Mitochondrial encephalomyopathies with the mutation of the mitochondrial tRNA(Leu(UUR)) gene.

Four families with mitochondrial encephalomyopathy are described. Probands of three families had typical clinical presentations of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), but the proband of family 4 lacked strokelike episodes. The mitochondrial DNA mutation of tRNA(Leu(UUR)) (transfer ribonucleic acid specific to leucine (UUR codon)) found in MELAS was examined in muscle DNA obtained from biopsy samples of the probands of four families and the maternal relatives of family 2. The mutation was detected in all muscle samples, and the degree of the mutated DNA was 68% to 84% by Southern blot analysis. However, the clinical patterns of the maternal relatives of family 2 were mild and distinctly different from MELAS. The same mutation was also detected in blood-derived DNA samples of all family members examined, including healthy mothers but not fathers, although the degree of mutation did not correlate with the clinical severity. These results confirmed the maternal inheritance of this disease and suggested that the mitochondrial DNA mutation (tRNA(Leu(UUR))) may cause clinical symptoms other than MELAS. The clinical findings of mitochondrial encephalomyopathy should be reinvestigated in terms of the mitochondrial gene mutation; the polymerase chain reaction method will be useful for screening for this mutation of mitochondrial DNA in blood samples.     

Magnetic resonance imaging using "fat-saturation" technique is useful for diagnosing small endometrioma: a case report.

Preoperative MRI using fat-saturation technique was performed in a patient with endometriosis. Endometrial implants as small as 3 mm were accurately diagnosed by MRI. The fat-saturation technique is useful for diagnosing small endometrioma.     

Fetal diaphragmatic hernia: prenatal evaluation of lung hypoplasia and effects of immediate operation

The outcome of fetuses with diaphragmatic hernia (CDH) has been reported to be related to the severity of lung hypoplasia. As an index of pulmonary hypoplasia, we attempted to measure the lung-thorax transverse area ratio (L/T) using ultrasonic echography in eight fetuses with left-sided CDH. Two cases with L/T more than 0.28 (controls: 0.52±0.04) were transported postnatally and recovered after early operation without episodes of persistent fetal circulation. Elective surgical repair was performed in six infants immediately after cesarean delivery at 35–37 weeks' gestation. In three cases with L/T between 0.21 and 0.24 who recovered with no complications, surgical reduction of the abdominal organs improved arterial blood gases and high-frequency oscillation ventilation (HFOV) was fully effective for respiratory management. In three with L/T between 0.11 and 0.17, extracorporeal membrane oxygenation (ECMO) was required from the 1st to the 12th postoperative day despite HFOV. Although two infants died of combined cardiovascular anomalies and airway bleeding caused by prolonged HFOV, respectively, one infant with minimal L/T survived. Measurement of L/T may help to predict the outcome of fetuses with CDH and to determine the indications for various treatments including immediate operation after cesarean delivery, HFOV, and ECMO. Offprint requests to: S. Kamata     

The Measles Outbreak in Chikuhou District, Fukuoka, Japan, 1990: Correlation between Herd Immunity Level and Outbreak Size

A measles outbreak occurred in the Chikuhou district of Fukuoka, Japan from May to October 1990, during which 71 patients were cared for at the Itoda Public Hospital. Hospital records revealed a large outbreak in the adjacent town of Kanada. In order to characterize the outbreak, questionnaires were sent to all preschool-age children in Itoda (73% effective response) and in Kanada (76% effective response) requesting information about their vaccination and/or history of measles. The number of patients was 22 (4%) in Itoda and 63 (14%) in Kanada, most of these being preschoolers, while the vaccination rate was 61% and 44%, respectively. The herd immunity levels in age-specific groups were compared between the two towns. Before the epidemic, the immunity level of 1 year old children in Kanada, who showed the higher attack rate, was lower (18%) than that in Itoda (39%), while after the epidemic it rose above 60% in both towns. When we studied the correlation between the attack rate and the vaccination rate, or the number of children susceptible to measles (susceptibility rate) in each preschool, the attack rate correlated negatively with the vaccination rate (correlation coefficient [CC] = - 0.818; P < 0.01), and positively with the susceptibility rate (CC 0.860; P < 0.01). The regressed equation on the correlation indicated that the immunity level should be more than 70% to keep the attack rate under 5% in preschoois. After the epidemic, the immunity levels of all preschoolers reached above 70%. Early vaccination should be given to at least 70% of young preschoolers in order to prevent a large outbreak of measles under the present vaccination program in Japan.     

Urinary Fibrin and Fibrinogen Degradation Products and the Origin of Hematuria

We examined urinary fibrin and fibrinogen degradation product (U-FDP) concentrations in pediatric patients with hematuria using the rapid and highly-sensitive latex particle agglutination test (LPAT), and assessed the value of this test for the localization of the site of hematuria. Patients with hematuria were divided into two groups: 60 with glomerular hematuria and 46 with non-glomerular hematuria. If U-FDP concentrations less than 0.25 μg/ml are Accepted as an indicator of glomerular bleeding, the sensitivity and specificity of localization of glomerular hematuria in the present study were 78% (47/60) and 89% (41/46), respectively. The high U-FDP concentrations observed in patients with non-glomerular hematuria may reflect direct bleeding into the urinary tract. Since all 13 patients with glomerular hematuria and U-FDP concentrations of 0.25 μg/ml or more had coexistent erythrocyte cylindruria, the U-FDP test seems to be compensated with combined urinalysis for the relatively lower sensitivity. We conclude that a knowledge of U-FDP concentrations by LPAT can be of help in localizing the site of bleeding in hematuria.     

Sex Hormone-dependent Renal Cell Carcinogenesis Induced by Ferric Nitrilotriacetate in Wistar Rats

Ferric nitrilotriacetate (Fe-NTA), an iron chelate, induces necrosis of renal proximal convoluted tubules as a consequence of lipid peroxidation, and a high incidence of renal cell carcinoma (RCC) is also observed in rats and mice. The incidence of RCC and the extent of lipid peroxidation are greater in males than females. In the present study, the effects of castration or ovariectomy, and sex hormone treatment on Fe-NTA-induced renal carcinogenesis in rats were examined. Male and female Wistar rats were each divided into 5 groups. In group 1, rats were sham-operated and treated intraperitoneally (i.p.) with nitrilotriacetate (NTA). In group 2, sham-operated rats were treated with Fe-NTA (5-10 mg iron/kg/day, i.p.). Castrated or ovariectomized rats treated with Fe-NTA served as group 3, Group 4 or 5 was treated in the same way as group 3, but in addition received either testosterone (group 4) or estradiol (group 5). NTA, Fe-NTA or sex hormone treatments were initiated 4 weeks after the operation. NTA or Fe-NTA treatments were conducted for 12 weeks, and sex hormones were administered for 10 months. After 10 months of treatment, all rats were autopsied and both kidneys were examined histopathologically. In NTA-treated groups, there was no pathological change in the kidneys. In Fe-NTA-treated groups (groups 2-5), testosterone treatment or ovariectomy increased the incidence of RCC, and estradiol treatment or castration decreased the incidence of RCC (male: sham operation, castration and testosterone treatment > castration > castration and estradiol treatment, female: ovariectomy and testosterone treatment > ovariectomy > sham operation, ovariectomy and estradiol treatment). These results indicate that sex differences observed in the incidence of RCC induced by Fe-NTA are dependent upon sex hormones.     

Absence of ras Mutations and Low Incidence of p53 Mutations in Renal Cell Carcinomas Induced by Ferric Nitrilotriacetate

Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe-NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H-, K-, and N- ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3'half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR-single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG-to-CTG transition at codon 199, and the other had an ATC-to-ATT transition at codon 229 and two nonsense C-to-T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe-NTA.     

Cytotoxic Activity of CD4+ T Cells against Autologous Tumor Cells

The ⁵¹Cr-release assay is mostly applied to detecting the cytotoxic activity of CD8⁺ T cells, and little is known about the activity of CD4⁺ T cells. Therefore, the correlation between the cytotoxic activity of CD4⁺ or CD8⁺ T cells and the incubation period with autologous tumor cells was analyzed by two methods. The incubation periods were 4 and 20 h (4 h and 20 h assay) for the ⁵¹Cr-release assay. Eight pairs of tumor cells and T cells were assayed. T cells were fractionated into CD4⁺ and CD8⁺ T cells by using magnetic beads and panning methods, and those cells were activated by culture with recombinant interleukin-2 and immobilized anti-CD3 monoclonal antibody. In 6 out of 8 cases, no cytotoxic activity of CD4⁺ T cells was detected by the 4 h assay, whereas cytotoxic activity was detected in all cases in the 20 h assay. The cytotoxic activities in 20 h assay of CD4⁺ T cells were increased 67-fold in comparison with the activities in 4 h assay (range: 5–197). In the case of CD8⁺ T cells, cytotoxic activities were detected in 6 out of 8 cases in the 4 h assay. The lytic unit ratio of CD4⁺ and CD8⁺ T cells was calculated as 1.5 in the 20 h assay (range: 0.2->7.2) versus 0.4 in the 4 h assay (range: < 0.1–1.3). Cytotoxic activities in colorimetric assay using Crystal Violet with a 24 h incubation were similar to those in the 20 h ⁵¹Cr-release assay in all eight cases. These results indicate that CD4⁺ T cells have cytotoxic activity as strong as that of CD8⁺ T cells towards autologous tumor cells.     

Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes

PURPOSE: To examine immunohistochemically the localization of myocilin/trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) protein in the glaucomatous and normal trabecular meshworks. METHODS: Trabecular tissues were used from one eye with late-onset goniodysgenetic glaucoma, three with primary open angle glaucoma (one of which had the MYOC/TIGR gene mutation), two with exfoliation glaucoma and one without glaucoma. For light microscopic immunohistochemistry, frozen sections were stained by the avidin-biotin complex method using anti-MYOC/TIGR polyclonal antibody. For electron microscopic immunohistochemistry, the pre-embedding method using the same antibody was performed. Double immunostaining using both anti-MYOC/TIGR and anti-type VI collagen antibodies was done by the immunofluorescence method. RESULTS: With light microscopy, immunoreactivity was seen in the whole trabecular meshwork of each of the specimens. No notable differences were detected in staining among the types of glaucoma, or between the eyes with and those without the gene mutation. Under electron microscopy, immunoreaction products were observed not only in the cytoplasm of the trabecular cells but also in the extracellular matrix, where staining was associated with the long-spacing collagen, fine granular materials and possibly microfibrils. With double immunohistochemistry, MYOC/TIGR was colocalized with type VI collagen in the trabecular meshwork. CONCLUSIONS: In glaucomatous and normal trabecular meshworks, the MYOC/TIGR protein is distributed in the extracellular matrix colocalizing with type VI collagen.     

Expression of involucrin by ocular surface epithelia of patients with benign and malignant disorders

PURPOSE: Keratinization of the ocular surface epithelium is associated with various disorders impairing vision. We immunohistochemically determined whether the ocular surface epithelia express involucrin, and whether its expression pattern may differ in benign vs. malignant disorders. Expression of cytokeratins was also examined to provide further information relative to the epithelial differentiation. METHODS: We evaluated 17 specimens; 6 specimens of the normal ocular surface epithelia, 3 specimens from cases of conjunctival intraepithelial neoplasia (CIN), 6 of conjunctival squamous cell carcinoma (SCC) and 2 of conjunctivae from cases of superior limbic keratoconjunctivitis (SLK). RESULTS: Corneal epithelium exhibited intracellular immunoreactivity for involucrin. Four of the 6 specimens of bulbar conjunctival epithelium showed involucrin immunoreactivity in the perimembranous region, whereas the fornical conjunctiva was negative. Cornified envelope in SLK specimens was positive for involucrin. The CIN showed its immunoreactivity in the perimembranous region in all levels of the hyperproliferative epithelium without keratinization, i.e., similar to the bulbar conjunctiva. The neoplastic cells of well-differentiated SCC showed involucrin in the perimembranous region, and those of moderately- to poorly-differentiated SCC have involucrin in their cytoplasm. The expression pattern of cytokeratins was unrelated to grade of malignancy in ocular SCC. CONCLUSION: The epithelia of normal subjects and of CIN expresses involucrin without keratinization. In contrary, the keratinized SLK epithelium markedly expresses involucrin in the cornified envelope. The subcellular immunolocalization of involucrin in the ocular SCC may help in evaluating the differentiation, i.e., malignancy, of neoplastic cells.