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.     

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.     

Monoclonal antibodies to feline lymphocyte membranes recognize the leukocyte-common antigen (CD45R).

By immunization of BALB/c mice with a feline T lymphoblastoid cell line, MYA-1 cells, two types of lymphocyte-specific monoclonal antibodies (mAbs) were obtained. The 220/205/190 kd protein defined by 2F11 mAb is highly expressed on the surface of MYA-1 cells and another feline T lymphoma cell line, FL74 cells. The protein is also expressed on normal feline thymocytes, splenocytes and feline peripheral blood mononuclear cells (PBMCs). Another mAb, 17B10, caused similar results as those of 2F11 except for its low reactivity with FL74 cells. The second type of mAb, 15B3, defined the 220 kd protein. The reactivities of this mAb with MYA-1 cells, FL74 cells, PBMCs and feline splenocytes were lower than the former two mAbs, and did not react to feline thymocytes. On the other hand, 17B10 and 15B3 defined partial populations of MYA-1 and FL74 cells recognized by 2F11. The cells defined by the 2F11 and 17B10 are all leukocytes in spleen and lymph node. In contrast, 15B3 defined most of the cells in B cell area and partially in T cell area. These results suggested that 2F11 and 17B10 recognized the specific antigen of 220/205/190 kd of the leukocyte-common antigen (L-CA) family, CD45R, with different epitopes, and that 15B3 defined the distinct antigen of 220 kd on CD45R.     

Adaptation of feline immunodeficiency virus subtype B strain TM2 to a feline astrocyte cell line (G355-5 cells)

Based on receptor usage during infection, feline immunodeficiency virus (FIV) isolates can be divided into two groups; those that require feline CD134 (fCD134) as a primary receptor in addition to CXCR4 to enter the cells, and those that require CXCR4 only. Most primary isolates, including strain TM2, belong to the former group and cannot infect a feline astrocyte cell line (G355-5 cells) due to a lack of fCD134 expression. In a previous study, we found that G355-5 cells transduced with fCD134 (termed G355-5/fOX40 cells) were susceptible to strain TM2 and the inoculated cells became persistently infected. In this study, we examined the phenotype of the virus prepared from the persistently infected cells (termed strain TM2PI). Intriguingly, strain TM2PI replicated well in na?ve G355-5 cells and the inoculated G355-5 cells (termed G355-5/TM2PI cells) became persistently infected. The infection of TM2PI in G355-5 cells was inhibited by CXCR4 antagonist AMD3100 and TM2PI infected other fCD134-negative, CXCR4-positive cell lines, FeTJ and 3201 cells. Four amino acid substitutions were found in the Env protein of the strain TM2PI when compared with that of the parental strain TM2. Among the substitutions, the Env amino acid position at 407 of TM2PI was substituted to lysine which has been known to be responsible for the FIV tropism for Crandell feline kidney cells. The strain TM2PI will be useful for studying the receptor switching mechanism and FIV pathogenesis in cats.     

Carrier-state infection of feline T-lymphoblastoid cells with feline calicivirus

The susceptibility of feline T lymphocytes to feline calicivirus (FCV) in vitro was investigated using feline T-lymphoblastoid cell lines, namely MYA-1 and FL74 cells. The virus titers of supernatants in FCV-infected MYA-1 and FL74 cell cultures increased rapidly, and FCV antigens were also detected in the FCV-infected cells. There were slight differences in the molecular weights of capsid proteins expressed in FCV-infected MYA-1, FL74 and Crandell feline kidney cells. MYA-1 and FL74 cells were productively and persistently infected with FCV, and FCV antigens were observed in the FCV-infected cells for more than one month. At 3 months post infection, FCV-infected FL74 cells that stopped producing infectious FCV could be reinfected with FCV. However, no cytopathic effects were observed.     

Generation of monoclonal antibody against canine neural-cell adhesion molecule

A monoclonal antibody, K9BYU, was generated using Escherichia coli recombinant extracellular domain of canine neural-cell adhesion molecule (N-CAM) as an antigen. Immunoreactivity of K9BYU to insect cell recombinant canine N-CAM was demonstrated by Western blotting using Sf9 insect cells transfected with the canine N-CAM gene. In Western blotting against canine brain tissue, K9BYU detected three isoforms of N-CAM that correspond to three major isoforms of human and mouse N-CAM (N-CAM-120, -140, and -180). From these results, K9BYU was considered to be a useful tool for research of canine N-CAM.     

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.     

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8⁺ peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8⁺ cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3⁺, CD4^?, CD21^?, CD5^lo, α/βTCR⁺, and γ/δTCR^? contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8^? cells. This subset of CD8⁺ lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8^? cells. In contrast, CD8⁺ cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8⁺ cells are an important subset associated with NK cytotoxic activity.     

Involvement of cytolytic and non-cytolytic T cells in the control of feline immunodeficiency virus infection

The appearance of non-cytolytic T cells that suppressed feline immunodeficiency virus (FIV) replication in vitro, and FIV-specific cytotoxic T cell (CTL) responses was compared in a group of seven, specific pathogen free (SPF) domestic cats following primary infection with the Glasgow(8) isolate of FIV (FIV(GL-8)). FIV proviral burdens were quantified in the blood and lymphoid tissues by real-time PCR. Non-cytolytic T cell suppression of FIV replication was measured by co-cultivating lymphoblasts prepared from the cats at different time-points during infection with FIV-infected MYA-1 cells in vitro. Non-cytolytic suppressor activity was detected as early as 1 week after infection, and was evident in all the lymphoid tissues examined. Further, this activity was present in subpopulations of T cells in the blood with normal (CD8(hi)) or reduced (CD8(lo)) expression of the CD8 molecule, and temporal modulations in non-cytolytic suppressor activity were unrelated to the circulating CD8(+) T cell numbers. Virus-specific CTL responses, measured by (51)Cr release assays, were not detected until 4 weeks after infection, with the emergence of FIV-specific effector CTLs in the blood. Throughout infection the response was predominantly directed towards FIV Gag-expressing target cells, and by 47 weeks after infection CTL responses had become localised in the lymph nodes and spleen. The results suggest that both non-cytolytic T cell suppression of FIV replication and FIV-specific CTL responses are important cellular immune mechanisms in the control of FIV replication in infected asymptomatic cats.     

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, −a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, −a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4⁺ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4⁺ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

Characterization of a monoclonal antibody identifying a CD45RA antigen on feline leukocytes

The antibody produced by a murine hybridoma obtained from the fusion of SP2/0 plasmacytoma cells with splenocytes of a mouse immunized with feline bone marrow was found to react with 60% of bone marrow cells and 80% of peripheral blood leukocytes (PBL); reactivity in the latter tissue was restricted almost entirely to mononuclear cells. Two-color FACScan analyses of this antibody with mAbs specific for feline lymphocytes revealed positive and negative populations of CD4 and CD8 cells. The reactivity for CD4 and CD8 cells was animal age dependent, binding to a higher percentage of the cells in young (2-9 months) versus older animals (> 4 years). In a mitogen driven assay for IgG production by PBL the addition of this antibody to the cultures enhanced the suppressor activity of CD8 cells, a function attributed to activation of a CD4 suppressor-inducer population; removal of CD8 cells negated any induction of suppression. Mild papain digestion of bone marrow and PBL completely removed the antigen detected by this antibody while not affecting reactivity of a pan-T antibody. Western blot analysis showed binding of the antibody to polypeptides of approximately 200 kDa on feline bone marrow and PBL. The data suggest that this mAb is identifying the feline homologue of the leukocyte common antigen of cells with a functional specificity characteristic of a CD45RA isoform.     

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain.

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.     

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.     

Molecular cloning and sequencing of feline CD7

Human CD7 is one of the earliest molecules to appear in T cell development. In this study, putative feline CD7 cDNA was identified based on its similarities with human and mouse CD7 genes. The feline CD7 cDNA contained an open reading frame consisting of 630 nucleotides. The amino acid sequence of feline CD7 had 47.7% identity with that of human CD7, and 52.9% with that of mouse CD7. In addition, the feline CD7 protein fused with histidine tag was expressed in 293T cells. The expression was confirmed by indirect immunofluorescence assay.     

Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

Enhanced tyrosine hydroxylase expression in PC12 cells co-cultured with feline mesenchymal stem cells

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson''s disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p<0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.     

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.