remission. In contrast to the mesangial matrix, mesangial cells significantly decreased in 23 patients, did not change in 16, and significantly increased in only two in the second biopsy. These findings suggest that mesangial matrix increase is usually an irreversible change and that persistent proteinuria is associated with matrix increase with worsening in glomerular morphology and clinical outcome. This study indicates the importance of serial renal biopsy in children with IgA nephropathy with persistent proteinuria.
We report our experience with ciclosporin (CS) treatment in 18 children with steroid-dependent nephrotic syndrome. CS was started at 3-5 mg/kg per day after the patients had attained remission with steroid therapy, and was adjusted to maintain a trough blood level of between 250 and 600 ng/ml, being administered for 6 months. Although 1 patient dropped out of the study because of renal dysfunction, the remaining 17 children completed the full trial. No relapse occurred during the 6-month period of CS treatment, and it was possible to discontinue steroid therapy in all patients. However, after discontinuation of CS treatment, nephrotic syndrome relapsed in 16 patients and 14 again had frequent relapses and became steroid-dependent, as before CS treatment. The effect of CS in maintaining remission from steroid-dependent nephrotic syndrome was thus dependent on continuation of CS treatment. Although several side-effects occurred during CS treatment, they were not so serious as to necessitate discontinuation of treatment, except in 1 patient, and all of these side effects were reversible. CS is therefore a new agent for the management of children with steroid-dependent nephrotic syndrome in place of corticosteroid and alkylating agents, although long-term maintenance therapy may be necessary for maintaining longer remission.
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The serum carnitine (total carnitine), total protein, amino acid, and triglyceride levels were determined in children on continuous ambulatory peritoneal dialysis (CAPD). Compared with levels in controls, serum carnitine levels were significantly decreased in patients on CAPD for more than 4 months, while those of patients on CAPD for 1-3 months were not decreased. Patients on CAPD for more than 4 months also showed lower serum total protein levels than in normal controls. The mean triglyceride levels in patients on CAPD for both 1-3 months and more than 4 months were higher than those in normal controls. Among the amino acids, the serum levels of tryptophan, isoleucine, leucine, tyrosine, valine, serine, and asparagine were significantly lower in patients treated with CAPD than in normal controls, whereas the levels of other amino acids were either increased or not changed. Isoleucine and leucine levels showed a strong correlation with serum carnitine. Our data suggest that malnutrition plays a role in the decrease of serum carnitine levels in patients receiving CAPD.
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Immunoglobulin production by peripheral blood mononuclear cells (PBMC) and lymphocyte subpopulations were studied in 56 children with IgA nephropathy (IgAN) and 22 healthy controls. All the patients had persistent proteinuria at the time of diagnosis, and were divided into three clinical groups on the basis of urinary findings at the time of examination: 27 patients had proteinuria with or without microscopic hematuria (group A; active stage), 9 had microscopic hematuria only (group B; healing stage) and 20 had normal urine (group C; remission stage). PBMC from the patients in group A cultured without mitogenic stimulation produced significantly more IgA and IgG than those from controls (p less than 0.05). After polyclonal B cell stimulation with pokeweed mitogen (PWM), PBMC from patients in group A produced significantly more IgA than those from group B (p less than 0.05), group C (p less than 0.05) or controls (p less than 0.01), and produced significantly more IgG than those from group B (p less than 0.05) or controls (p less than 0.01). However, there was no significant difference in PWM-stimulated IgG production between groups A and C. PWM-stimulated PBMC from patients in group C produced significantly more IgA and IgG than those from controls (p less than 0.05). There were no significant differences in lymphocyte subpopulations among groups A, B and C and controls. These findings show that the clinical course of childhood IgAN is correlated with IgA production by PBMC suggesting that overproduction of IgA might be responsible for the pathogenesis of IgAN in children.
The effects of T3 and T4 on the iodothyronine 5'-deiodinase (5'-D) activity in FRTL-5 rat thyroid cells were investigated. T3 and T4 stimulated the 5'-D activity in a dose-dependent manner. Kinetic studies showed that the stimulation of the 5'-D by T3 was associated with an increase in maximum velocity (Vmax) in [11.9 +/- 0.2 (mean +/- SE) and 25.4 +/- 0.9 pmol I-released/mg protein.min, respectively, in control and cultured with 10(-9) M T3 for four days] but without a change in apparent Michaelis-Menten constant (Km) (94.8 +/- 5.3 nM and 105.4 +/- 12.1 nM, respectively). Furthermore, cycloheximide (5 microM) completely abolished the stimulatory effect of T3 on the 5'-D activity. T3 and T4 also enhanced the 5'-D activity stimulated by TSH in a dose-dependent manner. Kinetic studies showed that the stimulatory effect of T3 on the 5'-D stimulated by TSH was again associated with an increase in Vmax (86.0 +/- 4.0 and 166.5 +/- 1.9 pmol I- released/mg protein.min, respectively, cultured with 0.3 U/liter TSH and cultured with TSH plus 10(-9) M T3 for four days) without a change in apparent Km (114.0 +/- 7.4 nM and 111.6 +/- 12.5 nM, respectively). Cycloheximide (5 microM) completely abolished the stimulatory effect of TSH plus T3 on the 5'-D activity. There were no significant differences observed between cells cultured with TSH and with TSH plus T3 in either the intra- or extracellular cAMP contents. Furthermore, T3 enhanced the 5'-D activity stimulated by (Bu)2 cAMP. These results strongly suggest that T3 or T4 was synergistic with TSH in stimulating the 5'-D activity in FRTL-5 cells, and that cAMP production would be an important component of the synergism.
To elucidate the effect of Graves' immunoglobulin G (IgG) on iodothyronine deiodination in the thyroid, we examined the characteristics of iodothyronine 5'-deiodinating (I-5'-D) activity in FRTL-5 rat thyroid cells and the effect of Graves' IgG on its activity. FRTL-5 cells were sonicated and incubated with 0.5 mumol/L rT3 with a tracer amount of [125I]rT3 in 0.1 mol/L phosphate buffer containing 1 mmol/L EDTA and 0.5 mmol/L dithiothreitol. The released 125I- was separated by Dowex-50WX2 column and counted. The amount of I- released was tissue, incubation time, temperature, and pH dependent, strongly suggesting that the reaction is enzymatic. The activity was completely inhibited by propylthiouracil. The Km value for rT3 was approximately 0.32 microM when analyzed by Lineweaver-Burk plot. TSH, dibutyl cAMP, and Graves' IgG induced I-5'-D activity in a dose-dependent manner. However, cycloheximide (5 mumol/L) abolished the stimulating effects of these agents on I-5'-D activity. These results suggest that TSH, dibutyl cAMP, and Graves' IgG induced I-5'-D activity through the synthesis of new enzyme protein. A significant positive correlation between thyroid-stimulating antibody activity assayed by measuring cAMP production using FRTL-5 cells and I-5'-D activity induced by the Graves' IgG in 10 patients with untreated Graves' disease was observed (r = 0.79; P less than 0.01). The present findings suggest that type I iodothyronine 5'-deiodinase exists in FRTL-5 rat thyroid cells and that Graves' IgG as well as TSH stimulate the activity at least in part by activating adenylate cyclase.
To study the possible correlation between the bioactivity ratios of the serum TSH and serum thyroid hormone concentrations in patients with primary hypothyroidism, the bioactivity of TSH in these patients was determined by means of a sensitive and practical bioassay and the results were compared to those in normal subjects. Sample sera were obtained from 11 patients with primary hypothyroidism and 9 normal subjects. TSH was extracted from the serum with anti-human TSH monoclonal antibody-coated tubes. The bioassay for TSH was performed by measuring the amount of cAMP released into the medium from cultured FRTL-5 cells incubated with the TSH extract. Linear relations were found between the serum TSH values, measured by the immunoassay, and the bioactivity of the TSH, measured by the present assay, in normal subjects (y = 1.13 x-0.3, r = 0.89, p less than 0.01) and in patients with primary hypothyroidism (y = 0.90 x + 0.7, r = 0.93, p less than 0.01). Moreover, the regression line in patients with primary hypothyroidism was not significantly different from that in normal subjects based on the analysis of covariance. The present findings suggest that the bioactivity of TSH is consistent with the immunoreactivity of TSH in patients with primary hypothyroidism as well as in normal subjects.
To ascertain the presence of thyroid growth-promoting activity (TGA) in the sera of pregnant women, we measured TGA in the sera of pregnant women by means of a bioassay based on [3H]-thymidine [( 3H]Tdr) incorporation in cultured rat FRTL-5 thyroid cells. Furthermore, to elucidate the mechanisms of human chorionic gonadotropin (hCG) in promoting the thyroid growth, we evaluated the effects of blocking type TSH receptor antibody (blocking IgGs) from patients with primary hypothyroidism on the activity of hCG. After the PEG-pretreated serum or the serum plus blocking IgGs was incubated for 72 h at 37 degrees C with FRTL-5 cells and [3H] Tdr, [3H] Tdr incorporated in the cells was counted. Although 9 normal pregnant women had normal TGA, two patients with hydatidiform mole, whose hCG levels were 966,500 and 497,100 IU/L, had positive TGA, but the activity showed normal when analyzed with the addition of a blocking IgG. hCG also showed a dose-dependent increase in [3H]Tdr incorporation, and it was inhibited by the addition of blocking IgGs. Furthermore, the inhibition of hCG-induced [3H]Tdr incorporation by 16 blocking IgGs correlated with their TBII and the inhibition activity of hCG-induced cAMP accumulation. Analysis by the Lineweaver-Burk plots of dose response curves of TSH- and hCG-induced [3H]Tdr incorporation showed the same inhibition pattern as with the addition of the same blocking IgGs. In conclusion, 1) hCG-related TGA exists in the sera of some patients with hydatidiform mole; and 2) hCG and the sera of some patients with hydatidiform mole promote thyroid growth, at least in a part, via TSH-receptors in FRTL-5 cells.
To ascertain the thyrotropic activity of human chorionic gonadotropin in sera of normal pregnant women, we examined the adenylate cyclase activation in the cultured FRTL-5 cells by extracted hCG from 7 normal pregnant women. hCG was extracted from the sera using anti-hCG-beta subunit monoclonal antibody-coated microwells, eluted with 2 mol/l guanidine-HCl, and reconstituted with hypotonic Hanks' solution. FRTL-5 cells were precultured in 5H medium, incubated for 2 h with the serum extracts, and the cAMP released into the medium was measured. hCG levels in serum extracts ranged from 1100 to 6800 IU/l; values corresponded to 1.4-19.8% compared with those in the original serum samples. Addition of the extracts to FRTL-5 cells resulted in significant increases in the cAMP accumulation, ranging from 9.8 to 59.0 nmol/l. cAMP levels were also increased in a dose-dependent manner by adding purified hCG as well as crude hCG and hTSH to FRTL-5 cells. These findings suggest that the thyroid gland of normal pregnant women may actually be stimulated by hCG itself.
Hepatocytes isolated from rat liver were immobilized within Ca-alginate. Immobilized hepatocytes could remove ammonia and other toxic substances causing hepatic coma, such as indole, phenol, bilirubin, and short chain fatty acids. Although free hepatocytes lost those activities within 2 days, immobilized hepatocytes maintained those activities for more than 7 days. Immobilized hepatocytes induced tyrosine aminotransferase (TAT) in the presence of dexamethasone and dibutyryl-cAMP and retained the ability to induce TAT for more than 7 days. Biologically active form of coagulation factor II, prothrombin could be synthesized and secreted into medium by immobilized hepatocytes. Moreover, immobilized hepatocytes produced glucose from lactate, alanine, fructose, and galactose. Like adult rat liver, growth-related function and liver-specific function in immobilized hepatocytes were reciprocally controlled by cell density. There are both alpha-, and beta-adrenergic receptors in membrane of liver cells, and the adrenergic action of epinephrine is alpha-predominant in adult rat liver. Monolayer-cultured hepatocytes can not maintain alpha-adrenergic response. However, immobilized hepatocytes maintained alpha-adrenergic response as shown in vivo. Those characteristics of immobilized and three-dimensionally cultured hepatocytes are regarded almost the same as liver cells in vivo. Furthermore, therapeutic effect of immobilized hepatocytes on the hepatic failure were confirmed in the experiment using hepatocytes damaged with D-galactosamine. Therefore, it is suggested that immobilized hepatocytes could be applied to a hybrid artificial liver support.
A new antibiotic, MK4588, structurally related to xanthocillin, was isolated from the culture broth of Leptosphaeria sp. L-179. Antibiotic MK4588 exhibited inhibitory activity against a limited range of Gram-positive and Gram-negative bacteria. The antibiotic was degraded by alkali to a more active product. The structures of MK4588 and the degradation product were determined to be (1R*,6S*,7S*)-7-(Z)-(1-isocyano-2-(4-methoxyphenyl]ethenyl-1-hydro xy-7- isocyanobicyclo[4,2,0]oct-2-en-4-one and (Z)-2,3-diisocyano-1-(4-methoxyphenyl)buta-1,3-diene, respectively, by NMR spectral analyses coupled with X-ray crystallographic analysis of MK4588.