Comparison of mouse and human ankles and establishment of mouse ankle osteoarthritis models by surgically-induced instability.

OBJECTIVE: Prevalence of ankle osteoarthritis (OA) is lower than that of knee OA, however, the molecular mechanisms underlying the difference remain unrevealed. In the present study, we developed mouse ankle OA models for use as tools to investigate pathophysiology of ankle OA and molecular characteristics of ankle cartilage. DESIGN: We anatomically and histologically examined ankle and knee joints of C57BL/6 mice, and compared them with human samples. We examined joints of 8-week-old and 25-month-old mice. For experimental models, we developed three different ankle OA models: a medial model, a lateral model, and a bilateral model, by resection of respective structures. OA severity was evaluated 8 weeks after the surgery by safranin O staining, and cartilage degradation in the medial model was sequentially examined. RESULTS: Anatomical and histological features of human and mouse ankle joints were comparable. Additionally, the mouse ankle joint was more resistant to cartilage degeneration with aging than the mouse knee joint. In the medial model, the tibiotalar joint was markedly affected while the subtalar joint was less degenerated. In the lateral model, the subtalar joint was mainly affected while the tibiotalar joint was less altered. In the bilateral model, both joints were markedly degenerated. In the time course of the medial model, TdT-mediated dUTP nick end labeling (TUNEL) staining and Adamts5 expression were enhanced at early and middle stages, while Mmp13 expression was gradually increased during the OA development. CONCLUSION: Since human and mouse ankles are comparable, the present models will contribute to ankle OA pathophysiology and general cartilage research in future.

Reactivity of synthetic SAG1 (p30) peptide sequences with RH, S273 and Beverley strain-induced anti-Toxoplasma gondii antibodies.

OBJECTIVES: We compared the reactivity of IgG1 and IgG2a antibodies in mouse sera after infection with virulent RH and low-virulent S273 and Beverley strains of Toxoplasma gondii against RH SAG1 recombinant p30 (rp30) and synthetic SAG1 peptides. METHODS: Infected mouse serum samples were collected 9 days after infection, and the level of total IgG, IgG1 and IgG2a against the RH SAG1 rp30 protein and twenty peptides of the RH SAG1 protein were assessed. The glycosylphosphatidylinositol (GPI) modification site, the hydrophilic-hydrophobic structure, the transmembrane region and the secondary structure of the SAG1 sequence of virulent and low-virulent strains were analyzed using software. RESULTS: The virulent strain-infected mice produced a higher level of IgG1 but a lower IgG2a against the rp30 antigen, while the low-virulent strain-infected mice produced a higher level of IgG2a than the virulent strain. The difference in the secondary structure of SAG1 protein between the virulent and low-virulent strain was largely confined to amino acid positions 291-336, showing mutations and GPI anchor site. CONCLUSION: The difference in the reactivity of IgG against the rp30 antigen and synthetic peptides between virulent and low-virulent strains points to the importance of the primary and secondary structure assumed by antigens in the activation of Th cells and, subsequently, in the induction of IgG and its subclasses.

Intra- and extracellular reactive oxygen species generated by blue light.

Blue light from dental photopolymerization devices has significant biological effects on cells. These effects may alter normal cell function of tissues exposed during placement of oral restorations, but recent data suggest that some light-induced effects may also be therapeutically useful, for example in the treatment of epithelial cancers. Reactive oxygen species (ROS) appear to mediate blue light effects in cells, but the sources of ROS (intra- versus extracellular) and their respective roles in the cellular response to blue light are not known. In the current study, we tested the hypothesis that intra- and extracellular sources of blue light-generated ROS synergize to depress mitochondrial function. Normal human epidermal keratinocytes (NHEK) and oral squamous cell carcinoma (OSC2) cells were exposed to blue light (380-500 nm; 5-60 J/cm(2)) from a dental photopolymerization source (quartz-tungsten-halogen, 550 mW/cm(2)). Light was applied in cell-culture media or balanced salt solutions with or without cells present. Intracellular ROS levels were estimated using the dihydrofluorescein diacetate (DFDA) assay; extracellular ROS levels were estimated using the leucocrystal violet assay. Cell response was estimated using the MTT mitochondrial activity assay. Blue light increased intracellular ROS equally in both NHEK and OSC2. Blue light also increased ROS levels in cell-free MEM or salt solutions, and riboflavin supplements increased ROS formation. Extracellularly applied ROS rapidly (50-400 muM, <1 min) increased intracellular ROS levels, which were higher and longer-lived in NHEK than OSC2. The type of cell-culture medium significantly affected the ability of blue light to suppress cellular mitochondrial activity; the greatest suppression was observed in DMEM-containing or NHEK media. Collectively, the data support our hypothesis that intra- and extracellularly generated ROS synergize to affect cellular mitochondrial suppression of tumor cells in response to blue light. However, the identity of blue light targets that mediate these changes remain unclear. These data support additional investigations into the risks of coincident exposure of tissues to blue light during material polymerization of restorative materials, and possible therapeutic benefits.

Toxoplasma gondii does not persist in goldfish (Carassius auratus).

Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish (Carassius auratus) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.

Expression of mRNA of chemokine receptor CXCR4 in feline mammary adenocarcinoma.

The expression of mRNA of the chemokine receptor CXCR4 in 65 surgically resected mammary adenocarcinomas from cats was investigated by in situ hybridisation. No expression of the receptor's mRNA was detectable in the mammary tissue of healthy cats, but it was expressed in areas adjacent to necrosis, surrounding blood vessels and cells infiltrating the lymphatics of 47 (72.3 per cent) of the 65 samples. There was a significant relationship between lymphatic infiltration by neoplastic cells and the expression of the receptor's mRNA (P < 0.005), but there was no significant relationship between its expression and the one-year survival of the cats.

Modified protection against Toxoplasma gondii lethal infection and brain cyst formation by vaccination with SAG2 and SRS1.

Numerous studies have supported the importance of immunity to SAG1, the most predominant antigen of Toxoplasma tachyzoite, in protection against Toxoplasma gondii infection. Nevertheless, vaccination with SAGI provides insufficient protection when compared with that of Toxoplasma lysate (TL). In order to screen the Toxoplasma antigens for immunogenic potential shown by modified protection or induction of specific immune response after infection, recombinant antigens were prepared in Eschericha coli using DNA fragments corresponding to SAG1, SAG2, SAG3, SRS1 and P54 of T. gondii RH strain maintained in our laboratory. Each of the recombinant antigen products or a mixture of the five antigens (Mix) was used to vaccinate mice. Mice then received a lethal dose of T. gondii. Up to 25% of the mice vaccinated with SAG2, SRS1, P54 and Mix survived, whereas there were no survivors in gene 10- (negative control), SAG1- and SAG3- vaccinated groups. In all the survivors, brain cysts were not observed. Conversely, vaccination with TL almost completely protected mice in the acute phase but permitted brain cyst formation and resulted in gradual decrease of survivors to 33% during 4 months of experiments. Western blot analysis on convalescent sera showed an extensive IgG induction to a 30 kDa antigen in TL-vaccinated mice, a 22 kDa in SAG2-vaccinated mice and a 55 kDa in P54-vaccinated mice. The protection modified by boost in specific antibody is suggestive of the immunogenic potential of SAG2, SRS1 and possibly P54 against T. gondii infection.

Naturally-occurring Neospora caninum infection in an adult sheep and her twin fetuses.

Neospora caninum tissue cysts were found in the brains of surgically delivered twin fetuses at 119 days of gestation. In the brains of both fetuses, there was an inflammatory reaction involving perivascular cuffings of mononuclear cells, glial nodules. The dam of these fetuses died because of metritis. Histopathological examination of the ewe revealed N. caninum tissue cysts and focal gliosis with mononuclear cell cuffings. A N. caninum-specific DNA fragment was detected in a brain homogenate of the ewe by the polymerase chain reaction method. This is the first report of N. caninum infection in twin ovine fetuses and in an adult sheep.

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase.

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B 1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116-134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.

Antibody reactivity in mice and cats to feline enteroepithelial stages of Toxoplasma gondii.

The reactivity of antibodies in mice and cats to feline enteroepithelial stages of Toxoplasma gondii was examined by means of an indirect immunofluorescent antibody test. Mice immunized with feline enteroepithelial stage (FES) parasites produced antibodies not only against FES, but also against tachyzoites, sporozoites/oocysts, tissue cysts and one part of the infected feline enterocytes. After absorption with tachyzoites, the titer of antibodies reactive to enterocytes was significantly reduced. In contrast, the titer of antibodies reactive to FES remained unchanged. The antibodies from cats immunized with FES, reacted specifically to FES, but not to tachyzoites, tissue cysts or enterocytes. These results suggest that FES parasites may have stage-specific antigen(s).

Roles of CD4(+) T cells and gamma interferon in protective immunity against Babesia microti infection in mice.

Babesia microti produces a self-limiting infection in mice, and recovered mice are resistant to reinfection. In the present study, the role of T cells in protective immunity against challenge infection was examined. BALB/c mice which recovered from primary infection showed strong protective immunity against challenge infection. In contrast, nude mice which failed to control the primary infection and were cured with an antibabesial drug did not show protection against challenge infection. Treatment of immune mice with anti-CD4 monoclonal antibody (MAb) diminished the protective immunity against challenge infection, but treatment with anti-CD8 MAb had no effect on the protection. Transfer of CD4(+) T-cell-depleted spleen cells resulted in higher parasitemia than transfer of CD8(+) T-cell-depleted spleen cells. A high level of gamma interferon (IFN-gamma), which was produced by CD4(+) T cells, was observed for the culture supernatant of spleen cells from immune mice, and treatment of immune mice with anti-IFN-gamma MAb partially reduced the protection. Moreover, no protection against challenge infection was found in IFN-gamma-deficient mice. On the other hand, treatment of immune mice with MAbs against interleukin-2 (IL-2), IL-4, or tumor necrosis factor alpha did not affect protective immunity. These results suggest essential requirements for CD4(+) T cells and IFN-gamma in protective immunity against challenge infection with B. microti.

Protective effect of lactoferricin against Toxoplasma gondii infection in mice.

The protective effect of lactoferricin against Toxoplasma gondii infection was examined in experimental murine toxoplasmosis. All mice orally administered 5.0 mg of lactoferricin, and challenged with cysts of T. gondii at a dose of LD90 survived until the end of experiment (35 days post challenge). Intraperitoneal administration of 0.1 mg of lactoferricin also prevented death in 100% of treated mice challenged with T. gondii cysts. In contrast, 80% of untreated mice died of acute toxoplasmosis within 14 days post challenge. In the mice treated perorally with lactoferricin, the number of cysts in the brain was significantly lower than that in untreated mice. Levels of interferon-r in the serum of infected mice treated perorally with lactoferricin showed a tendency to lower than those in the infected mice without treatment. These results demonstrate that oral administration of lactoferricin induces resistance to T. gondii infection in mice.

Identification of carbohydrates on Eimeria stiedai sporozoites and their role in invasion of cultured cells in vitro.

Invasion specificity of Eimeria stiedai sporozoites in cultured cells was examined. Intracellular sporozoites were observed in hepatobiliary epithelial cells, as early as 3 hours post inoculation (p.i.) but the infection rate was monitored for 6 hours. No intracellular parasites were found in rabbit parenchymal hepatocytes and rabbit kidney cells, even on prolonged culturing. In the hepatobiliary epithelial cells inoculated with fixed sporozoites, no intracellular parasite were found. Sporozoites attached on the cell surface of the hepatobiliary epithelial cells fixed with paraformaldehyde but did not penetrate. The carbohydrates present on Eimeria stiedai sporozoites and their functional role in the process of invasion of host cells were also examined. Lectin binding sites on the surface of sporozoites were detected by means of peroxidase-conjugated lectins. Sporozoites showed specific binding with UEA-I and PNA lectins, which bind L-fucose and D-galactose, respectively. Exposure of sporozoites to 100 microg/ml UEA-I significantly reduced their ability to invade primary rabbit hepatobiliary epithelial cells, but similar treatment with PNA had no such effect. Preincubation of these cells in Dulbecco's minimum essential medium containing 10% fetal bovine serum and 1% L-fucose suppressed the invasion activity of the sporozoites, but preincubation of the sporozoites in the same medium without L-fucose had no effect on cell penetration. D-galactose added to the medium had no effect on the invasion activity of sporozoites. These results indicate that L-fucose residues on E. stiedai sporozoites and L-fucose binding sites on host cells both are associated with recognition and/or invasion process.

Isolation of coccidian enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of cats by Percoll density-gradient centrifugation.

A method for isolation of enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of experimentally infected cats was developed using Percoll density-gradient centrifugation. Gamonts and merozoites were obtained essentially free of host-cell debris. A recovery rate of nearly 30% of the parasites in the original preparations was obtained by this method. Merozoites were separated from gamonts by filtration through a 3-micron polycarbonate filter.

Positive evidence that anti-Toxoplasma gondii IgA antibody exists in the intestinal tract of infected cats and exerts protective activity against the infection.

The presence of anti-Toxoplasma gondii IgA antibody in the feces and intestinal tract of cats infected with this parasite was demonstrated using an immunoblotting assay. Cats (n = 5) were inoculated orally with T. gondii cysts and supernatants of feces and washings of the intestinal tract were assayed for secretory IgA specific for the parasite. The secretory IgA detected recognized tachyzoite antigens of m.w. 24, 34, 38 and 43 kDa and one sporozoite antigen of m.w. 24 kDa. No reactivity was shown against bradyzoites or enteroepithelial stage parasites. Tachyzoites preincubated with washings of the intestinal tract of infected cats showed decreased activity in penetration of feline fibroblast cells, as compared to tachyzoites preincubated with similar washings derived from non-infected cats. The addition of either anti-cat IgA or anti-cat IgG to the washings had no effect on the inhibitory activity which reduced the parasite's cell-penetration activity. However, the addition of both anti-cat IgA and anti-cat IgG to the washings diminished the inhibitory activity. These results suggest that anti-T. gondii antibodies of both classes, secretory IgA and IgG, exist in the intestinal tract of infected cats and these may be capable of preventing infection.

Toxoplasma gondii: experimental infection in cats vaccinated with 60Co-irradiated tachyzoites.

The effects of 60Co-irradiation on the viability and immunogenicity of RH and Beverley strains of Toxoplasma gondii was examined. While 60Co-irradiated tachyzoites retained invasive activity in mouse embryonal cells and sustained antigenicity in mouse peritoneal macrophages, the parasites did not develop in host cells. Vaccination of mice with 60Co-irradiated tachyzoites of either RH or Beverley strains induced resistance to challenge with a highly virulent strain (RH strain). Vaccination of kittens with 60Co-irradiated tachyzoites of the Beverley strain partially prevented oocyst shedding when challenged with cysts (bradyzoites) from the Beverley strain. But kittens vaccinated with 60Co-irradiated or fixed tachyzoites of the RH strain shed oocysts when challenged with cysts (bradyzoites) from the Beverley strain. These results suggest that the development of protective immunity in feline toxoplasmosis seems to be strain and/or stage-specific.

Mycotoxin T-2 and aflatoxin B1 as immunosuppressors in mice chronically infected with Toxoplasma gondii.

The aim of this study was to determine whether repeated ingestion of mycotoxin T-2 (T2) or aflatoxin B1 (AFL) at low doses could contribute to the activation of toxoplasmosis in experimentally infected mice. Mice were divided into two groups: Control (C) and Infected (I). The cyst-forming Beverley strain of Toxoplasma gondii was used to produce the infection one month before treatment with mycotoxins. Mycotoxins were given intragastrically for a 50-day period. The average weight gain was reduced in the groups treated with mycotoxins. Mice developed specific IgG to T. gondii. Histopathological studies showed severe encephalitis in all groups infected. The number of unruptured and ruptured cysts was established and the severity of the lesions was evaluated, the groups treated with mycotoxins being the most severely affected. Immunohistochemical studies of the brain showed free antigen in tissues surrounding ruptured cysts. It is suggested that low and repeated doses of mycotoxins, necessary to produce a subclinical intoxication, precipitate Toxoplasma cyst rupture and consequently the activation of chronic toxoplasmosis.

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.

Interaction between cytochrome P450 2B1 and cytochrome bs: inhibition by synthetic peptides indicates a role for P450 residues Lys-122 and Arg-125.

Binding of cytochrome b5 to rat cytochrome P450 2B1 was inhibited (by 75%) by a synthetic peptide corresponding to P450 residues 116-134. The role of Lys-122 and Arg-125 were evaluated using peptides in which one or both of these basic residues were replaced with Glu. The Lys-122 substitution nearly abolished while the Arg-125 replacement decreased (by 20%) the inhibitory potential of the peptide. Substitution of both residues resulted in a peptide with no inhibitory activity. These results thus indicate a role for a specific P450 region as well as two basic residues within this region in the cytochrome P450-cytochrome b5 interaction.

Survey of Sarcocystis infection in cattle in east Hokkaido, Japan.

The prevalence of Sarcocystis infection was examined in older cows and imported cattle slaughtered in East Hokkaido. Samples of myocardial tissues were examined for Sarcocystis microscopically. Sarcocystis cysts were detected in 15.7% of 83 older cows, 48.4% of 91 imported cattle which were kept in East Hokkaido prior to slaughter and 51.1% of 94 imported cattle slaughtered immediately after quarantine check. Based on the morphology of the cyst wall and the establishment of infection in experimentally inoculated dog, the Sarcocystis species was identified as Sarcocystis cruzi.

Correlation between antibody levels in Toxoplasma gondii infected pigs and pathogenicity of the isolated parasite.

Sera and diaphragm muscle tissues were obtained from 109 commercial pigs between September 1991 and May 1992 from the slaughterhouse at La Plata, Provincia Buenos Aires, Argentina. Anti-Toxoplasma gondii IgG antibody reactivity to T. gondii antigens were assayed using sera by indirect immunofluorescence assay and immunoblotting technique. Anti-T. gondii IgG titers at serum dilutions of 1:1024 and higher were noted in 11.0% of the tested sera, and at dilutions of 1:16 and lower in 36.7% of the serum samples. Using mouse inoculation test, T. gondii was isolated from 14 pig diaphragm samples. Of five samples derived from pigs with antibodies at dilutions of 1:1024 and higher, four contained trophozoites which, when inoculated into mice intraperitoneally, killed all recipient hosts within 15 days post inoculation. Parasites detected in seven out of eight samples from pigs with antibodies at serum dilutions of 1:64 and lower formed cysts in the brain, and mice survived longer than 13 days post inoculation. Immunoblotting demonstrated antibody reactivity in pig sera samples with relatively high titers for parasite antigens. Results of the present study suggest that antibody production in infected pigs is apparently dependent on the pathogenicity of the parasite strain.