Lamellar events related to insulin-like growth factor-1 receptor signalling in two models relevant to endocrinopathic laminitis.

BACKGROUND: Insulin dysregulation, obesity, and exposure to high-nonstructural carbohydrate (NSC) forage are risk factors for equine metabolic syndrome-associated laminitis (EMSAL); high systemic insulin concentrations in EMSAL are proposed to induce cellular dysregulation in the digital lamellae through activation of the insulin-like growth factor-1 receptor. OBJECTIVES: To use a dietary challenge model (DCM) and a euglycaemic-hyperinsulinaemic clamp (EHC) model to assess lamellar growth factor-related signalling. STUDY DESIGN: Lamellar phospho (P)-protein concentrations of signalling proteins important in growth factor-related signalling were assessed in 2 models: 1) lean and obese ponies on a low- or high-NSC diet; and 2) EHC model using Standardbred horses. METHODS: Ponies stratified for body condition (lean [LN, n = 11] and obese [OB, n = 11]) were exposed to a low-NSC diet (LO, n = 5 per group for LN LO and OB LO) or a high NSC diet (HI, n = 6 per group for LN HI and OB HI groups) for 7 days. For the EHC model, horses were administered insulin (constant rate infusion [6 mIU/kg bwt/min] combined with 50% dextrose, EHC group, n = 8)] or saline (0.57 mL/kg bwt/h, CON group, n = 8) for 48 h. Immunoblotting was employed to assess concentrations of activated/phosphorylated and total protein for members of the PI3K/Akt/mTORC1 and Ras/ERK pathways in lamellar samples from both models. RESULTS: In the DCM, lamellar P-(Ser 240/244) RPS6 was increased in OB HI ponies (vs. OB LO, P<0.05); positive correlations existed (P<0.05; r>0.5) between Day 7 basal serum insulin concentrations and lamellar concentrations of P-p70S6K and P-(Ser 240/244) RPS6. In the EHC model, lamellar concentrations of P-Akt, P-p70S6K, P-ERK 1/2, P-p90RSK, and both P-(Ser 235/236) and P-(Ser 240/244) RPS6 were increased in the EHC group (vs. CON, P<0.05). MAIN LIMITATIONS: The primary limitations of this study are the small number of animals per group in the DCM study, and the fact that many animals did not develop laminitis as that was not the endpoint of either study. CONCLUSIONS: These results support further investigation of mTORC1/RPS6 signalling as a potential therapeutic target(s) in EMSAL. The Summary is available in Chinese - see Supporting Information.

Feline oncornavirus-associated cell membrane antigen. VI. Cytotoxic antibody in cats exposed to feline leukemia virus.

Feline leukemia virus (FeLV)-infected specific pathogen-free (SPF) cats, normal uninfected SPF cats, and healthy cats from leukemic households were tested for antibody reactive to the feline oncornavirus-associated cell membrane antigen (FOCMA)-containing target cell line FL-74 by microcytotoxicity and indirect membrane immunofluorescence. Of the infected SPF animals, 81% showed concordant reactivity for the two tests. In contrast, only 55% of the healthy cats known to be naturally exposed to FeLV for long periods showed such concordance. FOCMA antibody could not be detected in normal SPF cats by either indirect membrane immunofluorescence or microcytotoxicity. Most cats in the FeLV-infected SPF group developed antibody detectable by both procedures by the fifth week post inoculation. Antibody detectable by membrane immunofluorescence persisted in a high percentage (75-90%) of the animals throughout the observation period of 19 weeks; after 9 weeks, fewer cats had antibody that was also detectable by microcytotoxicity.

Feline leukemia virus infection: age-related variation in response of cats to experimental infection.

Sixty-seven specific-pathogen-free cats of various ages (newborn, 2 wk, 1 mo, 2 mo, 4 mo, and 1 yr) were inoculated ip with either the Rickard (R) or the Kawakami-Theilen (KT) strain of feline leukemia virus (FeLV). Susceptibility to FeLV was judged by induction of a) FeLV group-specific antigens (gsa) in leukocytes, b) FeLV-related disease, c) antibody to feline oncornavirus-associated cell membrane antigen (FOCMA), and d) virus-neutralizing (VN) antibody. Susceptibility to FeLV-decreased with age. Persistent viremia and FeLV-related disease developed in 100% of cats inoculated as newborns, in 85% of cats inoculated at 2 weeks to 2 months of age, and in 15% of cats inoculated at 4 months or 1 year of age. Cats susceptible to FeLV leukemogenesis became persistently FeLV gsa-positive (viremic) at 4 weeks post inoculation and thereafter and produced little or no FOCMA or VN antibody. Cats that resisted leukemogenesis by FeLV all developed persistent FOCMA and VN titers and never became FeLV gsa-positive. The disease in inoculated cats was influenced by virus strain; FeLV-R induced predominantly thymic lymphosarcoma, whereas FeLV-KT caused fatal nonregenerative anemia without concurrent neoplasia.

Infection of feline embryo adherent cells with feline leukemia virus: feline oncornavirus-associated cell membrane antigen expression and morphologic transformation.

Feline embryo adherent cells were infected with the Richard or Kawakami-Theilen strains of feline leukemia virus (FeLV) and examined for feline oncornavirus-associated cell membrane antigen (FOCMA), viral group-specific antigen (gsa) production, and in vitro evidence of transformation. As early as 10 days after infection, when more than half of the infected cells were gsa positive, FOCMA was detected on 5-10 percent of the cells. Transitory morphologic alterations (epithelioid appearance and rounding) were first noted in most cultures around 20-30 days post infection. At this time, approximately 50% of the cells in infected cultures expressed FOCMA. Morphologic characteristics of transformed fibroblastic cells (rounded shape, disordered alignment, and low adhesion to substratum), as well as enhanced agglutinability by plant lectins and ability to grow in agar, were demonstrated in one of four FeLV-infected, FOCMA-positive cultures. Findings showed that FOCMA may be expressed in FeLV-infected monolayer cells independent of transformation as assessed by in vitro criteria.

Inhibition of concanavalin A stimulation of feline lymphocytes by inactivated feline leukemia virus.

In a lymphocyte blast transformation assay, the response of feline lymphocytes to concanavalin A was suppressed 20 to 65 percent in the presence of inactivated feline leukemia virus. The decrease was not due to viral cytotoxicity, as determined by trypan blue viability counts, nor was the virus binding the concanavalin A and interfering with its mitogenic stimulation. The virus may be biochemically repressive in itself, interfering with cell-mediated immunity within the feline system.

Antibody in cats to mammalian RNA tumor virus interspecies antigens.

The studies were undertaken to determine whether the cat, a mammalian species that carries xenotropic endogenous C-type virus(es) and in addition undergoes horizontally transmitted oncogenic C-type RNA tumor virus infections, responds immunologically to the mammalian C-type virus interspecies antigens. Sera from normal cats and from cats with spontaneous or virus-induced neoplasms were examined for antibodies to interspecies antigen antigen by complement-fixation inhibition, by inhibition of the paired radioiodine-labeled antibody technique (PRILAT inhibition), and by two-step radioimmunoelectrophoresis. Using three separate complement-fixation inhibition systems designed to detect antibodies to interspecies antigen(s), 23 of 23 sera from tumor-bearing cats and 24 of 31 sera from normal cats were positive in both systems. The negative sera were from germ-free cats. Among the 49 positive sera, 47 yielded titers of 1:16 or greater by one or more complement-fixation inhibition tests. Of these 47 sera, 42 were positive by the paired radioiodine-labeled antibody technique inhibition test; the 5 paired radioiodine-labeled antibody technique-negative sera were from normal specific-pathogen-free cats. Direct reaction with the interspecies determinant on the p30 protein from Rauscher murine leukemia virus by immunoglobulin from cats immunized with feline leukemia virus was shown by two-step radioimmunoelectrophoresis. The feline antibody was also identified as an immunoglobulin by column chromatography and two-step radioimmunoelectrophoresis. These antibodies did not fix guinea pig complement during reaction with the interspecies antigen. That other mammals may produce similar noncomplement-fixing (guinea pig) antibodies to RNA tumor virus antigens is likely.

Inhibition of human lymphocyte mitogen and antigen response by a 15,000-dalton protein from feline leukemia virus.

Peripheral blood lymphocyte response of normal human subjects to mitogens and antigens was suppressed by a 15,000-dalton protein (p15) from a C-type feline leukemia virus. Four of six subjects were suppressed 70 to 96% when responding to concanavalin A or phytohemagglutinin in the presence of 5.0 microgram of p15. The three subjects who responded to streptokinase-streptodornase and Candida were suppressed 68 to 91% when cultured with 5.0 microgram of p15. The subviral protein did not appear to be cytotoxic at doses reported. Lymphocyte membrane studies with fluorescein isothiocyanate-concanavalin A revealed a reduction in concanavalin A-induced cap formation of 51 to 91% in the presence of the same dose of p15. These results demonstrate that in vitro immunological dysfunction in human lymphocytes can be induced by a C-type virion protein.

Relationship between feline leukemia virus antigen expression and viral infectivity in blood, bone marrow, and saliva of cats.

Correlation was greater than 90% between feline leukemia virus (FeLV), group-specific antigen (GSA) in leukocytes, and viral infectivity (VI) in serum or plasma from 132 cats infected with either the Rickard strain of FeLV, the Snyder-Theilen strain of feline sarcoma virus, or field strains of FeLV. Detection of GSA in blood cells was at least as sensitive as detection of VI in serum. In 45% of FeLV GSA-positive cats inoculated with FeLV-Rickard strain, VI was detected in saliva. No saliva samples from GSA-negative cats had VI. Sequential bone marrow biopsies from 34 cats inoculated with Snyder-Theilen feline sarcoma virus indicated that the correlation between FeLV GSA in bone marrow cells and blood cells was virtually 100%. FeLV GSA appeared in bone marrow leukocyte precursors 1 week before its appearance in peripheral blood leukocytes in 50% of the cats. The FeLV GSA-positive state was transient (3 to 6 weeks) in 34% of the Snyder-Theilen feline sarcoma virus-inoculated cats.

Mobility of lymphocyte surface membrane concanavalin A receptors of normal and feline leukemia virus-infected viremic felines.

The virus-associated depression of concanavalin A mitogenesis which accompanies feline leukemia virus-induced cat lymphoma was investigated by comparing lymphocyte surface receptor mobility of normal cats to that of viremic diseased animals. The mechanics of feline lymphocyte receptor mobility were studied using fluorescein-conjugated concanavalin A to quantitate lymphocyte capping. The results of a study of 21 disease-free animals showed that cat lymphocytes undergo appreciable concanavalin A capping, with a mean capping rate of 17% under conditions developed in this study. In contrast, morphologically normal peripheral blood lymphocytes of six feline leukemia virus-infected viremic cats, with or without lymphoma, exhibited a mean capping of only 7%, significantly less than that of the control animals (p less than 0.005). These findings suggest that a membrane-related lymphocyte deficiency accompanies the development of virus-induced lymphoma in the cat.

Abrogation of resistance to feline oncornavirus disease by immunization with killed feline leukemia virus.

Four-week-old specific-pathogen-free cats were immunized with a combined vaccine composed of killed feline leukemia virus and killed feline oncornavirus-associated cell membrane antigen-containing tumor cells. Immunization induced feline oncornavirus-associated cell membrane antigen antibody titers ranging from 1:32 to 1:256 but did not elicit detectable virus-neutralizing antibody titers. Kittens immunized with tumor cells alone developed higher feline oncornavirus-associated cell membrane antigen antibody titers (ranging from 1:512 to 1:2048) than those given the combined vaccine. All kittens were challenged with virulent Dynder-Theilen feline sarcoma virus at 12 weeks of age. Seventy-five % of the kittens vaccinated with combined vaccine and 67% of unvaccinated control kittens developed progressive fibrosarcomas after challenge. By contrast, none of the kittens vaccinated with killed tumor cells alone developed progressive fibrosarcomas after challenge. The combined vaccine did not, however, inhibit the induction of feline leukemia virus viremia.

Immunization against feline oncarnavirus disease using a killed tumor cell vaccine.

Specific pathogen-free cats were immunized with an inactivated feline oncornavirus tumor cell vaccine. Immunized cats produced high antibody titers to the feline oncornavirus-associated cell membrane antigen and were protected from oncogenic feline sarcoma virus challenge. However, immunization did not produce virus-neutralizing antibody nor did it prevent viremia.

Detection and evaluation of feline oncornavirus-induced cell surface antigen(s) shed from cells in vitro.

A method for preparation of soluble feline oncornavirus-induced cell surface antigens was described. This technique relied upon the natural release of antigen(s) from FL-74 feline lymphoblastoid cells during their maintenance at 37 degrees in serum-deficient medium. When concentrated and clarified spent medium from 4-day cultures was tested for its antigen content by inhibition of humoral cytotoxicity, it was found that this natural production of soluble antigen provided more feline oncornavirus-associated cell membrane antigen per cell than did a solubilization procedure in which papain was used. The shed antigen preparation was immunogenic in cats, eliciting humoral antibody that was reactive with the surface of FL-74 Cells and feline sarcoma virus-transformed nonproducer mink cells but was not reactive with feline leukemia virus in a virus neutralization assay.

Influence of adrenal corticosteroids on the susceptibility of cats to feline leukemia virus infection.

The natural resistance of adult specific-pathogen-free cats to feline leukemia virus (FeLV) was abrogated by treatment with various doses of a synthetic corticosteroid, methylprednisolone acetate (MPA), prior to either oral-nasal or i.p. inoculation of FeLV. Persistent viremia was induced in 82% (18 of 22) of MPA-treated cats versus 11% (1 of 9) of age-matched control cats. MPA-treated FeLV-inoculated cats developed prolonged lymphopenia (2 to 8 weeks postinoculation) and a delayed antibody response to the feline oncornavirus-associated cell membrane antigen. The distribution of FeLV group specific antigen in tissues of MPA-treated, FeLV-inoculated cats suggested that corticosteroids enhanced susceptibility to FeLV by impairing early viral containment in the reticuloendothelial and lymphoid tissues.

Increased susceptibility to feline leukemia virus infection in cats exposed to methylnitrosourea.

Exposure of adult specific-pathogen-free cats to methylnitrosourea resulted in increased susceptibility to infection by feline leukemia virus. A greater proportion of cats exposed to methylnitrosourea and feline leukemia virus (69%) became persistently viremic than those exposed to feline leukemia virus alone (17%). Segmented neutrophils were reduced by 90 to 99% within 3 days following exposure to methylnitrosourea, (15 to 20 mg/kg) whereas the effects on lymphocytes and erythrocytes, although less obvious, were also detected.

Inhibition of erythroid colony-forming cells by a Mr 15,000 protein of feline leukemia virus.

The effects of concentrated ultraviolet-inactivated feline leukemia virus (FeLV), the purified Mr 15,000 envelope protein (p15E) of FeLV, or the purified Mr 27,000 structural protein (p27) of FeLV on feline bone marrow mononuclear cells were studied in vitro in methylcellulose cultures. Whole virus and purified viral proteins were from the Kawakami-Theilen isolate of FeLV, which induces erythroid aplasia in cats. Bone marrow mononuclear cells from FeLV-negative young adult cats were preincubated with a medium control, ultraviolet-inactivated whole virus, or the p15E or p27 of FeLV, incubated in methylcellulose cultures for 2 days, and then observed for the formation of colony-forming units-erythroid (CFU-E) and colony-forming units-granulocyte/macrophage. The ultraviolet-inactivated Kawakami-Theilen isolate of FeLV at concentrations of 10 or 20 micrograms of viral protein/5 X 10(4) cells suppressed CFU-E to 66 to 56% of control values but had no significant effect on proliferation of colony-forming units-granulocyte/macrophage. p15E at concentrations of 0.1 to 0.2 micrograms/5 X 10(4) cells decreased CFU-E numbers to 0 to 1% of control values, whereas the same concentration of p27 did not alter CFU-E growth when compared with controls. Neither p15E nor p27 had a significant effect on growth of colony-forming units-granulocyte/macrophage. The erythrosuppressive effects of whole virus and an envelope-derived protein but not a structural core protein suggest that FeLV envelope proteins are important in the selective inhibition of erythrogenesis observed in vivo in FeLV-infected cats.

Immunosuppressive properties of a virion polypeptide, a 15,000-dalton protein, from feline leukemia virus.

The 15,000-molecular-weight polypeptide (p15) of feline leukemia virus (FeLV) was shown to impair normal lymphocyte function in vitro and to abrogate immunity to feline oncornavirus disease in vivo. FeLVp15 suppressed concanavalin A-induced blast transformation of normal feline lymphocytes by 68%, while other virion proteins had no effect. p15 suppression was not due to toxicity, nor was p15 a competitive inhibitor of concanavalin A binding. Capping of receptors for concanavalin A on normal feline lymphocytes also was inhibited by either inactivated FeLV or FeLV p15. Groups of cats were immunized with either killed feline oncornavirus-associated cell membrane antigen bearing tumor cells or tumor cells plus FeLV p15. After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats. The cats receiving p15 also had lower cytotoxic antibody titers against feline oncornavirus-associated cell membrane antigen (mean peak titer, 1:6) than did the non-p15 group (1:74). These data support the hypothesis that the immunosuppression in cats infected with FeLV is mediated by FeLV p15.

Experimental oncornavirus vaccines in the cat.

An experimental approach to the immunoprophylatic control of feline oncornavirus-mediated diseases has included induction of antivirus immunity and antibodies to the feline oncornavirus-associated membrane (tumor) antigens. A suitable model for exploring the effectiveness of killed oncornavirus vaccines in the cat has been provided by the use of feline sarcoma virus. Immunization of seven pregnant queens over a 6-week period with ultraviolet light-inactivated Gardner-Arnstein feline sarcoma virus resulted in significant protection among 12 kittens challenged with a tumor-forming Dose 90 at 7 days of age. This immunity was not present in kittens challenged at 35 days of age. Among 12 kittens born of queens immunized during pregnancy with ultraviolet light-inactivated Kawakami-Theilen feline leukemia virus and challenged with the same live virus at 4 days of age, significant protection was noted, ranging from prolongation of survival time to complete protection in 3 kittens. In general, the higher the antibody titer in the mother, the more effective the protection afforded the kittens. Immunization of 43 kittens during their first 5 weeks of life with the same vaccines used in adult cats did not immunize sufficiently to protect against feline sarcoma virus challenge at 5 weeks of age. Neutralizing antibody responses in these kittens were significantly lower than in pregnant queens. That kittens of this age are immunologically responsive was established, since complete protection of 9 kittens to feline sarcoma virus was obtained by immunization with a crude tumor extract inactivated with 5 to 7 megarads of gamma-irradiation. All these kittens developed feline oncornavirus-associated membrane antibodies while 3 developed demonstrable levels of virus-neutralizing antibodies. The results of these studies are believed indicative that killed virus vaccines and tumor vaccines can be effective immunoprophylatic measures in the control of RNA tumor virus oncogenesis in the cat. Developments in this model system should be relevant to any consideration given similar vaccines in humans.

Prophylactic ZDV therapy prevents early viremia and lymphocyte decline but not primary infection in feline immunodeficiency virus-inoculated cats.

Prophylactic zidovudine (ZDV) therapy was evaluated in the feline immunodeficiency virus (FIV)-inoculated cat model for HIV-1 infection in humans. ZDV treatment (30 mg/kg/day via continuous subcutaneous infusion) was initiated 48 h prior to virus inoculation and continued for 28 days. Transient plasma antigenemia evident in six of six untreated cats at week 2 post-inoculation (pi) was absent in the ZDV-treated cats although at 10 and 14 weeks pi (6 and 10 weeks after drug treatment), one of the ZDV-treated cats had low-level antigenemia. Both CD4 and CD8 lymphocyte numbers were consistently higher in the ZDV-treated cats when compared to both the FIV-inoculated untreated cats and the virus-naive, age-matched controls. CD4:CD8 ratios were lower for the ZDV-treated cats than either the FIV-inoculated untreated or virus-naive, control cats. The decreased CD4:CD8 ratios were the result of an increase in CD8 lymphocytes in the ZDV-treated cats while decreased ratios in the FIV-inoculated untreated cats were due to cell loss. Both ZDV-treated and untreated cats showed nearly identical FIV-specific antibody responses beginning 2 weeks pi. Polymerase chain reaction (PCR) results from blood lymphocytes showed that six of six ZDV-treated and six of six untreated cats were positive for FIV-specific gag sequences. Although primary infection was not prevented, these results suggest that prophylactic ZDV therapy deterred early systemic spread of infection mediated by viremia and delayed absolute CD4 and CD8 lymphocyte decline.

Recovery of soluble feline oncornavirus-associated cell membrane antigen from large volumes of tissue culture fluids.

Feline oncornavirus-associated cell membrane antigen (FOCMA), which is released spontaneously in tissue culture, was harvested from large volumes of culture fluid. Continuous-flow molecular filtration was the method of choice for concentrating the component. The yield of antigen recovered, as determined by cytotoxicity inhibition, was equal to that recovered with concentration in cellulose membrane tubing against polyethylene glycol and half that obtained by lyophilization. The latter two techniques were found to be impractical for handling large volumes of material. However, lyophilization was used for the final concentration step following volume reduction to approximately 300 ml in the molecular filtration system. Antigen yields were equivalent when removing salt by constant-volume molecular washing, prior to concentration in the filtration system, or when removing salt by conventional diffusion through cellulose membrane tubing.

In vivo evolution and selection of recombinant feline leukemia virus species.

Ecotropic feline leukemia viruses subgroup A (FeLV-A) is known to recombine with endogenous FeLV (enFeLV) env elements yielding polytropic FeLV-B viruses. However, scattered nucleotide differences exist between enFeLV env elements and corresponding sequences of exogenous FeLV-B isolates. To address this disparity, we examined recombinant FeLV (rFeLV) viruses obtained from three experimentally-induced feline thymic tumors, along with rFeLVs derived from one naturally-occurring thymic tumor. Two of the three experimental cats were challenged with a FeLV-A/Rickard preparation, while one cat received this FeLV-A along with a mixture of in vitro-generated rFeLVs. The FeLV-A/Rickard preparation employed in this study was shown to be free of detectable rFeLVs since no recombinant products were observed in this preparation following nested PCR analyses. For each of the four tumor DNAs, nucleotide sequence analysis was performed on multiple clones of rFeLV-specific PCR products derived from the surface glycoprotein (SU) portion of the recombinant proviral env gene. Relative to the parental enFeLV sequence used to generate the rFeLVs, a total of 19 nucleotide differences were found scattered within the SU region of the env gene in these in vivo-derived rFeLV clones. Most interestingly, this set of 19 differences led to complete sequence identity with natural FeLV-B isolates. Our results indicate these differences are present early in the in vivo evolution of recombinant viruses, suggesting that rFeLVs harboring these differences are strongly selected. We also present evidence indicating an in vivo selection pattern exists for specific recombinant species containing relatively greater amounts of enFeLV-derived SU sequence. This in vivo selection process appears to be gradual, occurring over the infection timecourse, yielding rFeLV species which have recombination structural motifs similar to those seen in natural FeLV-B isolates.