Expression of the T200 family of glycoproteins in canine malignant lymphoma.

High molecular weight surface proteins were examined in lymph node lymphocytes from five control dogs and 27 dogs with malignant lymphoma. Polyclonal rabbit antiserum was raised against a 210,000-dalton (210 K) membrane protein which was purified from a canine lymphoid tumor by preparative slab gel electrophoresis. Three high MW proteins at 210, 195 and 170 K and a common proteolytic fragment at 95 K were detected in the electrophoretically separated plasma membrane preparations by immunoblotting with polyclonal anti-210 K antiserum. Two antigenically distinct patterns were evident: 1) Type 1 lymphomas expressed a major 210 K peptide (with or without a minor 195 K component), and 2) Type 2 lymphomas lacked the 210 K form but had a 170 K or 195 K peptide singly or in combination. Immunoperoxidase staining of formalin-fixed, paraffin-embedded tissue sections demonstrated that the antigen was localized predominantly to the surface membrane of lymphocytes, and that canine lymphoma cells expressed a greater amount of the antigen than normal lymph node lymphocytes. It was concluded that these structurally and antigenically related high molecular weight proteins, based on their antigenic patterns, limited peptide analysis and tissue distribution, represent the canine homologue of the lymphocyte differentiation antigen known as T200.     

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.     

犬瘟热病毒自然感染犬淋巴组织中T、B细胞变化的研究

为了调查患犬瘟热病犬淋巴组织中T、B细胞变化的特点及淋巴细胞减少的发病机制,试验通过免疫组织化学的方法观察了T细胞(用CD3和CD45RO检测T细胞)、B细胞(用IgG、IgM抗血清检测B细胞)和犬瘟热病毒(抗犬瘟热病毒抗体)在病犬淋巴组织中的分布。结果表明:在淋巴组织中的淋巴细胞、淋巴小结中树突状细胞和巨噬细胞中均检出了抗病毒阳性反应细胞。在骨髓组织的前髓细胞中也发现抗病毒阳性反应细胞和嗜酸性胞浆内及核内包涵体的存在。与对照组相比,CD3和CD45RO阳性细胞主要存在于T细胞的分布域;但CD3和CD45RO阳性T细胞的数量较少。位于淋巴组织中的巨噬细胞有的被CD45RO染成阳性。在B细胞分布的区域中,IgG、IgM阳性细胞的数量明显减少;一些位于淋巴组织的浆细胞也被IgG或IgM染成阳性。在淋巴组织中淋巴细胞减少的顺序为:IgG阳性细胞减少最明显,其次为IgM和CD45RO阳性细胞,再次为CD3阳性细胞。依据试验结果,作者认为病犬淋巴组织中淋巴细胞减少主要是由B细胞缺乏所引起的;淋巴细胞的增殖能力减弱是引起淋巴组织中淋巴细胞减少的重要原因。     

Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes

Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1-10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.     

Lymphocyte responsiveness to mitogens and quantitation of T and B lymphocytes in canine malignant lymphoma.

Two canine malignant lymphoma cases were studied, one from the time of detection of enlarged palpable lymph nodes through the terminal stage and another at the terminal stage. Hematologic and histopathologic studies were confirmative of leukemia. The lymphocyte subpopulations, T and B cells, were quantitated as identified by the presence or absence of surface immunoglobulin and erythrocyte-antibody-complement-rosette formation. The average number of B cells in the peripheral blood lymphocytes throughout the study were approximately 80%. The B cells in the lymph node lymphocytes were 82%. There was considerable fluctuation in the number of blood lymphocytes, but the percentage of T and B lymphocytes remained nearly constant. There was marked impairment in the lymphocytic response to mitogens. The results of this study indicate that the canine malignant lymphoma is predominantly a B-lymphocyte type.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.     

Significance of tumor-infiltrating immune cells in spontaneous canine mammary gland tumor: 140 cases

The numbers of tumor infiltrating T lymphocytes, B lymphocytes and antigen presenting cells were evaluated in an immunohistochemical manner in 140 canine spontaneous mammary gland tumor (MGT) tissues. As a result, we found a statistically significant increase in the number of intratumoral T lymphocytes (23.2 ± 23.8) in the malignant MGT group (n=51) compared with the benign MGT group (14.0 ± 16.0, n=89; P<0.05). Moreover, the high T lymphocyte infiltration in the malignant group correlated with poor prognosis in multivariate analysis (P<0.05). This study indicated the relationship between increased infiltrating T lymphocytes and canine MGT malignancy.     

Development of the polymerase chain reaction assay based on the canine genome database for detection of monoclonality in B cell lymphoma

From the canine genome database and its bioinformatic analysis, we identified conserved sequences within the vast majority of 61 variable segments and 1 joining segment of the immunoglobulin heavy chain (IgH) gene, and designed optimal primers for polymerase chain reaction (PCR) amplification directed at these conserved sequences to evaluate the monoclonality of IgH in canine B cell lymphoma. Using the primers, a PCR-based assay was performed on fine-needle aspiration samples of normal, hyperplasia, and malignant lymph nodes and lymphoma cell lines. All fine-needle aspiration samples of five B cell lymphoma cases and the B cell lymphoma line GL-1 exhibited clonal amplification, whereas no amplification was observed in the samples from normal and hyperplasia lymph nodes, cases of T cell lymphoma, and the T cell lymphoma line CL-1. The primers we designed clearly distinguished malignant B lymphocytes from normal, reactive, and malignant T lymphocytes, indicating a potential utility of the primers for PCR-based routine clinical examination for canine B cell lymphoma.     

Phenotypical characterization of T and B cell areas in lymphoid tissues of dogs with spontaneous distemper

CD3, CD4, CD5, and CD8 antigen expression of T cells and IgG expression of B cells and canine distemper virus (CDV) antigen distribution were immunohistochemically examined in lymphoid tissues (lymph node, spleen, thymus, and tonsil) of control dogs and animals with spontaneous canine distemper. In addition, CNS tissue of all animals was studied for neuropathological changes and CDV antigen distribution. Based on the degree of depletion distemper dogs were classified into two groups. Group I represented animals with moderate to marked lymphoid depletion, while group II dogs displayed mild or no depletion. CDV antigen was mainly found in lymphocytes and macrophages of group I dogs, whereas CDV expression was most prominent in dendritic cells of group II animals. In group I dogs, a marked loss of CD3, CD4, CD5, CD8, and IgG expression was noticed, hereby loss of CD4+ cells was more prominent than depletion of CD8+ cells. In the lymphoid tissues of group II animals, a significant increase in the number of T and B cells was observed compared to group I dogs. The number of CD3+, CD4+, and CD8+ cells in group II dogs was similar to the findings in controls, however, CD5 and IgG expression was mildly reduced in T and B cell areas, respectively. Additionally, in groups I and II dogs, CD3+ and CD5- T cells were detected in T cell areas. Whether this cell population represents a cell type with autoimmune reactive potential remains to be determined. Surprisingly in group II animals, viral antigen was found predominantly in dendritic cells indicating a change in the cell tropism of CDV during chronic infection and a possible mechanism of viral persistence. The two patterns of lymphoid depletions correlated to two different types of canine distemper encephalitis (CDE). Group I dogs displayed acute non-inflammatory CDE, whereas group II dogs suffered from chronic inflammatory demyelinating CDE, indicating a pathogenic relationship between lymphocytic depletion and inflammatory brain lesions in distemper.     

Detection of intracellular canine distemper virus antigen in mink inoculated with an attenuated or a virulent strain of canine distemper virus

Using an indirect immunofluorescence technique, the distribution of viral antigen in various tissues and blood mononuclear leukocytes was studied in wild mink, either vaccinated with an attenuated vaccine strain of canine distemper virus (CDV) or experimentally inoculated with the virulent Snyder-Hill strain of CDV. Viral antigen was detected in cells of the lymphoid system 6 to 12 days after vaccination. From 2 to 3 days after inoculation with the virulent strain, CDV antigen was demonstrated in cells of the lymphoid system and, during the incubation period, the antigen had spread to the epithelia and brain at days 6 and 12, respectively. In clinical cases of acute fatal canine distemper, the viral antigen was detected in a wide variety of tissues, including the cells of the lymphoid system, epithelial cells of skin, mucous membranes, lung, kidney, and cells of the CNS. The diagnostic importance of CDV antigen detection is discussed on the basis of these findings.     

Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma

Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.     

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.     

Clinical, histopathological and immunophenotypical findings in five horses with cutaneous malignant lymphoma

This study documents the clinical, histopathological, immunohistochemical and flow-cytometric findings in five horses with cutaneous non-epidermotropic malignant lymphoma (ML). The median survival time after discovery of the first subcutaneous nodules was 3.8 years (range 2-5 years: n=4). Histologically, the cutaneous ML had a pleiomorphic structure and contained a mixture of large reticulo-endothelial cells, medium-large sized lymphoid cells with a rounded nucleus and small nucleoli, many medium sized lymphoid cells with irregular nuclei, and some small lymphoid cells. Immunohistochemically (IHC) the lymphoid cells were positive for the pan-T-lymphocyte marker CD3 but negative for the B-lymphocyte markers CD21 and kappa and lambda immunoglobulin light chains. Although routine haematological examination revealed no abnormalities in the horses with cutaneous ML, changes in the peripheral blood lymphocyte population were apparent flow-cytometrically. Compared to clinically healthy horses, a decreased total percentage of cells was recorded in the lymphocyte gate. In three horses with cutaneous ML, an increase in CD4 positive cells was noticed in the monocyte gate. Flow-cytometric analysis of tumour cells collected by fine needle aspiration (FNA) suggested that the cutaneous MLs consisted primarily of CD4 and CD8 positive T-lymphocytes. The results were compared to those of a monomorphic multicentric T- and a monomorphic multicentric B-cell lymphoma. The results of immunohistochemistry and flow-cytometry were largely but not completely in accordance. In conclusion, the results of this study suggest that cutaneous non-epitheliotropic malignant lymphomas in the horse are of T-cell origin and that, after improvement of its accuracy, flow cytometric analysis of FNAs might become a useful aid to rapid tumour identification.     

Molecular cloning and expression analysis of the canine chemokine receptor CCR9

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD.     

Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of P‐glycoprotein

Overexpression of ABC‐transporters including Pgp, MRP1, and BCRP has been associated with multidrug resistance (MDR) in both human and canine oncology. Therapeutic interventions to reverse MDR are limited, but include multidrug protocols and the temporary concomitant use of inhibitors of ABC‐transporters. Recently, the use of tyrosine kinase inhibitors has been proposed to overcome MDR in human oncology. One of the tyrosine kinase inhibitors, masitinib, is licensed for veterinary use in the treatment of canine mast cell tumors. Therefore, this study aimed to assess the potential of masitinib to revert MDR in canine malignant lymphoma using an in vitro model with canine lymphoid cell lines. Masitinib had a mild antiproliferative effect on lymphoid cells, inhibited Pgp function at concentrations equal to or exceeding 1 μm and was able to reverse doxorubicin resistance. The current findings provide the rationale for a combined use of masitinib with doxorubicin in the treatment of dogs with doxorubicin‐resistant malignant lymphoma but await confirmation in clinical trials.     

Characterization of a monoclonal antibody against desmoplakin 1 for use in dogs

Abstract Skin biopsy specimens from face, axilla, abdomen and thigh, mucocutaneous tissues from anus and vagina, and oral mucosa from six healthy Beagle dogs were examined for desmoplakin (Dsp) immunoreactivity using immunoblotting and immunohistochemical analysis. With immunoblotting using mouse antihuman Dsp 1 monoclonal antibody (DP2.17), a band was detected at 250 kDa in all the extracts as in normal human skin samples, although no band was detected at 210 kDa, suggesting that monoclonal antibody DP2.17 recognizes canine Dsp 1 but not Dsp 2. Moreover, the desmosome regions of all specimens were stained with DP2.17 using immunohistochemical analysis. From these results, DP2.17, developed for the examination of human skin, might be suitable for the investigation of Dsp-related skin disorders in dogs.     

Development of a monoclonal antibody for the detection of anti-canine CD20 chimeric antigen receptor expression on canine CD20 chimeric antigen receptor-transduced T cells

Chimeric antigen receptor (CAR) CAR-T cell therapy targeting CD20 can be a novel adoptive cell therapy for canine patients with B-cell malignancy. After injection of the CAR-T cells in vivo, monitoring circulating CAR-T cells is essential to prove in vivo persistence of CAR-T cells. In this study, we developed a novel monoclonal antibody against canine CD20 CAR, whose single-chain variable fragment was derived from the our previously reported anti-canine CD20 therapeutic antibody. Furthermore, we proved that this monoclonal antibody can detect therapeutic anti-canine CD20 chimeric antibody in the serum from healthy beagle dogs injected with the therapeutic antibody for safety study. This monoclonal antibody is a useful tool for monitoring both canine CD20-CAR-T cells and anti-canine CD20 therapeutic antibody for canine lymphoma.     

Parathyroid hormone-related protein (PTHrP) produced by dog lymphoma cells

Parathyroid hormone-related protein (PTHrP) was investigated in a canine lymphoma case with hypercalcemia by means of immunoradiomentric assay (IRMA) and molecular analysis. The plasma calcium level of the patient dog was 13.7 mg/dl. The PTHrP concentration examined by IRMA was 6.1 pmol/L in the plasma sample from the dog, but it was undetectable (< 1.1 pmol/L) in plasma samples from 4 lymphoma cases without hypercalcemia or 5 normal dogs. The PTHrP concentration examined in the culture supernatant of the lymphoma cells from this case was 1.3 pmol/L, whereas those of the lymphoma cells from a lymphoma case without hypercalcemia was undetectable. PTHrP mRNA was clearly detected not only in the lymphoma cells from this dog with hypercalcemia but also in lymphoma cells from 4 lymphoma cases without hypercalcemia and 2 canine lymphoma cell lines.     

Malignant lymphoma in nasal cavity and paranasal sinuses of a dog

A case of a canine large cell type T-cell lymphoma, with features of high-grade malignancy is described. The tumour was found confined in the nasal cavity and the paranasal sinuses of a crossbred German Shepherd dog. Histological examination revealed the features of a highly malignant large cell lymphoma. Ultrastructurally, the lymphoid tumour cells bore cytoplasmic protrusions that interdigitated tightly. From a panel of tumour markers used, the neoplastic cells were stained only for vimentin. Immunophenotyping of the tumour cells by means of CD3, CD79, kappa-light chains and lambda-light chains detection was undertaken. The tumour stained only for CD3 and was classified as T-cell lymphoma.