Influence of a new push-out test method on the bond strength of three resin-based sealers.

AIM: Compare the displacement resistance of AH Plus, Ad Seal and Real Seal on dentine discs (DDs) treated with 10% citric acid, 17% EDTA or 2.5% NaOCl, through a new push-out test method. METHODOLOGY: From the middle third of the roots of 15 maxillary central incisors, three dentine discs 1 ± 0.1 mm thick were obtained. On the axial surface of each dentine disc, three 1.2-mm-wide holes were drilled. In the third dentine disc from the same root, each hole was treated with one of three irrigating solutions for 30 s, rinsed with distilled water and dried. Each hole of the same dentine disc was filled with sealer, and the discs were maintained at 37 °C for 7 days. The dentine discs were fixed on a testing machine to test the shear strength. Data were compared using the univariate anova test with a 5% significance level. The Tukey test was used for multiple comparisons. RESULTS: The irrigating solutions did not affect the adhesion of the sealers (P > 0.05). There was no significant difference between the Ad Seal and AHPlus (P > 0.05); however, Real Seal had a significantly lower shear bond strength (P < 0.05). CONCLUSION: The use of different irrigating solutions did not affect resistance to the displacement of resin sealers. Real Seal sealer was less resistant than Ad Seal and AHPlus.

Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates.

Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.

Characterization and immortalization of woodchuck hepatocytes isolated from normal and hepadnavirus-infected woodchucks (Marmota monax).

Primary woodchuck (Marmota monax) hepatocytes from normal woodchucks and woodchucks with chronic woodchuck hepatitis virus (WHV) infection were cultured in either a conventional serum-containing medium or a serum-free medium. The de novo synthesis of the plasma proteins albumin, transferrin, fibrinogen, and complement C3 were identical under both conditions. However, expression of the WHV and the synthesis of nitric oxide were diminished under serum-free conditions. Primary woodchuck hepatocytes cultured in conventional, serum-containing medium were immortalized utilizing the simian virus 40 T antigen oncogene. Immortalized hepatic cell lines retained differentiated functions of nitric oxide synthesis and expression of complement C3. The woodchuck hepatocyte culture model will supplement current experimental methods, allowing investigation of hepadnaviral pathogenesis, including hepatocarcinogenesis in vitro.

Enhanced neutralization of feline immunodeficiency virus by complement viral lysis.

The ability of complement to inactivate feline immunodeficiency virus (FIV) was examined. Treatment of virus with complement plus sub-neutralizing titers of antiserum resulted in a significant reduction in the virus titer compared with treatment of the virus with complement or antibody alone. One of the mechanisms by which cat complement inactivates FIV was shown to be by viral lysis as determined by a reverse transcriptase release assay. Kinetic studies revealed that viral lysis is initiated soon after the addition of complement to a mixture of virus and antiserum. Treatment of FIV with normal non-complement-inactivated human serum resulted in virus inactivation and release of viral RT in the absence of specific antiserum. It appears that FIV activates complement directly through the classical pathway and that integrity of the membrane attack components is a requirement for FIV lysis by human serum. The vulnerability of two distinct isolates of FIV to complement lysis was compared using complement from different species. Oradell isolate was more sensitive to complement lysis than the Petaluma isolate as assessed by reverse transcriptase release. It appears that factors intrinsic to the virus isolate may influence the amplitude of complement-dependent viral lysis.

New enzyme immunoassays for the serologic detection of woodchuck hepatitis virus infection.

The woodchuck and the woodchuck hepatitis virus (WHV) have been used as a model of hepatitis B virus infection and its disease sequelas. Serologic responses to WHV infection have been described in previous reports from this laboratory by using virus-specific radioimmunoassays (RIAs) for WHV surface antigen, antibody to WHV core antigen, and antibody to WHsAg. In this study, we developed and evaluated new enzyme immunoassays (EIAs) for these WHV serologic markers. Relative to the established RIAs, the EIAs were either improved or comparable in their sensitivity and specificity, and in their utility for monitoring experimental WHV infection and classifying woodchucks into serological diagnostic categories. These EIA systems are amenable to the quantitative titration of antibodies and quantitation of WHV antigens in serum, and ultimately should allow improved resolution of virologic and humoral immune responses of woodchucks to WHV infection.

Characterization of two monoclonal antibodies against feline immunodeficiency virus gag gene products and their application in an assay to evaluate neutralizing antibody activity.

Monoclonal antibodies (MAbs) 3B7 and 1C11 were produced against the gag gene products of feline immunodeficiency virus (FIV). These MAbs reacted strongly with FIV p24 in Western blots (immunoblots) and recognized p50 with a lower intensity. They specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence and immunocytochemistry. Although neither MAb inhibited viral replication in vitro, they were useful in a simple assay for the detection and quantification of infectious virus and neutralizing antibody activity. The assay utilizes Crandell feline kidney cells and requires 4 days for completion. Neutralizing antibodies in cats were detected 3 to 4 weeks after experimental infection with FIV. Antibody titres progressively increased during the first year of infection reaching high titres which were maintained 2.5 years post-infection. The MAbs produced should be valuable reagents for the monitoring of viral replication in cells or tissues from FIV-infected cats and for other in vitro applications.

Antibody response to reverse transcriptase in cats infected with feline immunodeficiency virus.

The antibody response in cats to feline immunodeficiency virus (FIV) reverse transcriptase (RT) was followed for 3 years. Eight of the nine cats used in this study produced reverse transcriptase-inhibiting (RTI) antibodies. Relative inhibitory means of 2.9%, 18.4%, 33%, and 47% were found 6, 12, 24, and 36 months, respectively, after infection with FIV. The enzyme activity was suppressed by greater than or equal to 78% with the use of 100 micrograms of FIV-associated IgG. The RTI antibodies were FIV-specific, as they did not inhibit other mammalian retroviral polymerases, including feline leukemia virus RT. An RT-inhibition assay with sera in the presence of protein A and immunoblot analysis showed that antibody binding to FIV RT protein p62 is independent of antibody ability to block enzyme activity. Viral RT released by detergent-treated virus was stable for more than 6 weeks at 4 degrees C, whereas its activity was reduced by 50% after 2 weeks at 37 degrees C. Because significant concentrations of RTI antibodies are detected only at 1 to 2 years after infection, they can be used to determine the approximate time of virus infection and as a marker for disease progression.

Anti-idiotypic antibodies to feline leukemia virus: an approach for retroviral immunization strategies.

The present study describes an approach to the development and use of anti-idiotypic antibodies as a possible immunization strategy to prevent retroviral infection. The rationale for using anti-idiotypes (anti-Ids) to try to elicit an antigenic-specific immune response is examined, and the production and characterization of polyclonal and monoclonal anti-Ids are described. Several techniques were used to determine antigenic mimicry and anti-Id subtypes. The potential use of anti-Ids in feline leukemia virus (FeLV) receptor studies and vaccine trials in vivo were investigated. Results from these studies suggest that the anti-Id strategy is feasible for the FeLV model. Polyclonal Ab2 reagents were developed that blocked virus-receptor binding and thus inhibited viral infection in vitro and induced humoral immune responses in 6- to 8-week old kittens characterized by production of Ab3 with the ability to bind the original FeLV envelope protein gp70 as assessed by Western blot analysis.

Testing of nucleoside analogues in cats infected with feline leukemia virus: a model.

In the present communication we evaluate the feline leukemia virus (FeLV) infection of cats as a model for antiretroviral chemotherapy studies. Additionally, we report the results of testing the antiviral effect of the compounds, 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine, 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine against FeLV replication in vitro. Cumulative data from experiments in which FeLV-infected cats were treated with AZT at different stages of experimental infection are also evaluated.

3'-Azido-3'-deoxythymidine in feline leukemia virus-infected cats: a model for therapy and prophylaxis of AIDS.

Due to similarities between human immunodeficiency virus and feline leukemia virus, the etiological agents of acquired immunodeficiency syndromes in humans and cats, the feline system was used as a model to conduct preliminary investigations as to the efficacy of the thymidine analogue 3'-azido-3'-deoxythymidine (AZT) as a therapeutic and preventive agent against retroviruses. In vitro evaluations of AZT cytotoxicity and its antiviral effects were conducted. Subsequently, 50 6-week-old specific pathogen free kittens were inoculated with a highly immunosuppressive strain of Richard-Feline Leukemia Virus. These cats were randomly subdivided into smaller groups with initiation of AZT treatment at variable times postinfection. All animals were periodically monitored for circulating infectious virus particles and virus-neutralizing antibodies. Their clinical condition was closely followed throughout the 6-week AZT treatment phase and for several months thereafter. The results indicate that AZT prevents retrovirus infection if administered immediately following virus exposure, and may also reduce retrovirus replication if administered to previously infected animals. Some of the treated cats developed neutralizing antibodies against the virus and became resistant to subsequent viral challenge. Future trials with this drug, both for the prevention and treatment of retroviral diseases in humans and animals, are warranted.

Characterization of monoclonal antibodies directed against the envelope proteins of feline leukemia virus.

Monoclonal antibodies directed against the feline leukemia virus (FeLV) envelope proteins, gp70 and p15E, were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six of these monoclonal antibodies were specific for the gp70; two for the p15E. Enzyme-linked immunosorbent assay binding assays against FeLV subtypes A, B, and C showed that most of the monoclonal antibodies bound to more than one subtype but have a greater affinity for subtype B. One monoclonal bound exclusively to the FL74 isolate. These studies also indicate that antigenic variability exists between FeLV isolates previously classified as being the same subtype. One antibody was found to bind the gp70s of all FeLV isolates tested and to be directed against a viral neutralizing site. A p15E-specific monoclonal antibody, in addition to binding all the FeLV subtypes, also bound to Moloney and Rauscher murine leukemia viruses, suggesting a group determinant is involved. No binding was seen to human T-cell leukemia virus, bovine leukemia virus, equine infectious anemia virus, or RD114V proteins, however.

Experimental anterior uveitis after subcutaneous injection of feline sarcoma virus.

Feline sarcoma virus (FeSV) is a naturally occurring virus that causes spontaneous tumors in cats. The immunologic and morphologic characteristics of these tumors have been studied extensively. It was recently observed in experiments undertaken to induce systemic malignancy with this virus, that severe uveitis and clinical blindness occurred. An investigation of the ophthalmologic changes was undertaken. A fulminent anterior uveitis was produced in cats by a series of subcutaneous injections of live FeLV-FeSV. This intraocular inflammation occurred in five of six animals using high viral titers, and four of seven with lower titers, resulting from the freeze thaw process. On histopathologic examination, most animals demonstrated dysplastic changes of the ciliary body in addition to the iridocyclitis. The remainder of the eye was unaffected. These animals developed systemic tumors unaccompanied by local inflammation, many of which spontaneously regressed. Notable features of this potential model for uveitis are that (1) direct injection into the eye is unnecessary, and (2) intravenous administration inducing immune tolerance with antigenic overload presented to the spleen is avoided. This inflammatory reaction seems to be specific to the iris and ciliary body. Levels of live virus detected in the aqueous humor exceeded those in the serum. These results suggest that the virus may be actively secreted by the ciliary epithelium, or may preferentially proliferate within the eye.

Detection and localization of a phosphotyrosine-containing onc gene product in feline tumor cells.

Protein phosphorylation by a tyrosine-specific kinase is now recognized as a common event in retrovirus-transformed cells. We report in this communication that the feline sarcoma virus (FeSV) encoded transformation-specific proteins (gag-fes fusion proteins) and their associated protein kinases are also found in the FeSV in vivo induced tumor preparations, either in the form of fresh tumor homogenate or in the form of cultured cells. With the combined use of subcellular fractionation and detergent extraction we found that the protein kinase activity was present in both the membrane fraction (P100) and the cytosol (S100). The gag-fes proteins of two different strains of FeSV were found to associate with the cell framework to different degrees, suggesting that the specific conformational presentation of these proteins may be dictated by the unique portion of each polyprotein. The same gag-fes transformation related proteins could be immunoprecipitated with antiserum to phosphotyrosine.

Feline sarcoma virus-specific transformation-related proteins and protein kinase activity in tumor cells.

Polyproteins (gag-fes) encoded by the Synder-Theilen (ST) and the Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) were previously shown to be associated with mink or rat cells that were nonproductively transformed in vitro. In the present study we demonstrated that the same gag-fes proteins were found in cat cells transformed in vitro. Of greater importance, these transformation-related proteins were also in cells taken from fresh biopsies of FeSV-induced tumors. Cells from fibrosarcomas induced with ST-FeSV had gag-fes proteins that were characteristic of this strain. Fibrosarcomas and melanomas were induced with GA-FeSV and both types of tumors contained the protein that is characteristic of cells transformed in vitro with this virus. Expression of these proteins in cultured tumor cells appeared to be independent of the passage level. Based on two-dimensional tryptic peptide analysis, the gag-fes proteins of cat tumor cells appeared to be indistinguishable from those found in cells transformed in vitro. The polyproteins of the cat tumor cells have a closely associated protein kinase activity, as demonstrated in the in vitro assay, and phosphorylated tyrosine residues. Gag-fes proteins of either the ST or GA class were not present in cell cultures initiated from five spontaneous cat tumors.

Neutralization of feline leukemia virus with feline antisera to leukocyte alloantigens.

The possibility that normal cellular antigens might serve as targets for antibody neutralization of the feline leukemia virus was investigated. Xenospecific antiserum directed to normal feline cells was shown to inactivate feline leukemia virus grown in fibroblasts. Cat antisera to normal feline leukocyte alloantigens were then prepared, after which persistent viremia was induced in the donor cats. Such alloantisera neutralized virus taken from plasma of the appropriate cat but did not neutralize virus from a different cat. The virus neutralization was dependent on the presence of complement. These results indicate that alloantigens are present at the virus surface and raise the possibility that such antigens may play a role in the natural immune response directed to retrovirus infections.

Effect of bacille Calmette-Guerin immunotherapy on feline sarcoma virus-induced neoplasms in the cat.

Kittens infected experimentally with feline sarcoma virus (FeSV) were inoculated with emulsions of bacille Calmette-Guerin (BCG) cell wall preparations to determine the effect of BCG immunotherapy on FeSV-induced sarcoma-genesis. The BCG preparations or emulsificant control preparations were administered (i) subcutaneously at the same time and site as was the FeSV inoculation, (ii) at the same site but 1 week after FeSV inoculation, or (iii) with a mixture of viable autochthonous neoplastic cells approximately 35 days after FeSV inoculation. There was no difference in the percentage of kittens that developed neoplasms, the prepatent period for neoplastic development, the percentage of kittens with neoplastic regression, or the survival rate among groups given BCG, emulsificant control, or FeSV alone. Significantly (P less than 0.05) greater dissemination of neoplasms was seen in groups given BCG or emulsificant control preparations, compared with dissemination in groups given FeSV alone.

Protection of cats against progressive fibrosarcomas and persistent leukemia virus infection by vaccination with feline leukemia cells.

Young cats (3-6 mo old) were challenged with oncogenic Snyder-Theilen feline sarcoma virus (FeSV) after vaccination with live or killed FL74 cat lymphoma cells. Compared with controls immunized with normal cat fibroblasts, the FL74-vaccinated cats exhibited increased resistance to FeSV-induced progressive primary and disseminated secondary tumors. Maximum protection was achieved by vaccination with live FL74 cells or with a low dose of freeze-thawed cells, but tumor cells inactivated by glutaraldehyde or paraformaldehyde were also effective. Infectious helper feline leukemia virus (FeLV) was detected in the blood of all cats after FeSV challenge, but the duration and magnitude of this viremia were reduced in animals that had been previously vaccinated with live, freeze-thawed, or paraformaldehyde-fixed cells. Although immunized cats were resistant to FeSV-induced tumors and FeLV viremia, no evidence was obtained to suggest that vaccination with dead cells induced detectable circulating antibody prior to challenge with oncogenic virus. After FeSV challenge, complement-dependent antibody to feline oncornavirus-associated cell membrane antigen (CDA-FOCMA) appeared at high titer in cats that were destined either to survive tumor-free or to develop small, localized, and eventually regressing tumors. Cats immunized with live FL74 cells developed CDA-FOCMA prior to challenge, and antibody appeared in these cats following an episode of transient FeLV viremia induced by virus replicating from the injected tumor cells. Therefore, apparently, a state of transient or persistent FeLV viremia regularly preceded detection of CDA-FOCMA activity. Several individually derived feline lymphoma cell lines were used as targets for CDA-FOCMA, and the results suggested that lytic activity is directed to multiple antigen determinants expressed differently by individual feline lymphomas.