Kunio Yonemasu

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.

Seasonal distribution of adenoviruses, enteroviruses and reoviruses in urban river water

In the 63‐month period from January 1988 to March 1993, monthly levels of adenoviruses, enteroviruses (coxsackie B, polio, echo) and reoviruses in the urban river water in Nara Prefecture, Japan were in the range 0‐25, 0‐190 and 0‐325, plaque forming units per liter (PFU/liter), and the average levels were 2.4, 40.6 and 56.2 PFU/liter, respectively. The peak reovirus level was found in winter during the cold weather months (Nov. to Mar.). The peak enterovirus level was found in summer (May to Sept.) but continued to be found in autumn‐winter (Oct. to Jan.) from 1991 to 1993. The levels of adenoviruses were low throughout all 5 years, as compared to those of reoviruses and enteroviruses. Polioviruses were isolated following the administration of vaccine. Although a changing pattern of serotype prevalence was seen with the coxsackie B viruses and echoviruses from 1988 to 1993, this is not so for polioviruses, which remained almost unchanged for the five‐year period. Adenoviruses were isolated throughout all five years, though in small numbers. Reoviruses were isolated most frequently throughout five years.

A Novel Polymorphism in the Promoter Region for the Human Osteocalcin Gene: The Possibility of a Correlation with Bone Mineral Density in Postmenopausal Japanese Women

We present a polymorphism of the human osteocalcin gene (also known as BGP, for bone Gla protein) due to a 1 base pair (bp) substitution from cytosine to thymine at position 298 nucleotides (nt), which is at position 198 nt upstream from the BGP exon 1. This mutation was detected by single‐strand conformation polymorphism analysis after polymerase chain reaction for the osteocalcin gene fragment (326 bp) and sequencing analysis. The cytosine/thymine polymorphism can be defined by restriction fragment length polymorphism analysis using a modified primer pair and the restriction endonuclease HindIII. The osteocalcin genotype was determined in 160 postmenopausal Japanese women (age 48–80 years). Osteocalcin alleles were designated according to the absence (H) or presence (h) of the HindIII restriction site. There were 12 HH, 49 Hh, and 99 hh individuals, and the allele frequencies were 22.8% for H and 77.2% for h. To determine if genetic variation influences bone mineral density (BMD) and thus can be a determinant of susceptibility to osteoporosis in older women, we examined the association of BMD with the osteocalcin genotypes found in the present study. The subjects with genotype HH had the smallest BMD and those with hh had the greatest BMD among subjects, but these differences did not reach statistical significance. The HindIII genotype showed a significant effect on the prevalence of osteopenia in the subjects, that is, women with genotype HH had a 5.74 times greater risk for osteopenia (p < 0.05) and those with genotype Hh had a 1.59 times greater risk than women with genotype hh. We identified the osteocalcin gene polymorphism, detected with the HindIII genotype, which was suggested to influence bone density and is a possible genetic marker for bone metabolism.

Greater Trunk Muscle Torque Reduces Postmenopausal Bone Loss at the Spine Independently of Age, Body Size, and Vitamin D Receptor Genotype in Japanese Women

Bone mineral density (BMD) is affected by muscle strength. Recently, vitamin D receptor (VDR) genotype was reported to affect muscle strength as well as BMD in Caucasian women. The aim of this study was to evaluate independent effects of muscle strength of the trunk on BMD at the spine and its change over time in Japanese women. We followed 119 healthy postmenopausal women for 4 years and determined the change in BMD at the spine by dual energy X-ray absorptiometry. Isometric peak torque and isokinetic concentric and eccentric peak torque of the trunk flexors and extensors were measured. The VDR genotype was determined by the PCR-RFLP method based on Apa I and Taq I endonuclease digestions defining the absence/existence of the restriction sites as A/a and T/t, respectively. The subjects were 60.1 ± 6.6 years old, had 0.808 ± 0.159 g/cm2 of BMD at baseline. The mean annual change in BMD (DBMD) was ?5.6 ± 10.4 mg/cm2 during the follow-up period. The VDR genotype, defined by Taq I enzyme, significantly related to BMD at baseline and DBMD showing that the subjects with genotype TT had the lowest BMD at baseline and lost bone most rapidly. However, its effect on muscle strength was not significant. All the trunk muscle strength indices showed significant positive effects on DBMD, that is, the effects in increasing the gain and reducing the loss of BMD, after controlling for the effects of age, body size and the VDR genotype. The eccentric trunk extensor torque had a significant positive effect on DBMD in a dose-dependent manner. The effect of this torque was the greatest among all the muscle indices. The net effect of the trunk extensor torque on DBMD was greater than that of the VDR genotype. The trunk muscle strength was suggested to affect BMD change independently of age, body size, and the VDR genotype. Exercise programs to increase the strength of the trunk muscles would be beneficial for the prevention of osteoporosis regardless of the VDR genotypes.

Impact of metabolic syndrome on brachial- ankle pulse wave velocity in Japanese

The aim of this study was to determine the effect of metabolic syndrome on brachial-ankle pulse wave velocity (baPWV) by using the new guidelines for diagnosis of this syndrome in Japan. We examined 525 men and women without a history of cardiovascular disease or cancer, and an ankle-brachial index<0.9. The baPWV was measured using a device (Form PWV/ABI) that simultaneously monitored bilateral brachial and ankle pressure wave forms. Metabolic syndrome was defined as a waist circumference ≥85 (90) cm in men (women) and two or more of the following risk factors: hypertension, dyslipidemia, and glucose intolerance diagnosed by a 75 g oral glucose tolerance test. The baPWV showed a significant linear relationship with waist circumference, waist-to-hip ratio, body fat, systolic and diastolic blood pressure, triglycerides, fasting glucose, 2-h-postload glucose, fasting insulin, and glycosylated hemoglobin-A1c, after adjusting for sex and age. These factors were also strongly related to fasting insulin levels. When subjects were classified into six groups based on waist circumference and the number of risk factors for metabolic syndrome (0, 1, and ≥2), we found that more risk factors clearly increased the odds ratios for an elevated baPWV in those subjects in the highest quartile of the baPWV distribution in multivariate logistic models. An increase in odds ratio was observed despite a normal waist circumference and may well have been due to increased fasting insulin and blood pressure levels. An increase in the number of risk factors for metabolic syndrome was highly correlated with an increased baPWV, probably due to insulin resistance.

Stimulation of Locomotion of Peripheral Blood Monocytes by Human Plasma Fibronectin

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind‐well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nm, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nm. The monocyte migration‐inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nm–100 μm). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.

Pregnancy zone protein inhibits production of interleukin-2 but does not affect interleukin-2 receptor expression on T cell activation

The effect of pregnancy zone protein (PZP), which exhibits increased levels in the blood during pregnancy, on T cells was examined. PZP was found to suppress DNA synthesis following stimulation with phytohemagglutinin (PHA), concanavalin A (ConA) or CD3 antigen or in the mixed lymphocyte reaction (MLR). This effect of PZP was mediated by a reduction in interleukin-2 (IL-2) production, and was abolished by exogenous recombinant IL-2 administration. PZP did not affect the proliferation of T cells following stimulation with the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA). These results suggest that PZP acts on the T cell surface and reduces IL-2 production, but not IL-2R expression, and does not directly affect Ca2+ influx or protein kinase C.

Opsonic effect of fibronectin on staphylococcal phagocytosis by human polymorphonuclear leukocytes: its relative inefficiency in post-phagocytic metabolic activities and in intracellular killing.

The binding of 125I‐labeled human plasma fibronectin (FN) to two strains of live Staphylococcus aureus (S. aureus) (a coagulase‐positive Cowan I and a coagulase‐negative Newman D2C) and the opsonic effect of FN on phagocytosis of these bacteria by human polymorphonuclear leukocytes (PMN) have been studied. 125I‐FN bound to a similar extent in both staphylococcal strains. The 125I‐FN‐binding was significantly inhibited by human fibrinogen as well as unlabeled FN. The FN‐binding was also reduced markedly by trypsinization of these bacteria, but the extent of its decrease did not correlate with their tryptic susceptibility of protein A and clumping factor. FN enhanced the uptake of these bacteria by PMN. However, its binding had no effect on superoxide anion (O2−) generation. The FN‐binding definitely stimulated staphylococcal ingestion and intracellular killing by PMN, but the extent of such promotion was dissimilar between these two strains of bacteria. These results suggest that post‐phagocytic metabolic activities as well as intracellular killing of these Staphylococci may also be greatly influenced by FN‐unrelated factors as are other bacteria having no FN‐receptors.

Role of metallothionein isoforms in bone formation processes in rat marrow mesenchymal stem cells in culture.

Temporal changes in mRNAs for metallothionein (MT) isoforms in subcultures of rat marrow mesenchymal stem cells (MSCs) after treatment with dexamethasone were investigated. Both MT-1 and MT-2 mRNA expression in the cultured MSCs with dexamethasone showed maximum levels at d 1, whereas ALP and osteocalcin mRNAs peaked at d 12. MT-3 mRNA was not detected in the cultured MSCs at any time. The expression level of MT-2 mRNA at d 1 was 9.4-fold higher than that of MT-1 mRNA. Finally, osteoblast differentiation and mineralization of MSCs at d 14 was inhibited by the addition of a common antisense oligonucleotide for both MT-1 and MT-2 in the culture medium during the first 4 d. The results suggest that the large amounts of MT-2 are produced in the early stage of subculture of MSCs, and this might regulate their differentiation.

In vitro effects of Habu snake venom on cultured mesangial cells.

Background: Habu snake venom (HSV)-induced glomerulonephritis is a unique model showing a progressive course of mesangial proliferation. To elucidate the in vitro effects of HSV, we examined whether HSV itself could have direct effects on the cultured mesangial cells, such as cell proliferation and activation of chemokine gene expression. Methods: The incorporation of 5-[125I]iodo-2’-deoxyuridine was measured with a γ-counter, and gene expressions of growth factors, chemokines and cytokines were evaluated by a real time quantitative PCR. Results: We demonstrated that excessive or continuous HSV stimulation decreased a mesangial cell viability. However, adequate and temporary HSV stimulation induced proliferation of mesangial cells in vitro along with a significant elevation of monocyte chemoattractant protein-1 (MCP-1) mRNA levels. In addition to these in vitro results, we showed that MCP-1 mRNA levels increased in renal cortices of glomerulonephritis induced by HSV. Immunohistochemistry also showed a positive staining for MCP-1 in the marginal area of glomerulus with mesangiolysis. Conclusions: These data suggest that HSV itself may elicit direct biological effects on mesangial cells which may participate in pathophysiology of glomerulonephritis induced by HSV.