Kaoru Hatanaka

Immunohistochemical localization of C-reactive protein-binding sites in human atherosclerotic aortic lesions by a modified streptavidin-biotin-staining method.

One‐step fluorescein‐conjugated polyclonal antibody technique has shown that C‐reactive protein (CRP) was located only extracellularly in human atherosclerotic lesions. In this report a more sensitive streptavidin‐biotin technique was applied to detect the localization of CRP in human athere sclerotic lesions. lmmunohistochemical staining with polyclonal and monoclonal anti‐human CRP antibodies both produced a brown color extracellularly in the necrotic lesions, and intracelluarly in CD68+ foam cells. The latter suggests an uptake of CRP‐lipid complexes by macro‐phages. The staining is human CRP‐specific because it was eliminated by preabsorption of the monoclonal antibody with pure human CRP, or by substitution of the primary antibody with non‐immune rabbit serum. By overlaid CRP‐binding study, a positive stain was observed on intimal smooth muscle cells and foam cells, suggesting that they have CRP‐binding sites unless the CRP‐binding activity was generated de novo through the fixation procedure. Accordingly, it is hypothesized that CRP may facilitate the uptake of lipids by macrophages accumulating in atherosclerotic lesions. Further, CRP might participate in cytolysis, which enlarges the necrotic area, and/or in phagocytosis that scavenges the necrotic tissue.

Three autopsy cases of progression to left ventricular dilatation in patients with hypertrophic cardiomyopathy

The hearts of three cases of congestive heart failure with dilated left ventricles developing in patients with symptomatic hypertrophic cardiomyopathy (HCM) were morphologically investigated. The results showed that disproportionate hypertrophy and dilatation of the left ventricles, accompanied by massive fibrosis and myocardial disarray, were present in the three patients. The mean percent area of fibrosis of the left ventricle was 34.7% and 47.4% at the upper third and lower third levels, respectively, and was much more frequently associated with disarray (84.4 +/- 12.3%). Moreover, the fibrosis was most extensive in the lateral wall of the left ventricle, followed by the posterior, anterior, and interventricular walls. The fibrosis was also diffuse regardless of the subendocardial or subepicardial region of the heart. The findings in the present study suggest that the disarray in this particular series of HCM might be responsible for the mechanism of the fibrosis leading to dilatation of the left ventricle.

Response of mucosal mast cells to intestinal ischemia-reperfusion injury in the rat

The goals of this study were to investigate the in vivo effects of intestinal ischemia-reperfusion on mucosal mast cells, and to evaluate the morphological changes induced by standardized arterial occlusion in anesthetized rats. Complete segmental ileal ischemia was maintained for 15, 30, or 60 min, and was followed by a 30 min reperfusion period. Intestinal biopsies taken at the end of ischemia and in the 30th min of reperfusion were evaluated by image analysis, and the rate of release of type II rat mast cell protease, a marker of mast cell exocytosis, was determined from the venous effluent of the segment. Electron microscopy revealed cytoplasmic vacuolization of the mast cells of the villi after the 15 min ischemia. Ischemia induced a continuous diminution of the mucosal thickness and a significant fall in the number of mast cells in the villi; with immunoperoxidase staining with a monoclonal antibody that recognizes the AD1 mast cell surface antigen, the decrease was 57, 49, and 66% in the 15, 30, and 60 min ischemia groups, respectively. In these groups, the mucosal type II mast cell protease concentration increased to 2.4-, 2.5-, and 3.6-fold, respectively, and a significant increase in plasma protease levels was observed on reperfusion. These results lead us to conclude that mucosal mast cells are very sensitive to intestinal ischemia, with the majority of mast cells in the ileal villi already involved in the response to ischemia after a short period of arterial occlusion.

Ischemic heart disease in the WHHL rabbit: a model for myocardial injury in genetically hyperlipidemic animals.

Myocardial lesions were frequently found in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits. The lesions were of two types. One consisted of a dissolution of muscle cells with inflammatory cell infiltrate. The other consisted of replacement fibrosis. The more the lumens of at least one of the three main coronary arteries were narrowed by atherosclerosis, the more the myocardial lesions occurred. The greater the number of vessels that were occluded, the more the incidence of myocardial lesions increased. From the age distribution of rabbits which died of natural causes, the average life span was estimated to be markedly shorter in homozygous WHHL rabbits than in normal rabbits. These findings suggest that WHHL rabbits suffered from ischemic heart disease, including acute myocardial infarction. Thus, this study shows that WHHL rabbits may be useful not only as a model of familial hypercholesterolemia but also for investigating the mechanisms of various clinical forms of ischemic heart disease.

Binding of serum amyloid P component to heparin in human serum

It has been proposed that the function of serum amyloid P component (SAP) may closely relate with its binding to polysaccharides, especially glycosaminoglycans. We employed a quantitative immunoelectrophoresis (QIE) method and a native polyacrylamide gel electrophoresis (PAGE) method to characterize the SAP-heparin binding in soluble state. The SAP-heparin binding showed positive cooperativity. The apparent numbers of heparin molecules bound to SAP varied with the calcium concentration with a ratio of 1:1 (SAP/heparin), a Kd of 2.06 x 10(-7) M at 0.1 mM CaCl2 and a ratio of 1:1.6 (SAP/heparin), a Kd of 3.91 x 10(-7) M at 2 mM CaCl2, when estimated by the QIE method. No binding between SAP and heparin was observed in the absence of calcium. Magnesium and barium failed to induce the formation of SAP-heparin complex. Furthermore, they showed inhibitory effects on the calcium-mediated complex formation. We propose that heparin might be a regulator to modulate the anticoagulant activity of SAP and a useful drug to prevent SAP deposition on amyloid deposits.

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

SummaryA mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/Wvmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [3H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [3H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.

Hyperlipidemia in mast cell-deficient W/Wv mice

Approximately 70% of the W/WV mice lacking mast cells due to a genetic defect showed hypertriglyceridemia combined with hypercholesterolemia. Increases of various magnitudes in chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein were observed in the plasma of W/WV mice compared to those in the plasma of congenic normal mice. The increase in these lipoproteins was seen even in normolipidemic W/WV mice. Activities of both lipoprotein lipase and hepatic triacylglycerol lipase in the plasma after heparin injection were markedly lower in the W/WV mice than in the congenic normal mice, although activities of both lipoprotein lipase in the heart and adipose tissue and hepatic triacylglycerol lipase in the liver were not decreased. These results suggest that the W/WV mice have genetic defects in one or more of the following: secretion of both lipases from their synthesising cells, transport to the endothelium, and anchoring to the endothelial surface. Heparin deficiency in these mice may be responsible for the impairment and, thereby, may partially contribute to the hyperlipidemia.

CEREBRO-SPINAL INFARCTION CAUSED BY ATHEROMATOUS EMBOLI

Cholesterol embolization to the abdominal viscera is common, but rare in the central nervous system. Fourteen cases of atheromatous embolization to the central nervous system were morphologically investigated. Among the 800 consecutive autopsy cases, 38 cases had atheromatous emboli in various organs. Cerebro‐spinal infarction caused by atheromatous emboli was found in 11 cases. Infarction rate (11/14) was relatively higher than in other organs and 5 of these cases were thought to be due to direct injury to the erosive surface of the aorta; cardiac catheterization (2 cases), intra‐aortic baloon pumping (2 cases), and extra‐anatomical bypass graft operation (1 case). These 14 patients consisted of elderly patients (70.1 ±6.3 years old) usually associated with hypertension (78.6%) and diabetes mellitus (42.8%). Anatomically, aortic aneurysms were seen in 10 cases (71.4%), in which aortic arch aneurysm was seen in 6 cases. Hence, aortic mechanical procedure is of great importance for denuding atheromatous materials from erosive atherosclerosis to the central nervous system. ACTA PATHOL. JPN. 35 : 789–801, 1985.

Thrombus formation by the application of thrombin to the outer surface of mouse mesenteric vein: comparison with the application of ADP.

Mesenteries of mice under anesthesia were stretched over an inverted microscope. A micropipette filled with solution containing various concentrations of ADP or thrombin was brought into contact with the outside of a mesenteric vein by micromanipulation, and then poured over the outer surface of the vein. Morphological characteristics of the thrombi and the time needed for thrombus formation were examined. Application of either thrombin or ADP to the adventitia of mesenteric veins caused thrombus formation. Although thrombi by application of ADP seemed to be anchored by direct adhesion of platelets to the exposed subendothelium, thrombi by application of thrombin seemed to be anchored by deposited fibrin.

A sensitive peroxidase staining immunoblotting method for measuring total protein S in human plasma

In plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added to normal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.