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Biomedical subjects

C K Grant

Publications and source records attributed to C K Grant.

At least 37 records · Page 2Linked to original sources
PubMed1987

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.

Amino Acid Sequence↗
PubMed1987

Natural feline leukemia virus variant escapes neutralization by a monoclonal antibody via an amino acid change outside the antibody-binding epitope.

We have molecularly cloned a natural variant of feline leukemia virus subtype B. This isolate is unique in that it is not neutralized by a monoclonal antibody which neutralized all other feline leukemia virus isolates tested, including members of the A, B, and C subtypes. Western immunoblotting indicated that the monoclonal antibody was less able to bind to the gp70 of the resistant isolate (designated lambda B1) than to the gp70s of susceptible viruses. Nucleotide sequence analysis of the envelope gene of lambda B1 revealed a high degree of homology with the susceptible Snyder-Theilen, Gardner-Arnstein, and Rickard subtype B isolates, including the presence of a 5-amino-acid minimal binding epitope required for binding by the neutralizing monoclonal antibody. The only change within the vicinity of this epitope was in a single nucleotide, and this difference changed a proline residue to leucine three amino acids from the N terminus of the binding epitope. Competitive binding studies with synthetic peptides indicated that substitution of leucine for proline resulted in a 10-fold decrease in the ability of the peptide to compete for antibody binding to native antigen. The results are consistent with the interpretation that this amino acid change lowers the affinity of antibody binding, resulting in failure of the antibody to neutralize the variant virus.

Amino Acid Sequence↗
PubMed1987

Retrovirus-induced feline pure red cell aplasia. Hematopoietic progenitors are infected with feline leukemia virus and erythroid burst-forming cells are uniquely sensitive to heterologous complement.

Feline leukemia virus subgroup C/Sarma (FeLV-C) induces pure red cell aplasia (PRCA) in cats. Just before the onset of anemia, erythroid colony-forming cells (CFU-E) become undetectable in marrow culture, yet normal frequencies of erythroid burst-forming cells (BFU-E)- and granulocyte-macrophage colony-forming cells (CFU-GM) persist. To determine if erythroid progenitors were uniquely infected with retrovirus, marrow mononuclear cells from cats viremic with FeLV-C were labeled with monoclonal antibodies to gp70 and then analyzed with a fluorescence-activated cell sorter. Both erythroid and granulocyte-macrophage progenitors were among cells sorting positively, suggesting that infection of BFU-E alone did not result in PRCA. The results were confirmed by complement (C') lysis studies using baby rabbit or guinea pig sera as sources of C'. These studies also suggested that BFU-E from cats with PRCA were unusually sensitive to C' alone, without the addition of antibody. In further studies, we demonstrated that C' activation was via the classical pathway and that C' sensitivity was unique to BFU-E and not a property of CFU-E, CFU-GM, or progenitors that were capable of giving rise to BFU-E in suspension culture. As BFU-E from cats viremic with FeLV-A/Glasgow-1 or the Rickard strain of feline leukemia virus were not sensitive to C', this finding may relate to the pathogenesis of feline PRCA. We hypothesize that, in cats viremic with FeLV-C, the abnormal C' sensitivity of BFU-E leads to the absence of CFU-E and anemia.

Animals↗
PubMed1984

Lymphosarcoma and persistent feline leukemia virus infection of pet cats: a system to study responses during extracorporeal treatments.

Pet cats, persistently infected with feline leukemia virus and having lymphosarcoma, were treated by extracorporeal immunoadsorption using Staphylococcus aureus Cowan I (which has protein A on its surface) to remove immunoglobulin G and circulating immune complexes. Complete viral clearance and long-lasting tumor regression were achieved following such treatments in 9 of 16 cats. In addition, in vitro immunologic assays have been developed that are useful in the clinical evaluation of cats during treatment.

Animals↗
PubMed1984

Comparison of feline leukemia virus-infected and normal cat T-cell lines in interleukin 2-conditioned medium.

Following mitogen stimulation (phytohemagglutinin or concanavalin A), feline lymphocytes were maintained in medium containing feline interleukin 2. Lymphocyte lines derived from the blood of feline leukemia virus-infected cats contained cells 50 to 90% of which expressed virus antigens, but lymphocyte lines derived from uninfected cats remained virus free. Leukemia virus-positive and -negative lymphocyte lines retained total dependence upon the presence of interleukin 2 for finite life spans of 20 to 40 cell divisions. Cell-doubling times and surface properties (membrane immunoglobulin negative, guinea pig red cell rosette positive) of all T-cell lines were similar. All cat lymphocyte lines rapidly developed strong but nonspecific cytotoxic effects against a variety of established cat target lines, including normal and leukemia virus-infected fibroblasts and virus-producing lymphomas. Attempts to infect virus-negative lymphocytes with leukemia virus in vitro produced lines containing 1 to 4% of infected cells; subsequently, this level of infection remained constant. Within observation limits, the characteristics of feline leukemia virus-infected and normal cat T-cells were similar. Leukemia virus infection did not predispose target lymphocytes to exhibit properties in vitro that might be associated with preneoplastic change, such as rapid or infinite cell division or development of independence from interleukin 2 regulation.

Animals↗
PubMed1983

Feline cytotoxic immune mechanisms against virus-associated leukemia and fibrosarcoma.

Humoral and cellular cytotoxic immune mechanisms of cats were compared against feline leukemia virus (FeLV)- and feline sarcoma virus (FeSV)-transformed cells. The groups of animals studied were nonexposed control cats; FeLV-infected immune or viremic tumor-bearing cats; FeSV-inoculated tumor progressor or regressor cats, and cats immunized with FeSV-transformed autochthonous fibroblasts (ATF). Sera containing complement-dependent antibodies (CDA), which lysed FeLV-producer lymphoma lines, had no cytotoxic effects when tested against FeLV-producer FeSV-transformed fibroblasts. Sera with lytic CDA activity were also tested for antibody-dependent cellular cytotoxic (ADCC) effects with peripheral blood lymphocytes (PBL) from nonimmune cats. No ADCC activity was detected against either lymphoid or fibroblast target lines. To demonstrate that cat PBL contained ADCC effector cells, antibody-coated murine target cells were employed and positive results obtained. Natural killer (NK) assays were performed using PBL from normal and tumor-bearing cats. Cytotoxic effects were only detectable to FeLV-producer lymphomas, and comparable levels of NK activity were found in normal and lymphoid tumor-bearing animals. In cats immunized with ATF, a population of effector cells was found in peripheral blood which had functional characteristics of cytotoxic T lymphocytes (CTL). The killing of ATF by CTL-like cells was not inhibited by FeLV/FeSV immune sera or by sera from autochthonous immune cats. The comparative importance of humoral and cellular cytotoxic mechanisms against FeLV- and FeSV-induced tumors is discussed.

Animals↗
PubMed1983

Cellular immune response in the blood of cats is restricted to autochthonous feline sarcoma virus-transformed cells.

The blood-borne cytotoxic cellular immune response of cats to autochthonous fibroblasts transformed with feline sarcoma virus (FeSV) was examined. Peripheral blood lymphocytes (PBL) from immune animals were assayed for cytotoxic activity using a 51Cr release microcytotoxicity assay. The time courses for appearance of cytotoxic cells were similar for all cats: effector lymphocytes appeared in the blood 7 days after immunization; peak activity occurred about day 16, and cytotoxic PBL were no longer detectable by 35 days. The specificity of cytotoxic lymphocytes was studied using autochthonous and allogeneic targets. PBL from each of five immunized cats killed autochthonous transformed fibroblast (ATF) targets, but the immune lymphocytes were not cytotoxic for non-transformed autochthonous fibroblasts, for allogeneic fibroblasts transformed with FeSV, or for an allogeneic lymphoma cell line which produces feline leukemia virus (FeLV). Specific restriction of target-cell killing to the ATF occurred at all effector:target-cell ratios tested, and was maintained throughout prolonged assay incubation periods. The results suggest that for cytotoxicity to occur it is necessary for the immune lymphocytes to recognize a combination of virus-associated antigens and "self" antigens on the ATF. The similarities between this restricted killing and that caused by cytotoxic T lymphocytes in other mammalian systems are discussed.

Animals↗
PubMed1983

Feline leukemia virus envelope gp70 of subgroups B and C defined by monoclonal antibodies with cytotoxic and neutralizing functions.

Nine murine monoclonal antibodies (MAb) to the envelope proteins of feline leukemia virus (FeLV) are described. Eight MAb are directed to epitopes of the same molecular species of gp70 and the other MAb is directed to the p15E moiety. Six of the gp70 epitopes are discrete; two are closely associated or overlapping. Four anti-gp70 MAb (2 of IgG2A and 2 of IgG2B subclasses) were directly cytotoxic for FeLV-producer lymphoma cells with cat or with rabbit complement (C). Another MAb (IgG2B), which was not cytotoxic alone, specifically and synergistically increased the cytotoxic effects of both IgG2A MAb. Cytotoxic anti-gp70 MAb also had virus-neutralizing capacity; one MAb recognized a determinant common to all FeLV subgroups (A, B, and C), the others recognized gp70 epitopes not present on subgroup A but common to both B and C subgroups. Competitive inhibition of MAb binding was employed to map spatial distributions of the epitopes, and the results fitted a molecule shaped as an incomplete loop. According to the model, epitopes involved with cytotoxic and virus neutralizing antibody functions were closely associated; the region involved is approximately in the center of the molecule, and it contains epitopes that are variably expressed among individual isolates of FeLV derived from different cat lymphoma cell lines.

Animals↗
PubMed1981

Feline leukemia - unique and cross-relating antigens on individual virus-producing tumors identified by complement-dependent antibody.

Following exposure to feline leukemia virus (FeLV), cats that resist leukemia produce complement dependent antibodies (CDA) which lyse leukemia cells in the presence of cat complement. The CDA activity has been shown to be directed to feline oncornavirus-associated cell membrane antigen (FOCMA) found on the surface of most feline leukemias. Despite the retrovirus etiology of feline leukemia, and the ubiquity of FOCMA expression by individual tumors, we found that CDA activity did not cross-react when five feline leukemia lines were compared (FL74, F422, 3272, 3281, and 79-14940). Profiles for CDA mediated lysis of the leukemia target lines were determined for sera from 149 FeLV exposed cats, and animals were identified whose sera would selectively lyse almost any possible combination of tumors from one to five lines tested. Cross absorption and cold target inhibition studies were performed on selected sera, and a spectrum of different antigens was identified which included both individual determinants and antigens shared by some of the tumor lines. Leukemias FL74 and F422 carried a cross-reactive determinant which was distinct from a second antigen shared by 3272 and 3281; lines F422, 3272 and 3281 each also carried a unique antigen. Leukemia 79-14940 did not cross-react with other target tumor lines. Targets of CDA mediated lysis on leukemia cells maybe associated with normal feline cell surface antigens, because CDA titers were sometimes reduced after serum absorption with normal cat spleen cells. The relevance of multiple FOCMA determinants is discussed in terms of tumor escape from immunity.

Animals↗
PubMed1981

Rejection of allogeneic tumor cells growing in mouse cerebrospinal fluid. Functional analysis of the inflammatory process.

Mice injected intracerebrally (i.c.) with mastocytoma cells reject the tumor, which grows rapidly in cerebrospinal fluid (CSF), within 7 or 8 days. The characteristics of the inflammatory exudate have been examined by several different, functional criteria. Potent cytotoxic T-lymphocyte populations are readily demonstrated in CSF, and both phagocytic and non-phagocytic cells capable of mediating antibody-dependent cell-mediated cytotoxicity are also present. However, no tumor-specific antibody was detected in CSF of mice that had recently rejected the tumor, using sensitive complement-dependent and lymphocyte-dependent cytotoxic assay systems. The process of cell-mediated tumor rejection does not obviously compromise the blood-CSF barrier to immunoglobulins.

Animals↗
PubMed1980

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.

Animals↗
PubMed1980

Natural feline leukemia virus infection and the immune response of cats of different ages.

Forty-two kittens and 28 adult cats were placed as tracers in leukemia cluster environments in contact with resident cats, 30% of which were persistently infected with feline leukemia virus (FeLV). After 7 months exposure, FeLV viremia had been detected in 71% of the tracer kittens, although only 55% of these remained persistently infected; in the same period, 11% of tracer adults became infected, but by 2 years the figure reached 43%. Mean latent periods before detection of viremia were 3.4 +/- 1.8 (S.D.) and 13.0 +/- 5.9 months for kittens and adults, respectively. First detection of FeLV infection was accompanied by a sharp although transient drop in peripheral white blood cell numbers, and infection onset triggered the humoral immune response which was comprised of separate antibodies with virus-neutralizing and tumor lysis activities. High titers of virus-neutralizing antibody appeared in transiently viremic cats immediately following elimination of viremia; this antibody was rarely detected in cats that remained persistently viremic. Lytic complement-dependent antibody to feline oncornavirus-associated cell membrane antigen appeared in most cats 1 to 2 weeks after FeLV infection was first detected, and subsequently high titers of this antibody remained in both transiently and persistently infected cats. If the rate of FeLV infection was summarized by using viremia and/or antibody appearance, then 95% of the kittens became infected within 1 year and 61% of the adults within 2 years. Adult cats are, therefore, susceptible to FeLV infection following long-term natural exposure, and their apparent resistance cannot be attributed to a protective humoral immune response that developed immediately after exposure commenced.

Aging↗
PubMed1979

Leukemia specific antigens: FOCMA and immune surveillance.

In cats, horizontally transmitted viruses cause leukemia and lymphoma under natural conditions. As with other retroviruses, feline leukemia virus (FeLV) contains products of 3 major genes; the virus core gag gene products, the polymerase, and the virus envelope glycoprotein. When cells are transformed in vitro by the related feline sarcoma virus (FeSV), an additional protein, FOCMA is expressed at the cell membrane. FOCMA, which is FeSV-coded, is transformation and/or tumor specific and expressed regardless of whether or not the cells make virus or contain virus structural antigens. Lymphoid leukemia cells also express FOCMA, both when FeLV is used to induce the disease in laboratory cats and when the tumors occur under natural conditions. FOCMA is expressed on both T and B lymphoid leukemia cells, but not expressed on non-malignant lymphoid cells, even when they are infected with FeLV. About one-third of the naturally occurring lymphoid tumors of cats lack detectable FeLV proteins and varying portions of the FeLV provirus. Despite this, they regularly express FOCMA, which is the target of an immuno-surveillance response that functions effectively under most conditions. FOCMA thus provides a useful model for antigens that might be expressed in "virus-negative" leukemias of man.

Animals↗