Detection of mitogen-induced lymphocyte proliferation by bromodeoxyuridine (BrdU) incorporation in the chicken

3H-thymidine (3H-TdR) incorporation assay has been generally used to measure lymphocyte proliferation in the chicken. Disadvantages of this assay are that radioisotope is biological hazard to the person and environment and that it can not measure which subset of lymphocytes proliferates. In this study, bromodeoxyuridine (BrdU) incorporation assay by flow cytometry was compared with 3H-TdR incorporation assay. As a result, BrdU incorporation assay showed a strong correlation with 3H-TdR incorporation assay, and it could be applied simultaneously to detect BrdU incorporation and expression of cell surface marker antigens. These results suggest that the BrdU incorporation assay by flow cytometry is useful to analyze lymphocyte proliferation in detail.     

Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity

A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

A latex test for canine rheumatoid factor

Canine rheumatoid factor (RF) has been reported in several canine diseases, particularly in arthritis. Although RF can be assayed using IgG sensitized erythrocytes, the test has a number of disadvantages. As an alternative, latex sensitized with canine IgG was investigated as an assay of canine RF. The canine IgG-latex could be easily produced, was stable, and could be standardized with commercial antisera. The reagent detected RF of the IgM anti-canine-aggregated-IgG type. A comparison of the titres obtained using the canine IgG-latex reagent with those obtained using a rabbit IgG-erythrocyte reagent showed no correlation, suggesting that the two assays may detect RF of different specificities.     

Lymphocyte proliferation response during Eimeria tenella infection assessed by a new, reliable, nonradioactive colorimetric assay

The application of a tetrazolium salt, WST-8,2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt to the lymphocyte proliferation assay in the chicken system was evaluated. Proliferation of concanavalin (Con A)-induced splenic lymphocytes and peripheral blood lymphocytes (PBL) was evaluated with WST-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Coefficients of correlation (r) between these two reagents were 0.98 and 0.97 in splenic lymphocytes and PBL, respectively. In general, the sensitivity of the WST-8 assay was significantly higher than that of the MTT assay, and the standard deviations of the WST-8 assay were significantly lower than those of the MTT assay. The WST-8 assay was fast and highly reproducible and provided a good indication of mitogen-induced proliferation of spleen cells induced by Con A. With the use of the WST-8 assay, splenic mitogenic response of chickens infected with Eimeria decreased transiently at 7 days but increased significantly at 10 days after primary infection compared with that of uninfected chickens. Additionally, the measurement of interleukin (IL)-2 production with WST-8 was highly reproducible and showed a significant increase in IL-2 production upon stimulation of Eimeria tenella-immune spleen cells with Con A. After E. tenella infection, splenic IL-2 production increased significantly at 7 days post-primary and at 2 days post-secondary infection. The WST-8 assay is fast, simple, and more reproducible and sensitive than the MTT assay. This study demonstrates the effectiveness of the WST-8 assay to assess cell-mediated immune response of chickens in normal and disease states.     

Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.     

Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats

OBJECTIVES: To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferative activity of feline lymphocytes. SAMPLE POPULATION: Blood samples from 10 clinically normal domestic shorthair cats. PROCEDURE: Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with feline-specific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry. RESULTS: Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 microg of Con-A/ml were submitogenic, and 100 microg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 microg of Con-A/ml. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases.     

Production of interleukin 2 and expression of interleukin 2 receptors by pony peripheral blood lymphocytes after stimulation with a soluble fraction of Trypanosoma evansi

Pony peripheral blood lymphocytes (PBL) were stimulated with a soluble fraction of Trypanosoma (T.) evansi (SF). As determined by 3H-thymidine incorporation, the cells underwent a proliferative response and were able to: a) produce a factor having the biological activities of interleukin 2 (IL-2) since their supernatants could support the in vitro growth of pony PBL stimulated with concanavalin A (Con A-blasts); b) undergo a further proliferative response when incubated in short term cultures with SF, human recombinant IL-2 (hrIL-2), or both c) bind specifically radiolabelled hrIL-2 (125I-hrIL-2). The date described here indicate that a soluble fraction of T. evansi stimulated pony PBL which subsequently produced IL-2 and expressed IL-2 receptors (IL-2R).     

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.     

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.     

Evaluation of an automated colorimetric assay for the measurement of lipase activity in canine sera.

An automated colorimetric method for determining lipase activity in canine sera was evaluated for precision, linearity and correlation to existing assay methods. The colorimetric method was a commercial reagent that used a series of enzymatic reactions based on the hydrolysis of 1,2 diglyceride by pancreatic lipase. Within-run and between-run coefficients of variation were < 6.8% and < 8.3%, respectively. Linearity was determined to be at least 1366 U/L. Canine serum lipase concentrations attained using the colorimetric method were compared to both titrimetric and dry-film methods for measuring serum lipase activity, resulting in significant (P < or = 0.05) correlation coefficients of 0.92 and 0.77, respectively. Canine serum lipase concentrations measured using the colorimetric assay on 2 different automated analyzers had a significant (P < or = 0.05) correlation coefficient of 0.92. A laboratory reference range using serum samples from 56 healthy dogs (0-561 U/L) was established. There were no significant (P < or = 0.05) differences in mean serum lipase concentrations comparing male and female dogs or comparing young dogs (< or = 3 y) to mature (4-7 y) and older (> 7 y) dogs using this assay. It was concluded that the automated colorimetric assay was a reliable indicator of canine serum lipase activity and offered several advantages, including small sample volume and short analysis time.     

In vitro measurement of post‐natal changes in proliferating satellite cell frequency during rat muscle growth

Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat‐production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra‐peritoneal (ip) injection of huge dosage of mutagenic nucleosides, ³H‐labeled thymidine or bromodeoxyuridine (BrdU), at each age‐time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU‐incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind‐limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU‐incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3‐month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip‐injection method during the postnatal period examined from day‐2 to month‐11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age‐interval sampling from the same growing animals.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

Bovine leukosis virus in sheep,lymphocyte modification and surface-immunoglobulin-bearing cell numbers

Lymphocytes from sheep experimentally infected with bovine leukosis virus (BLV) and from non-infected normal sheep were examined for the presence of surface Ig by an immunofluorescence test. Surface Ig-bearing lymphocytes in blood from BLV-infected sheep increased when lymphocyte counts of blood were elevated in comparison with normal animals. The mitogen stimulation of cultured lymphocytes from BLV-infected sheep and from non-infected normal sheep was determined by measuring ³H-thymidine incorporation. Peripheral blood lymphocytes (PBL) from BLV-infected leukemic sheep with elevated PBL counts responded poorly to phytohemagglutinin M and concanavalin A but responded well to lipopolysaccharide compared with lymphocytes from uninfected animals. In BLV-infected preleukemic sheep with low PBL counts, stimulation indices of mitogen responses of lymphocytes with phytohemagglutinin M, concanavalin A, and pokeweed mitogen were low compared with those of lymphocytes from uninfected animals. The results indicated that B cells were affected by BLV infection in sheep as suggested by the increased number of surface Ig-bearing lymphocytes and that significant alteration of mitogen stimulation of lymphocytes occured in sheep with BLV infection.     

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。     

Immunosuppression by canine distemper virus: modulation of in vitro immunoglobulin synthesis, interleukin release and prostaglandin E2 production

In vitro or in vivo infection of canine mononuclear cells by canine distemper virus (CDV) in short-term microcultures resulted in suppression of lectin-induced 3H-thymidine incorporation. This suppressive effect was also evident in pokeweed mitogen-driven in vitro immunoglobulin synthesis and release. Lectin-induced interleukin-2 production by monocyte-depleted lymphocyte cultures was marginally affected by CDV, whereas interleukin-1 production by adherent mononuclear cells was significantly depressed. Monocyte cultures established from viremic dogs released prostaglandin (PG)E2. The results suggest that, in addition to a direct viral effect upon lectin responsive cellular population(s), CDV modulates monocyte functions by inhibition of interleukin-1 production and by enhancing PGE2 release.     

Immunohistochemical identification method of tumour cells in the S phase of mitotic cycle and its usefulness in diagnostics of mammary gland adenocarcinomas in bitches

The studies aimed at identification of neoplastic cells at the S phase of mitotic cycle in mammary gland adenocarcinomas of bitches. The material was sampled from bitches of various races, aging 6 to 12 years, in which the mammary gland tumours developed spontaneously. The tumours were verified histopathologically and, then, immunohistochemical reactions were performed in order to detect cells which had incorporated BrdU (bromodeoxyuridine), contained Ki-67 or PCNA antigen. The histological preparations were photographed and obtained pictures were subjected to computer-assisted image analysis using Axiophot microscope (Carl Zeiss) coupled to a computer and the Multi-ScaneBase V 8.08 software, working under Windows. Fifty percent of sections from mammary gland adenocarcinomas demonstrated BrdU labelling index of 4-5%, 40% of 1-3%, while in the remaining 10% of examined tumours no BrdU incorporation could be demonstrated. No evident relationship could be detected between the presence of BrdU incorporation and Ki-67 or PCNA antigen presence but a significant correlation was demonstrated between the expression of Ki-67 and PCNA.     

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

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