Expression of intercellular adhesion molecule-1 in murine hearts with acute myocarditis caused by coxsackievirus B3.

A cell-mediated autoimmune mechanism has been strongly implicated in the pathogenesis of viral myocarditis. Using a murine model of myocarditis caused by coxsackievirus B3 (CVB3), we previously reported that the heart is infiltrated first by natural killer cells, which express a cytolytic factor, perforin, and then by activated T cells. This action may play an important role in the pathogenesis of the observed myocardial cell damage. Cell-cell contact and adhesion is required in immune responses, and intercellular adhesion molecule-1 (ICAM-1), which is a ligand for lymphocyte function-associated antigen-1 (LFA-1), plays an important role in this process. To investigate the essential role of the ICAM-1/LFA-1 pathway in the cell-mediated cytotoxicity involved in viral myocarditis, we examined by immunofluorescence the expression of ICAM-1 in murine hearts with acute myocarditis caused by CVB3. We also evaluated the induction of ICAM-1 in cultured cardiac myocytes treated with cytokines by immunofluorescence and Northern blot hybridization. Furthermore, we analyzed the effects of in vivo administration of anti-ICAM-1 mAbs on the inflammation associated with acute viral myocarditis. We found that CVB3-induced murine acute myocarditis resulted in enhanced expression of ICAM-1 in myocardial cells. The expression of ICAM-1 in myocardial cells could be induced in vitro by IFN-gamma and TNF-alpha, which were shown to be synthesized by the infiltrating cells. In vivo treatment with F(ab')2 fragments of an anti-ICAM-1 mAb significantly reduced the myocardial inflammation induced by CVB3. These data strongly suggest that the expression of ICAM-1 in myocardial cells plays a critical role in the cell-mediated cytotoxicity involved in acute viral myocarditis.

Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu's arteritis.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of vascular cell injury in Takayasu's arteritis. To clarify the immunological mechanisms involved, we examined the expression of a cytolytic factor, perforin in infiltrating cells of aortic tissue samples from seven patients with Takayasu's arteritis. We also examined the expression of a 65-kD heat-shock protein (HSP-65), human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 in the aortic tissue. Immunohistochemical studies showed that the infiltrating cells mainly consisted of gamma delta T lymphocytes, natural killer cells, macrophages, cytotoxic T lymphocytes and T helper cells, and that perforin was expressed in gamma delta T lymphocytes, natural killer cells, and cytotoxic T lymphocytes. In situ hybridization analysis also revealed expression of perforin mRNA in the infiltrating cells. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly onto the surface of arterial vascular cells. We also found that expression of HSP-65, human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 was strongly induced in the aortic tissue and might facilitate the recognition, adhesion and cytotoxicity of the infiltrating killer lymphocytes. These findings provide the first direct evidence that the infiltrating cells in the aortic tissue mainly consist of killer cells, and strongly suggest that these killer cells, especially gamma delta T lymphocytes, may recognize HSP-65 and play a critical role in the vascular cell injury of Takayasu's arteritis by releasing perforin.

Restricted usage of T cell receptor V alpha-V beta genes in infiltrating cells in the hearts of patients with acute myocarditis and dilated cardiomyopathy.

Prolonged myocardial cell damage initiated by acute myocarditis is thought to be one of the most important etiology of dilated cardiomyopathy. To investigate the immunological mechanisms involved in the pathogenesis of dilated cardiomyopathy, we analyzed the phenotypes of infiltrating cells and examined the expression of perforin in infiltrating cells in the hearts of patients with dilated cardiomyopathy as well as acute myocarditis. We also examined the expression of HLA and intercellular adhesion molecule-1 (ICAM-1) in myocardial tissue of these patients. Furthermore, to evaluate the antigen specificity of infiltrating T cells and persistence of viral genomes in the myocardial tissue, we analyzed the expression of T cell receptor (TCR) V alpha and V beta genes as well as enterovirus genomes by PCR. We found infiltration of perforin-expressing killer cells and enhanced expression of HLA class I and ICAM-1 in the myocardial tissue. We also found that the repertoires of TCR V alpha as well as V beta gene transcripts were restricted, indicating that a specific antigen in the hearts was targeted. Because no enterovirus genomes were detected in all patients, it is strongly suggested that a cell-mediated autoimmune mechanism triggered by virus infection may play a critical role in the pathogenesis of dilated cardiomyopathy. However, we could not exclude the possibility that viruses other than enteroviruses could be pathogenic in these patients.

Expression of bFGF and TGF-beta 2 in experimental myopia in chicks.

PURPOSE: To determine factors that control ocular enlargement in experimental form-deprivation myopia and to clarify the mechanism of form-deprivation myopia. METHODS: After the left eyes of 20 chicks were monocularly occluded for 2 weeks, protein, basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 2 contents in samples of constant area (circular button, diameter = 8.5 mm) in the retina-retinal pigment epithelium (RPE)-choroid and the sclera in the posterior region of control and myopic eyes were determined by enzyme-linked immunosorbent assay. RESULTS: The bFGF content (per circular button) and bFGF concentration (per mg protein) were significantly lower in the sclera in the posterior region of the myopic eyes than in control eyes. The bFGF content and concentration were similar in the retina-RPE-choroid in myopic and control eyes. The TGF-beta 2 content and concentration were significantly higher in myopic eyes in both the retina-RPE-choroid and the sclera (P < 0.05). CONCLUSIONS: These results are consistent with the possibility that bFGF and TGF-beta 2 regulate ocular enlargement or respond to myopiagenic mechanisms in form-deprivation myopia.

Induction of vascular endothelial growth factor after application of mechanical stress to retinal pigment epithelium of the rat in vitro.

PURPOSE: To investigate the response to mechanical stress of vascular endothelial growth factor (VEGF) production by cultured retinal pigment epithelial (RPE) cells. METHODS: A pulsatile stretch device was used in vitro. RPE cells of the second passage were seeded onto flexible-bottomed culture plates; then, at subconfluent culture, the plates were subjected to pulsatile stretch. Culture plates prepared in the same way but not subjected to stretch were used as controls. After stretching for 1 hour or 24 hours, conditioned medium for measurement of VEGF production by RPE cells was collected using a mouse VEGF immunoassay. To study the expression of VEGF in RPE cells, passaged-cultured RPE cells were exposed to pulsatile stretch for 0, 1, 3, or 14 hours. Total cytoplasmic RNA was then prepared from the RPE cells. Northern blot analysis was performed for VEGF, with G3PDH used as an internal control. RESULTS: The expression and secretion of VEGF in RPE cells were increased by pulsatile stretching. CONCLUSIONS: Results indicate that stretching of the RPE could result in increased production of VEGF, with associated risk for neovascularization and changes in the blood-retinal barrier.

Reduced sensitivity of HeLa cells to cis-platinum by simultaneous overexpression of copper, zinc-superoxide dismutase and catalase.

The overexpression of catalase or Cu,Zn-superoxide dismutase (Cu,Zn-SOD) did not affect the sensitivity of HeLa cells to cis-platinum. However, the cytotoxicity of cis-platinum was depressed significantly by the simultaneous overexpression of catalase and Cu,Zn-SOD. We concluded that cis-platinum accelerated the generation of superoxide anion in the cells, and the superoxide anion produced was converted into H2O by the cooperative roles of catalase and Cu,Zn-SOD.

Overexpression of manganese-superoxide dismutase prevents methylmercury toxicity in HeLa cells.

HeLa cells were stably transformed with plasmid constructs that allowed constitutive expression of antioxidant enzymes such as catalase, glutathione peroxidase (GSH-Px), Cu,Zn-superoxide dismutase (Cu,Zn-SOD) or Mn-superoxide dismutase (Mn-SOD) to examine the involvement of reactive oxygen generation in methylmercury toxicity. Overexpression of catalase, GSH-Px or Cu,Zn-SOD did not affect the sensitivity of HeLa cells against methylmercury. However, the sensitivity of HeLa cells against methylmercury was decreased by overexpression of Mn-SOD, an enzyme localized in matrix of mitochondria and which decomposes superoxide anions. These results suggest that formation of superoxide anions in the mitochondria might be involved in the mechanism of the cytotoxicity of methylmercury.

Restricted usage of T-cell receptor Vgamma-Vdelta genes and expression of costimulatory molecules in Takayasu's arteritis.

To further investigate the immunological mechanisms involved in Takayasu's arteritis, we analyzed the T-cell receptor (TCR) Vgamma and Vdelta gene usage by infiltrating gammadelta T-cells and the expression of costimulatory molecules B7-1, B7-2, CD40, CD27 ligand (CD27L), CD30L, OX40L in the arterial tissue of a patient with Takayasu's arteritis. We found that the repertoires of TCR Vgamma as well as Vdelta gene transcripts of the infiltrating cells were restricted as compared with those of peripheral blood lymphocytes from a patient with Takayasu's arteritis. This strongly suggests that gammadelta T-cells as well as alphabeta T-cells, as we previously reported, were specifically involved in the pathogenesis of Takayasu's arteritis. We also found that B7-1, B7-2, CD40, CD27L, CD30L, and OX40L were expressed in the arterial tissue, suggesting the roles for these costimulatory molecules in T-cell-mediated vascular injury in Takayasu's arteritis. Our findings strongly support the involvement of T-cell-mediated immunological mechanisms in the pathogenesis of Takayasu's arteritis.

Active oxygen species generation and cellular damage by additives of parenteral preparations: selenium and sulfhydryl compounds.

We investigated the relationship between active oxygen species (AOS) generation and cultured vascular endothelial cellular damage caused by simultaneous exposure to selenium compounds and sulfhydryl compounds such as cysteine (Cys) or reduced glutathione (GSH). Selenium compounds, selenite, selenate or selenomethionine (SeMet), are added to total parenteral nutrition (TPN) and intravenously administered. We confirmed by luminol dependent chemiluminescence, an indicator of AOS generation, that selenite generates AOS in the presence of clinical concentrations of sulfhydryl compounds, 0.5 mM Cys or 0.5 mM GSH, and that the amount of AOS generated reaches the maximum when their mole ratio is 1:50. However, AOS generation was not observed after simultaneous administration of various concentrations of selenate or SeMet with sulfhydryl compounds. Moreover, simultaneous exposure to 10 microM selenite and sulfhydryl compounds was found to result in significant increases in the [3H]-adenine and lactate dehydrogenase (LDH) release rates from cells, a significant decrease in the amount of cellular protein, and enhancement of cellular damage as compared with after exposure to selenite alone. However, simultaneous exposure to 10 microM selenate or 10 microM SeMet together with sulfhydryl compounds did not induce cellular damage. These findings revealed that selenite generates AOS and causes cellular damage in the presence of sulfhydryl compounds. Accordingly, it seems better to choose selenate or SeMet instead of selenite when a selenium compound is to be added to TPN.

A new marker for estimation of bloodstain age by high performance liquid chromatography.

The applicability of a new marker for estimation of bloodstain age by reverse-phase high performance liquid chromatography (HPLC) is described. Using a microBondasphere C18 column with a two step linear gradient of 10.5-46.25% acetonitrile in 0.1% trifluoroacetic acid, an intriguing peak (unidentified) at a retention time of about 5 min was observed on chromatograms from human adult bloodstains and designated as 'X'. The area of this peak, which could be detected in extracts of bloodstains, but not in their fresh whole blood, increased with time. The ratios of the X area to heme area in bloodstains stored at room temperature and 4 degrees C for up to 52 weeks old linearly correlated with stain age by plotting on a double logarithmic scale. In bloodstains exposed to fluorescent light at room temperature, the regression equation calculated from the ratios (Rx) and the ages of stains in weeks (W) is ln(1000.Rx) = 1.1084 + 0.3937.ln(7.W), and the coefficient of correlation (r) is 0.9776 (n = 144, P < 0.001). When stains were stored at 37 degrees C, the ratio transformed into logarithms correlated linearly with stain age. The regression equation describing the relationship in bloodstains exposed to fluorescent light at 37 degrees C is ln(1000.Rx) = 2.4477 + 0.0866.W (r = 0.9826, n = 144, P < 0.001). The results of the present study suggest that the HPLC method may be applicable to the estimation of bloodstain age.

Right ventricular inflow obstruction due to giant hematoma formed by chronic constrictive pericarditis.

A 26-year-old man having chronic constrictive pericarditis with rare complications is described. Right ventricular inflow obstruction was caused by an intracavity giant mass which was surrounded by thick calcified pericardium. The mass consisted of old bloody fluid with some calcified tissue. The findings of auscultation closely mimicked those of tricuspid valvular stenosis.

Perforin-positive leukemic cell infiltration in the heart of a patient with T-cell prolymphocytic leukemia.

Here we report a rare case of T-cell prolymphocytic leukemia in whom leukemic killer cells, expressing a cytolytic factor perforin, infiltrated the heart. Perforin may have directly injured myocardial cells which showed marked expression of human leukocyte antigens (HLAs) and intercellular adhesion molecule-1 (ICAM-1) as well as costimulatory molecules B7 and B70, which are ligands for CD28 expressed on T-cells. In spite of chemotherapy against leukemic cells, this autoimmune process finally caused fatal congestive heart failure.

Perforin-positive leukemic cell infiltration in the aortic tissue of a patient with T-cell prolymphocytic leukemia.

Here we report a rare case of T-cell prolymphocytic leukemia in which leukemic killer cells, expressing a cytolytic factor perforin, infiltrated the aorta as well as the heart and may have directly injured aortic vascular cells which strongly expressed human leukocyte antigens (HLAs) and intercellular adhesion molecule-1 (ICAM-1) as well as costimulatory molecules B7 and B70, which are ligands for CD28 expressed on T-cells. In spite of chemotherapy against leukemic cells, this autoimmune process finally caused fatal multi-organ failure.

Restricted usage of T-cell receptor Valpha-Vbeta genes in infiltrating cells in the aortic tissue of a patient with atherosclerotic aortic aneurysm.

We report a rare case of an atherosclerotic aortic aneurysm with lymphocyte infiltration in which T-cell receptor (TCR) Valpha as well as Vbeta gene usage was restricted. Immunohistochemical studies showed that the infiltrating cells mainly consisted of macrophages, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) and T-helper (Th) cells, and that there were almost no infiltrating delta T lymphocytes, and human leukocyte antigen (HLA) class I and 65-kD heat-shock protein (HSP-65) was not strongly expressed in the aortic tissue. Although the immunohistochemical data were consistent with an ordinary atherosclerotic aortic aneurysm, in which TCR Valpha-Vbeta gene usage is known to be polyclonal, the restricted TCR gene usage suggests that a certain autoimmune mechanism was involved in the pathogenesis of this case similar to Takayasu's arteritis, in which massive infiltration of delta T lymphocytes and strong expression of HSP-65 in the aortic tissue are characteristic.

Effect of long-term treatment with vanadate in drinking water on KK mice with genetic non-insulin-dependent diabetes mellitus.

The glucose-lowering effect of vanadate, ammonium metavanadate (AMV), on diabetic KK mice was examined. Five-week-old male KK mice were administrated with a solution of AMV via drinking water at concentrations of vanadium (V) with 0.1, 1.0, 10 and 100 microg/mL for a period of 10 wk, respectively. Body weight, consumption of food and water, and blood glucose levels was measured every week for 10 wk. The results showed that food consumption and body weight in the experimental groups were similar to those in the control group. A statistically significant decrease of drinking water consumption and blood glucose levels in the group treated with 100 microg V/mL was observed. The glucose tolerance in the vanadate-treated mice with 10 and 100 microg V/mL was remarkably improved compared with the control group. Biochemical analyses at the end of experiments demonstrated that a distinct tendency for the glucose and hemoglobin A1c (HbA1c) levels to decrease with vanadate treatment in the blood was also observed. The glutamic pyruvic transaminase, glutamic oxaloacetate transaminase, blood urea nitrogen, triglyceride, high-density lipoprotein, and total cholesterol levels in plasma were lower in the higher vanadium groups than those in the control group. These results indicate that vanadium effectively produced the glucose-lowering effect at a higher dose than that at a low dose of vanadium in drinking water, without any overt signs of toxicity.

Active oxygen generation as a possible mechanism of selenium toxicity.

Selenium plays an important role in scavenging active oxygen (AO) species as an essential constituent of glutathione peroxidase. On the other hand, several reports proposed a possible induction of toxic AO by selenium compounds in vitro. However, some of these experiments including ours, were revealed to conclude on the basis of experimental artifacts, and to have problems in the interpretation of data. Methods or principles so far used for the detection of AO species generated by selenium compound were measurement of chemiluminescence from lucigenin or luminol by AO species, the spectrophotometric analysis of reduction of ferricytochrome c or nitroblue tetrazolium (NBT) by superoxide anion (O2-), electron spin resonance (ESR) spectra using dimethylpyrroline oxide (DMPO) as a spin trapping agent, the deoxyribose decomposition by hydroxyl radical (HO.), the salicylate hydroxylation by HO., and the strand breakage of DNA by AO. Many of these methods together with their principles seem to have some defects which prevent clear conclusion as stated below. (i) Lucigenin was found to mediate the formation of O2- in the presence of selenite and reduced glutathione (GSH). Therefore, lucigenin is not a suitable reagent. (ii) Luminol may also mediate O2- generation in the presence of HO.. (iii) ferricytochrome c can be reduced to ferrocytochrome c in the mixture of selenite and GSH in the absence of oxygen. Moreover, the spectrophotometric method is interfered by turbidity of elemental selenium formed under some conditions in the reaction mixture containing selenite and GSH. (iv) NBT is also reduced by selenium compounds in the absence of O2. (v) ESR signals of AO species were obtained in the reaction mixture containing selenite and GSH, or in the solution of hydrogen selenide in the presence of O2. However, selenide decomposed spin adduct of DMPO with HO. (DMPO-OH). Therefore, the intensity of the signals is not quantitative. (vi) CuZn-SOD is not necessarily a good tool to prove the involvement of O2- because it enhanced HO. generation in the reaction mixture containing selenite and GSH. Thus, we would like to emphasize that carefully designed experiments are required to further identify the molecular species of active oxygen induced by selenium compounds.

Processes of blue light-induced damage to retinal pigment epithelial cells lacking phagosomes.

PURPOSE: To experimentally clarify the processes of the changes induced by blue light directly on the retinal pigment epithelium (RPE) before the formation of phagosomes or the accumulation of lipofuscin. METHODS: We developed a new experimental method in which primary cultured cells of very young pigmented rats were exposed to several intensities and durations of blue light (wavelength = 440+/-10 nm). RESULTS: At 1.0 mW/cm2, the damage was limited to mitochondria. At 2.0 mW/cm2, the cytoplasm exhibited large whorls of membrane or whorled inclusions, which were consistent with autophagic vacuoles. At 4.0 mW/cm2, the RPE cells showed lysis of the cytoplasm and a nucleus that was consistent with necrosis. CONCLUSIONS: Our results suggested that damage induced by blue light to cultured RPE cells may originate in the mitochondria and end in necrosis. The type of cell death induced in the RPE by blue light seems to be determined mainly by the intensity of the light, but is also related to the duration of exposure.

Disturbance of electrolyte balance in vitreous of chicks with form-deprivation myopia.

PURPOSE: To investigate the changes in the electrolyte and protein concentrations in the vitreous of 3-week-old chicks with form-deprivation myopia (FDM). METHODS: FDM was induced in 2-day-old male white leghorn chicks by covering the left eye with a translucent plastic goggle and leaving the right eye uncovered to serve as control. After 19 days the animals were euthanized, and the axial dimensions of the eyes were measured with a caliper in an unfixed condition. The liquid vitreous and aqueous humor were removed by paracentesis, and blood was collected from the jugular vein. Sodium, potassium, and chloride concentrations were determined using ion-selective electrodes. Calcium and phosphate concentrations were determined by colorimetric assays using orthocresol phthalein complexone and bacterial xanthine oxidase, respectively. RESULTS: The concentrations of potassium and phosphate were decreased, whereas chloride concentration was increased in the vitreous of the FDM eyes (P < .01). Sodium and calcium concentrations were similar to those in the control eyes. No significant changes in the concentration of electrolytes were observed in the aqueous humor. No significant differences were found in the protein concentrations in the liquid vitreous, gel vitreous, and aqueous humor. CONCLUSIONS: Form-deprivation induced a significant increase of the volume of the liquid vitreous in the eye of the FDM chick. The increased liquid vitreous of the myopic eye was accompanied by an alteration of the electrolyte balance, by a mechanism that has not yet been clarified.

Clinicopathological report of retinitis pigmentosa with vitamin E deficiency caused by mutation of the alpha-tocopherol transfer protein gene.

PURPOSE: To discuss the clinicopathological findings in a patient with retinitis pigmentosa (RP) accompanied by a vitamin E deficiency caused by an H101Q mutation in the alpha-tocopherol transfer protein (alpha-TTP) gene. CASE: The clinical course of this patient was followed by conventional ophthalmological examinations over a 3-year period. After the patient died from pancreatic cancer, the eyes were obtained, and examined by light and electron microscopy. OBSERVATIONS: The patient complained of night blindness subsequent to adult-onset ataxia, although the ataxia was very mild. His visual acuity was 0.6 OU, and ophthalmoscopy revealed RP sine pigmento. Ring scotomas were detected, and the electroretinography, electro-oculography, and dark-adaptation were altered. Fluorescein angiography showed granular hyperfluorescence around the macula. No progression of the visual and neurological symptoms was observed during the 10 years he was taking oral vitamin E. Histopathological examination revealed the loss of the outer and inner segments of the photoreceptors in the area corresponding to the ring scotoma, as well as a disorganization and shortening of the outer segments in the peripheral retina. CONCLUSIONS: We conclude that the clinical and pathological findings in the eyes of this patient having RP with vitamin E deficiency caused by an H101Q mutation are similar to those of common autosomal recessive RP. However, special attention is required in making a diagnosis of RP with vitamin E deficiency because RP with vitamin E deficiency is medically treatable. The mild Friedreich-type ataxia accompanying the RP may be helpful in identifying this disease.

[Spontaneous reversion to sinus rhythm in a case of lone atrial fibrillation of fifteen years duration].

A 69-year-old male visited our clinic in 1973 because of atrial fibrillation noted during an annual check-up for the aged. Blood pressure, heart rate and CTR in chest X-ray films showed 110/80 mmHg, 150/min and 55%, respectively. There were no signs of valvular heart diseases, and a diagnosis of lone atrial fibrillation was convincing. Since then, repeated ECGs recorded twice or more a year had shown atrial fibrillation until 1988, when sinus rhythm with both first degree AV block and low P-wave amplitude appeared. The motion pattern of the anterior mitral leaflet on M mode echocardiography was abnormal with almost complete disappearance of the A-wave, whereas the motion pattern of the tricuspid valve was normal.