Phenotype Of Peripheral Blood Mononuclear Cells Research Articles

The Cheese Matrix Modulates the Immunomodulatory Properties of Propionibacterium freudenreichii CIRM-BIA 129 in Healthy Piglets.

Propionibacterium freudenreichii is a beneficial bacterium, used as a cheese starter, which presents versatile probiotic properties. These properties are strain-dependent. We hypothesized they may also be delivery vehicle-dependent. In this study, we thus explored in healthy piglets how the cheese matrix affects the immunomodulatory properties of P. freudenreichii. During 2 weeks, three groups of weaned piglets consumed, respectively, P. freudenreichii as a liquid culture (PF-culture), P. freudenreichii under the form of a cheese (PF-cheese), or a control sterile cheese matrix (Cheese-matrix). The in vivo metabolic activity of P. freudenreichii was assessed by determining short chain fatty acids (SCFA) concentration and bifidobacteria population in feces. Whatever the delivery vehicle, P. freudenreichii was metabolically active in piglets’ colon and enhanced both bifidobacteria and SCFA in feces. P. freudenreichii consumption decreased the secretion of TNFα and of IL-10 by peripheral blood mononuclear cells (PBMC). It did not alter IL-10, IFNγ, IL-17, and TNFα secretion in mesenteric lymph node immune cells (MLNC). PF-cheese enhanced significantly Treg phenotype, while PF-culture decreased significantly Th17 phenotype in PBMC and MLNC. Remarkably, only PF-cheese induced an increase of Th2 phenotype in PBMC and MLNC. Ex vivo stimulation of PBMC and MLNC by Lipopolysaccharides and Concanavalin A emphasized the difference in the immunomodulatory responses between PF-culture and PF-cheese group, as well as between PBMC and MLNC. This study shows the importance to consider the delivery vehicle for probiotic administration. It confirms the anti-inflammatory potential of P. freudenreichii. It opens new perspectives for the use propionibacteria-fermented products as preventive agents for inflammatory bowel diseases and intestinal infectious diseases.

Immune phenotype of peripheral blood mononuclear cells in patients with high-risk non-muscle invasive bladder cancer.

To explore the immune phenotype of peripheral blood mononuclear cells (PBMC) in patients with high-risk non-muscle invasive bladder cancer (NMIBC). We prospectively collected blood samples from patients with high-risk NMIBC treated at our institution. PBMC were analyzed by flow cytometry to determine the frequency of T cells and NK cells and the expression of immunoregulatory molecules (Tim-3, TIGIT and PD-1). PBMC from healthy donors (HD) were included for comparison, and associations with response to BCG were investigated. A total of 38 patients were included, 19 BCG responders and 19 BCG refractory. Compared to 16 PBMC from HD, the frequency of total NK cells was significantly higher in patients with NMIBC [15.2% (IQR: 11.4, 22.2) vs. 5.72% (IQR: 4.84, 9.79); p = 0.05], whereas the frequency of T cells was not statistically different. Both Tim-3 and TIGIT expressions were significantly higher in NMIBC compared to HD, particularly in NK cells [13.8% (11.0; 22.4) vs. 5.56% (4.20; 10.2) and 34.9% (18.9; 53.5) vs. 1.82% (0.63; 5.16), respectively; p < 0.001]. Overall, the expression of PD-1 in all cell types was low in both NMIBC patients and HD. The immune phenotype was not significantly different before and after initiation of BCG. However, the proportion of CD8+ T cells before BCG was significantly higher in responders. The immune phenotype of PBMC from patients with high-risk NMIBC was significantly different from HD, regardless of the presence of disease or the initiation of BCG. Peripheral CD8+ T cells could play a role in response to BCG.

Effect of Adalimumab on Peripheral Blood Mononuclear Cells in Non-Infectious Uveitis

ABSTRACTPurpose: This study analyzed the effect of adalimumab on peripheral blood mononuclear cells (PBMCs) in uveitis.Methods: PBMCs and serum S100A12 levels from 14 uveitis patients and 28 healthy controls were analyzed. Patient samples were taken before (w0), and 6 (w6) and 12 (w12) weeks after initiation of adalimumab therapy.Results: Monocytes expressing CD124, CD86, CD39, CD115, and MHCII were decreased in patients. Adalimumab induced CD86+ and CD39+ monocytes, and further decreased the frequency of MHCII- and CD124-positive cells. Patients (w0) had increased percentages of Th1-, Th17-, and Th2 cells and T cell subsets showed a pro-inflammatory polarization (p = 0.02 ratio Th17/Treg patients w0 vs controls), which was reduced upon adalimumab treatment (p = 0.05 w0 vs w6). S100A12 levels were increased in patients (p = 0.02) and reduced under treatment (p = 0.02 for w6/w12).Conclusions: The phenotype of PBMCs from uveitis patients is modified upon adalimumab treatment. Serum S100A12 levels reflect the systemic immune response.

Immunosenescence Induced by Plasma from Individuals with Obesity Caused Cell Signaling Dysfunction and Inflammation.

To evaluate the consequences of plasma from individuals with obesity on parameters associated with immunosenescence in unrelated healthy peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC were incubated in media supplemented with 10% of plasma from individuals with obesity or control subjects for the first 4 hours of 24 to 120 hours of culture. Plasma from individuals with obesity modulated the phenotype of healthy PBMC, leading to a higher rate of apoptosis, lower amounts of phospho-γH2AX and -p53, and mitochondrial dysfunction. After 120 hours, there was a higher secretion of inflammatory cytokines IL-1β and IL-8. CD8+ T lymphocytes presented decreased expression of CD28, which is associated with the immunosenescent phenotype. CD14+ macrophages showed increased expression of CD80 and CD206, suggesting a modulation in the activation of macrophages. These results demonstrate that chronic systemic inflammation observed in obesity induces dysfunctional features in PBMC that are consistent with premature immunosenescence.

Analysis of Peripheral Blood Mononuclear Cell Phenotypes during Treatment with Fingolimod: A 2-Year Study of a German Cohort of Patients with Relapsing-Remitting Multiple Sclerosis (P3.097)

Analysis of Peripheral Blood Mononuclear Cell Phenotypes during Treatment with Fingolimod: A 2-Year Study of a German Cohort of Patients with Relapsing-Remitting Multiple Sclerosis (P3.097)

Characterization of γδ T-cells via flow cytometry.

Stervbo et al. report on the influences of ageing on a variety of peripheral innate and adaptive immune populations in two publications in the October issue of this journal (Stervbo et al. 2015a; Stervbo et al. 2015b). These articles surveyed peripheral blood mononuclear cell phenotypes from donors recruited in the cross-sectional Protective Immunity in Ageing (PRIMAGE) study. The focus of that study was the investigation of fundamental immunologic questions of the natural ageing process using seasonal influenza vaccination as a model (PRIMAGE 2015). Both papers also nicely combined phenotypic and functional assessment of age-related differences in major compartments of the cellular immune system in peripheral blood. Unlike many earlier studies, Stervbo et al. also focused on the underinvestigated γδ T cell compartment (Stervbo et al. 2015a). This is valuable because there are very few data on the impact of age on this conserved minor T cell subset. It is therefore especially important to be certain of the exactitude of the published results. However, we would like to sound a note of caution in this regard due to the use of the pan-γδ antibody clone B1, which may not be able to identify every γδ T cell when used in complex antibody panels applied in polychromatic flow cytometry. These issues were addressed by us in a carefully reviewed and validated antibody panel recently published in the journal Cytometry Part A’s “OMIP” series of standardized antibody panels (Wistuba-Hamprecht et al. 2014). A possible explanation could be steric hindrance effects because the epitopes recognized by anti-CD3 antibodies and the B1-clone might be in close proximity, which is not the case for the 11F2-clone that for example reliably detects the γδ T-cell compartment. In parallel to publication of these findings, we informed the manufacturers and one of the largest companies immediately revised their technical data sheet in accordance with these results (Becton Dickinson 2015). It might therefore be problematic to perform subset analysis on the basis of a B1-positive cell population. We felt that readers of Stervbo et al. 2015a, 2015b should be aware of these potential limitations.

Mass cytometry analysis shows that a novel memory phenotype B cell is expanded in multiple myeloma.

It would be very beneficial if the status of cancers could be determined from a blood specimen. However, peripheral blood leukocytes are very heterogeneous between individuals, and thus high-resolution technologies are likely required. We used cytometry by time-of-flight and next-generation sequencing to ask whether a plasma cell cancer (multiple myeloma) and related precancerous states had any consistent effect on the peripheral blood mononuclear cell phenotypes of patients. Analysis of peripheral blood samples from 13 cancer patients, 9 precancer patients, and 9 healthy individuals revealed significant differences in the frequencies of the T-cell, B-cell, and natural killer-cell compartments. Most strikingly, we identified a novel B-cell population that normally accounts for 4.0% ± 0.7% (mean ± SD) of total B cells and is up to 13-fold expanded in multiple myeloma patients with active disease. This population expressed markers previously associated with both memory (CD27(+)) and naïve (CD24(lo)CD38(+)) phenotypes. Single-cell immunoglobulin gene sequencing showed polyclonality, indicating that these cells are not precursors to the myeloma, and somatic mutations, a characteristic of memory cells. SYK, ERK, and p38 phosphorylation responses, and the fact that most of these cells expressed isotypes other than IgM or IgD, confirmed the memory character of this population, defining it as a novel type of memory B cells.

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors. MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n=150) and RA patients (n=40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used. ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type. ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.

Altered NK cell function in obese healthy humans.

BackgroundObesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (<25 kg/m2) and obese (>30 kg/m2). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme+ and IFN-γ+ NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined.ResultsAs opposed to similar NK cell counts, the number of CD3+CD56+ cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells.ConclusionsNK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge.

Allogeneic bone marrow transplantation in two HIV-1 infected patients shows no detectable HIV-1 RNA or DNA, and a profound reduction in HIV-1 antibodies

<h3>Aims</h3> Two HIV-1 infected patients were studied post-haematopoietic stem cell transplant (HSCT) to determine effects on the viral reservoir. <h3>Methods</h3> HIV-1Ag/Ab was measured by chemiluminescence microparticle immunoassay (CMIA) and western blot (WB). Specific CD4+T-cell responses were measured by CD25/CD134 up-regulation. HIV-1 RNA was measured in serum and HIV-1 DNA was measured in peripheral blood mononuclear cells (PBMCs) and CD4+T-cells by PCR. CCR5 phenotype in PBMC was determined by PCR. <h3>Results</h3> Patient A and B underwent HSCT for non-Hodgkin's lymphoma and acute myeloid leukemia, respectively. Both remained on antiretroviral therapy. Patient A experienced systemic graft-versus-host disease (GVHD), whereas Patient B developed skin GVHD. Patient A was heterozygous for the CCR5Δ32 mutation and Patient B was wild-type. Following HSCT, both patients had undetectable HIV-1 RNA in plasma, and undetectable HIV-1 DNA in PBMCs and CD4+T-cells. Antigen-specific CD4+T-cell responses were absent. Pre-transplant, Ag/Abs to HIV-1 was detectable. Post-transplant, patients had low-level Ag/Abs on CMIA, but trace (Patient A) and absent (Patient B) antibody by WB. <h3>Conclusion</h3> HSCT reduced the HIV-1 reservoir size in these two patients with undetectable HIV-1 RNA in serum and DNA in PBMC/CD4+T-cells and absent antigen-specific CD4+T-cell responses. A profound reduction in Ab to HIV was demonstrated in both patients by WB.

Elevated Placental Growth Factor Levels after Allogeneic Hematopoietic Cell Transplantation: Clinical and Peripheral Blood Mononuclear Cell Receptor Expression Correlates

Background: Placental growth factor-1 (PlGF) is a member of the vascular endothelial growth factor (VEGF) family of angiogenic factors possessing inflammatory properties as well as a chemotactic effect for monocytes. Additionally, circulating PlGF levels are associated with disease severity in hypoxic conditions including sickle cell disease and coronary artery disease. We have recently identified that circulating levels of PlGF are significantly elevated in recipients of allogeneic hematopoietic cell transplantation (HCT), and most significantly at the onset of acute graft versus host disease [(aGVHD), Holtan, SG et al, ASH 2014]. It is currently unknown whether PlGF levels are associated with an alteration of expression levels of angiogenic factor receptors on mononuclear cells subsets or other clinical factors post-allogeneic HCT. Thus, as PlGF could play an immunomodulatory role on the developing donor-derived immune system, we sought to characterize peripheral blood mononuclear cell (PBMC) subset expression of the PlGF receptor (VEGFR1) using HCT donor cells collected prior to stem cell collection compared to HCT recipient PBMC collected at 3 months post-HCT. We also correlated clinical factors with PlGF levels and VEGF receptor expression on PBMCs at 3 months post-HCT.Patients and methods: Plasma and PBMCs from a cohort of 14 adult patients undergoing allogeneic HCT at Oregon Health & Science University were collected at 3 months post-HCT and compared to samples obtained from their matched sibling donors (MSD) prior to stem cell donation. Plasma levels of PlGF were quantified by ELISA. Lymphocyte and monocyte expression of VEGFR1 was determined by multiparameter flow cytometry. Comparison of donor and recipient plasma PlGF levels and PBMC phenotypes were performed by Wilcoxon signed rank. Correlations were determined using Spearman’s rho. Survival curves generated using Kaplan Meier estimates with differences between curves determined by log rank.Results: PlGF and clinical correlates: PlGF levels were 3.5-fold higher (25.1 versus 7.1 pg/mL, p=0.006) in recipients at 3 months post-HCT compared to their MSD. Donor PlGF levels were not associated with donor age or donor sex. Allogeneic HCT recipient PlGF levels were not associated with donor age, donor sex, recipient age, recipient sex, sex mismatch, conditioning intensity, disease risk index, or disease type. PlGF levels were not associated with the development of acute GVHD prior to the 3-month post-HCT blood draw (p=0.4); however, PlGF levels were negatively associated with steroid dose as a continuous variable (Spearman’s rho -0.6, p=0.02). Recipient PlGF levels >25.1 pg/ml at 3 months post-HCT were associated with improved overall survival. PBMC subsets: Allogeneic HCT recipients had significantly higher levels of circulating monocytes (15.4 vs 8.1%, p=0.05) and approximately double the percentage of CD8+ T-cell (1.5 vs 0.6%, p=0.009) and CD3-CD11b+DRlo/- subsets (6.0 vs 3.3%, p=0.01) expressing VEGFR1 as compared to their donors. CD16+ NK cells (r=0.62, p=0.02), CD14+16+ monocytes (r=0.64, p=0.01), and CD14+16+VEGFR1+ monocytes (r=0.61, p=0.02) correlated with PlGF levels in recipients.Conclusion: Elevated PlGF in HCT recipients is associated with an increase in monocyte percentage and an approximate doubling of VEGFR1-expressing lymphoid and myeloid cells compared to their MSD. Despite published association of elevated PlGF levels and chronic inflammatory states, in our small series, higher PlGF levels were associated with improved early survival. Studies are ongoing to correlate PlGF levels with later time points. Further research into the direct angiogenic and immune regulatory effects of PlGF in allogeneic HCT recipients, as well as larger scale studies of the impact of PlGF in late complications of HCT, are indicated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IMMUNE CHARACTERIZATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS OF THE DOGS RESTORED FROM INOCULATION OF CANINE TRANSMISSIBLE VENEREAL TUMOR CELLS

Canine transmissible venereal tumor (CTVT) is a tumor which can be transmitted naturally through mucosa contact between dogs. When CTVT cells are experimentally inoculated on dogs, they will grow rapidly (Progressive/P phase) and then regress (Regressive/R phase) spontaneously. Therefore, it is a good model to investigate the interactions between tumor cells and host immune system. Previous studies have shown that CTVT cells cannot grow in the dogs restored from CTVT inoculation. To investigate the possible mechanism, this study characterized the CTVT-specific immune response of the peripheral blood mononuclear cells (PBMCs) which isolated from the blood of "naïve" or "CTVT-restored" dogs. The phenotypes (CD3, CD4, CD8, or CD21) of PBMCs were examined by flowcytometry. In response to CTVT stimulation, proliferation, IFN-γ secretion, and cytotoxicity of PBMCs were analyzed. Expression level of proinflammatory cytokines (TNF-α, IL-1β, IL-6, TGF-β), Th1 (IL-2, IFN-γ), and Th2 cytokines (IL-4, IL-10) and cytotoxic proteins (Granzyme B, Perforin) in PBMCs was also evaluated by real-time RT-PCR. The results indicated that there is no significant difference between two groups on lymphocyte phenotypes. Proliferation, IFN-γ secretion, and cytotoxicity of PBMCs between two groups showed no significant difference, except naïve PBMCs present higher proliferation after Con-A stimulation. Production of IL-1β and IL-6 in naïve PBMCs was higher than that in CTVT-restored PBMCs (p &lt; 0.05). The production difference of IL-1β and IL-6 between two groups might be the reason why CTVT cannot be reinoculated on CTVT-restored dog. However, further investigations are necessary to explore the exact role of these cytokines in CTVT growth.

Liver injury in acute hepatitis A is associated with decreased frequency of regulatory T cells caused by Fas-mediated apoptosis

ObjectiveFoxp3+CD4+CD25+ regulatory T cells (Tregs) control immune responses, but their role in acute viral hepatitis remains elusive. Herein, we investigated alteration in the peripheral blood Treg population during acute hepatitis...

Select human milk oligosaccharides directly modulate peripheral blood mononuclear cells isolated from 10-d-old pigs

Infant formulas lack the complex mixture of oligosaccharides found in human milk. These human milk oligosaccharides (HMO) may be pivotal to the development of the neonatal immune system. Few comprehensive analyses of the effects of HMO on immune cells from neonates have been undertaken. Herein, the direct effects of HMO on immune cells were analysed ex vivo. Peripheral blood mononuclear cells (PBMC) isolated from 10-d-old sow-reared (SR) or colostrum-deprived formula-fed (FF) pigs were stimulated for 72 h with single HMO, mixtures of single HMO or a complex mixture of HMO isolated from human milk (iHMO). T-cell phenotype, cytokine production and proliferation were measured by flow cytometry, immunoassay and [³H]thymidine incorporation, respectively. Stimulation with HMO had direct effects on PBMC. For instance, cells stimulated with iHMO produced more IL-10 than unstimulated cells, and cells stimulated with fucosylated HMO tended to proliferate less than unstimulated cells. Additionally, co-stimulation with HMO mixtures or single HMO altered PBMC responses to phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) stimulation. Compared with PBMC stimulated with PHA alone, cells co-stimulated with iHMO and PHA proliferated more and had fewer detectable CD4⁺CD8⁺ T cells. Compared with PBMC stimulated by LPS alone, cells co-stimulated with a mixture of sialylated HMO and LPS proliferated more and tended to have fewer detectable CD4⁺ T cells. Differences in the baseline responses of PBMC isolated from the SR or FF pigs were observed. In summary, HMO directly affected PBMC populations and functions. Additionally, ex vivo measurements of PBMC phenotype, cytokine production and proliferation were influenced by the neonate's diet.

AB0159 Tnf membrane-bound and soluble receptors in health and in rheumatoid arthritis

BackgroundTNFα plays a critical role at all stages of the immunopathogenesis in rheumatoid arthritis (RA) [1]. Its soluble receptors potently regulate the cytokine activity in vivo, but the effects of...

Immunomodulatory effects of bevacizumab in metastatic malignant melanoma.

82 Background: Metastatic malignant melanoma is a nearly uniformly fatal disease. Systemic therapy has notable but limited success. Novel therapeutic strategies are urgently needed. To that end, we’ve pursued combinatorial therapeutics using conventional chemotherapy added to an inhibitor of angiogenesis (bevacizumab) with some evidence of clinical benefit. Preliminary analysis of one of our trials suggested a previously unrecognized immunomodulatory effect of bevacizumab when added to chemotherapy in patients with metastatic melanoma, and this may have a potential impact on clinical outcome. Herein we evaluate the immunomodulatory effect of bevacizumab when added to chemotherapy in patients with metastatic melanoma. Methods: We measured levels of peripheral blood mononuclear cell phenotypes in addition to 42 cytokines, chemokines and growth factors in plasma of 55 patients who received albumin-bound paclitaxel and carboplatin (AC) and of 73 patients who received AC with bevacizumab (ACB) on clinical trials N057e and N0775 respectively. The relative percent change in PBMC cell counts and cytokine levels were determined from baseline at their restaging evaluation after two cycles of therapy. The Mann Whitney U test was used to compare responses between the groups. Results: There was a significant increase in CD197+type 1 macrophages in the group that received ACB (35.3%, -11.8-142.7%) compared to AC (-11.6%, -45.9-38.3%; p=0.004). There was a significant decrease in IL-6 in the group that received ACB (-42.4%, -87.7-0.0%) compared to AC (28.4%, -37.4-480%; p=0.0018). Conclusions: The addition of bevacizumab to chemotherapy for the treatment of metastatic melanoma modulates cellular immunity and a pro-inflammatory cytokine. Patients who received bevacizumab had a significant increase in CD197+ type 1 macrophages and a significant decrease in IL-6. Clinical significance of these findings is the subject of ongoing work.

Mononuclear leukocyte fatty acid composition and inflammatory phenotype in periparturient and lactating sows1

Increased plasma NEFA concentrations and compromised immune responses are associated with increased disease susceptibility during farrowing and lactation. Increased plasma NEFA concentrations cause changes in the fatty acid (FA) content of plasma lipid fractions and peripheral blood mononuclear cells (PBMC) that could modify inflammatory responses. The goals of this study were to describe changes in plasma lipid composition and to characterize the FA content and proinflammatory phenotype of PBMC in periparturient and lactating sows. Blood samples from 10 sows were collected at 2 wk prefarrow, at 2 d after farrowing (hereafter referred to as farrowing), and at 18 d of lactation (hereafter referred to as lactation). Total lipids and lipid fractions were extracted from plasma and PBMC. Isolated PBMC also were assessed for gene expression of proinflammatory cytokines and enzymes involved in lipid mediator biosynthesis using quantitative PCR. The FA profile of plasma NEFA, phospholipids, neutral lipids, and PBMC phospholipids differed from the composition of total lipids in plasma. At farrowing and lactation, the proportion of palmitic and stearic acids increased (P<0.05) in the plasma NEFA and phospholipid fractions in comparison with prefarrowing concentrations. At the same time, the concentration of palmitic and linoleic acids increased (P<0.05) in the PBMC phospholipid fraction. Omega-3 FA, including docosapentaenoic and docosahexaenoic, increased (P<0.05) at farrowing in plasma and PBMC phospholipids compared with prefarrowing and lactation. Gene expression of IL-1β, IL-6, IL-8, and tumor necrosis factor-α (TNFα) decreased (P<0.05) after farrowing and in lactation. Similarly, cyclooxygenase expression was reduced during lactation when compared with farrowing (P<0.05). This study demonstrated changes in FA composition of serum lipid fractions and PBMC cellular membranes. Furthermore, it provided an initial assessment of inflammatory responses in mononuclear cells as a function of plasma and PBMC content of saturated and omega-3 FA. Future studies need to address the effect of increased NEFA concentrations, the main hallmark of lipid mobilization, and changes in plasma and cellular lipid profiles on immune function.

Ex vivo stimulation of neonatal porcine peripheral blood mononuclear cells with oligosaccharides found in human milk

Infant formulas lack the complex mixture of oligosaccharides (HMO) found in human milk, which may be pivotal to the development of the neonatal microbiota and immune system. Few comprehensive analyses of the direct effects of HMO on immune cells from neonates have been undertaken. Herein, peripheral blood mononuclear cells (PBMC) isolated from 10 and 23 day old (do) sow‐reared pigs were stimulated with HMO (2FL, 3FL, 3SL, 6SL, SL, LNFPIII, LnNT, sLeX and HMO isolated from human milk [iHMO]) for 72h. T cell phenotype, cytokine production and proliferation were measured by flow cytometry, ELISA and 3‐H thymidine incorporation. Of the HMO tested, none had significant effects on any parameters examined. However, developmental differences were found. PBMC from 23do pigs produced more cytokines, had a higher proportion of CD8+CD3+ T cells and proliferated less than those from 10do pigs. PBMC from 23do pigs made more IL‐10 in response to iHMO, PHA or PHA+iHMO than PBMC from 10do pigs. Only PHA or PHA+iHMO stimulation induced IL‐4 secretion, and 10do pigs produced nearly twice as much IL‐4 as 23do pigs. Lastly, PBMC from 10do pigs proliferated 6–10 times more in response to PHA stimulation than did PBMC from 23do pigs. In summary, while appropriate developmental responses were observed, direct effects of HMO on porcine PBMC phenotype, cytokine production or proliferation ex vivo were not detected. (Supported by Abbott Nutrition)

Gene expression profiling of PBMCs from Holstein and Jersey cows sub-clinically infected with Mycobacterium avium ssp. paratuberculosis

Infection of calves with intracellular Mycobacterium avium ssp. paratuberculosis (MAP) commonly results in a granulomatous, chronic inflammatory bowel disease known as Johne's disease. The asymptomatic stage of this infection can persist for the entire production life of an adult cow, resulting in reduced performance and premature culling, as well as transmission of MAP to progeny and herd-mates. It has been previously shown that the gene expression profiles of peripheral blood mononuclear cells (PBMCs) of healthy cows, and those chronically infected with MAP are inherently different, and that these changes may be indicative of disease progression. Since resistance to MAP infection is a heritable trait, and has been proposed to differ amongst domestic dairy cattle breeds, the objective of the present study was to compare gene expression profiles of PBMCs from healthy adult Holstein and Jersey cows to those considered to be sub-clinically infected with MAP, as indicated by serum ELISA. Microarray analysis using a platform containing more than 10,000 probes and ontological analysis identified differences in gene expression between a) healthy and infected cows, including genes involved in the inflammatory response, and calcium binding, and b) infected Holsteins and Jerseys, including genes involved in the immune response, and antigen processing and presentation. These results suggest a mixed pro- and anti-inflammatory phenotype of PBMCs from MAP-infected as compared to healthy control animals, and inherently different levels of immune and inflammatory-related gene expression between MAP-infected Holsteins and Jerseys.

Frequency of human T regulatory cells in peripheral blood is significantly reduced by cryopreservation

Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.