Dog Peripheral Blood Lymphocytes Research Articles

Influence of dietary fish oil supplementation on DNA damage in peripheral blood lymphocytes of nine healthy dogs.

BackgroundFish oil (FO) supplementation as a source of omega 3 fatty acids is associated with beneficial effects on health. However, high unsaturated fatty acid content in the diet could result in increased lipid peroxidation and damage to proteins, lipids and DNA. We evaluated the effect of dietary FO supplementation on DNA damage in peripheral blood lymphocytes of dogs. Additionally, we determined the effect of FO supplementation on lipid peroxidation and lipid profile of these dogs.MethodsHealthy male dogs (n = 9) were randomly assigned to one of two diets during 90 days: control (CG, n = 4), based on a commercial food, and FO (FOG, n = 5), the same food supplemented with 1000 mg FO. Blood samples were collected on days −1, 30, 60 and 90. DNA damage was assessed with the comet assay, and the damage index was obtained. Malondialdehyde (MDA) levels were determined as an indicator of lipid peroxidation. Lipid profile determination included serum triglyceride, cholesterol, low‐density lipoprotein and high‐density lipoprotein levels (HDL).ResultsDamage index values (arbitrary units) were lower in FOG on day 30 (CG, 13.7 ± 2.5; FOG, 6.5 ± 2.5), 60 (CG, 14.7 ± 2.5; FOG, 3.5 ± 2.5) and 90 (CG, 15.5 ± 2.5; FOG, 3.0 ± 2.5) compared with CG (treatment × time interaction, p < 0.01). Serum MDA and HDL concentrations were lower in FOG compared with CG on day 60 and 90 (treatment × time interaction, p < 0.05).ConclusionThese findings suggest that dietary FO supplementation did not induce DNA damage in peripheral blood lymphocytes of healthy dogs, but rather reduced it.

CHARACTERISTICS OF MAJOR HISTOCOMPATIBILITY COMPLEX EXPRESSION AND CYTOKINE PROFILES IN SPONTANEOUS CANINE TUMOURS

Recent studies have reported gene therapy as an alternative treatment for canine’s cancer. Fifty-three samples from canine patients were obtained and the expression of MHC classes I and II and CD-markers in tumour cells, subpopulations of tumour-infiltrating lymphocytes (TILs), peripheral blood lymphocytes (PBLs) through flow cytometry and tumour cell cytokines were examined through real-time reverse transcription polymerase chain reaction. These data were compared by using Biomarker Patterns Software. Mammary gland tumours (MGTs) showed low MHC I expression that can be correlated with tumour malignancy, with cut-off values of 72.58% (MGT, [Formula: see text]% and non-MGT, [Formula: see text]%). IL-8, showing a positive correlation with angiogenesis, expression in MGTs was significantly high and IL-12 expression in complex and tubulopapillary carcinomas were significantly lower than that in healthy mammary gland cells ([Formula: see text]). PBLs of dogs with MGTs had fewer T cells, particularly T helper cells, and B cells and more non-T, non-B cells, particularly malignant MGTs, than did PBLs of healthy dogs. Among all surface markers and cytokines detected, high CD3 TIL was significantly correlated with a more favourable prognosis. Cytokines, such as IL-8, -12, and -15, can be the indicators of therapeutic targets because of their specific expression patterns in specific MGT subtypes.

Influence of General Anesthesia with Isoflurane Following Propofol-Induction on Natural Killer Cell Cytotoxic Activities of Peripheral Blood Lymphocytes in Dogs

To investigate influence of general anesthesia on immunological anti-tumor activity, the natural killer (NK) cytotoxic activity of peripheral lymphocytes (PBLs) was measured in 7 dogs anesthetized for 3 hr with isoflurane following propofol-induction (anesthesia group) and 6 dogs without anesthesia (control group). Blood samples were collected before (baseline) and 24, 120 and 192 hr after the anesthesia. The PBLs were isolated via centrifugation with Ficoll-Hypaque solution (density, 1.073), and adherent cells were removed. The NK cytotoxic activity of the isolated PBLs against canine thyroid cancer cells was detected by the colorimetric rose Bengal assay. Significant decrease in the NK cytotoxic activity was observed at 24 hr after the anesthesia, compared with the baseline values and the control group. The NK cytotoxic activities were recovered to the baseline values until 120 hr after the anesthesia. The general anesthesia with isoflurane following propofol-induction decreased the NK cytotoxic activities of PBLs in dogs. This finding has a clinical relevance to the risk of tumor recurrence or metastasis induced by the suppression of immunological anti-tumor activity after general anesthesia in dogs. The results further emphasized the importance of the need to evaluate immune suppression following general anesthesia in animals.

Phenotypic analysis of hepatic lymphocytes from healthy dogs.

Phenotypes of lymphocytes from laparoscopically biopsied liver tissues of eleven healthy beagle dogs were analyzed. The proportion of CD3(+) lymphocytes (T cells), CD3 (-)CD21(+) lymphocytes (B cells) and CD3 (-)CD21(-) lymphocytes (non-T non-B lymphocytes), and the CD4(+)/CD8(+) ratio in the canine hepatic lymphocytes were 54.8 +/- 11.9%, 4.7 +/- 3.1%, 40.7 +/- 13.2%, and 0.33 +/- 0.12, respectively, while those in peripheral blood lymphocytes were 85.4 +/- 6.5%, 9.3 +/- 6.1%, 5.3 +/- 1.8%, and 1.64 +/- 0.36, respectively. These results indicated that the constitution of hepatic lymphocytes quite differed from that of peripheral blood lymphocytes in dogs, and suggested that the regional immunity in canine liver might be specific.

Cytometric evaluation of peripheral blood lymphocytes in dogs with lymphoma during chemotherapy.

Twenty dogs with clinically diagnosed multicentric lymphoma were evaluated for percentages of peripheral blood lymphocyte subpopulations. Cytometric analysis was performed before and during chemotherapy. The results were compared to those obtained from a control group of healthy dogs. The percentages of CD5+, CD4+ and CD8+ cells were markedly decreased and CD21-like+ cells markedly increased in dogs with lymphoma in comparison with the control group. During the course of chemotherapy these values returned to ranges observed in healthy animals.

Reduction in DNA damage in brain and peripheral blood lymphocytes of elderly dogs after treatment with dehydroepiandrosterone (DHEA)

Steady state levels of DNA damage are substantial in vertebrate animals as a consequence of exposure to endogenous and environmental mutagens. DNA damage may contribute to organismal senescence and an increased risk for specific age-related diseases. In this study, we determined if treatment with the neuroactive adrenal steroid, dehydroepiandrosterone (DHEA), which exhibits antioxidant and anticarcinogenic properties in rodents, would reduce DNA damage in the brain and peripheral blood lymphocytes (PBLs) of elderly dogs. Elderly male dogs, physiologically equivalent to 59–69-year-old men, were randomly assigned to receive no treatment ( n=9 dogs) or DHEA at 100 mg/kg PO daily ( n=8 dogs). Extent of DNA damage in brain cells and PBLs was measured using alkaline comet assay. The effect of DHEA treatment on the susceptibility of PBLs to H 2O 2-induced DNA damage was also measured. We found that elderly male dogs receiving daily DHEA treatment for 7 months had significantly less DNA damage detectable in their brain compared to age-matched control dogs. After 7 months treatment, DHEA-treated dogs also had a significant reduction in DNA damage in PBLs compared to pre-treatment levels. We also found that PBLs of dogs treated with DHEA were more resistant to H 2O 2-induced DNA damage than PBLs of untreated dogs. Our results did not show that basal DNA damage in PBLs was strongly correlated with DNA damage within the brain. The results of this study suggest that DHEA supplementation can significantly reduce steady state levels of DNA damage in the mammalian brain. Further evaluation of DHEA as a neuroactive agent and its effects on DNA damage and gene expression in other tissues and species is warranted.

Identification and characterization of a mouse monoclonal antibody (M10) directed against canine (dog) CD8+ lymphocytes

Monoclonal antibodies (mAb) were produced by immunizing BALB/c mice with non-adherent dog lymphocytes. M10 was specific for a subset of dog lymphocytes. M10 belonged to the IgG1 subclass and reacted with 26% of dog peripheral blood lymphocytes, 24% of spleen lymphocytes, 81% of thymus cells, 1.2% of bone marrow cells (5.8% of bone marrow lymphocytes) and 23% of PHA-stimulated lymphocytes. Immunohistology of snap-frozen thymus and spleen showed that the spleen B-cell area stained negative, whereas the spleen T-cell area and the thymus medulla exhibited positive reaction in 20–30%. The thymus cortex was strongly positive. M10 diminished cell lysis by 58% in cell mediated lysis assays (CML). Immunoblot assays revealed that M10 recognized an antigen with a molecular weight of 76 kD under non-reducing and 33 kD under reducing conditions. Finally, M10 bound to a canine CD8α transfected rat T-cell line (NB2). These findings characterize M10 as an antibody directed against the dog CD8 antigen.

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 hrlL-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 hrlL-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 hrlL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.

In vitro lymphocyte stimulation by Concanavalin A and with histamine as a co-mitogen in dogs with atopic dermatitis

Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentrations of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indices neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37°C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubation in cell-culture medium between normal and atopic dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type 1 hypersensitivities such as atopy, can modulate lymphocyte function. At 10 −4 and 10 −8 M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H 1 and H 2 antagonists, we concluded that histamine enhanced mitogenesis via H 1, receptors and suppressed it via H 2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.

Immunochemical analysis of glycosylated and nonglycosylated DLA class I antigens.

Dog peripheral blood lymphocytes, when cultured with 35S-methionine in the presence of tunicamycin, synthesize DLA molecules consisting of beta 2-microglobulin and a heavy chain approximately 3000 daltons lower in apparent mol. wt. than observed in control cases. This difference in mol. wt. is consistent with the fact that a single N-linked carbohydrate side chain is present on the heavy chain of DLA class I antigens. There is no evidence of polymorphism in the DLA light chain (beta 2m). Both glycosylated and nonglycosylated forms of the heavy chain, however, show microheterogeneity, which can be related to tissue-type. Analysis by two-dimensional electrophoresis shows that the biochemical heterogeneity in the DLA heavy chain is less than expected from DLA serology, and less than found in HLA class I antigens. The data are consistent with the fact that the products of only a single DLA class I locus are detected.

Technical aspects of low 3H-thymidine incorporation by mitogen stimulated canine peripheral blood lymphocytes [formula omitted

The value of [ 3H]-thymidine incorporation as a measurement for mitogen induced proliferation of dog peripheral blood lymphocytes (PBL) has been examined. The cells were cultured in RPMI 1640, enriched with 10% autologous plasma for 48 hours at 37°C, 5% CO 2 and 95% relative humidity. Under these conditions a great variability in [ 3H]-thymidine incorporation was observed. By analysis of CPM and number of activated cells (G 1), it was found that comparable number of G 1 cells were generated in human and dog PBL. Also, the membrane transport of thymidine was very similar for lymphocytes of the two species. Nevertheless, a low [ 3H]-thymidine incorporation by dog PBL was frequently seen, and this phenomenon could be related to a release of soluble substance(s) within the cultures. When the cultured cells were washed and resuspended in fresh medium immediately before pulsing, the expected CPM per G 1 cell could be obtained. Since it has been described in the literature that macrophages can produce cold thymidine in macrophage enriched lymphocyte cultures, the in vitro response of non-adherent dog PBL was analyzed. Mitogen stimulation of such non-adherent cells resulted in CPM per G 1 cells very similar to those obtained with washed cells. Based on these data, it is suggested that the production of cold thymidine might be one of the technical problems related to cultures of lectin stimulated dog PBL in vitro and it should be taken into consideration, if [ 3H]-thymidine incorporation is used as the only measure of lymphocyte proliferation.

Radiosensitivity and life span of dog peripheral blood lymphocytes

Observations made after 48 h of cultivation show that 52% of dog lymphocytes are already in M 2 and 17% in M 3 at that fixation time. Evidently, the large discrepancy observed between man and dog with respect to the yield of dicentric chromosomes induced by an exposure to 200 rad of X-rays and observed after a culture time of 48 h does not correspond to a difference in chromosomal radiosensitivity. There is, indeed, no statistically significant difference between the yield of dicentrics observed in M 1 human lymphocytes (32.0 per 100 cells) and that in dog lymphocytes after correction of the values obtained at 48 h (28.6 per 100 cells). From the observations performed on whole-body irradiated dogs one can estimate that the half-time of dog lymphocytes carrying chromosome aberrations is less than 30 days.

Responses of canine lymphocytes to allogeneic and autologous Islets of Langerhans in mixed cell cultures.

Because successful allotransplantation of islets of Langerhans isolated by collagenase digestion has been difficult in many animal species, we asked whether isolated islet preparations might have tissue specific determinants conferring amplified immunogenicity in vitro. Lymphocyte proliferative responses ([(3)H]thymidine uptake) were studied in beagle dogs in mixed culture combinations of lymphocyte vs. lymphocyte (MLC) and lymphocyte vs. islet (MLIC). In five MLC responder and five nonresponder pairs, peripheral blood lymphocytes of dogs A and B were used as responding cells, and dog B x-irradiated lymphocytes (Bx), x-irradiated (or nonirradiated) islets (BI), or hepatic cells (BH) were used as stimulating cells in primary and secondary reactions. For the secondary reactions, A + Bx, A + BI, or B + BI were incubated for 9 d (A'B, A'BI, B'BI, respectively) before addition of new stimulating cells. The results showed that islets were autostimulatory, eliciting a tissue-specific lymphoproliferative response in a primary MLIC. Thus, B + BI reactivity was evident at 3,5, and 7 d in primary culture, whereas collagenase-digested liver cells, or lymphocytes obtained from collagenase-digested lymph nodes did not stimulate autologous lymphocytes. A separate reactivity was observed in the allogeneic A + BI combination in MLC responder pairs, and the peak response of A + BI at 9 d was markedly greater than that of B + BI, suggesting the presence of major histocompatibility complex lymphocyte-defined locus determinants in the islet preparations, in addition to islet-specific determinants. A secondary reaction was observed if lymphocytes were primed with islets and challenged with islets (A'BI + BI or B'BI + BI), but not if they were challenged with lymphocytes (A'BI + Bx, B'BI + Bx) or hepatic cells (A'BI + BH, B'BI + AH). Furthermore, priming of lymphocytes with autologous islets (B'BI) led to exclusion of any reactivity against allogeneic lymphocytes, i.e., B'BI suppressed A + Bx, and B'BI also markedly suppressed phytohemagglutinin-stimulated lymphoproliferative responses. Experiments were performed that excluded the possibility that the insulin levels present in the MLIC, the presence of passenger lymphocytes in the islets, or the maintenance of islets in tissue culture for 1-7 d affected the observations. These results provide evidence for the existence of alloantigens as well as tissue-specific antigens on collagenase-isolated islets of Langerhans.

Secondary responses of alloantigen-primed dog lymphocytes.

The usefulness of Primed Lymphocyte Typing (PLT) for recognizing Lymphocyte Defined (LD) determinants of the Major Histocompatibility Complex of the dog (DLA) was assessed in 10 separate experiments. Application of a technique described for human cell to peripheral blood lymphocytes of dogs gave reproducible, informative results. DLA LD determinants were shown to be of major importance in secondary responses of alloantigen "primed" lymphocytes, but other, a yet undefined, factors also appeared to play a role in the assay.

Erythroid colony stimulating and inhibiting cells in peripheral blood of transfused dogs: separation of function by velocity sedimentation

We have previously shown that the addition of normal dog peripheral blood lymphocytes (PBL) to cultures of allogeneic marrow increases the number of marrow-derived erythroid colonies (EC), but that PBL from transfused dogs usually inhibit EC growth from marrow of the transfusion donor. In this study, the cells in normal dog PBL responsible for stimulating EC growth were shown to sediment in a narrow peak at 4.30 mm/hr. A similar population of stimulating cells exists in transfused dogs and can be separated, on the basis of size, from cells that inhibit EC growth. EC-stimulating cells from transfused dog PBL sediment at 3.3--5.0 mm/hr, while cells responsible for inhibition are larger and sediment more rapidly at 5.4--8.1 mm/hr. These data demonstrate that cells capable of stimulating allogeneic EC are present in transfused dogs, but their stimulating ability is masked by the presence of EC-inhibiting cells. Thus, coculture experiments designed to test lymphocyte/marrow cell interactions may miss significant but opposing effects if unfractionated cells are used.

Erythroid Colony Stimulating and Inhibiting Cells in Peripheral Blood of Transfused Dogs: Separation of Function by Velocity Sedimentation

We have previously shown that the addition of normal dog peripheral blood lymphocytes (PBL) to cultures of allogeneic marrow increases the number of marrow-derived erythroid colonies (EC), but that PBL from transfused dogs usually inhibit EC growth from marrow of the transfusion donor. In this study, the cells in normal dog PBL responsible for stimulating EC growth were shown to sediment in a narrow peak at 4.30 mm/hr. A similar population of stimulating cells exists in transfused dogs and can be separated, on the basis of size, from cells that inhibit EC growth. EC-stimulating cells from transfused dog PBL sediment at 3.3-5.0 mm/hr, while cells responsible for inhibition are larger and sediment more rapidly at 5.4-8.1 mm/hr. These data demonstrate that cells capable of stimulating allogeneic EC are present in transfused dogs, but their stimulating ability is masked by the presence of EC-inhibiting cells. Thus, coculture experiments designed to test lymphocyte/marrow cell interactions may miss significant but opposing effects if unfractionated cells are used.

Characterization of inhibitor(s) of lymphocyte activation in serum from rats with adjuvant arthritis.

Serum from adjuvant arthritic rats inhibits the concanavalin A- (Con A) and lipopolysaccharide-induced stimulation of lymph node cells, leaving the basal and phytohemagglutinin-stimulated 3H-thymidine incorporation unaffected. Con A-stimulated 3H-thymidine uptake is also inhibited in rat spleen and peripheral blood lymphocytes and in dog peripheral blood lymphocytes. The intensity of the inhibitory activity in serum is positively correlated with the intensity of the secondary lesions of adjuvant arthritis. Inhibitory activity was not found in serum from rats bearing nystatin-induced inflammation. Serum fractionation studies indicated that the inhibitory activity cannot be attributed to low molecular weight alpha2-glycoproteins or to gamma-globulins and alpha2-macroglobulins, but it is present in a fraction migrating with beta-globulins. The inhibitory activity in arthritic rat serum is reduced by treatment with non-steroidal anti-inflammatory drugs, but is unaltered by D-penicillamine. It is suggested that this inhibitory activity is part of the systemic response to an immunologically mediated inflammation.

Role of the regional lymph node in neoplasia: cellular mediated reactivity in vitro by autologous regional or distant lymph nodes or peripheral blood lymphocytes of dogs with spontaneous neoplasms.

This report concerns the in vitro reactivity of lymphocytes obtained from regional (draining) lymph nodes, distant nodes and peripheral blood against autochthonous spontaneous neoplasms. Tumors, blood and lymph nodes were collected aseptically prior to necropsy. Primary tumor cultures were established. Viable tumor cells were first incubated with media or autologous sera and then various numbers of lymphocytes were added. The mixtures were rotated for 60 minutes and then plated. Two and/or five days later cultures were terminated and viable target cells counted. The results of the tests were similar regardless of the lymphocyte source utilized. In vitro tumor cell cytotoxicity by high numbers (1000:1 lymphocytes to tumor cells) of any autologous lymphocytes and interference of such reactivity by autologous sera were demonstrated. Low numbers (100:1) of any lymphocyte source lead to tumor growth stimulation. Animals with spontaneous neoplasms are probably the best model for the study of human clinical oncology. Our data demonstrate that in these animals lymphocytes obtained from regional nodes were not unique in their reactivity to tumors. It is possible that early in the disease the draining node may play a vital role in initiation of host response; however, later its importance probably diminishes.

Stimulated lymphocyte cultures: responder recruitment and cell cycle kinetics.

Lymphocytes, stimulated with different doses of plant mitogens or allogeneic cells, incorporate varying amounts of [(3)H]thymidine. Theoretically, this may be due to different numbers of responding cells, to earlier proliferative response of these cells, and/or to their more or less rapid transit through the cell cycle. Dog peripheral blood lymphocytes were stimulated in vitro with different doses of phytohemagglutinin (PHA), or with allogeneic lymphocytes. After their synchronization by incubation with hydroxyurea (4 mM), the mean durations of the cell cycle, and of the different cell cycle phases were constant and unrelated to strength or type of stimulation. PHA-stimulated lymphocyte cultures were maintained in the presence of colchicine, to prevent clonal proliferation of responding lymphocytes. DNA uptake in this setting, attributed to first generation responders, was related to the strength of proliferative lymphocyte response in control cultures without colchicine. Furthermore, cell proliferation occurred earlier with greater stimulation. It is concluded that higher [(3)H]thymidine uptake in vitro by stimulated lymphocytes is due to greater numbers of responding cells, which are triggered into proliferative response earlier, and not to a more rapid transit of the responding cells through the cell cycle.