Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults' physiology. Incorrect processing of the IGF2 precursor, pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.
Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of R-phycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con A-stimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE+ B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.
- MeSH
- B-Lymphocytes immunology MeSH
- Phycoerythrin immunology metabolism MeSH
- Mitogens immunology MeSH
- Antibodies, Monoclonal immunology MeSH
- Mice, Inbred BALB C MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- T-Lymphocytes immunology MeSH
- Binding Sites immunology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Pathogenesis of amyotrophic lateral sclerosis (ALS) involves several mechanisms resulting in a shift from a neuroprotective to a neurotoxic immune reaction. A promising tool for ALS treatment is represented by mesenchymal stem cells (MSCs), which possess both regenerative potential and immunomodulatory properties. In this study, we aimed to compare the immunomodulatory properties of MSCs isolated from the bone marrow of patients suffering from ALS and healthy donors. Moreover, the influence of proinflammatory cytokines on the immunoregulatory functions of MSCs was also evaluated. We found that MSCs from ALS patients and healthy donors comparably affected mitogen-stimulated peripheral blood mononuclear cells and reduced the percentage of T helper (Th)1, Th17 and CD8+CD25+ lymphocytes. These MSCs also equally increased the percentage of Th2 and CD4+FOXP3+ T lymphocytes. On the other hand, MSCs from ALS patients decreased more strongly the production of tumour necrosis factor-α than MSCs from healthy donors, but this difference was abrogated in the case of MSCs stimulated with cytokines. Significant differences between cytokine-treated MSCs from ALS patients and healthy donors were detected in the effects on the percentage of CD8+CD25+ and CD4+FOXP3+ T lymphocytes. In general, treatment of MSCs with cytokines results in a potentiation of their effects, but in the case of MSCs from ALS patients, it causes stagnation or even restriction of some of their immunomodulatory properties. We conclude that MSCs from ALS patients exert comparable immunomodulatory effects to MSCs from healthy donors, but respond differently to stimulation with proinflammatory cytokines. Graphical Abstract Treatment of mesenchymal stem cells (MSCs) with cytokines results in a potentiation of their effects, but in the case of MSCs from amyotrophic lateral sclerosis (ALS) patients, it causes stagnation (an equal reduction of the percentage of CD8+CD25+ T lymphocytes) or even restriction (no increase of proportion of CD4+FOXP3+ T lymphocytes) of some of their immunomodulatory properties. It means that MSCs from ALS patients exert comparable immunomodulatory effects to MSCs from healthy donors, but respond differently to stimulation with proinflammatory cytokines.
- MeSH
- Amyotrophic Lateral Sclerosis immunology MeSH
- Bone Marrow Cells immunology MeSH
- Cytokines metabolism MeSH
- Immunologic Factors pharmacology MeSH
- Immunomodulation MeSH
- Leukocytes, Mononuclear drug effects immunology MeSH
- Middle Aged MeSH
- Humans MeSH
- Mesenchymal Stem Cells immunology MeSH
- Mitogens pharmacology MeSH
- T-Lymphocytes, Helper-Inducer drug effects immunology MeSH
- Tumor Necrosis Factor-alpha biosynthesis MeSH
- Healthy Volunteers MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Regulation of μ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express μ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both μ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of μ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized μ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of μ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gβ subunits were unchanged. The level of β-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and β-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.
- MeSH
- Concanavalin A pharmacology MeSH
- Rats MeSH
- Lymphocytes drug effects metabolism MeSH
- Mitogens pharmacology MeSH
- Rats, Wistar MeSH
- Receptors, Opioid, delta biosynthesis drug effects MeSH
- Receptors, Opioid, kappa biosynthesis drug effects MeSH
- Receptors, Opioid, mu biosynthesis drug effects MeSH
- Spleen cytology drug effects metabolism MeSH
- Up-Regulation MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Keywords
- paměťový T lymfocyt,
- MeSH
- Lymphocyte Activation * immunology MeSH
- Hypersensitivity * diagnosis classification MeSH
- Rubber adverse effects MeSH
- Immunologic Tests classification MeSH
- Insulin administration & dosage MeSH
- Metals classification adverse effects MeSH
- Humans MeSH
- Mitogens MeSH
- Aged MeSH
- Sensitivity and Specificity MeSH
- T-Lymphocytes MeSH
- Dental Amalgam adverse effects MeSH
- Check Tag
- Humans MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
Germ-free animals have been used to define the vital role of commensal bacteria on the maturation of the host immune system. However, the role of bacterial residues in diet in this setting is poorly understood. Here we investigated the effect of bacterial contamination in sterile diet on the level of allergic sensitization in germ-free mice. Sterile grain-based diets ST1 and R03 were tested for the level of bacterial contamination. ST1 contained higher amount of bacterial DNA, approximately ten times more endotoxin, and induced higher, TLR4-dependent, cytokine production in dendritic cells compared to R03. In a germ-free mouse model of sensitization to the major birch pollen allergen Bet v 1, feeding on ST1 for at least two generations was associated with decreased production of allergen-specific IgE and IgG1 antibodies in sera in comparison to R03. Furthermore, reduced levels of allergen-specific and ConA-induced cytokines IL-4, IL-5 and IL-13 accompanied by increased levels of IFN-γ were detected in splenocytes cultures of these mice. Our results show that contamination of experimental diet with bacterial residues, such as endotoxin, significantly affects the development of allergic sensitization in germ-free mice. Therefore, careful selection of sterile food is critical for the outcomes of germ-free or gnotobiotic experimental models of immune-deviated diseases.
- MeSH
- Hypersensitivity immunology pathology MeSH
- Antigens, Plant immunology MeSH
- Cell Differentiation drug effects MeSH
- Breeding MeSH
- Cytokines biosynthesis MeSH
- Dendritic Cells drug effects MeSH
- Diet * MeSH
- DNA, Bacterial analysis MeSH
- Endotoxins toxicity MeSH
- Epitopes immunology MeSH
- Germ-Free Life MeSH
- HEK293 Cells MeSH
- Immunization * MeSH
- Immunoglobulin A immunology MeSH
- Immunoglobulin G immunology MeSH
- DNA Contamination MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Ligands MeSH
- Mitogens pharmacology MeSH
- Mice, Inbred BALB C MeSH
- Spleen pathology MeSH
- Toll-Like Receptor 4 metabolism MeSH
- Digestive System immunology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Chronic periodontitis (CP) is an inflammatory disease of the teeth-supporting tissues in which genetic predisposition, dental plaque bacteria, and immune mechanisms all play important roles. The aim of this study was to evaluate the occurrence of IL-4 gene polymorphisms in chronic periodontitis and to investigate the association between polymorphisms and cytokines production after bacterial stimulation. Sixty-two subjects (47 CP patients and 15 healthy controls) with detected two polymorphisms in the IL-4 gene (-590C/T and intron 3 VNTR) were examined. Production of cytokines (IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17, TNFα, INFγ, and VEGF) was studied after in vitro stimulation of isolated peripheral blood by mitogens (Pokeweed mitogen, Concanavalin A), dental plaque bacteria (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Prevotella intermedia), and Heat Shock Protein (HSP) 70 by the Luminex multiplex cytokine analysis system. The results were correlated with IL-4 genotypes in patients with CP and healthy controls. The mononuclear cells isolated from peripheral blood of CP patients with selected IL-4 polymorphisms significantly altered the production of IFNγ, IL-10, IL-1β, IL-1α, TNFα, and IL-6 after stimulation by HSP 70 or selected bacteria (from P < 0.001 to P < 0.05). IL-4 gene polymorphisms may influence the function of mononuclear cells to produce not only interleukin-4 but also other cytokines, especially in patients with CP.
- MeSH
- Aggregatibacter actinomycetemcomitans MeSH
- Chronic Disease MeSH
- Cytokines metabolism MeSH
- Adult MeSH
- Genotype MeSH
- Immune System MeSH
- Interleukin-4 genetics metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- Mitogens chemistry MeSH
- Periodontitis metabolism MeSH
- Polymorphism, Genetic * MeSH
- Porphyromonas gingivalis MeSH
- Prevotella intermedia MeSH
- Sequence Analysis, DNA MeSH
- Aged MeSH
- Case-Control Studies MeSH
- Inflammation MeSH
- Dental Plaque microbiology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Administration, Oral * MeSH
- Biomedical Research * trends MeSH
- Chitosan therapeutic use MeSH
- Insulin * administration & dosage adverse effects therapeutic use MeSH
- Humans MeSH
- Meta-Analysis as Topic MeSH
- Mitogens adverse effects MeSH
- Mitosis physiology drug effects MeSH
- Nanotechnology methods trends MeSH
- Intestinal Mucosa metabolism drug effects MeSH
- Drug Administration Routes MeSH
- Check Tag
- Humans MeSH
OBJECTIVES: We studied a) mitogen lectin (PHA) evoked changes of Na+/K+-ATPase activity in functionally different lymphocytes or brain cortex cells and b) quantitative relationship between PHA- evoked early enzyme activation and late lymphocyte proliferation were analyzed. MATERIALS AND METHODS: We performed biochemical analyses of Pi released from ATP by Na+/K+-ATPase activity. Lymphocyte proliferation was assayed by 3H-thymidine incorporation. RESULTS: We demonstrated PHA stimulated Na+/K+-ATPase activity of mouse spleen lymphocytes or freshly isolated brain cortex cells. Besides this, we estimated high stimulation of Na+/K+-ATPase activity and subsequent late 3H-thymidine incorporation into pig lymphocytes as both PHA dose and K+ ion concentration dependent. CONCLUSIONS: Thus, early PHA dose-dependent stimulation of Na+/K+-ATPase activity is a more general response in different animal species and functionally different cells. We measured both cell type- and PHA-dose dependent enzyme activity stimulation. We can suggest that intensity of early PHA induced Na+/K+-ATPase activation could be in relationship to subsequent elevated level of T lymphocyte proliferation. The Na+/K+-ATPase can be a part of mitogen lectin evoked signal transduction mechanisms.
- MeSH
- Lymphocyte Activation drug effects MeSH
- Cell Culture Techniques MeSH
- Potassium pharmacology MeSH
- Phytohemagglutinins pharmacology MeSH
- Mitogens pharmacology MeSH
- Cerebral Cortex drug effects metabolism MeSH
- Mice, Inbred CBA MeSH
- Mice MeSH
- Swine MeSH
- Spleen drug effects metabolism MeSH
- Sodium-Potassium-Exchanging ATPase metabolism MeSH
- T-Lymphocytes metabolism MeSH
- Thymus Gland drug effects metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The Th1/Th2 dysbalance plays an important role in the pathogenesis of human allergy.. The study was performed on 18 allergic patients, 5 healthy patients without any clinical history served as negative controls. Characterization of T cell response based on cytokine production was studied in whole blood ex vivo stimulation. IL-2, IL-4, IL-5, IL-10, TNF-alpha and IFN- cytokine response both spontaneous and to mitogens PHA and ConA was done by multiplexed ALBIA (Adressable Laser Bead Immunoaasays) with CBA Human Th1/Th2 Cytokine Kit (BD Bioscience). Compared with controls patients suffering from allergy had higher spontaneous production of IL 4 and higher TNFalpha and IL 10 production both spontaneous and after PHA and ConA stimulation. Multiplex technique becomes more complex and can quantify and detect dynamic changes in cytokine profiles. High effectivity and a good price make the method acceptable for cytokine assessment in laboratory routine.
- Keywords
- multiplexová analýza,
- MeSH
- Hypersensitivity MeSH
- Cytokines analysis MeSH
- Humans MeSH
- Mitogens MeSH
- Th1-Th2 Balance MeSH
- Check Tag
- Humans MeSH
