Proliferation Of Peripheral Blood Mononuclear Cells Research Articles

Human umbilical cord-derived mesenchymal stem cells suppress proliferation of PHA-activated lymphocytes in vitro by inducing CD4+CD25highCD45RA+ regulatory T cell production and modulating cytokine secretion

Bone marrow-derived mesenchymal stem cells (MSCs) are promising candidate cells for therapeutic application in autoimmune diseases due to their immunomodulatory properties. Unused human umbilical cords (UC) offer an abundant and noninvasive source of MSCs without ethical issues and are emerging as a valuable alternative to bone marrow tissue for producing MSCs. We thus investigated the immunomodulation effect of umbilical cord-derived MSCs (UC-MSCs) on human peripheral blood mononuclear cells (PBMCs), T cells in particular, in a co-culture system. We found that UC-MSCs efficiently suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated PBMCs (p<0.01). Kinetic analysis revealed that UC-MSCs primarily inhibited the division of generation 3 (G3) and 4 (G4) of PBMCs. In addition, UC-MSCs augmented the expression of CD127+ and CD45RA+ but reduced the expression of CD25+ in PBMCs stimulated by PHA (p<0.05). Furthermore, UC-MSCs inhibited PHA-resulted increase in the frequency of CD4+CD25+CD127low/− Tregs significantly (p<0.01) but augmented PHA-resulted increase in the frequency of CD4+CD25highCD45RA+ Tregs to about three times in PBMCs. The levels of anti-inflammatory cytokines, PEG2, TGF-β, and IL-10 were greatly up-regulated, accompanied by a significant down-regulation of pro-inflammatory IFN-γ in the co-culture (p<0.01). Our results showed that UC-MSCs are able to suppress mitogen-induced PBMC activation and proliferation in vitro by altering T lymphocyte phenotypes, increasing the frequency of CD4+CD25highCD45RA+ Tregs, and modulating the associated cytokine production. Further studies are warranted to investigate the therapeutic potential of UC-MSCs in immunologically-diseased conditions.

Immunomodulatory effects ofEchinacea laevigataethanol tinctures produced from different organs

Echinacea supplements may prevent or reduce symptoms of upper respiratory infections by immunomodulation, possibly by altering the cytokine profile. Compared with other species in the genus, the immunomodulatory properties of Echinacea laeviagata are poorly characterized. The purposes of this study were to compare the diversity and quantity of known bioactive compounds from aboveground organs of E. laevigata, and to characterize the in vitro immumodulatory properties of ethanol tinctures produced from those structures. High-performance liquid chromatography (HPLC) was used to determine the levels of alkamides and caffeic acid derivatives. Peripheral blood mononuclear cells (PBMCs) were obtained from 16 adults and challenged in vitro with extracts. PBMC proliferation and production of the cytokines interleukin-2 (IL-2), IL-10 and tumour necrosis factor-α (TNF-α) were measured. Fresh flower, leaf and root extracts were able to augment TNF and IL-10 and prolif eration, whereas fresh stem extract was only able to augment TNF. Extracts produced from flowers contained the greatest bioactive compound quantities and diversity. Caftaric acid was the most abundant compound and correlated with some (but not all) observed immune effects. These results suggest that of all aboveground parts, flowers have the greatest abundance and diversity of known bioactive compounds, and both flower and leaf extracts were immunomodulators.

Adipose-derived mesenchymal stem cells from infrapatellar fat pad of patients with rheumatoid arthritis and osteoarthritis have comparable immunomodulatory properties

Adipose-derived mesenchymal stem cells (ASCs) possess immunosuppressive properties, but their activity is dependent on stimuli provided by local environment. It is possible that proinflammatory milieu of rheumatoid joint affects ASCs function. To verify this hypothesis, rheumatoid ASCs (RA-ASCs) and osteoarthritic ASCs (OA-ASCs) derived from infrapatellar fat pad (IPFP) of the knee joint have been compared. RA- and OA-ASCs isolated from patients were cultured in vitro. Their secretory and proliferative activity was measured. Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with ASCs. Then, PBMCs proliferation was measured by 3H-thymidine incorporation method, cytokines secretion by immunoassays, T cells activation and regulatory T cells (Tregs) percentage – by flow cytometry. RA- and OA-ASCs properties in vitro were comparable, however, some differences in secretory activity occurred. RA- and OA-ASCs inhibited PBMCs proliferation and induced interleukin 10 production but up-regulated interleukin 17 A secretion and failed to limit release of other proinflammatory mediators (tumor necrosis factor [TNF], interferon γ [IFNγ], CCL5) by PBMCs. RA- and OA-ASCs did not suppress activation markers expression on T cells and did not trigger Tregs expansion. The present study shows that IPFP-ASCs from RA and OA patients have comparable functions in vitro. Their immunosuppressive activity seems to be impaired comparing to available data.

Vitamin A Decreases Cytotoxicity of Oxidized Low-Density Lipoprotein in Patients with Atherosclerosis

ABSTRACTBackground: Oxidized low-density lipoprotein (ox-LDL) is implicated in initiation and progression of atherosclerosis. Previously, we found that ox-LDL increases vulnerability of peripheral blood mononuclear cells (PBMCs) in atherosclerotic patients compared to controls. Vitamin A induces proliferation of PBMCs. The aim of this study was to determine the effect of vitamin A supplementation on PBMC survival against LDL and different doses of ox-LDL.Method: In this double-blind placebo-controlled trial, we recruited 35 atherosclerotic patients and 38 healthy controls and randomly allocated them into placebo and vitamin A groups, which received either placebo or 25,000 IU/day of vitamin A for 3 months. PBMCs were isolated, cultured, and stimulated by 1 µg/mL LDL as well as 1 µg/mL and 50 µg/mL ox-LDL. The stimulation indexes (SIs) of PBMCs were calculated to identify cell viability. Additionally, the circulating ox-LDL levels were measured by ELISA.Results: Viability of PBMCs stimulated by 50 µg/mL ox-LDL significantly increased following vitamin A supplementation in patients (p < 0.01). The levels of circulating ox-LDL were not changed by vitamin A treatment. Ox-LDL levels were strongly and positively correlated to SI of PBMCs stimulated by 1 µg/mL LDL and1 µg/mL ox-LDL in all groups.Conclusion: Vitamin A decreases cytotoxicity of high-dose ox-LDL and improves PBMC viability. The protective effect of vitamin A is not mediated by an antioxidative mechanism, but may instead have been due to intracellular protection of the apoptotic machinery or induction of proliferation of the cells. Higher levels of ox-LDL increase PBMC irritability in all participants.

Clinical efficacy of a new CD28-targeting antagonist of T cell co-stimulation in a non-human primate model of collagen-induced arthritis.

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co-stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen-induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA-4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C-reactive protein (CRP) and interleukin (IL)-6 and anti-collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII-induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.

The Comparison of the Immunologic Properties of Stem Cells Isolated from Human Exfoliated Deciduous Teeth, Dental Pulp, and Dental Follicles.

Aim. To compare the effects of various mesenchymal stem cells, those isolated from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and dental follicle stem cells (DFSCs), on human peripheral blood mononuclear cells (PBMCs). Method. Mesenchymal stem cells were isolated from three sources in the orofacial region. Characterization and PCR analyses were performed. Lymphocytes were isolated from healthy peripheral venous blood. Lymphocytes were cocultured with stem cells in the presence and absence of IFN-γ and stimulated with anti-CD2, anti-CD3, and anti-CD28 for 3 days. Then, lymphocyte proliferation, the number of CD4+FoxP3+ T regulatory cells, and the levels of Fas/Fas ligand, IL-4, IL-10, and IFN-γ in the culture supernatant were measured. Results. The DFSCs exhibited an enhanced differentiation capacity and an increased number of CD4+FoxP3+ T lymphocytes and suppressed the proliferation and apoptosis of PBMCs compared with SHEDs and DPSCs. The addition of IFN-γ augmented the proliferation of DFSCs. Furthermore, the DFSCs suppressed IL-4 and IFN-γ cytokine levels and enhanced IL-10 levels compared with the other cell sources. Conclusion. These results suggest that IFN-γ stimulates DFSCs by inducing an immunomodulatory effect on the PBMCs of healthy donors while suppressing apoptosis and proliferation and increasing the number of CD4+FoxP3+ cells.

Adiponectin Isoforms and Leptin Impact on Rheumatoid Adipose Mesenchymal Stem Cells Function.

Adiponectin and leptin have recently emerged as potential risk factors in rheumatoid arthritis (RA) pathogenesis. In this study we evaluated the effects of adiponectin and leptin on immunomodulatory function of adipose mesenchymal stem cells (ASCs) derived from infrapatellar fat pad of RA patients. ASCs were stimulated with leptin, low molecular weight (LMW) and high/middle molecular weight (HMW/MMW) adiponectin isoforms. The secretory activity of ASCs and their effect on rheumatoid synovial fibroblasts (RA-FLS) and peripheral blood mononuclear cells (PBMCs) from healthy donors have been analysed. RA-ASCs secreted spontaneously TGFβ, IL-6, IL-1Ra, PGE2, IL-8, and VEGF. Secretion of all these factors was considerably upregulated by HMW/MMW adiponectin, but not by LMW adiponectin and leptin. Stimulation with HMW/MMW adiponectin partially abolished proproliferative effect of ASC-derived soluble factors on RA-FLS but did not affect IL-6 secretion in FLS cultures. ASCs pretreated with HMW/MMW adiponectin maintained their anti-inflammatory function towards PBMCs, which was manifested by moderate PBMCs proliferation inhibition and IL-10 secretion induction. We have proved that HMW/MMW adiponectin stimulates secretory potential of rheumatoid ASCs but does not exert strong impact on ASCs function towards RA-FLS and PBMCs.

Zinc enhances the number of regulatory T cells in allergen-stimulated cells from atopic subjects.

The trace element zinc is essential for immune function and its regulation. Since zinc deficiency and allergic hyperresponsive reactions are often accompanied, the influence of zinc on allergen-induced cell growth, CD4+ regulatory T (Treg) cell numbers and cytokine expression during allergic immune reactions was investigated. Peripheral blood mononuclear cells (PBMCs) from non-atopic and atopic subjects were treated with timothy grass allergen pre-incubated with or without zinc. Proliferation was determined by analyzing the incorporation of 3H-thymidine. Intracellular zinc and Foxp3 levels and cell surface antigens were measured by FACS, cytokine expression by ELISA and real-time PCR. Incubation with 50μM zinc sulfate (Zn50) enhances cytosolic zinc concentrations in CD3+ T cells. The data also reveal that the combination of Zn50 plus allergen significantly reduces PBMC proliferation of atopic subjects. Additionally, Zn50 plus allergen enhances Th1 cytokine responses shown by increased interferon (IFN)-γ/interleukin (IL)-10 ratios as well as enhanced tumor necrosis factor-α release. In response to allergen, zinc increases Treg cells and upregulates the mRNA expression of cytotoxic T-lymphocyte antigen-4 in atopic subjects. Interestingly, Zn50 alone leads to an increase of CD4+CD25high(hi)+ cells in atopic and non-atopic subjects. Zinc may regulate unwanted hyperresponsive immune reactions by suppressing proliferation through a significant shift from IL-10 to the Th1 cytokine IFN-γ, and enhanced regulatory T cell numbers. Therefore, zinc supplementation may be a promising tool for the therapy of allergies, without negatively affecting the immune system.

Immunomodulatory activity of Tityus serrulatus scorpion venom on human T lymphocytes.

BackgroundTityus serrulatus scorpion venom (TsV) contains toxins that act on K+ and Na+ channels and account for the venom’s toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells.MethodsCytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations.ResultsTsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4+ and CD8+ T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8+ CD25+ T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses.ConclusionsTsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-015-0046-3) contains supplementary material, which is available to authorized users.

Solution-Phase Crosstalk and Regulatory Interactions Between Multipotent Adult Progenitor Cells and Peripheral Blood Mononuclear Cells.

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in clinical trials for acute graft versus host disease with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Anti-CD3/anti-CD28 (3/28) activation of T cells within the peripheral blood mononuclear cell (PBMC) compartment was performed in the presence or absence of MAPCs. Liquid chromatography-coupled tandem mass spectrometry was used to characterize the differential secretion of proteins, and transcriptional profiling was used to monitor mRNA expression changes in both cell populations. Overall, 239 secreted and/or ectodomain-shed proteins were detected in the secretomes of PBMCs and MAPCs. In addition, 3/28 activation of PBMCs induced differential expression of 2,925 genes, and 22% of these transcripts were differentially expressed on exposure to MAPCs in Transwell. MAPCs exposed to 3/28-activated PBMCs showed differential expression of 1,247 MAPC genes. Crosstalk was demonstrated by reciprocal transcriptional regulation. Secretome proteins and transcriptional signatures were used to predict molecular activities by which MAPCs could dampen local and systemic inflammatory responses. These data support the hypothesis that MAPCs block PBMC proliferation via cell cycle arrest coupled to metabolic stress in the form of tryptophan depletion, resulting in GCN2 kinase activation, downstream signaling, and inhibition of cyclin D1 translation. These data also provide a plausible explanation for the immune privilege reported with administration of donor MAPCs. Although most components of the major histocompatibility complex class II antigen presentation pathway were markedly transcriptionally upregulated, cell surface expression of human leukocyte antigen-DR is minimal on MAPCs exposed to 3/28-activated PBMCs. This study documents experiments quantifying solution-phase crosstalk between multipotent adult progenitor cells (MAPCs) and peripheral blood mononuclear cells. The secretome and transcriptional changes quantified suggest mechanisms by which MAPCs are hypothesized to provide both local and systemic immunoregulation of inflammation. The potential impact of these studies includes development of a robust experimental framework to be used for preclinical evaluation of the specific mechanisms by which beneficial effects are obtained after treatment of patients with MAPCs.

Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

Abstract 1041: PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis

Abstract Background: PTHrP is produced by various osteotropic malignancies and promotes progression of bone metastasis. PTHrP(12-48) is a unique, proteolytically-derived fragment of full length PTHrP and is the first PTHrP fragment, identified in the serum of breast cancer patients. Serum PTHrP(12-48) levels in breast cancer patients, correlate with a high sensitivity and specificity with the presence of bone metastasis. The purpose of this study was to define the role of PTHrP(12-48) in the metastatic breast tumor microenvironment. PTHrP(12-48) activity at the cellular level, was examined by PTHrP(12-48) agonist/antagonist activity at the PTH1 receptor (PTH1R), examination of subcellular localization and the effects of PTHrP(12-48) on cell proliferation, survival and osteoclast differentiation. Methods: To test the agonist/antagonist activity of PTHrP(12-48), c-AMP and IP3 assays were performed in cells expressing the PTH1R, in the presence of potent PTH1R agonists (PTH 1-34) and antagonists (PTH7-34). Immunolocalization of PTHrP(12-48) was examined by immunohistochemistry (IHC) using a specific anti-PTHrP(12-48) antibody and by immunofluorescence in PTH1R expressing human cells and human peripheral blood mononuclear cell (PBMC)-derived osteoclasts, which lack PTH1R. Cellular proliferation was measured in PTH1R expressing and PTH1R lacking cells. In addition, the effect of PTHrP(12-48) on human osteoclast precursor survival and osteoclast differentiation was assessed in receptor activator of nuclear factor kappa beta ligand (RANKL) treated PBMC cultures. Results: The data demonstrate that PTHrP(12-48) is not an agonist or an antagonist of the PTH1R receptor. IHC demonstrated positive immunostaining in primary breast tumors and bone metastases, whereas normal breast tissue was negative. Immunofluorescence successfully identified the cytoplasmic localization of PTHrP(12-48), 90 minutes after addition of ligand in PTH1R expressing cells. In contrast, no cytoplasmic staining was observed in PTH1R lacking cells. In addition PTHrP(12-48) significantly inhibited serum-induced proliferation of PTH1R expressing cells, but not PTH1R lacking cells. Interestingly, although human osteoclast precursors lack PTH1R expression, PTHrP(12-48) treatment strongly inhibited the survival and proliferation of PBMCs as well as their differentiation into osteoclasts. Conclusions: PTHrP(12-48) is a circulating bone metastasis biomarker that is expressed by primary breast tumors as well as bone metastasis. PTHrP(12-48) does not interact productively with the PTH1R in terms of second messenger activation. However, treatment of cells with PTHrP(12-48) results in cellular uptake by PTH1R expressing cells, with a resultant inhibition of serum-induced cell proliferation and osteoclastogenesis. Citation Format: Varinder Kaur, Archana Kamalakar, Nisreen Akel, Kim Leitzel, Allan Lipton, Larry J. Suva. PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2015-1041

Analysis of stability of human urine derived stem cells during serial subcultures

This study was conducted to assess stabilities of the human urine derived stem cells (USCs) during serial subcultures to demonstrate clinical feasibility for future stem cell therapy. For this work, we compared to cell morphology, proliferation rate, immunophenotype, multi-lineage differentiation potentials and immunomodulatory effect of USCs. USCs maintained cell morphology, cell proliferation ability through serial culture (till passage 7). In immunophenotypical analysis, USCs revealed positive for embryonic/mesenchymal stem cell marker (stage specific embryonic antigen, SSEA4) and mesenchymal stem cell markers (CD44, CD73, CD90 and CD105), while negative for hematopoietic stem cell markers (CD34, CD45 and C-kit) and immunogenic marker (HLA-DR). The USC had multi-differentiation potencies into adipocytes and osteocytes, and these abilities maintained into passage 7. The immunomodulatory effect of USCs with phytohemagglutinin stimulated peripheral blood mononuclear cell proliferation was analyzed with two systems, co-culture or separated culture system. The peripheral blood mononuclear cells’ proliferation was effectively inhibited on both systems, and the value was more effective with co-culture system than separate one. The present study suggests that sequential sub-passages till passage 7 could maintain stem cell characteristics, multipotent properties, and immune suppressive effect of USCs which should be considered in their therapeutic application in regenerative medicine.

Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine

Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals.We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitro has the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitro investigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

Traditional preparation of Phaleria nisidai, a Palauan tea, reduces exposure to toxic daphnane-type diterpene esters while maintaining immunomodulatory activity

Ethnopharmacological relevanceThe leaves of Phaleria nisidai Kaneh. (Thymelaeaceae) are brewed into a tea commonly used as a tonic, strengthening beverage and immune enhancer in Palau, Micronesia. Recently, the leaves of P. nisidai have been shown to contain toxic daphnane diterpene esters which may pose a public health threat to Palauans. Aims of the studyThis project documents the use frequency, preparation and side effects of P. nisidai. The content of daphnane diterpene esters in aqueous and methanol extracts and infusions prepared by healers in Palau is compared to assess the risk of daphnane ingestion associated with traditional consumption. Quantitative results are correlated with an in vitro assessment of the immunomodulating activity of the extracts. Materials and methodsResearch participants, comprising traditional healers and laypeople, were interviewed concerning use patterns and side effects of P. nisidai. Several traditional healers prepared and provided boiled tea samples for chemical analysis. Leaves were collected and methanolic and aqueous extractions were prepared in the laboratory. Peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of methanol and aqueous leaf extracts and their output of IFNγ was measured using ELISA. Cell proliferation was also assessed using the MTT assay. The concentration of selected daphnane diterpene esters in healer-prepared infusions, lab methanol and lab aqueous extracts was quantified using ultraperformance liquid chromatography-mass spectrometry-triple quadrupole detection (UPLC-MS-TQD). ResultsThrough structured interviews it was determined that P. nisidai tea was used frequently, with many participants drinking it daily. The reported side effects were mild, and with the exception of diarrhea (n=2), no side effect was mentioned more than once. Methanol extracts contained 4.0μg simplexin, 17.6μg acetoxyhuratoxin and 2.3μg huratoxin per g dry leaf material. In traditional water infusions provided by healers and in standardized lab-prepared aqueous extracts all three compounds were below the limit of detection (16.3ng/mL) using our UPLC-MS-TQD method. Methanol and aqueous extracts increased the release of IFNγ by PBMCs (p<0.05); however, methanol extracts were significantly more active than aqueous extracts (p<0.05). Methanol and aqueous extracts significantly increased proliferation of PBMCs, causing at least 60% more cell proliferation than negative control (p<0.05). ConclusionsThe presence of daphnane diterpene esters in a frequently consumed traditional beverage was initially viewed as a public health concern, though interview data reveal that Palauans do not observe toxicity or side effects associated with their use of P. nisidai tea. Concurrently, daphnanes are present in methanolic extracts but not detected in aqueous preparations indicating that the traditional method of preparation avoids the extraction of these potentially toxic compounds, while still maintaining immunostimulant activity.

Retraction Note To: Functional activation of Proline-rich Tyrosine Kinase2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease characterized by activated T cells and polyclonally activated B cells that produce autoantibodies. Activation of autoreactive T and B cells plays a pivotal role in the pathogenesis of this disease. A role of focal adhesion kinase (FAK) in the pathogenesis has been suggested. Proline-rich tyrosine kinase2 (PYK2) is structurally related to FAK, however, the functional activation of PYK2 in SLE remains unclear. In the present study, we showed that PYK2 is significantly increased and activated in peripheral blood mononuclear cells (PBMCs) of patients with SLE. In addition, we showed the involvement of PYK2 proteins in the up-regulation of CD40L and CTLA4 expression and PBMC proliferation.

Endocrine and immunological responses to adrenocorticotrophic hormone (ACTH) administration in juvenile harbor seals (Phoca vitulina) during winter and summer

There is increasing interest in measuring endocrine and immune parameters in free-ranging seals and sea lions, but there is a lack of understanding in how an acute stress response, often associated with capture and handling, influences these parameters of interest. The main objective of this study was to assess the impact of a simulated stressor on both endocrine and immune parameters. During two seasons, exogenous adrenocorticotrophic hormone (ACTH) was administered to seven female juvenile harbor seals and the response of several hormones (cortisol, aldosterone, total and free thyroxine and total triiodothyronine) and immunological parameters (total and differential leukocyte counts and peripheral blood mononuclear cells (PBMC) proliferation) were assessed. Cortisol peaked at 165min (winter 203.1±84.7ng/ml; summer 205.3±65.7ng/ml) and remained significantly elevated 240min after ACTH infusion in both seasons. Aldosterone peaked at 90min (winter 359.3±249.3pg/ml; summer 294.1±83.7pg/ml) and remained elevated 240min after administration of ACTH in both seasons. An increase in circulating total white blood cells was driven primarily by the increase in neutrophils which occurred simultaneously with a decrease in lymphocytes leading to an overall increase in neutrophil to lymphocyte ratio. These findings demonstrate that a simulated stress response in juvenile harbor seals results in a predictable increase in both cortisol and aldosterone concentrations, and were associated with altered immunological parameters.

Human Papillomavirus Vaccine-Induced Cytokine Messenger RNA Expression in Vaccinated Women.

The objective of this work was to evaluate the influence of human papillomavirus (HPV) vaccination on peripheral blood mononuclear cell (PBMC) proliferation and cytokine gene transcription. PBMCs isolated after HPV immunization were incubated with HPV vaccine, phytohemagglutinin, or buffer. Cell proliferation was assessed by MTT reduction assay. RNA was extracted from PBMCs, and the relative concentration of cytokine messenger RNA (mRNA) transcripts (IFN-β, IFN-γ, IL-12, TNF-α, IL-6, IL-17, or IL-10) relative to transcription of the β-actin gene was determined by real-time polymerase chain reaction. PBMC proliferation in response to HPV vaccine and PHA were greater than that observed in unstimulated cells (p<0.001). Cytokine mRNAs were upregulated in stimulated PBMC cultures. The median increase in vaccine-stimulated cultures was: IFN-β=334.4-fold; IL-12=46.33-fold; IFN-γ=12.64-fold; IL-6=9.07-fold; IL-17=7.33-fold; IL-10=6.47-fold; and TNF-α=2.36-fold. The IFN-β expression was significantly higher (p<0.05). Proliferative PBMC responses and multiple cytokine gene expression were detected in women who received the HPV vaccine.

Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

BackgroundCystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis.MethodsMorphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs).ResultsDespite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs.ConclusionsOur results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

Evaluation of Methanol Extract Activity of Leaf, Seed, Skin and Juice from Five Iranian Grape Cultivars on Lymphocyte Proliferation and their Mutagenicity in the Ames Test

The present study investigated the mutagenicity of fruit and leaf extracts of five Iranian grape cultivars and their effect on human peripheral blood mononuclear cells (PBMC) proliferation. The grape cultivars studied were: Bidane sefid , Shahroodi , Halvaie , Yaghoti and Ghermez yaghoti dorosht . The effect of the methanol extracts of Vitis vinifera cultivars on PBMC proliferation were measured using MTT assay at different concentrations (100, 1000, 2000, 3000 µg/ml). The mutagenicity of these extracts at two concentrations (1000, 3000 µg/ml) was also investigated on Salmonella typhimurium strain TA98. Among these five species, methanol old leaf and seed extracts strongly increased PBMC proliferation. The grape juices of all cultivars did not have any effect on PBMC proliferation. The grape juices of all species were also found to have potent mutagenicity on S. typhimurium. But skin, seed and leaf extract of all cultivars didn’t show any mutagenicity on this strain. This study provides data concerning the Immune-enhancing activity of fruit, leaf extracts of grape as well as weak mutagenic potency of grape juice.