RAP may induce cytokine production of RAW264.7 cells through TLR4-mediated activation of MAPKs and NF-κB.
A crude polysaccharide fraction (cDOP) has been determined to be the characteristic marker of Dendrobium officinale, an expensive tea material in Asia, but its chemistry and bioactivity have not been studied. In work reported here, cDOP was destarched (DOP, 90% yield) and separated into two subfraction polysaccharides, DOPa and DOPb, which were characterized by monosaccharide composition and methylation analyses and spectral analyses (FT-IR and (1)H and (13)C NMR). Both are composed of mannose and glucose at similar ratios and have a similar structure with a backbone of 1,4-linked β-D-mannopyranosyl and β-D-glucopyranosyl residues. Significant differences were observed only in their molecular weights. Bioassay using mouse macrophage cell line RAW264.7 indicated that DOP and its two subfractions enhance cell proliferation, TNF-α secretion, and phagocytosis in a dose-dependent manner. They also induced the proliferation of lymphocytes alone and with mitogens. DOPa and DOPb are thus proven to be major, active polysaccharide markers of D. officinale.
Purpose: The purpose of this study was to determine if an Astragalus polysaccharide (RAP) can protect immune cells from the toxic side effects of paclitaxel (Taxol), a powerful anti-tumor drug whose equally powerful side effects limit its clinical use.Methods: We hypothesized that RAP can reduce the toxic effects induced by Taxol. To test this hypothesis, we conducted a series of studies in vivo and in vitro. First, we confirmed RAP’s effects in vivo utilizing BALB/c mice inoculated with 4T1 mouse breast cancer cells as the tumor model. Mice were treated with RAP and/or Taxol, and the differences in the life spans were recorded. Second, a co-culture cell model was used to study the protective effect of RAP on cells vis-a-vis Taxol. The cell cycle and apoptosis of RAW 264.7 cells that were treated with RAP with/without Taxol were checked by flow cytometry and Hoechst staining. Proteins involved in the cell cycle and apoptosis were also tested by Western blot to reveal the probable mechanism.Results: RAP prolonged the life span of tumor-bearing mice treated with Taxol. The in vitro experiments showed that Taxol suppressed the proliferation of RAW 264.7 cells while RAP protected the RAW 264.7 cells from Taxol-induced suppression. The protection is selective because RAP had no effect on 4T1 cells. Furthermore, Taxol clearly led to cell cycle arrest mainly at the G2/M phase and generated cytotoxicity against RAW 264.7 cells, while RAP blocked cell cycle arrest and protected cells from apoptosis. Taxol up-regulated the protein levels of P-H2A, PARP, Chk1, p53, and p21 and down-regulated Bcl-Xl and Mcl-1, and RAP reversed the expression of all these proteins.Conclusion: These results suggested that RAP can protect immune cells from Taxol-induced toxicity, by changing the cell cycle and apoptosis.
Fragment 450-650 of the spike (S) protein (S450-650) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains epitopes capable of being recognized by convalescent sera of SARS patients. Vaccination of mice with recombinant S450-650 (rS450-650) can induce Abs against SARS-CoV, although the titer is relatively low. In the present study, a fusion protein linking a fragment (residues 39-272) of murine calreticulin (CRT) to S450-650 in a prokaryotic expression system was created. Compared with target antigen alone, the recombinant fusion product (rS450-650-CRT) has much improved hydrophilicity and immunogenicity. The S450-650-specific IgG Abs of BALB/c mice subcutaneously immunized with rS450-650-CRT were in substantially higher titer (approximately fivefold more). Furthermore, the fusion protein, but not rS450-650 alone, was able to elicit S450-650-specific IgG responses in T cell deficient nude mice. Given that rCRT/39-272 can drive the maturation of bone-marrow-derived dendritic cells, directly activate macrophages and B cells, and also elicit helper T cell responses in vivo, we propose that fragment 39-272 of CRT is an effective molecular adjuvant capable of enhancing target Ag-specific humoral responses in both a T cell-dependent and independent manner. Fusion protein rS450-650-CRT is a potential candidate vaccine against SARS-CoV infection.Key words Spike protein, severe acute respiratory syndrome-associated coronavirus, calreticulin, antibody.Severe acute respiratory syndrome is an infectious disease caused by SARS-CoV (1). The genome of SARS-CoV encodes several structural proteins, including the S glycoprotein, N protein, M glycoprotein and small E protein, which play synergistic roles in viral infectivity and pathogenicity (1, 2). S protein, with 1255 amino acid residues, is the largest structural protein in the virus. It is a type I transmembrane glycoprotein consisting of two domains, List of Abbreviations: APC, antigen-presenting cell; CD, cluster of differentiation; CRT, calreticulin; DC, dendritic cell; E, envelope; E. coli, Escherichia coli; EGFP, enhanced green fluorescence protein; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte macrophage colony-stimulating factor; IPTG, isopropyl b-D-thiogalactoside; IL, interleukin; LPS, lipopolysaccharide; M, membrane; N, nucleocapsid; OPD, orthophenylenediamine; PBST, PBS containing 0.05% Tween-20; rCRT/39-272, recombinant murine CRT amino acid 39-272; rS450-650, recombinant S protein amino acid 450-650; rS450-650-CRT, recombinant fusion protein between S450-650 and CRT/39-272; rEGFP, recombinant enhanced green fluorescence protein, S, spike; SARS-CoV, severe acute respiratory syndrome-associated coronavirus; s.c., subcutaneous. S1 and S2 (2). The former contains a receptor-binding domain responsible for viral binding to the receptor on target cells (3-7). Former studies have shown that the S protein is a major antigen recognized by protective neutralizing antibodies in patients (3-6). We have previ...
Although caltreticulin (CRT) is mainly a residential ER protein, it is also expressed on the membrane surface of various types of cells exhibiting multiple functions. We report here that intraperitoneal administration of a soluble recombinant CRT fragment (rCRT/39-272) led to a substantial decrease in delayed type hypersensitivity (DTH) responses in BALB/c mice and EAE in C57BL/6 mice. In the recall response against keyhole limpet hemocyanin (KLH) in vitro, draining lymph node cells from the rCRT/39-272-treated mice produced less IFN-γ but more IL-4 as compared with the cells from the control group. The immunomodulating effect of intraperitoneally administered rCRT/39-272 was attributed to anti-CRT Abs thereby induced, because, in passive transfer experiments, the CRTspecific antiserum could suppress DTH in BALB/c mice. B-cell-deficient μMT mice were not susceptible to rCRT/39-272-mediated DTH suppression. Furthermore, CRT appears on the surface of murine T cells soon after activation and remains detectable (at relatively low level) by flow cytometry for approximately 5 days in vitro. Anti-CRT Abs were able to inhibit AKT phosphorylation, proliferation, and cytokine production by activated murine T cells. We propose that cell surface CRT could play a role in the function of effector T cells and may be considered a target for immunological manipulation.Keywords: Anti-CRT antibodies r Calreticulin r Immunoregulation Supporting Information available online IntroductionCalreticulin (CRT) is a multifunctional glycoprotein of 46 kDa, and is most abundant in the ER of the cell [1][2][3][4][5][6]. It folds into three domains: (i) a lectin-like N domain, (ii) a proline-rich P domain, and (iii) a Ca 2+ -binding C-domain. CRT has well-recognized physiological roles in intracellular Ca 2+ storage and signaling and also as a molecular chaperone [1,5,6]. It has been detected in soluble form in the sera of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus [7,8]. We have shown that a recombinant CRT fragment 39-272 containing its partial NCorrespondence: Prof. Xiao-Ming Gao e-mail: xmgao@suda.edu.cn and P-domains (CRT/39-272) exhibits potent immunostimulatory activity and strong adjuvanticity [7], implying soluble CRT may be involved in immunopathological and inflammatory reactions leading to the development of RA and/or systemic lupus erythematosus. Moreover, CRT can be exposed at the membrane surface of various types of cells and exhibits multiple cellular functions as a surface molecule [6]. For example, cell-surface CRT (csCRT) mediates thrombospondin (TSP) triggered disassembly of focal contacts, exhibits antithrombotic effects, and inhibits melanoma cell spreading and angiogenesis [5,9,10]. Ligation of neuronal csCRT by complement C1q triggers increased levels of cellular * These authors contributed equally to this work. Eur. J. Immunol. 2012. 42: 2419-2430 reactive oxygen species [11]. There is also ample evidence showing that surface exposure of CRT enhances the immunogenicity of tumor cells and ...
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