Short-term effect of cyclophosphamide and azathioprine on selected aspects of the canine blastogenic response

The short-term, in vitro responses of canine peripheral blood lymphocytes to mitogenic stimulation and serum immunoglobulin concentrations were evaluated following treatment with currently recommended doses of cyclophosphamide and azathioprine. Cyclophosphamide had no significant effect on either the serum immunoglobulin concentrations or the blastogenic response of lymphocytes to mitogenic stimulation. Serum immunoglobulin concentrations remained unchanged following azathioprine treatment. The blastogenic response was significantly suppressed following one week of azathioprine therapy and returned to baseline values one week following cessation of treatment. The response to phytohemagglutinin was most suppressed, followed, in order, by the response to concanavalin A, and to pokeweed mitogen. These results suggest that the short-term use of azathioprine, but not cyclophosphamide, in clinically used dosages, does suppress selective aspects of the canine immune system, and the T cells appear to be more susceptible than B cells to the immunosuppressive effect of this drug.     

Effect of administration of nonsteroidal anti-inflammatory drugs before surgery on renal function in clinically normal dogs

OBJECTIVES: To investigate renal function in clinically normal dogs undergoing general anesthesia for ovariohysterectomies that received nonsteriodal antiinflammatory drugs (NSAID) before surgery. ANIMALS: 40 clinically normal dogs. PROCEDURE: After induction of anesthesia, dogs were given an analgesic. Renal function was assessed before surgery and 24 and 48 hours after surgery by means of serum urea and creatinine concentrations, fractional clearance of sodium (FC(Na)), urine gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) activities, and urine analysis. Ten dogs in each of 4 groups received ketorolac tromethamine (0.5 mg/kg of body weight), ketoprofen (1 mg/kg), carprofen (4 mg/kg), or morphine (0.1 mg/kg; control group). RESULTS: Duration of general anesthesia ranged from 1.75 to 5 hours, with a mean of 3 hours. Two ketorolac- and 2 ketoprofen-treated dogs had transient azotemia. A significant decrease in the FC(Na) between before surgery and 24 hours after surgery, and between before surgery and 48 hours after surgery, was found in ketoprofen- and carprofen-treated dogs. Ketorolac-, ketoprofen-, and morphine-treated dogs had a decrease in urine specific gravity. Two ketorolac, 1 ketoprofen-, 1 carprofen-, and 4 morphine-treated dogs had increases in renal tubular epithelial cells on urine sediment examination 24 hours after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: In clinically normal dogs undergoing general anesthesia and elective surgery, the use of NSAID as analgesics is not contraindicated. Compared with ketorolac or ketoprofen, carprofen had the least effect on renal function and integrity.     

Effects of etomidate on adrenocortical function in canine surgical patients

Adrenocortical function in canine surgical patients given etomidate at 1 of 2 dosages (1.5 mg/kg of body weight or 3 mg/kg, IV) was evaluated and compared with that of dogs given thiopental (12 mg/kg, IV). The adrenocortical function was evaluated by use of adrenocorticotropic hormone (ACTH) stimulation tests and determination of plasma cortisol concentrations at 0 minute (base line) and 60 minutes after ACTH administration. At 24 hours before administration of either drug (ie, induction of anesthesia), each dog had an increase in plasma cortisol concentration when given ACTH. The ACTH stimulation tests were repeated 2 hours after induction of anesthesia. Dogs given thiopental had base-line plasma cortisol concentrations greater than preinduction base-line values, but did not increase plasma cortisol in response to ACTH stimulation. Postinduction ACTH stimulation tests in dogs given etomidate at either dose indicated base-line and 60-minute plasma cortisol concentrations that were not different from preinduction base-line values. Therefore, adrenocortical function was suppressed 2 and 3 hours after the administration of etomidate in canine surgical patients.     

Determination of thyroxine, triiodothyronine, and cortisol changes during simultaneous adrenal and thyroid function tests in healthy dogs

Changes in thyroxine (T4), triiodothyronine (T3), and cortisol during a combined adrenal (dexamethasone suppression/adrenocorticotrophic hormone response test) and thyroid function tests (thyroid-stimulating hormone [TSH] response test) were determined in 20 healthy hospitalized pet dogs. The effect of dexamethasone on T4 and T3 changes was evaluated during a simultaneous TSH response/dexamethasone suppression adrenocorticotrophic hormone response test. Greater ranges in basal cortisol concentrations and slower changes after dexamethasone was administered were observed in healthy pet dogs kenneled in a hospital setting than those reported for conditioned laboratory dogs. Pet dogs were observed to demonstrate cortisol suppression more reliably at 4 hours than at 2 hours after dexamethasone was administered. Dexamethasone had no effect on the response to TSH as assessed by T4 and T3 assays, thus supporting the validity of combining adrenal and thyroid response tests in a 5-hour period.     

Thyroid function tests in dogs using radioimmunoassay kits

Thyroid function has been assessed using radioimmunoassay kits for total and free thyroxine (T4). A radioimmunoassay kit for total T4, designed for human samples, was unable to detect total T4 in 35 per cent of normal canine serum tested, due to the kit being designed to measure total T4 over the normal human range which is four times the canine normal range. The canine normal range for free T4 (7.5–50 pmol/1) was found to be of the same order as the human range and therefore a free T4 kit was suitable for measuring the free hormone in dogs. In response to thyroid stimulating hormone in normal dogs, total T4 rose 6.2 times whereas free T4 rose only 2.6 times. Hypothyroid animals could be distinguished from normal dogs by free T4 assay whereas they were indistinguishable from many normal dogs by total T4 assay. Response to thyroid stimulating hormone was used to confirm hypothyroidism, but also revealed one animal with normal total and free T4 but which had a negligible response on stimulation.     

In vitro sensitization and stimulation of bovine lymphocytes with encapsulated or nonencapsulated Smith strain of Staphylococcus aureus

An in vitro immunization procedure was developed, whereby bovine blood and mammary gland lymphocytes were isolated, and their blastogenic responses (measured by [3H]thymidine uptake) to sensitization and subsequent stimulation with selected antigens were determined. Capsular extracts of Staphylococcus aureus, encapsulated S aureus, and nonencapsulated S aureus were used as test antigens. Differences were observed between kinetics of the secondary response to encapsulated and nonencapsulated S aureus. The secondary response to nonencapsulated S aureus peaked in 48 hours, whereas the secondary response to the encapsulated S aureus peaked in 72 hours. The delayed peak response to encapsulated S aureus was observed only when encapsulated S aureus was used for sensitization of lymphocytes, regardless of whether the encapsulated or nonencapsulated strain was used for stimulation. Sensitization of lymphocytes with the nonencapsulated strain and stimulation with the encapsulated strain did not alter the kinetics of the secondary response of cells sensitized with the nonencapsulated strain. Seemingly, T and B cells were responsive to in vitro immunization with S aureus antigens.     

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.     

Cellular immune response of pigeons in the conditions of endotoxin fever and pyrogenic tolerance

The aim of this study was to investigate changes in selected parameters of cellular immune response in the conditions of endotoxin fever and pyrogenic tolerance in pigeons. On the first day of observation the experimental birds (n = 18) were intravenously injected with Escherichia coli LPS at a dose of 10 microg/kg b.w., while the control animals (n = 6) received apyrogenic physiological saline also in the form of injection. On the second and the third day of the experiment LPS was injected additionally at 24 h intervals. Four and a half hours after the saline and pyrogen administration blood samples were collected from the control and experimental pigeons. The following immunological assays were performed: WBC, leucogram and immunophenotyping of lymphocyte subsets in peripheral blood, i.e. CD 3+ (T lymphocytes), CD 4+ (T helper lymphocytes) and CD 8+ (T suppressor/cytotoxic lymphocytes) cells. In the conditions of endotoxin fever (i.e. after the first LPS injection) leucopenia, monocytopenia, heterophilia and eosinophilia were observed. Additionally, the immunophenotyping of peripheral blood lymphocytes indicated an increase in percentage of CD 3+, CD 4+ and CD 8+ cells in response to the single injection of LPS. In contrast, the consecutive injections of LPS, which created a pyrogenic tolerance effect, caused a decrease in WBC value, heteropenia, eosinopenia and lymphocytosis. Moreover, during this state an increase in percentage of CD 3+ and CD 8+ cells was demonstrated in contrast to the percentage of CD 4+ lymphocytes. The general tendencies in cellular immune response of the affected pigeons in the conditions of endotoxin fever and pyrogenic tolerance aim at activation of defence mechanisms against LPS for its prompt elimination from the animal's organism.     

Brucella canis induces canine CD4+ T cells multi-cytokine Th1/Th17 production via dendritic cell activation

Brucella canis is a small intracellular Gram-negative bacterium that frequently leads to chronic infections highly resistant to antibiotic therapy in dogs. Also, it causes mild human brucellosis compared to other zoonotic Brucella spp. Herein we characterize the cellular immune response elicited by B. canis by analysing human and canine CD4⁺ T cells after stimulation with autologous monocyte-derived dendritic cells (MoDCs). Human and canine B. canis-primed MoDCs stimulated autologous CD4⁺ T cells; however, a Th1 response was triggered by human MoDCs, whereas canine MoDCs induced Th1/Th17 responses, with increased CD4⁺ T cells producing IFN-γ and IL-17A simultaneously. Each pattern of cellular response may contribute to host susceptibility, helping to understand the differences in B. canis virulence between these two hosts. In addition, other aspects of canine immunology are unveiled by highlighting the participation of IL-17A-producing canine MoDCs and CD4⁺ T cells producing IFN-γ and IL-17A.     

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.     

Phytomitogen- and antigen-induced blast transformation of feline lymphocytes.

A semiautomated microassay of blast transformation of feline lymphocytes was developed and used in a study of the response of normal cats to phytomitogen- and antigen-induced stimulation. Peripheral blood lymphocytes were isolated from defibrinated blood by ficolldiatrizoate gradient centrifugation and incubated at a concentration of 1 X 10(5) cells/culture. Blast transformation was measured by the incorporation of tritiated thymidine, to which the cells were exposed during the last 18 hours of incubation. Phytomitogens induced maximal transformation after 3 days' incubation. Greatest stimulation produced by 10 mug of concanavalin A (38- to 183-fold) followed by 4 mug of pokeweek mitogen (nine- to 51-fold), and finally phytohemagglutinin-P (three- to sixfold). Three of 4 cats sensitized to keyhole limpet hemocyanin responded when exposed to 50 mug of keyhole limpet hemocyanin in vitro, maximal transformation occurring after 4 days' incubation. Lymphocyte blast tranformation provides a convenient technique to assess lymphocyte function serially during experimentally induced immunologic alterations in the cat.     

Tumoricidal effect of interleukin-2-activated killer cells in canines

We investigated the effect of both partially purified (TCGF) and recombinant interleukin-2 (rIL-2) on the tumor-directed cytotoxic activity of canine peripheral blood mononuclear cells (PBMC) using three normal canines. No cytotoxic activity was displayed by unstimulated effector cells at 3 h of incubation; however, the cytotoxic effect was observed in a 16-h assay. PBMC of all canines displayed significant levels of lytic activity after stimulation for 4 to 7 days with both types of IL-2 against a variety of allogeneic and xenogeneic neoplastic cells in 3-h 51Cr release assay. The cytotoxic activity of cultured cells increased proportionally in the 16-h assays. Morphological examination of the May-Grünwald and Giemsa stained cytocentrifuged slides of cultured cells on each day of assay showed an increase in large granular lymphocytes (LGLs) beginning on day 4 and reaching a peak on day 7 of culture.     

Responses of enriched populations of feline T and B lymphocytes to mitogen stimulation.

Lymphocytes from healthy adult cats were separated into T and B cell-enriched subfractions by centrifuging rosetted cells on sodium metrizoate/Ficoll gradients. The responsiveness of unseparated lymphocytes (T + B), and T and B cell-enriched subfractions to stimulation with mitogens (phytohemagglutinin-P (PHA-P), concanavalin A (ConA), and pokeweed mitogen (PWM) was tested. Cultures of unseparated lymphocytes and those enriched in T cells showed similar responsiveness of PHA, ConA, and PWM stimulation; however, only a weak response to ConA and PWM was observed in B cell-enriched cultures. The mitogenic effects of PHA-P, ConA, and PWM on feline lymphocytes appeared to be due primarily to T-cell activation.     

Kinetics of IL-2 receptor expression on lymphocyte subsets from goats infected with Mycobacterium avium subsp. paratuberculosis after specific in vitro stimulation

Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.     

Effect of storage time on automated cell count and cytological interpretation of body cavity effusions

The effect of 24- and 48-hour storage at room temperature on automated total nucleated cell count (TNCC), differential cell count (DCC) and cell morphology was assessed, and the effect of initial total protein concentration on canine and feline body cavity effusion samples (2 to 5 ml) was evaluated. At 24 and 48 hours, TNCC and absolute numbers of neutrophils, macrophages and small lymphocytes were significantly decreased and numbers of unrecognisable cells were significantly increased. Neoplastic cells and intracellular bacteria identified in fresh samples were missed at 24 and 48 hours. The initial total protein concentration was associated with an effect on percentage of unrecognisable cells and small lymphocytes over time. Change in TNCC over time would have resulted in misclassification of the effusion type in four of 47 samples.     

Expression of procoagulant activity by equine lung macrophages: stimulation by blood lymphocytes

Increases in procoagulant activities (PCA) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed PCA which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the PCAs expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater PCA increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced PCA induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of PCA in only two of five specimens tested. The failure to induce PCA in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, PCA response of equine lung macrophages might be lymphocyte-stimulated in which case PCA might be a useful tool for monitoring the processes of cell-mediated immunity in horses.     

Modulation of canine lymphocyte blastogenesis via histamine

The effect of histamine on in vitro T cell blastogenic responses of canine peripheral blood lymphocytes to phytohemagglutinin-P (PHA-P) was investigated. A dose dependent inhibition of blastogenesis was observed; an effect which could be blocked by cimetidine, a type II histamine receptor antagonist, but not by diphenhydramine, a type I receptor antagonist, suggesting that histamine's inhibitory effect is mediated through a type II histamine receptor. The inhibitory effect of histamine on blastogenesis was also reversible by indomethacin, a prostaglandin synthetase inhibitor, implicating prostaglandin involvement in histamine suppression. Histamine release at sites of inflammation may result in down regulation of local immune responses by activation of specific immunoregulatory cells. This could permit the escape of certain neoplasia from local immunosurveillance mechanisms. Cimetidine may block activation of histamine responsive regulatory cells bearing type II receptors, which may help explain the beneficial effect cimetidine therapy has on regression of certain human tumors (i.e., malignant melanomas).     

The effect of glucocorticoids on canine lymphocyte marker expression and apoptosis

BACKGROUND: Glucocorticoids are commonly administered to dogs for the treatment of inflammatory disorders, autoimmunity and cancers such as lymphoma. Despite evidence of clinical efficacy, understanding of the effects of glucocorticoids on cells of the canine immune system is limited. HYPOTHESIS: Glucocorticoids affect the expression of phenotypic markers on canine lymphocytes and induce apoptosis. ANIMALS: Fifteen healthy mixed breed dogs. METHODS: Prospective randomized study. Prednisone was administered orally for 3 days, and cells aspirated from the popliteal lymph node before prednisone administration, and on days 1, 3, 10, 17, 24, and 38, were labeled with antibodies against canine CD3, CD4, CD8alpha, CD18, CD21, CD45, CD45RA, and CD90 molecules, and analyzed by flow cytometry. Additional samples were cultured in media with prednisolone for 24 hours and analyzed by cytometry for marker expression, and by gel electrophoresis for DNA fragmentation. RESULTS: Treatment of dogs with glucocorticoids resulted in reduced (p < or = .05) proportions of CD3 (days 1, 3, 17, and 24), CD4 (days 3 and 10), CD21 (day 1, 3, and 38), CD45RA (day 17) and CD90 (days 1, 10, and 17) expressing lymphocytes, and reduced intensity of CD18 (day 17) and CD45 (day 17 and 24) molecules on nodal lymphocytes. Culture oflymphocytes with prednisolone for 24 hours caused a significant reduction in the expression of all markers (p < or = .05) and DNA fragmentation. CONCLUSIONS AND CLINICAL IMPORTANCE: Glucocorticoids significantly alter the expression of phenotypic markers on canine lymphocytes, and in vitro induce apoptosis. These findings identify potential mechanisms for clinical immunosuppression from glucocorticoid treatment.     

Cell surface markers on canine lymphocytes.

Reference values for T and B lymphocytes were determined on lymphocytes from canine thymus, spleen, lymph node, bone marrow, and peripheral blood by use of erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosette assays, plus a direct fluorescent technique for assay of surface immunoglobulins. Numbers of T lymphocytes, indicated by E rosette formation with human erythrocytes, ranged from a low of 1% in the thymus to 13% in the peripheral blood, whereas B-lymphocyte numbers ranged from 3% (thymus) to 41% (bone marrow) and from 6% (thymus) to 36% (bone marrow), as indicated by EAC rosette formation or presence of surface immunoglobulins respectively. Stimulation of peripheral blood lymphocytes with either phytohemagglutinin or concanavalin A increased the total number of E-rosetting cells two to threefold, whereas the number of EAC-rosetting cells decreased by half. Further, the percentage of cells bearing Fc receptors increased after phytohemagglutinin stimulation. These results indicate the E rosette technique can be used to identify and to monitor a population of canine T lymphocytes.     

Effects of corticosteroids on responses of bovine peripheral blood lymphocytes cultured with phytohemagglutinin.

Corticosteroids given in vivo altered the response of lymphocytes in the peripheral blood of calves. Lymphocytes were cultured and stimulated in vitro with phytohemagglutinin (PHA). After an initial suppression of lymphocyte responses to PHA, there was a rapid return to normal. It is concluded that, in calves, short-term, high-dose immunosuppressive therapy with corticosteroids produces a population of lymphocytes resistant to corticosteroids, possibly by destruction of corticosteroid-sensitive lymphocytes.