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Microorganisms Nov 2022We sought to investigate the influence of SARS-CoV-2 infection on the cytokine profiles of peripheral blood mononuclear cells (PBMCs) and neutrophils from coronavirus...
Influence of SARS-COV-2 Infection on Cytokine Production by Mitogen-Stimulated Peripheral Blood Mononuclear Cells and Neutrophils in COVID-19 Intensive Care Unit Patients.
We sought to investigate the influence of SARS-CoV-2 infection on the cytokine profiles of peripheral blood mononuclear cells (PBMCs) and neutrophils from coronavirus disease 2019 (COVID-19) intensive care unit (ICU) patients. Neutrophils and PBMCs were separated and stimulated with the mitogen phytohemagglutinin. Culture supernatants of mitogen-stimulated PBMCs and neutrophils from 88 COVID-19 ICU patients and 88 healthy controls were evaluated for levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-2, -4, -5, -6, -9, -10, -12, -17A, and tumor necrosis factor (TNF)-α using anti-cytokine antibody MACSPlex capture beads. Cytokine profiles of PBMCs showed significantly lower levels of GM-CSF, IFN-γ, IL-6, IL-9, IL-10, IL-17A, and TNF-α (p < 0.0001) in COVID-19 ICU patients. In contrast, COVID-19 ICU patients showed higher median levels of IL-2 (p < 0.001) and IL-5 (p < 0.01) by PBMCs. As for neutrophils, COVID-19 ICU patients showed significantly lower levels of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-17A, IL-12, TNF-α (p < 0.0001), and IFN-α (p < 0.01). T-helper (Th)1:Th2 cytokine ratios revealed lower inflammatory cytokine for PBMCs and neutrophils in COVID-19 ICU patients. Cytokine production profiles and Th1:Th2 cytokine ratios suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has an immunomodulatory effect on PBMCs and neutrophils. This study also suggests that the increased levels of several cytokines in the serum are not sourced from PBMCs and neutrophils.
PubMed: 36363785
DOI: 10.3390/microorganisms10112194 -
Biology Nov 2022Although impaired mitochondrial function has been proposed as a hallmark of multiple sclerosis (MS) disease, few studies focus on the mitochondria of immune cells. We...
Although impaired mitochondrial function has been proposed as a hallmark of multiple sclerosis (MS) disease, few studies focus on the mitochondria of immune cells. We aimed to compare the mitochondrial function of the peripheral blood mononuclear cells (PBMCs) from MS patients with (M+) and without (M-) lipid-specific oligoclonal immunoglobulin M bands (LS-OCMB), and healthydonors (HD). We conducted an exploratory cross-sectional study with 19 untreated MS patients (M+ = 9 and M- = 10) and 17 HDs. Mitochondrial superoxide anion production and mitochondrial mass in PBMCs were assessed without and with phytohemagglutinin by flow cytometry. The PBMCs' mitochondrial function was analyzed using Seahorse technology. Superoxide anion production corrected by the mitochondrial mass was higher in MS patients compared with HDs ( = 0.011). Mitochondrial function from M+ patients showed some impairments compared with M- patients. Without stimulus, we observed higher proton leak ( = 0.041) but lower coupling efficiency ( = 0.041) in M+ patients; and under stimulation, lower metabolic potential ECAR ( = 0.011), and lower stressed OCR/ECAR in the same patients. Exclusively among M+ patients, we described a higher mitochondrial dysfunction in the oldest ones. The mitochondrial impairments found in the PBMCs from MS patients, specifically in M+ patients, could help to better understand the disease's physiopathology.
PubMed: 36358334
DOI: 10.3390/biology11111633 -
BMC Biotechnology Oct 2022Leukocyte phytohemagglutinin (PHA-L), derived from the L4 tetramer of PHA, has been frequently employed as a mitogen to induce T lymphocyte proliferation in vitro. The...
BACKGROUND
Leukocyte phytohemagglutinin (PHA-L), derived from the L4 tetramer of PHA, has been frequently employed as a mitogen to induce T lymphocyte proliferation in vitro. The biological application of PHA-L in cancer diagnosis and treatment has gained traction in recent years. However, it has been noted that PHA-L obtained using traditional procedures has a massive amount of impurities or toxic components, which interfere with the activity of PHA-L. Preparation of a monoclonal antibody against active PHA-L is a significant tool for studying PHA-L's function and therapeutic potential.
RESULTS
We successfully prepared monoclonal antibodies against the active components of PHA-L based on the whole PHA-L protein as an antigen, and found that monoclonal antibody 3C1C6G11 can be employed in western blot, immunofluorescence, and immunohistochemistry detection. Importantly, preliminary result shows that the mAb 3C1C6G11 may prevent PHA-L-induced cell aggregation and AICD (activation-induced cell death).
CONCLUSIONS
The monoclonal antibody mAb 3C1C6G11 prepared in this study can be used as an effective tool for detecting PHA-L active components, investigating PHA-L's function and antineoplastic application.
Topics: Phytohemagglutinins; Antibodies, Monoclonal; Phaseolus; Lymphocyte Activation
PubMed: 36309691
DOI: 10.1186/s12896-022-00761-7 -
Journal of Clinical Immunology Feb 2023Hypoparathyroidism-retardation-dysmorphism (HRD) syndrome is a disease composed of hypoparathyroidism, growth retardation, severe developmental delay, and typical...
BACKGROUND
Hypoparathyroidism-retardation-dysmorphism (HRD) syndrome is a disease composed of hypoparathyroidism, growth retardation, severe developmental delay, and typical dysmorphic features caused by the tubulin-specific chaperone E gene variant. Many patients succumb in infancy to HRD due to overwhelming infections mainly caused by Pneumococcus spp. Knowledge related to the immune system in these patients is scarce.
PURPOSE
To define the immune phenotype of a cohort of HRD patients including their cellular, humoral, and neutrophil functions.
METHODS
The study included HRD patients followed at Soroka University Medical Center. Clinical and immunological data were obtained, including immunoglobulin concentrations, specific antibody titers, lymphocyte subpopulations, lymphocyte proliferation, and neutrophil functions.
RESULTS
Nine patients (5 females and 4 males) were enrolled, aged 6 months to 15 years. All received amoxicillin prophylaxis as part of a routine established previously. Three patients had bacteremia with Klebsiella, Shigella spp., and Candida. Three patients had confirmed coronavirus disease 19 (COVID-19), and two of them died from this infection. All patients had normal blood counts. Patients showed high total IgA and IgE levels, low anti-pneumococcal antibodies in spite of a routine vaccination schedule, and reduced frequency of naive B cells with increased frequency of CD21lowCD27- B cells. All patients had abnormal T-cell population distributions, including reduced terminally differentiated effector memory CD8, inverted CD4/CD8 ratios, and impaired phytohemagglutinin (PHA)-induced lymphocyte proliferation. Neutrophil superoxide production and chemotaxis were normal in all patients tested.
CONCLUSION
HRD is a combined immunodeficiency disease with syndromic features, manifesting in severe invasive bacterial and viral infections.
Topics: Male; Female; Humans; Tubulin; COVID-19; Growth Disorders; Hypoparathyroidism
PubMed: 36258138
DOI: 10.1007/s10875-022-01380-9 -
Iranian Journal of Allergy, Asthma, and... Aug 2022A decrease in T cell count or reduced T cell function can be indicative of T cell immunodeficiency. In the present study, T-cell function was assessed using...
A decrease in T cell count or reduced T cell function can be indicative of T cell immunodeficiency. In the present study, T-cell function was assessed using Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution test after stimulation with commonly used Phytohaemagglutinin (PHA) or anti-CD3/anti-CD28 coated beads in pediatric patients with recurrent infections. Seven infants with recurrent infections and seven sex/age-matched healthy infants were included in this study. A blood cell count, immunophenotyping, and serum immunoglobulin level were performed. The proliferation of T cells was also assessed with CFSE dilution after stimulation with PHA or anti-CD3/anti-CD28 coated beads. This study showed increased IgA, IgG, and IgM levels in patients compared to the controls. In contrast to the controls, the immunophenotyping results showed a significant decline in the number of CD4+ T cells in patients. Although there was no difference in CD3+ T cell proliferation between patients and controls, the CD4+ and CD8+ T cell proliferation rates were significantly decreased in patients when stimulated with PHA. As a mitogen with the potential for maximum proliferation of T cells, PHA is better able to distinguish between patients with recurrent infections and controls than anti-CD3/anti-CD28, which mimics only the TCR pathway for stimulation of T cells.
Topics: CD28 Antigens; Cell Proliferation; Child; Fluoresceins; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Infant; Lymphocyte Activation; Mitogens; Phytohemagglutinins; Receptors, Antigen, T-Cell; Reinfection; Succinimides
PubMed: 36243934
DOI: 10.18502/ijaai.v21i4.10293 -
The Journal of Comparative Neurology Feb 2023The orbital cortex (ORB) of the rat consists of five divisions: the medial (MO), ventral (VO), ventrolateral (VLO), lateral (LO), and dorsolateral (DLO) orbital...
The orbital cortex (ORB) of the rat consists of five divisions: the medial (MO), ventral (VO), ventrolateral (VLO), lateral (LO), and dorsolateral (DLO) orbital cortices. No previous report has comprehensively examined and compared projections from each division of the ORB to the thalamus. Using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin, we describe the efferent projections from the five divisions of the ORB to the thalamus in the rat. We demonstrated that, with some overlap, each division of the ORB distributed in a distinct (and unique) manner to nuclei of the thalamus. Overall, ORB projected to a relatively restricted number of sites in the thalamus, and strikingly distributed entirely to structures of the medial/midline thalamus, while completely avoiding lateral regions or principal nuclei of the thalamus. The main termination sites in the thalamus were the paratenial nucleus (PT) and nucleus reuniens (RE) of the midline thalamus, the medial (MDm) and central (MDc) divisions of the mediodorsal nucleus, the intermediodorsal nucleus, the central lateral, paracentral, and central medial nuclei of the rostral intralaminar complex and the submedial nucleus (SM). With some exceptions, medial divisions of the ORB (MO, VO) mainly targeted "limbic-associated" nuclei such as PT, RE, and MDm, whereas lateral division (VLO, LO, DLO) primarily distributed to "sensorimotor-associated" nuclei including MDc, SM, and the rostral intralaminar complex. As discussed herein, the medial/midline thalamus may represent an important link (or bridge) between the orbital cortex and the hippocampus and between the ORB and medial prefrontal cortex. In summary, the present results demonstrate that each division of the orbital cortex projects in a distinct manner to nuclei of the thalamus which suggests unique functions for each division of the orbital cortex.
Topics: Animals; Rats; Prefrontal Cortex; Thalamus; Midline Thalamic Nuclei; Hippocampus; Intralaminar Thalamic Nuclei; Phytohemagglutinins; Neural Pathways
PubMed: 36226328
DOI: 10.1002/cne.25419 -
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 -
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 -
Animal Models and Experimental Medicine Jun 2023Amarogentin (AMA) is a secoiridoid glycoside extracted from Swertia and Gentiana roots and exhibits many biological effects such as antioxidative, anti-inflammatory, and...
BACKGROUND
Amarogentin (AMA) is a secoiridoid glycoside extracted from Swertia and Gentiana roots and exhibits many biological effects such as antioxidative, anti-inflammatory, and antitumor activities. Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by disorders in the regulation of multiple inflammatory cytokines. No effective cure has been found for AD now.
METHODS
We constructed the HaCat and splenocyte model and tested the inhibitory effect of AMA on IL-4, IL-6, and IL-13 secretions using enzyme-linked immunosorbent assay (ELISA). The AD mouse model was constructed and treated with AMA, the severity of skin lesions was observed, epidermal tissue was collected, and epidermal thickness and mast cell infiltration were observed using hematoxylin and eosin and toluidine blue staining, respectively. The expression of kallikrein-related peptidase 7 (KLK7) and filaggrin (FLG) was detected using immunostaining and Western blot analysis. The mRNA expression of KLK7 and FLG was detected using quantitative polymerase chain reaction (qPCR). Blood immunoglobulin E (IgE) secretion was detected.
RESULTS
AMA inhibited IL-6 secreted by tumor necrosis factor (TNF)-α-induced HaCaT cells and reduced IL-4 and IL-13 secreted by phytohemagglutinin (PHA)-induced primary cells in the mice spleen. It was found that the treatment of AMA with 2,4-dinitrochlorobenzene-induced AD-like mice could promote the recovery of dermatitis, reduce the score of dermatitis severity and the scratching frequency, treat the skin lesions, reduce the epidermal thickness, decrease the infiltration of mast cells, reduce the IgE level in serum, decrease the expression levels of AD-related cytokines, increase protein and mRNA expression of FLG, and reduce the protein and mRNA expression of KLK7 in the skin tissues of AD-like mice.
CONCLUSION
In conclusion, AMA inhibits inflammatory response at the cellular level, and AMA reduces the validation response of specific dermatitis mice, relieves pruritus, and repairs the damaged skin barrier.
Topics: Animals; Mice; Humans; Dermatitis, Atopic; Dinitrochlorobenzene; Interleukin-13; Interleukin-6; HaCaT Cells; Interleukin-4; Cytokines; Tumor Necrosis Factor-alpha; Anti-Inflammatory Agents; Immunoglobulin E; RNA, Messenger
PubMed: 36131559
DOI: 10.1002/ame2.12260 -
Journal of Immunological Methods Nov 2022As the interest in cell-based therapies continue to increase, so does the need for assays detailing potency and providing platforms for identifying mechanisms of action....
Functional in vitro models of the inhibitory effect of adipose tissue-derived stromal cells on lymphocyte proliferation: Improved sensitivity and quantification through flow cytometric analysis.
As the interest in cell-based therapies continue to increase, so does the need for assays detailing potency and providing platforms for identifying mechanisms of action. For most clinical implications of mesenchymal stromal cells, the immunomodulatory effect is crucial. While the suppressive potential on lymphocyte proliferation is well-described in literature, reproducible and standardized assays to document and quantify it varies from research group to research group and between methodologies. The aim of the present study was to utilize flowcytometry to quantify proliferation and identify measurements to increase the assay sensitivity to treatment with adipose tissue-derived stromal cells (ASC). Lymphocyte proliferation was induced by the unspecific mitogen phytohemagglutinin or by alloreactivity towards an irradiated donor in a mixed lymphocyte reaction. Addition of ASC did not change the composition of T cells, B cells, NK cells, NKT cell types considerably; likewise, no increases in proliferation were observed upon inclusion of ASC, demonstrating that ASC does not evoke an additive response. On the contrary, the suppressive effect of ASC was documented. By applying different gating strategies and curve fitting, the sensitivity was increased, and dose-response relationships established. Flow cytometric evaluation allows for more detailed identification of the lymphocytes affected by ASC and constitute a significant asset in future unraveling of modes and mechanisms of action, as well as quantification of potency.
Topics: Adipose Tissue; Cell Proliferation; Cells, Cultured; Mitogens; Phytohemagglutinins; Stromal Cells
PubMed: 36130659
DOI: 10.1016/j.jim.2022.113360