Relationship between immunological rejection and matrix GLA protein in cryopreserved vascular allografts.

INTRODUCTION: Cryopreserved tissue allografts used for cardiovascular diseases become calcified as a late complication after transplantation, probably caused by immunological rejection. Recent attention has been focused on the inhibitory effect of matrix Gla protein (MGP) on ectopic vascular calcification, but the behavior of MGP in cryopreserved allografts is uncertain. In this study we examined the relationship between immunological rejection and MGP in cryopreserved rat aortic grafts after transplantation. METHODS: Cryopreserved rat aortae were isografted or allografted intraperitoneally. Fresh isografts were also tested. The grafts were retrieved 9 days after transplantation and the intragraft MGP mRNA was measured by a real-time quantitative PCR method. The effect of daily administration of FK506 on MGP mRNA levels in cryopreserved isografts and allografts after transplantation was also evaluated. RESULTS: There was no significant difference in intragraft MGP mRNA levels between fresh and cryopreserved isografts 9 days after transplantation. MGP expression levels in cryopreserved allografts were significantly lower as compared to those in cryopreserved isografts (P < .01). Daily administration of FK506 enhanced intragraft MGP mRNA (ninefold) in cryopreserved allografts (P < .01), but not in cryopreserved isografts. CONCLUSIONS: Immunological rejection is likely to inhibit MGP expression in cryopreserved vascular allografts, resulting in late-onset calcification.

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 (delta BMD) 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 delta BMD 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 delta BMD, 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 delta BMD 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 delta BMD 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.

Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis.

In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR METHOD: Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0.005), while AM mRNA levels were decreased (P < 0.05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.

Recombinant human bone morphogenetic protein-2 potentiates the in vivo osteogenic ability of marrow/hydroxyapatite composites.

A composite of marrow mesenchymal stem cells (MSCs) and porous hydroxyapatite (HA) has bone-forming capability. To promote the capability, we added recombinant human bone morphogenetic protein-2 (BMP) to the composite. The bone formation was assessed by rat subcutaneous implantation of 4 different kinds of implants, i.e., HA alone, BMP/HA composites, MSCs/HA composites, and the composites containing BMP (MSCs/BMP/HA). Both HA and the BMP/HA composites did not show bone formation at any time after implantation. The MSCs/HA composites showed moderate bone formation at 4 weeks and extensive bone formation at 8 weeks. The MSCs/BMP/HA composites showed obvious bone formation together with active osteoblasts at 2 weeks and more bone formation at 4 and 8 weeks. The MSCs/BMP/HA composites demonstrated high alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of MSCs, porous HA, and BMP synergistically enhances osteogenic potential and provides a rational basis for their clinical application in bone reconstruction surgery.

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.

Trends in oral cancer mortality in Japan: 1950-1993.

OBJECTIVES: To investigate trends in mortality rates of oral cancer patients in Japan between 1950 and 1993 by sex, age and cancer site, and compare the results with previous studies to determine whether there are any common characteristics of oral cancer patterns between Japan and European countries. MATERIAL AND METHODS: The mortality data obtained from the Japanese Vital Statistics were analyzed using the 5-year moving average method, and the mortality rates were adjusted to the 1990 world population by age and sex. Age-specific mortality rates were analyzed by birth cohort. RESULTS: The age-standardized mortality rates among the males increased from 1.14 per 100,000 person--years in 1952 to 1.84 in 1991, whereas the corresponding rate among females changed little over the same period. Cancer of the tongue was the most common cause of death in Japan among the five studied oral regions: lip, tongue, floor of the mouth, major salivary glands and oropharynx, males aged under 54 born in 1920 or later were found to have an increased risk of such disease. CONCLUSION: Further epidemiological investigations are necessary to clarify the etiology of oral cancer in Japan.

A historical cohort mortality study of workers exposed to asbestos in a refitting shipyard.

To investigate the risks of developing asbestos-related diseases we conducted a historical cohort mortality study on 249 ship repair workers (90 laggers and 159 boiler repairers) in a single U.S. Navy shipyard in Japan. We successfully identified the vital status of 87 (96.7%) laggers and 150 (94.3%) boiler repairers, and, of these, 49 (56.3%) and 65 (43.3%) died, respectively, during the follow-up period from 1947 till the end of 1996. Our in-person interviews with some of the subjects clarified that asbestos exposure was considered to be substantially high in the 1950-60s, decreased thereafter gradually but remained till 1979 in the shipyard. The laggers, who had handled asbestos materials directly, showed a significantly elevated SMR of 2.75 (95% C.I.: 1.08-6.48) for lung cancer. The risk developing the disease was greater in the laggers after a 20-year latency (SMR = 3.42). Pancreatic cancer yielded a greater SMR than unity (7.78, 90% C.I.: 2.07-25.19) in a longer working years group. Four laggers died from asbestosis. The boiler repairers, who had many chances for secondary exposure to asbestos and a few for direct exposure, showed no elevation of the SMR of lung cancer overall, but there was a borderline statistically significant SMR of 2.41 (90% C.I.: 1.05-5.45) in a longer working years group. One boiler repairer died from mesothelioma and four from asbestosis.

Time trends in the mortality rates for tobacco- and alcohol-related cancers within the oral cavity and pharynx in Japan, 1950-94.

Mortality data of oral cancer over 40 years in Japan were analyzed to investigate time trends of the disease site-specifically and discuss the relation between these trends and the changing patterns of consumption of tobacco and alcohol beverages. Mortality rates were adjusted to the world standard population. In the males, overall oral cancer (ICD-9: 141-149) mortality rates have increased consistently from the lowest value of 1.25 (per 100,000 per year) in 1956 to 2.40 in 1992. The rates for females were constantly lower than those for males, and formed a modest peak of 0.96 in 1979. Regarding site-specific mortality rates, tongue cancer (141) presented a decreasing trend, while oro/hypopharyngeal (146, 148) and mouth (143-145) cancers showed increasing patterns, particularly in males. When the changing patterns of male truncated rates for ages 35-64 were compared with those of the annual consumption of cigarette and alcohol per capita, the time trend of oro/hypopharyngeal cancer mortality was analogous to cigarette consumption rather than to alcohol consumption, mouth cancer vice versa, and tongue cancer was not related to tobacco or alcohol consumption. The present findings suggest that tobacco and alcohol have different site-specific effects on the development of cancers within the oral cavity and pharynx.

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.

Development of a simple and rapid elisa of urinary cotinine for epidemiological application.

Highly stereospecific polyclonal antibodies (anti-CN) to cotinine (CN), a major metabolite of nicotine, were prepared from rabbit antisera to CN-linked keyhole limpet hemocyanin (KLH) by removing the antibodies to KLH and to its binding regions of CN. This was achieved by using immunoadsorbents consisting of insolubilized KLH onto CNBr-activated-Sepharose 4B. A new simple and rapid enzyme-linked immunosorbent assay (ELISA) of urinary CN was developed using the anti-CN. A brief outline of the method is as follows: CN-bovine thyroglobulin complex is coated onto wells of microtiter plates (1 ng/well), and then aliquots of urine samples or standard CN solutions were added followed by appropriate dilution of the anti-CN. The bound anti-CN antibodies are quantified spectrophotometrically with horseradish peroxidase-labelled anti-rabbit IgG and 2,2'-azino-di (3-ethylbenz- thiazoline)-6-sulfonic acid. Measurement of CN concentration in urine samples can be read off on a calibration curve drawn by using standard CN solutions. The standard curve ranged from 1 ng to 4 µ g /ml with an estimated lower limit of sensitivity of 7-8 ng/ml, resulting in within/between-assay CV (coefficient of variation) of lower than 10%. The method allowed one to assay more than 40 samples in duplicate by using just one plate, and is thus easily applicable to epidemiological investigations into exposure status to tobacco smoke.

Active and passive exposure status to tobacco smoke of department store employees measured by cotinine ELISA.

Quantitation of urinary cotinine, a major metabolite of nicotine, by an enzyme-linked immunosorbent assay (ELISA), was performed in parallel with questionnaires containing items on smoking status, such as active and/or passive smokers, the number of cigarettes smoked, and the presence or absence of active smokers in the surroundings in a department store (517 employees). The cotinine values corrected by creatinine (cotinine-creatinine ratios, CCRs) approximately conformed to the extent of self-recognition of their exposure status to tobacco-smoke, and were low in the order of active smokers, passive smokers and non-smokers who felt they were not exposed to tobacco-smoke. Occupational differences of the CCRs were not found in the employees.In the active smokers, the CCRs were increasing according to the number of cigarettes per day they smoked, and the values were nearly proportional to nicotine contents of cigarette in the moderate smokers who smoked 11-20 cigarettes per day. The CCRs of males were higher than those of females in the active smokers, which also agreed well with the numbers of cigarettes they smoked per day. In the passive smokers, the CCRs were remarkably and significantly higher in subjects who felt they were exposed to tobacco-smoke both in their workplaces and homes.Urinary CCRs measured by ELISA are thus found to be a reliable and excellent objective indicator of both active and passive exposure-status to tobacco-smoke.

Effects of transfer of hybridomas producing various isotypes of immunoglobulins on the C1q metabolism in mice.

In order to elucidate possible effects of immunoglobulin on C1q metabolism at the anabolic steps, serum C1q levels and C1q mRNA of peritoneal exudate cells (PEC) and spleen cells were measured in female BALB/c mice implanted intraperitoneally with complement-(C)-fixing IgG2b- or non-C-fixing class IgG3-producing hybridomas and/or with immunoglobulin-non-productive myeloma cells (p3x63-Ag.8.653)(myeloma 653)(2 x 10(6)/0.2 ml) or without any treatment as controls. In the IgG2b-hybridoma-treated mice, the serum C1q levels and C1q mRNA in PEC increased conspicuously as compared with those in the controls, but C1q mRNA in spleen cells was almost equal to that in the control mice. On the other hand, in the IgG3-hybridoma-treated mice, the serum C1q levels decreased significantly, but the extent of such decrease and the level of C1q mRNA in their PEC were almost equivalent to those in the myeloma 653-implanted mice. The serum C1q levels and C1q mRNA in PEC fluctuated similarly in mice injected intraperitoneally with highly purified IgG2b and/or IgG3 preparations. These results suggest some anabolic interaction, as well as catabolic interaction, between the C-fixing class of immunoglobulin and C1q.

Collagen cross-reactive antigen of Sarcocystis cruzi.

Collagen cross-reactive antigenic substance(s) in Sarcocystis cruzi cysts were examined with immunologic techniques using anti-bovine collagen type-specific, but non-species-specific, antibodies. By immunoperoxidase test, anti-bovine collagen type-specific, but non-species-specific, antibodies. By immunoperoxidase test, anti-bovine type IV collagen antibody showed higher reactivity to the cysts than other antibodies tested. Cyst wall rupture was induced by collagenase treatment and digestion was inhibited with EDTA supplementation. With immunoblotting analysis, one band of the cyst extract, which exhibited specific reactivity to anti-bovine type IV collagen antibody, was detected. The band had a molecular weight of approximately 66 kDa. These results suggest that sarcocysts of S. cruzi may be comprised of bovine collagen type IV cross-reactive antigenic substances.

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.

Antiviral activity of trichothecene mycotoxins (deoxynivalenol, fusarenon-X, and nivalenol) against herpes simplex virus types 1 and 2.

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC50) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.

Intraglomerular expressions of IL-1 alpha and platelet-derived growth factor (PDGF-B) mRNA in experimental immune complex-mediated glomerulonephritis.

Both PDGF and IL-1 play important roles as autocrine growth factors for cultured mesangial cells, and may be closely associated with the progression of glomerulonephritis. In this study we investigated intraglomerular expressions of PDGF-B and IL-1 alpha mRNA in mice with bovine serum albumin (BSA) nephritis, a model of immune complex-mediated glomerulonephritis, using the reverse transcription-polymerase chain reaction (RT-PCR) method. We also quantified intraglomerular PDGF-B mRNA by the competitive PCR and studied the correlation between the level of intraglomerular PDGF-B mRNA expression and the degree of observed glomerular injury. While expression of neither PDGF-B nor IL-1 alpha mRNA was detected in glomeruli from control mice, both were strongly expressed in glomeruli from mice with BSA nephritis. IL-1 alpha mRNA in glomeruli showed low accumulation in mice with mild glomerular injury, and was increased in mice with moderate glomerular injury. In contrast, high intraglomerular expression of PDGF-B mRNA occurred in all mice with mild glomerular injury and continued throughout the course of the disease. We observed no correlation between the level of PDGF-B mRNA expression and the histologic grade of renal damage. These results suggest that PDGF and IL-1 have different growth properties, and PDGF might play a role as a competence factor rather than a progression factor in the pathogenesis of immune complex-mediated glomerulonephritis.