Culture conditions for blastogenic responses of bovine mammary mononuclear cells

Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.     

天门冬多糖对小鼠脾脏淋巴细胞体外增殖的影响

本试验旨在研究天门冬多糖对小鼠脾脏淋巴细胞体外增殖的影响。从正常小鼠分离脾脏淋巴细胞体外培养,在细胞培养体系中分别加入终浓度为50、100、200、400 mg/L天门冬多糖或配合刀豆素(5 mg/L)作用24、487、2 h,以MTT法测定细胞的增殖程度。结果表明,天门冬多糖可显著促进体外培养的小鼠脾脏淋巴细胞增殖,并与ConA有协同作用。     

不同组分、不同纯度中药复方多糖对小鼠脾淋巴细胞增殖影响

目的：研究中药复方多糖分级组分及不同纯度对小鼠脾脏淋巴细胞的增殖作用。方法：用ConA诱导T淋巴细胞增殖,用MTT法测定淋巴细胞的增殖能力。结果：水煎剂、总多糖、分级组分2、纯化组分2均可极显著促进小鼠脾脏淋巴细胞增殖,并能增加ConA诱导T淋巴细胞增殖。结论：中药复方的不同活性部位具有不同的免疫调节活性。     

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.     

Effect of Different Mulberry Leaf Extracts on Spleen Lymphocyte Proliferation in Mice

This experiment was aimed to compare the effects of different mulberry leaf extract on spleen lymphocyte proliferation in mice. Mulberry leaf polysaccharides (MLP-1, MLP-2, MLP-3, MLP-4 and MLP-5) were prepared with mulberry leaf aqueous extract (MLAE) by different concentration of alcohol deposit, polysaccharides concentration of all extracts were determined by phenol sulfuric acid method. Five different MLPs and MLAE were as the experimental drug, when polysaccharide concentration was 15.625, 31.25, 62.5, 125 and 250 μg/mL, the changes of spleen B and T lymphocytes proliferation in mice which were stimulated by MLPs and MLAE in single or synergistical stimulation of drugs with LPS and PHA were determined by MTT method. The results showed that when polysaccharide concentration was in the range of 15.625 to 250 μg/mL, MLPs and MLAE in single or synergistical stimulation with LPS and PHA could stimulate the spleen B and T lymphocytes proliferation, MLP-1 at low concentration could stimulate significantly spleen B lymphocyte proliferation remarkably (P<0.05), and MLP-3 and MLP-5 at low concentrations showed strong stimulation ability of T lymphocyte proliferation. When MLAE was in synergistical stimulation with LPS at most concentrations points, the spleen B lymphocytes proliferation in mice were significantly higher than those of the cell control and LPS control groups, which revealed that MLAE could significantly improve the humoral immune function (P<0.05).     

不同桑叶提取物对小鼠脾脏淋巴细胞增殖能力的影响

试验旨在比较不同桑叶提取物对小鼠脾脏淋巴细胞增殖作用的影响。用不同浓度乙醇醇沉桑叶水提物,制备桑叶粗多糖MLP-1、MLP-2、MLP-3、MLP-4、MLP-5,采用苯酚硫酸法测定各提取物多糖含量。以5种桑叶粗多糖和桑叶水提物（MLAE）为试验药物,采用MTT法比较多糖含量分别在15.625、31.25、62.5、125、250 μg/mL浓度时各提取物单独刺激及协同LPS、PHA共同刺激小鼠脾脏B、T淋巴细增殖的变化。结果显示,多糖浓度在15.625~250 μg/mL范围内,各提取物无论单独还是协同LPS和PHA时均能促进小鼠脾脏B、T淋巴细胞的增殖,其中,MLP-1多糖含量在低浓度时能显著刺激脾脏B淋巴细胞增殖（P<0.05）;MLP-3和MLP-5在低浓度时表现出较强的刺激T淋巴细胞增殖的能力;MLAE协同LPS在大部分浓度点的脾脏B淋巴细胞的增殖能力均显著高于细胞对照组和LPS对照组,可显著提升体液免疫功能（P<0.05）。     

The Effects of Salivary Gland Extracts from <Emphasis Type="Italic">Boophilus microplus</Emphasis> Ticks on Mitogen-stimulated Bovine Lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.     

天门冬多糖对猪脾淋巴细胞体外增殖的影响

试验旨在研究不同浓度的天门冬多糖(ASP),在ConA或LPS的协同刺激下对猪脾淋巴细胞体外增殖的影响。猪脾淋巴细胞体外培养体系中加入不同浓度的天门冬多糖使其终浓度为400、200、100、50、25、12.5μg/ml,在ConA(5μg/ml)或者LPS(10μg/ml)的协同刺激作用下,细胞培养24、48、72 h时,观察猪脾淋巴细胞增殖情况。结果表明,天门冬多糖及其协同ConA或LPS能显著或极显著的促进猪脾淋巴细胞体外增殖(P<0.05或P<0.01)。     

猪外周血T淋巴细胞增殖反应MTT检测方法的建立

T细胞增殖反应是宿主T细胞识别病原的结果,也是宿主细胞免疫应答的重要指标之一。为了便于检测猪群在病原感染或者疫苗免疫过程中产生的细胞免疫应答,本研究应用MTT法建立了体外检测猪外周血T细胞增殖反应的研究方法。通过密度梯度离心法从外周血分离得到外周血单个核细胞（PBMC）,然后利用单核细胞和淋巴细胞不同的生长特性（贴壁与否）,弃掉贴壁的单核细胞,获得外周血淋巴细胞(PBL)。外周血淋巴细胞的流式分析结果显示,分离获得的PBL中T细胞所占比例达到了80%以上。应用MTT法分析了非特异性刺激物刀豆蛋白A（ConA）的浓度和细胞培养密度对T细胞增殖的影响。结果显示,ConA的工作浓度为5 μg/mL、细胞培养密度为2×106/mL时T细胞的增殖反应最强烈。本研究所建立的猪外周血T细胞增殖反应检测法可以为研究猪针对病原或疫苗的细胞免疫反应提供参考。     

Mobiloization of intracellular calcium ions in chicken and rat lymphocytes induced by T cell mitogens

Cytosolic Ca²⁺ is known to be an important factor in intracellular signaling pathways that regulate several cellular functions. The present study was designed to measure the intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) in T cell mitogen-stimulated chicken lymphocytes, and to compare the results with those in rat lymphocytes. [Ca²⁺]_i was increased in the thymocytes, splenocytes and bursacytes of chickens, and in the thymocytes and splenocytes of rats following exposure to the mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Increases were greatest in the thymocytes followed by the splenocytes and bursacytes. The PHA-induced changes in the thymocytes and splenocytes were similar in chickens and rats, but the ConA-induced increases were significantly lower in the chickens than rats. Pretreatment with EGTA before the application of PHA and ConA completely suppressed the rise in [Ca²⁺]_i in all the chicken lymphocytes, indicating that the increases that occurred in PHA- and ConA-treated chicken lymphocytes could be entirely attributed to the influx of extracellular Ca²⁺. On the other hand, the PHA- and ConA-induced increase in [Ca²⁺]_i in rat lymphocytes was not completely suppressed by EGTA, indicating the recruitment of Ca²⁺ from the intracellular Ca²⁺ pool. The results suggest species differences in the Ca²⁺-based responses to T cell mitogens between chicken lymphocytes and rat lymphocytes.     

Production of interleukin-2 mRNA by bovine lymph node lymphocytes in response to concanavalin A, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin

Interleukin-2 (IL-2) is a lymphokine which, upon binding to its receptor, leads to the proliferation and differentiation of T-cells (helper, suppressor, and cytotoxic) and B-cells. While human and murine IL-2 have been extensively studied, less is known about bovine IL-2. In order to understand the induction of bovine IL-2 at the molecular level, we have examined IL-2 mRNA induction. The dose-responses and time courses of the production of IL-2 mRNA in response to Concanavalin A (ConA), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin in lymph node lymphocytes (LNC) were determined. We found that high levels of IL-2 mRNA were produced in response to 1 μg ml⁻¹ ConA plus 10⁻⁸ M TPA, but that even higher levels were produced in response to 1 μM ionomycin plus 10⁻⁸ M TPA. We also found that LNC stimulated with ConA displayed two phases of IL-2 mRNA production, one occurring approximately 2–4 h after stimulation and one occurring approximately 10 h after stimulation. However, in the presence of ConA plus TPA or ionomycin plus TPA the response was monophasic. IL-2 mRNA was detected within 2 h of addition of ConA plus TPA (the earliest time examined), reached maximum levels within 6 h, and declined to low levels after 12 h. IL-2 mRNA from LNC incubated with ionomycin plus TPA appeared within 2 h, and reached maximum levels at about 9 h. In contrast to the decrease seen after 12 h with ConA plus TPA, IL-2 mRNA from these cells remained high for 18 h and declined to low levels after 24 h.     

人工感染新城疫病毒对雏鸡淋巴细胞功能的影响

确定了ＭＴＴ法测定鸡脾淋巴细胞转化实验的最佳条件,并用此法检测了人工感染新城疫病毒雏鸡和ＬａＳｏｔａ免疫雏鸡受到强毒攻击时脾淋巴细胞的转化率。实验表明,在两种情况下,雏鸡脾淋巴细胞对ＣｏｎＡ和ＰＨＡ的反应性与对照组比较有显著的差异,提示在新城疫感染和免疫中,淋巴细胞具有一定作用。     

Cell titration assay for measuring blastogenesis of bovine lymphocytes

The blastogenic response of bovine peripheral blood lymphocytes to phytohemagglutinin (PHA) and to microbial antigens was measured using a lymphocyte titration assay. Culture conditions, including lymphocyte concentrations, incubation periods and medium formulation, were established which produced linear or nearly linear responses over a range of cell concentrations. These conditions were established by testing lymphocytes from unimmunized cattle and from heifers infected with Brucella abortus with PHA and a B. abortus extract. Four cell concentrations in 2-fold increments were selected for measuring responses to PHA (3.125 X 10(3) to 2.5 X 10(4) cells/well) and to antigens (5.0 X 10(4) to 4.0 X 10(5) cells/well). The strength of response varied among animals and also over time for individual animals, but the titration assay allowed exponential proliferation to be distinguished from decline, which may have been due to overcrowding of microtiter wells, exhaustion of nutrients or induction of regulatory events. This assay provided a more reliable and discriminating method of evaluating lymphocyte proliferation responses than that achieved by single point assays. The displacement of the titration curves could be used to estimate the relative frequency of lymphocytes responding to antigens or mitogens.     

Development of optimal conditions for lymphokine production by chicken lymphocytes

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine-containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50% cytostasis-inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)-stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41 degrees C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 micrograms ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 micrograms and 45 micrograms ConA/ml.     

Suppression of bovine lymphocyte responses to mitogens following in vivo and in vitro treatment with dexamethasone.

Gnotobiotic calves given intramuscular injections of dexamethasone (DM, 0.5 mg kg-1 day-1) showed marked changes in haematological parameters including a neutrophilia and a lymphopaenia. Not only was there a reduction in the numbers of circulating mononuclear cells, but there was also a significant (P less than 0.01) decrease in the in vitro responsiveness of the remaining circulating peripheral blood lymphocytes to the mitogens, phytohaemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM). Responses to all three mitogens were suppressed to a similar degree. Analysis of the circulating mononuclear cell sub-populations before and during DM treatment demonstrated a selective depletion of B cells; the T lymphocyte sub-population that expresses the gamma/delta form of T cell receptor, are CD2-, CD5+, CD8-, CD4- and constitute a major population in peripheral blood of calves. In vitro studies in gnotobiotic and conventional calves confirmed that DM was highly inhibitory for PHA responses but, in contrast to the in vivo findings, showed little effect of DM on ConA responses. Expression of surface antigens after 72 h in vitro culture in the presence of DM were little affected with the exception of BoCD8 and MHC II, which showed increased and decreased expression, respectively. These observations would suggest that distinct mechanisms are involved in glucocorticosteroid suppression of the responses to these two mitogens.     

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.     

Proliferation of peripheral blood mononuclear cells from healthy piglets after mitogen stimulation as indicators of disease resilience

Disease resilience refers to the productivity of an animal under disease. Given the high biosecurity of pig nucleus herds, traits that can be measured on healthy pigs and that are genetically correlated with disease resilience, that is, genetic indicator traits, offer a strategy to select for disease resilience. Our objective was to evaluate mitogen stimulation assays (MSAs) on peripheral blood mononuclear cells (PBMCs) from young healthy pigs as genetic indicators for disease resilience. Data were from a natural disease challenge in which batches of 60 or 75 naïve Yorkshire × Landrace piglets were introduced every 3 wk into a continuous flow barn that was seeded with multiple diseases. In this environment, disease resilience traits, including growth, treatment, and mortality rates, were recorded on 3,136 pigs that were genotyped with a high-density marker panel. PBMCs from 882 of these pigs from 19 batches were isolated from whole blood collected prior to the disease challenge and stimulated with five mitogens: concanavalin A (ConA), phytohemagglutinin (PHA), pokeweed mitogen (PWM), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). The proliferation of cells was evaluated at 48, 72, and 96 h and compared with unstimulated samples (rest count). Heritabilities of cell proliferation were estimated using a model with batch as a fixed effect and covariates of entry age; rest count; complete blood count proportions of lymphocytes, monocytes, eosinophils, and basophils; and pen, litter, and animal genetics as random effects. Heritability estimates were highest for response to ConA (0.30 ± 0.09, 0.28 ± 0.10, 0.17 ± 0.10, and 0.25 ±0.10 at 48, 72, and 96 h after stimulation and for area under the curve across the three time points, respectively). Estimates were in a similar range for response to PHA and PMA but low for PWM and LPS. Responses to ConA, PHA, and PMA were moderately genetically correlated with several disease resilience traits and in the expected direction, but individual estimates were not significantly different from zero due to large SEs. In conclusion, although validation is needed, MSAss, in particular based on ConA, show promise as genetic indicator traits for disease resilience.     

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.     

Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study

Objective

The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection.

Animals Studied

Three adult horses with no evidence of ophthalmic or systemic disease.

Procedure

Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4′,6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test.

Results

No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations.

Conclusions

Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from “safe” to “retinotoxic” in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.