Influence of the anatomical form of the posterior maxilla on the reliability of superior maxillary repositioning by Le Fort I osteotomy.

Certain patients with facial deformities require superior repositioning of the maxilla via Le Fort I osteotomy; however, the magnitude of superior repositioning of the maxilla is often less than expected. In this study, the correlation between the accuracy of superior repositioning of the maxilla and the anatomical form of the maxillary posterior region was examined. Seventy-five patients who underwent Le Fort I osteotomy without forward movement of the maxilla but with superior repositioning of the maxilla were included in this study. The bone volume around the descending palatine artery (DPA), the angle of the junction between the pterygoid process and the tuberosity, and the distance between the upper second molar and the pterygoid process were measured via three-dimensional analysis. A significant negative correlation (r=-0.566) was found between the bone volume around the DPA and the ratio of repositioning (actual movement divided by expected movement). It is possible that the superior repositioning of the maxilla expected prior to surgery was not sufficiently attained because of the large volume of bone around the DPA. The results of this study show that in some patients, superior repositioning was not achieved at the expected level because of bone interference attributable to the anatomical form of the maxillary posterior region.

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells.

Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO(2))-mediated oxidation, diazene dicarboxylic acid bis-N,N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO(2) > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO(2) and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.

In vitro detection of equine arteritis virus from seminal plasma for identification of carrier stallions.

Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU/ml. In vitro detection of EAV by virus isolation supplemented with RT-PCR using seminal plasma was proved to be an effective alternative to the standard test mating as a diagnostic method for carrier stallions.

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein.

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.

Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay.

A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.

The effect of hydroxyapatite coating on the bonding of bone to titanium implants in the femora of ovariectomised rats.

We have studied the effect of hydroxyapatite (HA) coating in 15 ovariectomised and 15 normal rats which had had a sham procedure. Twenty-four weeks after operation, HA-coated implants were inserted into the intramedullary canal of the right femur and uncoated implants into the left femur. The prostheses were removed four weeks after implantation. Twelve specimens in each group had mechanical push-out tests. Sagittal sections of the other three were evaluated by SEM. The bone mineral density (BMD) of the dissected left tibia was measured by dual-energy x-ray absorptiometry. The difference in BMD between the control and ovariectomised tibiae was 35.01 mg/cm2 (95% CI, 26.60 to 43.42). The push-out strength of the HA-coated implants was higher than that of the uncoated implants in both groups (p < 0.0001), but the HA-coated implants of the ovariectomised group had a reduction in push-out strength of 40.3% compared with the control group (p < 0.0001). Our findings suggest that HA-coated implants may improve the fixation of a cementless total hip prosthesis but that the presence of osteoporosis may limit the magnitude of this benefit.

Pathogenesis of Babesia caballi infection in experimental horses.

The present study was designed to investigate the role of cytokines in the pathogenesis of Babesia caballi in experimentally infected horses. The expression of cytokine mRNA was determined by using reverse transcription-polymerase chain reaction in two B. caballi-infected horses for 2 weeks after the infection. In one horse, there was up-regulation of interferon-gamma, tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 mRNAs, while in the second horse, expression of only TNF-alpha mRNA was up-regulated. No change was observed in interleukin-4 mRNA in both of the horses. To know the relation between nitric oxide (NO) production and pathogenesis, NO production was assayed in three dexamethasone treated-B. caballi-infected horses. Production of NO in all 3 horses increased significantly before death, although the parasitemia level remained very low. Treatment with NO inhibitor resulted in the suppression of NO production and increased parasitemia level in a horse, which died of the infection. The pathological examination showed that the main cause of the death was dyspnoea and pulmonary edema. Histopathologically, diffuse global mesangial proliferative glomerulonephritis was also observed. These results suggested that NO may be a critical effector molecule of immune defense against parasite. TNF-alpha and NO might be contributing to the pathogenesis in B. caballi infection.

Identification of B cell epitopes of a 30 kDa Babesia equi merozoite surface protein.

A 30 kDa immunodominant surface antigen (p30) of Babesia equi has been used as a diagnostic antigen. The B cell epitopes on this molecule recognized by horse sera and monoclonal antibody (MAb) against p30, 36/133.97, were determined. A synthetic peptide of p30 with amino acid sequence of 123FYQEVLFKGFEAV135 exhibited strong positive reaction with the infected horse sera. In contrast, MAb 36/133.97 recognized different region of p30, as peptide synthesized with amino acid sequence of 27ASGAVVDFQLESI39 reacted strongly. In competitive inhibition ELISA, the binding of MAb 36/133.97 to recombinant p30 was inhibited by horse antibodies, although they did not recognize same or an overlapping epitope. The data on B cell epitopes in this study may be important in improving serodiagnostic methods of B. equi infection.

Hydroxyapatite-coating on titanium arc sprayed titanium implants.

We developed a new titanium spray technique using an inert gas shielded arc spray (titanium arc spray). Hydroxyapatite (HA)-coating can be applied to the implant without any surface pore obstruction after the rough surface is made by this technique. Scanning electron microscopy (SEM) of various porous implant surfaces after HA-coating revealed that the bead and fiber metal-coated implants had either a pore obstruction or an uneven HA-coating. On the other hand, the titanium arc sprayed implant demonstrated an even HA-coating all the way to the bottom of the surface pore. In the first set of animal experiments (Exp. 1), the interfacial shear strength to bone of four kinds of cylindrical Ti-6A1-4V (Ti) implants were compared using a canine transcortical push-out model 4 and 12 weeks after implantation. The implant surfaces were roughened by titanium arc spray (group A-C) and sand blasting (group D) to four different degrees (roughness average, Ra = group A: 56.1, B: 44.9, C: 28.3, D: 3.7 microns). The interfacial shear strength increased in a surface roughness-dependent manner at both time periods. However, the roughest implants (group A) showed some failed regions in the sprayed layers after pushout test. In the second set of animal experiments (Exp. 2), four kinds of Ti implants; HA-coated smooth Ti (sHA) with Ra of 3.4 microns, bead-coated Ti (Beads), titanium arc sprayed Ti (Ti-spray) with Ra of 38.1 microns and HA-coated Ti-spray (HA + Ti-spray) with Ra of 28.3 microns were compared using the same model as that in Exp. 1. The interfacial shear strength of HA + Ti-spray was significantly greater than that of sHA and Beads at both time periods, and that of Ti-spray at 4 weeks. Although a histological examination revealed that HA-coating enhanced bone ingrowth, sHA showed the lowest shear strength at both time periods. SEM after pushout test showed that sHA consistently demonstrated some regional failure at the HA-implant substrate interface. HA + Ti-spray had many failed regions either at the HA-bone interface or within the bone tissue rather than at the HA-implant substrate interface. These results suggested that the HA-coated smooth surfaced implants had a mechanical weakness at the HA-substrate interface. Therefore, HA should be coated on the rough surfaced implants to avoid a detachment of the HA-coating layer from the substrate and thus obtain a mechanical anchoring strength to bone. HA-coating on this new type of surface morphology may thus lead to a solution to the problems of conventional HA-coated and porous-coated implants.

Improved in vitro cultivation of Babesia caballi.

Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O2, 5% CO2 and 93% N2). All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels. However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air, with parasitemia ranging from 8-10%.

Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge.

Twelve foals, between 27 and 83 days old, were infected with 2 strains of Rhodococcus equi by intratracheal administration. Ten of the 12 foals were inoculated with 10(4)-10(10) colony forming units (cfu) of ATCC 33701 strain. The other 2 foals were inoculated with 10(9) cfu of a plasmid-cured derivative of the ATCC 33701 strain (ATCC 33701P-). All of the 10 foals challenged with the ATCC 33701 strain showed clinical signs of pulmonary disease within 5-13 days, such as gross lesions associated with acute bronchopneumonia and microscopic lesions associated with granulomatous pneumonia. The two foals challenged with the ATCC 33701P- strain showed neither clinical signs of disease nor gross lesions. Apparently, when lacking plasmid, the virulent Rhodococcus equi lost its pathogenicity.

Venereal infection of mares by equine arteritis virus and use of killed vaccine against the infection.

Venereal infection with equine arteritis virus (EAV) was established in each of seven mares by inoculation via the cervix with 20 ml of viral suspension (> or = 8 x 10(6) plaque-forming units; PFU), following treatment with prostaglandin and oestradiol. A dose of < or = 8 x 10(5) PFU produced infection in only five of eight mares. Serum neutralizing antibody developed in mares manifesting clinical signs of equine viral arteritis (EVA), and a weak antibody was detectable in one apparently healthy mare inoculated with 8 x 10(5) PFU. Virus isolation was demonstrated not only in the buffy coat but also in nasal swabs of infected mares. EAV was isolated frequently from the body tissues of the mares (killed 10 to 34 days post-inoculation) up to day 12, but rarely from the reproductive tissues later than day 12. The virus persisted longest in the splenic and deep inguinal lymph nodes, followed by the spleen and internal iliac lymph nodes. Four mares immunized with a killed vaccine for EVA showed no clinical disease after venereal challenge with EAV; the virus was recovered from the buffy coat of three mares and from the nasal swab of one of them, but not from the remaining animal.

Histopathological and immunofluorescent studies on transplacental infection in experimentally induced abortion by equine arteritis virus.

Five pregnant mares, at between 6 and 8 months gestation, were experimentally infected with the Bucyrus strain of equine arteritis virus (EAV). Of the five mares, four aborted and one died. The pathogenesis of the abortions was studied, using histopathologic techniques, tissue immunofluorescence and virus isolation. Common microscopic lesions in the maternal reproductive organs indicated myometritis with a degeneration of the myocytes and an infiltration of the mononuclear cells. Epithelial cells of the endometrial gland showed sporadic degeneration. Lesions in the fetal tissue included an atrophy of the lymphoid follicles in the spleen and lymph nodes with degenerated lymphocytes. The placentae were oedematous and degenerated fibroblasts were observed in the subvillous layers. Immunofluorescence detected EAV antigen in the myometrium and the endometrial gland in the dams, in the subvillous layer of the placentae, and in the aborted fetuses. EAV was recovered from the maternal uteri, placentae and fetuses. The placentae yielded the greatest amounts of the virus. Transplacental infection of the fetus was clearly demonstrated in the EAV infection.

Ultrastructure and immuno-cytochemistry of BHK-21 cells infected with a modified Bucyrus strain of equine arteritis virus.

Morphogenesis of a modified Bucyrus strain of equine arteritis virus (EAV) in BHK-21 cells was studied. Bacillary tubules were first detected in the cytoplasm 8 h after infection, and mature virions 79 to 122 nm in diameter, 101 nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (RER) at 12 h or later. They had isometrical cores and morphological subunits in the outer layer. Budding occurred from the RER and the outer nuclear membrane, but not from the cell surface. Structural linkage was detected between the tubule and the virus core. Aberrant strands were occasionally demonstrated within the nucleus 12 h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus.

Use of the serum neutralisation test for equine viral arteritis with different virus strains.

Serum cross neutralisation tests were conducted with a recent American isolate (84KY-A1) and a European isolate, (Wroclaw-2) and compared with the prototype and modified viruses of the Bucyrus strain of equine arteritis virus by using virus specific immune horse sera. The modified Bucyrus strain was neutralised and showed high neutralisation titres with all the immune sera. The prototype Bucyrus strain was also substantially neutralised, followed by the 84KY-A1 strain. As a result of the tests with the modified Bucyrus strain as the antigen, 20 seropositive horses were discovered among home-bred horses which had no previous record of clinical equine viral arteritis. Heat inactivation of the sera at 62 degrees C for 30 minutes caused the disappearance of all but the most positive reactions. When the prototype Bucyrus strain was used instead of the modified virus, no positive reactions were detectable. A serological survey, using the Bucyrus strain in a microtitre neutralisation test, was conducted with 1656 horse sera collected between 1988 and 1990 in Japan. The test disclosed only eight foreign-bred horses positive to the virus; they had been imported as competition horses. No circumstantial evidence of an effect of such horses as a source of infection for horse populations free of the virus was obtained.

Complement-dependent serum neutralization with virulent and avirulent Bucyrus strains of equine arteritis virus.

Virulent and avirulent strains of Bucyrus equine arteritis virus (EAV) were used to raise antiserum in horses. Serum neutralization (SN) tests were performed with and without the addition of guinea pig complement. The inclusion of ten percent guinea pig serum in the virus suspension was sufficient for optimal enhancement of SN titres at any immune stages after immunization. Immune serum prepared against avirulent virus reacted only with homologous virus and there was no complement enhancement. Immune sera raised against live or inactivated virulent virus neutralized both virulent and avirulent virus. The reaction with virulent virus demonstrated complement enhancement. There was also moderate potentiation in the presence of complement when serum raised against inactivated virulent virus reacted with avirulent virus.

Protein characterization of Babesia equi piroplasms isolated from infected horse erythrocytes.

Proteins of Babesia equi piroplasms were characterized. The piroplasms of B. equi were purified by lysis of infected horse erythrocytes with N2 gas cavitation followed by separation in Percoll density-gradient centrifugation. The relative molecular weights (Mr) of major proteins separated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 18, 28, 30, 41, 43, 54, 66.5, and 96 kDa. Immunoblot analysis using serum from an experimentally infected horse revealed six immunodominant proteins of 15, 18, 28, 30, 41, and 96 kDa. Two immunodominant proteins of 18 and 28 kDa were membrane-bound proteins as revealed by Triton X-114 phase partitioning.

Meteorological observations at Hiroshima on days with weather similar to that of the atomic bombing.

An attempt to explain discrepancies between measured neutron-induced radioactivities (i.e., 152Eu, 60Co) and calculated yields based on the Dosimetry System 1986 was made by considering moist air densities at different altitudes over Hiroshima, Japan. The investigation checked the validity of moist air density estimates used in the Dosimetry System 1986 computer codes. Meteorological observations were conducted to obtain atmospheric temperature and pressure profiles for the Hiroshima area. By coupling these observations with surface measurements taken on 6 August 1945 at Ebayama Park (3.6 km from the hypocenter), estimates of temperature, pressure, and humidity at the time of detonation were derived. This allowed the calculation of densities for dry air, moist air, and water vapor. The results proved similar to the Dosimetry System 1986 estimates. Water vapor density calculations showed the largest difference (at most 7%). These results imply that the 152Eu yield contradiction in the Dosimetry System 1986 calculations vs. real measurements for large ground distances (> 900 m) is not the result of an erroneous estimate of moist air density in Hiroshima.