Changes in maxillofacial morphology due to improvement of nasal obstruction in rats.

OBJECTIVES: To investigate the effect of release of experimentally introduced nasal obstruction on maxillofacial morphology and percutaneous arterial oxygen saturation (SpO2 ) in rats. MATERIALS AND METHODS: Six-week-old male Wistar rats (n = 36) were divided into a control group (n = 6) and a nasal obstruction group (n = 30). In the nasal obstruction group, the right nostril was occluded with silicon, which was subsequently removed after a given experimental period (days 7, 21, 35, 49 and 63). These animals were then divided into groups D7, D21, D35, D49 and D63 (each n = 6), according to the day at which the obstruction was released. The SpO2 was measured in rats with nasal obstruction at five experimental points. The maxillofacial morphology in rats on the first day and 63 days after the start of the experiment was evaluated by microcomputed tomography. RESULTS: The SpO2 was still lower at 2 weeks after the improvement of the nasal obstruction in the D49 group than in the control group. In addition, the height of the nasal maxillary complex of the D35, D49 and D63 groups was significantly decreased compared with the control group. CONCLUSIONS: The results of this study suggest that long-term unilateral nasal obstruction in growing rats may affect the growth of the nasomaxillary complex and reduce the SpO2 permanently. Therefore, early improvement of nasal obstruction in rats during the growth period may improve the SpO2 and cranial development and promote normal growth and development.

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts.

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IkappaBalpha and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam(3)CSK(4), but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.

Surface components of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Research on Porphyromonas gingivalis, a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis. MATERIAL AND METHODS: Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. RESULTS: Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly. CONCLUSION: Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases.

Detection of epidemics in their early stage through infectious disease surveillance.

BACKGROUND: Surveillance of infectious diseases is done in many countries. The aims of such surveillance include the detection of epidemics. In the present study, the possibility of detecting an epidemic in its early stage using a simple method was evaluated for 16 infectious diseases. METHODS: We used as an index the number of cases per week per sentinel medical institution in the area covered by a health centre in infectious disease surveillance in Japan in 1993-1997. Periods of epidemics in health centre areas were determined according to the reported indices. The simple method used for detecting the early stage of an epidemic is that if the index exceeds a critical value, then an epidemic will begin in the following 4 weeks. The sensitivity, specificity and positive predictive value for this epidemic warning were evaluated for given critical values. RESULTS: When the specificity of the epidemic warning was more than 95%, the sensitivity was more than 60% in ten diseases, and more than 80% in four diseases (influenza-like illness, rubella, hand-foot-and-mouth disease, and herpangina). The positive predictive value was between 15.6% and 31.4% in these ten diseases. CONCLUSION: The early stage of epidemics of some infectious diseases might be detectable using this simple method.

Distribution of PASII/PMP22 and connexin 32 proteins in the peripheral nervous system.

Various mutations of PO, PASII/PMP22 and connexin 32 genes were recently reported in hereditary neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine Sottas disease (DS). However, physiological roles of the proteins in PNS are not well understood. To address the functions of the proteins, we examined their localization in PNS comparatively by immunohistochemical methods. In Western blotting, a polyclonal antibody against the carboxyl terminal peptide of PASII/PMP22 reacted to 20-24 kD bands of PASII/PMP22 in mammalian PNS myelin, but produced no reaction in either mammalian or carp CNS myelin proteins. Monoclonal anti-connexin 32 antibody recognised connexin 32 of a dimer or monomer form in rat and human PNS myelin. By histological examination, PASII/PMP22 expressed dominantly in rat PNS compact myelins, while connexin 32 localized exclusively in the nodes of Ranvier, but not in compact myelins. In cell culture, axonal contact induced a remarkable increase of PASII/PMP22 in the Schwann cell in contrast to faint staining in immature Schwann cells. While localization of connexin 32 is quite different from that of PASII/PMP22, the mutations of the two proteins often induce similar phenotypes of hereditary neuropathies.

Control of complement activities for immunoisolation.

Immunoisolation of cells by semipermeable membranes is a most promising approach to transplant xenogeneic cells. Although membranes which allow xenotransplantation have been reported, ambiguity remains as to their long term effectiveness. In this review, we would like to reconsider the immuno-isolative effectiveness of membranes reported from the standpoint of permeability and present our strategy to prepare membranes that can realize long-term functioning of xenograft. There are distinct different types of semi-permeable membranes, hydrogel membranes and ultrafiltration membranes. Studies on their permeability indicated that neither of these membranes effectively fractionate solutes on the basis of molecular size under a diffusion-controlled process, nor thus can they immuno-isolate xenograft for a long time. Humoral immunity including antibodies and complement proteins is suspected of playing a major role in the rejection of xenografts. Control of complement cytolytic activities, not antibody permeation, may be a key factor determining the fate of the xenograft enclosed in membranes. We found that the microbead containing poly(styrene sulfonic acid) can consume complement cytolytic activities and thus can effectively protect xenogeneic islets of Langerhans in diabetic mice from the humoral immunity.

Functional comparison of the single-layer agarose microbeads and the developed three-layer agarose microbeads as the bioartificial pancreas: an in vitro study.

In this study, the insulin secretory characteristics of the microencapsulated hamster islets were studied during long-term culture. The hamster islets were encapsulated as single-layer agarose microbeads or three-layer agarose microbeads with agarose and agarose containing poly(styrene sulfonic acid) (PSSa), respectively. The influence of PSSa on the function of the rat islets microencapsulted in three-layer microbeads was primarily monitored. The aim of this study was to examine the influence of the PSSa on the in vitro function of the islets encapsulated in the agarose/PSSa microbeads compared with single-layer agarose microbeads during long-term culture. The microbeads were cultured for 30 days in medium of Eagle's MEM at 37 degrees C in 5% CO2 and 95% air. The basal insulin secretion into the culture medium was measured daily during the first 12 days and two times per week until 30 days. The microbeads were subjected to static incubation test on the 10th, 20th, and 30th day during culture. The basal insulin secretion level of the agarose/PSSa microbeads was significantly higher than that of single-layer agarose microbeads. The static incubation tests revealed a similar pattern of insulin secretion from both microbeads when they were exposed to high glucose challenge. In the static incubation test, both could significantly increase insulin release to more than 6.61 times (stimulation index) in response to high glucose stimulation and could significantly decrease when glucose concentration returned from high glucose to low glucose on the 10th, 20th, and 30th day of culture. This study demonstrated that the hamster islets enclosed in agarose/PSSa hydrogel not only continuously secreted basal amounts of insulin, but also maintained their response to high glucose stimulation similar to the agarose microbeads. The above results together with those of our previous in vivo study suggest that the three-layer microbeads (agarose/PSSa) are well suitable for xenotransplantation of islets for the clinical application.

Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis.

Inhibitory effects of synthetic fragments in histatin 8, having the sequence Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, on hemagglutination by Porphyromonas gingivalis 381 were examined. The hemagglutinating activity was reduced much more by the peptide Lys-His-His-Ser-His-Arg-Gly-Tyr than by the peptides Lys-His-His-Ser-His and/or Lys-Phe-His-Glu-Lys. These results suggest that the arginine residue may have an important role in the inhibition of hemagglutination by P. gingivalis.

Binding of a histidine-rich peptide to Porphyromonas gingivalis.

Porphyromonas gingivalis 381 cells were incubated with 125I-histidine-rich polypeptide (histatin) 5 in the presence or absence of unlabeled histatin 5, to evaluate the histatin-binding capacity of the cells. The binding of histatin 5 was rapid, reversible, saturable and specific. The number of histatin 5-binding sites per cell was 3,600, and the dissociation constant (Kd) was in the order of 10(-6) M. These findings suggest that histatin interacts with certain bacterial cells through specific binding sites on their surface, and will allow the development of a histatin radioreceptor assay.

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination of Bacteroides gingivalis 381.

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.

New modification of the Damus-Kaye-Stansel operation.

We report a successful modification of the Damus-Kaye-Stansel operation for transposition of the great arteries when the coronary arteries are unsuitable for transfer. The procedure includes creation of a neoaorta with end-to-end anastomosis of the proximal pulmonary artery to the distal ascending aorta and creation of an aortopulmonary window between the proximal great arteries. A valved conduit is interposed between the right ventricle and the distal pulmonary artery.

Fibronectin in saliva inhibits Porphyromonas gingivalis fimbria-induced expression of inflammatory cytokine gene in mouse macrophages.

The aim of this study was to examine whether fibronectin in saliva plays a regulatory role in Porphyromonas gingivalis fimbria-mediated pathogenesis in adult periodontal disease. Our previous study demonstrated that fibronectin is one of the binding proteins of P. gingivalis fimbrillin. In the present study, we observed that fibronectin in saliva binds specifically to the fimbrillin. The fibronectin content in saliva of adult periodontal patients was significantly lower than that of healthy subject. In addition, the inhibitory action of salivary fibronectin from adult periodontal patients toward P. gingivalis fimbria-induced expression of the neutrophil chemoattractant KC gene in mouse macrophages was clearly weak compared with that of the fibronectin from healthy subjects. These results suggest that fibronectin in saliva plays an important role as a regulator in the pathogenesis of P. gingivalis fimbriae in adult periodontal disease.

Tumor accumulation of poly(vinyl alcohol) of different sizes after intravenous injection.

The body distribution and tumor accumulation of polymers were evaluated using poly(vinyl alcohol) (PVA) which has the simplest chemical structure among water-soluble polymers, similar to poly(ethylene glycol). To study the effect of polymer size on the tumor accumulation, we used not only water-soluble PVA with different molecular weights but also PVA microgels prepared through gamma-irradiation of aqueous PVA solutions. The PVA specimens in the aqueous solution were intravenously injected to mice carrying a tumor mass at their footpad. Both types of PVA (water-soluble and microgel) of larger size were retained in the blood circulation for longer time periods and excreted more slowly from the kidney than those of smaller size. The plasma half-life period of PVA became longer with increasing size both for the water-soluble and microgel PVA, indicating that the body fate of PVA is governed only by the size. Both the water-soluble PVA and PVA microgels were accumulated in tumor tissue to a significantly greater extent than in normal tissue. The size dependence of the plasma half-life period and tumor accumulation was similar between the water-soluble PVA and the PVA microgels and the tumor accumulation became maximum around the size of 60 nm both for water-soluble and microgel PVA. A pharmacokinetical study demonstrated that the tumor uptake rate index of PVA decreased with the increase in PVA size. On the other hand, the greater the size, the larger the value of the area under the blood concentration-time curve (AUC). In addition, the PVA around 60 nm in diameter showed the smallest liver clearance. It was concluded that the balance between the uptake rate and the AUC as well as the liver clearance resulted in the maximum accumulation of PVA with the size of 60 nm.

Inhibitory effect of human plasma and saliva on co-aggregation between Bacteroides gingivalis and Streptococcus mitis.

The effect of human plasma and saliva on co-aggregation between Bacteroides gingivalis and Streptococcus mitis was studied by means of a turbidimetric assay. The co-aggregation activity was obtained from the maximum slope of the absorbance vs. time curve. Its dependence on pH, temperature, and ionic strength was examined, and the number of Bacteroides cells in relation to the number of Streptococcus cells resulting in optimal co-aggregation was established. Co-aggregation inhibition experiments showed that the co-aggregation activity was inhibited by l-arginine and l-lysine, although the activity was unaffected by the sugars tested. Human plasma and saliva were able to inhibit the co-aggregation in a dose-dependent reaction. Plasma exhibited the most potent inhibitory activity in these fluids. Fibrinogen was the most potent inhibitor of the plasma-derived proteins tested. These data suggest the possibility that the oral fluids may modulate the attachment of B. gingivalis to Gram-positive bacteria in periodontal pockets.

Relationship between enzyme activities involved in oxygen metabolism and oxygen tolerance in black-pigmented Bacteroides.

In this study, the relationship between enzyme activities involved in oxygen metabolism and the degrees of oxygen tolerance in black-pigmented Bacteroides was investigated. All strains of Bacteroides tested possessed the activities of NADH oxidase and superoxide dismutase, whereas the activities of catalase and peroxidases were not detected in the cell-free extracts. There were relatively high correlations between oxygen tolerance and activity of either NADH oxidase or superoxide dismutase. The activity of superoxide dismutase showed a higher correlation with oxygen tolerance than with that of NADH oxidase. Among the species tested, Bacteroides gingivalis showed the highest activities of both the enzymes and was the most tolerant in the presence of oxygen. Furthermore, the activities of these two enzymes increased during aeration of the oxygen-tolerant strain Bacteroides gingivalis 381, but not in the oxygen-sensitive strain Bacteroides denticola ATCC 33185. These results suggest that superoxide dismutase and NADH oxidase might be important for protection of black-pigmented Bacteroides against the toxic effect of oxygen.

Effects of erbium,chromium:YSGG laser irradiation on canine mandibular bone.

BACKGROUND: Only relatively few reports have described the morphological effects on bone produced by erbium,chromium: yttrium,scandium,gallium,garnet (Er,Cr:YSGG) laser irradiation, and none has investigated the atomic changes or estimated the temperature increases involved. The objectives of this study were to investigate the morphological, atomic, and temperature changes in irradiated areas during and after laser irradiation, and to evaluate the cutting effect on canine mandibular bone in vitro. METHODS: Two canine mandibular bones were cut into 3 to 5 cm pieces and irradiated by an Er,Cr:YSGG laser utilizing a water-air spray at 5 W and 8 Hz for 10 or 30 seconds. During and after laser irradiation, temperature increases in the irradiated areas were measured by thermography. The samples were then observed by stereoscopy and scanning electron microscopy to determine morphological changes and by energy dispersive x-ray spectroscopy to evaluate atomic alterations. RESULTS: Regular holes or grooves having sharp edges and smooth walls were produced, but no melting or carbonization was observed. The maximum temperature increase was an average 12.6 degrees C for 30-second irradiation. The continuous time of a temperature increase of more than 10 degrees C was consistently less than 10 seconds. An atomic analytical examination revealed that the calcium:phosphorus ratio was not significantly changed between the lased and unlased areas (P>0.0 1). CONCLUSION: These results showed that the Er,Cr:YSGG laser cuts canine mandibular bone effectively without burning, melting, or altering the calcium:phosphorus ratio of the irradiated bone.

Inhibitory effects of synthetic histidine-rich peptides on haemagglutination by Bacteroides gingivalis 381.

The haemagglutinating activity of Bacteroides gingivalis 381 was significantly inhibited by the synthetic peptide, Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His- Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. However, bradykinin potentiator C, which does not contain cationic amino acids such as L-histidine, L-arginine and L-lysine, had no inhibitory effect.

Cloning and sequence analysis of the poly (3-hydroxyalkanoic acid)-synthesis genes of Pseudomonas acidophila.

Pseudomonas acidophila can grow with CO2 as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H2-oxidizing bacterium Alcaligenes hydrogenophilus. H2-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO2, but recombinant P. acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA). In this study, the pha genes of P. acidophila were cloned and a sequence analysis was carried out. A gene library was constructed using the cosmid vector pVK102. A recombinant cosmid carrying the pha genes was selected by the complementation of a PHB-negative mutant of Alcaligenes eutrophus H16. The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert. Subcloning was done. and the recombinant plasmid pEH74 was selected by hybridization with the A. eutrophus H16 pha genes. Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained the pha genes of P. acidophila. The nucleotide sequences of the PHA-synthesis genes phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase) in pEH74 were determined. The homologies of phaA, phaB, and phaC between P. acidophila and A. eutrophus H16 were 64.7, 76.1 and 56.6%, respectively.

Preparation of virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus from inactivated vaccine.

Virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus was prepared from an inactivated FMD vaccine. The VIA antigen coupled with an adjuvant of aluminium hydroxide gel supplemented vaccine was efficiently eluted by suspending and stirring in high concentration of phosphate buffer solution (0.3M, pH 7.6). The final elute purified by DEAE-Sephadex A50 from the vaccine was concentrated in 1/500-1/1,000 of the original volume. VIA antigens prepared from two kinds of vaccine (Type O and Asia-1) were antigenically identical to one prepared from a cell culture infected with live virus. Two cattle were infected with FMD virus (Type O) and sera were obtained from each cattle every week. The VIA antigens from the inactivated vaccine were compared with the ones from the cell culture infected with live virus in agar gel diffusion tests using sera from cattle infected with live virus. The antibodies against the VIA antigen were detected between the second week and the 13th or 14th week after infection. The VIA antigen from an inactivated vaccine would be very useful in FMD free countries like Japan to avoid the risk of using live FMD viruses.

Application of latex beads agglutination test for the detection of the antibody against virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus.

Latex beads agglutination (LA) for the detection of the antibody against virus infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus was estimated using experimentally infected animals. The VIA antibody titer by the LA test were compared with the neutralization titer and the titer by agarose gel diffusion (AGD) test, which has been used as a standard method for VIA antibody titration. The latex beads were coated with VIA antigen in carbonate buffer solution (0.5 M, pH 9.6) for the test. The sensitivity of the LA test was clearly higher than that of the AGD test in the results for cattle and swine infected experimentally. The antibody was detected in the bovine serum obtained at the 13th week after inoculation by the LA but not by the AGD test. The LA test appears to be simple, rapid and sensitive for the detection of the antibody of FMD virus in the surveillance of FMD and the FMD quarantine of imported animals.