Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy.

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), Akt and glycogen synthase kinase-3beta (GSK-3ß) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3ß in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3ß signaling pathway.

Expression of a major plant allergen as membrane-anchored and secreted protein in human cells with preserved T cell and B cell epitopes.

BACKGROUND: Expression of allergens in human cells is a prerequisite for the development of antigen-specific cell therapy in IgE-mediated allergy. We developed a strategy how the clinically relevant major grass pollen allergen Phl p 5 can be efficiently secreted or expressed on the surface of human cells with preserved allergenic activity. METHODS: The cDNA of Phl p 5 was fused to a leader peptide with or without a transmembrane domain and both constructs were ligated into a mammalian expression vector. Transfection of these plasmids into human cells resulted in a membrane-anchored or secreted version of Phl p 5, respectively, as determined by ELISA or flow cytometric analysis. RESULTS: Both the secreted and membrane-anchored Phl p 5 proteins bound IgE from allergic patients in an immunoblot assay and induced specific histamine release and CD203c upregulation in basophils of grass pollen-allergic patients. Proliferation of peripheral blood mononuclear cells from Phl p 5-allergic individuals was induced upon stimulation with both variants of Phl p 5 expressed in human cells similar to recombinant Phl p 5. CONCLUSIONS: Secreted and membrane-anchored Phl p 5 expressed in human cells preserved B cell as well as T cell epitopes and may be used to develop and test various cell-based strategies for allergen-specific immunomodulation and to delineate the tolerance mechanisms involved therein.

Oestrogen modulates experimental autoimmune encephalomyelitis and interleukin-17 production via programmed death 1.

The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17beta-oestradiol (E(2)) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4(+) FoxP3(+) regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E(2) at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E(2) treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E(2) treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E(2) failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E(2)-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.

Ursolic acid enhances the cellular immune system and pancreatic beta-cell function in streptozotocin-induced diabetic mice fed a high-fat diet.

This study investigated the effects of ursolic acid on immunoregulation and pancreatic beta-cell function in type 1 diabetes fed a high-fat diet for 4 weeks. Male mice were divided into non-diabetic, diabetic control, and diabetic-ursolic acid (0.05%, w/w) groups, which were fed a high-fat (37% calories from fat). Diabetes was induced by injection of streptozotocin (200 mg/kg B.W., i.p.). Ursolic acid significantly improved blood glucose levels, glucose intolerance, and insulin sensitivity compared to the diabetic group. The plasma insulin and C-peptide concentrations were significantly higher in the diabetic-ursolic acid group than in the diabetic group. Ursolic acid significantly elevated the insulin levels with preservation of insulin staining of beta-cells in the pancreas. In splenocytes, concanavalin (Con) A-induced T-cell proliferation was significantly higher in the diabetic-ursolic acid group compared to the diabetic group, but liposaccharide (LPS)-induced B-cell proliferation did not differ between groups. Ursolic acid enhanced IL-2 and IFN-gamma production in response to Con A stimulation, whereas it inhibited TNF-alpha production in response to LPS stimulation. In this study, neither streptozotocin nor ursolic acid had effects on lymphocyte subsets. These results indicate that ursolic acid exhibits potential anti-diabetic and immunomodulatory properties by increasing insulin levels with preservation of pancreatic beta-cells and modulating blood glucose levels, T-cell proliferation and cytokines production by lymphocytes in type 1 diabetic mice fed a high-fat diet.

Therapeutic potential of various beta-glucan sources in conjunction with anti-tumor monoclonal antibody in cancer therapy.

Combined beta-glucan with anti-tumor mAb therapy has demonstrated therapeutic efficacy in murine tumor models. The current study was designed to compare the therapeutic efficacy of various sources of beta-glucans. Our studies demonstrated that yeast beta-glucan, in combination with anti-tumor mAb, resulted in significantly smaller tumor burdens and achieved enhanced long-term survival compared to mAb alone or beta-glucan extracts from mushrooms. Further studies indicated that yeast beta-glucan particle was superior to mushroom extracts in inducing cytokine secretion, particularly IL-12 production in dendritic cells (DCs). In addition, results showed that cytokine production was markedly decreased in MyD88-deficient macrophages and DCs but not in complement receptor 3 (CR3)-deficient mice. Our data suggest that yeast beta-glucan demonstrates much stronger adjuvant activity compared to mushroom beta-glucan extracts in tumor therapy. This effect of yeast beta-glucan may be in part ascribed to the cytokine secretion by DCs and macrophages and bioavailability of active beta-glucan moiety.

Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties.

OBJECTIVE: Pancreatic cancer is among the most aggressive solid malignancies. It is possible that pancreatic cancer contains cancer stem cells responsible for its malignancy. The purposes of this study were (1) to establish an assay in which a subset of pancreatic cancer cell line (PANC-1) cells with stem cell properties can propagate, and (2) to identify the cells obtained from this assay. METHODS: The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 supplemented with epidermal growth factor, basic fibroblast growth factor, insulin, transferrin, selenium, and bovine serum albumin at a density of 1000 cells/mL for 10 to 14 days to form spheres. Cells of spheres were cultured in different conditions to evaluate their ability of self-renewal and differentiation. Clone formation assay and tumor formation assay were used to identify the ability of propagation in vitro and in vivo. The cells and spheres were also stained by using Hoechst 33342 dye to evaluate their capacity of excluding Hoechst dye. Real-time polymerase chain reaction was used to detect expressions of LY6E, c-Met, TACSTD1, CD34, and CD44 mRNA. RESULTS: A subpopulation of PANC-1 cells could propagate to form spheres in this assay. Cells of obtained spheres had the hallmark of excluding Hoechst 33342 dye. Cultured in serum-free medium, the dissociated single cells of primary spheres could form filial spheres again; cultured in serum-containing medium, these cells generated both the cells with the ability of excluding Hoechst 33342 dye and the cells without that ability. The propagation capacity of PANC-1 spheres was higher than that of cells cultured in serum-containing medium both in vitro and in vivo. In addition, LY6E, TACSTD1, and CD44 mRNA were overexpressed in PANC-1 spheres. CONCLUSIONS: A subpopulation of PANC-1 cells can propagate to form spheres with properties of stem cells in this assay; enough of these cells can be obtained for further study. Considering the overexpression of mRNA, it was tentatively suggested that LY6E, TACSTD1, and CD44 proteins may act as surface markers for sorting pancreatic cancer stem cells with fluorescence-activated cell sorter/magnetic-activated cell sorter.

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers.

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Only MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.

TNF/LTalpha double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury.

Following acute liver injury, hepatocytes divide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LTalpha). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LTalpha are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LTalpha knockout (TNF-/-LTalpha-/-) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF-/-LTalpha-/- mice. Lymphocyte clusters were evident in all TNF-/-LTalpha-/- livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF-/-LTalpha-/- but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LTalpha signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF-/-LTalpha-/- animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.

Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells.

A major function of macrophages is to engulf apoptotic cells to prevent them from releasing noxious materials as they die. Milk fat globule-EGF-factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages that works as a bridge between apoptotic cells and phagocytes by specifically recognizing phosphatidylserine exposed on apoptotic cells. In this study, we found that developmental endothelial locus-1 (Del-1), originally identified as an embryonic endothelial cell protein that binds alphavbeta3 integrin, is structurally and functionally homologous to MFG-E8. That is, both consist of a signal sequence, two epidermal growth factor domains and two factor VIII-homologous domains (C1 and C2). Del-1 bound to the apoptotic cells by recognizing phosphatidylserine via the factor VIII-homologous domains with an affinity similar to that of MFG-E8. The phagocytic activity of NIH 3T3 cells against apoptotic cells was enhanced by Del-1 through an interaction between the epidermal growth factor domain in Del-1 and alphavbeta3 integrin expressed in the NIH 3T3 cells. Screening of primary macrophages and macrophage cell lines for the expression of MFG-E8 and Del-1 indicated that MFG-E8 and Del-1 are expressed in different sets of macrophages. These results suggest the existence of macrophage subsets that use MFG-E8 or Del-1 differently to engulf apoptotic cells.

Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues.

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers.

Langerhans cells are the most potent antigen-presenting cells in the skin and play a critical role in the induction of contact allergy. Research on the phenotypical and functional changes of LCs occurring after application of skin sensitizers indicated their use as an in vitro model for the screening of chemicals. In the present investigations, LCs from human skin explants served as the test system. The application of this cell system has been aggravated by the difficulty in isolating sufficient numbers of live LCs from skin. This disadvantage was overcome by the culture of immature dendritic cells from peripheral mononuclear blood cells. These cells can serve as a replacement for LCs as they bind haptens and show phenotypical and functional changes similar to LCs. The sensitizers NiSO(4), dinitrochlorobenzene, 2,4,6-trinitrobenzene sulfonic acid, alpha-hexylcinnamaldehyde and eugenol were applied. Both the expression of surface markers and the induction of intracellular interleukin-1 beta (IL-1 beta) were analyzed. No clear-cut results could be established for intracellular cytokine production, only NiSO(4) induced a remarkable number of IL-1 beta-positive cells. However, all skin sensitizers caused an up-regulation of the co-stimulatory molecule CD86, of intercellular adhesion molecule CD54 and of the HLA-DR antigen. The irritant sodium dodecyl sulfate (SDS) and the vehicle dimethyl sulfoxide (DMSO) had no effect.

The influence exerted by a restricted phospholipid microenvironment on the expression of tissue factor activity at the cell plasma membrane surface.

Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 cells, showing a reduced ability to externalize PhtdSer, were transfected with human TF cDNA. PhtdSer exposure and TF activity were examined in transfected cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externalization potency. TF expression was evidenced by flow cytometry and its activity measured using functional assays. PhtdSer exposure was monitored by enzymatic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noticeable TF expression level, clone DC9 presented only a weak TF activity even after ionophore stimulation. The apparent Km, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for THP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 than THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts of available PhtdSer abolished this difference. The DC9 clone appears suitable for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Such cells should enable further assessment of the role of TF as a receptor coupled to intracellular signaling pathways and its fate during apoptotic cell death.

[Expression of eukaryotic expression plasmid pcDNA3/pacA and pcDNA3/pacP in mammalian cells].

OBJECTIVE: Prior to the use of pcDNA3/pacA and pcDNA3/pacP in vivo, the transcription and expression products of these two eukaryotic expression plasmids in mammalian cells were detected. METHODS: The eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP were transfected into COS-7 cells respectively with liposome according to the manufacturer's protocol. In order to generate stable transfectants, after a 48 h incubation period in regular Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum, cells were treated with neomycin (1 mg.ml-1 of G418) and maintained under continuous selective pressure. G418-resistant colonies, which became visible after 2 weeks, were isolated and screened for expression of the vector-encoded protein. The control transfections were performed with the pcDNA3 vector and without recombinant as well as vector plasmid DNA. The mRNA transcriptions of the two insertional genes were detected by RT-PCR assay and their expression products were analysed by labelled-avidin-biotin enzymed-linked immunosorbent assay, flow cytometry and Western blotting. The cells transfected by pcDNA3 were used as the negative control. RESULTS: 1. The two eukaryotic expression plasmids could be correctly transcripted and translated under the control of the CMV immediate early promoter in mammalian cells. 2. The protein products could be detected in cell plasma, cell membrane and the culture supernatant. CONCLUSION: The two eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP can express protein products which were encoded by insertional gene pac-A and pac-P in mammalian cells.

Human retinoblastoma: in vitro differentiation and immunoglobulin superfamily antigen modulation by retinoic acid.

Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 microM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 microM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 microM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours.

The effect of beta-carotene supplementation on the immune function of blood monocytes from healthy male nonsmokers.

Although there is strong epidemiologic evidence that diets rich in carotenoids such as beta-carotene are associated with a reduced incidence of cancer, the cellular mechanisms underlying this phenomenon remain unknown. This article describes the effect of dietary beta-carotene supplementation on both the expression of functionally associated surface molecules on human monocytes and on the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha) by monocytes, all of which are involved in the initiation and regulation of immune responses involved in tumor surveillance. A double-blind, placebo-controlled, crossover study was undertaken in which 25 healthy, adult male nonsmokers were randomly assigned to receive beta-carotene (15 mg daily) or placebo for 26 days, followed by the alternative treatment for a further 26 days. The expression of functionally related monocyte surface molecules was quantified by flow cytometry, and ex vivo secretion of TNF-alpha was quantified by an enzyme-linked immunosorbent assay, before and after each treatment period. After dietary supplementation there were significant increases in plasma levels of beta-carotene and in the percentages of monocytes expressing the major histocompatibility complex class II molecule HLA-DR and the adhesion molecules intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. In addition, the ex vivo TNF-alpha secretion by blood monocytes was significantly increased after supplementation. These findings suggest that moderate increases in the dietary intake of beta-carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anticarcinogenic properties attributed to beta-carotene.

Effects of petrochemicals and ultraviolet radiation on epidermal IA expression in vitro.

We previously demonstrated that combined treatment of mice with crude oil and longwave ultraviolet radiation (UVA) led to the depletion of IA-positive cells from the epidermis. In the present study, we have developed an in vitro screening assay for combined effects of purified petrochemicals and UVA on epidermal IA and Thy-1 expression. This method involves removal of skin from donor mice prior to treatment with chemicals and UVA (20,000 J/m2), followed by in vitro culture and subsequent immunoperoxidase staining. In this study, a complete correlation was observed in terms of IA-positive cell density among similarly treated cultured skin and live mice. In vivo and in vitro studies both indicated that anthracene but not phenanthrene or benzo[a]pyrene led to significant depletion of both epidermal Langerhans cells and Thy-1-positive dendritic cells when followed by UVA treatment. The in vitro assay developed for this study should prove to be a valuable tool for the screening of a wide variety of chemicals for contact photosensitizing activity.

T cell surface markers expression by immature human thymocytes in in vitro culture: role of Ia+ accessory cells.

Previous studies have indicated that the human thymus is composed of several discrete compartments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most medullary cells, unreactive with anti-T6, stain brightly with anti-T3 antibody, which defines mature T cell populations. By using an indirect immune rosette method, we isolated the minor thymocyte population (1 to 2% of all thymocytes) lacking both T3 and T6 but expressing T11 antigens. These cells could be maintained in culture supplemented with recombinant IL 2 (Rec-IL 2) for several days. Under these conditions, T3-T6- cells were shown to undergo phenotypic changes. In the absence of thymic macrophage (Mo), T3+ and T8+ thymocytes appeared in culture, whereas the development of T4+ cells strictly required the presence of Mo. The expression of T4 antigen could be largely prevented by the addition of anti-HLA-DR antibody, further indicating that Ia+ accessory cells had the ability to promote in vitro development of T4+ thymocytes. In the presence of Mo, not only T4+ but also T8+ cells were obtained. Double fluorescence staining with anti-T8-FITC and anti-T4-biotin demonstrated that after 12 days of culture, T4 and T8 antigens were mutually exclusive. Furthermore, during the course of these studies, we observed that under the culture conditions utilized (e.g., presence or absence of Mo), T3-T6-thymocytes failed to express the T6 antigen. Thus, the in vitro development of T cells bearing a mature phenotype could be obtained in the absence of intermediate expression of cortical (T6+) thymocytes.