Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes

A microculture technique was developed for the in vitro blastogenesis of feline lymphocytes. Blastogenesis of ficoll-diatriazoate gradient separated mononuclear cell, washed blood and whole blood were compared. In general the whole blood cultures yielded higher stimulation indices (SI) than the washed blood or separated mononuclear cell cultures.The effect of several variables on the stimulation of lymphocyte cultures was examined. A cell concentration of 3 × 10⁵ cells/well and a 1:20 dilution of washed and unwashed whole blood gave optimal stimulation with concanavalin A (Con A). Phytohaemagglutinin-P (PHA-P) did not give significant levels of stimulation. Inactivated fetal calf serum (FCS) at levels of 2.5% (for washed blood) and 5% (for separated mononuclear cell and whole blood) gave highest SI. Supplementation with FCS was preferable to autologous, homologous or horse sera for all cultures. Optimal SI was obtained in all cultures incubated for 3 days and labelled with 1 μCi tritiated thymidine (³H-TdR) for the last 16 hours. The highest SI were in the range of 70 to 105 (18,764 to 42,681 counts per minute (CPM) for separated mononuclear cell culture, 100 to 165 (28,403 to 45,334 CPM) for washed blood culture and 105 to 186 (41,076 to 69,999 CPM) for whole blood culture.     

Leptin inhibits mitogen-induced proliferation of peripheral T lymphocytes from Holstein cows

This study examined the mitogenic response of bovine peripheral T lymphocytes to leptin, a pleiotropic hormone regulating food intake and energy expenditure. Leptin alone slightly suppressed proliferation of T lymphocytes in the presence of concanavalin A (ConA). Leptin also inhibited proliferation of T lymphocytes induced by anti-CD3 antibody. ConA treatment activated some protein kinases, including p44/p42(MAPK) and Akt/PKB, while anti-CD3 antibody treatment increased mRNA expression of suppressor of cytokine signalling (SOCS) 3, interferon (IFN)gamma, interleukin (IL) 2 and IL4 in T lymphocytes. Leptin alone increased only SOCS3 mRNA expression. Simultaneous treatment with mitogens and leptin enhanced IFNgamma mRNA expression but decreased IL2 mRNA expression, without any synergistic effect on phosphorylation of protein kinases or mRNA expression of SOCS3 and IL4. These results suggest that leptin modulates bovine T lymphocyte functions.     

A whole blood method for measuring mitogen-induced transformation of sheep lymphocytes

A simple and reproducible micromethod for determination of in vitro mitogenic responses of sheep peripheral blood lymphocytes is described. The test uses (i) whole blood diluted in RPMI 1640 medium to give a cell count of 0.5 x 106-1 x 106 lymphocytes/ml, (ii) mitogens in the range of 5-20 micrograms of phytohaemagglutinin/ml, 20-80 micrograms of lipopolysaccharide/ml or 20-80 microliters of poke weed mitogen/ml, and (iii) a stimulation time of 42 to 90 h. A considerable variation in mitogenic response was observed both between animals and on different occasions in the same animal. Because of the large periodic variation it was suggested that the test should be repeated using blood drawn at different times in order to determine the mitogenic response of an animal.     

Phagocytic activity in blood and proliferation of peripheral blood lymphocytes during the perinatal period in primiparous sows

The objective of this study was to determine whether phagocytic activity in blood and proliferation of peripheral blood lymphocytes are impaired during perinatal period. The study comprised 18 primiparous sows (Landras × Large White) free from clinical signs of diseases. During the experiment blood samples were collected three times from each sow. Sampling was performed on three different dates, always from all sows at once. At the first date of sampling sows were 21 ± 3 days before parturition, at the second date ± 1 day around parturition time and at the third date 21 ± 3 days after parturition. Phagocytic activity of monocytes and granulocytes was assessed in heparinized whole blood with addition of fluorescein isothiocyanate (FITC)-labelled opsonized bacteria Escherichia coli and the percentage of phagocytes which have ingested bacteria was measured as fluorescence activity by flow cytometry. The percentage of phagocyting monocytes and granulocytes was lowest at parturition (72.6 ± 16.37, 52.4 ± 20.59) and significantly increased within the next 21 ± 3 days (86.5 ± 6.16, 69.89 ± 5.80). Similarly, the phytohemagglutinin (PHA) (10 μg/ml) stimulated in vitro lymphocyte response was suppressed by parturition in primiparous sows (p < 0.001).     

两种方法测定羊外周血淋巴细胞增殖的研究

本试验比较~3H胸腺嘧啶核苷(~3H-TdR)渗入法和BrdU-ELISA方法测定羊外周血淋巴细胞增殖。分离健康羊外周血淋巴细胞,与不同浓度的非特异刺激因子刀豆素A(concanavalinA,1.0,0.5,0.25,0.125,0.0625,0.0313,0.0156,0.0078,0.0039μg/孔)培养,分别用~3H胸腺嘧啶核苷(~3H-TdR)和5-溴-2’-脱氧尿苷(BrdU)标记细胞,测定刺激细胞和对照细胞的每分钟脉冲数cpm或光吸收值OD,计算刺激指数SI和光吸收值差△OD,统计学方法分析SI和△OD的相关程度。同样的方法测定羊试验感染肝片吸虫(Fasciola hepatica)后,在感染早期阶段(PIWO-PIW4)其外周血淋巴细胞在特异性抗原-片形吸虫分泌排泄产物(ESP,5μg/孔)刺激下的增殖。结果表明,刺激指数SI和光吸收值差△OD在两个试验中的相关系数分别为0.946和0.924,SI和OD用均呈强正线性相关。BrdU-ELISA方法无同位素放射物,且敏感、快速、方便,和~3H-TdR渗入法一样可以用于测定淋巴细胞增殖。     

Monocyte-derived dendritic cells enhance cell proliferation and porcine circovirus type 2 replication in concanavalin A-stimulated swine peripheral blood lymphocytes in vitro

Dendritic cells (DCs) are professional antigen presenting cells cooperating with other immune cells for the activation of innate and adaptive immune responses. The objective of the present study was to investigate the replication activity of porcine circovirus type 2 (PCV2) in DCs and/or lymphocytes during their cross talk and its possible mechanism. Two models were set, herein. Swine blood monocyte (Mo)-derived DCs (MoDCs) or peripheral blood lymphocytes (PBLs) were inoculated with PCV2 prior to their co-cultivation. Bacterial lipopolysaccharide (LPS) and concanavalin A (Con A) were used to stimulate MoDCs and PBLs, respectively. During 6 days of cultivation, a high PCV2 antigen-containing rate without detectable intranuclear signals and a slight but significant increase in the copy number of PCV2 genome were detected in PCV2-inoculated MoDCs. The presence of LPS alone or PCV2-free PBLs, however, had no effect on the location of PCV2 antigens or copy number of PCV2 genome in PCV2-inoculated MoDCs. On the contrary, active PCV2 replication occurred in Con A-stimulated PCV2-inoculated PBLs. When compared with blood Mos, MoDCs induced significantly higher cell proliferation and intensified PCV2 replication in Con A-stimulated PCV2-inoculated PBLs, for which direct contact between MoDCs and lymphocytes was required. Among the cytokines secreted by Con A-activated PBLs, interleukin (IL)-2, but not IL-4 or interferon-γ, could induce cell proliferation and PCV2 replication in PCV2-inoculated PBLs. The findings suggest that although MoDCs support only limited PCV2 replication in themselves, their accessory cell function is required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes.     

In vitro response of purified ovine peripheral blood lymphocytes to phytohemagglutinin-M.

Peripheral blood was collected from three normal yearling castrated male lambs. Lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation. Lymphocyte response to phytohemagglutinin was evaluated in an isotopic uptake stimulation test under varying culture conditions in order to standardize the assay. Results were analyzed as log10 counts per minute and stimulation indices. Culture conditions consisting of 2 microL (20 micrograms) phytohemagglutinin-M/culture, 1 X 10(6) cells/mL and a 72 hour incubation period were selected for use in further studies.     

Ochratoxin A as a suppressor of mitogen-induced blastogenesis of porcine blood lymphocytes

The in vitro effect of ochratoxin A on pig lymphocytes stimulated by the mitogen Concanavalin A was studied by measuring the rates of ³H-thymidine incorporation in DNA.Ochratoxin A inhibited the mitogenic response to Concanavalin A in a dose dependent way. An almost total inhibition was obtained with ≥ 1 mg ochratoxin A/1, approximately 60 % inhibition was produced by 0.5 mg ochratoxin A/1 and approximately 10 % inhibition by 0.06 mg ochratoxin A/1.The immunosuppressive effect of ochratoxin A was not altered much by different contents of bovine serum albumin, 0.1 or 0.3 %, in the cell culture medium.     

Analysis of pokeweed mitogen-induced in vitro proliferative and antibody responses of bovine lymphocytes.

The functional role of subpopulations of bovine cells in the regulation of pokeweed mitogen (PWM)-induced proliferative and antibody responses of peripheral blood mononuclear cells (PBM) was analysed. Subpopulations of bovine PBM identified by specific monoclonal antibodies (mAbs) were isolated by sorting them through the fluorescence activated cells sorter (FACS). The depletion from PBM of T cells bearing the CD4 marker, recognised by mAb IL-A12, resulted in a reduction of PWM-induced responses, which could be partly reversed by the addition of CD4 positive T cells. The depletion of cells belonging to the macrophage/monocyte lineage also resulted in a reduction of PWM-induced proliferative responses. PBM depleted of CD8 positive T cells, recognised by mAb IL-A51, showed increased PWM-induced responses, which in turn were reduced by the addition of mAb IL-A51 positive cells. Proliferative and antibody responses were also obtained by PWM stimulation of FACS-purified B cells, in the presence of bovine T cell growth factor.     

Effect of surgery on the in vitro response of canine peripheral blood lymphocytes to phytohemagglutinin

The effects of surgery (ovario-hysterectomy) and anesthesia on phytohemagglutinin-induced lymphocyte blastogenesis were studied in vitro in 12 dogs. Four dogs had depressed lymphocyte blastogenic responses after surgery. This suppression was transient with normal blastogenic responses occurring in cells from all dogs 24 hours after surgery. Seemingly, T-lymphocyte function may be depressed, only transiently, after surgery.     

In vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes from ponies.

The in vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes (PBL) from ponies were studied. Prednisolone inhibited lymphocyte stimulation by phytohemagglutin (PHA) in a dose-dependent manner, without inducing lysis even at large doses. The PBL from horses heterozygous for the combined immunodeficiency trait responded to corticosteroid treatment the same as did PBL from normal ponies. Removal of the corticosteroid after incubation with PBL from normal ponies partially restored responsiveness of these cells to PHA. Chronic in vivo treatment of ponies with corticosteroids caused a marked decrease in the absolute numbers of circulating lymphocytes. Most remaining lymphocytes had detectable surface immunoglobulin and C3 receptors, suggesting a greater decrease in the T-lymphocyte population. In spite of this, there was little change in the in vitro PHA- or keyhole limpet hemocyanin-sensitized ponies. In general, the corticosteroid effects of lysis, as well as the mitogenic and antigenic responses of PBL from ponies, were similar to those previously reported for human lymphocytes.     

Human recombinant interleukin-2(125) induced in vitro proliferation of equine, caprine, ovine, canine and feline peripheral blood lymphocytes

Equine, caprine, ovine, canine and feline peripheral blood lymphocytes were evaluated in a short term dose-response study for their in vitro blastogenic responsiveness to human recombinant interleukin-2(125) (HrIL-2(125] alone or in combination with phytohemagglutinin-P, concanavalin-A, and pokeweed mitogen. HrIL-2(125) induced lymphocyte proliferation in all of the animals tested. The magnitude of the proliferative response varied among the species of animal tested. In all cases the proliferative response was dependent on the concentration of HrIL-2(125). HrIL-2(125) at a minimum concentration of 10(2) Cetus Units (CU)/ml produced a significant proliferative response in isolated horse, goat and sheep lymphocytes. In cat and dog lymphocytes, a concentration of 10(3) CU/ml was necessary to induce a significant proliferative response. Maximal lymphocyte proliferation was reached in horses and sheep at a concentration of 10(4) CU/ml of HrIL-2(125). In goats, cats, and dogs a maximum proliferative response was found to be at a concentration equal to or greater than 10(4) CU/ml of HrIL-2. Co-stimulation of lymphocytes with mitogens and submaximal concentrations of HrIL-2(125) (10 CU/ml) induced a synergistic proliferative response which in nearly all cases was significantly greater (P less than 0.05) than the arithmetic sum of the responses induced by the same concentration of the mitogens and HrIL-2(125) alone. The two exceptions were co-stimulation of feline lymphocytes with concanavalin-A and co-stimulation of canine lymphocytes with pokeweed mitogen.     

Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells

Synthetic oligodeoxyribonucleotides (ODN) may prove useful immune modulators in equine medicine. It is however important to assess the effects of each specific ODN in the species it is intended to be used in. The present study therefore aimed to evaluate some ODN for induction of cytokine production; i.e. type I interferons (IFN), IFN-γ, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and proliferation of equine peripheral blood mononuclear cells (PBMC). A panel of four ODN containing unmethylated cytosine-guanosine sequences (CpG) was used: ODN 1 and ODN 8 representing A-class; ODN 2006 representing B-class and ODN 2395 representing C-class-ODN. In addition, two ODN where CpG-motifs were reversed to GpC were included; ODN 2137 otherwise identical to ODN 2006 and ODN 5328 otherwise identical to ODN 2395. Cytokine concentrations were measured in cell culture supernatants after 24h of induction and proliferation was determined after 72 h of induction. Each ODN was tested with PBMC from at least 5 individual horses with and without the addition of lipofectin to cell cultures. Type I IFN, IFN-γ and TNF-α production was readily induced by ODN 1, ODN 2006 and ODN 2395 both in the presence and absence of lipofectin and all three types of ODN induced similar levels of cytokines. Proliferation of PBMC was clearly induced by ODN 2006 and ODN 2395 while ODN 1 only induced low-level proliferation. The levels of proliferation induced were not influenced by the presence of lipofectin. TGF-β production was not induced by any of the tested ODN. ODN 8, ODN 2137 and ODN 5328 were largely inactive in all assays. Thus, responses seemed dependent on or increased by CpG-motifs but presence of CpG-motifs did not necessarily confer activity since ODN 8 was inactive despite its CpG-motifs. Taken together, with equine PBMC distinctions in induction of different leukocyte functions between A-, B-, and C-class ODN were less obvious than what has been observed for human cells. These observations further stress the presence of species differences in ODN-induced responses.     

Technical aspects of low 3H-thymidine incorporation by mitogen stimulated canine peripheral blood lymphocytes in vitro

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 degrees C, 5% CO2 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 (G1), it was found that comparable number of G1 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 G1 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 G1 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.     

Mitogen-induced transformation of sheep and goat peripheral blood lymphocytes in vitro: The effects of varying culture conditions and the choice of an optimum technique

Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H³]-thymidine ([H³]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 10⁵ viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H³]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.     

In vitro stimulation of bovine peripheral blood lymphocytes: effect of short-term storage of blood prior to lymphocyte culture

Storage of peripheral blood from Mycobacterium bovis-sensitized cattle from 1 to 48 hours at 4, 22, and 37 C was shown not to alter markedly the lymphocyte blastogenic response to M bovis-purified protein derivative. Concanavalin A-induced lymphocyte mitogenic responses were unaffected by storage of blood for 1, 24, or 48 hours at 22 C and 37 C; however, storage of blood for 48 hours at 4 C significantly lowered (P less than 0.05) mitogenic responses to concanavalin A, as compared with responses to blood kept at 22 C. Mononuclear cell recovery from stored blood at all temperatures was markedly less than that from freshly drawn blood samples. Cell recoveries were most affected by storage of blood at 4 C and 37 C.