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Journal of Chromatography. B,... Aug 2021B lymphocytes ('B cells') are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation...
B lymphocytes ('B cells') are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely the CD27 B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B cells provides high number of cells, hence increases the number of subsequent experiments feasible with identical cell material, consequently improving comparability of results. The cultivability of the isolated B cells was demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time that the proliferative response of tonsillar B cells to mitogens declines with the age of the donor. Furthermore, we observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some terminal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure preserves the proliferative capability as well as the differentiation capacity of the B cells.
Topics: Adult; B-Lymphocytes; Cell Culture Techniques; Cell Proliferation; Cell Separation; Cells, Cultured; Centrifugation; Child; Humans; Nylons; Palatine Tonsil; Pokeweed Mitogens
PubMed: 34325309
DOI: 10.1016/j.jchromb.2021.122853 -
Journal of Animal Science Aug 2021Disease resilience refers to the productivity of an animal under disease. Given the high biosecurity of pig nucleus herds, traits that can be measured on healthy pigs...
Disease resilience refers to the 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, that is, genetic indicator traits, offer a strategy to select for disease resilience. Our objective was to evaluate mitogen stimulation assays (MSAs) on peripheral blood mononuclear cells (PBMCs) 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 × Landrace piglets were introduced every 3 wk 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 3,136 pigs that were genotyped with a high-density marker panel. PBMCs 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). The proliferation of cells was evaluated at 48, 72, and 96 h and compared with unstimulated samples (rest count). Heritabilities of cell proliferation were estimated using a model with batch as a fixed effect and covariates of entry age; rest count; 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 h 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 SEs. In conclusion, although validation is needed, MSAss, in particular based on ConA, show promise as genetic indicator traits for disease resilience.
Topics: Animals; Cell Proliferation; Leukocytes, Mononuclear; Lymphocyte Activation; Mitogens; Phytohemagglutinins; Pokeweed Mitogens; Swine
PubMed: 33944943
DOI: 10.1093/jas/skab084 -
Cytokine May 2021Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African...
Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African elephant (Loxodonta africana), is limited however due to lack of information on immune responses. Since many immunoassays require both identified biomarkers of immune activation as well as species-specific reagents, it is crucial to have knowledge of basic immunological responses in the species of interest. Cytokine gene expression assays (GEAs) used to measure specific immune responses in wildlife have frequently shown that targeted biomarkers are often species-specific. Therefore, the aim of this study was to identify elephant-specific cytokine biomarkers to detect immune activation and to develop a GEA, using pokeweed mitogen stimulated whole blood from African elephants. This assay will provide the foundation for the development of future cytokine GEAs that can be used to detect antigen specific immune responses and potentially lead to various diagnostic tests for this species.
Topics: Animals; Cytokines; Elephants; Gene Expression Regulation; Immunoassay
PubMed: 33548797
DOI: 10.1016/j.cyto.2021.155453 -
Biomolecules & Therapeutics Mar 2021This study aimed to investigate whether the antidiabetic drugs dipeptidyl peptidase 4 (DPP4) inhibitors such as evogliptin and sitagliptin affect the membrane DPP4...
This study aimed to investigate whether the antidiabetic drugs dipeptidyl peptidase 4 (DPP4) inhibitors such as evogliptin and sitagliptin affect the membrane DPP4 (mDPP4) enzymatic activity and immune function of T helper1 (Th1) cells in terms of cytokine expression and cell profiles. The mDPP4 enzymatic activity, cytokine expression, and cell profiles, including cell counts, cell viability, DNA synthesis, and apoptosis, were measured in pokeweed mitogen (PWM)-activated CD4CD26 H9 Th1 cells with or without the DPP4 inhibitors, evogliptin and sitagliptin. PWM treatment alone strongly stimulated the expression of mDPP4 and cytokines such as interleukin (IL)-2, IL-10, tumor necrosis factor-alpha, interferon-gamma, IL-13, and granulocyte-macrophage colony stimulating factor in the CD4CD26 H9 Th1 cells. Evogliptin or sitagliptin treatment potently inhibited mDPP4 activity in a dose-dependent manner but did not affect either the cytokine profile or cell viability in PWM-activated CD4CD26 H9 Th1 cells. These results suggest that, following immune stimulation, Th1 cell signaling pathways for cytokine expression function normally after treatment with evogliptin or sitagliptin, which efficiently inhibit mDPP4 enzymatic activity in Th1 cells.
PubMed: 33148870
DOI: 10.4062/biomolther.2020.150 -
Veterinary Immunology and... Sep 2020The establishment of a panel of immune markers is of paramount importance to understand the different transcription patterns of infectious diseases in livestock. The...
The establishment of a panel of immune markers is of paramount importance to understand the different transcription patterns of infectious diseases in livestock. The array of commercially available immunological assays for cattle and sheep is currently limited, due to the lack of antibodies for these species. Even though SYBR Green based real time quantitative PCR (qPCR) is the most commonly used method to study cytokine transcription in ruminants, a lack of standardization impairs its implementation in the study of different ruminant diseases. In order to obtain reliable qPCR results, several variables need to be considered: choice of reference genes for optimal normalization, variation of annealing temperature among primer sets, and assay specificity and sensitivity. In this study, we developed and validated a panel of immune markers in bovine and ovine samples using SYBR Green based qPCR in a cost-effective way with multiple primer sets optimised to amplify at a common thermal cycling temperature. Twenty primer sets were designed to quantify immune markers (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, TNF-α, IFN-γ, IFN-α, Ki-67, NFkB-65, TLR-3, TLR-4, TLR-8 and Rig-1) in ovine and bovine templates. For optimal normalization and selection of suitable reference genes, primer sets that measure the transcription of five reference genes were also included in the panel. The amplification efficiency, linearity and specificity was validated for all target genes. Optimal amplification conditions were achieved in both ovine and bovine samples for all gene targets, with the exception of Ki67. Relative quantification studies were performed on ovine and bovine mRNA obtained from sheep peripheral blood mononuclear cells (PBMCs) stimulated with three different treatments (PMA/Ionomycin, Concanavalin A (Con A) and pokeweed mitogen (PWM)). Pokeweed and ConA efficiently induced gene transcription of most of the targeted genes, while PMA/Ionomycin showed a weaker induction. Finally, we further assessed usability of our panel by running it on bovine monocyte derived dendritic cells (MoDCs) stimulated with different vaccines. Results confirmed the induction of a specific pro-inflammatory gene transcription pattern by rabies vaccine, which resembles the one occurring during viral infection. Altogether, we validated the efficiency and usability of an extended real-time PCR panel that gives the possibility to rapidly measure a broad spectrum of ovine and bovine immune markers by using a single set of reagents and protocol thus representing a valid and cost-effective tool for research purposes.
Topics: Animals; Biomarkers; Cattle; Cells, Cultured; Cytokines; Gene Expression; Gene Expression Profiling; Leukocytes, Mononuclear; Real-Time Polymerase Chain Reaction; Sheep
PubMed: 32673891
DOI: 10.1016/j.vetimm.2020.110092 -
Peptides Oct 2020The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro-Pro fragment are...
The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro-Pro fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed. The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu-Val-(R)-(αMe)Ser(ΨPro)-Pro-Phe-Phe-Leu-Ile-Ile) and c(Leu-Val-Pro-(R)-(αMe)Ser(ΨPro)-Phe-Phe-Leu-Ile-Ile), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders.
Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Circular Dichroism; Female; Humans; Immune System Diseases; Immunosuppressive Agents; Lymphocytes; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred CBA; Peptides, Cyclic; Proline; Sheep; Spleen; Structure-Activity Relationship; Thiazoles
PubMed: 32622694
DOI: 10.1016/j.peptides.2020.170365 -
Role of cell autophagy in the generation of IgM and hepatic fibrosis in primary biliary cholangitis.Clinical Rheumatology Nov 2020Autophagy in the immune and autoimmune diseases has been concerned more and more. We investigated the potential role of cell autophagy of B cells generating IgM in...
OBJECTIVE
Autophagy in the immune and autoimmune diseases has been concerned more and more. We investigated the potential role of cell autophagy of B cells generating IgM in primary biliary cholangitis (PBC), and explored the relationship between the cell autophagy of hepatic stellate cells (HSCs) and hepatic fibrosis in PBC.
METHODS
We examined the aggregation degree of microtubule-associated protein light chain 3II (LC3II) in B cells of PBC (n = 21), health control (HC, n = 15) and disease control (DC, n = 8, active hepatitis B). The expression level of p62/SQSTM1 (p62) in B cells was detected by flow cytometry. ELISA was adopted to detect the level of IgM in the B cell culture supernatant under the basal condition, activated condition(stimulated by pokeweed mitogen, PWM) and inhibited condition(inhibited by autophagy inhibitor, 3-methyladenine, 3-MA) respectively. We detected the expression of α-smooth muscle actin (α-SMA), the infiltration level of LC3II, transforming growth factor-β (TGF-β), platelet-derived growth factor-bb (PDGF-bb), and collagen I in the hepatic tissues of five PBC patients and two HC individuals.
RESULTS
When B cells were stimulated and activated by PWM, the expression of p62 increased, and the mean fluorescence intensity (MFI, 2404.8 ± 689.0) of p62 in PBC was lower than that in HC (2966.8 ± 488.9,P = 0.0227). Under 3-MA treatment, the MFI expressed of p62 in B cells weakened. The reduction difference in PBC (466.4 ± 214.9) was smaller than that in HC (1166.6 ± 231.2, P = 0.0000) and DC (1184.8 ± 197.7, P = 0.0001). The level of generating IgM in the PBC group was obviously higher than that in the HC group (65.7 ± 15.4 vs. 49.5 ± 20.4 ng/ml, P = 0.0357). Before and after B cells were treated with 3-MA, the peak level of IgM did not significantly diminish in PBC and DC (65.7 ± 15.4 vs. 59.6 ± 18.7 ng/ml, P = 0.2965; 50.1 ± 17.4 vs. 48.4 ± 12.3 ng/ml, P = 0.8336). The immunohistochemical result revealed that the expression level of collagen I, α-SMA, TGF-β and PDGF-bb in PBC was higher than that in HC, but the expression of LC3II was lower. Similarly, immunofluorescence assay revealed that the fluorescence intensity of collagen I was higher but LC3II was lower in PBC than that in HC.
CONCLUSION
The high level autophagy of B cells from PBC patients is one important factor to synthesize and secrete IgM. Hepatic fibrosis of PBC is probably associated with the weakened autophagy of HSCs. Key Points • High-level autophagy of B cells in PBC patients is an important factor to synthesize and secrete IgM. • Pro-fibrogenic cytokines in PBC influence the increase of collagen-I through HSC autophagy, and promote the occurrence and development of hepatic fibrosis.
Topics: Autophagy; Hepatic Stellate Cells; Humans; Immunoglobulin M; Liver Cirrhosis; Liver Cirrhosis, Biliary
PubMed: 32382831
DOI: 10.1007/s10067-020-05111-6 -
Veterinary Immunology and... May 2020CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow...
CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than 90 % for both stained and unstained cells. Based on these data, CellTrace Violet™ is not recommended for the assessment of lymphocyte proliferation in equine cells. The mechanism of inhibition of equine lymphocyte proliferation by CellTrace Violet™ is unknown.
Topics: Animals; Cell Proliferation; Cell Survival; Concanavalin A; Flow Cytometry; Fluorescent Dyes; Horses; Lymphocyte Activation; Lymphocytes; Pokeweed Mitogens
PubMed: 32229340
DOI: 10.1016/j.vetimm.2020.110037 -
Research in Veterinary Science Jun 2020Stimulation with polyclonal activators is a tool to increase antibody secretion in B cells. The aim of the present study was to select the most effective common...
Stimulation with polyclonal activators is a tool to increase antibody secretion in B cells. The aim of the present study was to select the most effective common commercially available polyclonal activators of rabbit B cells. Specifically, type B oligodeoxynucleotides with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODN), recombinant rabbit interleukin-2 (rrIL-2), lipopolysaccharide (LPS), pokeweed mitogen (PWM) and Resiquimod (R848) were tested on B cells isolated from blood and spleen by fluorescence-activated cell sorting. Based on the obtained data, stimulation with CpG-ODN induced the highest antigen-specific antibody levels detected by ELISA in supernatants when a single activator was used. In contrast, LPS, PWM and R848 showed a weak or no stimulatory effect. Stimulation with a mix of activators was more effective than CpG-ODN alone, which indicates a synergistic effect in the stimulation of antibody production.
Topics: Animals; Antibody Formation; B-Lymphocytes; Female; Male; Oligodeoxyribonucleotides; Rabbits; alpha-Macroglobulins
PubMed: 32199178
DOI: 10.1016/j.rvsc.2020.03.022 -
Frontiers in Immunology 2020Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite...
Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite in the kynurenine pathway of tryptophan catabolism leading to the synthesis of nicotinamide adenine dinucleotide (NAD). As a precursor for NAD, Quin can direct a portion of tryptophan catabolism toward replenishing cellular NAD levels in response to inflammation and infection. Intracellular Quin levels increase dramatically in response to immune stimulation [e.g., lipopolysaccharide (LPS) or pokeweed mitogen (PWM)] in macrophages, microglia, dendritic cells, and other cells of the immune system. NAD serves numerous functions including energy production, the poly ADP ribose polymerization (PARP) reaction involved in DNA repair, and the activity of various enzymes such as the NAD-dependent deacetylases known as sirtuins. We used highly specific antibodies to protein-coupled Quin to delineate cells that accumulate Quin as a key aspect of the response to immune stimulation and infection. Here, we describe Quin staining in the brain, spleen, and liver after LPS administration to the brain or systemic PWM administration. Quin expression was strong in immune cells in the periphery after both treatments, whereas very limited Quin expression was observed in the brain even after direct LPS injection. Immunoreactive cells exhibited diverse morphology ranging from foam cells to cells with membrane extensions related to cell motility. We also examined protein expression changes in the spleen after kynurenine administration. Acute (8 h) and prolonged (48 h) kynurenine administration led to significant changes in protein expression in the spleen, including multiple changes involved with cytoskeletal rearrangements associated with cell motility. Kynurenine administration resulted in several expression level changes in proteins associated with heat shock protein 90 (HSP90), a chaperone for the aryl-hydrocarbon receptor (AHR), which is the primary kynurenine metabolite receptor. We propose that cells with high levels of Quin are those that are currently releasing kynurenine pathway metabolites as well as accumulating Quin for sustained NAD synthesis from tryptophan. Further, we propose that the kynurenine pathway may be linked to the regulation of cell motility in immune and cancer cells.
Topics: Animals; Biomarkers; Cell Movement; Gerbillinae; HSP90 Heat-Shock Proteins; Hippocampus; Immunity; Indoleamine-Pyrrole 2,3,-Dioxygenase; Inflammation; Kynurenine; Lipopolysaccharides; Liver; Male; Mice; Mice, Inbred C57BL; NAD; Pokeweed Mitogens; Poly(ADP-ribose) Polymerases; Quinolinic Acid; Rats; Spleen; Tryptophan
PubMed: 32153556
DOI: 10.3389/fimmu.2020.00031