Murine Spleen Cells Research Articles

Toxicity, Antibacterial, Antioxidant, Antidiabetic, and DNA Cleavage Effects of Dextran-Graft-Polyacrylamide/Zinc Oxide Nanosystems.

Synthesis of metal oxide nanoparticles-polymer nanocomposites is an emerging strategy in nanotechnology to improve targeted delivery and reduce the toxicity of nanoparticles. In this study, we report biological effects of previously described hybrid nanocomposites containing dextran-graft-polyacrylamide/zinc oxide nanoparticles (D-PAA/ZnO NPs) prepared from zinc sulfate (D-PAA/ZnONPs(SO42-)) and zinc acetate (D-PAA/ZnONPs(-OAc)) focusing primarily on their antimicrobial activity. D-PAA/ZnONPs(SO42-) and D-PAA/ZnONPs(-OAc) nanosystems were tested in a complex way to assess their antioxidant activity (DPPH assay), antidiabetic potential (α-amylase inhibition), DNA cleavage activity, antimicrobial, and antibiofilm activity. In addition, the toxicity of D-PAA/ZnONPs(SO42-) and D-PAA/ZnONPs(-OAc) nanosystems against primary murine splenocytes was tested using MTT assay. The studied nanosystems inhibited E.coli growth. For all the investigated strains, minimum inhibitory concentrations (MICs) of D-PAA/ZnONPs(SO42-) and D-PAA/ZnONPs(-OAc) were in the range of 8mg/L-128mg/L and 16mg/L-128mg/L, respectively. The nanocomposites demonstrated effective antibiofilm properties as 94.27% and 86.43%. The compounds showed good antioxidant, anti-α-amylase, and DNA cleavage activities. D-PAA/ZnONPs(SO42-) and D-PAA/ZnONPs(-OAc) nanosystems reduced cell viability and promoted cell death of primary murine spleen cells at concentrations higher than those that proved to be antibacterial indicating the presence of therapeutic window. D-PAA/ZnONPs(SO42-) and D-PAA/ZnONPs(-OAc) nanosystems show antioxidant, antidiabetic, DNA cleavage, antimicrobial, and antibiofilm activity against the background of good biocompatibility suggesting the presence of therapeutic potential, which should be further investigated in vivo.

Protective immunity induced through two calving seasons following administration of live epizootic bovine abortion agent (EBAA) vaccine

A live, infectious vaccine candidate for epizootic bovine abortion, designated EBAA Vaccine, USDA-APHIS Product code #1544.00, has been reported to be both safe and effective. Previous studies established that a single dose of EBAA vaccine administered to cows at potencies of either 2000 or 500 live P. abortibovis-infected murine spleen cells (P.a.-LIC) induced protective immunity for a minimum of 5 months. The current study employed 19 pregnant cows that were challenged with P. abortibovis in their 2nd trimester of gestation; 9 were vaccinated 17.2-months earlier as 1-year-olds with 2000 P.a.-LIC and 10 served as negative controls. Eighty-nine percent of the vaccinates gave birth to healthy calves as compared to 10% of challenge controls. Vaccine efficacy was significant when analyzed by prevented fractions (87.7%; 95% CI=0.4945–0.9781). Serologic data supports previous findings that pregnant cows with detectable P. abortibovis antibodies are immune to P. abortibovis challenge as demonstrated by the birth of healthy calves.

Acupoint Autohemotherapy Attenuates DNCB-Induced Atopic Dermatitis and Activates Regulatory T Cells in BALB/c Mice.

Acupoint autohemotherapy (A-AHT) has been proposed as an alternative and complementary treatment for atopic dermatitis (AD), yet the exact role of its blood component in terms of therapeutic efficacy and mechanism of action is still largely unknown. This study aimed to evaluate the therapeutic efficacies and action mechanisms of intramuscular injections of autologous whole blood (AWB) and mouse immunoglobulin G (IgG) (autologous or heterologous) at acupoints on 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse models. Serum levels of total immunoglobulin E (IgE), IgG, interleukin-10 (IL-10), and interferon-gamma (IFN-γ) were measured, as well as mRNA expression levels of Forkhead box P3 (FoxP3), IL-10 and IFN-γ in dorsal skin lesions, and IL-10+, IFN-γ+ and FoxP3+CD4+T cells in murine spleen. It showed that repeated acupoint injection of AWB, autologous total IgG (purified from autologous blood in AD mice) or heterologous total IgG (purified from healthy blood in normal mice) effectively reduced the severity of AD symptoms and decreased epidermal and dermal thickness as well as mast cells in skin lesions. Additionally, AWB acupoint injection was found to upregulate FoxP3+, IL-10+ and IFN-γ+ CD4+T cells in murine spleen, suppressing the production of IgE antibodies and increasing that of IgG antibodies in the serum. Furthermore, both AWB and autologous total IgG administrations significantly elevated FoxP3 expression, mRNA levels of IL-10 and IFN-γ in dorsal skin lesions. However, acupoint injection of heterologous total IgG had no effect on regulatory T (Treg) and Th1 cells modulation. These findings suggest that the therapeutic effects of A-AHT on AD are mediated by IgG-induced activation of Treg cells.

Mechanisms of interleukin-10 induction in murine spleen and RAW264 cells by Latilactobacillus curvatus K4G4 isolated from fermented Brassica rapa L.

Lactic acid bacteria (LAB) are commonly used in fermented foods, and some LAB modulate the immune response. We aimed to investigate the mechanism by which LAB isolates from fermented Brassica rapa L. induce the production of anti-inflammatory interleukin (IL)-10 by the murine spleen and RAW264 cells. Spleen cells from BALB/c mice or the mouse macrophage cell line RAW264 were cultured with heat-killed LAB isolated from fermented B. rapa L., and the IL-10 level in the supernatant was measured. Latilactobacillus curvatus K4G4 provided the most potent IL-10 induction among 13 isolates. Cell wall components of K4G4 failed to induce IL-10, while treatment of the bacteria with RNase A under a high salt concentration altered K4G4 induction of IL-10 by spleen cells. In general, a low salt concentration diminished the IL-10 induction by all strains, including K4G4. In addition, chloroquine pretreatment and knock down of toll-like receptor 7 through small interfering RNA suppressed K4G4 induction of IL-10 production by RAW264 cells. Our results suggest that single-stranded RNA from K4G4 is involved, via endosomal toll-like receptor 7, in the induction of IL-10 production by macrophages. K4G4 is a promising candidate probiotic strain that modulates the immune response by inducing IL-10 from macrophages.

Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody.

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

Inhibitors of the Actin-Bundling Protein Fascin-1 Developed for Tumor Therapy Attenuate the T-Cell Stimulatory Properties of Dendritic Cells.

Simple SummaryExpression of the actin-bundling protein Fascin-1 (Fscn1) is largely restricted to neuronal cells and to activated dendritic cells (DCs). DCs are important inducers of (antitumor) immune responses. In tumor cells, de novo expression of Fscn-1 correlates with their invasive and metastatic activities. Pharmacological Fscn1 inhibitors, which are currently under clinical trials for tumor therapy, were demonstrated to counteract tumor metastasis. Within this study, we were interested in better understanding the effects of Fscn1 inhibitors on DCs and discovered that two distinct Fascin-1 inhibitors affect the immune-phenotype and T-cell stimulatory activity of DCs. Our results suggest that systemic application of Fscn1 inhibitors for tumor therapy may also modulate antitumor immune responses.Background: Stimulated dendritic cells (DCs), which constitute the most potent population of antigen-presenting cells (APCs), express the actin-bundling protein Fascin-1 (Fscn1). In tumor cells, de novo expression of Fscn1 correlates with their invasive and metastatic properties. Therefore, Fscn1 inhibitors have been developed to serve as antitumor agents. In this study, we were interested in better understanding the impact of Fscn1 inhibitors on DCs. Methods: In parallel settings, murine spleen cells and bone-marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide in the presence of Fscn1 inhibitors (NP-G2-044 and BDP-13176). An analysis of surface expression of costimulatory and coinhibitory receptors, as well as cytokine production, was performed by flow cytometry. Cytoskeletal alterations were assessed by confocal microscopy. The effects on the interactions of BMDCs with antigen-specific T cells were monitored by time lapse microscopy. The T-cell stimulatory and polarizing capacity of BMDCs were measured in proliferation assays and cytokine studies. Results: Administration of Fscn1 inhibitors diminished Fscn1 expression and the formation of dendritic processes by stimulated BMDCs and elevated CD273 (PD-L2) expression. Fscn1 inhibition attenuated the interaction of DCs with antigen-specific T cells and concomitant T-cell proliferation. Conclusions: Systemic administration of Fscn1 inhibitors for tumor therapy may also modulate DC-induced antitumor immune responses.

Glutamate in the medium of Lactiplantibacillus plantarum FL-664 affects the production of IL-12(p40) on murine spleen cells.

Lactic acid bacteria (LAB) have been attracting attention for their effects on innate immunity, and therefore, it is required to develop an efficient culturing method while maintaining their functionality. In this study, first, we compared the growth and functionality of LAB cultured on food grade (FG) medium with those on standard LAB medium and found that LAB cultured in the FG medium were smaller in cell size with high yield and had a higher ability to induce IL-12(p40) production by murine spleen cells in vitro. Moreover, the higher the glutamate concentration in the medium, the smaller the cell size, and the higher the yield and the higher the ability to induce IL-12 production. Addition of glutamate to the culture medium changes the size of LAB and affects their ability to induce IL-12(p40) production. In conclusion, regulating the concentration of glutamate would be important in the efficient culturing of functional LAB.

Effect of Poly(I:C) and melanoma B16-F10 on the immunophenotype of murine spleen cells

Introduction. It is known that the agonist of TLR-3 Poly(I:C), used as an adjuvant in a number of models of antitumor vaccines, causes inhibition of melanoma B16 growth, but the immunological aspects involved in this process have not been fully studied.The aim of the study was to evaluate changes of the immunophenotype of the spleen cells of C57BL / 6 mice caused by the tumor load and / or Poly(I:C), which is necessary for better understanding of the processes occurring during Poly(I:C) inhibition of melanoma B16-F10.Materials and methods. The immunophenotype of splenocytes of C57Bl / 6 mice was studied by flow cytometry asfollowing: the group 1 was a control (intact animals), the group 2 was mice with subcutaneously transplanted melanoma B16-F10, the group 3 was mice without a tumor treated with Poly(I:C) and the group 4 – mice with subcutaneously transplanted melanoma B16-F10 treated with Poly(I:C).Results. Median values of parameters such as the CD4 / CD8 immunoregulatory index, the percentage of CD69+ CD4+ and CD8+ T cells, the number of B and NK cells for the group of mice with melanoma treated with Poly(I:C) were between the values in the control group and in the group of mice with B16-F10. when comparing the results, the number of B and NK cells, the percentage of CD69+ on CD4+ and CD8+ T cells, their median in the group of mice with melanoma treated with Poly(I:C) was closer to the control than to the values obtained in the B16-F10 group and in the group of healthy mice receiving Poly(I:C). At the same time, we found that the total number of CD3+ cells, the number of naive CD4+ and CD8+ T cells was higher in the group of mice with melanoma treated with Poly(I:C) compared to all other groups.Conclusion. The analysis revealed the changes of the immunophenotype of murine spleen cells (CD4 / CD8, the percentage of CD69+ CD4+ and CD8+ T cells, the number of B and NK cells), which were affected by the tumor load and / or the administration of Poly adjuvant (I:C). Changes in the immunophenotype of murine splenocytes were associated with the tumor load and its size. It was also found that the splenocyte immunophenotype was affected by the repeated administration of Poly(I:C) during the tumor growth.

Maca against Echinococcosis?-A Reverse Approach from Patient to In Vitro Testing.

Drug-based treatment of alveolar echinococcosis (AE) with benzimidazoles is in most cases non-curative, thus has to be taken lifelong. Here, we report on a 56-year-old male AE patient who received standard benzimidazole treatment and biliary plastic stents, and additionally self-medicated himself with the Peruvian plant extract Maca (Lepidium meyenii). After 42 months, viable parasite tissue had disappeared. Based on this striking observation, the anti-echinococcal activity of Maca was investigated in vitro and in mice experimentally infected with Echinococcus multilocularis metacestodes. Albendazole (ABZ)-treated mice and mice treated with an ABZ+Maca combination exhibited a significantly reduced parasite burden compared to untreated or Maca-treated mice. As shown by a newly established UHPLC-MS/MS-based measurement of ABZ-metabolites, the presence of Maca during the treatment did not alter ABZ plasma levels. In vitro assays corroborated these findings, as exposure to Maca had no notable effect on E. multilocularis metacestodes, and in cultures of germinal layer cells, possibly unspecific, cytotoxic effects of Maca were observed. However, in the combined treatments, Maca inhibited the activity of ABZ in vitro. While Maca had no direct anti-parasitic activity, it induced in vitro proliferation of murine spleen cells, suggesting that immunomodulatory properties could have contributed to the curative effect seen in the patient.

Evaluation of Red and Green Colored Bell peppers for the Production of Polyclonal IgM and IgG Antibodies in Murine Spleen Cells

Immunostimulants are greatly required for the upregulation of immunity to fight against viral and bacterial infections and cancers. Bell peppers (Capsicum annuum L.), eaten as vegetables, are rich sources of vitamin C and E, provitamin A, β-carotene, and numerous phenolic compounds. Antimicrobial, antioxidant, anti-mutagenic and anti-inflammatory properties of Bell peppers were reported. Our research group for the first time reported the immunomodulatory activities of Bell peppers. In this study, we evaluated the antibody production abilities of two different colored Bell peppers (red and green) in the culture of antibody producing splenic B cells of mice. Antibodies and the number of viable cells were determined by an ELISA and MTT assays, respectively. Red Bell pepper Extract (RBPE) at the doses of (0.375, 0.75, 1.5, and 2.25 mg/mL) significantly augmented the production of polyclonal IgM and IgG antibodies in-vitro. The highest amount of IgM antibody production was observed by the dose of 1.5 mg/kg which was 3 times higher than that of the untreated cells. Similarly, RBPE also enhanced the production of IgG antibody in the culture of murine splenic B cells. On the contrary, cultural treatment of murine splenic B cells with Green Bell pepper Extract (GBPE) could not stimulate the B cells, and hence, failed to produce neither IgM nor IgG antibody. Thus the current findings suggest that consumption of Red Bell Pepper extract or its vegetables, not green pepper, may be beneficial to strengthen humoral immune responses. Bangladesh Pharmaceutical Journal 24(1): 45-53, 2021

The Role of Low-Dose Radiation in Association with TNF-α on Immunomodulatory Properties of Mesenchymal Stem Cells.

Ionizing radiation (IR) is an important medical tool. Despite the effects associated with high-dose radiation during or after treatment, as well as in accidental exposures, the direct or indirect effect of low-dose IR in cells remain poorly documented. IR can affect the tissue microenvironment, including mesenchymal stem cells (MSCs), which have high regenerative and immunomodulatory capacities. This study aimed to investigate the effect of low-dose IR in association with the inflammatory stimuli of TNF-α on the immunomodulatory capacity of MSCs. MSCs were irradiated with a low-dose IR, stimulated with TNF-α, and cultivated in a bystander system with murine spleen cells. The results showed that TNF-R1 is expressed in MSCs and is not affected, even in irradiated MSCs. However, irradiated MSCs produced reduced amounts of IL-6 and increased amounts of IL-10. The levels of PGE2 and NO• in MSCs were also increased when stimulated with TNF-α. Furthermore, conditioned media from irradiated MSCs reduced the proliferation of bystander lymphocytes and reduced the metabolic activity of macrophages. In addition, conditioned media from irradiated MSCs modulated the profile of cytokines in bystander spleen cells (lymphocytes and macrophages), reducing inflammatory and increasing anti-inflammatory cytokines, also increasing Treg cells. In conclusion, low-dose IR in association with an inflammatory stimulus affects the immunomodulatory properties of MSCs. In this way, the immunosuppressive capability of MSCs can be explored for several disease treatments where IR usually part of the context of the treatment. However, a complete understanding of the mechanisms underlying these interactions need further investigation. Graphical Abstract.

Hepatitis B envelope antigen increases Tregs by converting CD4+CD25- T cells into CD4+CD25+Foxp3+ Tregs.

Hepatitis B virus (HBV) can establish a lifelong chronic infection in humans, leading to liver cirrhosis, liver failure and hepatocellular carcinoma. Patients with chronic hepatitis B (CHB) exhibit a weak virus-specific immune response. Regulatory T cells (Tregs) play a key role in regulating the immune response in patients with CHB. Patients with hepatitis B envelope antigen (HBeAg)-positive CHB harbored a higher percentage of Tregs in their peripheral blood than those with HBeAg-negative CHB. However, whether and how HBeAg manipulates the host immune system to increase the population of Tregs remains to be elucidated. The present manuscript describes a preliminary immunological study of HBeAg in a mouse model. Multiple potential CD4+ T cell epitopes in HBeAg were identified using Immune Epitope Database consensus binding prediction. It was demonstrated that HBeAg treatment increased the numbers of Tregs in mouse spleens in vitro and in vivo. Furthermore, it was indicated that the HBeAg-mediated increase in Tregs occurred through the conversion of CD4+CD25- T cells into CD4+CD25+Foxp3+ Tregs. Additionally, in vitro study illustrated that HBeAg stimulated murine spleen cells to produce increased transforming growth factor-β, which is required to enable HBeAg to convert T cells into Tregs. The results of the present study may provide further evidence of the effect of HBeAg on Tregs and aid in the development of novel HBeAg-based immunotherapy for CHB.

Purification and characterisation of a xylose-specific mitogenic lectin from Fusarium sambucinum

A xylose-specific intracellular lectin, showing hemagglutination only with rabbit erythrocytes was purified from mycelium of Fusarium sambucinum which was designated as FSL. An array of anion exchange chromatography on Q-Sepharose and gel-exclusion chromatography on Sephadex G-100 resulted in 84.21% yield and 53.99-fold purification of lectin with specific activity of 169.53 titre/mg. Molecular weight of FSL determined by SDS-PAGE was 70.7 kDa, which was further confirmed by gel-exclusion chromatography. Native-PAGE analysis of FSL showed its monomeric nature. FSL was observed to be a glycoprotein containing 2.9% carbohydrate. Hapten inhibition profile of FSL displayed its strong affinity towards D-xylose (MIC 1.562 mM), L-fucose (MIC 6.25 mM), D-mannose (MIC 3.125 mM), fetuin (MIC 15.62 μg/mL), asialofetuin (MIC 125 μg/mL) and BSM (MIC 3.125 μg/mL). Affinity of FSL towards xylose is rare. FSL was found stable over a pH range 6.0–7.5 and upto 40 °C temperature. Hemagglutination activity of FSL remained unaffected by divalent ions. Lectin concentration of 5 μg/mL was found sufficient to stimulate proliferation of murine spleen cells and its concentration 75 μg/mL exhibited highest mitogenic potential. FSL exhibited maximum mitogenic stimulatory index of 14.35. The purification, characterisation and mitogenicity of F. sambucinum lectin has been reported first time.

Transition metal complexes of a multidentate Schiff base ligand containing guanidine moiety: Synthesis, characterization, anti-cancer effect, and anti-microbial activity

Abstract Guanidine Schiff bases are famous for their distinctive features and medicinal benefits with potential applications for the control of bacterial infections and cancer development. The current study was performed with a prospect to the dinuclear series of Cu(II), Ni(II), Co(II), Mn(II), Fe(III), Cr(III), Bi(III), and Zn(II) complexes of new guanidine Schiff base ligand. These complexes have been arranged and examined by 1H NMR, mass spectra, IR, UV–Vis, ESR spectra, magnetic moments, and conductance measurements, as well as, elemental and thermal analyses (DTA and TGA). The cytotoxicity, the anti-cancer effect, and the anti-microbial activity of these complexes were assessed. The IR spectral data showed that the Schiff base behaved as a neutral bidentate or monobasic bidentate ligand bonded to the metal particles through the azomethine nitrogen atom and the hydroxyl oxygen atom in protonated or deprotonated form embracing tetragonally distorted octahedral geometries. The new complexes were non-hygroscopic dissolvable in DMSO but insoluble in common natural solvents. A molar conductance, in DMF arrangement, demonstrated that the complexes were non-electrolytes. There was a variation in the cytotoxic activity of the complexes on murine spleen cells. Also, cytotoxic and apoptotic effects, indicative of the anti-cancer activity, of the twelve complexes, were noticed against the human hepatoma (HepG2) cell line. Additionally, the newly synthesized complexes showed enhanced anti-tumor and anti-microbial effects in comparison to the parent ligand and to the standard drug.

Synthesis and biological activities of amino acids functionalized α-GalCer analogues

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were synthesized by incorporation of l-amino acid methyl esters in the C-6′ position of glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated that they were potent CD1d ligands and could stimulate murine spleen cells into a higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3 showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as efficient X-ray crystallographic probes to reveal the relationship between glycolipids and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new potent immunostimulating agents.

Selective Histone Deacetylase 6 Inhibition Normalizes B Cell Activation and Germinal Center Formation in a Model of Systemic Lupus Erythematosus.

Autoantibody production by plasma cells (PCs) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. Histone deactylase 6 (HDAC6) is a unique cytoplasmic HDAC that modifies the interaction of a number of tubulin- associated proteins; inhibition of HDAC6 has been shown to be beneficial in murine models of SLE, but the downstream pathways accounting for the therapeutic benefit have not been clearly delineated. In the current study, we sought to determine whether selective HDAC6 inhibition would abrogate abnormal B cell activation in SLE. We treated NZB/W lupus mice with the selective HDAC6 inhibitor, ACY-738, for 4 weeks beginning at 20 weeks-of age. After only 4 weeks of treatment, manifestation of lupus nephritis (LN) were greatly reduced in these animals. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune, and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggest that a critical event might be modulation of cellular metabolism.

MicroRNA‑146a as a potential regulator involved in the pathogenesis of atopic dermatitis.

Previous studies have demonstrated that microRNA (miR)-146a is involved in the inflammatory response of atopic dermatitis (AD). The aim of the present study was to investigate the expression of miR-146a in the serum of patients with AD and in skin lesions of AD animal models. In addition, we aimed to predict and verify the target genes of miR-146a. miR-146a expression was measured in AD patient serum via reverse transcription-quantitative PCR. T-helper (Th)1 [CD4+; interferon (IFN)-γ+] and Th2 [CD4+; interleukin (IL)-4+] expression in peripheral blood mononuclear cells was evaluated using flow cytometry. Following the establishment of a 2,4-dinitrofluorobenzene-induced C57BL/6 mouse AD model, Th1 (CD4+IFN-γ+) and Th2 (CD4+IL-4+) expression was analyzed in murine spleen cells via flow cytometry. Plasmids were transfected into 293T cells and at 48 h post-transfection, cells were analyzed using a luciferase assay system. The results revealed that the AD group had a significantly lower Th1/Th2 ratio and a significantly higher miR-146a expression compared with the control group (P<0.05). Furthermore, a decreased Th1/Th2 ratio and a significantly increased miR-146a expression were observed in the model group compared with the control group (P<0.01). We also conducted a dual-luciferase assay to determine whether small ubiquitin-related modifier 1 (SUMO1) if the target gene of miR-146a. We observed a ~30% decrease in the relative luciferase activity in cells containing the 3′-untranslated region of SUMO1 + miR-146a). The results of the luciferase assay indicated that may be a direct mRNA target of miR-146a; however, the quantification of band density of SUMO1 expression following western blotting did not significantly differ. The development of animal models in AD research is of vital importance. The results revealed that miR-146a may be a potential regulator involved in the pathogenesis of AD. Furthermore, the current study determined that miR-146a could be a valuable marker of AD and thus, may be applied in the development of therapeutic strategies for treating AD.

Effect of Welsh onion (Allium fistulosum L.) green leaf extract on immune response in healthy subjects: a randomized, double-blind, placebo-controlled study

Background: Welsh onion belongs to genus allium. Although its medicinal benefit such as cold prevention is well known by tradition, the underlying mechanism and active components have never been elucidated. The recent study revealed that Welsh onion mucus enhances tumor necrosis factor-α and monocyte chemotactic protein-1 production from RAW 264 cells and natural killer (NK) cell activity in murine spleen cells. This mucilage was abundant in the leaf blade of Welsh onion. Thus, we investigated the effect of Welsh onion green leaf extract (GLE) on immune competence in the human clinical trial. Methods: A randomized, double-blind, placebo-controlled trial was conducted. Fifty-five healthy subjects were enrolled after the screening based on NK cell activity, and each sample (placebo, low-dose GLE or high-dose GLE) was supplemented for four weeks. The immune competence activity was evaluated through the primary endpoints that were NK cell activity and the score of immunological vigor (SIV). The SIV was calculated from lymphocyte subset analysis by flow cytometry method.Results: NK cell activity was enhanced in all supplemented groups including placebo, especially, it was significant in both GLE groups. The result of immune score showed that the immunity of high-dose GLE group did not change although it was significantly lowered in placebo group (p&lt;0.01), suggesting that GLE ameliorated the immunity suppression. In addition, when SIV was compared between pre- and post-intake, the ratios of the improved subjects in two GLE groups were higher than that of placebo group while those of the aggravated subjects showed the opposite result. In particular, they were remarkable in high-dose GLE group.Conclusion: These results suggest that the intake of low- or high-dose GLE might positively regulate immune competence.Keywords: welsh onion, immunity, natural killer cell, clinical trial

Progesterone modulates the T-cell response via glucocorticoid receptor-dependent pathways.

Steroid hormones such as progesterone and glucocorticoids rise during pregnancy and are accountable for the adaptation of the maternal immune system to pregnancy. How steroid hormones induce fetal tolerance is not fully understood. We hypothesized that steroid hormones selectively regulate the T-cell response by promoting T-cell death. We incubated murine spleen cells isolated from non-pregnant and pregnant mice with physiological concentrations of steroid hormones in vitro and analyzed T-cell subsets after 48h of incubation. Results We found that progesterone and the synthetic glucocorticoid dexamethasone induce T-cell death. CD4+ regulatory T (Treg ) cells were refractory toward progesterone-induced cell death, in contrast to conventional CD4+ T cells, which resulted in a preferential enrichment of CD4+ Treg cells in culture. T cells isolated from pregnant mice at early and late gestation showed comparable sensitivity to steroid-induced cell death. The target receptor for progesterone in immune cells is controversially discussed. We provide here support of progesterone binding to the glucocorticoid receptor as only T cells lacking the glucocorticoid but not the progesterone receptor showed resistance against progesterone-induced death. Conclusions Our results indicate that high levels of progesterone during pregnancy can induce selective T-cell death by binding the glucocorticoid receptor. Although physiological hormone concentrations were used, due to different bioavailability of steroid hormones in vivo these results have to be validated in an in vivo model. This mechanism might ensure immunological tolerance at the feto-maternal interface at gestation.

IFN-γ stimulates CpG-induced IL-10 production in Bcells via p38 and JNK signalling pathways.

The production of IL-10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN-γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL-10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN-γ on Toll-like receptor (TLR)-induced IL-10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN-γ inhibited IL-10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL-10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN-γ on TLR9-induced IL-10 was restricted to Bcells. In line with the increased IL-10, Bcells stimulated with CpG and IFN-γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen-activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 - an inhibitor of p38 and JNK activity - is downregulated after combined stimulation with IFN-γ and CpG. Our data may represent a novel immunoregulatory role of IFN-γ in Bcells after triggering of TLR9, by stimulating IL-10 production.