Proliferation of Peripheral Blood Mononuclear Cells From Healthy Piglets After Mitogen Stimulation As Indicators of Disease Resilience

Disease resilience refers to 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, i.e. genetic indicator traits, offer a strategy to select for disease resilience. Our objective was to evaluate mitogen stimulation assays on peripheral blood mononuclear cells 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 x Landrace piglets were introduced every three weeks 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 3136 pigs that were genotyped with a high-density marker panel. Peripheral blood mononuclear cells 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). Proliferation of cells was evaluated at 48, 72, and 96 hrs and compared to unstimulated samples (rest count). Heritabilities of cell proliferation were estimated using a model with batch as a fixed effect, covariates of entry age, rest count, and 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 hrs 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 standard errors. In conclusion, although validation is needed, mitogen stimulation assays, in particular based on ConA, show promise as genetic indicator traits for disease resilience.

PSVI-31 The Effect of Aging on Peripheral Blood Mononuclear Cell Proliferation in Labrador Retrievers

It is well documented that companion animals are living longer and as animals age their cells undergo immuno-aging or senescence, an age associated process of deterioration. In an effort to see how aging plays a role in immune cell proliferation, twelve healthy Labrador Retrievers (6-Young, 3Male/3Female, 6-Senior, 3Male/3Female) were selected to participate in a trial to determine what effect aging might have on the proliferative rate of peripheral blood mononuclear cells (PBMC). Mean age for Senior group was 8.74yrs, Young 2.38yrs (P.< 0.0001). Peripheral blood mononuclear cells (PBMC) were isolated, counted, seeded at a density of 5x104 in 96-well MP, cultured in RPMI-1640 complete medium (5% FBS), and mitogenic stimulation via 2-fold serial dilutions of Phytolacca Americana lectin (pokeweed mitogen-PWM) ranging from 0.8 -0.025 µg/mL. Following 48hr incubation in 5% CO2/95% humidity, 100µM Bromodeoxyuridine (5-bromo-2’-deoxyuridine, BrdU; a synthetic analog nucleoside of thymidine), was added to each culture well and further incubated 24hrs to allow DNA incorporation. Cells were harvested, denatured, and BrdU incorporated in newly synthesized DNA was bound by monoclonal antibodies (anti-BrdU-POD). The immune complexes were detected by tetramethyl-benzidine (TMB), reaction stopped with 1M Sulfuric acid (H2SO4), and quantified by measuring absorbance at 450nm. Proliferation was higher in Young dogs than Senior dogs at every concentration, signifying overall statistical significance (Mean: Young-0.972, Senior-0.697±0.057 SEM, P = 0.001). Rate difference by concentration was as follows: PWM 0.00 µg/mL: Young-0.109, Senior-0.194±0.031 (P = 0.099); 0.025 µg/mL: Young-0.788, Senior-0.481±0.201 (P = 0.321); 0.05 µg/mL: Young-1.002, Senior-0.610±0.207 (P = 0.227); 0.1 µg/mL: Young-1.113, Senior-0.776±0.195 (P = 0.244); 0.2 µg/mL: Young-1.249, Senior-0.915±0.172 (P = 0.217); 0.4 µg/mL: Young-1.271, Senior-0.965±0.148 (P = 0.191); 0.8 µg/mL: Young-1.252, Senior-0.936±0.117 (P = 0.101). These results coincide with other published literature, and these data suggest age plays a critical role in immunity and proliferative ability of PBMCs in the Labrador Retriever.

Comparative Analysis of Cellular Immune Responses in Conventional and SPF Olive Baboons (Papio anubis)

Olive baboons (P. anubis) have provided a useful model of human diseases and conditions, including cardiac, respiratory, and infectious diseases; diabetes; and involving genetics, immunology, aging, and xenotransplantation. The development of a immunologically defined SPF baboons has advanced research further, especially for studies involving the immune system and immunosuppression. In this study, we compare normal immunologic changes of PBMC subsets, and their function in age-matched conventional and SPF baboons. Our results revealed that both groups have comparable numbers of different lymphocyte subsets, but phenotypic differences in central and effector memory T-cell subsets are more pronounced in CD4+ T cells. Despite equal proportions of CD3+ T cells among the conventional and SPF baboons, PBMC from the conventional group showed greater proliferative responses to phytohemagglutinin and pokeweed mitogen and higher numbers of IFNγ-producing cells after stimulation with concanavalin A or pokeweed mitogen, whereas plasma levels of the inflammatory cytokine TNFα were significantly higher in SPF baboons. Exposure of PBMC from conventional baboons to various Toll-like (TLR) ligands, including TLR3, TLR4, and TLR8, yielded increased numbers of IFNγ producing cells, whereas PBMC from SPF baboons stimulated with TLR5 or TLR6 ligand had more IFNγ-producing cells. These findings suggest that although lymphocyte subsets share many phenotypic and functional similarities in conventional and SPF baboons, specific differences in the immune function of lymphocytes could differentially influence the quality and quantity of their innate and adaptive immune responses. These differences should be considered in interpreting experimental outcomes, specifically in studies measuring immunologic endpoints.

Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss)

The lymphocyte proliferation assay is a valuable method used for the evaluation of the fish immune system. However, there are many variations and optimal results are not always obtained. Unification is necessary to ensure the comparability between different studies. The aim of this study was to optimise the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). This goal included the determination of the optimal incubation length, serum type, incubation temperature, type of mitogen and its concentration, and anticoagulant. The peripheral blood and head kidney lymphocytes were isolated by density gradient centrifugation. Subsequently, the cells were incubated for 3–8 days with different mitogens (pokeweed mitogen 5, 10 and 50 µg/ml, concanavalin A 1, 10 and 20 µg/ml, phytohaemagglutinin 25, 50 and 100 µg/ml, lipopolysaccharide 1, 50 and 100 µg/ml). The use of the different serum types (foetal bovine serum, trout serum), incubation temperatures (10–20 °C) and anticoagulants (heparin, EDTA) was compared. Labelled thymidine was used to evaluate the assay. The best results were obtained after seven days of incubation at 15 °C with foetal bovine serum (FBS). The head kidney lymphocytes showed the highest proliferative response with 50 µg/ml phytohaemagglutinin. With the peripheral blood lymphocytes (heparin and EDTA), the best results were obtained with 50 µg/ml pokeweed mitogen. The highest proliferation levels were detected with heparinised blood. In conclusion, optimisation of this assay contributes to the improved assessment of the rainbow trout immune function.

THE COMPARATIVE STUDY OF HUMAN ENDOGENOUS RETROVIRUS HERV-E λ 4-1 EXPRESSION IN BLOOD IMMUNE CELLS OF MULTIPLE SCLEROSIS PATIENTS

In order to investigate the function of human endogenous retrovirus HERV-E λ 4-1 in multiple sclerosis pathogenesis, the comparative research of frequency of this retrovirus expression and mRNA level on different types of blood immune cells of progredient course multiple sclerosis patients have been conducted with using of the reverse - transcriptase polymerase chain reaction method. Peripheral blood mononuclear cells were isolated on Ficoll density gradient centrifugations. Monocytes were separated by the adhesion to plastic Petri dishes. For the estimation of the mitogen-induced HERV-E λ 4-1 expression, blood mononuclear cells were incubated with adding of phytogemagglutinin or pokeweed mitogen during 72 hours in CO2 incubator at 37 °C and 5% CO2. Results of HERV-E λ 4-1 env gene expression estimation demonstrate that both monocytes and lymphocytes express HERV-E λ 4-1. The level of the HERV-E λ 4-1 env mRNA was higher in lymphocytes than in monocytes. The main source of HERV-E λ 4-1 in progredient course MS patients between blood immune cells are lymphocytes, especially B lymphocytes.

KCTD15 is overexpressed in human childhood B-cell acute lymphoid leukemia

Leukemic cells originate from the malignant transformation of undifferentiated myeloid/lymphoid hematopoietic progenitors normally residing in bone marrow. As the precise molecular mechanisms underlying this heterogeneous disease are yet to be disclosed, the identification and the validation of novel actors in leukemia is of extreme importance. Here, we show that KCTD15, a member of the emerging class of KCTD ((K)potassium Channel Tetramerization Domain containing) proteins, is strongly upregulated in patients affected by B-cell type acute lymphoblastic leukemia (B-ALL) and in continuous cell lines (RS4;11, REH, TOM-1, SEM) derived from this form of childhood leukemia. Interestingly, KCTD15 downregulation induces apoptosis and cell death suggesting that it has a role in cellular homeostasis and proliferation. In addition, stimulation of normal lymphocytes with the pokeweed mitogen leads to increased KCTD15 levels in a fashion comparable to those observed in proliferating leukemic cells. In this way, the role of KCTD15 is likely not confined to the B-ALL pathological state and extends to activation and proliferation of normal lymphocytes. Collectively, data here presented indicate that KCTD15 is an important and hitherto unidentified player in childhood lymphoid leukemia, and its study could open a new scenario for the identification of altered and still unknown molecular pathways in leukemia.

Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [ days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis ( n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV ( P = 0.01), OKT3 ( P = 0.02), and PWM ( P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points ( P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis. NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Effects of Transportation and Relocation on Immunologic Measures in Cynomolgus Macaques (Macaca fascicularis)

NHP are a small, but critical, portion of the animals studied in research laboratories. Many NHP are imported or raised at one facility and subsequently moved to another facility for research purposes. To improve our understanding of the effects of transportation and relocation on the NHP immune system, to minimize potential confounds associated with relocation, and to maximize study validity, we examined the phenotype and function of PBMC in cynomolgus macaques (Macaca fascicularis) that were transported approximately 200 miles by road from one facility to another. We evaluated the phenotype of lymphocyte subsets through flow cytometry, mitogen-specific immune responses of PBMC in vitro, and plasma levels of circulating cytokines before transportation, at approximately 24 h after arrival (day 2), and after 30 d of acclimation. Analyses of blood samples revealed that the CD3+ and CD4+ T-cell counts increased significantly, whereas NK+, NKT, and CD14+ CD16+ nonclassical monocyte subsets were decreased significantly on day 2 after relocation compared with baseline. We also noted significantly increased immune cell function as indicated by mitogen-specific proliferative responses and by IFNγ levels on day 2 compared with baseline. After 30 d of acclimation, peripheral blood CD4+ T-cells and monocyte counts were higher than baseline, whereas B-cell numbers were lower. The mitogen-induced responses to LPS and IFNγ production after stimulation with pokeweed mitogen or phytohemagglutinin remained significantly different from baseline. In conclusion, the effects of transportation and relocation on immune parameters in cynomolgus monkeys are significant and do not fully return to baseline values even after 30 d of acclimation.

Mitogen-Induced Interferon Gamma Production in Human Whole Blood: The Effect of Heat and Cations

Background: Interferon-gamma release assays (IGRAs) are blood tests used to measure the amount of interferon-γ (IFN-γ) released by T lymphocytes after stimulation by antigens specific for the diagnosis of latent tuberculosis infection. A mitogen serves as a positive control to assess the immune function in IGRAs. Methods: This in vitro study was conducted to evaluate IFN-γ production by human whole blood stimulated with heat-treated and/or cation-supplemented phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), using QuantiFERON-TB Gold Kit ELISA tests. Results: The optimal concentrations of PWM, Con A and PHA for IGRAs were 2 µg/mL, 5 µg/mL and 10 µg/mL, respectively. The results showed that IFN-γ production in response to PWM was the highest and PHA was the lowest amount. The median values of three mitogens were in the following order: PWM≥Con A≥ positive control>PHA-P>negative control. PWM and PHA were heat stable, while Con A was heat sensitive. The mitogen response of lymphocytes to untreated or heat-treated PWM and heat-treated Con A was increased in 1 mM Ca2+-supplemented groups, whereas the response to heat-treated PHA was decreased. Exposure to 1 mM Mg2+ had no effect on untreated or heat-treated PWM, and a concentration of 1 mM Zn2+ inhibited the stimulation of un-treated PWM. We found that calcium supplementation improved the PWM-induced production of IFN-γ. Conclusion: Therefore, PWM is an appropriate mitogen for use as a positive control in IGRAs. It is a potential indicator of cytokine production in the diagnostic as well as research settings, and calcium supplementation improved stimulation.