Impact of salivary contamination during placement of implants with simultaneous bony augmentation in iliac bone in sheep.

Our aim was to investigate the possible impact of contamination with saliva on osseointegration during placement of implants with simultaneous bone augmentation. Six hemispheric shape bone defects (8mm in diameter×4mm deep) were prepared in each iliac bone of six sheep. A dental implant (2.9mm in diameter×10mm long) was placed in the centre of each defect, and then pairs of defects were filled with one of the following bone augmentation materials: autogenous bone, autogenous bone plus bovine bone, or resorbable biphasic ceramic bone substitute. One site in each augmentation group was impregnated with saliva (contaminated group), while the other was not (non-contaminated group). Bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) within implant threads were measured after a healing period of five weeks, both in respect of the implant inserted in the augmented bone and in that inserted in the residual bone. Overall results showed that there was a significant difference between the contaminated and non-contaminated group for BIC in the augmented implant (p=0.028), while there were no significant differences in the implant in residual bone (p=0.722). For BAFO, there were no significant differences between the contaminated and non-contaminated groups among the different augmentation materials. The results showed that contamination with saliva during placement of an implant with simultaneous bone augmentation had a serious deleterious effect on osseointegration of the aspect of the implant within the augmented defect. Contamination with saliva during placement of an implant with simultaneous bone augmentation should therefore be avoided.

Impact of surface contamination of implants with saliva during placement in augmented bone defects in sheep calvaria.

Our aim was to try and find out whether contamination with saliva during insertion of dental implants affects osseointegration in bone that has been augmented with different grafts. Six bony defects were created in each of the calvaria of six sheep, and then augmented with three different materials (autogenous bone, bovine bone, and resorbable biphasic ceramic bone substitute) After five weeks of healing, three implants contaminated with saliva (contaminated group) and three not contaminated (uncontaminated group) were placed in the centre of the augmented areas. For histomorphometric analysis, bone implant contact, bone area fraction occupancy, bone and material area, and bony area were measured after a healing period of five weeks. There was a significant difference between the contaminated and uncontaminated groups (p=0.036) for bone implant contact only in the augmented areas, but there were no significant differences in bone area fraction occupancy, bone and material area, and bony area. We conclude that contamination with saliva during placement of dental implants can significantly compromise bone implant contact in augmented areas, but had no significant effect on the formation of bone in areas more distant from the surface of the implant. We suggest that salivary contamination should be avoided during placement of dental implants in augmented areas.

Simvastatin enhances bone formation around titanium implants in rat tibiae.

Statins are cholesterol-lowering drugs that have been reported to promote bone formation. The purpose of this study was to investigate the effect of simvastatin on the enhancement of bone formation around titanium implants. Thirty-week-old female rats received pure titanium implants in both tibiae. The animals were intra-peritoneally administered 0, 0.125, 1, 5 or 10 mg kg(-1) of simvastatin daily. After 30 days, the animals were sacrificed, and specimens were prepared. The bone contact ratio of the implant, bone density in the medullary canal and percentage of cortical bone were obtained. Markers for bone turnover were also measured using sera collected at the time of euthanasia. In the medullary canal, a scanty amount of bone was observed in the 0, 0.125 and 1 mg kg(-1) groups. In contrast, in both the 5 and 10 mg kg(-1) groups, thicker bone trabeculae were abundant. Histometric observations showed that the bone contact ratio and the bone density of both groups were significantly greater than those of the other groups (anova, P < 0.01). However, no significant difference in the percentage of cortical bone was found between groups. Serum chemistry showed that statin increased bone formation markers and decreased bone resorption markers. In conclusion, although the dose equivalent to that used in human patients with hypercholesterolemia was not effective, a simvastatin dose of 5 mg kg(-1) or higher increased medullary bone formation around the titanium. In contrast, no effect of simvastatin on pre-existing cortical bone was indicated.

MEFV mutation analysis of familial Mediterranean fever in Japan.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever with serosal inflammation. FMF gene (MEFV) mutations have been identified primarily in patients from Mediterranean populations. Although several clinical cases have been reported in Japan, there have been few reports to date on mutation analysis. We studied FMF patients and their relatives to examine the clinical and genetic features of this disease in the Japanese population. METHODS: Twelve Japanese FMF patients who met the Tel Hashomer criteria and a total of 17 relatives from 5 of 10 families underwent molecular genetic studies to detect MEFV mutations. The characteristics of these Japanese FMF patients and geno-phenotypical correlations were examined. RESULTS: Almost all of our patients had been suffering for a long time from fever of unknown origin and one patient also had systemic amyloidosis. In our 12 FMF patients, we detected the substitutions E84K, L110P, E148Q, R761H and M694I. We also newly diagnosed 2 relatives as having FMF based on clinical symptoms and the existence of FMF mutations. One patient was homozygous for E148Q, the patient with systemic amyloidosis was a homozygote for M694I and 4 patients from 3 families were compound heterozygotes for E148Q and M694I. Three patients in one family were compound heterozygotes for E148Q, L110P and M694I. There were 3 patients who were heterozygous for E84K, L110P-E148Q or M694I and had no other nucleotide changes in the exons of MEFV. On the other hand, 2 relatives who had never experienced symptoms of FMF were homozygous for L110P-E148Q as well as compound heterozygous for E148Q/E148Q-R761H. E148Q and M694I were the most frequently detected substitutions in our study. CONCLUSIONS: MEFV mutations occur in Japanese FMF patients though FMF is rare in Japan. The identification of MEFV mutations could be a reliable diagnostic test for FMF. The results of genetic analyses on 14 Japanese FMF patients in this study revealed that E148Q and M694I are frequent alleles.

Cellular localization of placenta-specific human endogenous retrovirus (HERV) transcripts and their possible implication in pregnancy-induced hypertension.

We previously investigated tissue specificity and temporal changes in expression of five human endogenous retroviruses (HERVs), including syncytin/ERVWE1 and syncytin 2. In the current study, we examined the cellular localization and quantified the transcripts of five HERVs, syncytin, syncytin 2, HERV-H7/F(XA34), HERV-Fb1, and HERV-HML6-c14, in order to elucidate their physiological and etiological roles in the placenta and in pregnancy-induced hypertension (PIH), respectively. In situ hybridization revealed trophoblast-specific transcription of all five HERVs. Consistent with a previous immunohistochemical analysis, syncytin 2 transcripts were detected only in cytotrophoblasts. All the HERVs except HERV-HML6-c14 (HML6-c14) were predominantly localized in the cytoplasm of syncytiotrophoblasts and/or cytotrophoblasts. Quantitative analysis showed that transcriptional levels of these four HERVs were lower in placentas obtained from pregnant women with PIH (n=22) than in those from normotensive pregnant women (n=87) and that the differences were statistically significant (p=0.001, 0.01, <0.001, and 0.04 for syncytin, syncytin 2, HERV-H7/F(XA34), and HERV-Fb1, respectively). In contrast to the other HERVs, HML6-c14 transcripts localized to the nucleus and the average transcriptional level of HML6-c14 was higher in PIH placentas than in control placentas from normotensive pregnant women, although the differences did not reach significance (p=0.19). These results suggest that placenta-specific HERVs may have some function in the human placenta and that their reduced expression in PIH placentas is not merely a secondary effect derived from the pathology of PIH placentas.

Augmented expression of secondary lymphoid tissue chemokine and EBI1 ligand chemokine in Crohn's disease.

BACKGROUND: A dominant T helper type 1 (Th1) immune response is thought to be involved in Crohn's disease (CD). SLC/CCL21 and ELC/CCL19, chemokines that regulate T cell homing and promote recirculating T and dendritic cell (DC) interactions, help control antigen specific T cell responses. AIMS: To investigate the Th1 response and SLC and ELC in CD pathogenesis. METHODS: Surgically resected intestine and mesenteric lymph nodes (MLNs) from controls and patients with CD and ulcerative colitis (UC) were investigated. CD3, CD83, HECA452, VEGFR3, SLC, ELC, and CCR7 expression was studied immunohistochemically. CCR7 mRNA was quantified using real time RT-PCR. RESULTS: ELC was almost undetectable in intestinal samples. SLC was found sporadically in lymphoid follicles, lymphoid aggregate venules, and lymphatic vessels. In MLNs, SLC was highly expressed in high endothelial venules (HEVs), lymphatic vessels, and stromal DCs, predominantly in T cell areas. ELC was highly expressed in mature DCs. There were significantly more SLC positive HEVs and ELC positive mature DCs, important components of T cell areas, in CD. SLC, ELC, and CCR7 mRNA was significantly higher in CD MLNs compared with UC. CD MLNs had increased expression of SLC and ELC, mainly in HEVs, mature DCs, and lymphatic vessels, inducing T cell hyperplasia. CCR7 mRNA was increased in T cell areas. CONCLUSION: The dominant Th1 immune response is facilitated by interaction of SLC positive HEVs/lymphatic vessels, ELC positive mature DCs, and CCR7 positive T cells in hyperplastic T cell areas. In CD, memory T cells and mature DCs may home to MLN.

Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study.

The goal of the present study was to investigate inter-individual and age-dependent variation of global DNA methylation in human tissues. In this work, we examined 5-methyldeoxycytidine ((met)C) content by HPLC in human peripheral blood leukocytes obtained from 76 healthy individuals of ages varying from 4 to 94 years (yr), and 39 human placentas from various gestational stages. The HPLC analysis revealed a significant variation of (met)C across individuals and is consistent with the previous findings of age-dependent decrease of global methylation levels in human tissues. The age-dependent decrease of (met)C was relatively small, but statistically highly significant (p= 0.0002) in the aged group (65.9 +/- 8.9 [mean age +/- SD] yr; n = 22) in comparison to the young adult group (19.3 +/- 1.4 yr; n = 21). Males showed a subtle but statistically significant higher mean (met)C content than females. In contrast to the peripheral blood samples, DNA extracted from placentas exhibited gestational stage-dependent increase of methylation levels that appeared to inversely correlate with the expression levels of human endogenous retroviruses. These data may be helpful in further studies of DNA methylation, such as inheritance of epigenetic patterns, environment-induced changes, and involvement of epigenetic changes in disease.

Search for active endogenous retroviruses: identification and characterization of a HERV-E gene that is expressed in the pancreas and thyroid.

To elucidate possible physiological functions of human endogenous retroviruses (HERVs) and their role in the pathogenesis of human diseases, we have developed a strategy to identify transcriptionally active HERV genes. By this approach, we have identified and isolated an active HERV-E gene that was mapped to 17q11. Although the gene was predicted to produce no intact viral particles due to the presence of stop codons, long open reading frames were retained in each gag and pol region. Northern blot analyses revealed in the pancreas (and thyroid) two major transcripts, 3.3 and 4.1 kb in size, associated with 500- to 600-nucleotide-longer minor bands. Preferential expression in pancreas and thyroid gland tissues may suggest a role for this gene in physiological functions common to these tissues.

Transcriptionally active HERV-K genes: identification, isolation, and chromosomal mapping.

Preceding the isolation of transcriptionally active HERV-K genes, expression status was examined by RT-PCR and sequence analysis of mRNA from various tissues. In addition to the detection of IDDMK(1,2)22/HERV-K18 expression in peripheral leukocytes, three novel members of the family, which are expressed in multiple tissues, were identified. The novel HERV-K genes (HGMW-approved symbols ERVK4 and ERVK5) were isolated from a BAC library using oligonucleotide probes and assigned by RH mapping to chromosomal regions 3q21-q25.2, 3cen-q13, and 1q21-q23. Although their expression could not be confirmed in any normal tissues by Northern blot analysis, substantial promoter activity of their 5' LTRs was demonstrated in luciferase assays using teratocarcinoma cell lines. Thus, they seem to have the potential to be actively transcribed. The results, combined with those of the expression analysis by RT-PCR and subsequent sequencing of cloned products, also suggest that LTR sequences with subtle base changes might play a role in gene regulation, such as tissue specificity of HERV-K expression.

IgG subclasses of anti Saccharomyces cerevisiae antibody in inflammatory bowel disease.

Elevation of serum anti Saccharomyces cerevisiae antibody (ASCA) has been reported in patients with Crohn's disease. We analysed the subclasses of Immunoglobulin (Ig) G reaction in ASCA in sera from patients with inflammatory bowel disease, healthy controls, and patients with intestinal Behçet's disease. Serum samples were obtained from 29 patients with Crohn's disease, 30 patients with ulcerative colitis, 7 patients with intestinal Behçet's disease, and 12 healthy controls. Serum IgG subclasses IgG1, IgG2, IgG3, and IgG4 of ASCA were analysed using ELISA. IgG4 ASCA was significantly increased in patients with inflammatory bowel disease. In patients with intestinal Behçet's disease, IgG1, IgG3, and IgG4 ASCA were increased. Differential responses, in terms of subclasses in ASCA, were found in patients with inflammatory bowel disease and patients with intestinal Behçet's disease, which may represent different pathophysiologies of these intestinal inflammatory diseases.

Identification of nonsynonymous polymorphisms in the superantigen-coding region of IDDMK1,2 22 and a pilot study on the association between IDDMK1,2 22 and type 1 diabetes.

To investigate the possible involvement of IDDMK1,2 22/HERV-K18 in childhood type I diabetes mellitus, we identified two nonsynonymous A/G polymorphisms in the superantigen-coding region of IDDMK1,2 22 at the 290- and 461-nucleotide (nt) positions from the initial methionine codon and compared their frequencies in 74 Japanese patients with type 1 diabetes and in 54 nondiabetic controls. Although the G substitution was observed more frequently at either site in the patients than it was in the controls (7% vs. 4% at 290 nt, and 29% vs. 20% at 461 nt), the differences were not statistically significant. A weak significance of difference in the frequency of 461G was obtained only in an early-onset group of patients manifesting the disease at 5 years of age or less (n = 24) when compared with controls (38% vs. 20%; P = 0.03). However, in addition to the common absence of a particular allele among the expected four alleles, remarkable differences in allele frequencies were present between Japanese and European populations. This first trial investigating the association of IDDMK1,12 22 with type 1 diabetes presents intriguing suggestions for the role of this region in the etiology of autoimmune and infectious diseases.

A novel imprinted gene, KCNQ1DN, within the WT2 critical region of human chromosome 11p15.5 and its reduced expression in Wilms' tumors.

WT2 is defined by a maternal-specific loss of heterozygosity on human chromosome 11p15.5 in Wilms' and other embryonal tumors. Therefore, the imprinted genes in this region are candidates for involvement in Wilms' tumorigenesis. We now report a novel imprinted gene, KCNQ1DN (KCNQ1 downstream neighbor). This gene is located between p57(KIP2) and KvLQT1 (KCNQ1) of 11p15.5 within the WT2 critical region. KCNQ1DN is imprinted and expressed from the maternal allele. We examined the expression of KCNQ1DN in Wilms' tumors. Seven of eighteen (39%) samples showed no expression. In contrast, other maternal imprinted genes in this region, including p57(KIP2), IMPT1, and IPL exhibited almost normal expression in these samples, although some samples expressed IGF2 biallelically. These results suggest that KCNQ1DN existing far from the H19/IGF2 region may play some role in Wilms' tumorigenesis along with IGF2.

Are cerebrovascular factors involved in Alzheimer's disease?

Recent epidemiological studies have shown that vascular risk factors may be involved in Alzheimer's disease (AD) as well as dementia in general. To investigate the relation between a vascular disorder and AD pathology, current criteria are defective because most depend on exclusion of a cerebrovascular disorder. Epidemiological studies have indicated the possibilities that arteriosclerosis, abnormal blood pressure, diabetes mellitus and smoking may be related to the pathogenesis of AD. As for the mechanism that vascular disorders influence AD, it is presumed that amyloid deposition may be caused by a vascular disorder. Alternatively, a vascular event may cause progression of subclinical AD to a clinical stage. Insulin resistance and apolipoprotein E may also be involved in these mechanisms. Our studies show that ischemia-induced the Alzheimer-associated gene presenilin 1 (PS1) and endoplasmic reticulum-stress, generated from a vascular disorder, may unmask clinical AD symptoms caused by presenilin mutation, suggesting that a vascular factor might be involved in the onset of familial AD.

Prediction of short-term outcome for patients with active ulcerative colitis.

We report results of a retrospective chart review to evaluate factors predicting short-term outcome of patients with ulcerative colitis treated by corticosteroids. Between January 1992 and December 1997, we treated 71 patients with ulcerative colitis (44 with severe and 27 with moderately severe disease). Forty-nine patients were treated by conventional prednisolone therapy and 22 patients by steroid pulse therapy. There were no differences in clinical or endoscopic improvement between the two treatments. Clinical examination showed that 41 patients entered remission, 17 patients improved, and 13 patients did not respond. Endoscopically, 26 patients entered remission, 30 patients improved, and 15 patients did not respond. Extent of disease, type of disease (first attack, relapsing, or chronic active type), and endoscopic findings were factors useful in predicting short-term outcome of medical treatment.

Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in Igf2/H19 imprinting.

A differentially methylated region (DMR) and endoderm-specific enhancers, located upstream and downstream of the mouse H19 gene, respectively, are known to be essential for the reciprocal imprinting of Igf2 and H19. To explain the same imprinting patterns in non-endodermal tissues, additional enhancers have been hypothesized. We determined and compared the sequences of human and mouse H19 over 40 kb and identified 10 evolutionarily conserved downstream segments, 2 of which were coincident with the known enhancers. Reporter assays in transgenic mice showed that 5 of the other 8 segments functioned as enhancers in specific mesodermal and/or ectodermal tissues. We also identified a conserved 39-bp element that appeared repeatedly within the DMR and formed complexes with specific nuclear factors. Binding of one of the factors was inhibited when the target sequence contained methylated CpGs. These complexes may contribute to the presumed boundary function of the unmethylated DMR, which is proposed to insulate maternal Igf2 from the enhancers. Our results demonstrate that comparative genomic sequencing is highly efficient in identifying regulatory elements.

Effects of the K+ channel opener KRN4884 on the cardiovascular metabolic syndrome model in rats.

We examined the effects of the potassium channel opener KRN4884 (5-amino-N-[2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarboxamidine ) on cardiovascular metabolic syndrome (i.e., syndrome X), in rats. High-fructose diet rats developed hypertension, hypertriglyceridemia, increased total cholesterol/HDL (high-density lipoprotein)-cholesterol ratio, and hyperinsulinemia, KRN4884 (0.3-3.0 mg/kg, twice a day for 14 days, p.o.) alleviated the risk factors in fructose-fed rats. Furthermore, fructose-fed rats exhibited impairment of glucose tolerance and excess insulin secretion when loaded with glucose orally. Treatment with KRN4884 (1.0 mg/kg, twice a day for 14 days, p.o.) improved the glucose intolerance and inhibited hypersecretion of insulin in the glucose-loaded, fructose-fed rats. In contrast, KRN4884 (0.3-1.0 mg/kg, twice a day for 10 days, p.o.) did not affect serum triglyceride, cholesterol, glucose, or insulin concentrations in normal rats. LPL (lipoprotein lipase) activities in skeletal muscle and adipose tissue, and HTGL (hepatic triglyceride lipase) activity in liver were measured after administration of KRN4884 or vehicle twice a day for 14 days in fructose-fed rats. KRN4884 caused a significant increase in LPL activity in muscle and tended to increase LPL activity in adipose tissue in fructose-fed rats. HTGL was decreased in fructose-fed rats as compared with normal controls and was unaffected by KRN4884. These findings suggested that KRN4884 enhances insulin sensitivity and LPL activity, which are related to glucose and lipid metabolism and may be useful for the treatment of syndrome X.

Decreased anti-Saccharomyces cerevisiae antibody titer by mesalazine in patients with Crohn's disease.

BACKGROUND: Saccharomyces cerevisiae may contribute to the pathophysiology of Crohn's disease. We determined serum anti-Saccharomyces cerevisiae antibody (ASCA) levels in patients with inflammatory bowel disease. METHODS AND RESULTS: Immunoglobulin G (IgG) ASCA was measured by using an ELISA in serum samples from 19 patients with ulcerative colitis, 18 patients with Crohn's disease and 7 healthy controls. The ASCA level was significantly higher in patients with ulcerative colitis and patients with Crohn's disease than in controls, and was significantly higher in patients with Crohn's disease compared with patients with ulcerative colitis. Age, gender, disease activity, extent of disease and small bowel involvement each did not affect ASCA levels. The use of elemental or polymeric diet therapy for Crohn's disease and administration of corticosteroids to patients with inflammatory bowel disease also did not affect ASCA levels. The ASCA titer was significantly lower in patients with Crohn's disease taking mesalazine than in those not taking it, although, serum IgG levels did not differ between these two groups, which might imply a suppression of IgG production by mesalazine at the intestinal level. CONCLUSIONS: The finding of increased serum ASCA titers in patients with inflammatory bowel disease suggests that Saccharomyces cerevisiae may play a role in the pathophysiology of this condition.