Cytokines In Culture Supernatants Research Articles

Inflammatory Response to Wear Particles: Comparisons Between Zirconia and Titanium In Vitro.

The objective of this study was to compare the inflammatory responses from peripheral blood mononuclear cells PBMCs subjected to Titanium (Ti) and/or Zirconia (Zr) particles while growing on Ti or Zr discs. In total, 240 discs were fabricated in the size of 2 mm height and 5 mm diameter. 120 discs were made of Ti (Commercially pure (CP grade 4) and 120 discs were made of Zr (3 Mol% Yttria-Stabilized Zirconia Polycrystals (3Y TZP). The PBMCs were cultured on the two-disc materials and particles with a size up to 20 μm Ti (99, 5 % Ti) and 0, 1 -2 μm Zr (3Y TZP) were added to the cultures. The concentration levels of inflammatory cytokines in culture supernatants were measured through Bioplex assay and light microscopic analysis was performed to detect cell attachment and characterize particle shape and cell-particle interaction. The inflammatory responses from PBMCs were generally higher when cells were cultured on a Ti surface compared to a Zr surface. In addition, higher cytokine levels were generally seen when cells were cultured in the presence of Ti particles compared to Zr particles in the absence of discs. However, there were only significantly increased levels for three cytokines (MCP-1, IFN-g, and TNF-a) when particles were added to the Ti discs. Higher release of NETS from neutrophils were seen in presence of Zr particles compared to Ti particles. And a lowering of cell death was seen when adding Zr particles compared to Ti particles and unstimulated control samples. Type of growth material and presence of particle affects PBMCs in vitro. Cells seeded on Ti disc and together with Ti particles generated higher levels of inflammatory cytokines compared to the Zr counterparts.

Characterization of CD4+ t cell subsets during Chlamydia muridarum genital infection

Characterization of CD4+ T cell subsets in a stressed mouse model during Chlamydia muridarum genital infection. Katrina Davis, Tesfaye Belay. Department of Applied Sciences and Mathematics.  Chlamydia genital infection caused by Chlamydia trachomatis is highly prevalent in low socioeconomic status populations. Whether stress is a risk factor for the high incidence of chlamydia remains to be investigated. CD4+ T cells are the major immune cells that fight chlamydia genital infection; however, the dynamics of CD4+ T cells during stressful conditions and chlamydia infection are unclear. The profiles of CD4+ T cell signature cytokines: T helper 1 (IL-12, Interferon-gamma (IFN-γ), Thelper2 (IL-4, IL-5, IL-13), Thelper17(IL-17), and Treg (IL-10). Mice were stressed for 5 minutes daily for 21 days and infected with 1x 105 IFU/ml of Chlamydia muridarum intravaginally. Non-stressed mice were infected as controls. Mice were sacrificed 7 days post-infection, the spleen and lymph nodes were harvested, and T cells were purified and proliferated for 72 h. Determination of the levels of cytokines in culture supernatants of T cells from stressed and non-stressed mice are underway for poster presentation.     Supported by the NIH grant # 1R15AI124156-01 awarded to BSU

Activation of murine macrophages by membrane proteins from Tritrichomonas foetus grown on iron- and calcium-rich conditions.

Tritrichomonas foetus is a protozoan parasite that causes a venereal disease in cattle limiting reproduction by abortions and sterility. The immune response against this parasite is poorly understood. Since the iron and calcium ions are important regulators of the microenvironment of the urogenital tract in cattle, we decided to evaluate the role of these divalent cations on the antigenicity of membrane proteins of T. foetus on macrophage activation as one of the first inflammatory responses towards this pathogen. Colorimetric methods and ELISA were used to detect the nitric oxide and oxygen peroxide production and expression of cytokines in culture supernatant from macrophage incubated with membrane proteins from T. foetus cultured in iron- and calcium-rich conditions. qRT-PCR assays were used to evaluate the transcript expression of genes involved in the inflammatory response on the macrophages. The membrane proteins used for in vitro stimulation caused the up-regulation of the iNOS and NOX-2 genes as well as the generation of NO and H2 O2 in murine macrophages on a dependent way of the metal concentrations. Additionally, after stimulation, macrophages showed a considerable rise in pro-inflammatory cytokines and a downregulation of anti-inflammatory cytokines, as well as up-regulation in the transcription of the TLR4 and MyD88 genes. These data suggest that membrane proteins of T. foetus induced by iron and calcium can activate an inflammatory specific macrophage response via TLR4/MyD88 signalling pathway.

Immunoprotective effect and mechanism of rEg.P29 against CD4 + T cell-deficient mice with Echinococcus multilocularis infection.

Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by infection with the larval stage of Echinococcus multilocularis and a major challenge to human public health. Vaccines are the most effective way to prevent and control infectious diseases. We previously revealed that the Echinocuccus granulosus recombinant protein P29 is a good vaccine candidate against E. granulosus. However, the protective and immunological mechanism of rEg.P29 against E. multilocularis remain unclear. In this study, CD4 + T cell-deficient mice are transferred with spleen CD4 + T cells isolated from wild-type mice and subjected to rEg.P29 immunization, and then these immunized mice are infected with E. multilocularis. The cyst inhibition rate is calculated by weighing the body and cyst weights. The level of antibody is detected by ELISA. Flow cytometry is used to detect the level of IFN-γ production by CD4 + T and CD8 + T cells. The cytokines in culture supernatant are detected by ELISA. The expressions of CD44 and CD62L on memory T cells are determined by flow cytometry. The results show the cyst inhibition rate is 41.52% after adoptive transfer of CD4 + T cells. Furthermore, the levels of IgG, IgM, IgA and IgE in serum are significantly increased compared with those in the PBS group. The IFN-γ-secretion by CD8 + T cells and the level of IFN-γ in culture supernatant are obviously increased; and the number of CD4 + T cells is increased, but the number of IFN-γ producing CD4 + T cells has no significant difference compared with PBS group. In addition, the number of CD44 +CD62L ‒CD8 + memory T cells in the spleen is significantly increased, while the number of CD44 ‒CD62L + CD8 + memory T cells is not significantly altered. Collectively, rEg.P29 can alleviate E. multilocularis infection by inducing humoral immune responses and CD8 + T cell responses.

Preclinical Assessment of Immunogenicity and Protectivity of Novel ROR1 Fusion Proteins in a Mouse Tumor Model

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a new tumor associated antigen (TAA) which is overexpressed in several hematopoietic and solid malignancies. The present study aimed to produce and evaluate different fusion proteins of mouse ROR1 (mROR1) to enhance immunogenicity and protective efficacy of ROR1. Four ROR1 fusion proteins composed of extracellular region of mROR1, immunogenic fragments of TT as well as Fc region of mouse IgG2a were produced and employed to immunize Balb/C mice. Humoral and cellular immune responses and anti-tumor effects of these fusion proteins were evaluated using two different syngeneic murine ROR1+ tumor models. ROR1-specific antibodies were induced in all groups of mice. The levels of IFN-γ, IL-17 and IL-22 cytokines in culture supernatants of stimulated splenocytes were increased in all groups of immunized mice, particularly mice immunized with TT-mROR1-Fc fusion proteins. The frequency of ROR1-specific CTLs was higher in mice immunized with TT-mROR1-Fc fusion proteins. Finally, results of tumor challenge in immunized mice showed that immunization with TT-mROR1-Fc fusion proteins completely inhibited ROR1+ tumor cells growth in two different syngeneic tumor models until day 120 post tumor challenge. Our preclinical findings, for the first time, showed that our fusion proteins could be considered as a potential candidate vaccine for active immunotherapy of ROR1-expressing malignancies.

Novel Th17 Lymphocyte Populations, Th17.1 and PD1+Th17, are Increased in Takayasu Arteritis, and Both Th17 and Th17.1 Sub-Populations Associate with Active Disease.

PurposeWe evaluated T helper lymphocyte profile, including novel Th17 subsets Th17.1 (secrete IFN-γ, associate with corticosteroid resistance) and PD1+Th17 (secrete TGF-β1, implicated in fibrosis), and related cytokines in peripheral blood of Takayasu arteritis (TAK).Materials and MethodsWe evaluated circulating Th1, Th2, Th17, Th17.1, PD1+CD4+ T lymphocytes, PD1+Th17, and Treg lymphocytes, inflammatory (IFN-γ, IL-4, IL-6, IL-17A, IL-23, IL-1β, TNF-α) and regulatory (IL-10, TGF-β1) cytokines in peripheral blood of TAK (n = 57; median age 35 (interquartile range 26–45) years; 40 females) in a cross-sectional design. We studied inflammatory and regulatory cytokines in culture supernatant of peripheral blood mononuclear cells (PBMCs) from TAK following stimulation with anti-CD3/anti-CD28 and their modulation by tacrolimus (immunosuppressive) with/without tadalafil (anti-fibrotic). Furthermore, we followed up immunosuppressive-naïve active TAK (n = 16) and compared T helper lymphocyte populations and cytokines before and after immunosuppressive therapy. Healthy controls (HC, n = 21) and sarcoidosis (disease control, n = 11) were compared against TAK.ResultsTAK had higher Th17, Th17.1 and PD1+Th17 lymphocytes than HC (p < 0.001), and higher PD1+CD4+ T lymphocytes than sarcoidosis (p < 0.001). Th17 lymphocytes associated with active TAK after multivariable-adjusted logistic regression (p = 0.008). TAK had greater cytokine secretion from PBMCs (IFN-γ, IL-17A, IL-10 versus HC; IL-6, TNF-α, IL-1β versus HC or sarcoidosis) (p < 0.05). In-vitro, PBMCs from TAK showed reduced secretion of all inflammatory cytokines with tacrolimus, with synergistic reduction in IL-17A, IL-6, IL-1β and IL-10 following addition of tadalafil to tacrolimus. Serial follow-up of immunosuppressive-naïve TAK (n = 16) showed reduction in serum IL-6 and TGF-β1 (p < 0.05) and IL-6 in culture supernatant (p < 0.05) following immunosuppressive therapy.ConclusionNovel Th17 sub-populations (Th17.1 and PD1+Th17) are elevated in TAK. Th17 lymphocytes associate with active TAK. In-vitro experiments on cultured PBMCs suggest promise for further evaluation of a combination of immunosuppressive tacrolimus with anti-fibrotic tadalafil (or other anti-fibrotic therapies) in clinical trials of TAK.

Proliferation and Directional Differentiation of iNKT Cells Derived from DBA/1 Mice Thymus.

The rates of invariant natural killer T (iNKT) cells in vivo are very low, and the amounts of cells obtained directly from the body are hard enough to fulfill their potential in clinical application. To overcome this problem, we subcutaneously injected alpha-galactosylceramide (α-GalCer) into DBA/1 mice and thymic single cells were isolated and cultured in vitro. Fluorescence-activated cell sorting was used to detect the iNKT cells and their subsets in the thymus after the injection of α-GalCer by different methods. In addition, in vitro changes of single-cell suspensions and their cytokines in culture supernatants were assessed. Compared with the α-GalCer multiple subcutaneous injection group, the rates of iNKT cells in the α-GalCer single subcutaneous injection group were markedly higher at each time point, while the highest levels of iNKT1 and iNKT2 cells were observed on day 4 and 8, respectively. In α-GalCer single subcutaneous injection for 8 days and thymic mononuclear cell cultured for 14 days group, the expansion rate of iNKT cells was significantly faster than the other groups, while it reached a peak for iNKT1 cells. Interferon-gamma was consistent with the development of iNKT1 cells, however no difference was found between the cultured iNKT cells in vitro and the natural iNKT cells in vivo in terms of cytokine production. Herein, we introduced a method in which antigenic stimulation in vivo and directed induction in vitro yielded high levels of iNKT cells with specific functions.

CMV Infection Is Directly Related to the Inflammatory Status in Chronic Heart Failure Patients.

High levels of inflammation play an important role in chronic heart failure (CHF). Patients with CHF have elevated levels of pro-inflammatory cytokines circulating systemically, mainly TNF and IL-6. However, there are almost no studies that relate these levels to the functional status of patients in CHF, much less to their CMV serostatus. In this study, patients with CHF (n=40; age=54.9 ± 6.3; New York Heart Association functional classification (NYHA, I-III) and healthy controls (n=40; age=53.5 ± 7.1) were analyzed. The serum concentrations of nine pro- and anti-inflammatory cytokines were measured by Luminex® xMap Technology and the basal level of mRNA expression of some immune molecules was quantified by TaqMan™ Array in CD4+ T-lymphocytes. The concentration of these cytokines in culture supernatants in response to anti-CD3 and LPS was also measured. The percentage of CD28null T-cells was determined, as well as the antibody titer against CMV. We found a higher concentration of all cytokines studied in CHF serum compared to healthy controls, as well as a direct correlation between functional status in CHF patients and levels of inflammatory cytokines. Moreover, the highest cytokine concentrations were found in patients with higher concentrations of lymphocytes lacking CD28 molecule. The cytokine production was much higher in CMV+ patients, and the production of these cytokines was found mainly in the T-lymphocytes of CMV+ patients in response to anti-CD3. Anti-CMV antibody levels were positively correlated with cytokine levels. The baseline expression of specific mRNA of the main molecules involved in the Th1 response, as well as molecules related to the CD4+CD28 null subset was higher in CMV+ patients. The cytokine concentrations are higher in CHF CMV+ patients and these concentrations are related to the production of antibodies against CMV. These high levels of cytokines are also associated with the more differentiated CD28null lymphocyte populations. All this, together with the dynamics of the pathology itself, makes CMV+ patients present a worse functional status and possibly a worse evolution of the pathology.

Porphyromonas gingivalis lipopolysaccharide promotes T-hel per17 cell differentiation by upregulating Delta-like ligand 4 expression on CD14+ monocytes.

BackgroudTo investigate the effect and mechanism of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on Th17 cell differentiation mediated by CD14+ monocytes. Methods P. gingivalis LPS-activated CD14+ monocytes were co-cultured with CD4+T cells in different cell ratios. An indirect co-culture system was also established using transwell chambers. Furthermore, anti- Delta-like ligand 4 (Dll-4) antibody was used to investigate the role of Dll-4 in Th17 cell response. The mRNA expression was analyzed using quantitative reverse transcription-polymerase chain reaction, and secreted cytokines in culture supernatant were detected using enzyme-linked immunosorbent assay. Flow cytometry was used to determine the frequencies of Th17 cells. IL-17 protein expression levels were determined using western blotting assay. ResultsP. gingivalis LPS increased the expressions of interleukin (IL)-1β, IL-6, IL-23 and transforming growth factor (TGF)-β in CD14+ monocytes. Th17 cell frequency upregulated, which is not solely cytokine-dependent but rather requires cell-cell contact with activated monocytes, particularly in the 1:10 cell ratio. Furthermore, P. gingivalis LPS increased t he expression of Dll-4 on CD14+ monocytes, whereas the anti- Dll-4 a ntibody decreased the response of Th17 cells. The results suggest that P. gingivalis LPS enhances Th17 cell response via Dll-4 upregulation on CD14+ monocytes.

PDCD4 regulates apoptosis in human peritoneal mesothelial cells and promotes gastric cancer peritoneal metastasis.

Programmed cell death 4 (PDCD4) is a tumor suppressor gene, however, the function and regulatory mechanism remain to be discovered. The connection between tumorigenesis and apoptosis is one of the most important foci of cancer research. Our study aimed to explore the connections between PDCD4-mediated apoptosis of human peritoneal mesothelial cells (HPMC) and peritoneal metastasis in gastric cancer. The PDCD4 expression in 31 pairs of HPMC and tumor tissues was assessed by immunohistochemistry and RT-PCR. In cell experiments, we monitored gastric cancer cell migration with a Transwell chamber assay when PDCD4 was silenced in HPMC. Subsequently, apoptosis of HPMC was detected by a flow cytometric assay and western blotting. After analyzing cytokines in culture supernatants from gastric cancer with enzyme-linked immunosorbent assays (ELISAs), transforming growth factor-beta 1 (TGF-β1) was abundant in the culture supernatants of gastric cancer. Then, PDCD4 expression in HMrSV5 cells was analyzed by western blotting after retreatment with different concentrations of TGF-β1. Moreover, apoptosis of peritoneal mesothelial cells treated with TGF-β1 was detected according to the above methods. In human metastatic peritoneal tissues, the expression of PDCD4 was significantly lower than that in normal tissues. At the same time, decreased expression of PDCD4 in HPMC was associated with increased migration capacity of gastric cancer cells. Moreover, suppressing the expression of PDCD4 promoted apoptosis in mesothelial cells which may be regulated by TGF-β secreted from gastric cancer cells. These data suggested that decreased expression of PDCD4 significantly promoted apoptosis in human peritoneal mesothelial cells, thus inducing peritoneal metastasis, and that TGF-β1 secreted from gastric cancer cells may have played a crucial role.

Botanical formulation, TADIOS, alleviates lipopolysaccharide (LPS)-Induced acute lung injury in mice via modulation of the Nrf2-HO-1 signaling pathway

Ethnopharmacological relevanceTADIOS is an herbal formulation prepared from a mixture of Taraxacum officinale (L.) Weber ex F.H.Wigg, Dioscorea batatas Decaisne and Schizonepeta tenuifolia (Benth.) Briquet. These plants have traditionally been used in Asia to treat a variety of respiratory diseases. A bulk of literature on traditional Korean medicine describe their activities and functions for respiratory problems. Therefore, we hypothesized that the combination of these plants might be effective in alleviating respiratory symptoms. Aim of the studyIn this study, we investigated whether TADIOS ameliorates LPS-induced acute lung injury via regulation of the Nrf2-HO-1 signaling pathway. Materials and methodsThe LPS-induced acute lung injury mouse model was used to determine the anti-inflammatory and anti-oxidative stress effects of TADIOS. The amount of marker compounds contained in TADIOS was quantified using high-performance liquid chromatography (HPLC) analysis. The protein level of pro-inflammatory cytokines in culture supernatant was measured by ELISA. Changes in the RNA level of pro-inflammatory cytokines in mice lungs and RAW264.7 cells were measured by quantitative RT-PCR. The relative amounts of reactive oxygen species (ROS) were measured by DCF-DA assay. Western blot analysis was used to evaluate expression of cellular proteins. Effects of TADIOS on antioxidant responsive elements (AREs) were determined by luciferase assay. The severity of acute lung injury was evaluated by Hematoxylin & Eosin (H&E) staining. To test the effects of TADIOS on LPS-induced oxidative stress, myeloperoxidase (MPO) activity and the total antioxidant capacity were measured. ResultsTADIOS was prepared by extraction of a blend of these three plants by ethanol, and quality control was performed through quantification of marker compounds by HPLC and measurement of bioactivities using cell-based bioassays. In the murine macrophage cell line RAW264.7, TADIOS effectively suppressed the production of pro-inflammatory cytokines such as IL-6 and IL-1β, and also ROS induced by LPS. When RAW264.7 cells were transfected with a luciferase reporter plasmid containing nucleotide sequences for AREs, TADIOS treatment increased the level of relative luciferase units in a dose-dependent manner. In the LPS-induced acute lung injury mouse model, orally administered TADIOS alleviated lung damage and neutrophil infiltration induced by LPS. Consistent with the in vitro data, treatment with TADIOS inhibited the LPS-mediated expression of pro-inflammatory cytokines and oxidative stress, and activated the Nrf2-HO-1 axis. ConclusionOur data suggest the potential for TADIOS to be developed as a safe and effective therapeutics for the treatment of acute respiratory distress syndrome.

Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells (CYNK-001) Mediate Protection Against Influenza A Viral Infection

Background: Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells provide the first line of innate defense against IAV by killing infected epithelial cells, by producing antiviral cytokines and affecting adaptive immunity. Preclinical studies have demonstrated that NK cells play a pivotal role in reducing IAV-induced pulmonary infection; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Celularity Inc. is developing human placental hematopoietic stem cell-derived allogeneic, off-the-shelf NK cell therapy (CYNK-001) for the treatment of viral infections, including coronavirus disease of 2019. Here, we report the evaluation of antiviral activities of CYNK-001 against IAV infection.Methods:In vitro antiviral activities of CYNK-001 were evaluated using human alveolar epithelial cell line A549, infected with IAV strain A/PR/8/34 (H1N1) at variable multiplicity of infection (MOI). The expression of ligands for NK cell receptors was analyzed on infected A549 cells using Fc-coupled recombinant proteins. CYNK-001 was added to A549 cells 16 hours post infection. CYNK-001 degranulation was measured after 4 hours of coculture, and CYNK-001 cytotoxicity against IAV-infected A549 was measured real-time using impedance-based xCELLigence platform. In vivo antiviral and immunomodulatory activities of CYNK-001 were assessed in A/PR/8/34 (H1N1)-induced severe acute lung injury mouse model. Mice were intranasally infected with 2500 PFU IAV. PBS or 1 x 107 CYNK-001 cells were intravenously administered twice at 1 and 3 days post infection (dpi). At 6 dpi, lungs were collected for the evaluation of viral load by qPCR, lung injury and immune cell profiling by histology. Bronchoalveolar lavage fluid (BALF) was collected at 6 dpi for cytokine analysis by multiplex assays, total protein concentration by ELISA and immune cell profiling by flow cytometry.Results:In vitro, IAV infection corresponded with dose-dependent expression of ligands to NK cell-activating receptors, including NKp44, NKp46 and NKG2D. CYNK-001 cells exhibited increased IFNγ, TNFα and GM-CSF production, and elevated level of degranulation upon coculture with IAV-infected A549 cells. Cytokines in culture supernatant and CD107a expression in CYNK-001 cells were upregulated in a virus dose-dependent manner. Consistent with this finding, CYNK-001 cytotoxicity against IAV-infected A549 cells increased from 35% at 0 MOI to 50%, 60% and 75% at 0.001, 0.01 and 0.1 MOI, respectively. These data indicate that CYNK-001 cells recognize virally infected cells, resulting in specific cytotoxic elimination of the source of infection. In vivo, treatment of IAV-infected mice with CYNK-001 reduced weight loss and increased their likelihood of survival. PBS control group developed a severe disease and 37.5% mortality was observed as early as day 4. In the group treated with CYNK-001, disease onset was delayed by 2 days. qPCR analysis of viral RNA showed that CYNK-001-treated mice had lower viral load in the lung than vehicle-treated mice, demonstrating antiviral function of CYNK-001 in vivo. CYNK-001-treated mice had reduced lung injury as assessed by lower total protein concentration in BALF. Moreover, CYNK-001 reduced BALF murine cytokines and chemokines, including IFNγ (p<0.001), IL-6, TNFα, MCP-1 (p<0.05), CXCL2 and CXCL9. Lastly, immunohistochemical analysis of the lung showed that CYNK-001-treated mice had an altered immune response to IAV with higher number of CD68+ macrophages and CD8+ T cells at 6 dpi.Conclusions: Our in vitro and in vivo data show the promising antiviral activities of CYNK-001 against IAV infection. In a severe IAV infection mouse model, CYNK-001 treatment demonstrates lower mortality rate, lower weight loss, lower lung viral load and reduced lung injury along with reduced inflammation. These results support our hypothesis that the adoptive transfer of CYNK-001 could reduce the burden of viral infection through the elimination of infected epithelial cells, coordinate a more effective immune response, and result in a clinical benefit in patients with severe viral infection.DisclosuresHe:Celularity Inc.: Current Employment. Mahlakõiv:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. Van Der Touw:Celularity Inc.: Current Employment. Kang:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.

Th2-Immune Polarizing and Anti-Inflammatory Properties of Insulin Are Not Effective in Type 2 Diabetic Pregnancy.

Background The implication of the immune system in the physiopathology of pregnancy complicated by diabetes has been reported. Here, we investigated the effects of insulin treatment on the frequencies of immune cell subpopulations as well as T cell-derived cytokines in type 2 diabetic (T2D) pregnancy compared to gestational diabetes mellitus (GDM). Methods Fifteen (15) women with GDM, twenty (20) insulin-treated T2D pregnant women, and twenty-five (25) pregnant controls were selected. Immune cell subpopulation frequencies were determined in blood using flow cytometry. The proliferative capacity of T cells was performed, and serum and cell culture supernatant cytokine levels were also quantified. Results The frequencies of total CD3+ and CD4+ T cells and nonclassical monocytes significantly increased in insulin-treated T2D pregnant women compared to pregnant controls. The proportions of CD4+ T cells as well as B cells were significantly higher in women with GDM than in pregnant controls. GDM was associated with high frequencies of total CD3+ and CD4+ T cells and B cell expansion, suggesting a concomitant activation of cellular and humoral immunity. Concomitantly, Th1/Th2 ratio, determined as IFN-γ/IL-4, was shifted towards Th1 phenotype in women with GDM and insulin-treated T2D pregnant women. Besides, isolated T cells elicited similar proliferative capacity in the three groups of women. Insulin-treated T2D pregnant women and women with GDM exhibited a low serum IL-10 level, without any change in the number of Treg cells. Conclusion Our study showed that, despite insulin treatment, pregnant women with T2D displayed a proinflammatory status consistent with high proportions of CD3+ and CD4+ T cells, upregulation of Th1 cytokines, and low IL-10 production, suggesting a reduced immune-suppressive activity of regulatory T cells. However, GDM, although associated with proinflammatory status, has shown increased humoral immunity consistent with high proportion of CD19+ B cells. Thus, the lack of response to insulin in diabetes during pregnancy and clinical implications of these immunological parameters deserves further investigations.

Suppression of miR-203-3p inhibits lipopolysaccharide induced human intervertebral disc inflammation and degeneration through upregulating estrogen receptor α.

Accumulating evidence demonstrates that estrogen receptor α (ERα) and microRNAs (miRNAs) play crucial roles in intervertebral disc degeneration (IDD). However, the specific miRNA that related with ERα during IDD development remains unknown. Therefore, we aimed to explore the role of ERα-related miRNA in the IDD model. Nucleus pulposus (NP) cells were isolated from IDD patients. ERα-related miRNAs were selected and verified in NP tissues from IDD patients using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Also, the related cytokine mRNA levels were detected by qRT-PCR. Protein levels were determined by Western blot. The concentrations of inflammatory cytokines in culture supernatants were detected by enzyme-linked immunosorbent assay. MiR-203-3p was found to be upregulated in NP tissues of high-grade IDD patients compared with low-grade IDD patients, and negatively associated with ERα expression. MiR-203-3p directly targeted ERα in NP cells of IDD patients. After lipopolysaccharides (LPS) stimulation, miR-203-3p expression increased, while ERα expression decreased in NP cells. MiR-203-3p inhibition suppressed the effect of LPS on ERα expression and IDD related genes, while ERα downregulation rescued the effect of LPS. In conclusion, suppression the expression of miR-203-3p could inhibit LPS-induced human intervertebral disc inflammation and degeneration through upregulating ERα.

Activation of IL-10+ B cells: A novel immunomodulatory mechanism for therapeutic bacterial suspensions.

Objectives:Bacterial components are used to improve immune responses in patients with respiratory infections. Pharmacological formulations of bacterial components include a mixture of bacterial antigens, some of which are complete inactivated bacteria, that is, named bacterial suspensions; while others are fragments of bacteria, which are presented as bacterial lysates. Although bacterial lysates have been broadly used as immune-stimulators, the biological support for the therapeutic effectiveness of bacterial suspension has not yet been studied. Thus, the aim of our study was to investigate the immunological activity induced by bacterial suspension.Methods:This work was an exploratory translational study. Peripheral blood mononuclear cells were obtained from healthy donors and cultured in time–dose dependent assays with a commercial bacterial suspension. Flow cytometry was used for phenotypic analysis and for determining soluble cytokines in culture supernatants.Results:We observed that bacterial suspension activates B cells in a dose-dependent manner. Peripheral blood mononuclear cells were able to secrete IL-6 and IL-10 after 24 h of bacterial suspension stimulation. TLR2 expression was observed mainly on CD19+ CD38Lo B cells after 72 h of culture; remarkably, most of the TLR2+ CD19+ cells were also IL-10+.Conclusion:Our findings suggest that bacterial suspension induces the activation of B cell subsets as well as the secretion of IL-6 and IL-10. Expression of TLR2 on CD19+ cells could act as an activation loop of IL-10+ B regulatory cells. The clinical implications of these findings are discussed at the end of this article.

Differentiation plasticity of human monocytes in culture

&amp;lt;p&amp;gt;Introduction. Human monocytes are heterogeneous and plastic cell population with the ability to undergo phenotypic and functional changes as a response to a stimulus from a local microenvironment. Our aim was to determine the potential of human monocytes to differentiate into different cell populations depending on two different cytokines (IL-4 and IL-6) added to cultures as well as to compare their phenotypical and functional characteristics. Methods. Peripheral blood mononuclear cells (PBMNC) were isolated from buffy coats of healthy donors. Monocytes, which were separated from PBMNC by plastic adherence, had been cultivated in Dendritic cell (DC), serum free medium for 5 days, either with granulocyte/macrophage colony-stimulating factor (GM-CSF) alone or with GM-CSF, with addition of interleukin 4 (IL-4) or interleukin 6 (IL-6), respectively. After cultivation, phenotypic characteristics of these cells were analyzed by flow cytometry, whereas the levels of produced cytokines in culture supernatants were quantified by ELISA. The potential of differentiated cells to modulate the proliferation of allogeneic T cells was examined by co-cultivation of these cells with PBMNC. Results. GM-CSF differentiated monocytes into M0/M1 macrophages (M&amp;amp;Oslash;). The combination of GM-CSF and IL-4 favoured differentiation of immature DC, whereas GM-CSF and IL-6 transformed monocytes into monocytic myeloid derived suppressor cells (M-MDSC). All cell populations expressed typical monocyte/macrophage markers such as CD14, CD11b, CD16 and CD33, HLA-DR, CD209 and CD86, a co-stimulatory marker. DC and M-MDSC expressed CD1a and CD11c, in contrast to M0/M1 M&amp;amp;Oslash;. The expression of HLA-DR, CD1a, CD209 and CD86 was highest on DC. The expression of CD33 and CD16 was highest on M-MDSC, followed by lowest expression of HLA-DR. The potential of promoting T-cell proliferation was highest in cultures of PBMNC with DC, whereas M-MDSC had the opposite, suppressive, effect. These differences correlated with highest production of immunosuppressive cytokines such as IL-10, IL-27 and TGF-b by M-MDSC. Conclusion. This study confirmed the differentiation plasticity of human monocytes, which are influenced by cytokines added in cultures. Phenotypic characteristics of these cells correlated with the production of cytokines involved in modulation of T-cell proliferation.&amp;lt;/p&amp;gt;

Simvastatin Enhances the Immune Response Against Mycobacterium tuberculosis.

Tuberculosis remains a serious threat worldwide. For this reason, it is necessary to identify agents that shorten the duration of treatment, strengthen the host immune system, and/or decrease the damage caused by the infection. Statins are drugs that reduce plasma cholesterol levels and have immunomodulatory, anti-inflammatory and antimicrobial effects. Although there is evidence that statins may contribute to the containment of Mycobacterium tuberculosis infection, their effects on peripheral blood mononuclear cells (PBMCs) involved in the immune response have not been previously described. Using PBMCs from 10 healthy subjects infected with M. tuberculosis H37Rv, we analyzed the effects of simvastatin on the treatment of the infections in an in vitro experimental model. Direct quantification of M. tuberculosis growth (in CFU/mL) was performed. Phenotypes and cell activation were assessed via multi-color flow cytometry. Culture supernatant cytokine levels were determined via cytokine bead arrays. The induction of apoptosis and autophagy was evaluated via flow cytometry and confocal microscopy. Simvastatin decreased the growth of M. tuberculosis in PBMCs, increased the proportion of NKT cells in culture, increased the expression of co-stimulatory molecules in monocytes, promoted the secretion of the cytokines IL-1β and IL-12p70, and activated apoptosis and autophagy in monocytes, resulting in a significant reduction in bacterial load. We also observed an increase in IL-10 production. We did not observe any direct antimycobacterial activity. This study provides new insight into the mechanism through which simvastatin reduces the mycobacterial load in infected PBMCs. These results demonstrate that simvastatin activates several immune mechanisms that favor the containment of M. tuberculosis infection, providing relevant evidence to consider statins as candidates for host-directed therapy. They also suggest that future studies are needed to define the roles of statin-induced anti-inflammatory mechanisms in tuberculosis treatment.

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor (M-CSF) to prepare macrophages. The purity of CD14+ CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-infected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pretreating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2.66±0.53, P<0.05) and then decreased. The purity of macrophages detected by flow cytometry was (89.16±2.07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16.10±0.17 and 29.76±0.58, while the expression levels at 24 h in the uninfected group were 1.46±0.67 and 1.60±0.54, respectively. Expression of IL-6, IL-8, TNF-α and IL-1β at mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-α and IL-1β expression at 24 h were 45.82±3.72, 52.34±1.69 (12 h), 8.94±1.75 and 30.96±1.44 in the infected macrophages, and 1.16±0.22, 1.15±0.21, 1.11±0.09 and 1.47±0.31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infected HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-culture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0.01) and that of IL-6, IL-8, TNF-α and IL-1β in macrophages (P<0.01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-α and IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent. Key words: Dengue virus type 2 (DENV-2); Human umbilical vein endothelial cell (HUVEC); Macrophage; Cytokine; Immunoregulation

Cold-induced stress alters the dynamics of T helper 1 and T helper 2 cytokine production in a mouse model during Chlamydia muridarum genital infection.

Chlamydia caused by Chlamydia trachomatis is the leading cause of bacterial STDs worldwide and the USA. Although there are more clinical and epidemiological studies of chlamydia genital infection, the effect of stress on chlamydia disease is not well explored. Previous results from our lab show that cold-induced stress results in increased intensity of chlamydia genital infection in mice; however, the mechanisms are unknown. Thelper 1(Th1) is known to be protective against chlamydia, whereas Th2 leads to infection.  However, the differentiation of Th1 and Th2 during exposure to cold stress is not well understood. Several studies show GATA-3 and T-bet are associated with Th1 and Th2 cytokine production. The purpose of this study was to evaluate the effect of cold water on the activity off Th1 and Th2 during Chlamydia muridarum genital infection. We hypothesized that cold stress alters the differentiation and variation in gene expression profile of transcription factors and cytokine productions in T cells. T cells isolated from the genital tract were proliferated for 48 h incubated at 37OC and 5% CO2. The mRNA levels and detection of cytokines in culture supernatants was determined by quantitative real time PCR and ELISA, respectively. Our results showed increased IL-4 production, which was associated with upregulated expression of transcription factor GATA-3. In contrast, decreased gamma interferon production was associated with decreased T-bet gene expression in T cells. Based on this data, cold stress modulates the dynamics of signature Th1 and Th2 cytokine productions that are regulated by expression of GATA-3 and T-bet.  This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.

Production of immunoregulatory cytokines in clinically asymptomatic periapical lesions depends on the lesions size

Bacground/Aim. Development of periapical lesions (PLs) involves a complex cross-talk between pathogenic microorganisms from the root canal and host immune mechanisms. In these processes proinflammatory cytokines are involved in stimulation of inflammation and osteodestructive mechanisms, whereas anti-inflammatory cytokines, with the immunoregulatory functions, have the opposite effects. How this balance is controlled is still the subject of numerous studies. The aim of this study was to examine whether the local production of interleukin (IL)-10, IL-27 and transforming growth factor (TGF)-? in human asymptomatic PLs depends on the lesion size and how levels of these immunoregulatory cytokines correlate with the cellular composition of PLs. Methods. The study was conducted on 30 PLs which were collected at the Clinic for Stomatology of the Military Medical Academy, Belgrade, Serbia. The PLs were divided according to their size into small- and largesize lesions (n = 12 and n = 18, respectively). The inflammatory cells (PL-ICs) were isolated from PLs and cultivated for 24 hours in culture medium supplemented with phorbol myristate acetate and Ca2+ ionophore. Cytospins were processed for immunocytology by using monoclonal antibodies to cell subsets. The levels of cytokines in culture supernatants were determined by the ELISA method. Statistical analysis was performed using the Student t-test and the Spearman?s correlation test. The values of p &lt; 0.05 were considered to be significant. Results. The levels of IL-10 and TGF-? were significantly higher in the PL-ICs cultures of large-size lesions than in small ones (p &lt; 0.01 and p &lt; 0.05, respectively). In contrast, the levels of IL-27 were higher in the cultures of small-size lesions than in small ones (p &lt; 0.05). Although the total number of PL-ICs and the proportion of mononuclear phagocytes were higher in the large-size PLs (p &lt; 0.01 and p &lt; 0.05, respectively), their main composition was not significantly different between the groups. The proportions of B cells/plasma cells (CD19/CD38+ cells), CD8+ T cells and CD14+ cells were significantly higher in the large-size PLs (p &lt; 0.005; p &lt; 0.05; p &lt; 0.05, respectively). In contrast, the proportion of total T cells (CD3+ cells) was higher in the small-size lesions (p &lt; 0.05). No statistically significant correlation was found between the levels of these cytokines and the composition/ phenotype of PL-ICs. Conclusion. This study suggests that IL-10, IL-27 and TGF-? may play different roles in suppression of unwanted immune/inflammatory reactions in asymptomatic PLs, depending on the extension of pathological process as judged by the size of lesions.