Pokeweed Mitogen Research Articles

Use of serum-free media for peripheral blood mononuclear cell culture and the impact on T and B cell readouts

IntroductionAs part of a wider programme of work developing next-generation risk assessment approaches (NGRA) using non-animal methods (NAMs) for safety assessment of materials, Unilever SEAC is exploring the use of a peripheral blood mononuclear cell (PBMC) system to investigate how cells from different arms of the human immune system are impacted by different treatments. To maximise human relevance, the cell cultures are supported by human serum, but this came with some challenges, including an inability to measure induced levels of immunoglobulins due to high background levels. Therefore, a study comparing use of human sera containing media with three different chemically defined serum-free media was undertaken.Materials and MethodsPBMC were isolated from healthy donors and cultured in the absence (media alone) or presence of stimulation reagents (CpG-ODN plus IL-15, Pokeweed Mitogen (PWM) or Cytostim (CS)), in RPMI plus human serum, AIM-V, CTS OpTmizer T cell expansion SFM or X-VIVO 15 media. T cell (CD4+ and CD8+) and B cell proliferation and viability were measured after 6 days, along with levels of total IgG in the cell culture supernatants.ResultsEach of the serum-free media tested supported good levels of viable and proliferating T cells and B cells over the 6 days of culture, with only a few, small differences across the media, when there was no stimulation. They also enabled detection of a stimulation-evoked increase in IgG levels. There were however some differences in the viability and proliferation responses of T and B cells, to different stimuli, across the different media.DiscussionThe serum-free media formulations tested in this study offer defined systems for. measuring B cell IgG responses, in vitro, in either a ‘T cell-independent’ (CpG + IL-15) or “T cell-dependent” (PWM or CS) manner and for assessing B cell proliferation, particularly in response to a “T cell-independent” stimulus. However, there are some characteristics and features endowed by human serum that appear to be missing. Therefore, further work is required to optimise animal-free, chemically defined culture conditions for PBMC based assays for inclusion in tiered safety assessments.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15–1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15–1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.

Granulocyte- Macrophage Colony-Stimulating Factor Reverses Immunosuppression Acutely Following a Traumatic Brain Injury and Hemorrhage Polytrauma in a Juvenile Male Rat Model.

Traumatic brain injury (TBI) is a common cause of morbidity and mortality in children. We have previously shown that TBI with a concurrent extracranial injury reliably leads to post-injury suppression of the innate and adaptive immune systems. In patients with post-injury immune suppression, if immune function could be preserved, this might represent a therapeutic opportunity. As such, we examined, in an animal injury model, whether systemic administration of granulocyte macrophage colony-stimulating factor (GM-CSF) could reverse post-injury immune suppression and whether treatment was associated with neuroinflammation or functional deficit. Prepubescent male rats were injured using a controlled cortical impact model and then subjected to removal of 25% blood volume (TBI/H). Sham animals underwent surgery without injury induction, and the treatment groups were sham and injured animals treated with either saline vehicle or 50 μg/kg GM-CSF. GM-CSF was administered following injury and then daily until sacrifice at post-injury day (PID) 7. Immune function was measured by assessing tumor necrosis factor-α (TNF-α) levels in whole blood and spleen following ex vivo stimulation with pokeweed mitogen (PWM). Brain samples were assessed by multiplex enzyme-linked immunosorbent assay (ELISA) for cytokine levels and by immunohistochemistry for microglia and astrocyte proliferation. Neuronal cell count was examined using cresyl violet staining. Motor coordination was evaluated using the Rotarod performance test. Treatment with GM-CSF was associated with a significantly increased response to PWM in both whole blood and spleen. GM-CSF in injured animals did not lead to increases in levels of pro-inflammatory cytokines in brain samples but was associated with significant increases in counted astrocytes. Finally, while injured animals treated with saline showed a significant impairment on behavioral testing, injured animals treated with GM-CSF performed similarly to uninjured animals. GM-CSF treatment in animals with combined injury led to increased systemic immune cell response in whole blood and spleen in the acute phase following injury. Improved immune response was not associated with elevated pro-inflammatory cytokine levels in the brain or functional impairment.

Genetic profile of adaptive immune traits and relationships with parasite resistance and productivity in Scottish Blackface sheep

Gastrointestinal (GI) parasites cause significant production losses in grazing ruminants which can be mitigated by breeding animals resistant to disease. Lymphocyte cytokine production and parasite-specific Immunoglobulin A (IgA) are adaptive immune traits associated with immunity to GI parasites. To explore the utility of these traits for selective breeding purposes, this study estimated the genetic parameters of the immune traits in sheep and assessed their relationship with disease and productivity traits. Whole blood stimulation assays were performed on 1 040 Scottish Blackface lambs at two months of age in 2016–2017. Blood was stimulated with either pokeweed mitogen (PWM), a non-specific activator of lymphocytes, and Teladorsagia circumcincta (T-ci) larval antigen to activate parasite-specific T lymphocytes. The type of adaptive immune response was determined by quantifying production of cytokines interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10, which relate to T-helper type (Th) 1, Th2 and regulatory T cell responses, respectively. Serum T-ci specific IgA was also quantified. Heritabilities were estimated for each immune trait by univariate analyses. Genetic and phenotypic correlations were estimated between different immune traits, and between immune traits vs. disease and productivity traits that were recorded at three months of age. Disease phenotypes were expressed as faecal egg counts (FEC) of nematode parasites (Strongyles and Nematodirus), faecal oocyst counts (FOC) of coccidian parasites, and faecal soiling score; production was measured as lamb live weight. Significant genetic variation was observed in all immune response traits. Heritabilities of cytokine production varied from low (0.14 ± 0.06) to very high (0.77 ± 0.09) and were always significantly greater than zero (P < 0.05). IgA heritability was found to be moderate (0.41 ± 0.09). Negative associations previously identified between IFN-γ production and FOC, and IL-4 production and strongyle FEC, were not evident in this study, potentially due to the time-lag between immune and parasitology measures. Instead, a positive genetic correlation was found between FOC and PWM-induced IFN-γ production, while a negative genetic correlation was found between FOC and T-ci induced IL-10. Live weight was negatively genetically correlated with IFN-γ responses. Overall, IFN-γ and IL-4 responses were positively correlated, providing little evidence of cross-regulation of Th1 and Th2 immunity within individual sheep. Furthermore, T-ci specific IgA was highly positively correlated with PWM-induced IL-10, indicating a possible role for this cytokine in IgA production. Our results suggest that while genetic selection for adaptive immune response traits is possible and may be beneficial for parasite control, selection of high IFN-γ responsiveness may negatively affect productivity.

PSX-8 Effect of Feeding Additional Whey Protein to Senior Labrador Retrievers for Maintaining Lean Mass and Minimizing Inflammation

Abstract We previously reported increases in lean mass and lean:fat ratio in senior Labrador Retrievers fed a commercial whey protein supplement after a 26-week trial with exercise regimen. Further work on this project examined the impacts of this supplement on immunity and inflammation. Thirty-six senior (&amp;gt;7 yr) Labrador Retrievers received a basal diet meeting AAFCO crude protein (CP) requirements (45g/1,000 kcal, 18% CP on DM basis) and were randomly assigned to one of three supplements: 1) an additional 22.5 g CP/1,000 kcal (27% CP on DM basis) sourced from hydrolyzed pea protein isolate (Pea); 2) additional 22.5 g CP/1,000 kcal (27% CP on DM basis) sourced from whey protein (Whey; Hilmar Protelyze Pet; Hilmar, CA) or 3) poultry fat and sucrose to equalize calories but provide no additional CP (Control). All dogs participated in an exercise regimen consisting of twice weekly 2.2 km runs. Blood samples were collected via jugular venipuncture at week 0 (baseline) and week 26 (end of trial) for analysis of cytokines (IL-2, IL-6, IL-8, IL-10, IL-12, NGF-β, IFN-γ, TNF-α, MCP-1, VEGFA, and SCF) and immune cell proliferation. Cytokines were measured with Luminex MAGPIX analyzer. Peripheral blood mononuclear cells were isolated from whole blood, plated, challenged with pokeweed mitogen. Proliferation was evaluated with a 5-bromo-2’-deoxyuridine proliferation assay kit. Gait analysis was evaluated at weeks 0 (baseline), 4, 8, 16 and 26 using a Gait4Dogs pressure walkway (CIR Systems, Inc; Franklin, NJ). Gait data were processed to generate a FRK Total Gait Inflammation Index, calculated by the sum of the differences from the ideal scores for gait lameness score, total pressure index, step/stride ratio, and hind reach. Data were analyzed using one-way ANOVA with Tukey’s post-hoc adjustments. We observed increases in IL-12 (P = 0.004), MCP-1 (P = 0.04), VEGFA (P = 0.05), and SCF (P = 0.04) in Control vs Whey at week 26, with Pea intermediate. Spontaneous immune cell proliferation was less in both Pea and Whey compared with Control (P &amp;lt; 0.01). Proliferation after challenge was less in Whey compared with Pea (P = 0.05), with Control intermediate. The FRK Total Gait Inflammation Index significantly increased after exercise in Control and Pea dogs, but not Whey (P &amp;lt; 0.01). Scores of Control dogs remained increased compared with Whey dogs from weeks 4 to 26 (P &amp;lt; 0.01). Dietary supplementation with whey protein at 1.5 x AAFCO minimum proved beneficial for Labrador Retrievers by maintaining more lean mass with improved lean/fat ratio, reduced inflammatory biomarkers, immune cell proliferation, and FRK Total Gait Inflammation Index after an exercise regimen when compared with seniors only consuming a 18% protein basal diet. Supplemental whey protein may better maintain senior dogs experiencing chronic inflammation from aging or disease.

Mathematical Model to Predict Polyclonal T-Cell-Dependent Antibody Synthesis Responses

Mathematical models are becoming indispensable tools to explore the complexities of biological systems at cellular levels. We present a model to explore the baseline immune cell interactions for in vitro polyclonal antibody synthesis via B-cells regulated by helper and regulatory T-cells. The model incorporates interactions of antigen-presenting cells, T-cells, regulatory T-cells, and B-cells with each other and predicts time-dependent trajectories of these cells and antibody synthesis stimulated by pokeweed mitogen. We used an ordinary differential equation-based approach to simulate the dynamic changes in the cells and cytokines numbers due to the cellular and humoral response to pokeweed mitogen stimulation. The parameters of the ordinary differential equations model are determined to yield a normal immune response as observed in the pokeweed mitogen-stimulated in vitro antibody synthesis via normal T, B, and antigen-presenting cells. The dose effects of antigen load and basal values of regulatory T-cells on the profiles of various immune response variables are also evaluated.

Shiga Toxin, Stx2e, Influences the Activity of Porcine Lymphocytes In Vitro.

Oedema disease (OD) in piglets is one of the most important pathologies, as it causes significant losses due to the high mortality because of the Shiga toxin family, which produces Escherichia coli (STEC) strains. The main toxin responsible for the characteristic pathologies in pigs is Shiga toxin 2 subtype e (Stx2e). Moreover, there is growing evidence that Stx's family of toxins also targets immune cells. Therefore, this study evaluated the effect of different concentrations of Stx2e on porcine immune cells. Porcine peripheral blood mononuclear cells were pre-incubated with Stx2e, at three different concentrations (final concentrations of 10, 500, and 5000 CD50/mL) and with a negative control group. Cells were then stimulated with polyclonal mitogens: concanavalin A, phytohemagglutinin, pokeweed mitogen, or lipopolysaccharides. Cell proliferation was assessed by BrdU (or EdU) incorporation into newly created DNA. The activation of the lymphocyte subsets was assessed by the detection of CD25, using flow cytometry. The toxin significantly decreased mitogen-driven proliferation activity, and the effect was partially dose-dependent, with a significant impact on both T and B populations. The percentage of CD25+ cells was slightly lower in the presence of Stx2e in all the defined T cell subpopulations (CD4+, CD8+, and γδTCR+)-in a dose-dependent manner. B cells seemed to be the most affected populations. The negative effects of different concentrations of Stx2e on the immune cells in this study may explain the negative impact of the subclinical course of OD.

Phylogeny and expression of ADAM10 and ADAM17 homologs in lamprey

The ADAMs (a disintegrin and metalloproteinase) play regulatory roles in cell adhesion, migration and proteolysis. To explore the origin and evolution of ADAMs, this study identified the homologs of adam10 and adam17 in Lampetra morii and Lampetra japonica. Sequence analysis revealed that they share the same genomic structures with their counterparts in jawed vertebrates. The putative proteins possess conserved motifs, including a furin cut site (RXXR) for precursor processing, an enzyme catalytic motif (HEXGEHXXGXXH) for hydrolysis, and a Ca2+-binding motif (CGNXXXEXGEXCD) for stabilizing protein structure. In addition, a substrate recognition domain is present at the membrane-proximal region of lamprey ADAM17. The cytoplasmic region of lamprey ADAM10 contains a potential threonine phosphorylation site which has been shown to be activated by protein kinase C (PKC) in mammals. Both the adam10 and adam17 genes were constitutively expressed in the brain, kidney, and gills and were differentially regulated in the primary blood leukocytes by lipopolysaccharide (LPS) and pokeweed mitogen (PWM). Adam10 was induced by LPS but not PWM; conversely, adam17 was induced by PWM but not LPS. Taken together, our results suggest that the activation pathways and functions of ADAM10 and ADAM17 are conserved in agnathans.

Characterization of the immunomodulatory protein isolated from guduchi (Tinospora cordifolia) churna, an ayurvedic rejuvenator

Objective: Guduchi [Tinospora cordifolia (Willd.) f. and Thoms.], a rasayana (rejuvenator) in the traditional system of Indian medicine (Ayurveda), is known for its diverse bioactivities. The aim of this study was to isolate the immunomodulatory protein (ImP) from commercial guduchi churna (stem powder) and characterize it. Methods: Guduchi churna extract following 80% ammonium sulfate precipitation was chromatographed on Q-Sepharose. Purified guduchi ImP was evaluated for its lymphoproliferative and macrophage activation properties, structural relatedness to certain plant lectins, amino acid composition, stability to trypsin, and N-terminal sequencing. Results: The purified protein, obtained in a yield of 140[Formula: see text]mg/100[Formula: see text]g guduchi churna, showed a molecular mass of [Formula: see text] kDa by SDS-PAGE. Guduchi ImP (10 [Formula: see text]g) showed seven-fold increase over control in mitogenic activity of murine splenocytes, and a four-fold increase over control in nitric oxide (NO) release from murine peritoneal exudates cells (PECs). These activities were abolished in pronase-treated guduchi ImP. Guduchi ImP does not agglutinate rabbit erythrocytes. Rabbit antiserum to guduchi stem ImP strongly reacted with purified guduchi ImP, and did not recognize common plant lectins (concanavalin A (Con A), poke weed mitogen (PWM) and garlic lectin). Amino acid composition of guduchi ImP shows high content of acidic amino acids, serine (12.4%) and cysteine (10.2%); guduchi ImP is highly stable to trypsin and has a blocked N-terminus. Conclusion: Guduchi ImP is an acidic, non-lectin ImP with a high content of serine and cysteine, and appears to have an immense potential for future structural, immunological, and therapeutic studies.

Optimizing an immunomodulatory potency assay for Mesenchymal Stromal Cell.

The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 via TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.

B cell phenotypes and maturation states in cows naturally infected with Mycobacterium avium subsp. Paratuberculosis.

Little is known about the role that B cells play in immune responses to infection with the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). Traditionally, the role of B cells has been constrained to their function as antibody-producing cells, however, antibodies are not thought to play a protective role in mycobacterial infections. The present study was designed to characterize B cell subpopulations as well as activation/maturation states in cattle with paratuberculosis. Peripheral blood mononuclear cells (PBMCs) were isolated from noninfected control cows (n = 8); as well cattle naturally infected with MAP in the subclinical (n = 8) and clinical (n = 7) stage of infection and stimulated with MAP antigen for 6 days. MAP infection resulted in greater numbers of total B cells for clinical cows compared to control noninfected cows. The major subpopulation in freshly isolated PBMCs in clinical cows was B-1a B cells, but this shifted to a composite of both B-1a and B-2 B cells upon stimulation of PBMCs with either MAP antigen or pokeweed mitogen, with higher numbers of B-2 B cells. Early B cells were observed to predominate the population of B cells in PBMCs, with lesser populations of germinal B cells, memory B cells and plasma cells. These subpopulations were elevated in clinical cows upon stimulation of PBMCs with MAP antigen, except for plasma cells which were lower compared to control noninfected cows. Increased numbers of B cells in clinical cows aligned with higher expression of B cell markers such as MAPK1/3, BTG1, Bcl2, CD79A and SWAP70, depending upon in vitro stimulation with either mitogen or antigen. This would indicate that the B cells were capable of activation but were anti-apoptotic in nature. The shift to B-2 B cells in the periphery of clinical cows seems to be indicative of an expansion of memory B cells, rather than plasma cells. This may be a last attempt by the host to control the rampant inflammatory state associated with advanced clinical disease.

Cellular assays to evaluate B-cell function

Inborn errors of immunity (IEI) that present with recurrent infections are largely due to antibody (Ab) deficiencies. Therefore, assessment of the B-cell and Ab compartment is a major part of immunologic evaluation. Here we provide an overview about cellular assays used to study B-cell function and focus on lymphocyte proliferation assay (LPA), opsonophagocytic assay (OPA), and the Enzyme-linked Immunosorbent Spot Assay (ELISPOT) including clinical applications and limitations of these techniques. LPAs assess ex-vivo cell proliferation in response to various stimuli. Clinically available LPAs utilize peripheral blood mononuclear cells and mostly assess T-cell proliferation with pokeweed mitogen considered the most B-cell specific stimulus. In the research setting, isolating B cells or using more B-cell specific stimuli such as CD40L with IL-4/IL-21 or the TLR9 ligand CpG can more specifically capture the proliferative ability of B cells. OPAs are functional in-vitro killing assays used to evaluate the ability of IgG Ab to induce phagocytosis applied when assessing the potency of vaccine candidates or along with avidity assays to evaluate the quality of secreted IgG. The B-cell ELISPOT assesses Ab production at a cellular level and can characterize the Ab response of particular B-cell subtypes. It can be used in patients on IgG therapy by capturing specific Abs produced by individual B cells, which is not affected by exogenous IgG from plasma donors, and when assessing the vaccine response in patients on immunomodulatory drugs that can affect memory B-cell function. Emerging approaches that are only available in research settings are also briefly introduced.

Effects of fatty acid mixtures on proliferation of peripheral blood mononuclear cells in dairy cows.

This in vitro study was performed to assess the effects of three different mixtures of nonesterifed fatty acids (NEFA) on mitogen-driven proliferation of peripheral blood mononuclear cells (PBMC) in dairy cows. Substantially, the three mixtures differed for n-6 to n-3 fatty acids (FA) ratio and were intended to mimic plasma NEFA composition of cows given fat supplements with different n-6 to n-3 FA ratio. PBMC from six Holstein heifers were cultured in media containing three different mixtures of oleic, palmitic, stearic, linoleic, palmitoleic, or linolenic acid at concentrations mimicking different degree of lipomobilisation. Proliferation of PBMC was stimulated by concanavalin A or pokeweed mitogen (PWM). Low concentrations of the three mixtures (62.5 and 125 µmol/l), did not affect the ability of PBMC to proliferate. Concentrations of the three mixtures mimicking medium-intense lipomobilisation (500, 1,000 and 1,500 µmol/l) impaired to the same extent proliferation of PBMC. The addition to cultures of the three mixtures at concentration of 250 µmol/l, impaired the proliferation only in PBMC stimulated with PWM. Even in this case, the three mixtures did not exert differential effects on PBMC proliferation. Present results reinforce the hypothesis that high concentrations of plasma NEFA play a role in the immunosuppression taking place in cows undergoing intense lipomobilisation, and authorize to suggest that under these conditions different composition of plasma NEFA in terms of different n-6 to n-3 FA ratio cannot prevent their negative effects on lymphocyte proliferation.

FISH Analysis of CDKN2A/B Deletion in Chronic Lymphocytic Leukemia

Introduction: The course of chronic lymphocytic leukemia (CLL) ranges from indolent to rapid disease progression, including transformation to an aggressive lymphoma (Richter transformation, RT). A subset of CLL pts have been shown to acquire deletions of the tumor suppressor CDKN2A/B. RT has been associated with deletion of CDKN2A/B, however, prior studies’ sample populations were limited or not primarily focused on those with this deletion. Due to the rarity of this genetic abnormality in CLL and severity of RT, additional studies concerning this subgroup should be investigated. Here, we examine a larger cohort of CLL pts with CDKN2A/B deletion by fluorescence in situ hybridization (FISH) to provide further clarity on this genetic lesion. Methods: We conducted a retrospective case study for all pts for whom cytogenetic clinical samples were submitted for extended CLL FISH panel analysis over a 3.5 year (yrs) period, between 2015 and 2018 at The Ohio State University. Pts were screened for CDKN2A/B deletion with a new or follow up diagnosis of CLL or small lymphocytic lymphoma (SLL). Cytogenetic testing was performed on cells stimulated with pokeweed mitogen, phorbol myristate acetate (PMA), and CpG oligonucleotides. FISH for D13S319, D12Z3, ATM, TP53, BCL6, MYC, REL, SEC63, MYB, and CDKN2A/B was performed via manufacturer's recommendations. IGHV mutational status was determined using PCR. A 50-gene hematologic sequencing panel was performed via Ion Torrent. Overall survival (OS) from first detection of abnormal CDKN2A/B results were examined. Cox regression model was used to evaluate OS post CDKN2A/B deletion. The Fine and Gray Model was used to examine correlation of variables with regard to RT, treating death without RT as a competing risk. Results: 636 pts had sample(s) submitted for extended CLL FISH panel analysis. Of these, 43 (6.8%) pts with confirmed CLL or SLL were found to have loss of CDKN2A/B by FISH, including 6 pts within one year of diagnosis. Median age at diagnosis was 54.9 yrs (range 41.9-77.7). Loss of CDKN2A/B was detected at a median of 7.8 yrs (range diagnosis-26.1) from diagnosis. Of those with known IGHV status (n=33), 91% were unmutated. A complex karyotype (>3 clonal chromosomal abnormalities) was found in 90% percent of pts. In regard to CDKN2A/B deletion, 77%, 16%, and 7% of pts had heterozygous, homozygous, or subclonal populations of heterozygous as well as homozygous loss, respectively. Additional FISH testing showed 23% had gain of REL, 16% had BCL6 gain/rearrangement, 16% had deletion 6q, 37% had gain/rearrangement of MYC, 16% had loss of ATM, 14% had trisomy 12, and 58% had deletion 13q. Deletion of TP53 was seen in 63%. Sequencing data was available for 15 pts (35%), of note, 10 pts had mutations in TP53, with 4 also having mutations in SF3B1. In terms of OS, there were 30 deaths in the cohort, with median OS of 10.8 yrs from diagnosis (95% CI 8.0-14.4). With a median follow-up of 4.9 yrs among survivors, the median OS from time of detection of CDKN2A/B deletion was 1.7 yrs (95% CI 0.3-4.7). Multivariable analysis of OS post CDKN2A/B detection was significant for increasing beta-2-microglobulin measured at first visit at our institution (p=0.0044, hazard ratio (HR) 1.32 (95% CI 1.09-1.60)) and CDKN2A/B subclonal heterozygous and homozygous loss (p=0.0024, HR 9.81 (95% CI 2.25-42.73)). Of the 43 pts with loss of CDKN2A/B, 21 (49%) progressed via RT (n=20) or prolymphocytic leukemia (PLL) (n=1). The Cumulative Incidence Rate of RT development 12-months post CDKN2A/B deletion was 23.5% (95% CI 10.9-38.9), at 24-months, it was 29.4% (15.1-45.3). Deletion of TP53 was seen in 85.7%. Albeit there was limited pts with detection of CDKN2A/B deletion prior to RT, the multivariable model for RT found loss of TP53 (p=0.02, HR 4.53 (95% CI 1.33-15.43)) and homozygous and heterozygous loss of CDKN2A/B (p=0.0008, HR 6.08 (95% CI 2.13-17.39)) were independently significant. Conclusion: Here we show that deletion of CDKN2A/B in CLL is a very rare occurrence and was seen predominantly in pts with highly aggressive disease characteristics. Close to half of our cohort progressed with RT or PLL. Additional investigation of serial samples is needed to establish when CDKN2A/B deletion occurs relative to RT. Our results indicate that prospective FISH analysis for CDKN2A/B deletion, in conjunction with other high-risk genetic alterations, to establish RT prognostication in high-risk CLL is warranted.

Low-fat dairy consumption improves intestinal immune function more than high-fat dairy in a diet-induced swine model of insulin resistance.

To understand the effects of consuming high-fat and low-fat dairy products on postprandial cardiometabolic risk factors and intestinal immune function, we used an established low birthweight (LBW) swine model of diet-induced insulin resistance. LBW piglets were randomized to consume one of the 3 experimental high fat diets and were fed for a total of 7weeks: (1) Control high fat (LBW-CHF), (2) CHF diet supplemented with 3 servings of high-fat dairy (LBW-HFDairy) and (3) CHF diet supplemented with 3 servings of low-fat dairy (LBW-LFDairy). As comparison groups, normal birthweight (NBW) piglets were fed a CHF (NBW-CHF) or standard pig grower diet (NBW-Chow). At 11weeks of age, all piglets underwent an established modified oral glucose and fat tolerance test. At 12weeks of age, piglets were euthanized and ex vivo cytokine production by cells isolated from mesenteric lymph node (MLN) stimulated with mitogens was assessed. Dairy consumption did not modulate postprandial plasma lipid, inflammatory markers and glucose concentrations. A lower production of IL-2 and TNF-α after pokeweed mitogen (PWM) stimulation was observed in LBW-CHF vs NBW-Chow (P < 0.05), suggesting impaired MLN T cell function. While feeding high-fat dairy had minimal effects, feeding low-fat dairy significantly improved the production of IL-2 and TNF-α after PWM stimulation (P < 0.05). Irrespective of fat content, dairy had a neutral effect on postprandial cardiometabolic risk factors. Low-fat dairy products improved intestinal T cell function to a greater extent than high-fat dairy in this swine model of obesity and insulin resistance.

SARS-CoV-2 pneumonia and bacterial pneumonia patients differ in a second hit immune response model

Secondary infections have been shown to complicate the clinical course and worsen the outcome of critically ill patients. Severe Coronavirus Disease 2019 (COVID-19) may be accompanied by a pronounced cytokine release, and immune competence of these patients towards most pathogenic antigens remains uncompromised early in the disease. Patients with bacterial sepsis also exhibit excessive cytokine release with systemic hyper-inflammation, however, typically followed by an anti-inflammatory phase, causing immune paralysis. In a second hit immune response model, leukocyte activation capacity of severely ill patients with pneumonia caused by SARS-CoV-2 or by bacteria were compared upon ICU admission and at days 4 and 7 of the ICU stay. Blood cell count and release of the pro-inflammatory cytokines IL-2, IFNγ and TNF were assessed after whole-blood incubation with the potent immune stimulus pokeweed mitogen (PWM). For comparison, patients with bacterial sepsis not originating from pneumonia, and healthy volunteers were included. Lymphopenia and granulocytosis were less pronounced in COVID-19 patients compared to bacterial sepsis patients. After PWM stimulation, COVID-19 patients showed a reduced release of IFNγ, while IL-2 levels were found similar and TNF levels were increased compared to healthy controls. Interestingly, concentrations of all three cytokines were significantly higher in samples from COVID-19 patients compared to samples from patients with bacterial infection. This fundamental difference in immune competence during a second hit between COVID-19 and sepsis patients may have implications for the selection of immune suppressive or enhancing therapies in personalized medicine.

The immune system ofthe ferret (Mustela putorius furo) - Areview.

The basic information dealing with the anatomy ofthe ferret's immune system, cross-reactivity oftheferret leukocytes with polyclonal and monoclonal antibodies invitro and immune response tothe mitogens and various infections are presented. The leukocyte numbers inthe peripheral blood inthe ferrets are lower compared toother species and only one subclass ofIgG has been identified inferrets sofar. Lymphocytes make up 12-67% ofall theleukocytes inthe peripheral blood ofthe healthy adult ferrets. Lymphocyte subpopulations are similar toother mammals and include T-and B-lymphocytes. T-lymphocytes differentiate into helper (Th) lymphocytes and cytotoxic (Tc) lymphocytes. Currently, ferret granulocytes (CD11), B-lymphocytes (CD79α), T-lymphocytes(CD3), Th-lymphocytes (CD3, CD4), Tc-lymphocytes (CD3, CD8), and CD30, CD45 subpopulations are detected withtheuse ofanumber ofpolyclonal aswell aswith monoclonal antibodies. Inalymphocyte transformation assay, themitogen response ofthe peripheral blood mononuclear cells toconcanavalinA(ConA), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM) isthe greatest atday2, 2 and 3, respectively. Serious lymphopenia isobservedinferrets during adistemper infection. Asignificant decrease inthe lymphocyte transformation activity isobserved onday5 and reaches amaximal decrease ondays 8-11, with full recovery ondays 23-30 after theinoculation oflaboratory ferrets with the distemper virus. Ferrets have also been used instudies related tothe function oftheimmune system inHelicobacter pylori infections, Crohn's disease and bronchial asthma.

Comparison of the oxidative respiratory burst and mitogen-induced leukocyte responses of camels, goats, sheep, and cows.

Background and Aim:The reports from the Ministry of Agriculture and Fisheries suggest that camels suffer less compared to goats, sheep, and cows from a number of common infectious diseases in Oman. However, there is no immunological evidence to substantiate this claim. This present study is, therefore, an attempt to study the immunological responses of camels, goats, sheep, and cows by comparing their oxidative respiratory burst of peripheral blood leukocytes (PBLs) as a marker of innate immunity occurring during phagocytosis and the mitogenic responses of their peripheral blood mononuclear leukocytes (PBMLs) as a marker of their adaptive immune response.Materials and Methods:Ten female adult animals (n = 10) were selected from each species (goats, sheep, and cows). The goats, sheep, and cows were maintained at the Agricultural Experiment Station, while camels were kept at the Royal Camel Corps (RCC). Blood samples were collected from the jugular vein in 7 mL of heparin and ethylenediaminetetraacetic acid vacutainer tubes. The oxidative respiratory burst of PBLs was measured using a chemiluminescence (CL) assay. Reactants consisted of 75 mL of whole blood diluted (1:50), 75 mL of luminol/isoluminol, and 75 mL of zymosan opsonized with non-heat inactivated serum/heat-inactivated serum or non-opsonized zymosan. CL responses were measured as relative light units and expressed as the mean count per minute and peak CL values. The mitogenic response of PBMLs to concanavalin A (Con-A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) was tested using a WST-8 assay and read spectrophotometrically at 450 nm.Results:The present findings showed that camel PBLs generate significantly higher CL responses, both intracellularly as well as extracellularly, with zymosan opsonized with autologous serum. Camel PBLs demonstrated a significantly higher (p = 0.001) response when stimulated with zymosan opsonized with heat-inactivated serum compared to those of goat, sheep, and cow lymphocytes from camels exhibited significantly higher (p = 0.001) stimulation indices (SI) with Con-A, PHA, and PWM.Conclusion:The present study suggests that camels are capable of mounting both superior innate as well as adaptive immune responses and provide immunological evidence supporting the belief of some authors, who have proposed that camels are less susceptible to a number of common infectious diseases than other domesticated ruminants.

Comparison of [3H]-Thymidine, Carboxyfluorescein Diacetate Succinimidyl Ester and Ki-67 in Lymphocyte Proliferation.

BackgroundPatients with T cell deficiency <10% of normal proliferation are indicated to receive immune reconstruction by hematopoietic stem cell transplantation (HSCT). This study aimed to investigate whether non-radioactive assays can be used to quantitatively detect the lymphocyte proliferation <10% of normal as radioactive [3H]-thymidine.”MethodsRadioactive [3H]-thymidine, non-radioactive carboxyfluorescein diacetate succinimidyl ester (CFSE), and Ki-67 protein expressions were used to measure the lymphocyte proliferation as calculated using the stimulation index (SI), subtraction percentage, and proliferation index (FlowJo software). Normal references were established for comparison in the absence of parallel healthy controls.ResultsNormal ranges of mitogen-stimulated lymphocyte proliferation were established as a SI of 15–267 (CSFE 47–92%, Ki-67 42–79%) with phytohemagglutinin (PHA) 5 μg/ml stimulation; 19–139 (CFSE 62–83%, 45–74% Ki-67) with concanavalin-A (ConA) 5 μg/ml stimulation; 7–53 (CFSE 6–23%, Ki-67 10–24%) with pokeweed mitogen (PWM) 0.1 ug/ml stimulation; 3–28 (CFSE 4–10%, Ki-67 5–14%) with candida 10 ug/ml stimulation; and 2–27 (CFSE 6–41%, Ki-67 6–30%) with bacille Calmette-Guerin (BCG) 0.02 ng/ml stimulation. The normalized CFSE-proliferation index was between 2.1 and 3.0. Although there was no significant correlation between these three assays in the healthy controls, the SI value for <10% [3H]-thymidine proliferation in those with T cell deficiency was compatible with CFSE- and Ki-67-stained lymphocyte percentages, and validated in patients with IL2RG, RAG1, and ZAP70 mutations. When calculating [3H]-thymidine <10% of normal lymphocyte proliferation, the threshold of parallel controls was more reliable than previously established normal references.ConclusionThe large quantitative value of radioactive [3H]-thymidine was more easily recognizable than that for non-radioactive CFSE and Ki-67. Even though the correlation was not significant, those identified to have <10% of normal proliferation by [3H]-thymidine could be consistently detected by CFSE and Ki-67, and consequently indicated for HSCT.

Bovine Peripheral Blood Derived Lymphocyte Proteome and Secretome Show Divergent Reaction of Bovine Immune Phenotypes after Stimulation with Pokeweed Mitogen

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation.