Development of antibody against epitope of lipoprotein(a) modified by oxidation: evaluation of new enzyme-linked immunosorbent assay for oxidized lipoprotein(a).

BACKGROUND: Recently, the biological effects of oxidized lipoprotein(a) [Lp(a)] have been reported to be more potent than Lp(a), the arteriosclerosis-relevant lipoprotein. Thus, investigations with oxidized Lp(a) are expected to provide viewpoints different from the conventional ones based on Lp(a). METHODS AND RESULTS: An anti-Lp(a) monoclonal antibody (161E2) was produced against synthetic peptide antigen (Arg-Asn-Pro-Asp-Val-Ala-Pro). This epitope was characterized as having various properties because its external exposure was induced as a result of oxidative modification. Using 161E2 antibody, we developed a new enzyme-linked immunosorbent assay to measure Lp(a) modified by oxidative stress. The present data demonstrated that oxidized Lp(a) that contains the epitope of 161E2 antibody was present in the serum of humans. Therefore, we used this new enzyme-linked immunosorbent assay to evaluate the role of oxidized Lp(a) in patients with hypertension, which induces oxidative stress. Interestingly, hypertensive patients with complications showed a significantly higher level of oxidized Lp(a) in serum than did normotensive subjects (P:<0.01), whereas there was no significant difference in native Lp(a) between normotensive and hypertensive subjects. Importantly, positive immunostaining with 161E2 monoclonal antibody was found in the human arteriosclerotic tissue. CONCLUSIONS: We developed a new antibody against an epitope in Lp(a) as a result of oxidation treatment but not in native Lp(a). The present data demonstrated in vivo the presence of oxidized Lp(a) in the atherosclerotic tissue and its elevation in hypertensive patients. The presence of oxidized Lp(a) may be important in understanding the role of Lp(a) in cardiovascular disease.

Atherosclerosis in a rhesus monkey with genetic hypercholesterolemia and elevated plasma Lp(a).

A female rhesus monkey with a marked elevation of total plasma cholesterol LDL and Lp(a) while on a low-fat, low-cholesterol diet, died at 22 years of age. Her spontaneous hypercholesterolemia was related to a genetically determined LDL receptor deficiency (Scanu, A.M. et al., J. Lipid Res., 29 (1988) 1671). Autopsy revealed grossly visible multifocal to diffuse raised yellow plaques predominantly in the aorta and, to a lesser extent, in the coronary arteries. Microscopically, the plaques in the aorta and in the coronary arteries showed heavy lipid deposition. Some had features seen in advanced human atherosclerotic plaques, including a fibrous cap and a necrotic core. Immunohistochemical staining showed a co-localization of apo(a) with apo B in lesion sites, a pattern seen frequently in advanced human atherosclerotic plaques. Evidence of fibrinogen/fibrin in the plaque areas was also seen, but was not co-localised with either Lp(a) or apo B. This monkey developed progressive atherosclerosis which was not induced by diet, but rather was dependent on the LDL receptor deficiency with a possible contribution by the elevated plasma levels of Lp(a).

Second nation-wide study of atherosclerosis in infants, children and young adults in Japan.

This paper reports the results of the second nation-wide cooperative study of atherosclerosis in young Japanese, aged from 1 month to 39 years, who were autopsied between 1991 and 1995. Atherosclerotic lesions in 1066 aortas and 974 coronary arteries were classified into fatty streaks, fibrous plaques and complicated lesions and quantificated with the point-counting method. The results of this study were compared with those of the former study, which was conducted 13 years earlier in almost the same fashion as this study. Atherosclerosis of aorta, which was determined by surface involvement (SI) of atherosclerotic lesions and atherosclerotic index (AI), increased with age in both sexes of the former and the present studies and their tendency for the progression of the extent of atherosclerotic lesions appeared to be similar. In the coronary arteries, the mean values of SI and AI in the males of the present study were greater significantly than those in the male of the former studies and in the female of the both studies in the third and fourth decades. This difference suggests that atherosclerotic lesions are increasing in young Japanese males. It also suggests that these subjects may be increasingly susceptible to atherosclerotic cardiovascular disease with increasing age.

Expression of peroxisome proliferator-activated receptor subtypes in human atherosclerosis.

To clarify the involvement of peroxisome proliferator-activated receptors (PPARs) in atherosclerotic plaque formation, we investigated the expression patterns of mRNA and protein of PPARalpha and PPARgamma in human aorta. Atheromatous plaque, fatty streak, and diffuse intimal thickening (DIT) were separated macroscopically, and each sample was divided into halves. Half of them were used for analysis of mRNA expression with reverse transcription-polymerase chain reaction and the others were used for histologic analysis. Both PPARalpha and PPARgamma mRNA were detected in all atheromatous plaques, all fatty streaks, and in some DIT. However, expressions of PPARalpha and PPARgamma were obviously less frequently found in DIT than in atheromatous plaques, and the intensity of these expressions was stronger in the atheromatous plaques than in the DIT. Compared with PPARalpha, PPARgamma mRNA was expressed more frequently in atheromatous plaques. In atheromatous plaques, PPARgamma mRNA was expressed independently, whereas PPARalpha mRNA was coexpressed with PPARgamma. PPARgamma protein was obviously found in the nuclei of endothelial cells, macrophages, mononuclear cells, and smooth muscle cells in the aortic intima. These results suggest that expressions of PPARalpha and PPARgamma in human aortic wall are involved in atherogenesis from the early stages.

Inhibitory effect of fluvastatin at doses insufficient to lower serum lipids on the catheter-induced thickening of intima in rabbit femoral artery.

The anti-atherosclerotic effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors at doses insufficient to lower serum cholesterol was investigated in rabbit femoral artery denuded by balloon catheter. Fluvastatin and pravastatin were given orally at doses of 4 and 8 mg/kg per day, respectively, for 2 weeks after the catheterization. There was little change in serum cholesterol, triglyceride and phospholipid by chronic treatment with the drugs. The cross-sectional area of the intima, expressed as relative values to media (I/M ratio), was increased by the catheterization, showing intimal thickening in the denuded arteries. The I/M ratio was reduced by fluvastatin but not pravastatin: 0.327 +/- 0.060 for control, 0.116 +/- 0.035 for 4 mg/kg fluvastatin, 0.088 +/- 0.027 for 8 mg/kg fluvastatin and 0.22 +/- 0.069 for 8 mg/kg pravastatin. Fluvastatin (8 mg/kg)-induced effect on the I/M ratio, was prevented by the combined administration with 40 mg/kg per day mevalonate, a metabolite in the HMG-CoA reductase pathway. These results suggest that fluvastatin inhibits intimal thickening after catheterization-induced injury through percutaneous transluminal coronary angioplasty (PTCA) and that the inhibition is presumably attributed to reduced migration and proliferation of smooth muscle cells but not secondarily to a lowering of serum lipid.

Effect of alpha-ketobutyrate on microvascular thickness in the diabetic rat kidney.

The first objective of this study was to examine the intermediary metabolism of plasma amino acids and keto acids in streptozotocin (STZ)-treated rats. Plasma alpha-aminobutyrate (alpha-ABA) concentration in STZ rats was 1.5-fold greater than in control (CNT) animals at 1 month. In contrast, the level of plasma alpha-ketobutyrate (KB), which is transaminated to alpha-ABA, did not differ significantly between STZ and CNTs at 1 month, and also increased with age. Additionally, HPLC analysis revealed consistent profiles containing peaks of unknown origin. Two pathways exist for the formation of alpha-KB, either from the action of threonine dehydratase or via homocysteine, the latter metabolite being closely associated with the development of cardiovascular disease. These observations suggest that uncharacterized metabolites, including plasma alpha-KB, may be potential risk factors for the development of diabetic complications. We carried out preparatory experiments on non-diabetic rats to investigate the influence of alpha-KB and confirmed this metabolite had no adverse effects. The second aim of the study was to compare vascular wall thickness in diabetic rats treated or untreated with alpha-KB with CNT animals in order to determine the effects of alpha-KB on the renal microvasculature. The thickness of the medial wall of arterioles and small arteries differed significantly among all groups and was increased, especially in the small arterial walls of the diabetic rats treated with alpha-KB. Plasma renin activities (PRA) in both diabetic rats treated or untreated with alpha-KB were decreased significantly compared to CNT animals, while diabetic rats treated with alpha-KB had higher angiotensin converting enzyme (ACE) activity than the CNT group (p < 0.01). These results suggest that alpha-KB may have a role in the renal microvascular complications of diabetes.

Studies of glomerular permeability factor (GPF) in focal segmental glomerular sclerosis and the relationship between GPF and vascular permeability factor (VPF).

BACKGROUND: We previously demonstrated that the supernatants of cultured concanavalin-A (con-A) stimulated peripheral blood mononuclear cells (PBMC) from patients with minimal change nephrotic syndrome (MCNS) increased the urinary protein excretion in injected rats and suggested that PBMC released a factor, which we called glomerular permeability factor (GPF), changes in the glomerular permeability and thus resulted in proteinuria in MCNS. MATERIAL AND METHODS: In this study we investigated the GPF activity in focal segmental glomerular sclerosis (FGS) and other conditions of chronic glomerulonephritis (CGN), and also the relationship between GPF and vascular permeability factor (VPF). In experiment 1 the supernatants of the cultured con-A stimulated PBMC from patients with 10 FGS, 5 other CGN and 10 controls were tested regarding their ability to produce GPE The GPF activity was defined as positive when the 8-hour urinary protein excretion after the injection of the supernatant in Sprague-Dawley rats exceeded the mean value plus 2 standard deviations (M + 2 SD) of that before injection. RESULTS: Three out of 10 FGS patients and 1 membranous nephropathy patient out of the 5 other CGN patients were positive for GPF activity. In experiment 2 the relationship between GPF and VPF was analyzed using culture supernatants of PBMC from 10 nephrotic MCNS patients and 15 controls. The VPF activity was measured following the method developed by Ovary [1975]. All 7 cases that were positive for GPF activity were simultaneously positive for VPF activity. On the other hand, 16 cases that were positive for VPF activity were not always positive for GPF activity (7 cases were positive and 9 were negative for VPF activity). CONCLUSION: Experiments 1 and 2 thus suggested that GPF was not active in MCNS alone, but also in other CGN conditions and it was therefore not considered to be the same factor/substance(s) as VPF.

Expression of muscarinic receptor genes in the human coronary artery.

The authors investigated the role of muscarinic receptors in functional control of coronary arteries affected by intimal thickening due to arteriosclerosis. They first examined the genetic subtypes of muscarinic receptors expressed in human coronary arteries. Twelve samples of human coronary artery, obtained by autopsy from eight subjects, were examined for the expression of four genetic subtypes of muscarinic receptor, m1 to m4, by reverse transcription and polymerase chain reaction (RT-PCR). Two subtypes, m2 and m3, were found to be expressed in the coronary artery. The m2 gene was expressed in seven of the 12 vessels, and m3 in eight of the 12. Expression of both m2 and m3 genes was observed in five of the 12 vessels. Neither the m1 nor m4 was expressed in these samples. These results indicate that the m2 and m3 genes are mainly expressed in the coronary arteries and suggest that these patterns of expression are differentially controlled to induce the diversity of contraction/relaxation reactions induced in the coronary arteries by acetylcholine.

[Clinical evaluation of cefmenoxime in chronic complicated urinary tract infection].

The efficacy of cefmenoxime (CMX), which is a third generation, beta-lactamase-resistant cephem with a broad antibacterial spectrum, was examined in 43 patients with chronic complicated urinary tract infections. The usual dosage regimen was given 2 approximately 4 g/day of CMX by intravenous drip infusion over 1 hour. The duration of treatment was 5 days. Fifteen patients were cured and 21 improved, and the effective rate was 83.7%. Bacterial eradication rate in these cases was 88.2%, especially eradication of the original pathogens such as Serratia marcescens, Proteus species and Klebsiella species, occurred in high frequency. Laboratory abnormalities were slight elevation of serum GOT and GPT in 2 cases. From these findings, CMX was considered to be very effective in complicated urinary tract infections.

[A case of angiographically occult arteriovenous malformation with metaplasia (so-called brain stone)].

The patient was a 69-year-old female. Right hemiparesis occurred on April 25, 1996, and then was relieved a day later. Because headache (dull pain in the left) persisted subsequently, she consulted our department on April 26. Head CT showed, without enhancement effect, osseous high density on the surface of the left frontal area. MRI showed high intensity on T1 and low intensity on T2 with flow-void like findings. Cerebral angiography showed a pooling of contrast medium in the same region. 123I-IMP-SPECT revealed reduced cerebral blood flow in the left frontal and parietal lobes just under the same region. On June 11, the patient underwent surgery during which a tumor with arachnoid hypertrophy was extracted en block. Histopathologically, there were abnormal blood vessels with elastic fibers, expanding to an ossified site, and AVM accompanying ossification was thus diagnosed. Postoperative 123I-IMP-SPECT showed improved cerebral blood flow in the left frontal and parietal lobes. The patient was discharged on June 22. The TIA pathologic condition, a symptom of its onset, was considered attributable to cerebral blood flow steal due to AVM.

[Application of crush cytology preparation for rapid diagnosis of brain tumors].

The crush cytology perform during the surgery for 22 cases with brain tumors at Surugadai Nihon University Hospital, being compared with frozen and permanent histology sections. Crush cytology, which was generally and less damaged by artefacts than frozen sections. Immunocytochemistry used crush cytology were useful tools for differential diagnosis between undifferentiated glioma and carcinoma. Crush cytology were thought to be useful using together with the frozen sections for intraoperative diagnosis.

[Roles of pathology laboratories on medical care service and quality assessment system for pathological diagnosis].

In practical medicine, pathologists provide highly specialized medical information (pathological diagnosis) to clinicians. Pathological diagnosis includes histopathology, cytology, intraoperative consultation (rapid diagnosis by frozen section) and autopsy service. In particular, histopathological diagnosis of biopsy specimens, nearly always gives a qualitative diagnosis to unknown pathological process, in other words, a final diagnosis. In addition, pathological diagnosis contributes to supporting medical audit. Thus it is quite important that to establish the quality assessment system for pathological diagnosis which is hard to be judged with the value.

[Stimulatory effects of a streptococcal protein, preabsorbing antigen (PA-Ag), on human lymphocytes].

The possible role of a streptococcal protein, preabsorbing antigen (PA-Ag), in human cellular immunity was examined by a lymphocyte stimulation test. Lymphocytes were obtained from 12 healthy adult donors who had no history of acute poststreptococcal glomerulonephritis nor of other renal diseases, and from the blood of 4 umbilical cords. In addition to PA-Ag, pre-purified material from ruptured cell supernatant (RCS) was also tested for lymphocyte stimulation, and their time-response relationships were compared to those with stimulation by phytohemagglutinin (PHA), a nonspecific mitogen. Results showed that the lymphocytes of the 12 adult donors proliferated in a dose-dependent manner in response to PA-Ag or RCS stimulation. In the response to these proteins, the peak responsiveness of lymphocyte proliferation was characteristically seen between the 5th and 7th days of cultivation, while it was seen between the 3rd and 5th days in PHA stimulation. Surprisingly, lymphocytes derived from umbilical cord bloods also significantly proliferated in the presence of PA-Ag. These results indicate that PA-Ag stimulates human lymphocytes to proliferate in a different manner than when stimulated by PHA, and the induction of the stimulatory effects is not dependent on antigen-specific response, since it does not require pre-sensitization of the host.

[Beneficial effect of salazosulfapyridine (SASP) in a patient with secondary renal amyloidosis].

A 55-year old female with rheumatoid arthritis (RA) had presented with proteinuria since July 1996. She was referred to us for persistent edema on her face and legs in November 1996. On admission, her 24-hour urinary protein excretion was 4.4 g/day, total serum protein level was 5.3 g/dl, and serum level of amyloid A protein (SAA) was elevated to 45.8 mg/ml. A percutaneous renal biopsy was performed, and light microscopy revealed varying degrees of amyloid deposits in the mesangial areas and arteriolar walls. The diagnosis of secondary amyloidosis (AA amyloidosis) was based on immunohistochemical staining for amyloid A protein using monoclonal antibody against SAA. Four weeks after treatment with salazosulfapyridine (SASP) and dipyridamole, proteinuria began to decrease and the edema had disappeared. Finally she recovered from nephrotic syndrome. AA amyloidosis has been thought to have a poor prognosis, with progression to renal failure. Since there is no specific effective therapy for the disease, it is very important to reduce the activity of the underlying cause. In our patient with renal amyloidosis following RA, SASP was evidently effective for arthritis and improvement of renal function. SASP might have a beneficial effect on AA amyloidosis by suppressing inflammatory cytokines.