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.

Levels of cardiorespiratory fitness in men exerts strong impact on lymphocyte function after mitogen stimulation

Relationship between lymphocyte function and cardiorespiratory fitness (CRF) is well documented at rest; however, upon mitogen stimulation the proliferation and cytokine production alters but are incipient the lymphocyte responses front mitogens according to the CRF. So, the purpose of the present study was to analyze the lymphocyte function according to the physical fitness status of healthy young men. The study is divided in two experiments being the first analyzing the lymphocyte phenotypes profile and the inflammatory responses, according to CRF, in lymphocyte cell culture treated for 48 hours with Concanavalin A (ConA). And the second experiment analyzed the proliferation, reactive oxygen species production, viability, and mitochondrial polarization state in lymphocytes treated with ConA in different concentrations, considering the CRF levels. The results showed a difference in the percentage of total lymphocytes expression between groups (p=0.011) observing a lower lymphocytes T expression in the group with High when compared with Moderate group. When treated with ConA, the lymphocytes of the Low group released higher TNF-α concentration (p=0.032), reflecting an elevated TNF-α/IL-10 ratio (p=0.055), parallel with lower IL-6 production (p=0.027), mainly when compared with Moderate group. In addition, there is a positive relationship between and IL-6 production (r=0.507; p=0.016) whereas the percentage of total lymphocytes (LyT%) shows a negative association tendency with (r=-0.497; p=0.060). Also, individuals with lower showed reduced absolute and relative ROS production, lower cell proliferation, and higher mitochondrial membrane depolarization. In conclusion, cardiorespiratory fitness degree exerts a strong impact on lymphocyte function after mitogen stimulation.

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling

Key Points Genome-wide RNAi screen provides the first comprehensive list of putative hepatic hepcidin regulators. Hepcidin suppression is linked to the control of mitogen stimulation and nutrient status via components of Ras/RAF MAPK and mTOR signaling.

Mitogen Stimulated Direct Antiglobulin Test Compared with Traditional Methods for the Detection of Anti-RBC Autoimmunity.

Abstract 5091 Background The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed by various methods with different sensitivity. Recently mitogen stimulated (MS)-DAT was suggested able to disclose a latent anti-erythrocyte autoimmunity Aims: To compare different traditional (tube, microcolumn and solid phase) and new (mitogen stimulated, MS)-DAT methods in different diagnostic conditions: 54 consecutive cases of hemolytic anemia of suspected autoimmune origin, 28 idiopathic AIHA in clinical remission, and 12 difficult cases of DAT-negative AIHA, and 2) to correlate results with hematologic and hemolytic parameters. Methods DAT tube, microcolumn, solid phase, and eluates were performed by standard techniques. MS-DAT was performed by stimulating whole blood with PHA, PMA and PWM and antibodies were detected by competitive solid phase ELISA. Results Forty out of 54 consecutive cases of suspected AIHA were positive by one or more tests namely 14 DAT-tube, 19 microcolumn, 19 solid phase and 35 MS-DAT. Among the 14 DAT-tube positive cases, 11 were confirmed by all test, and 3 by one or more (1 microcolumn, 1 solid phase, and 1 MS-DAT), eluates were positive in 11, and the majority of patients (10/14) showed hemolytic anemia. As regards the 26 DAT-tube negative cases, 7 were positive in microcolumn and solid-phase (eluates positive in 2/8, panreactive), and 16 in MS-DAT, although in both groups anemia and hemolytic signs were less clear. Mitogen stimulation increased the amount of RBC-bound IgG in all groups, suggesting that MS-DAT could disclose a latent autoimmunity. Tube-negative/other methods positive cases included patients with B-CLL, myelodysplasia/aplasia, and thalassemia intermedia, in which autoimmune phenomena are more frequently observed than overt clinical autoimmune diseases. MS-DAT failed to detect anti-erythrocyte antibodies in half cases AIHA in clinical remission which still were tube-positive. Finally, MS-DAT was the only positive test in 10 cases of AIHA of difficult diagnosis, and mitogen stimulation allowed the identification of autoantibody specificity in culture supernatants of 2 cases which gave weak positive results in microcolumn/solid phase only. Conclusion We concluded that a battery of tests rather than a single test is recommended for the diagnosis of AIHA, depending on the specific clinical context: tube is still a good choice for first screening, microcolumn and solid-phase, which are the automated routine, should be confirmed by a positive eluate to diagnose AIHA, although positivity without a clinical equivalent may be taken into account as part of an autoimmune habitus. MS-DAT could be considered as an additional test for selected cases of difficult diagnosis. Disclosures No relevant conflicts of interest to declare.