Facilitation of bone resorption activities in synovial lavage fluid patients with mandibular condyle fractures.

The aim of this study was to investigate the bone resorption effect of the mediators delivered in joint cavity of patients with mandibular condyle fractures by detecting osteoclast markers using cellular biochemistry methods, and by analysing bone resorption activities via inducing osteoclast differentiation of the infiltrated cells from arthrocentesis. Sixteen joints in 10 patients with mandibular condyle fractures were evaluated. The control group consisted of synovial fluid (SF) samples from seven joints of four volunteers who had no clinical signs or symptoms involving the temporomandibular joint (TMJ) or disc displacement. We collected SF cells from all patients during therapeutic arthrocentesis. The infiltrating cells from TMJ SF were cultured, differentiated into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and examined bone resorption activities. We also investigated factors related to osteoclast induction of SF, using ELISA procedures. Osteoclast-like cells were induced from the SF cells obtained from all patients with condylar fractures. These multinucleated giant cells were positive for TRAP and actin, and had the ability to absorb dentin slices. The levels of macrophage colony-stimulating factor (M-CSF), prostaglandin E2 (PGE2), soluble form of receptor activator of nuclear factor kappa-B ligand (sRANKL) and osteoprotegerin (OPG), in SF samples from the patients, were significantly higher than in the controls. These findings indicate that bone resorption activities in SF from patients with mandibular condyle fractures were upregulated and may participate in the pathogenesis and wound healing.

Effect of salt and heating on a mesoscopic structure composed of ovalbumin globules in aqueous solution.

Mesoscopic structural changes of an ovalbumin solution by heating and adding NaCl have been investigated with a small-angle neutron scattering method. In the natural solution, a broad peak at q = 0.057 A(-1), which disappeared by adding NaCl, indicates the existence of an electrostatic long-range interaction between the ovalbumin globules. Along with the broad peak, a prominent intensity increase in a very small q region was observed on heating except for the initial and final stages, indicating the coexistence of a disordered structure of the denatured ovalbumin and a regular-interspacing structure of the natural ovalbumin globules. Though the macroscopic feature in the final stage, whether the solution forms a gel (10 wt %) or not (5 wt %), strongly depended on the concentration of the ovalbumin, the scattering profiles showed a common characteristic feature: the appearance of a new peak around q = 0.023 A(-1), which indicates the emergence of another regular structure.

Urinary trypsin inhibitor levels in the urine of patients with haematological malignancies.

The urinary trypsin inhibitor (UTI) levels in the urine of patients with various haematological malignancies were determined, using automated latex agglutination immunoturbidimetry. The mean UTI levels in urine in acute non-lymphocytic leukaemia, myelodysplastic syndrome, non-Hodgkin's lymphoma, and multiple myeloma groups were significantly elevated, compared with the normal control group. It was found that the UTI level in urine changed from an elevated value to a normal value with haematological improvement by chemotherapy in a patient with myelodysplastic syndrome included in a previous study. These results suggest tha

Impaired glucose homeostasis and neonatal mortality in hepatocyte nuclear factor 3alpha-deficient mice.

Hepatocyte nuclear factors 3 (HNF-3) belong to an evolutionarily conserved family of transcription factors that are critical for diverse biological processes such as development, differentiation, and metabolism. To study the physiological role of HNF-3alpha, we generated mice that lack HNF-3alpha by homologous recombination in embryonic stem cells. Mice homozygous for a null mutation in the HNF-3alpha gene develop a complex phenotype that is characterized by abnormal feeding behavior, progressive starvation, persistent hypoglycemia, hypotriglyceridemia, wasting, and neonatal mortality between days 2 and 14. Hypoglycemia in HNF-3alpha-null mice leads to physiological counter-regulatory responses in glucocorticoid and growth hormone production and an inhibition of insulin secretion but fails to stimulate glucagon secretion. Glucagon-producing pancreatic alpha cells develop normally in HNF-3alpha-/- mice, but proglucagon mRNA levels are reduced 50%. Furthermore, the transcriptional levels of neuropeptide Y are also significantly reduced shortly after birth, implying a direct role of HNF-3alpha in the expression of these genes. In contrast, mRNA levels were increased in HNF-3 target genes phosphofructo-2-kinase/fructose-2,6-bisphophatase, insulin growth factor binding protein-1, and hexokinase I of HNF-3alpha-null mice. Mice lacking one or both HNF-3alpha alleles also show impaired insulin secretion and glucose intolerance after an intraperitoneal glucose challenge, indicating that pancreatic beta-cell function is also compromised. Our results indicate that HNF-3alpha plays a critical role in the regulation of glucose homeostasis and in pancreatic islet function.

Specific fluorescence assay for advanced glycation end products in blood and urine of diabetic patients.

Late rearrangement products that accumulate by glycation of proteins, known as advanced glycation end products (AGEs), have been implicated in the pathogenesis of complications related to diabetes. Circulating AGEs, especially in the form of a small peptide (AGE-peptide) of less than 10 kd, increase in the blood of diabetic patients with end-stage renal disease (ESRD). The aim of the study was to evaluate AGE-peptide levels by measuring AGE-specific fluorescence (excitation at 370 nm and emission at 440 nm) and to examine the relationship between AGE-peptide and diabetic nephropathy. AGE-specific fluorescence in serum and urine were examined in diabetic subjects with various levels of renal complications of varying severity: normoalbuminuria (N), microalbuminuria (Mi), macroalbuminuria (Ma), chronic renal failure (C), and hemodialysis (HD). We also assessed correlations among the AGE-peptide level and age, duration of diabetes, hemoglobin A1c (HbA1c), serum creatinine, and creatinine clearance. Serum and urine AGE-peptide levels in C and HD were significantly higher than in N, Mi, and Ma. Serum AGE-peptide levels were significantly correlated with serum creatinine (r=.866, P < .0001) and creatinine clearance (r=-.720, P < .0001) but not with duration of diabetes or age. There was a significant correlation between AGE-peptide levels measured by enzyme-linked immunosorbent assay (ELISA) and levels determined from the specific fluorescence intensity (r=.688, P < .0001). These findings suggest that renal function may play a greater role in the accumulation of AGEs than persistent hyperglycemia in diabetic patients. Measurement of AGE-specific fluorescence (ie, AGE-peptide) may serve as a simple and useful test to assess circulating AGE levels and monitor AGE excretion.

Aneurysmal bone cyst of the clavicle in a child.

We report a case of an aneurysmal bone cyst (ABC) of the clavicle in a 9-year-old boy, which initially presented as a pathological fracture of a benign cystic lesion. ABC of the clavicle is rare in children less than 10 years old and radiological diagnosis may prove difficult in the early stages of presentation.

The role of advanced glycosylation end-products in the pathogenesis of atherosclerosis.

Coronary artery disease and cerebrovascular disease due to the rapid progression of atherosclerosis is the principal cause of death in diabetes mellitus. Modification of low-density lipoproteins (LDL) by advanced glycosylation end-products (AGE) may play a central role in the development of atherosclerosis, especially in diabetic patients. An AGE-modified form of LDL (AGE-LDL) has been found to circulate in human plasma, and AGE modifications have been identified as being present on both the apoprotein (ApoB) and the phospholipid components of LDL. By utilizing an AGE-specific ELISA, we measured the AGE attached to the ApoB and lipid components of LDL from normal controls and diabetic patients with or without end-stage renal disease (ESRD), as well as lipid oxidation. AGE-ApoB, AGE-lipid and oxidized LDL (Ox-LDL) in diabetic patients were significantly higher than those in patients without diabetes. The correlation between AGE-ApoB and AGE-lipid were highly significant. An especially marked elevation of AGE-LDL was found in diabetic patients with ESRD. The correlation between the serum total cholesterol and the AGE-LDL (AGE-ApoB and AGE-lipid) was significant. In addition, based on the known biological properties of AGE-modified peptide (AGE-peptide), we have proposed that these chemically reactive circulating AGE-peptides contribute to tissue injury by reattaching to susceptible target proteins both within and outside the vasculature, and that this process accelerates vascular pathology in diabetic patients. These data indicate that AGE-modified LDLs may represent a particularly atherogenic form of LDL, and AGE-LDLs as well as AGE-peptides are likely to contribute to the development of atherosclerosis in diabetic patients.

Advanced glycation endproducts and diabetic nephropathy.

Diabetic nephropathy is currently the single largest cause of endstage renal disease (ESRD) in the United States and many European countries. The primary cause for the development of diabetic complications (including diabetic nephropathy) is persistent exposure to hyperglycemia, although genetic and other incompletely understood factors also play an important role. Although much consideration has been given to the pathogenesis and genetics of the disease itself, the mechanisms by which persistent exposure to hyperglycemia cause biochemical and metabolic alterations have been very sketchily understood. Recently, a growing body of evidence has linked the accumulation of the late products of glucose-protein interaction to a variety of chronic complications, including diabetic nephropathy. The formation of irreversible advanced glycosylation endproducts (AGEs) resulting from the spontaneous reaction between glucose and proteins occur most noticeably on long-lived structural proteins. Recent studies demonstrate that the pathogenesis of diabetic nephropathy is caused by the hyperglycemia-accelerated formation of AGEs. Also, reactive AGE peptides in the circulation are thought to play a role as a new version of so called middle molecule toxic substances. This evidence is opening a new window for our understanding of the pathogenesis of diabetic nephropathy.

Peritoneal dialysis using a recycling system in dogs.

In this study, Recirculation Peritoneal Dialysis (RPD) apparatus using a pump to circulate the dialysate was devised and its dialysis efficiency was investigated. In the experiment, 15 mongrel dogs with body weights of 6.6 +/- 0.7 kg were used. The surgery to induce azotemia was performed on the dogs, a disk catheter for drainage was placed between the liver and diaphragm, and a straight catheter for infusion was placed on the abdominal side of the bladder. RPD was conducted using 2 l of dialysate containing 1.3% glucose, and the dialysate was circulated by a pump through the peritoneal cavity and the dialysate chamber at the circulation rates of 30, 60, 90 and 120 ml/min. RPD was conducted for 3 hr and the clearances of urea nitrogen, creatinine, inorganic phosphorus and potassium were recorded every hour. The clearances in RPD became higher with an increase in the circulation rate. Although RPD requires two catheters in the peritoneal cavity, it has no appreciable technical difficulty in handling and is considered to be easier than hemodialysis. RPD enabled higher dialysis efficiency to be achieved in a short time compared with conventional peritoneal dialysis. It seems reasonable to conclude that RPD is a useful new dialysis technique for animals.

[Immunochemical determination of advanced glycation end products in erythrocyte peripheral-membrane proteins from diabetic patients].

Nonenzymatic glycation of protein leads to changes in protein structure and function, including browning, crosslinking and polymerization. There are several functional abnormalities in erythrocytes of diabetic patients. However, nothing definite is known about the relationship between these abnormalities and nonenzymatic glycation products, especially advanced glycation end products (AGEs). In this view, the present study was aimed to determine the amount of AGEs in diabetic erythrocyte peripheral-membrane proteins (EPMPs) by an ELISA system using AGE-specific antibodies. Erythrocyte membrane proteins from 19 control and 48 diabetic subjects were treated with 0.1 N-NaOH solution and then, the supernatants of each sample, containing EPMPs, were used to measure the amount of AGEs. The amount of AGEs in EPMPs was approximately 3 times greater in diabetic patients than in controls (70.7 +/- 51.8 AU vs. 22.5 +/- 7.7 AU (mean +/- SD), p < 0.001). There was a significant correlation between levels of AGEs in EPMPs and HbA1C (r = 0.779, p < 0.001). In SDS-PAGE analysis of EPMPs, two major bands, 221 kDa and 242 kDa were observed. These two bands were defined to be spectrin by immunoblotting with mouse monoclonal anti-human spectrin antibody. Based on these findings, it was speculated that the formation of AGEs in erythrocyte peripheral-membrane proteins is related to the functional abnormalities of erythrocytes, including reduced deformability and lowered membrane fluidity, frequently observed in diabetic patients.

Immunochemical detection of advanced glycation end products in renal cortex from STZ-induced diabetic rat.

To reassess the accumulation of advanced glycation end products in diabetic renal cortex, we used a newly developed enzyme-linked immunosorbent assay to measure AGEs in renal cortex from STZ-induced diabetic and age-matched control rats. Kidneys and aortas were obtained from rats after 5 and 20 wk of STZ injection. At 5 wk of diabetes, the mean AGE content in collagenase-digested materials of renal cortex was > 16-fold higher in diabetic animals compared with controls (1044.4 +/- 151.8 vs. 64.3 +/- 5.7 arbitrary units, P < 0.01). At 20 wk of diabetes, it was > 45-fold higher in diabetic compared with control animals (3841.0 +/- 1077.3 vs. 83.8 +/- 12.8 AUs, P < 0.01). These increases were surprisingly large compared with the < 1.5-fold increase in the fluorescence levels both after 5 and 20 wk of diabetes. In control animals, neither the AGE content nor the fluorescence level increased during this period. Moreover, at 20 wk of diabetes, the AGE content was 39-fold higher in renal cortex compared with aorta. This study provided the first immunochemical evidence that collagenase-digested materials of renal cortex, as well as aorta, contained AGE products and that these products were present in much higher levels in diabetic animals than in control animals. With duration of diabetes, the AGE contents increased significantly both in renal cortex and aorta. The excessive accumulation of AGEs was most apparent in the diabetic kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient preparation of monospecific anti-canine C-reactive protein serum and purification of canine C-reactive protein by affinity chromatography.

The canine C-reactive protein (CRP) fraction isolated from canine acute-phase serum on a phosphorylcholine-Sepharose 4B column was further subjected to Sephacryl S-300 gel filtration. A canine CRP fraction not containing IgM was then obtained. The antisera, obtained after several immunizations with this canine CRP fraction, contained nonspecific antibodies that reacted with albumin, transferrin and IgG in addition to CRP. This antiserum could be easily changed to monospecific canine CRP serum, when it was subjected to absorption for only 15 min using glutaraldehyde-insolubilized normal canine serum protein containing 3.5 micrograms ml-1 of CRP. Pure canine CRP was isolated with a recovery rate of 95% from canine acute-phase serum by affinity chromatography using specific anti-canine CRP antibody.

Immunochemical detection of advanced glycation end products in lens crystallins from streptozocin-induced diabetic rat.

To reassess the significance of AGEs in cataract formation in diabetic animals, we measured amounts of AGEs in lens crystallins from STZ-induced diabetic animals with a newly developed ELISA. Lenses were removed at 5 and 20 wk after STZ injection. In 20-wk diabetic rats, all lenses were cataractous but not in control rats. In 20-wk diabetic compared with control rats, significant increases were observed in AGEs (172.3 +/- 18.3 vs. 14.3 +/- 1.7 AU, P < 0.01) and fluorescence (2.04 +/- 0.22 vs. 1.27 +/- 0.10 AU, P < 0.05). The amounts of AGEs in lens crystallins, measured by the ELISA, were > 12-fold higher in diabetic rats. In agreement with earlier studies, we found that fluorescence in lens crystallins increased by 61% in diabetic rats. In 5-wk diabetic rats, all lenses were noncataractous. In 5-wk diabetic compared with control rats, significant increases were observed in AGEs (84.1 +/- 7.7 vs. 9.4 +/- 1.5 AU, P < 0.01) and fluorescence (1.45 +/- 0.06 vs. 1.05 +/- 0.06 AU, P < 0.01). Analysis of the AGE content by ELISA showed that accumulation of AGEs in diabetic lens crystallins does markedly occur with time, and a large amount of AGEs exists in the diabetic (cataractous) lens crystallins. The disproportionate elevation of AGEs, measured by the ELISA, compared with fluorescence suggests that the actual levels of AGEs in cataractous lens crystallins from diabetic animals are higher than previously anticipated, and nonfluorescent AGEs may exist in diabetic lens crystallins.(ABSTRACT TRUNCATED AT 250 WORDS)

[Urinary trypsin inhibitor as an acute phase reactant].

Early in this century, trypsin inhibiting activity has already been recognized in patients with acute infection or renal disease. In addition to these, conditions such as coronary thrombosis, surgical operation, artificial fever by heat-killed bacilli, malignancy, leukemia, later stage of normal pregnancy, etc. have been known to cause the elevated excretion of UTI in urine. Typically, maximal excretion of UTI has been observed within one or two days after the onset. It appears that recent studies have overcome the complexity of UTI molecule. Automated measurement of urinary trypsin inhibitor (UTI) in urine sample was carried out by either enzymic or immunologic method. UTI as well as erythrocyte sedimentation rate and C-reactive protein enables us to monitor acute phase response, being confirmed in cases of abdominal surgery.

Characterization of antibodies to advanced glycosylation end products on protein.

Antibodies directed against advanced glycosylation end products (AGEs) formed during a Maillard reaction have been generated and characterized. Since protein-bound AGEs recognized by the antibodies were labile to acid hydrolysis, the antibodies were further characterized by using the AGE-alpha-acetyl-L-lysine methyl ester (AGE-ALME) with a brown and fluorescent property as well as the AGE-proteins. The antibodies reacted with fluorescent compounds, rather than brown pigment compounds, in the AGE-ALME. The fluorescent compounds in the AGE-ALME were separated into four fluorescent compounds by reversed-phase thin layer chromatography (TLC). Of the fluorescent compounds tested, compound 3 (Rf = 0.63), as designated on a TLC plate, showed the highest affinity for the antibodies. In addition, the antibody recognition to the cross-linked oligomers with fluorescence in the AGE-protein was investigated by using bovine pancreatic ribonuclease A (RNase), which is known as a model protein for studying AGE-induced cross-linking. Fluorescence in the AGE-RNase existed in both of the oligomers and the monomer. The cross-linked oligomers exhibited higher affinity to the antibodies than did the monomer, which has a similar degree of fluorescent intensity. These results indicate that our antibodies against cross-linked protein-bound AGEs may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.

Automated measurement of trypsin inhibitor in urine with a centrifugal analyzer: comparison with other acute phase reactants.

Automated measurement of trypsin inhibitor in urine was performed with good precision using the COBAS FARA. Elevated levels of both trypsin inhibitor in urine and acute phase proteins in serum were shown in most cases of major abdominal surgery. We suggest that the automated assay of urinary trypsin inhibitor might be useful for the clinical diagnosis of acute phase response.

New ligands for human C-reactive protein: calcium-dependent binding with epsilon-aminocaproic acid-agarose and calcium-independent binding with omega-aminohexyl-agarose.

Human C-reactive protein (CRP) is an acute phase protein which increases in concentration in response to inflammation. CRP has been known to bind with phosphorylcholine in a calcium-dependent manner. In this study, CRP is found to bind to affinity chromatography of negatively-charged epsilon-aminocaproic acid-agarose when calcium ions are present, and to the affinity chromatography of positively-charged omega-aminohexyl-agarose if a normal ionic strength of NaCl exists but calcium ion is not present.

Production and characterization of antibodies to advanced glycation products on proteins.

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.