Kimio Yasuhira

Spontaneous age-associated amyloidosis in senescence-accelerated mouse (sam).

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R). Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%). Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm. The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.

Cataract and other ophthalmic lesions in senescence accelerated mouse (SAM). Morphology and incidence of senescence associated ophthalmic changes in mice

In a murine model of accelerated senescence (SAM), grading score and incidence in cataract, periophthalmic lesions, opacity and ulcer of the cornea were determined in mice from 4 to 24 months of age. From 4 to 6 months of age, incidence and grading score of these four categories began to increase in both the accelerated senescence prone (SAM) and resistant series with normal aging, and these increases continued with aging. As compared with the resistant series, there was a higher incidence and grading score of the four categories and a higher rate of increase in the prone series. The prone 3 series in particular showed a much higher incidence and grading score on cataract, the rate being 27.5% and 70.6% at 12 and 16 months, respectively. Histologically, the cataract was classified into two types. In one, degeneration of lens fibers, disintegration of lens cortex, and at an advanced stage, liquefaction of the lens cortex and proliferation of the anterior lens epithelial cells occurred. In the other type, lens fibers lost their distinct shapes and a homogenous mass formed at the anterior and posterior superficial cortex. The anterior lens epithelial cells had shrunk. There was an opacity and ulcer of the cornea with keratitis and the corneal epithelium was lost in case of the latter. Periophthalmic lesions included catarrhal changes of the skin of the eyelids and face and blepharitis. There were no lesions specific to each of the prone and resistant series. Thus, SAM should prove to be a suitable murine model for investigation of age-related ophthalmic lesions, including cataract in humans.

Carcinoembryonic antigen producing cultured cell lines enable detection of autoantibodies in sera from patients with gastrointestinal cancer

Of 16 human malignant tumor cell lines established in our laboratory, seven lines, including three gastric cancer cell lines derived from patients with cancer, were found to carry carcinoembryonic antigen on their cell surface, as determined by radioimmunoassay and indirect immunofluorescence. The existence of antibodies (IgG class) against the gastric cancer cell lines (HPE‐GAC‐T, ‐2, ‐3) and lung cancer cell line (HPL‐Ad‐K) in the sera of patients with gastrointestinal cancer (incidence 70.8%) was demonstrated by indirect immunofluorescence. In nonmalignant cases and healthy controls, the incidence was 7.7 and 3.2%, respectively. Specificity of the antibodies detected in the sera of patients with gastric cancer was examined by absorption and blocking test methods of immunofluorescence. Even though there was an apparent heterogeneity of the specificity among the antibodies, the detection of such antibodies may be a feasible and practical approach to a clinical diagnosis of malignancy. Cancer 50:1775‐1782, 1982.

d-Penicillamine toxicity in mice I. Pathological findings

Abstract DDD mice were fed 4, 2, or 0.5 g of d -penicillamine/kg of commercial diet from 3 weeks of age for periods ranging from 10 to 48 weeks. In male mice fed 4 g/kg diet, subcutaneous hemorrhage, dorsal hernia, priapism, and paralysis of the digits were observed. In a few mice, there was a ruptured aortic aneurysm. In most advanced cases, ataxia due to hindleg paralysis was observed. In male mice fed 2 g/kg diet, priapism and paralysis of the digits were noted at a lower incidence. No abnormalities were observed also in the mice fed 0.5 g/kg diet. Subcutaneous hemorrhage, dorsal hernia, and priapism were observed exclusively in males and orchidectomy significantly reduced the incidence. The incidence of digit paralysis, however, showed no sex difference and was not prevented by orchidectomy and administration of estrogen. Histologically, nerves, such as the optic and sciatic, showed moderate to marked demyelination and axonal degeneration, and spinal ganglion cells showed degenerative changes in mice fed the 4 g/kg diet. Back muscle covering the herniated area became thin or almost disappeared, and was replaced by a thin layer of collagenous tissue. When the onset of penicillamine feeding was delayed until 3 months of age, all the toxicities were remarkably reduced. Copper supplementation significantly prevented the toxicity while pyridoxine hydrochloride did not.

d-Penicillamine toxicity in mice. II. Concentrations of Cu, Zn, and Fe related to development of toxicity

Abstract Three-week-old DDD mice were fed 4 g of d -penicillamine/kg added to a commercial diet for 3 to 12 weeks. The copper content of various organs was determined at 6, 9, 12, and 15 weeks of age. The copper concentration in the skin, liver, brain, and spinal cord was significantly decreased 3 weeks after the initiation of penicillamine feeding, and at this time, clinical signs began to appear. In suckling offspring of dams fed 4 g of d -penicillamine/kg commercial diet throughout gestation and lactation, the copper content of various organs was determined at 1, 7, 14, and 21 days of age. Liver and spinal cord concentrations were significantly decreased at 1, 7, 14, and 21 days of age, and brain, serum, kidney, and skin copper deficiency was seen at 7, 14, and 21 days of age. Copper in the aorta was also decreased in these suckling mice. Stomach milk copper levels in penicillamine-treated sucklings was very low at 1, 7, and 14 days of age. These suckling mice showed signs of neurological dysfunction. The copper content in these tissues, in the sera of both the adult and suckling mice, and in the milk of lactating mice rose following copper supplementation; the various clinical signs all but disappeared. Zinc and iron levels in the liver and brain remained unaltered in the treated adult mice. In the suckling mice, zinc and iron concentrations increased significantly in the liver but not in the brain. There was a close correlation between clinical signs and the level of copper in the tissues particularly between neurological abnormalities and the brain and spinal cord copper levels.

d-Penicillamine toxicity in mice. III. Pathological study of offspring of penicillamine-fed pregnant and lactating mice

Abstract Mice were divided into six experimental diet groups during pregnancy and lactation: (1) C + C, control diet; (2) P(2) + (2), 2 g of d -penicillamine/kg of diet; (3) P(4) + P(4), 4 g of d -penicillamine/kg of diet (these groups received diets throughout pregnancy and lactation); (4) P(4) + P(4)Cu, the test diet was fed during pregnancy, and 5 × 10 −3 , m copper sulfate solution was added to replace drinking water after delivery; (5) C + P(4), the test diet was given only during lactation; (6) P(4) + C, the test diet was given only during pregnancy. Highest mortality, most severe and variegated neurological abnormalities, and rupture of aortic aneurysms were observed in the P(4) + P(4) offspring. Supplementation with copper (P(4) + P(4)Cu) resulted in no abnormalities. In C + P(4), mortality was higher and abnormalities were more variegated than in P(4) + C or P(2) + P(2). No abnormality was detected in C + C. The aortas in P(4) + P(4) showed dissecting aneurysms and breaks in the elastic lamellae; neuronal degeneration was noted in the cerebral cortex, thalamic nuclei, and spinal ganglion on the 14th and 21st postnatal days, but not on the 1st or 7th postnatal days. No neuronal degeneration was observed in the copper-supplemented group. In C + P(4), there were a few abnormal neurons in the cerebral cortex on the 21st postnatal day. No neuronal degeneration was detected in C + C, P(2) + P(2), or P(4) + C. These observations suggest that abnormal signs and gross lesions are related to copper deficiency induced by the metal-chelating action of d -penicillamine especially postnatally through two basic mechanisms: maturation defect in fibrous protein and neuronal degeneration in the brain and spinal ganglia.

Microsomal hydroxylation of 3-methylcholanthrene: analysis by computerized gas chromatography—mass spectrometry

Microsomal metabolism of 3-methylcholanthrene (MC) was examined by computerized gas chromatography-mass spectrometry. Five mono-, four di- and thirteen trihydroxylated metabolites were found after incubation of MC in mouse liver microsomal fraction for 15 min, in the presence of NADPH. Among these metabolites, three mono- and three dihydroxylated metabolites were identified by means of authentic samples. The chemical structures of the other metabolites were deduced from their characteristic mass spectral fragmentations. This is the first description of trihydroxylated metabolites in MC metabolism in vitro and in vivo.

Experimental induction of lung cancer in rat and mouse with 20-methylcholanthrene in Freund's adjuvant.

I. Wistar and Sprague-Dawley strains of rats and CFI strain of mice were subjected to a single direct application into the lung of 20-methylcholanthrene suspended in complete or incomplete Freunds adjuvant. 2. Five mg of the Carcinogen induced squamous cell carcinomas and fibrosarcomas in the lungs of about 62 percent of the treated rats. 3. The squamous cell carcinomas arose from the proliferating metaplartic bronchial epithelium which wos regenerating on granulomatous tissue surrounding central necrotic foci. These were produced by Freunds adjuvant with the carcinogen which retained prolonged stimulus to the epithelium and produced eventually the tumor of invasive nature. Soon after the treatment and prior to the proliferation of regenerating bronchial epithelium, prominent epidermoid foci oppeared which resulted from metaplastic proliferation of the bronchial and alveolar epithelia. The foci underwent necrosis and none developped directly into the tumor. 5. Two stage hypothesis on lung cancer production has been proposed on the basis of these observations. 6. One mg of the carcinogen was introduced into lungs of mice and this resulted in rather small number of tumors; nonepithelial and lepithelial. The farmer was fibrosarcoma and the latter adenoma, adenocarcinoma and quamous cell carcinoma. The squamous cell carcinomas were induced in the same manner as those in rats, but without evident appearance of initial epidermoid foci. 7. Hemoblastosis occurring in mice seemed to be attributed to Freunds adjuvant introduced as a vehicle. 4.

Effect of Mycobacterial Water-Soluble Adjuvant (MAF3) on the Immune Response of Guinea Pigs to the Hapten-Carrier Conjugates

To clarify the complex mechanisms of the immune responses, many adjuvants have been used as immunologic tools. A classical example of such materials is Freunds complete adjuvant (FCA). It is well known that the heat-killed tubercle bacilli in mineral oil are the most important constituents for the adjuvant activity of FCA. Recently, it has been demonstrated that the tubercle bacilli in FCA can be replaced with water-soluble fractions from mycobacteria (1, 7, 10-13) and other Gram-positive bacteria (8). These water-soluble fractions probably share common chemical constituents. MAF3, which was prepared from the delipidated cells of Mycobacterium tuberculosis strain Aoyama B by hydrogenolysis and gel filtration (10), was found to be one of such water-soluble components responsible for the adjuvant activity. Although adjuvant activity of MAF3 on the antibody response, especially of the IgG2 class, and on the induction of delayed-type hypersensitivity (DTH) reaction to ovalbumin (OA) have been observed in guinea pigs, its effect on the immune response to other types of antigens such as hapten-carrier conjugates are not fully investigated yet. In this paper, we describe the adjuvant effect of MAF3 on the antibody response and the induction of DTH reaction in guinea pigs immunized with 2, 4-dinitrophenyl (DNP)-conjugates, DNP-bovine serum albumin (BSA), DNP-dextran or DNP-guinea pig serum albumin (GPA) emulsified with Freunds incomplete adjuvant (FIA) which lacks mycobacterial components. Female Hartley/s guinea pigs, weighing about 300 to 500 g, were used in all experiments. BSA (fraction V, Seikagaku-Kogyo, Tokyo), OA ( •~ 5 crystalline, ICN Pharmaceuticals Inc., Ohio) and GPA (Cohns fraction V from normal guinea