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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 -
Scientific Reports Aug 2022Patterns of human immune responses to viruses and bacteria and how this impacts risk of infections or onset/exacerbation of chronic respiratory diseases are poorly...
Patterns of human immune responses to viruses and bacteria and how this impacts risk of infections or onset/exacerbation of chronic respiratory diseases are poorly understood. In a population-based birth cohort, we measured peripheral blood mononuclear cell responses (28 cytokines) to respiratory viruses and bacteria, Toll-like receptor ligands and phytohemagglutinin, in 307 children. Cytokine responses were highly variable with > 1000-fold differences between children. Machine learning revealed clear distinction between virus-associated and bacteria-associated stimuli. Cytokines clustered into three functional groups (anti-viral, pro-inflammatory and T-cell derived). To investigate mechanisms potentially explaining such variable responses, we investigated cytokine Quantitative Trait Loci (cQTLs) of IL-6 responses to bacteria and identified nine (eight novel) loci. Our integrative approach describing stimuli, cytokines and children as variables revealed robust immunologically and microbiologically plausible clustering, providing a framework for a greater understanding of host-responses to infection, including novel genetic associations with respiratory disease.
Topics: Bacteria; Child; Cytokines; Humans; Leukocytes, Mononuclear; Toll-Like Receptors; Viruses
PubMed: 35931775
DOI: 10.1038/s41598-022-16509-4 -
Molecules (Basel, Switzerland) Nov 2022Our recent study has shown that pomegranate peel extract (PEx) showed significant immunomodulatory activity, which might be caused by ellagitannins. The aim of this work...
BACKGROUND
Our recent study has shown that pomegranate peel extract (PEx) showed significant immunomodulatory activity, which might be caused by ellagitannins. The aim of this work was to test the hypothesis that ellagitannin components act synergistically in the modulation of cytokine production.
METHODS
Human peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with phytohemagglutinin and treated with different concentrations of PEx or punicalagin (PG), punicalin (PN), and ellagic acid (EA), alone or with their combinations. Cytotoxicity, cell proliferation, and cytokine production were determined.
RESULTS
Non-cytotoxic concentrations of all compounds significantly inhibited cell proliferation. IC50 values (μg/mL) were: EA (7.56), PG (38.52), PEx (49.05), and PN (69.95). PEx and all ellagitannins inhibited the levels of TNF-α, IL-6, and IL-8, dose-dependently, and their combinations acted synergistically. PEx and all ellagitannins inhibited Th1 and Th17 responses, whereas the lower concentrations of PEx stimulated the production of IL-10, a Treg cytokine, as did lower concentrations of EA. However, neither component of ellagitannins increased Th2 response, as was observed with PEx.
CONCLUSIONS
The combination of PG, PN, and EA potentiated the anti-inflammatory response without any significant synergistic down-modulatory effect on T-cell cytokines. The increased production of IL-10 observed with PEx could be attributable to EA, but the examined ellagitannins are not associated with the stimulatory effect of PEx on Th2 response.
Topics: Humans; Hydrolyzable Tannins; Ellagic Acid; Interleukin-10; Pomegranate; Lythraceae; Leukocytes, Mononuclear; Plant Extracts; Cytokines
PubMed: 36431972
DOI: 10.3390/molecules27227871 -
Clinical and Experimental Immunology Oct 2019Hepatic stellate cells (HSCs) have a central role in liver inflammation and fibrosis by producing inflammatory and fibrotic mediators. Their activation is regulated...
Hepatic stellate cells (HSCs) have a central role in liver inflammation and fibrosis by producing inflammatory and fibrotic mediators. Their activation is regulated through direct cell-cell interactions, but also through systemic and local effects of soluble factors such as cytokines. The effects of the proinflammatory cytokines interleukin (IL)-17 and tumor necrosis factor (TNF)-α and cell interactions with hepatocytes on HSC activation were assessed. Human HSC and HepaRG cells were exposed to IL-17 and/or TNF-α. IL-17 and TNF-α contribution from immune cells was determined in a co-culture model with phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC), HSC and/or hepatocytes. IL-17 enhanced TNF-α effects on the induction of IL-6, IL-1β, and the chemokine IL-8, chemokine (C-C motif) ligand 20 (CCL20) and monocyte chemoattractant protein-1 (MCP-1) expression/secretion in isolated HSC cultures. HSC-hepatocyte interactions did not enhance IL-6, IL-8 and CCL20 production compared to hepatocyte alone. However, HSC-hepatocyte interactions increased C-reactive protein expression. IL-17 and/or TNF-α had no direct profibrotic effects on collagen 1 α1, tissue inhibitor of matrix metalloproteinase (TIMP) and matrix metalloproteinase (MMP) 2 gene expression, whereas mRNA levels of MMP3, an enzyme involved in matrix destruction, were up-regulated in HSCs. The use of specific inhibitors of IL-17 and TNF-α indicated their contribution to the strong increase of IL-6 and IL-8 production induced by PBMC, HSC and/or hepatocyte interactions. As chronic liver inflammation leads to liver fibrosis, IL-17 and/or TNF-α neutralization can be of interest to control liver inflammation and therefore its effects on fibrosis.
Topics: Cells, Cultured; Chemokine CCL2; Chemokine CCL20; Collagen Type I; Cytokines; Hepatic Stellate Cells; Hepatocytes; Humans; Inflammation; Interleukin-17; Leukocytes, Mononuclear; Liver; Liver Cirrhosis; Tissue Inhibitor of Metalloproteinase-1; Tumor Necrosis Factor-alpha
PubMed: 31102558
DOI: 10.1111/cei.13316 -
Frontiers in Endocrinology 2023The retinoic acid-related orphan receptor alpha (RORα) protein first came into the limelight due to a set of staggerer mice, discovered at the Jackson Laboratories in... (Review)
Review
The retinoic acid-related orphan receptor alpha (RORα) protein first came into the limelight due to a set of staggerer mice, discovered at the Jackson Laboratories in the United States of America by Sidman, Lane, and Dickie (1962) and genetically deciphered by Hamilton et al. in 1996. These staggerer mice exhibited cerebellar defects, an ataxic gait, a stagger along with several other developmental abnormalities, compensatory mechanisms, and, most importantly, a deletion of 160 kilobases (kb), encompassing the ligand binding domain (LBD). The discovery of the staggerer mice and the subsequent discovery of a loss of the LBD within the gene of these mice at the genetic level clearly indicated that RORα's LBD played a crucial role in patterning during embryogenesis. Moreover, a chance study by Roffler-Tarlov and Sidman (1978) noted reduced concentrations of glutamic acid levels in the staggerer mice, indicating a possible role for the essence of a nutritionally balanced diet. The sequential organisation of the building blocks of intact genes, requires the nucleotide bases of deoxyribonucleic acid (DNA): purines and pyrimidines, both of which are synthesized, upon a constant supply of glutamine, an amino acid fortified in a balanced diet and a byproduct of the carbohydrate and lipid metabolic pathways. A nutritionally balanced diet, along with a metabolic "enzymatic machinery" devoid of mutations/aberrations, was essential in the uninterrupted transcription of RORα during embryogenesis. In addition to the above, following translation, a ligand-responsive RORα acts as a "molecular circadian regulator" during embryogenesis and not only is expressed selectively and differentially, but also promotes differential activity depending on the anatomical and pathological site of its expression. RORα is highly expressed in the central nervous system (CNS) and the endocrine organs. Additionally, and the genes are core components of the circadian rhythmicity, with the expression of fluctuating in a night-day-night sigmoidal pattern and undoubtedly serves as an endocrine-like, albeit "molecular-circadian regulator". Melatonin, a circadian hormone, along with tri-iodothyronine and some steroid hormones are known to regulate RORα-mediated molecular activity, with each of these hormones themselves being regulated rhythmically by the hypothalamic-pituitary axis (HPA). The HPA regulates the circadian rhythm and cyclical release of hormones, in a self-regulatory feedback loop. Irregular sleep-wake patterns affect circadian rhythmicity and the ability of the immune system to withstand infections. The staggerer mice with their thinner bones, an altered skeletal musculature, an aberrant metabolic profile, the ataxic gait and an underdeveloped cerebellar cortex; exhibited compensatory mechanisms, that not only allowed the survival of the staggerer mice, but also enhanced protection from microbial invasions and resistance to high-fat-diet induced obesity. This review has been compiled in its present form, more than 14 years later after a chromatin immunoprecipitation (ChIP) cloning and sequencing methodology helped me identify signal transducer and activator of transcription 5 (STAT5) target sequences, one of which was mapped to the first intron of the gene. The 599-base-long sequence containing one consensus TTCNNNGAA (TTCNGAA) gamma-activated sequence (GAS) and five other non-consensus TTNAA sequences had been identified from the clones isolated from the STAT5 target sites (fragments) in human phytohemagglutinin-activated CD8+ T lymphocytes, during my doctoral studies between 2006 and 2009. Most importantly, preliminary studies noted a unique expression profile, during a time-course study on the ribonucleic acid (RNA), extracted from human phytohemagglutinin (PHA) activated CD8+ T lymphocytes stimulated with interleukin-2 (IL-2). This review mainly focuses on the "staggerer mice" with one of its first roles materialising during embryogenesis, a molecular-endocrine mediated circadian-like regulatory process.
Topics: Animals; Nuclear Receptor Subfamily 1, Group F, Member 1; Mice; Mice, Neurologic Mutants; Humans
PubMed: 38766309
DOI: 10.3389/fendo.2023.1300729 -
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 -
Journal of Veterinary Research Jun 2022Ochratoxin A (OTA) is a mycotoxin notably produced by and spp. fermentation extract (BSFE) contains specific enzymes which hydrolyse OTA. This study evaluated the...
INTRODUCTION
Ochratoxin A (OTA) is a mycotoxin notably produced by and spp. fermentation extract (BSFE) contains specific enzymes which hydrolyse OTA. This study evaluated the efficiency of BSFE in ameliorating the immunotoxic and nephrotoxic effects of OTA in broiler chickens.
MATERIAL AND METHODS
Day-old broiler chicks were divided equally into four groups of ten: control, OTA (0.5 mg/kg feed), BSFE product (1 mL/L water) and OTA + BSFE at the same concentrations. The chicks were vaccinated against avian influenza, Newcastle disease, and infectious bronchitis, and lymphoproliferation was induced in all birds by phytohaemagglutinin-P (PHA-P). Serum samples were taken before sacrifice and organ tissue samples were taken after, in which renal function biomarkers were assayed and the presence of OTA residue was evaluated by high-performance thin-layer chromatography. Protein markers of apoptosis were determined by qPCR, and tissue lesions were examined histopathologically.
RESULTS
Exposure to OTA significantly decreased the antibody response to the vaccines and the lymphoproliferative response to PHA-P, and significantly elevated the renal function indicators: serum urea, uric acid and creatinine. It also induced oxidative stress (reduced catalase activity and glutathione concentration), lipid peroxidation (increased malondialdehyde content), apoptosis (increased and and decreased gene levels) and pathological lesions in kidney, bursa of Fabricius, spleen and thymus tissue. Residues of OTA were detected in the serum and tissue. BSFE mitigated most of these toxic effects.
CONCLUSION
BSFE counters OTA-induced immunotoxicity and nephrotoxicity because of its content of carboxypeptidase and protease enzymes.
PubMed: 35892096
DOI: 10.2478/jvetres-2022-0030 -
Journal of Obstetrics and Gynaecology... Apr 2022To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates. (Randomized Controlled Trial)
Randomized Controlled Trial
OBJECTIVE
To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates.
METHODS
This prospective double-blind randomized parallel clinical trial included 213 patients undergoing IUI at the Fertilys clinic. PBMC were isolated on the day of ovulation (day 0; D0) and stimulated with phytohemagglutinin (PHA) and human chorionic gonadotropin (hCG) for 48 hours (day 2; D2). Patients in the PBMC group (n = 108) underwent in utero administration of 1.10 cells on D2, while patients in the control group (n = 105) were administered sperm-washing medium. Distribution of CD4 T lymphocyte populations (n = 61) was assessed on D0 and D2. Pregnancy and live birth rates were also evaluated.
RESULTS
Demographic and clinical characteristics, pregnancy rates, and live birth rates were not significantly different between the PBMC and control groups. Significantly higher levels of T helper (Th) 2, Th22, and T regulatory cells (P < 0.0001) and lower levels of Th17 cells were observed in hCG-activated PBMC at D2 than at D0.
CONCLUSION
Intrauterine administration of PBMC was not beneficial in IUI patients. New clinical approaches to better identify patients requiring endometrium immunomodulation needs to be addressed.
Topics: Chorionic Gonadotropin; Female; Fertilization in Vitro; Humans; Insemination; Leukocytes, Mononuclear; Male; Ovulation Induction; Pregnancy; Pregnancy Rate; Prospective Studies
PubMed: 34848351
DOI: 10.1016/j.jogc.2021.11.010 -
Frontiers in Veterinary Science 2022Silver nanoparticles (AgNPs) interact with the microbes and host immune system to protect against diseases. Fertile broiler eggs ( = 900) were allotted to six groups:...
Silver nanoparticles (AgNPs) interact with the microbes and host immune system to protect against diseases. Fertile broiler eggs ( = 900) were allotted to six groups: un-injected control, sham (sterile water), AgNPs (50 μg), AgNPs+Amino acids (Methionine-10 mg + Arginine-25 mg), AgNPs+Vitamins (Vit B1-72μg + Vit B6-140μg), and AgNPs+Trace Elements (Zn-80 μg and Se-0.3 μg) and incubated for 18 days. On 18th embryonic day, 0.6 ml test solution was injected at the broad end of egg using 25 mm needle and transferred to hatcher. Post-hatch, half of the chicks from each group were vaccinated with Newcastle disease (ND) vaccine, and the other half were kept as unvaccinated unit and reared for 42 d with standard management practices. Hatchability, 1st and 42nd d body weight, feed intake, and feed conversion ratio were similar between treatment groups in both vaccinated and unvaccinated units. The relative weight of bursa Fabricius and thymus was similar, but spleen weight was higher ( ≤ 0.05) in AgNPs, AgNPs+Vits, and AgNPs+TEs chicks than control group. Cellular immune response (against mitogen phytohemagglutinin-P) was higher ( ≤ 0.05) in AgNPs+TEs chicks, whereas HA titer against sheep red blood cells antigen, serum IgG, IgM, and HI titer against ND vaccine was apparently higher in AgNPs+Vits group chicks than control. No clinical symptoms were observed in the vaccinated groups except for a few control birds 6 days postchallenge (PC). Three days PC, unvaccinated birds show depression, off feed, greenish diarrhea, and nasal discharge and the control group started dying. The highest cumulative infection (CI) was observed in sham (79.17%) and un-injected control (75%), but lowest in AgNPs+AAs birds (58.33%) on 3rd dpi. The CI reached 100% on 5th dpi in control groups and AgNPs, and 91.67% and 93.75% in AgNPs+TEs and AgNPs+AAs group, respectively. The AgNPs+TEs and AgNPs+AAs group birds lived for more than 90 h compared to 75 h in control groups and also had higher IL-6 and IL-2 gene expressions at 24 h PC. It was concluded that 50 μg/egg AgNPs with vitamins (B1 and B6) and trace elements (Zn and Se) improved performance, but AgNPs with trace elements and amino acids enhanced immune response and resistance against vND virus challenge in broilers.
PubMed: 36187823
DOI: 10.3389/fvets.2022.948069