Mycobacterium marinum infection in Japanese forest green tree frogs (Rhacophorus arboreus).

Four Japanese forest green tree frogs (Rhacophorus arboreus) were presented with emaciation, abdominal distention and ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen. Another three frogs had been found dead in the same tank 1 year previously. Necropsy examination of these seven frogs revealed splenomegaly and hepatomegaly, with multiple tan-yellow nodular foci present in the liver, spleen, heart, lungs, ovaries and kidneys. Microscopically, five frogs had necrosis and surrounding granulomatous inflammation in the liver, spleen, kidneys, lungs, intestine and ovaries, with numerous acid-fast bacilli in the areas of necrosis. Two frogs had granulomatous lesions in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall. These lesions had no or minimal necrosis and few acid-fast bacilli. Mycobacterium spp. was cultured from three frogs and identified as Mycobacterium marinum by colony growth rate and photochromogenicity and DNA sequencing. This is the first report of M. marinum infection in Japanese forest green tree frogs.

Spontaneous T/NK-cell lymphoma associated with simian lymphocryptovirus in a Japanese macaque (Macaca fuscata).

A 5-year-old female Japanese macaque (Macaca fuscata) was humanely destroyed because of severe anaemia with poor response to treatment. At necropsy examination, marked splenomegaly and systemic enlargement of lymph nodes were observed. Microscopical examination revealed diffuse proliferation of neoplastic lymphoid cells in the spleen and lymph nodes with infiltration of the liver, lung, gastrointestinal tract, kidney and bone marrow. Immunohistochemically, the neoplastic cells expressed CD3 and CD4, but not CD20, CD79α or CD8, consistent with a T helper phenotype. A portion of neoplastic cells expressed the natural killer (NK) cell marker CD56. In-situ hybridization detected Epstein-Barr virus (EBV)-encoded small RNAs in the neoplastic cells, indicating the involvement of simian lymphocryptovirus (LCV). This is the first report of simian LCV-associated T/NK-cell lymphoma with the predominant expression of T-cell antigens in non-human primates.

[Coronary artery bypass grafting in the patients with renal dysfunction].

We studied coronary artery bypass grafting (CABG) in patients with renal dysfunction. From April 1994 to October 1999, 59 patients with renal dysfunction underwent CABG. The patients were divided into 2 groups: group A: 18 patients with end stage renal disease who were receiving hemodialysis, group B; 41 patients with creatinine levels higher than 1.5 mg/dl who were not supported by dialysis. We compared and analyzed the findings of there 2 groups. Regarding preoperative factors, the incidence of old myocardial infarctions, diabetes mellitus and old cerebral infarctions did not differ significantly between the 2 groups. Regarding perioperative factors, the incidence of the number of vessel diseases, emergency operations, operation times and blood transfusions did not differ significantly between the 2 groups. Regarding the post-operative course, the hospital mortality rates demonstrated low levels in 2 groups. The graft patency of group A was 95%, while it was 99% in group B. The post-operative in-hospital days was 24.8 days in the group A, while it was 30.1 days in the group B. No significant difference was observed between the 2 groups. As a results, post-operative hemodialysis was needed in 8 of the patients who underwent on-pump CABG from group A. The actual survival rates were 75.3% in group A and 84.3% in group B at 4 years. The cardiac event free rate for group A was 93.3%, while it was 97.5% in group B at 4 years. In conclusion, CABG may improve the post-operative outcome in renal dysfunction patients. In addition, the use of off-pump CABG is also considered to achieve a better renal function than on-pump CABG.

Evidence for a link between glycoxidation and lipoperoxidation in patients with chronic renal failure.

AIM: The purpose of this study is to examine whether or not there is a relationship between glycoxidation and lipid peroxidation in patients with chronic renal failure. SUBJECTS AND METHODS: Dermal samples from 26 living or autopsied subjects were sequentially extracted with NaCl, pepsin, collagenase, and NaOH to obtain four fractions (salt-soluble fraction: SSF; pepsin-soluble fraction: PSF; collagenase-soluble fraction: CSF; and insoluble fraction: ISF). The glycoxidation product was measured by pentosidine-linked fluorescence (ex: 335/em: 385) and the levels of lipid peroxide, malondialdehyde (MDA), were assessed by determining the MDA-linked fluorescence (ex: 390/em: 460) which was further confirmed by HPLC. RESULTS: In patients undergoing hemodialysis, MDA-linked fluorescence markedly increased in collagen-rich fractions, PSF, CSF, and ISF, while pentosidine-linked fluorescence increased in PSF and CSF, in comparison to the controls and the pre-dialysis patients with CRF. Interestingly, the increase in the lipid peroxides strongly correlated with the level of glycoxidation product in PSF, CSF, and ISF (p < 0.0001 in PSF, CSF; p < 0.01 in ISF). The HPLC data of MDA in the PSF was in good correlation with logistic levels of both MDA- (n = 9, r = 0.738, p = 0.023) and pentosidine-linked fluorescence (n = 9, r = 0.721, p = 0.028). In contrast, in SSF, the collagen-poor fraction (collagen content: less than 3% of the total extracted collagen), the data showed a significant increase in the MDA-linked fluorescence only in the pre-dialysis patients with CRF, but not in the HD patients with no correlation with the glycoxidation products. CONCLUSION: These findings suggest that both the lipid peroxidation and glycoxidation increased in close relation to each other in the matrix collagen and thus demonstrate a synergetic contribution to the tissue damage observed in patients with CRF

Increased advanced glycation end products in atherosclerotic lesions of patients with end-stage renal disease.

Although advanced glycation end products (AGEs) are increased in the serum and tissues of patients with end-stage renal disease, little is known about the role of AGEs in atherogenesis. We therefore carried out an immunohistochemical study on the accumulation of AGEs and apolipoprotein B in the human aortas of diabetic and nondiabetic subjects with end-stage renal disease. The atherosclerotic lesions included diffuse intimal thickening, fatty streaks and atherosclerotic plaque. We used antibodies against two different epitopes of AGE structures, i.e. an Nepsilon-(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML). The area that was positive for an antigen as a percentage of the total area (%Ar) was determined morphometrically, using an NIH-image program. In diffuse intimal thickening, atherosclerotic plaque and tunica media, the %Ar of CML and nonCML was significantly greater in diabetic or nondiabetic subjects with end-stage renal disease than in control subjects without end-stage renal disease. In fatty streaks, the %Ar of nonCML was significantly greater in nondiabetic subjects with end-stage renal disease than in control subjects, while no difference in the %Ar of CML was found between the subjects with or without end-stage renal disease. Nondiabetic subjects with end-stage renal disease showed a significantly increased %Ar of apolipoprotein B in fatty streaks and atherosclerotic plaque than the control subjects. The %Ar of CML and nonCML significantly correlated with the duration of hemodialysis in diffuse intimal thickening and atherosclerotic plaque of subjects with end-stage renal disease, but not in fatty streaks. On the other hand, the %Ar was not related to the duration of diabetes in any of the lesions in the diabetic subjects with end-stage renal disease. In diffuse intimal thickening and atherosclerotic plaque, subjects with end-stage renal disease showed a significant correlation between the %Ar of apolipoprotein B and AGEs (CML and nonCML), as well as their immunohistochemical colocalization. These results suggest that impaired AGE clearance may cause the increased accumulation of AGEs in the aortic wall of subjects with end-stage renal disease, thus resulting in the rapid progression of atherosclerosis. The accumulation of AGEs may be related to an enhanced LDL deposition in atherosclerotic lesions of subjects with end-stage renal disease.

Immunohistochemical localization of different epitopes of advanced glycation end products in human atherosclerotic lesions.

To better understand the role of advanced glycation end products (AGEs) in atherogenesis, we developed specific antibodies against different immunological epitopes of AGE structures, including Nepsilon-(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML), and demonstrated the immunohistochemical localization of CML- and nonCML-epitopes in atherosclerotic lesions of human aorta, which were obtained at autopsy from 20 nondiabetic patients (12 males and eight females; mean age, 60.8+/-16.7 years). Monoclonal anti-CML antibody (6D12) recognized not only AGE-modified proteins, but also CML-modified proteins. On the other hand, polyclonal anti-nonCML antibody reacted to AGE-modified proteins, but not to CML-modified proteins. Both antibodies were unreactive to the early-stage products of glycation, including fructose-modified butyloxycarbonyl-lysine and fructose-epsilon-aminocaproic acid. Atherosclerotic lesions included diffuse intimal thickening (DIT), fatty streaks (FS), atherosclerotic plaques (AP) and complicated lesions. An immunohistochemical analysis showed both CML- and nonCML-epitopes to be found along the collagen fibers in DIT in subjects more than 40 years old, but not in subjects less than 40 years old. CML-epitopes accumulated mainly in the cytoplasm of macrophage/foam cells, while nonCML-epitopes accumulated exclusively in the extracellular spaces in FS. APs showed the CML-epitope stored macrophage/foam cells, and the accumulation of both CML- and nonCML-epitopes in the lipid-rich fibrous area. An immunohistochemical analysis with a monoclonal antibody against oxidized low density lipoprotein (FOH1a/DLH3) showed the presence of this antigen within the cytoplasm of the macrophage/foam cells in atherosclerotic lesions, which were also positive for the CML-epitopes. These findings thus suggest that the heterogeneous localization of AGEs in atherosclerotic lesions depends on their different epitopes, and that a close link, therefore, exists between the peroxidation of LDL and the formation of AGEs in atherosclerotic lesions.

Oxidative damage of vascular smooth muscle cells by the glycated protein-cupric ion system.

To clarify the mechanism of cellular injury through the nonenzymatic reaction of glucose with proteins, we studied the cytotoxic effect of glycated bovine serum albumin on cultured smooth muscle cells in the presence of cupric ion. Glycated proteins were prepared by incubating bovine serum albumin with 0.5 M D-glucose in 0.3 M sodium phosphate buffer at 37 degrees C for 2, 4 and 16 weeks (g-BSA-2, g-BSA-4 and g-BSA-16, respectively). Early glycation products, such as fructosamine, were formed more than two weeks after incubation. However, the immunoreactivity of glycated proteins to anti-AGE antibody was 12-fold higher in g-BSA-16 than in g-BSA-2. Both g-BSA-2 and g-BSA-16 showed a concentration-dependent cytotoxicity in smooth muscle cells in the presence of 80 microM cupric ion by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye reduction assay and dye exclusion test. Flow cytometry and spectrofluorophotometry using dihydrorhodamine 123 showed that the extracellular generation of oxidants was dose-dependently enhanced with increasing concentrations of g-BSA-2 or g-BSA-16 in the presence of cupric ion. However, no difference was observed in the intracellular generation of oxidants between the presence and absence of glycated proteins by flow cytometry using 2', 7'-dichlorofluorescein diacetate. Cytotoxicity and oxidant generation were prevented by catalase and tiron, but not by superoxide dismutase or mannitol, a hydroxyl radical scavenger. These results indicate that smooth muscle cells may be damaged by reactive oxygen species which are produced extracellularly by the interaction with the early glycation products and cupric ion, and suggest that hydrogen peroxide may be a candidate for reactive oxygen species which contribute to such oxidative damage of smooth muscle cells.

Coronary artery bypass grafting without cardiopulmonary bypass for high-risk patients.

Between January 1991 and June 1993, coronary artery bypass grafting was performed without either cardiopulmonary bypass or cardiac arrest in 23 patients. Most patients had several surgical risk factors, including age > or = 70 years, poor left ventricular function, left main coronary artery stenosis, chronic renal failure, and aortic aneurysm. Distal anastomoses were made under temporary interruption of coronary flow. A total of 37 distal anastomoses to the left anterior descending coronary artery and/or right coronary artery (mean 1.6 per patient) were made, 24 of which were internal thoracic arteries. The coronary occlusion time ranged from 7-14 min (mean 9.8 min). Combined cardiac or vascular operations were carried out in six patients (abdominal aortic aneurysm repair, thoracic aortic aneurysm repair, carotid endarterectomy, and coronary endarterectomy). There was one hospital death. Postoperative angiography was performed in 22 patients and showed a patency rate of 89%. In summary, coronary artery bypass grafting without cardiopulmonary bypass may improve the postoperative outcome of high-risk patients.

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana).

To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1(-1). These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.

Sequential internal mammary artery grafts: clinical and angiographic assessment.

Between January 1988 and August 1992, the internal mammary artery was used as a sequential graft to the left anterior descending artery and/or diagonal branch in 34 patients. One patient died in hospital. After surgery all survivors were free from angina for a follow-up of up to 4 years. Recatheterization was performed in 33 patients within 1 year of surgery. Postoperative angiography showed that 65 anastomoses (98%) were patent, but three patent grafts (5%) between the proximal and distal sequential anastomoses showed 'string sign'. It is important to prevent 'string sign' in sequential grafting. It is considered that sequential internal mammary artery grafting should be limited to coronary arteries with severe stenosis that divides anastomosed coronary arteries into two.

Early and late results with combined mitral and aortic valve replacement.

From February 1977 to January 1993, a total of 52 patients have undergone combined mitral and aortic valve replacement (MAVR) at the Fukuoka University Hospital. The ages at operation ranged from 35 to 72 years (mean, 55.3 years) for 16 males and 36 females. Twenty-five patients received two bioprosthetic valves (group BB); 6 received a combination of bioprosthetic valve and mechanical valve (group BM); and 21 received two mechanical valves (group MM). The previous intra-cardiac operations were performed on 24 patients (46.2%). The purpose of this study was to learn about the long-term clinical results of MAVR and to analyze the difference among three groups. Seven patients died within 30 days of operation or during initial hospitalization (early mortality: 13.5%). Two patients died at late period, 5.8 years and 6.9 years after operation, respectively (linearized occurrence rate: 0.9%/pt-yr). Patient survival rate including operative death was 82.9% +/- 5.7% at 5 years and 79.0% +/- 6.7% at 8 years in the total number of patients. Thromboembolism occurred in 2 patients in group MM (0.9%/pt-yr). Anticoagulant-related hemorrhage occurred only in 1 patient (0.4%/pt-yr in overall), in whom two bioprosthetic valves were implanted (group BB), in spite of being in good control with anticoagulants. No infective endocarditis was encountered in any patients during any of the periods. Reoperations were performed in 7 patients (3%/pt-yr in overall), 5 in group BB and 2 in group MM. Percentage freedom from reoperation was 86.4% +/- 5.9% at 5 years and 78.2% +/- 7.7% at 8 years in all. There was no difference among the three groups. Percentage freedom from overall morbidity and mortality was 74.5% +/- 6.9% at 5 years in all. Group MM showed higher morbidity and mortality at 8 years than other groups, but there were no significance (MM: 52.4% +/- 17.6%, BB: 76.5% +/- 9.5%, BM: 83.3% +/- 15.2%). We conclude that there were no significant differences in long-term results of NAVR which consisted of the following combinations of prostheses such as BB, BM, and MM. Redoing MAVR with New York Heart Association functional class IV and emergency cases were considered as in-hospital risk factors.

[Treatment of postoperative mediastinitis using an omental pedicle flap].

Four patients with postoperative mediastinitis who were treated by omentopexy at the Fukuoka University Hospital between 1989 and 1990. Three of the 4 patients healed successfully, another one died of multiple organ failure 83 days after surgery. All patients were received coronary artery bypass surgery harvesting a left internal thoracic artery for ischemic heart disease. Three patients had diabetes mellitus, one patient had renal failure preoperatively. Recognition of mediastinitis was made by sternal wound purulent discharge and sternal dehiscence. Culture of the discharge fluid yielded methicillin-resistant Staphylococcus aureus in three, and Enterococcus cloacae in one. Irrigation with popidone-iodine or blonopol were ineffective. Thus, the wound was treated with debridement and omentopexy with an omental pedicle flap, respectively. Postoperative course after omentopexy were excellent, had no complications. We conclude that the omentopexy is useful in the treatment of postoperative refractory anterior mediastinitis.

[Long-term splinting of a traumatic upper permanent incisor with root fracture: report of a case].

Splinting was administered in a case of a traumatic upper right permanent incisor with root fracture. The patient was a boy, 11 years and 10 months old. The radiographical examination indicated that the injured tooth, that had already completed the apical growth, fractured horizontally at the middle 1/3 part of root. Clinical findings showed severe mobility of the coronal fragment of the injured tooth and a small amount of bleeding from the gingival sulcus, but there was almost no disposition of the coronal fragment and it was found to be vital in the electric pulp test. Immobilizing the coronal fragment of the injured tooth with the resin splint bonded directly to the tooth surfaces was prescribed. After 31 months, the pulp of the injured tooth remained vital, and after the elimination of the line of fracture and no symptoms of ankylosis were radiographically confirmed, the splint was then removed. From the case reported above the following implications were obtained: Although the previous investigators reported that the term of splinting teeth with root fractures was for 2-3 months, and prognosis of the injured teeth with root fractures having severe mobility of the coronal segments might be unfavorable, it was also indicated that the healing process by calcification might be possible with the use of long term splinting, as the pulp remained vital. In this case, it was found that the repair by calcification appeared initially on the proximate portion of the pulp at the fractured line, and slowly proceeded into the direction of the site of the outer surface of the root along the fractured line.