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Biotechnology Letters Dec 2019Although several methods have been reported and used for in vitro T cell amplification, there are no consistent reports on the optimal stimulation conditions and the... (Comparative Study)
Comparative Study
OBJECTIVE
Although several methods have been reported and used for in vitro T cell amplification, there are no consistent reports on the optimal stimulation conditions and the characterization of these stimulated T cells. The current study aimed to determine the optimal conditions for efficient T cell amplification by two commonly used methods involving CD3/CD28 antibody and phytohemagglutinin (PHA), respectively.
RESULTS
Orthogonal design and CCK8 assay showed that 5 μg/mL CD3, 5 μg/mL CD28, and 100 ng/mL IL2 for the first method and 50 μg/mL PHA for the second method was optimal for T cell stimulation. Flow cytometry demonstrated that the percentage of CD8 in the stimulated groups significantly increased, while the percentage of CD4/CD8 was significantly decreased compared with the unstimulated group. The percentage of CD4 showed no significant difference among the three groups. Notably, there was no significant difference between the two stimulated groups. In addition, the percentage of apoptotic cells was significantly increased in the stimulated groups compared with the unstimulated group, but showed no remarkable difference between the PHA and CD3/CD28 stimulation groups. Glycolysis analysis showed that the glycolytic capacity and glycolytic reserve were both significantly increased in the PHA and CD3/CD28 groups compared with the unstimulated group, with no significant difference noted between the stimulated groups.
CONCLUSIONS
Although both stimulation methods showed similar efficacies, we suggest the PHA method might be better considering its easy application and cost-effective nature.
Topics: Antibodies; CD28 Antigens; CD3 Complex; Cell Proliferation; Cells, Cultured; Cytological Techniques; Flow Cytometry; Healthy Volunteers; Humans; Lymphocyte Activation; Phytohemagglutinins; T-Lymphocytes
PubMed: 31631231
DOI: 10.1007/s10529-019-02743-w -
Ecology Letters Oct 2019Immunosenescence, the decline in immune defense with age, is an important mortality source in elderly humans but little is known of immunosenescence in wild animals. We... (Meta-Analysis)
Meta-Analysis Review
Immunosenescence, the decline in immune defense with age, is an important mortality source in elderly humans but little is known of immunosenescence in wild animals. We systematically reviewed and meta-analysed evidence for age-related changes in immunity in captive and free-living populations of wild species (321 effect sizes in 62 studies across 44 species of mammals, birds and reptiles). As in humans, senescence was more evident in adaptive (acquired) than innate immune functions. Declines were evident for cell function (antibody response), the relative abundance of naïve immune cells and an in vivo measure of overall immune responsiveness (local response to phytohaemagglutinin injection). Inflammatory markers increased with age, similar to chronic inflammation associated with human immunosenescence. Comparisons across taxa and captive vs free-living animals were difficult due to lack of overlap in parameters and species measured. Most studies are cross-sectional, which yields biased estimates of age-effects when immune function co-varies with survival. We therefore suggest longitudinal sampling approaches, and highlight techniques from human cohort studies that can be incorporated into ecological research. We also identify avenues to address predictions from evolutionary theory and the contribution of immunosenescence to age-related increases in disease susceptibility and mortality.
Topics: Aging; Animals; Birds; Cross-Sectional Studies; Immunosenescence; Inflammation; Mammals; Reptiles
PubMed: 31321874
DOI: 10.1111/ele.13343 -
Journal of Molecular Endocrinology Jun 2021Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds,...
Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds, and to verify whether they can reduce the body weight gain and investigate their molecular mechanisms. By using drug-screening methods, Phytohemagglutinin (PHA) was found to be the most anti-obesity candidate natural compound. Six-week-old C57BL/6J mice were fed with a high-fat diet (HFD) and intraperitoneally injected with 0.25 mg/kg PHA everyday for 8 weeks. The body weight, glucose homeostasis, oxygen consumption and physical activity were assessed. We also measured the heat intensity, body temperature and the gene expression of key regulators of energy expenditure. Prevention study results showed PHA treatment not only reduced the body weight gain but also maintained glucose homeostasis in HFD-fed mice. Further study indicated energy expenditure and uncoupling protein 1 (UCP-1) expression of brown adipose tissue (BAT) and white adipose tissue (WAT) in HFD-fed mice were significantly improved by PHA. In the therapeutic study, a similar effect was observed. PHA inhibited lipid droplet formation and upregulated mitochondrial-related gene expression during adipogenesis in vitro. UCP-1 KO mice displayed no differences in body weight, glucose homeostasis and core body temperature between PHA and control groups. Our results suggest that PHA prevent and treat obesity by increasing energy expenditure through upregulation of BAT thermogenesis.
Topics: Adipose Tissue, Brown; Adipose Tissue, White; Animals; Biological Products; Cell Differentiation; Diet, High-Fat; Energy Metabolism; Glucose; Homeostasis; Male; Mice, Inbred C57BL; Mice, Knockout; Obesity; Phytohemagglutinins; Thermogenesis; Uncoupling Protein 1; Weight Gain; Mice
PubMed: 33983894
DOI: 10.1530/JME-20-0349 -
Journal of Clinical and Translational... Jan 2020Whole blood is processed to derive a red cell concentrate, plasma, and buffy coat (BC) (from which platelets can be further extracted). Unused plasma and BCs are common... (Review)
Review
BACKGROUND AND AIM
Whole blood is processed to derive a red cell concentrate, plasma, and buffy coat (BC) (from which platelets can be further extracted). Unused plasma and BCs are common in most blood establishments and considered a liability. The redirection of these products to xeno-free applications is not complicated or time-consuming and cannot only benefit the research recipients but also the blood establishment suppliers interested in research collaboration. The aim of this study is to describe a diverse yet by no means an exhaustive list of options for preparing blood products for research applications.
MATERIALS AND METHODS
Plasma and BCs from healthy donors were processed using basic laboratory techniques under aseptic conditions and tested for their ability to support the culture of mesenchymal stem cells in both 2D and 3D cultures using fibrin clots. The white blood cells (WBC) from the BCs were induced by phytohemagglutinin and CD marker expression was monitored using quantitative polymerase chain reaction.
RESULTS
All the methods tested for preparing blood products were successful but the applicability to different settings varied greatly with the most successful being the supplementation of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 with 20% cryodepleted plasma and 0.1 mg/mL platelet lysate, the formation of fibrin clots using a ratio of 3:1 (medium: plasma) and the culturing of WBCs with 5 µg/mL phytohemagglutinin.
CONCLUSIONS
Using the wastes and by-products of blood establishments for xeno-free cell culturing of stem cells will reduce the reliance on commercially available, ready-made products, and increasing the potential for therapeutic stem cell research. Despite the benefits presented both in terms of cost and applications, further characterization and optimization of each blood product for reproducibility of results is required.
RELEVANCE FOR PATIENTS
The availability of low-cost xeno-free reagents will speed up therapeutic stem cell research and allow patients to receive treatments of the expected high standards at lower costs.
PubMed: 32377579
DOI: No ID Found -
Veterinary Immunology and... Jan 2022Ferrets are nowadays frequently used as animal models for biomedical purposes; in many cases, immunosuppression of experimental animals is necessary. The aim of this...
Ferrets are nowadays frequently used as animal models for biomedical purposes; in many cases, immunosuppression of experimental animals is necessary. The aim of this study was to evaluate the effect of intramuscular dexamethasone administration (2 mg/kg as the initiation dose continued with 1 mg/kg q 12 h applied 5 times) on ferret's immune system. In comparison with ferrets which received the saline (n = 5), significantly lower total counts of leukocytes (P < 0.01), lymphocytes (P < 0.01) and monocyte (P < 0.05), as well as absolute numbers of CD4+CD8- (P < 0.01) and CD4-CD8+ (P < 0.01) subsets were noted in dexamethasone treated ferrets (n = 5) the first day after the treatment (D1). Absolute number of CD79+ lymphocytes remained unchanged throughout the experiment. The proliferation activity of lymphocytes in dexamethasone treated ferrets was lower only in D1 using concanavalin A (conA), phytohemagglutinin (PHA) and pokeweed mitogen (PWM); statistical significance was noted using PHA 40 (P < 0.05) and PWM 10 (P < 0.01). Lower neutrophil activity (P < 0.01) was detected in D1 after the dexamethasone treatment in both production of reactive oxygen species (chemiluminescence test) and ingestion of particles (phagocytosis assay). The dexamethasone treatment proved to be useful for short-term immunosuppression in ferrets. The results closely resembled data previously reported in human studies and indicate classification of ferrets as steroid-resistant species.
Topics: Animals; Dexamethasone; Ferrets; Immunosuppression Therapy; Lymphocyte Activation; Models, Animal; Phytohemagglutinins; Pokeweed Mitogens
PubMed: 34826685
DOI: 10.1016/j.vetimm.2021.110362 -
Journal of Virus Eradication Apr 2017Latently infected resting CD4 T cells represent a major barrier to HIV-1 eradication efforts. The standard assays used for measuring this reservoir induce activation of...
AIMS
Latently infected resting CD4 T cells represent a major barrier to HIV-1 eradication efforts. The standard assays used for measuring this reservoir induce activation of resting CD4 T cells with either phytohaemagglutinin (PHA) with irradiated feeder cells, or with anti-CD3 antibodies. We designed a study to compare the sensitivity of a new assay (based on the stimulation of CD4 T cells with anti-CD3 and anti-CD28 coated microbeads) with that of the traditional PHA- and feeder-based viral outgrowth assay.
METHODS
Resting CD4 T cells from 10 HIV-1-infected patients on suppressive combination antiretroviral therapy (cART) regimens were cultured in the traditional PHA/feeders viral outgrowth assay and the new CD3/CD28 bead-based assay. Flow cytometry was used to assess the kinetics of activation of resting CD4 T cells in the two different assays.
RESULTS
There was no significant difference in the sensitivity of the two assays. The median frequency of latently infected cells was 0.83 infectious units per million (IUPM) for the PHA/feeders assay and 0.54 IUPM with the CD3/CD28 bead-based assay. However, while virus was obtained from all 10 patients with the traditional PHA/feeders outgrowth assay, no virus was obtained from two of 10 patients with the novel anti-CD3/CD28 bead-based viral outgrowth assay (IUPM < 0.02).
CONCLUSION
The new CD3/CD28 bead-based assay has comparable sensitivity to the PHA/feeders assay and does not require the addition of feeders, making it a simpler and less labour-intensive alternative to the standard PHA/feeders-based assay.
PubMed: 28435692
DOI: No ID Found -
Iranian Journal of Immunology : IJI Sep 2017Brucella is a well-known intracellular bacterium entailing acute and chronic illnesses in humans and domestic animals. The infection chronicity may be affected by the... (Comparative Study)
Comparative Study
BACKGROUND
Brucella is a well-known intracellular bacterium entailing acute and chronic illnesses in humans and domestic animals. The infection chronicity may be affected by the cell-mediated immunity and cytokine patterns.
OBJECTIVE
To evaluate the patterns of T-helper cytokines in patients suffering from chronic and acute brucellosis.
METHODS
In this cross-sectional study, 22 individuals with acute brucellosis, 21 individuals with chronic brucellosis, and 21 healthy individuals with the same genetic background were recruited from October 2015 to April 2016. Peripheral lymphocytes were isolated and stimulated by phytohemagglutinin (PHA) and brucella antigen in cell culture. The lymphocyte proliferation was detected by MTT assay. After collecting the supernatants, and through the use of ELISA method, we quantified the interferon gamma (IFN-γ), interleukin (IL)-5, IL-17 and transforming growth factor-beta (TGF-β).
RESULTS
Patients with chronic brucellosis had a lower level antigen-specific stimulation index compared to those suffering from acute brucellosis (p=0.0001). Cases with chronic brucellosis had a lower level of IFN-γ compared to cases with acute brucellosis (p=0.001). Finally, patients with chronic brucellosis had higher levels of IL-5 and TGF-β in comparison with the acute group (p=0.01 and p=0.04, respectively).
CONCLUSION
Chronic brucellosis reduces lymphocyte proliferation and TH1 cytokine secretion, but it enhances IL- 5 and TGF-β production. Polarizing the immune responses plays a crucial part in the progression and development of chronic diseases.
Topics: Acute Disease; Adult; Brucella; Brucellosis; Cell Proliferation; Cells, Cultured; Chronic Disease; Cross-Sectional Studies; Cytokines; Female; Humans; Immunity, Cellular; Interferon-gamma; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; Th1 Cells; Th1-Th2 Balance; Young Adult
PubMed: 28919584
DOI: No ID Found -
Foods (Basel, Switzerland) Nov 2020Lectins or carbohydrate-binding proteins are widely distributed in seeds and vegetative parts of edible plant species. A few lectins from different fruits and vegetables... (Review)
Review
Lectins or carbohydrate-binding proteins are widely distributed in seeds and vegetative parts of edible plant species. A few lectins from different fruits and vegetables have been identified as potential food allergens, including wheat agglutinin, hevein (Hev b 6.02) from the rubber tree and chitinases containing a hevein domain from different fruits and vegetables. However, other well-known lectins from legumes have been demonstrated to behave as potential food allergens taking into account their ability to specifically bind IgE from allergic patients, trigger the degranulation of sensitized basophils, and to elicit interleukin secretion in sensitized people. These allergens include members from the different families of higher plant lectins, including legume lectins, type II ribosome-inactivating proteins (RIP-II), wheat germ agglutinin (WGA), jacalin-related lectins, GNA ( agglutinin)-like lectins, and Nictaba-related lectins. Most of these potentially active lectin allergens belong to the group of seed storage proteins (legume lectins), pathogenesis-related protein family PR-3 comprising hevein and class I, II, IV, V, VI, and VII chitinases containing a hevein domain, and type II ribosome-inactivating proteins containing a ricin B-chain domain (RIP-II). In the present review, we present an exhaustive survey of both the structural organization and structural features responsible for the allergenic potency of lectins, with special reference to lectins from dietary plant species/tissues consumed in Western countries.
PubMed: 33255208
DOI: 10.3390/foods9121724 -
Tissue Engineering. Part A May 2021Mammalian platelets participate in the immediate tissue injury response by initiating coagulation and further promoting tissue injury mitigation and repair. The latter...
Mammalian platelets participate in the immediate tissue injury response by initiating coagulation and further promoting tissue injury mitigation and repair. The latter properties are deployed following platelet release of presynthetized morphogens, cytokines, and growth and chemotactic factors, which launch a tissue regenerative, angiogenic, and anti-inflammatory program. Several blood-derived biologic products, like platelet-rich plasma (PRP) and platelet lysate (PL), are currently on the market to allow proper healing and tissue regeneration. However, not all growth factors are released from the platelets and the final products contain plasma proteins such as albumin, fibrinogen, complement, and immunoglobulins, increasing the risks of serum sickness or allergic reaction. To address this problem, we developed a new platelet extract where equine blood platelets are concentrated, washed, and thereafter lysed by detergent Triton X-114. Distinct from PRP, this extract is devoid of albumin, fibrinogen, and immunoglobulins and is 266-fold enriched in platelet-derived growth factor content relative to PRP. Washed equine platelet extract (WEPLEX) is amenable to lyophilization without loss of biological activity. , WEPLEX significantly inhibits human and equine T cell proliferative response to phytohemagglutinin and also polarizes murine CD45/CD11b peritoneal macrophages to an IL-10 M2-like phenotype. , WEPLEX substantially improves clinical outcome of murine experimental dextran sulfate sodium colitis. We propose that equine-sourced, zoonosis-free WEPLEX may serve as an anti-inflammatory biological therapy across mammalian species.
Topics: Animals; Anti-Inflammatory Agents; Biological Products; Blood Platelets; Horses; Humans; Mice; Plant Extracts; Platelet-Rich Plasma
PubMed: 32854583
DOI: 10.1089/ten.TEA.2020.0160 -
Journal of Dairy Science Apr 2023We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon...
We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the "commensal" Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4 and CD8 subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T- or B-cell proliferation. Furthermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-γ production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multiparous cows also produced significantly more IL-17A and IFN-γ. In contrast to concanavalin A, phytohemagglutinin M-form selectively stimulated T-cell proliferation.
Topics: Female; Cattle; Animals; Phytohemagglutinins; Interleukin-17; Concanavalin A; Leukocytes, Mononuclear; Staphylococcus aureus; Staphylococcal Infections; Antibodies, Monoclonal; Cell Proliferation; Mastitis, Bovine; Milk; Cattle Diseases
PubMed: 36870844
DOI: 10.3168/jds.2022-22529