Patterns of expression of feline cytokeratins in healthy epithelia and mammary carcinoma cells.

Expression of keratins (cytokeratins, CK) in healthy feline epithelia and 2 established feline mammary carcinoma cell lines was examined immunohistochemically and by use of immunoblotting analysis. A panel of specific anti-CK monoclonal antibodies (MAb) identifying epitopes unique to individual keratins or shared by 2 (or 3) CK polypeptides was used. Besides already available anti-human CK MAb, this panel of MAb consisted of 9 newly generated anti-human CK MAb and 1 newly generated anti-feline CK MAb. Immunohistochemical analysis on normal epithelia revealed that most of the anti-human CK MAb and the anti-feline CK MAb reacted with both feline and human epithelia, with a comparable tissue distribution pattern. However, slight differences in CK tissue distribution pattern between human beings and cats were detected by one MAb. Immunoblotting analysis revealed that all anti-human CK MAb that were immunohistochemically reactive with feline tissues detected analogous CK in cats, indicating the presence of a number of common epitopes on human and feline CK. Two continuous cell lines derived from 2 distinct feline mammary adenocarcinomas, K248C and K266, were analyzed with respect to their CK phenotype. Although no difference in CK expression between the 2 cell lines was detected in vitro, a difference in CK phenotype was detected on subcutaneous transplantation of the 2 cell lines into nude mice. Although the K248C-induced adenocarcinomas maintained the same CK phenotype as observed in vitro, the CK pattern of the K266 heterotransplants, growing as adenosquamous carcinomas, changed with squamous differentiation. Our findings confirm the high degree of homology between mammalian CK, and on the basis of those findings, we suggest that CK proteins provide a set of markers valuable for the characterization of normal and neoplastic feline tissues and for studies of squamous metaplasia.     

Characterization of feline CD56 molecule expressed on insect cells by the baculovirus expression system.

Recently we cloned 140 kDa form of feline CD56 cDNA. In this study, we expressed the feline CD56 molecule by the baculovirus expression system. We found that the molecule was expressed on the cell surface when examined by the indirect immunofluorescence assay using an anti-human CD56 monoclonal antibody. Immunoblotting analysis revealed that the molecular weight of the major expressed product was 140 kDa. Interestingly we found that the insect cells expressing the feline CD56 molecule aggregated, indicating that the expressed molecule mediates homophilic adhesion.     

The detection of conventional class I and class II I-E homologue major histocompatibility complex molecules on feline cells

The presence on feline cells of class I and class II I-E type major histocompatibility complex (MHC) homologues was demonstrated using cross-reacting monoclonal antibodies (mAb). The feline class I antigen homologues were detected with both immunofluorescent and biochemical techniques, using the anti-human class I mAb W6/32. The class I antigens were detected on in vitro cultured feline fibroblasts and lymphoid cells, but not on fresh lymphoid cells, apparently as a result of the association of bovine beta-2 microglobulin with feline class I heavy chains which generated the determinant(s) recognized by mAb W6/32. Class II I-E-like molecules could be detected with immunofluorescent techniques using the species cross-reactive anti-mouse I-E antibody 40D only when peripheral blood mononuclear cells were activated, for example, with the mitogens staphylococcus enterotoxin A or lipopolysaccharide. The predominant expression of I-A-like molecules by resting class II-positive feline cells could explain some of the functional difference we have seen in comparison with those of most other mammalian species.     

Feline Hodgkin's-like lymphoma: 20 cases (1992-1999)

We identified 20 cases of feline lymphadenopathy that conform to many clinical and histologic manifestations of human Hodgkin's disease. Histologic subtypes encountered included lymphocyte predominance (nine cases), mixed cellularity (nine cases), and nodular sclerosis (two cases). Two cases were not easily classified; fibrous bands were present, but the absence of nodules supported a subclassification of mixed cellularity Hodgkin's disease. Immunohistochemical staining of the tissues using antibodies against the pan T-cell antigen CD3, the human B-lymphocyte antigen 36 (BLA.36), the pan B-lymphocyte and plasma cell marker CD79a, and a myeloid antigen (MAC387) confirmed the phenotypic heterogeneity of the tumor. Classic Reed-Sternberg (RS) cells and mononuclear, multinucleate, and lacunar cell variants did not stain with any of the antibodies used. In contrast, lymphohistiocytic RS variants (L+H cells) reacted positively to BLA.36 and CD79a B-cell markers. Eighteen of 20 affected cats were > or = 6 years of age (range, 1-14 years). A sex predilection could not be identified. These findings support the existence of Hodgkin's-like lymphoma in the cat. Proper identification of this disease in the cat will enable further characterization of clinical features and biologic behavior to determine whether there are significant differences in the treatment and prognosis of feline Hodgkin's-like lymphoma compared with non-Hodgkin's lymphoma.     

Feline one-way mixed leukocyte reaction

The feline one-way mixed leukocyte reaction (MLR) was accomplished by a microtechnique assay using gradient purified mononuclear leukocytes from the peripheral blood of specific-pathogen-free (SPF) cats. An eight day assay was required with an appropriate serum supplement of rabbit or cat serum and obtainable cell concentrations of 1×10⁵ mononuclear cells per well of each responder and stimulator population. This assay was devised as an $\text{in vitro}$ correlate of cell-mediated immunity and to demonstrate that feline histocompatibility differences can be detected in culture.     

Development of a monoclonal antibody-based sandwich ELISA for detection of guinea pig interleukin-2

Interleukin 2 (IL-2) is a T cell proliferation factor released by Th0- and Th1-type helper T cells and is an essential cytokine for immune responses. In the present study, recombinant glutathione S-transferase (GST)-guinea pig IL-2 (GPIL-2) fusion protein was prepared by Escherichia coli (E. coli) and by using this protein as an immunogen, monoclonal antibodies (mAbs) against GPIL-2 were produced to establish a basis for a research on immune responses in guinea pigs. Three stable hybridoma cell lines were established, and specific binding of each mAb to recombinant GPIL-2 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that all three mAbs were IgG1 and had kappa chain. Furthermore, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to three different epitopes. Thus, a sandwich ELISA based on the two mAbs specific to different GPIL-2 epitopes was developed for detection of GPIL-2, which had a sensitivity threshold of about 0.3 ng/ml of GPIL-2.     

p53 protein expression in conjunctival squamous cell carcinomas of domestic animals

The expression of p53 protein was investigated in eight formalin-fixed, paraffin-embedded conjunctival squamous cell carcinomas of five horses and one cow, dog and cat each by an immunohistochemical procedure in order to evaluate protein overexpression. Anti-human p53 protein mouse monoclonal antibodies known to be cross-reactive with p53 protein of the animal species examined were used. Positive p53 nuclear immunostaining was detected in five equine, one bovine and one feline cases. Conversely, no p53 immunostaining was found in the only canine case examined. These results demonstrate a frequent p53 overexpression in conjunctival squamous cell carcinoma that could be related to UV-induced mutations of the p53 tumor suppressor gene.     

Erythrophagocytic low‐grade extranodal T‐cell lymphoma in a cat

Abstract: A 13‐year‐old male castrated domestic shorthair cat was presented to the referring veterinarian with a 2‐month history of weight loss and lethargy. Splenomegaly, hepatomegaly, nonregenerative anemia, neutropenia, and hyperbilirubinemia were noted. Results of testing for feline immunodeficiency virus, feline leukemia virus, Toxoplasma gondii, and Mycoplasma sp. were negative. On cytologic examination of aspirates from the enlarged spleen and liver, a population of erythrophagocytic round cells was observed. Splenectomy and a liver biopsy were done which revealed a population of CD3+/CD79a– erythrophagocytic mononuclear round cells localized in the hepatic and splenic sinusoids. T‐cell PARR (PCR for antigen receptor gene rearrangements) analysis of bone marrow and spleen demonstrated a single band indicative of a clonal proliferation of T cells. Based on the marked splenomegaly, sinusoidal infiltration, lack of lymphadenopathy, and results of cytology, PARR, and immunophenotyping, a diagnosis of low‐grade extranodal T‐cell lymphoma was made. The cat was treated with chlorambucil and prednisolone; clinical and laboratory abnormalities resolved and the cat has remained clinically normal for 2.5 years. To our knowledge, this report documents the first case of an erythrophagocytic T‐cell lymphoma in a cat. The clinicopathologic findings were suggestive of hepatosplenic T‐cell lymphoma, a neoplasm described previously only in humans and dogs.     

Porcine lymphoblastoid cell lines of B-cell origin

Two lymphoblastoid cell lines were isolated from different pigs and were maintained in culture for over 100 passages or 20 months. These cell lines were characterized by their cell surface antigens, ability to stimulate a mixed lymphocyte reaction and production of immunoglobulin. When tested against a panel of monoclonal anti-cell surface antigen antibodies, only those monoclonal antibodies which detect porcine class I or II molecules reacted against the lymphoblastoid cell lines in a microcytotoxicity assay. The two pig cell lines could stimulate peripheral blood mononuclear cells in a mixed lymphocyte reaction. P-SC(1) and P-16(2) also demonstrated a dependency upon the presence of 2-mercaptoethanol for cell division. The secretion of pig immunoglobulin by P-SC(1) or P-16(2) was first demonstrated by ELISA using a polyclonal anti-swine IgG (heavy and light chain) serum. By the use of monoclonal anti-IgA, IgG or IgM antibodies in an enzyme-linked assay on Western blots of P-SC(1) or P-16(2) lysate/supernatant, the two cell lines were demonstrated to be producing a whole monomeric IgA molecule and a mu chain.     

Progress in the discovery and definition of monoclonal antibodies for use in feline research

The practice of veterinary medicine and research into both animal diseases and animal models of human disease are restricted by the scarcity of monoclonal antibodies (mAb) that react with animal proteins. One way to enlarge the repertoire of mAb to animal leukocyte differentiation antigens (LDA) is to test mAb specific to other species for cross-reactivity to the species of interest. We have tested a panel of 380 commercially available anti-human mAb for cross-reactivity to feline LDA. Twenty-six of these mAb cross-react with cat LDA and 19 others are of questionable cross-reactivity. Definition of mAb specificity in the cat is being investigated by multi-color flow cytometry (FCM) to compare test mAb specificity with that of mAb to known feline LDA. The addition of these cross-reactive mAb to the anti-feline mAb currently available will enhance studies in comparative medicine.     

Evaluation of Pax5 expression and comparison with BLA.36 and CD79αcy in feline non‐Hodgkin lymphoma

Paired box gene 5 (Pax5) is a widely used B‐cell marker for human and canine non‐Hodgkin's lymphoma (nHL); however, in the literature there is only one case report using Pax5 in a cat B‐cell lymphoma. The purposes of this study were to investigate the expression and detection of B‐cell specific activator protein (BSAP) using a monoclonal anti‐Pax5 antibody in feline nHL (FnHL) tissue samples to evaluate its diagnostic relevance as a B‐cell marker. A total of 45 FnHL samples in 45 cats were evaluated. B‐cell lymphoma was the most common immunophenotype (51.1%) for all the samples and T‐cell the most common immunophenotype (64.3%) for the gastrointestinal (GI) form. Pax5 stained 82.6% of all B‐cell lymphomas and no expression was found in any of the T‐cell lymphomas. Anti‐Pax5 antibody staining in FnHL is similar to that reported in human and canine counterparts and may offer an excellent B‐cell marker in cats.     

Evaluation of P-glycoprotein expression in feline lymphoma and correlation with clinical outcome

P-glycoprotein (Pgp) is a transmembrane protein pump involved in drug resistance in canine and human lymphoma. There are no published clinical studies evaluating Pgp expression in feline lymphoma. The purpose of this study is to evaluate the level of Pgp expression in feline lymphoma and correlate it with clinical outcome. Two human Pgp monoclonal antibodies, C219 and C494, were used to detect Pgp expression in tissue samples from 63 cats with lymphoma. Demographic results appear comparable to recently published feline lymphoma studies. The Kaplan–Meier median remission and survival times were 164 and 571 days, respectively. Fourteen cats had positive expression of Pgp using MAb C219, and 40 were positive with C494. Variables statistically associated with survival included bone marrow involvement, stage, substage, and use of radiation therapy as a part of treatment. Pgp expression as assessed by MAb C219 and C494 is not predictive of remission or survival time in cats with lymphoma.     

Monoclonal antibodies to human antigens recognise feline myeloid cells

Immunological techniques have been used to study the expression of a series of cell surface antigens in cat haemopoietic tissues. Forty-two monoclonal antibodies raised against well-defined antigens of human origin were tested by indirect immunofluorescence on feline blood, bone marrow, spleen and thymus. Myeloid cells from all tissues reacted with antibodies to CD9, CD10 and CD18 antigens. No antibodies specific for T or B lymphocytes were found to react with cat lymphoid cells. Osteoclasts, isolated from juvenile bone marrow, were found to express the 23C6 human osteoclast-specific antigen. The potential use of such antibodies in experimental and diagnostic veterinary haematology are discussed.     

Expression of recombinant feline serum amyloid A (SAA) protein.

Feline serum amyloid A (SAA) cDNA clone was isolated from a hepatic mRNA of a mixed-breed cat. The feline SAA cDNA clone contains 333 nucleotides and deduced amino acid sequence shows 87.4%, 73.9%, and 65.8% homology with dog, human and mouse SAA respectively. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified in the feline cDNA clone. Recombinant feline SAA (rfSAA) was expressed at high levels using pGEX bacterial expression system. Cleavage from the fusion moiety, and purification using glutathione-sepharose yielded pure soluble form of rfSAA. Antibodies generated against rfSAA were specific for feline SAA and showed no cross-reactivity with human SAA. Furthermore, antibodies against human SAA did not react with feline SAA. These results indicate that antigenicity of feline SAA is totally different from human SAA.     

Polymorphic expression in the CD8alpha chain surface receptor of African lions (Panthera leo).

Free-ranging African lion (Panthera leo) peripheral blood mononuclear cells (PBMC) were examined using flow cytometry and antibodies developed for use in the domestic cat to determine if phenotypic changes occurred in lion lymphocytes as a result of feline immunodeficiency virus (FIV) infection. The percentage of CD8 cells from lion peripheral blood was considerably lower than in the domestic cat. Lions with elevated levels of CD8+ cells were typically infected with FIV, similar to observations in the domestic cat. Antibodies against the alpha chain of the CD8 receptor (monoclonal antibody (mAb) 3.357) did not react consistently in all lions examined. Flow cytometric analysis determined that approximately 82 and 80% of the animals from Kruger and Hluhluwe-Umfolozi National Parks in South Africa reacted with the monoclonal antibody against the alpha chain of CD8 receptor, while only 17% of the lions in Etosha National Park in Namibia cross-reacted with the CD8alpha chain. There was no apparent correlation between FIV status and CD8alpha chain reactivity. The relative isolation of Etosha from the other two parks could explain the marked difference in CD8alpha chain expression and suggests that lions similar to other mammalian species demonstrate polymorphic expression of the CD8alpha chain (197).