Effect of anti-Ia monoclonal antibodies on in vitro immune responses of canine cells

Four murine monoclonal antibodies (MAB) B1F6, ISCR3, HB10a, and 7.2, specific for framework determinants of murine and human Ia, were tested for their effect on mixed lymphocyte culture, cell-mediated lympholysis, and mitogen assays of canine cells. All 4 MAB were reactive with monocytes and surface immunoglobulin-positive, as well as Thy-1 antigen-positive mononuclear cells, as determined by immunocytofluorescence. The mixed lymphocyte culture response was inhibited when B1F6 or HB10a was added to cultures at the beginning of the assay (day 0). However, cultures underwent a normal response in the presence of ISCR3 or 7.2. None of the MAB was able to inhibit cell-mediated lympholysis or mitogen reactivity to concanavalin A, pokeweed mitogen, or phytohemagglutinin. When effector cell populations of the 3 assays were cytolytically treated with each of the 4 MAB and complement, immune reactivity was consistently lost, indicating that effector cells or accessory cells supporting the cell populations had been eliminated. These data indicate that Ia-like antigens are expressed on canine mononuclear cells and that antigens recognized by different MAB may have different functional relevance, as defined by the mixed lymphocyte culture assay.     

Phenotypic characterization of canine lymphoma, using monoclonal antibodies and a microlymphocytotoxicity assay

Cells acquired from lymph node biopsy specimens obtained from 58 dogs scheduled to undergo chemotherapy for lymphoma were immunophenotyped, using a microlymphocytotoxicity (MLCT) assay comprising a panel of well-characterized monoclonal antibodies (MAB) specific for canine cell surface antigens. Cells from 54 of the dogs concurrently were tested cytofluorometrically, using surface immunoglobulin (SIg) as a marker for B cells and the MAB DT2 specific for peripheral blood T cells. The MLCT results indicated frequent coexpression of antigens identified by DT2 antibody and, to a lesser extent, by 1A1 antibody on SIg-positive cells, suggesting that these antigens may be associated with other types of less-differentiated lymphoid cells, in addition to being associated with mature T cells. Class-II major histocompatibility antigens, as recognized by MAB H81.98.71, HB10a, and H40.315.7, were detected on most SIg-positive cells, but generally were lacking on SIg-negative, DT2-negative cells. The MAB Wig4, reactive with canine monocytes, recognized relatively few cells (11 of 58). Response to chemotherapy was not correlated with reactivity to MAB DLy6 specific for resting lymphocytes or to MAB W3G10 specific for a polymorphic antigen associated with the canine major histocompatibility complex. The MLCT assay appears to be efficient, rapid, and inexpensive for immunophenotyping cells from lymphoma biopsy specimens.     

Immunohistochemical staining and radionuclide imaging of canine tumors, using a monoclonal antibody recognizing a synthetic carbohydrate antigen

The in vitro and in vivo binding of a monoclonal antibody (MAB) that recognizes a tumor-associated carbohydrate antigen was studied in dogs. Monoclonal antibody 155H.7 was raised in response to innoculation of mice with beta-galactose(1-3)beta N-acetylgalactosamine conjugated to human serum albumin. Avidin-biotin-complex immunohistochemical staining of cryostat sections of normal and neoplastic canine tissue specimens revealed heterogenous binding of MAB 155H.7 to the cells of many canine mammary and lung carcinomas and homogenous staining of many sarcomas, including osteogenic sarcoma. In addition, there was variable staining of a variety of normal tissues including some ductual epithelium, peripheral nerve fibers, and some endothelial cells and fibroblasts. Immunoscintigraphy with 131I-labeled MAB 155H.7 was used to study the in vivo distribution of the antibody. The 131I-labeled MAB 155H.7 was administered to 1 clinically normal dog, 7 dogs with osteogenic sarcoma, 1 dog with undifferentiated sarcoma, and 2 dogs with mammary tumor. Scintigraphy revealed concentration of radioactivity in 8 of 10 tumor sites within 24 hours after MAB administration. The ratio of 131I in tumor sites to 131I in the surrounding normal tissues, compared with the similar ratio of 99mTc-labeled erythrocytes ranged from 1.1 to 4.3, in tumor vs normal tissue with a mean value of 2, confirming tumor localization of the radiolabeled MAB in excess of that associated with enhanced tumor vascularization.     

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.     

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.     

CD34 protein is expressed in murine,canine, and porcine lungs

The cell surface protein CD34 is expressed in various human tissues and cells, including hematopoietic stem cells, vascular endothelial cells, mucosal dendritic cells, mast cells, eosinophils, microglia, fibrocytes, muscle satellite cells, and platelets. There is a lack of data on the expression of CD34 in canine and porcine tissues. Therefore, we designed a series of immunoblotting, immunohistochemistry, and immunofluorescence experiments to observe CD34 expression in murine, canine, and porcine lungs. We used a rabbit antibody (clone EP373Y) to target the conserved human CD34 C-terminal region and validated its immunoreactivity against mouse lung homogenates. The data showed diffuse bronchiolar and alveolar epithelial localization of CD34 protein in normal murine, canine, and porcine lungs. At 9 or 24 h after bacterial endotoxin exposure, murine CD34 protein shifted to specific bronchoalveolar cells with a punctate pattern, as quantified by CD34 fluorescence. Specific porcine bronchoalveolar cells and leukocytes had significant CD34-positive immunostaining after H3N1 influenza infection. Thus, our study provides fundamental data on the expression of CD34 in lungs and validates an antibody for use in further experiments in these animal species.     

Immunohistochemical identification of B and T lymphocytes in formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site, and patient age.

The lymphocyte phenotype of 70 formalin-fixed, paraffin-embedded feline lymphosarcomas (LSAs) was determined immunohistochemically using a T cell polyclonal antibody, and a B cell monoclonal antibody. Forty-seven of 70 (67%) tumors were T cell, 19/70 (27%) were B cell, and 4/70 (6%) did not stain with either marker. Thirty-eight of 70 (54%) tumors were positive for feline leukemia virus (FeLV) antigen by immunohistochemistry (IHC), and 52/70 (74%) tumors were positive for FeLV DNA using the polymerase chain reaction (PCR). B cell tumors were as frequently FeLV-positive as T cell tumors using either IHC or PCR. Intestinal tumors were more likely to be B cell than T. The incidence of B and T cell tumors was not different among young (< or = 3 y), middle-aged (> 3 y to < or = 8 y), and old (> 8 y) cats. Both B and T cell tumors from old cats were FeLV-positive more often by PCR than by IHC. Feline leukemia virus DNA but not antigen, was detected in B cell tumors and intestinal tumors from cats > 8 y as often as it was detected in B cell tumors and intestinal tumors from cats < or = 8 y. Previously, most B cell and intestinal tumors from old cats were considered to be negative for FeLV. Here, the results suggest involvement of latent or replication-defective forms of the virus in such tumors from old cats. This study supports a role for FeLV in feline B cell as well as T cell tumorigenesis.     

Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns on microbes, and ligand recognition results in production of pro-inflammatory cytokines, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. The purpose of this study was to test antibodies specific for human or murine TLR2, 3, 4, 5 and 9 for their use in dogs. Twenty-two antibodies were assessed for recognition of canine monocytes (Mo), granulocytes (Gr) or lymphocytes (Ly) by flow cytometry, and results were compared with isotype-matched controls and other antibodies against the same TLR. Nine monoclonal antibodies detected canine leukocyte subpopulations. Antibodies TL2.1 and TL2.3 (specific for human TLR2), HTA125 (specific for human TLR4), as well as 85B152.5 and 19D759.2 (specific for human TLR5) recognized Mo and Gr but not Ly without permeabilization, and putatively cross-react with canine TLR2, 4 and 5, respectively. Antibodies 40C1285.6 and TLR3.7 (specific for human TLR3) as well as 26C593.2 and 5G5 (specific for human and murine TLR9) recognized Mo, Gr and Ly after their permeabilization and putatively cross-react with canine TLR3 and 9, respectively, inside the cell. None of these nine antibodies recognized paraformaldehyde-treated (4%) canine leukocytes but all except 40C1285.6, TLR3.7 and 5G5 recognized methanol-fixed cells, suggesting that they might be useful also in immunohistochemistry.     

Cross-reactivity between a monoclonal antibody that recognizes a tumor-associated antigen on bovine lymphosarcoma cells and blood lymphocytes from various mammalian species.

Tumor-associated antigens that are expressed in lymphosarcoma B cells of cattle with enzootic bovine leukosis had been analyzed in terms of their reactivity with 13 monoclonal antibodies (MAB). By use of flow cytometry and radioimmunoprecipitation, 1 of the MAB (c143) that recognized a tumor-associated antigen cross-reacted with blood lymphocytes (BL) from various mammalian species. By use of flow cytometry, the c143 MAB reacted with 10 to 49% of BL derived from human beings, mice, dogs, horses, pigs, llamas, sheep, goats, and cattle. Titer of the c143 MAB with BL from horses, pigs, human beings, and llamas ranged between 1:6.0 x 10(4) and 1:5.3 x 10(5); titer associated with BL of goats and sheep was 1:1.6 x 10(6); and that associated with BL of cattle was 1:4.3 x 10(7). The c143 MAB specifically immunoprecipitated 3 homologous proteins from cell extracts of caprine, ovine, and bovine BL (32-, 34-, and 36- to 37-kDa bovine proteins; 31-, 32-, and 36- to 37-kDa caprine proteins; and 31.5-, 33-, and 36- to 37-kDa ovine proteins), but none was immunoprecipitated from human, murine, canine, porcine, and llama BL. These results indicate that the avidity of the c143 MAB in binding to BL from ruminants (eg, goats, sheep, and cattle) is higher than that to BL from human beings, mice, dogs, horses, pigs, and llamas. In sheep, the c143 MAB could immunoprecipitate the aforementioned proteins from BL of the Suffolk breed, but not BL from the Corriedale breed, whereas the c143 MAB immunoprecipitated apparently identical proteins from BL of 4 breeds of cattle.     

Feline Lymphoma (145 Cases): Proliferation Indices, Cluster of Differentiation 3 Immunoreactivity, and Their Association with Prognosis in 90 Cats

Paraffin-embedded, formalin-fixed tissue samples from 145 cats with lymphoma were analyzed for cluster of differentiation 3 (CD3, a surface antigen) immunoreactivity, argyrophilic nucleolar organizer region (AgNOR) frequency, and proliferating cell nuclear antigen labeling index (PCNA-LI). This information along with signalment, anatomic site, and feline leukemia virus (FeLV) antigen status was used to determine the potential of these indicators to predict response to therapy, remission, and survival times, and to characterize cats with lymphoma in the era of general availability of FeLV testing and vaccination. Alimentary lymphoma, primarily occurring in older, FeLV-negative cats, was the most common site of involvement. Although the majority of tumors from FeLV-positive cats were CD3-immunoreactive, only one half of CD3-immunoreactive tumors occurred in FeLV-positive cats. Median remission duration and survival times were 126 days and 143 days, respectively, for all cats. Measures of tumor cell proliferation (AgNOR frequency and PCNA-LI) and CD3-immunoreactivity were not predictive of outcome. When all prognostic factors were accounted for by multivariate analysis, response to therapy, FeLV status, and clinical substage were predictive of outcome. FeLV-negative cats that achieved a complete response following induction therapy were likely to have durable (ie, > 6-month) responses, particularly when doxorubicin was included in the chemotherapy protocol. However, FeLV-positive cats had significantly shorter remission and survival times with available chemotherapeutic protocols.     

Chemical reactions in cells from leukemic cats

Cytochemical staining for leukocyte alkaline phosphatase(LAP), nonspecific esterase (NSE), nonspecific esterase with fluoride inhibition (NSE-F), periodic acid Schiff (PAS) reactivity, and peroxidase (PO) was valuable in identification of the neoplastic cell type in 10 leukemic cats. Staining both blood and bone marrow smears was often necessary for making the correct diagnosis. Cytochemical staining resulted in changing the morphologic diagnosis of leukemia in two of the 10 cats. Also, increased LAP activity, probably a marker for myelocytic leukemia in the cat, was observed in bone marrow cells from three nonleukemic, FeLV-positive cats.     

Malignant fibrous histiocytomas in dogs and cats: an immunohistochemical study.

Immunohistochemical staining was performed on seven canine and 10 feline soft tissue tumours histologically diagnosed as malignant fibrous histiocytomas (MFHs) or MFH-like tumours, and eight other histologically specified tumours (non-MFH). This was done to determine if commercially available antibodies that are used routinely in human diagnostic pathology for MFHs would express the same immunohistochemical patterns in canine and feline MFHs and MFH-like tumours. The antibodies were directed against human alpha 1-anti-trypsin (AT), human alpha 1-anti-chymotrypsin (ACT), human lysozyme, bovine S-100 protein and human desmin. AT did not show any immunoreactivity in the tissues investigated. Except for one MFH, all canine MFHs and other soft tissue tumours with a 'histiocytic' character stained for lysozyme and not for S-100. Six out of seven canine MFHs and MFH-like tumours stained positive for desmin as did most non-MFH sarcomas. Most of the canine and feline MFHs and MFH-like tumours were positive for ACT. These findings for ACT staining in canine and feline MFHs and MFH-like tumours are in agreement with the findings in human MFHs. The immunohistochemical results of canine MFHs and MFH-like tumours were different from those in cats. Feline MFHs differed from canine MFHs for both lysozyme and desmin staining.     

Comparative examination of cats with feline leukemia virus-associated enteritis and other relevant forms of feline enteritis

Cats with feline leukemia virus (FeLV)-associated enteritis (FAE), enteritis of other known viral etiology (parvovirus [PV], enteric coronavirus [CoV]), and enteritis of unknown etiology with histologic features similar to those of FAE and PV enteritis (EUE) and FeLV-negative and FeLV-positive cats without enterocyte alterations were examined. Amount and types of infiltrating leukocytes in the jejunum and activity and cellular constituents of mesenteric lymph nodes, spleen, and bone marrow were determined. PV and CoV infections were confirmed by immunohistologic demonstration of PV and CoV antigen, ultrastructural demonstration of viral particles in the intestinal content, and in situ hybridization for PV genome. FeLV infection was detected by immunohistology for gp70, p27, and p15E. Latent FeLV infection was excluded by polymerase chain reaction methods for exogenous FeLV DNA. Enterocyte lesions involved the crypts in cats with PV enteritis, FAE, and EUE and the villous tips in cats with CoV enteritis. Inflammatory infiltration was generally dominated by mononuclear cells and was moderate in the unaltered intestine and in cats with PV enteritis and marked in cats with FAE, CoV enteritis, and EUE. In cats with EUE, myeloid/histiocyte antigen-positive macrophages were relatively numerous, suggesting recruitment of peripheral blood monocytes. Lymphoid tissues were depleted in cats with PV enteritis and with EUE but were normal or hyperplastic in cats with FAE. Bone marrow activity was decreased in cats with PV enteritis; in cats with FAE or EUE and in FeLV-positive cats without enterocyte alterations, activity was slightly increased. In cats with FAE and PV enteritis, a T-cell-dominated response prevailed. EUE showed some parallels to human inflammatory bowel disease, indicating a potential harmful effect of infiltrating macrophages on the intestinal epithelium.     

Retrospective serologic survey for the presence of feline immunodeficiency virus antibody: a comparison of ELISA and IFA techniques.

A total of 878 samples from the New York State Diagnostic Laboratory (NYSDL), dating from January 1984 to May 1987, were examined to detect antibodies to feline immunodeficiency virus (FIV). We used 2 screening methods; an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA). Of these, 211 samples were from cats that tested negative for feline leukemia virus (FeLV) and exhibited disease signs consistent with immunodeficiency disease; 19 (9.0%) serum samples were determined to be positive. An additional 508 samples were from cats that tested FeLV-negative and were asymptomatic; 6 (1.2%) sera were determined to be positive. The final 159 samples were from FeLV-positive cats and included symptomatic and asymptomatic animals; this population of cats produced 6 (3.8%) positives. Additionally, 521 samples from the Cornell Feline Health Center (CFHC) serum bank, dating back to 1966, were tested to determine the earliest sample in which FIV antibodies could be detected. Five (2.7%) 1971 and 3 (3.3%) 1969 CFHC samples tested positive. The IFA for FIV antibody proved to be a sensitive (97.4%) and specific (100%) test. The ELISA also had high sensitivity (100%) and specificity (99.6%); however, the IFA proved to be more specific than the ELISA when assaying FeLV-positive cats.     

Isolation of Canine Mammary Cells With Stem Cell Properties and Tumour-Initiating Potential

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.     

The presence of leukaemia (lymphosarcoma) and feline leukaemia virus (FeLV) in cats in The Netherlands

To study the presence and spread of feline leukaemia virus (FeLV) in The Netherlands, seven different groups of cats were examined. The indirect fluorescent antibody (IFA) test was used to detect FeLV-antigen in blood smears. Of cats with lymphosarcoma/leukaemia 73.2% were positive and 32.4% with infectious peritonitis were positive. Only one of sixty-six cats with other tumours–a cat with mammary carcinoma–was positive.
Forty-two (7.5%) of 557 cats with various complaints were positive for FeLV-antigen. The IFA-test appeared to be an important diagnostic supplement.
Of all stud males which had had contact with FeLV-positive cats 24.7% were positive for FeLV-antigen, whereas all stud males which had not had this contact, were negative.
There was a distinct difference between the percentages of FeLV-positive individuals in the groups of cats which had had (20.6%) and which had not had (0.4%) contact with FeLV-positive cats.
From the follow-up study it was found that 67.3% of the FeLV-positive cats died from, or were destroyed because of, FeLV-associated diseases within a period of 20 months.     

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets,China

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.     

A functional comparison of canine and murine bone marrow derived cultured mast cells

Disorders involving mast cells are extremely common in dogs, ranging from allergic diseases to neoplastic transformation resulting in malignant mast cell tumors. Relatively little is known regarding the basic biologic properties of normal canine mast cells, largely due to the difficulty in reliably purifying large numbers from canine skin. In vitro generated bone marrow derived cultured mast cells (BMCMCs) are routinely used in both human and murine studies as a ready source of material for in vitro and in vivo studies. We previously developed a technique to generate canine BMCMCs from bone marrow derived CD34+ cells and demonstrated that these cells exhibit the phenotypic properties characteristic of mast cells and release histamine in response to IgE cross-linking. The purpose of the following study was to characterize the functional properties of these canine BMCMCs and contrast these with the functional properties of murine BMCMCs. Our work demonstrates that both IL-4 and IL-10 promote canine BMCMC proliferation, possibly through upregulation of Kit expression, while TGFbeta inhibits proliferation. The canine BMCMCs produce a variety of cytokines and chemokines in response to IgE cross-linking and chemical stimulation including IL-3, IL-4, IL-13, GM-CSF, RANTES, and MIP1alpha. Interestingly, the canine BMCMCs released significantly larger amounts of MCP-1 and tryptase and significantly smaller amounts of IL-6 following chemical stimulation and IgE cross-linking when compared to murine BMCMCs. Lastly, the canine BMCMCs produced larger amounts of active MMP9 than their murine counterparts. In summary, canine BMCMCs exhibit unique functional properties that distinguish them from murine BMCMCs and provide insight into the contribution of these cells to mast cell disorders in the dog.     

Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4

Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.     

Immunocytochemical study of tissues from clinically normal dogs and of neoplasms, using keratin monoclonal antibodies.

Three commonly used keratin monoclonal antibodies (MAB)--AE1:AE3, CAM 5.2, and MAK-6--were compared with routinely used cytokeratin antibody. The expression of these antibodies was analyzed in several tissues obtained from clinically normal dogs and in a variety of neoplasms from dogs. Using appropriate enzymatic digestion, paraffin-embedded tissues processed in routine manner retained their typical keratin expression. Differentiated and poorly differentiated epithelial neoplasms, lymphomas, and melanomas were studied by use of the avidin-biotin-peroxidase technique. All 4 of the aforementioned antibodies had similar staining profiles. Of 3 anaplastic carcinomas, 2 had positive reaction to all 4 antibodies. All lymphomas, plasma cell tumors, and amelanotic melanomas had negative reaction to MAK-6, CAM 5.2, AE1:AE3, and cytokeratin MAB. Three basal cell epitheliomas had positive reaction to all 4 antibodies, whereas 1 basal cell tumor with a solid pattern had negative staining reaction. Two carcinoids had negative reaction to all markers and 1 of 2 malignant chemodectomas and 1 transitional cell carcinoma had staining reaction to only AE1:AE3 MAB. Comparing the 4 antibodies, use of AE1:AE3 MAB produced the strongest staining intensity followed by cytokeratin, MAK-6, and CAM 5.2 MAB. All 4 antibodies had low background staining. In conclusion, AE1:AE3 and MAK-6 MAB are as useful as cytokeratin MAB for identification of poorly differentiated epithelial neoplasms in dogs and cats.