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International Journal of Biological... Aug 2021Inflammation is a common manifestation of body immunity and mediates a cascade of cytokines. Tumor necrosis factor-α (TNF-α), as a multi-effect cytokine, plays an...
Molecular characterization, expression analysis and function identification of Pf_TNF-α and its two receptors Pf_TNFR1 and Pf_TNFR2 in yellow catfish (Pelteobagrus fulvidraco).
Inflammation is a common manifestation of body immunity and mediates a cascade of cytokines. Tumor necrosis factor-α (TNF-α), as a multi-effect cytokine, plays an important role in the inflammatory response by interacting with its receptor (TNFR). In this study, Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 genes were cloned from yellow catfish (Pelteobagrus fulvidraco), and bioinformatics analyses showed that the three genes were conserved and possessed similar sequence characteristics as those of other vertebrates. The qPCR results showed that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 mRNAs were constitutively expressed in 14 tissues and the lymphocytes of four tissues from healthy adults. The mRNA expression levels of Pf_TNF-α and Pf_TNFR1 genes were significantly up-regulated in the spleen, liver, trunk kidney, head kidney and gill after Edwardsiella ictaluri infection, while the mRNA expression of Pf_TNFR2 was significantly up-regulated in the spleen, and down-regulated in the liver and gill. In the isolated peripheral blood leukocytes (PBLs) of yellow catfish, the expression of Pf_TNF-α mRNA was notably up-regulated and the two Pf_TNFR transcripts were distinctly down-regulated after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly I:C) and phytohaemagglutinin (PHA). After stimulated by recombinant (r) Pf_sTNF protein, the mRNA expressions of various inflammatory factors genes were up-regulated in the PBLs. Meanwhile, rPf_sTNF promoted the phagocytic activity of leukocytes, whereas the activity mediated by rPf_sTNF could be inhibited by rPf_TNFR1CRD2/3 and rPf_TNFR2CRD2/3. The up-regulation of TNF-α and IL-1β mRNAs expression triggered by rPf_sTNF could be inhibited by MAPK inhibitor (VX-702) and NF-κB inhibitor (PDTC). rPf_sTNF induced the expression of FADD mRNA in PBLs and increased the apoptotic rate of PBLs, and inhibiting the NF-κB and MAPK signal pathways could enhance the apoptosis of PBLs. The results indicate that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 play important roles in the immune response of yellow catfish to bacterial invasion.
Topics: Animals; Catfishes; Cloning, Molecular; Computational Biology; Female; Fish Proteins; Gene Expression Regulation; Lipopolysaccharides; Male; Organ Specificity; Peptidoglycan; Phylogeny; Phytohemagglutinins; Poly I-C; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha
PubMed: 34144067
DOI: 10.1016/j.ijbiomac.2021.06.090 -
Cytokine Jul 2021A single nucleotide polymorphism (SNP), 251 bases upstream from the IL-8 transcription start (-251A>T, rs4073), has been extensively investigated in cancers and...
A single nucleotide polymorphism (SNP), 251 bases upstream from the IL-8 transcription start (-251A>T, rs4073), has been extensively investigated in cancers and inflammatory and infectious diseases in predominantly European and Asian populations. We sequenced the IL-8 gene of 109 black and 32 white South African (SA) individuals and conducted detailed characterization of gene variation and haplotype structure. IL-8 production in phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) of a subset (black: N = 22; white: N = 32) of these individuals was measured using ELISA. Select variants were genotyped for additional black individuals (N = 141), and data from the 1000 Genomes Project were used for haplotype analysis and comparative purposes. In white individuals, the -251A>T SNP formed part of a prevalent six-variant haplotype [haplotype frequency (HF): 61%], Hap-1C, involving the following variants: -251A>T; +394T>G (rs2227307); +780C>T (rs2227306); +1240->A (rs2227541); +1635C>T (rs2227543) and +2770A>T (rs2227543). Hap-1C (-251T+394T+780C+1240+A+1635C+2770A) was composed of two three-variant sub-haplotypes [Hap-1Ca: -251T+394T+1240+A; Hap-1Cb: +780C+1635C+2770A) sharing similarities with haplotypes identified in the black population. Hap-1C was found to be present in European, East and South Asian populations. Four haplotypes were identified in the black population with the two prevalent haplotypes each comprised of two variants: Hap-1B [-251A>T and +1240->A; -251T+1240+A; HF: 14%] and Hap-2B [-743T>C (rs2227532) and +2452A>C (rs2227545); -743C+2452C; HF: 13%]. Populations did not differ in unstimulated PBMC IL-8 production. Upon PHA stimulation, PBMCs from white individuals produced more IL-8 (P = 0.04), suggesting the -251T allele is responsible for higher production, however further analysis revealed that Hap-1C (and constituent sub-haplotypes), did not associate with IL-8 production. Populations did however differ in monocyte number with the white population having significantly more monocytes compared to the black population (P = 0.025), and furthermore monocyte number strongly correlated with IL-8 production in both population groups (black: p = 0.0002, r = 0.71; white: P = 0.0005, r = 0.59). Hap-1B, Hap-2B, and a SNP located one base pair upstream of the IL-8 ATG start codon, +100C>T SNP (rs2227538), all associated with higher IL-8 production in the black population - individuals harbouring at least one of these haplotypes/variant associated with higher IL-8 production (P = 0.003) compared to individuals without. The black population was enriched for individuals harbouring Hap-1B and/or Hap-2B compared to the 1000 Genomes project sub-Saharan African population (P = 0.006), suggesting that SA black individuals may be high IL-8 producers. Given the paucity of IL-8-related studies that have been conducted in populations from sub-Saharan Africa, this study has significantly increased our understanding of this important chemokine in the South African population.
Topics: Adult; Africa South of the Sahara; Alleles; Black People; Ethnicity; Female; Gene Frequency; Genetic Variation; Genetics, Population; Haplotypes; Humans; Interleukin-8; Leukocytes, Mononuclear; Linkage Disequilibrium; Male; Middle Aged; Monocytes; Phytohemagglutinins; South Africa; White People; Young Adult
PubMed: 33814271
DOI: 10.1016/j.cyto.2021.155489 -
Chemico-biological Interactions Jan 2022Ochratoxin A (OTA) is a fungal secondary metabolite produced by certain species of Aspergillus and Penicillium, and exerts immunosuppressive effect on humans and...
Ochratoxin A (OTA) is a fungal secondary metabolite produced by certain species of Aspergillus and Penicillium, and exerts immunosuppressive effect on humans and animals. Quercetin (QUE) is one of the flavonoids produced as a plant-secondary metabolite. The present study was designed to evaluate the efficacy of QUE against the immunotoxic hazard of OTA in broiler chickens. Forty one-day-old broiler chicks were randomly and equally allocated into four groups; control, OTA (0.5 mg/kg feed), QUE (0.5 g/kg feed) and OTA + QUE (0.5 mg/kg OTA + 0.5 g/kg QUE). The results revealed that dietary OTA induced a significant decrease in the antibody response to Newcastle Disease (ND), Infectious Bronchitis (IB) and Avian Influenza (AI) vaccination and in the lymphoproliferative response to Phytohemagglutinin-P (PHA-P). Ochratoxin A also induced oxidative stress and lipid peroxidation in the bursa of Fabricius, spleen and thymus tissues of chickens as demonstrated by decreased CAT and GSH levels and increased TBARS content. In addition, administration of OTA resulted in apoptosis, which was evident by the increased expression level of PTEN, Bax and Caspase-3 genes and decreased expression level of PI3K, AKT and Bcl-2 genes. Furthermore, exposure to OTA resulted in various pathological lesions in the bursa of Fabricius, spleen and thymus of chickens. On the other hand, administration of QUE ameliorated most of the immunotoxic effects of OTAby its immunomodulatory, antioxidant and anti-apoptotic activities. Taken together, the results suggested that QUE potentially alleviated the OTA-induced immunotoxicity in broiler chickens, probably through amelioration of oxidative stress and activation of the PI3K/AKT signaling pathway.
Topics: Animals; Antibody Formation; Antioxidants; Avian Proteins; Bursa, Synovial; Chickens; Gene Expression; Immunologic Factors; Ochratoxins; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quercetin; Signal Transduction; Spleen; Thymus Gland
PubMed: 34717913
DOI: 10.1016/j.cbi.2021.109720 -
Psychoneuroendocrinology Feb 2021This study examined whether early life adversity (ELA) limited to infancy was associated with an increase in circulating levels of proinflammatory cytokines and cellular...
This study examined whether early life adversity (ELA) limited to infancy was associated with an increase in circulating levels of proinflammatory cytokines and cellular cytokine responses to three stimulants [lipopolysaccharide (LPS), phytohemagglutinin (PHA), and phorbol myristate acetate plus ionomycin (PMA/IO)]. Participants were previously institutionalized (PI) youth (N = 45, 56 % female) who had spent their first years in institutional care (e.g., orphanages, baby homes) before being adopted into well-resourced homes (median age at adoption = 13 mos) and non-adopted comparisons (NA; N = 38, 55 % female). Their age range was 13.3-21.2 years (M = 16.3 years). This analysis followed up an earlier report on these youth (Reid et al., 2019a) that identified an increase in terminally differentiated CD8 + CD57 T cells among the PI relative to the NA youth. Cytokine levels in circulation were not highly correlated and thus examined separately. PI youth had higher circulating levels of Tumor Necrosis Factor-alpha (TNFα), but not Interleukin-1β (IL-1β) or Interleukin-6 (IL-6). Cytokine responses to in vitro activation within each stimulant condition were highly correlated and were thus combined to generate an index of the inflammatory reaction to each stimulant. Using Multivariate Analysis of Covariance, there was a highly significant multivariate effect of group, which was carried primarily by the PMA/IO condition, with PI youth exhibiting a larger inflammatory response than NA youth. Tests of mediation showed that both the early rearing effects on circulating TNFα and the composite inflammatory index of PMA/IO responsiveness were mediated in the statistical model by the percentage of CD8 + CD57+ TEMRA cells in circulation, a marker of replicative senescence in T cells. Sex differences were also found in circulating levels of IL-6 and TNFα, with males having higher levels than females.
Topics: Adolescent; Adult; Cytokines; Female; Humans; Infant; Interleukin-6; Male; Orphanages; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Young Adult
PubMed: 33278786
DOI: 10.1016/j.psyneuen.2020.105065 -
Frontiers in Immunology 2022The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The...
The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.
Topics: Leukocytes, Mononuclear; Cells, Cultured; Mesenchymal Stem Cells; Immunomodulation; Stromal Cells; Mitogens
PubMed: 36578497
DOI: 10.3389/fimmu.2022.1085312 -
The International Journal of... Aug 2022Pluripotent stem cell derived-hepatocytes depict fetal -hepatocyte characteristics/maturity and are immunogenic limiting their applications. Attempts have been made to...
Pluripotent stem cell derived-hepatocytes depict fetal -hepatocyte characteristics/maturity and are immunogenic limiting their applications. Attempts have been made to derive hepatocytes from mesenchymal stem cells using developmental cocktails, epigenetic modulators and small molecules. However, achieving a stable terminally differentiated functional state had been a challenge. Inefficient hepatic differentiation could be due to lineage restrictions set during development. Hence a novel lineage reprogramming approach has been utilized to confer competence to adipose-mesenchymal stem cells (ADMSCs) to efficiently respond to hepatogenic cues and achieve a stable functional hepatic state. Lineage reprogramming involved co-transduction of ADMSCs with hepatic endoderm pioneer Transcription factor (TF)-FOXA2, HHEX-a homeobox gene and HNF4α-master TF indispensable for hepatic state maintenance. Lineage priming was evidenced by endogenous HFN4α promoter demethylation and robust responsiveness to minimal hepatic maturation cues. Induced hepatocytes (i-Heps) exhibited mesenchymal-to-epithelial transition and terminal hepatic signatures. Functional characterisation of i-Heps for hepatic drug detoxification systems, xenobiotic uptake/clearance, metabolic status and hepatotropic virus entry validated acquisition of stable hepatic state and junctional maturity Exhaustive analysis of MSC memory in i-Heps indicated loss of MSC-immunophenotype and terminal differentiation to osteogenic/adipogenic lineages. Importantly, i-Heps suppressed phytohemagglutinin-induced T-cell blasts, inhibited allogenic mixed-lymphocyte reactions (MLRs) and secreted immunomodulatory- indoleamine 2,3-dioxygenase in T-cell blast co-cultures akin to native ADMSCs. In a nutshell, the present study identifies a novel cocktail of TFs that reprogram ADMSCs to stable hepatic state. i-Heps exhibit adult hepatocyte functional maturity with robust immune-modulatory abilities rendering suitability for rigorous drug testing, hepatocyte-pathogen interaction studies and transplantation in allogenic settings.
Topics: Adipose Tissue; Adult; Cell Differentiation; Cells, Cultured; Hepatocytes; Humans; Mesenchymal Stem Cells
PubMed: 35772664
DOI: 10.1016/j.biocel.2022.106256 -
Developmental and Comparative Immunology Feb 2022The novel tumor necrosis factor (TNF-New or TNFN) gene has been identified only in teleost such as zebrafish, medaka (Oryzias latipes), fugu (Takifugu rubripes), and...
The novel tumor necrosis factor (TNF-New or TNFN) gene has been identified only in teleost such as zebrafish, medaka (Oryzias latipes), fugu (Takifugu rubripes), and rainbow trout (Oncorhynchus mykiss). In this study, a putative TNFN gene in rock bream (named RB-TNFN) was cloned and its functional expression in the immune system was analyzed. Although it was previously reported to share a high degree of homology with mammalian lymphotoxin (LT)-β, in silico analysis revealed that RB-TNFN differed slightly from mammalian LT-β in its genomic structure, phylogenetic relationship, and predicted protein tertiary structure, whereas the genomic location of TNFN (immediately behind TNF-α) was the same as that of LT-β. In healthy rock bream, RB-TNFN gene expression was the highest in the liver and the lowest in the head kidney. In vitro, it was significantly upregulated in head kidney cells following polyinosinic:polycytidylic acid, concanavalin A, phytohemagglutinin, or calcium ionophore (CI) stimulation and in spleen cells by lipopolysaccharide (LPS), CI, and rock bream iridovirus (RBIV). In vivo, it was upregulated in the spleen, liver, and gut on day 1 and in the blood on day 3 following LPS injection, and in the blood, head kidney, and liver following RBIV vaccination. Post-RBIV infection, the vaccinated group showed a significantly higher TNFN gene expression in the head kidney and blood than the unvaccinated group. Treatment with recombinant TNFN protein (RB-rTNFN) resulted in significantly upregulated interleukin-1β expression in the head kidney, spleen, blood, liver, and peritoneal cells. It also enhanced IL-8 gene expression in the head kidney, blood, and peritoneal cells, and interferon γ gene expression in the gut and gills on day 1. TNFN and cyclo-oxygenase-2 gene expression was upregulated in peritoneal cells on day 3. Flow cytometry analysis revealed a significant increase in the peritoneal lymphocyte population after the intraperitoneal (i.p.) injection of RB-rTNFN. These results suggest that RB-TNFN mediated innate and adaptive immunity in rock bream.
Topics: Animals; Fish Diseases; Fish Proteins; Mammals; Perciformes; Phylogeny; Tumor Necrosis Factors; Zebrafish
PubMed: 34600021
DOI: 10.1016/j.dci.2021.104269 -
Journal of Immunological Methods Jan 2021Investigational cell-based therapeutics are rapidly heading towards pivotal clinical trials. The premise is that the scientific rationale is well defined, and that... (Comparative Study)
Comparative Study
Investigational cell-based therapeutics are rapidly heading towards pivotal clinical trials. The premise is that the scientific rationale is well defined, and that product quality reflects exactly this. In vitro potency assays are necessary tools for evaluating cell products, and with potency assays comes high demands for standardization and reproducibility of the methods involved. For demonstrating principles of cell therapeutics for allogeneic use or with claimed immunosuppressive efficacies, assays involving peripheral blood mononuclear cells (PBMC) are critical. Establishment of a cryopreserved bank of PBMC favors standardization, as it allows repeated use of a single donor and simultaneous testing of several donors. The first step to fulfil such potential is to ensure optimum conditions for preservation of PBMC function, and secondly to design assays which heightens the reproducibility. Emphasis should be put on application of the assay. The objective of the present study was to establish a methodological foundation for cell therapeutics to be tested, and several aspects were factored in, including cell concentrations and partial changes of medium. PBMC were isolated and cryopreserved in six formulations of cryoprotective medium consisting of fetal bovine serum (90%, 60%, and 30%) in combination with dimethyl sulfoxide (10% or 5%). The proliferative capacity of the cryopreserved cells was assayed by labeling with carboxyfluorescein succinimidyl ester and stimulation by phytohemagglutinin or in mixed lymphocyte reactions, analyzed by flow cytometry. To counter an eventual lag phase post thaw, the assays were designed to include two durations and to explore the possibility of reducing cell numbers, two cell concentrations. Qualitative and quantitative aspects of the staining were affected by formulation as well as design, stressing the importance of basic optimization for assay development. We conclude that the established methods allow for optimized preservation of function and will serve as a platform for further development of robust functional assays.
Topics: Cell Proliferation; Cells, Cultured; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Flow Cytometry; Humans; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Mitogens; Phytohemagglutinins; Reproducibility of Results; Serum Albumin, Bovine; Time Factors
PubMed: 33049298
DOI: 10.1016/j.jim.2020.112897 -
Environmental Science and Pollution... Sep 2021Aflatoxin B1 (AFB1) is a secondary metabolite of some Aspergillus species that contaminate the agricultural commodities intended for animal and human consumption. The...
Aflatoxin B1 (AFB1) is a secondary metabolite of some Aspergillus species that contaminate the agricultural commodities intended for animal and human consumption. The present in vivo study aimed to evaluate activated charcoal (AC) for its ability to reduce AFB1-induced immune suppressive effects in broiler chickens. One-day-old broiler chicks were divided into 12 groups (n = 30) and raised until 42 days of age. One control group was offered basal broiler feed. Three AFB1 groups were kept on AFB1-contaminated basal broiler feed (0.1, 0.2, and 0.6 mg/kg AFB1, respectively), whereas two AC groups were offered AC-added basal broiler feed (2.5 and 5.0 g/kg AC, respectively). Six combination groups were maintained on a combination of different doses of AFB1 and AC. The immune protective efficacy of AC was assessed by anti-sheep RBC's antibodies, phagocytic activity of the reticuloendothelial system, phytohemagglutinin-P (PHA-P)-induced cutaneous basophil response, and histopathological and morphometric analysis of lymphoid organs. Dietary exposure to AFB1 alone resulted in dose-dependent suppression of immune responses and degenerative and necrotic changes in the bursa of Fabricius and thymus. The dietary addition of AC reduced the toxic effects of 0.1 and 0.2 mg/kg dietary AFB1 on immune responses and histological lesion on lymphoid organs; however, at higher dietary level of AFB1 (0.6 mg AFB1/kg), the dietary addition of AC was not effective to prevent the immunotoxic effects. The results of this study suggested that dietary inclusion of AC has the ability to prevent immunotoxic effects induced by AFB1 at lower dietary contaminations levels in broiler chickens.
Topics: Aflatoxin B1; Animal Feed; Animals; Carbon; Chickens; Dietary Supplements; Humans; Liver; Sheep
PubMed: 33932208
DOI: 10.1007/s11356-021-14048-5 -
Shock (Augusta, Ga.) Feb 2023Background: There is currently no standard definition of a severe burn in the pediatric patient population to identify those at higher risk of infectious complications.... (Observational Study)
Observational Study
Background: There is currently no standard definition of a severe burn in the pediatric patient population to identify those at higher risk of infectious complications. Our aim was to correlate total burn surface area (TBSA), burn depth, and type of burn injury to nosocomial infection rates and systemic immune system responses to better define risk factors associated with adverse outcomes. Methods: A prospective observational study at a single-center, quaternary-care, American Burn Association-verified pediatric burn center was conducted from 2016 to 2021. Blood was collected within 72 h of injury from 103 pediatric patients. Whole blood was incubated with lipopolysaccharide or phytohemagglutinin stimulation reagent to measure innate and adaptive immune response, respectively. Flow cytometry was performed on whole blood samples to measure both innate and adaptive immune cells. Unstimulated plasma was also extracted, and IL-6 and IL-10 as well as soluble proteins B- and T-lymphocyte attenuator, CD27, and T-cell immunoglobulin mucin 3 were quantified. Results: There was a significant increased risk for nosocomial infection in pediatric patients with TBSA burns of ≥20%, full-thickness burn injuries ≥5%, or flame burn injuries. There was an overall decrease in both innate and adaptive immune function in patients with TBSA burns ≥20% or full-thickness burn injuries ≥5%. Both burn injury characteristics were also associated with a significant increase in unstimulated IL-6 and IL-10 and soluble immunoregulatory checkpoint proteins. We observed a significant decrease in soluble B- and T-lymphocyte attenuator for those with a flame injury, but there were no other differences between flame injury and scald/contact burns in terms of innate and adaptive immune function. Conclusion: Burns with ≥20% TBSA or ≥5% full thickness in pediatric patients are associated with systemic immune dysfunction and increased risk of nosocomial infections.
Topics: Child; Humans; Interleukin-10; Interleukin-6; Burn Units; Cross Infection; Demography; Retrospective Studies
PubMed: 36730756
DOI: 10.1097/SHK.0000000000002037