Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.     

In vitro modulating effects of porcine immunoglobulin G on mitogens-induced lymphocyte response in precolostral, suckling and weaned piglets

The influence of allogeneic IgG on in vitro reactivity of peripheral blood lymphocytes (PBL) of neonatal colostrum-deprived piglets as well as of suckling and weaned piglets was studied. PBL were preincubated with purified allogeneic IgG for 24 h before their ability to respond to PHA, Con A or PWM was tested. PBL of precolostral piglets pretreated with allogeneic IgG exhibited higher response to PHA (P less than 0.01) than untreated control cells. An increased response of PBL treated with IgG was also observed in suckling piglets as compared to their respective control cells (P less than 0.01). Responsiveness of PBL treated with IgG to PWM was suppressed. No differences in response to Con A regardless of the sources of lymphocytes was observed as compared to IgG untreated controls. The results suggest that pretreatment of lymphocytes of piglets with allogeneic IgG modulates their reactivity to mitogens, suppressing the response to PWM and stimulating the response to PHA, respectively.     

The distributions of phytohemagglutinin-P and concanavalin A binding sites on equine, bovine and canine peripheral blood lymphocytes

The distributions of phytohemagglutinin-P (PHA) and concanavalin A (ConA) binding sites were investigated for equine, bovine and canine peripheral blood lymphocytes (PBL). Non-B lymphocytes were collected from each PBL using a fluorescence-activated cell sorter (FACS), and the numbers of PHA and ConA binding sites on their surfaces were counted. Most PHA binding sites on PBL of the three species were shown on the surfaces of non-B lymphocytes. On the other hand, the ConA binding sites on equine and canine PBL existed mainly on the surfaces of non-B lymphocytes, but B lymphocytes of these two species had many ConA binding sites. These results were confirmed by the results of two-parameter fluorescence analysis using FACS. It is, therefore, concluded that the different optimum concentrations of PHA and ConA in PBL blastogenic responses of each animal depended on the different distributions of their binding sites.     

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.     

Dog lymphocyte cultures facilitate the isolation and growth of virulent canine distemper virus.

Optimal conditions for the isolation and growth of virulent canine distemper virus (CDV) in canine thymic and peripheral blood lymphocyte cultures were determined. Peak virus titers were seen from 3 to 6 days postinoculation of lymphocytes and depended on the multiplicity of infection. Dog lymphocytes were at least as susceptible as canine macrophages to infection with virulent CDV. Virus replication in lymphocytes resulted in higher virus titers than in dog lung macrophages. Peripheral blood lymphocytes (PBL) from CDV-immune dogs were as susceptible to CDV as were PBL from susceptible dogs.     

Role of lymphocytes in dogs experimentally re-challenged with canine transmissible sarcoma

The role of lymphocytes in dogs re-challenged with canine transmissible sarcoma (CTS) was investigated histologically and immunologically. Tumors were detected on the 3rd day, began to regress on the 9th day and disappeared on approximately the 15th day after the secondary transplantation (AST). The CTS cells were surrounded by lymphocytes which had infiltrated from the early stage, degenerated and ultimately disappeared. Most of the lymphocytes expressed thymocyte antigen (Ta) on the cell membrane surface. The numbers of lymphocytes and Ta-positive lymphocytes in peripheral blood increased gradually AST. The blastogenic responses of peripheral blood lymphocytes (PBL) induced by 3 kinds of mitogens were elevated strikingly from the early stage AST. These results suggest that Ta-positive lymphocytes play an important role in vivo in the regression of tumors and that the immunological activity of PBL is stimulated immediately AST.     

Immunosuppression in dogs by pretransfusions with ultraviolet (UV)-irradiated whole blood.

Ultraviolet (UV)-irradiated whole dog blood prevented the initiation of proliferative responses in allogeneic mixed lymphocyte reactions (MLR). When dogs were given 4 weekly intravenous transfusions of UV-irradiated allogeneic donor whole blood, peripheral blood lymphocytes (PBL) of the recipients responded less significantly to donor PBL in MLR for over one week after the final transfusion. Red blood cell (RBC)-crossmatching of these dogs was negative. Dogs treated with UV-irradiated blood did not produce anti-donor PBL antibody, or IgG, IgM and C3 determined by the indirect Coombs test. These dogs also had negligible delayed type hypersensitivity (DTH) responses to donor PBL at the end of the treatment period, recognized as slightly suppression of skin graft rejection. In contrast, six dogs receiving injections of untreated allogeneic whole blood evidenced high proliferation in MLR, agglutination in RBC-crossmatching, acute rejection of skin grafts and three of them produced C3 in the Coombs test.     

Cell-mediated immune response to rabies virus in dogs following vaccination and challenge

Peripheral blood lymphocytes (PBL) from non-vaccinated dogs and from dogs either vaccinated intramuscularly (IM) or subcutaneously (SC) with an inactivated rabies virus vaccine (Rabguard-TC, Norden Laboratories, Lincoln, NE) or intramuscularly with an attenuated rabies virus vaccine (Endurall-R, Norden Laboratories, Lincoln, NE) were exposed in vitro to rabies virus. Blastogenesis of PBL was measured by incorporation of 3H-thymidine into the DNA of proliferating cells in the presence of a suboptimal concentration of phytohemagglutinin (PHA). Following the first vaccination, there was no difference in the blastogenic response of lymphocytes from dogs vaccinated IM with either the inactivated or attenuated rabies virus vaccines. The inactivated rabies vaccine stimulated as great or greater blastogenic response when it was given SC. The PBL from non-vaccinated control dogs were not stimulated by rabies virus. Dogs vaccinated with the inactivated vaccine developed a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus. Nonvaccinated control dogs did not develop a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus.     

Results of hyperamplification of centrosomes in naturally developing tumors of dogs.

OBJECTIVE: To evaluate results of centrosome hyperamplification in naturally developing tumors of dogs. SAMPLE POPULATION: Tumor specimens from 9 dogs with tumors (rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myxosarcoma, and mammary gland tumor) and 2 canine osteosarcoma cell lines. PROCEDURE: 3 antibodies for centrosome proteins (ie, anti-gamma-tubulin, anti-BRCA1, and anti-pericentrin) were used for immunohistochemical analysis. Double immunostaining for centrosomes was used to confirm the specificity of these antibodies for centrosomes. Mutational analysis of the canine p53 gene was carried out by polymerase chain reaction-single-strand conformation polymorphism analysis, and expression of canine MDM2 protein was evaluated by use of immunohistochemical analysis, using anti-MDM2 antibody. RESULTS: Immunohistochemical analysis of dog osteosarcoma cell lines with apparent aneuploidy revealed frequent hyperamplification of centrosomes in the osteosarcoma cell lines. Similar hyperamplified centrosomes were detected in the tumor tissues from all of the 9 tumors. The frequency of cells with hyperamplified centrosomes (3 to 20/cell) in each tumor tissue ranged from 9.50 to 48.1%, whereas centrosome hyperamplification was not observed in normal lymph nodes from these dogs. In 8 of the 9 tumors, mutation of p53 gene or overexpression of MDM2, or both, was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Various types of naturally developing tumors in dogs often have hyperamplification of centrosomes associated with chromosome instability. Hyperamplification of centrosomes is a novel tumor marker for use in cytologic and histologic examinations of clinical specimens obtained from dogs.     

Cytotoxic T-lymphocyte activity specific for hemagglutinin (H) protein of canine distemper virus in dogs

Cytotoxic T-lymphocyte (CTL) responses to hemagglutinin (H) protein of canine distemper virus (CDV) were evaluated in dogs using the replication-deficient adenovirus protein expression system. Skin fibroblasts were isolated from two dogs and were infected with recombinant adenovirus bearing the CDV-H gene (Ade-CDVH). CTL assay was performed using fibroblasts expressing CDV-H protein as target cells and peripheral blood lymphocytes (PBL) collected from the same dogs one week after immunization of CDV as effector cells. Specific cytotoxic activity was observed against autologous but not heterologous fibroblasts expressing CDV-H protein. These results indicate that the CTL epitope(s) were localized in the H protein.     

Assessment of serum antibody titers against canine distemper virus, canine adenovirus type II, and canine parvovirus in Alaskan sled dogs before and after a long-distance race

OBJECTIVE: To determine serum antibody titers against canine distemper virus (CDV), canine adenovirus type II (CAV-2), and canine parvovirus (CPV) in trained sled dogs prior to and after completion of a long-distance race. DESIGN: Prospective cohort study. ANIMALS: 195 Alaskan sled dogs (from 18 kennels) that participated in the 2006 Iditarod Trail Race. PROCEDURES: All 1,323 dogs participating in the race had been vaccinated against the 3 viruses at 19 to 286 days prior to initial blood sample collection (obtained within the month preceding the race). Within 12 hours of race completion, blood samples were collected from 195 dogs (convenience sample) and matched with each dog's prerace sample. Serum antibody titers (90% confidence intervals [CIs]) were determined via serum neutralization assays. RESULTS: After racing, geometric mean titers against CDV and CPV were significantly higher (2,495 [90% CI, 321 to 16,384] and 6,323 [90% CI, 512 to 32,768], respectively) than prerace values (82 [90% CI, 11 to 362] and 166 [90% CI, 32 to 1,024], respectively). Sixty-one of 194 (31.4%) dogs had > or = 4-fold increases in anti-CPV antibody titers after racing. Prerace serum antibody titers against CDV, CPV, and CAV-2 varied significantly by sled team but were not associated with time since vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Postrace increases in serum anti-CDV and anti-CPV antibody titer might reflect exposure of dogs to these agents immediately before or during racing. Dogs had no clinical signs of CDV-, CAV-2-, or CPV-associated disease; therefore, the clinical importance of these titer changes is uncertain.     

Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs.

To clarify the immunologic effects of vaccination in dogs, we monitored total leukocyte and lymphocyte counts, humoral antibody responses, blastogenic responses of lymphocyte, and delayed type hypersensitivity (DTH) responses after vaccination. Mixed vaccines were administered on day 0 except for canine parvovirus (CPV) vaccine which was readministered on day 21. The puppy and adult dogs had a significant decrease in leukocyte and lymphocyte counts on day 7. The puppies showed a significant increase in the blastogenesis of lymphocytes after each vaccination, whereas the adult dogs had no significant changes. However, the adult dogs were divided into two groups, high responders and low responders in blastogenesis of lymphocytes. The dogs with higher or lower response in SI values on day 0 tended to show decrease or increase after the first vaccination, respectively. Since almost all dogs developed high titers of humoral antibody, it is considered that vaccination acts in an immunomodulative fashion. DTH responses to phytohemagglutinin (PHA) and CPV vaccine monitored at 0, 3, and 8 weeks after the first vaccination produced strong reactions, in particular those to CPV vaccine rose significantly after vaccination and maintained the higher responses for at least 2 months. These results suggest that DTH responses to PHA and CPV vaccine are helpful to monitoring non-specific and specific immune functions in vivo, therefore, DTH could be used as simple and rapid immunologic tests in canine practice.     

Mitogen stimulation of canine normal and myasthenia gravis lymphocytes

Responses of canine lymphoid tissues to mitogens were studied in five normal dogs and in two dogs with acquired myasthenia gravis (MG). In the normal dogs, lymph-node-derived lymphocytes gave the most consistent proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), as determined by thymidine incorporation; and in most cases PHA, lipopolysaccharide (LPS), and PWM stimulated total IgG production, as determined by ELISA. Splenic lymphocytes had the greatest capacity for increased total IgG production. In the myasthenic dogs total IgG production by unstimulated lymph-node-derived lymphocytes was 88 micrograms/ml and 153 micrograms/ml, much higher than that of unstimulated normal dog lymphocytes (mean less than 1.0 microgram/ml). All mitogens resulted in suppression rather than stimulation of IgG production by lymphocytes from dogs with MG. Production of antibodies to acetylcholine receptors (AChRs) was detected in the supernatants of lymphocyte cultures from one of the dogs with MG at a rate of 78 fmol/5 x 10(5) cells per week and was not detected in culture supernatants of control dogs. This study demonstrates that lymph nodes may be an important site of antibody production in myasthenic dogs and provides the necessary groundwork for future studies of the cellular immunology of canine MG.     

Serum antibody response to canine parvovirus, canine adenovirus-1, and canine distemper virus in dogs with known status of immunization: study of dogs in Sweden

Serum antibody titers to canine parvovirus (CPV), canine adenovirus-1 (CAV-1), and canine distemper virus (CDV) were measured in dogs with known immunization status. The dogs represented 3 groups: nonvaccinated dogs less than 12 months old; vaccinated dogs less than 12 months old; and adult dogs greater than 12 months old. For practical reasons, the population from which the specimens were obtained could be considered as free from natural infection with CAV-1 and CDV. In nonvaccinated dogs less than 12 months old, antibodies against all 3 viruses were measured at the time the dogs were given their first vaccination. Altogether, 50.7% of the dogs had titer greater than or equal to 1:10 to CPV, and 26.1 and 46.2% had titer greater than or equal to 1:8 to CAV-1 and CDV, respectively. The concentration of maternal antibody seemed to be of major importance for failure of immunization with use of inactivated CPV vaccine, but not with CAV-1 and CDV vaccination. In dogs less than 12 months old and vaccinated against CPV infection with inactivated virus, only 11.5% had titer greater than or equal to 1:80. In dogs vaccinated against infectious canine hepatitis and canine distemper, 63.2 and 78.3%, respectively, had titer greater than or equal to 1:16. In adult dogs greater than 2 months old and vaccinated against CPV infection, less than 50% had titer greater than or equal to 1:80, regardless of time after vaccination. There was no significant difference in titer between vaccinated and nonvaccinated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells.

The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibit potent cytolytic activity against an equine tumor cell line, EqT8888. Cytolytic activity against the EqT8888 cells required 3 days of incubation with IL-2, was mediated primarily by T-cells, and was not restricted by major histocompatibility complex antigens. Though LAK activity could only be demonstrated using equine-derived target cells, xenogeneic targets could be lysed in a lectin-mediated cytotoxicity assay. The xenogeneic targets also failed to block LAK cell-killing of the EqT8888 cells in a cold-target competition assay. These results indicate that LAK cells in the horse appear to utilize a species-specific recognition mechanism during target cell lysis.     

Lymphocyte 5'-nucleotidase activities in normal dogs and in dogs with malignant lymphoma

Plasma membrane 5'-nucleotidase (5'NT) activity was assayed in lymph node lymphocytes from seven normal control dogs and in malignant lymphocytes from 25 dogs with lymphoma. The lymphoid tumors were classified according to the NCI Working Formulation into five histologic subtypes. When compared with normal controls significantly lower 5'NT activities were found in the lymphoblastic, small lymphocytic and diffuse large (noncleaved) subtypes while no significant differences were observed in the immunoblastic or small noncleaved groups. In addition, dogs with hypercalcemia or paraproteinemia had decreased 5'NT levels. However, no significant differences were found between prognostic groups or histologic subtypes of the NCI classification. In conclusion, canine lymphoma subtypes had generally decreased 5'NT activities which appeared to reflect the B or T cell lineage, degree of maturation and enzyme activity of the cell of origin.     

Expression of receptor activator of nuclear factor kappa-B ligand (RANKL) in neoplasms of dogs and cats

BACKGROUND: Receptor activator of nuclear factor kappa-B (RANK), RANK-ligand (RANKL), and the soluble decoy receptor osteoprotegerin (OPG) form a key axis modulating osteoclastogenesis. In health, RANKL-expressing bone stromal cells and osteoblasts activate osteoclasts through RANK ligation, resulting in homeostatic bone resorption. Skeletal tumors of dogs and cats, whether primary or metastatic, may express RANKL and directly induce malignant osteolysis. HYPOTHESIS: Bone malignancies of dogs and cats may express RANKL, thereby contributing to pathologic bone resorption and pain. Furthermore, relative RANKL expression in bone tumors may correlate with radiographic characteristics of bone pathology. ANIMALS: Forty-two dogs and 6 cats with spontaneously-occurring tumors involving bones or soft tissues were evaluated. METHODS: A polyclonal anti-human RANKL antibody was validated for use in canine and feline cells by flow cytometry and immunocytochemistry. Fifty cytologic specimens were collected from bone and soft tissue tumors of 48 tumor-bearing animals and assessed for RANKL expression. In 15 canine osteosarcoma (OSA) samples, relative RANKL expression was correlated with radiographic characteristics of bone pathology. RESULTS: Expression of RANKL by neoplastic cells was identified in 32/44 canine and 5/6 feline tumor samples. In 15 dogs with OSA, relative RANKL expression did not correlate with either radiographic osteolysis or bone mineral density as assessed by dual energy x-ray absorptiometry. CONCLUSIONS AND CLINICAL IMPORTANCE: In dogs and cats, tumors classically involving bone and causing pain, often may express RANKL. Confirming RANKL expression in tumors is a necessary step toward the rational institution of novel therapies targeting malignant osteolysis via RANKL antagonism.     

Evaluation of a killed feline panleukopenia virus vaccine against canine parvoviral enteritis in dogs

Immunogenic potency of a killed feline panleukopenia virus vaccine against canine parvoviral enteritis in dogs was examined. The vaccine elicited hemagglutination-inhibition antibodies to canine parvovirus (CPV) in all of the 72 dogs which were vaccinated. The vaccine was protective in dogs against both experimentally induced and naturally occurring CPV-induced disease. By statistical analysis, 4 weeks was found to be the optimal spacing between 2 vaccinal doses resulting in hemagglutination-inhibition antibody titers up to 1:5,120. Adverse reactions to the vaccine were not observed. Atypical lymphocytes were found consistently in the CPV-infected control dogs.     

Retrospective serological analysis of spontaneous CDV infection in 192 dogs

Spontaneous cases of canine distemper virus (CDV) infection were serologically evaluated. The 192 dogs in which CDV antigen was confirmed from tonsil by immunohistological examination were 2- to 4-months old, of various breeds, and unvaccinated. The prognoses were good in 74 dogs with significantly high levels of anti-CDV passive hemolytic aggregation (PHA) titer. In the other 118 dogs with poor prognoses, anti-CDV PHA titer was not detected. Anti-CDV PHA titer had the most significant association with the prognoses of CDV infection, and could be the most reliable and useful indicator for evaluating such prognoses.     

Serological responses of adult dogs to revaccination against distemper, parvovirus and rabies

Serum antibody titres to canine distemper virus (CDV), canine parvovirus (CPV) and rabies were measured in dogs that had not been revaccinated annually and compared with the titres in a control group of regularly vaccinated animals; 83 per cent (171 of 207) of the dogs vaccinated against CDV one or more years earlier had serum neutralising antibody titres equal to or greater than 16; 64 per cent (136 of 213) of the dogs vaccinated against CPV one or more years earlier had haemagglutination inhibiting titres equal to or greater than 80; and 59 per cent (46 of 78) of the dogs vaccinated against rabies two or more years earlier had serum neutralising antibody titres equal to or greater than 0.5 iu/ml. Three weeks after a single booster vaccination the dogs' antibody titres against CDV had increased above the threshold level in 94 per cent of the dogs, against CPV in 68 per cent, and against rabies in 100 per cent.