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Biological Chemistry Nov 2017We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the...
We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca2+]i. For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton's tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.
Topics: Dose-Response Relationship, Drug; Humans; Phospholipase C gamma; Phytohemagglutinins; Plant Lectins; Platelet Aggregation; Structure-Activity Relationship; Wheat Germ Agglutinins
PubMed: 28779561
DOI: 10.1515/hsz-2017-0115 -
BioFactors (Oxford, England) Mar 2018It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived...
It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158-167, 2018.
Topics: Adult; Amniocentesis; Amniotic Fluid; Anti-Inflammatory Agents; Apoptosis; Cell Proliferation; Culture Media, Conditioned; Exosomes; Female; Hepatocyte Growth Factor; Humans; Leukocytes, Mononuclear; Phytohemagglutinins; Pregnancy; Pregnancy Trimester, Second; Primary Cell Culture; Regenerative Medicine; Stem Cells; Transforming Growth Factor beta
PubMed: 29341292
DOI: 10.1002/biof.1407 -
The Journal of Nutritional Biochemistry Nov 2017Common beans (Phaseolus vulgaris L.) are enriched in non-digestible fermentable carbohydrates and phenolic compounds that can modulate the colonic microenvironment... (Comparative Study)
Comparative Study
Common beans (Phaseolus vulgaris L.) are enriched in non-digestible fermentable carbohydrates and phenolic compounds that can modulate the colonic microenvironment (microbiota and host epithelial barrier) to improve gut health. In a comprehensive assessment of the impact of two commonly consumed bean varieties (differing in levels and types of phenolic compounds) within the colonic microenvironment, C57Bl/6 mice were fed diets supplemented with 20% cooked navy bean (NB) or black bean (BB) flours or an isocaloric basal diet control (BD) for 3 weeks. NB and BB similarly altered the fecal microbiota community structure (16S rRNA sequencing) notably by increasing the abundance of carbohydrate fermenting bacteria such as Prevotella, S24-7 and Ruminococcus flavefaciens, which coincided with enhanced short chain fatty acid (SCFA) production (microbial-derived carbohydrate fermentation products) and colonic expression of the SCFA receptors GPR-41/-43/-109a. Both NB and BB enhanced multiple aspects of mucus and epithelial barrier integrity vs. BD including: (i) goblet cell number, crypt mucus content and mucin mRNA expression, (ii) anti-microbial defenses (Reg3γ), (iii) crypt length and epithelial cell proliferation, (iv) apical junctional complex components (occludin, JAM-A, ZO-1 and E-cadherin) mRNA expression and (v) reduced serum endotoxin concentrations. Interestingly, biomarkers of colon barrier integrity (crypt height, mucus content, cell proliferation and goblet cell number) were enhanced in BB vs. NB-fed mice, suggesting added benefits attributable to unique BB components (e.g., phenolics). Overall, NB and BB improved baseline colonic microenvironment function by altering the microbial community structure and activity and promoting colon barrier integrity and function; effects which may prove beneficial in attenuating gut-associated diseases.
Topics: Animals; Biomarkers; Cell Proliferation; Cellular Microenvironment; Colon; Dietary Carbohydrates; Dietary Fiber; Dysbiosis; Feces; Fermentation; Functional Food; Gastrointestinal Microbiome; Gene Expression Regulation, Developmental; Intestinal Mucosa; Male; Mice, Inbred C57BL; Molecular Typing; Phytohemagglutinins; Prevotella; Random Allocation; Ruminococcus; Seeds
PubMed: 28915390
DOI: 10.1016/j.jnutbio.2017.08.002 -
Viruses Apr 2021The multimammate mouse () has been identified as a major reservoir for multiple human pathogens including Lassa virus (LASV), spp., spp., and spp. Although are...
The multimammate mouse () has been identified as a major reservoir for multiple human pathogens including Lassa virus (LASV), spp., spp., and spp. Although are related to well-characterized mouse and rat species commonly used in laboratory models, there is an absence of established assays and reagents to study the host immune responses of . As a result, there are major limitations to our understanding of immunopathology and mechanisms of immunological pathogen control in this increasingly important rodent species. In the current study, a large panel of commercially available rodent reagents were screened to identify their cross-reactivity with Using these reagents, ex vivo assays were established and optimized to evaluate lymphocyte proliferation and cytokine production by lymphocytes. In contrast to C57BL/6J mice, lymphocytes from were relatively non-responsive to common stimuli such as phytohaemagglutinin P and lipopolysaccharide. However, they readily responded to concanavalin A stimulation as indicated by proliferation and cytokine production. In summary, we describe lymphoproliferative and cytokine assays demonstrating that the cellular immune responses in to commonly used mitogens differ from a laboratory-bred mouse strain.
Topics: Animals; Biomarkers; Cytokines; Host-Pathogen Interactions; Humans; Immunity, Cellular; Immunophenotyping; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Murinae; Rats; Rodent Diseases; Species Specificity; T-Lymphocyte Subsets
PubMed: 33922222
DOI: 10.3390/v13050729 -
International Journal of Biological... Sep 2022The aim of our studies was to determine the influence of a low-frequency electromagnetic field (EMF) on the phagocytosis of latex beads (LBs) and the expression level of...
The aim of our studies was to determine the influence of a low-frequency electromagnetic field (EMF) on the phagocytosis of latex beads (LBs) and the expression level of proteins/genes in the human monocytic macrophage Mono Mac 6 (MM6) cell line in in vitro conditions. Before phagocytosis assay cells were pre-stimulated with infectious agents such as lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB), or the proliferatory agent phytohaemagglutinin (PHA), and then exposed to EMF (30 mT, 7 Hz, 3 h). The expression of cytoplasmic proteins like iPLA, cPLA, iNOS, NLR3/4, and Hsp70 involved in the immune response pathways to phagocytosed particles were evaluated with the usage of the Western blot analysis. mRNA encoding the iNOS protein was detected by reverse transcription PCR method. The most meaningful changes were observed for PLA2 and NLC4 proteins level and between iNOS protein expression and mRNA encoding iNOS protein amount. The EMF exposure exerted the strongest effect on iNOS encoding mRNA in cells pre-stimulated with LPS or SEB and phagocytosing LBs. The influence of EMF on phagocytosis was experimentally proved for the first time and there is a need for further investigations in term of the usage of EMF as a prospect, supportive therapy.
Topics: Electromagnetic Fields; Humans; Lipopolysaccharides; Macrophages; Phagocytosis; RNA, Messenger
PubMed: 35841960
DOI: 10.1016/j.ijbiomac.2022.07.080 -
The Journal of Experimental Biology Jun 2017Small mammals in temperate areas face seasonal fluctuations of temperature and food availability, both of which may influence their immune responses, which are critical...
Small mammals in temperate areas face seasonal fluctuations of temperature and food availability, both of which may influence their immune responses, which are critical to survival. In the present study, we tested the hypothesis that low temperature and food restriction suppress immune function in striped hamsters (). Thirty-seven adult male hamsters were randomly assigned to warm (23±1°C) and cold (5±1°C) treatment groups, which were further divided into fed and food-restricted groups. Body mass was not affected by cold stress, food restriction or the interaction cold stress×food restriction. Cold stress decreased total body fat mass, haematological parameters including white blood cells, lymphocytes and neutrophilic granulocytes, and immunoglobin (Ig) M titres 5 days after injecting keyhole limpet haemocyanin (KLH). However, cold temperature increased bacterial killing capacity, indicative of innate immunity, and did not affect the mass of the thymus and spleen, intermediate granulocytes, the phytohaemagglutinin (PHA) response and the levels of blood glucose and serum leptin. Corticosterone concentration was affected significantly by the interaction cold stress×food restriction but not by cold stress or food restriction alone. Food restriction reduced thymus mass, but other immunological parameters including body fat mass, spleen mass, haematological parameters, innate immunity, PHA response, the titres of IgM and IgG, and the levels of blood glucose and serum leptin were all not affected by food restriction or the interaction cold stress×food restriction. Innate immunity was positively correlated with leptin levels, whereas no significant correlations were observed in the levels of blood glucose, serum leptin, corticosterone and all the detected immune parameters. Our results show that cold stress suppressed humoral immunity but enhanced innate immunity and did not affect cellular immunity in striped hamsters. Most immunological indices were not influenced by food restriction. Blood glucose, leptin and corticosterone could not explain the changes of innate, cellular and humoral immunity upon cold stress or food restriction in striped hamsters.
Topics: Animals; Cold Temperature; Cricetinae; Cricetulus; Food Deprivation; Immunity, Cellular; Immunity, Humoral; Immunity, Innate; Male; Random Allocation
PubMed: 28381582
DOI: 10.1242/jeb.153601 -
Frontiers in Cell and Developmental... 2022Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the...
Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly ( < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.
PubMed: 36158223
DOI: 10.3389/fcell.2022.948778 -
American Journal of Respiratory and... Aug 2018Late immune suppression is associated with nosocomial infection and mortality in adults and children with sepsis. Relationships between early immune suppression and... (Observational Study)
Observational Study
RATIONALE
Late immune suppression is associated with nosocomial infection and mortality in adults and children with sepsis. Relationships between early immune suppression and outcomes in children with sepsis remain unclear.
OBJECTIVES
Prospective observational study to test the hypothesis that early innate and adaptive immune suppression are associated with longer duration of organ dysfunction in children with severe sepsis or septic shock.
METHODS
Children younger than 18 years of age meeting consensus criteria for severe sepsis or septic shock were sampled within 48 hours of sepsis onset. Healthy control subjects were sampled once. Innate immune function was quantified by whole blood ex vivo LPS-induced TNF-α (tumor necrosis factor-α) production capacity. Adaptive immune function was quantified by ex vivo phytohemagglutinin-induced IFN-γ production capacity.
MEASUREMENTS AND MAIN RESULTS
One hundred two children with sepsis and 35 healthy children were enrolled. Compared with healthy children, children with sepsis demonstrated lower LPS-induced TNF-α production (P < 0.0001) and lower phytohemagglutinin-induced IFN-γ production (P < 0.0001). Among children with sepsis, early innate and adaptive immune suppression were associated with greater number of days with multiple organ dysfunction syndrome and greater number of days with any organ dysfunction. On multivariable analyses, early innate immune suppression remained independently associated with increased multiple organ dysfunction syndrome days (adjusted relative risk, 1.2; 95% confidence interval, 1.03-1.5) and organ dysfunction days (adjusted relative risk, 1.2; 95% confidence interval, 1.1-1.3).
CONCLUSIONS
Critically ill children with severe sepsis or septic shock demonstrate early innate and adaptive immune suppression. Early innate and adaptive immune suppression are associated with longer durations of organ dysfunction and may be useful markers to help guide future investigations of immunomodulatory therapies in children with sepsis.
Topics: Adolescent; Biomarkers; Child; Child, Preschool; Critical Illness; Female; Humans; Immunity, Innate; Infant; Interferon-gamma; Male; Multiple Organ Failure; Peptide Fragments; Prospective Studies; Sepsis; Time Factors; Tumor Necrosis Factor-alpha
PubMed: 29470918
DOI: 10.1164/rccm.201710-2006OC -
Advanced Biomedical Research 2022Adoptive T-cell therapy is a promising treatment strategy for cancer immunotherapy. The ability of immunotherapy based on the adoptive cell transfer of genetically...
BACKGROUND
Adoptive T-cell therapy is a promising treatment strategy for cancer immunotherapy. The ability of immunotherapy based on the adoptive cell transfer of genetically modified T cells to generate powerful clinical responses has been highlighted by recent clinical success. Techniques which are used to expand large numbers of T cells from different sources are critical in adoptive cell therapy. In this study, we evaluated the expansion, proliferation, activation of T lymphocytes, in the presence of various concentrations of interleukin-2, phytohemagglutinin (PHA), and insulin.
MATERIALS AND METHODS
The effect of different supplemented culture media on T cell expansion was evaluated using MTT assay. The expression level of the Ki-67 proliferation marker was evaluated by real-time polymerase chain reaction. In addition, flow cytometry analysis was performed to access T cell subpopulations.
RESULTS
Our results showed that supplemented culture media with an optimized concentration of PHA and interleukin-2 increased total fold expansion of T cells up to 500-fold with approximately 90% cell viability over 7 days. The quantitative assessment of Ki-67 in expanded T cells showed a significant elevation of this proliferation marker. Flow cytometry was also used to assess the proportion of CD4+ and CD8+ cells, and the main expanded population was CD3+ CD8+ cells.
CONCLUSIONS
Based on these findings, we introduced a low-cost and rapid method to support the efficient expansion of T cells for adoptive cell therapy and other experiments.
PubMed: 36518860
DOI: 10.4103/abr.abr_349_21 -
Scientific Reports Nov 2022Affordable therapeutics are vitally needed for humans worldwide. Plant-based production of recombinant proteins can potentially enhance, back-up, or even substitute for...
Affordable therapeutics are vitally needed for humans worldwide. Plant-based production of recombinant proteins can potentially enhance, back-up, or even substitute for the manufacturing capacity of the conventional, fermenter-based technologies. We plastome-engineered a tobacco cultivar to express high levels of two "plantakines" - recombinant human cytokines, interleukins IL-37b and IL-38, and confirmed their native conformation and folding. Assessment of their biological functionality was performed ex vivo by analyzing the effects exerted by the plantakines on levels of 11 cytokines secreted from human peripheral blood mononuclear cells (PBMCs) challenged with an inflammatory agent. Application of the plant-produced IL-37b and IL-38 in PBMCs stimulated with Lipopolysaccharide or Phytohaemagglutinin resulted in significant, and in particular cases-dose-dependent modulation of pro-inflammatory cytokines secretion, showing attenuation in two-thirds of significant level modulations observed. Plantakine treatments that increased inflammatory responses were associated with the higher dosage. Our results demonstrate feasibility of manufacturing functional recombinant human proteins using scalable, cost-effective and eco-friendly plant-based bioreactors.
Topics: Humans; Leukocytes, Mononuclear; Interleukins; Cytokines; Immunologic Factors; Lipopolysaccharides
PubMed: 36376518
DOI: 10.1038/s41598-022-23828-z