Effects of hyaluronic acid on differentiation of human amniotic epithelial cells and cell-replacement therapy in type 1 diabetic mice.

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the ß cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into ß cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes.

Hyaluronic acid enhances proliferation of human amniotic mesenchymal stem cells through activation of Wnt/ß-catenin signaling pathway.

This study investigated the pro-proliferative effect of hyaluronic acid (HA) on human amniotic mesenchymal stem cells (hAMSCs) and the underlying mechanisms. Treatment with HA increased cell population growth in a dose- and time-dependent manner. Analyses by flow cytometry and immunocytochemistry revealed that HA did not change the cytophenotypes of hAMSCs. Additionally, the osteogenic, chondrogenic, and adipogenic differentiation capabilities of these hAMSCs were retained after HA treatment. Moreover, HA increased the mRNA expressions of wnt1, wnt3a, wnt8a, cyclin D1, Ki-67, and ß-catenin as well as the protein level of ß-catenin and cyclin D1 in hAMSCs; and the nuclear localization of ß-catenin was also enhanced. Furthermore, the pro-proliferative effect of HA and up-regulated expression of Wnt/ß-catenin pathway-associated proteins - wnt3a, ß-catenin and cyclin D1 in hAMSCs were significantly inhibited upon pre-treatment with Wnt-C59, an inhibitor of the Wnt/ß-catenin pathway. These results suggest that HA may positively regulate hAMSCs proliferation through regulation of the Wnt/ß-catenin signaling pathway.

Viologen-mediated assembly of and sensing with carboxylatopillar[5]arene-modified gold nanoparticles.

Carboxylatopillar[5]arene (CP[5]A), a new water-soluble macrocyclic synthetic receptor, has been employed as a stabilizing ligand for in situ preparation of gold nanoparticles (AuNPs) to gain new insights into supramolecular host-AuNP interactions. CP[5]A-modified AuNPs with good dispersion and narrow size distributions (3.1 ± 0.5 nm) were successfully produced in aqueous solution, suggesting a green synthetic pathway for the application of AuNPs in biological systems. Supramolecular self-assembly of CP[5]A-modified AuNPs mediated by suitable guest molecules was also investigated, indicating that the new hybrid material is useful for sensing and detection of the herbicide paraquat.

Mechanized silica nanoparticles based on pillar[5]arenes for on-command cargo release.

Mechanized silica nanoparticles, equipped with pillar[5]arene-[2]pseudorotaxane nanovalves, operate in biological media to trap cargos within their nanopores, but release them when the pH is lowered or a competitive binding agent is added. Although cargo size plays an important role in cargo loading, cargo charge-type does not appear to have any significant influence on the amount of cargo loading or its release. These findings open up the possibility of using pillar[n]arene and its derivatives for the formation of robust and dynamic nanosystems that are capable of performing useful functions.

One-pot synthesis of pillar[n]arenes catalyzed by a minimum amount of TfOH and a solution-phase mechanistic study.

A practical and effective trifluoromethanesulfonic acid (TfOH)-catalyzed cyclooligomerization strategy was developed for the synthesis of functionalized pillar[n]arenes and copillar[5]arenes from 1,4-dialkoxybenzenes with paraformaldehyde under mild reaction conditions, and the reaction mechanism of solution-phase catalytic synthesis of pillararenes was investigated by room-temperature X-band ESR spectroscopy, mass spectroscopy, NMR and control experiments, suggesting a free radical process initially and a Friedel-Crafts alkylation process during the consequent coupling and ring-closure stage.

Polysaccharides from the Medicinal Mushroom Cordyceps taii Show Antioxidant and Immunoenhancing Activities in a D-Galactose-Induced Aging Mouse Model.

Cordyceps taii, an edible medicinal mushroom native to south China, is recognized as an unparalleled resource of healthy foods and drug discovery. In the present study, the antioxidant pharmacological properties of C. taii were systematically investigated. In vitro assays revealed the scavenging activities of the aqueous extract and polysaccharides of C. taii against various free radicals, that is, 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and superoxide anion radical. The EC(50) values for superoxide anion-free radical ranged from 2.04 mg/mL to 2.49 mg/mL, which was at least 2.6-fold stronger than that of antioxidant thiourea. The polysaccharides also significantly enhanced the antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase) and markedly decreased the malondialdehyde production of lipid peroxidation in a D-galactose-induced aging mouse model. Interestingly, the immune function of the administration group was significantly boosted compared with the D-galactose-induced aging model group. Therefore, the C. taii polysaccharides possessed potent antioxidant activity closely associated with immune function enhancement and free radical scavenging. These findings suggest that the polysaccharides are a promising source of natural antioxidants and antiaging drugs. Consequently, a preliminary chemical investigation was performed using gas chromatography-mass spectroscopy and revealed that the polysaccharides studied were mainly composed of glucose, mannose, and galactose. Fourier-transform infrared spectra also showed characteristic polysaccharide absorption bands.

Jiangxienone, a new compound with potent cytotoxicity against tumor cells from traditional Chinese medicinal mushroom Cordyceps jiangxiensis.

A new compound, named jiangxienone, has been isolated from a culture of the traditional Chinese medicinal mushroom Cordyceps jiangxiensis, and its chemical structure was established on the basis of spectroscopic and chemical techniques. Jiangxienone showed potent cytotoxic effects against human gastric adenocarcinoma SGC-7901 cell and human lung carcinoma A549 cell with IC(50) values ranging from 1.38 to 2.93 µM, i.e., with at least approximately six-fold stronger cytotoxicity than cisplatin, a first-line chemotherapy drug for cancer patients.

Hyaluronic acid promotes osteogenic differentiation of human amniotic mesenchymal stem cells via the TGF-ß/Smad signalling pathway.

AIMS: This study investigated the effects of hyaluronic acid (HA), a commonly used osteogenic medium referred to as DAG, and the combined administration of HA and DAG (CG) on the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs), and the underlying mechanism. MAIN METHODS: The phenotype of hAMSCs was detected by flow cytometry and immunocytochemical staining. Alkaline phosphatase (ALP) and calcium deposition assays were employed for evaluating the osteogenic differentiation of hAMSCs. The expression of osteogenesis-related genes and proteins was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Meanwhile, the molecular mechanism of osteogenic differentiation of hAMSCs was detected by PCR array and qRT-PCR. KEY FINDINGS: The results showed that treatment with CG could significantly stimulate hAMSC ALP activity and calcium deposition compared to treatment with DAG, while HA had little effect. The expression of osteogenesis-related molecules and stemness-related molecules was up-regulated at the mRNA and protein levels in all three groups, and this up-regulation was most significant in the CG group. In addition, treatment with CG significantly increased the gene expressions involved in regulation of the TGF-ß/Smad signalling pathway compared to treatment with DAG. Furthermore, the pro-osteogenic differentiation effects as well as the up-regulated expression of genes observed in the CG treatment group were significantly inhibited when the cells were pre-treated with SB431542, an inhibitor of the TGF-ß/Smad pathway. SIGNIFICANCE: These results suggest that HA in combination with DAG could significantly enhance the osteogenic differentiation of hAMSCs, potentially via the TGF-ß/Smad signalling pathway.

[Epidemiological investigation of Angiostrongylus cantonensis in Jiangmen of Guangdong Province].

OBJECTIVE: To make an epidemiological survey on Angiostrongylus cantonensis in Jiangmen City of Guangdong Province. METHODS: From October 2006 to November 2007, the characteristics of A. cantonensis infection were investigated in Jiangmen district in various hosts, including the third stage larva infection in the snails Achatina fulica and Pomacea canaliculata by digestion method, and the adult A. cantonensis in rats by the dissection of heart and lungs. Relevant symptoms and dietary habits in Jiangmen residents who were randomly recruited were also investigated by questionnaire, and the specific IgG and IgM antibodies against A. cantonensis in their sera were detected by ELISA. RESULTS: 695 A. fulica and 720 P. canaliculata were examined. The infection rate of third stage larva of A. cantonensis were 45.0% and 1.8% respectively, with an infectivity of 53.74+/-147.30 and 5.23+/-8.51 respectively. Natural infection rate of A. cantonensis in all 229 rats was 4.4%. Among the 300 people surveyed, 11.3% had a history of eating raw or undercooked fish and shrimp, 5.3% directly or indirectly exposed to A. fulica or P. canaliculata. The positive rate of specific IgG antibody against A. cantonensis for serum samples among residents was 14.0% (42/300), and 5 serum samples in the 42 positive samples showed specific IgM antibody, with a positive rate of 1.7%. CONCLUSION: Jiangmen district is an endemic area of A. cantonensis, and the local residents are under the risk of infection.

[Investigation on antitumor mechanism of polysaccharide from medicinal fungus Penicillium jiangxiense].

OBJECTIVE: To investigate the possible antitumor mechanism of polysaccharide from medicinal fungus Penicillium jiangxiense. METHODS: The cytotoxic effect was measured by MTT assay, and the cell cycle was analyzed by flow cytometry with Propidium iodide (PI) staining. To apoptotic detections, Hoechst 33258 staining for chromatin, annexin-V FITC/PI double staining for early phase cell apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for late phase cell apoptosis. RESULTS: Both MPPJ4 and MPPJ5, the fine polysaccharide fractions from P. jiangxiense, showed slight cytotoxic effects to inhibit human gastric adenocarcinoma SGC-7901 cells proliferation, but significantly caused cell cycle arrest at the S phase, and the rate of cell population at the subG1 phase was evidently increased. In apoptotic assays, MPPJ5 was more potent than MMPJ4 in inducing tumor cells in a time-dependent manner at the range from 0 to 72 hours, comparable to the negative control. CONCLUSION: The antitumor acting mechanism of P. jiangxiense polysaccharide is associated with the induction of apoptotic cell death and cell cycle arrest.

Synergistic antitumor efficacy of antibacterial helvolic acid from Cordyceps taii and cyclophosphamide in a tumor mouse model.

The antibacterial agent helvolic acid, which was isolated from the active antitumor fraction of Cordyceps taii, showed potent cytotoxicity against different human cancer cells. In the present study, the in vivo antitumor effect of helvolic acid was investigated in murine sarcoma S180 tumor-bearing mice. Doses of 10 and 20 mg/kg/day helvolic acid did not exert significant antitumor activity. Interestingly, co-administration of 10 mg/kg/day helvolic acid and 20 mg/kg/day cyclophosphamide (CTX) - a well-known chemotherapy drug - showed promising antitumor activity with a growth inhibitory rate of 70.90%, which was much higher than that of CTX alone (19.5%). Furthermore, the combination markedly prolonged the survival of tumor-bearing mice. In addition, helvolic acid enhanced the immune organ index. The protein expression levels of ß-catenin, cyclin D1, and proliferating cell nuclear antigen were significantly suppressed in mice treated with 20 mg/kg/day helvolic acid and in those receiving combination therapy. Taken together, these results indicated that helvolic acid in combination with CTX showed potent in vivo synergistic antitumor efficacy, and its mechanism of action may involve the Wnt/ ß-catenin signaling pathway.

Effects of histamine on immunophenotype and notch signaling in human HL-60 leukemia cells.

Surface molecules are important biomarkers for cell proliferation and differentiation and play important roles in cell function and cell interaction. Notch is a transmembrane receptor that regulates developmental processes and cell-fate decision. Histamine is used as an adjunct to immunotherapy in myelogenous leukemia, and regulates hematopoietic cell development. Thus, we investigated the effects of histamine on immunophenotype and Notch signaling in human HL-60 leukemia cells. Histamine (0.1-10 microM) inhibited the colony-forming efficiency of HL-60 cells in a dose-dependent fashion and shifted the growth curve to the right. HL-60 cells were treated with histamine 0.1-1.0 microM for 6 days, and surface molecules were analyzed by flow cytometry. Histamine decreased CD49d positive cells by 74% while increasing CD31 positive cells by 53% as compared to controls. Histamine did not affect the expression of CD11b, CD14, CD34, CD44, CD54, CD49e, and CD62L. To examine Notch signaling in histamine-induced immunophenotype alterations in HL-60 cells, total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. The expressions of Notch1, Notch4, the ligands Jagged1, Delta4, and the downstream hairy enhancer of split 1 gene (HES1) were not significantly altered by histamine. In summary, this study demonstrated that histamine inhibited HL-60 cell growth and regulated immunophenotypes of CD49d and CD31. These effects are not mediated through the Notch signaling.

[Value of the antigen with molecular mass of 32 000 in immunodiagnosis of Angiostrongylus cantonensis].

OBJECTIVE: To evaluate the specificity and sensitivity of immunodiagnosis with the antigen with molecular mass of 32 000 of Angiostrongyliasis cantonensis(AC32). METHODS: The major antigenic protein AC32 was purified from the antigens of A.cantonensis adult worm by electroelution from SDS-polyacrylamide gels. AC32-enzyme linked immunosorbent assay (ELISA) and adult worm antigen (AWA)-ELISA were used to detect the specific IgG in the sera of normal rats (n=5) and rats with angiostrongyliasis (n=61), sera of healthy individuals (n=50) and patients (n=50) with angiostrongyliasis, schistosomiasis and other parasitosis. RESULTS: The positivity rate in AC32-ELISA of the sera from rats with angiostrongyliasis was 100%; without false positive results or cross reaction between AC32 and the sera of the normal control and patients infected with parasites other than A.cantonensis. CONCLUSION: AC32 of A.cantonensis is a valuable candidate antigen for immunodiagnosis of angiostrongyliasis with high sensitivity and specificity.

Supramolecular assembly-induced yellow emission of 9,10-distyrylanthracene bridged bis(pillar[5]arene)s.

9,10-Distyrylanthracene has been introduced to bridge two pillarenes to form a dimeric host, which can assemble into a linear supramolecular polymer upon cooperatively binding to a neutral guest linker, exhibiting yellow fluorescence emission in solution and solid states.

Stimuli-responsive metal-organic frameworks gated by pillar[5]arene supramolecular switches.

Spurred on by recent advances in materials chemistry and drug delivery, a new stimuli-responsive theranostic hybrid platform, based on mechanized monodisperse nano metal-organic frameworks (NMOFs) gated by carboxylatopillar[5]arene (CP5) switches with bio-friendly pH-triggered cargo release capabilities, has been constructed for the first time. This nanoscale smart cargo delivery system showed pH- and/or competitive binding agent-triggered controlled cargo release with negligible premature release, large pore sizes for drug encapsulation, low cytotoxicity, good biodegradability and biocompatibility, and potential application in cell imaging, which offers a new tool in targeted drug delivery and the controlled release of therapeutic agents.

Purification of heat shock protein 70-associated tumor peptides and their antitumor immunity to hepatoma in mice.

AIM: To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect. METHODS: By ConA-sepharose affinity chromatography, ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42 degrees. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF. RESULTS: The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent. Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1 x 10(7) HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF. CONCLUSION: High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF. This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.

Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection.

BACKGROUND: A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj). METHODS: A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients. RESULTS: Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%. CONCLUSIONS: The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

[Screening and identification of phage antibodies from phage display library against Schistosoma japonicum].

OBJECTIVE: To obtain single chain variable fragment(ScFv) against the circulating antigen(CAg) from Schistosoma japonicum(Sj). METHODS: Metabolic antigen of adult worm of Sj (Sj-MAg) was used in the panning of phage library against Schistosoma japonicum. The activity of Sj-MAg-binding phage clones was assayed by ELISA. The specificity of expression products of the positive clones was analyzed by ELISA, SDS-PAGE and Western blotting. RESULTS: Seventy-two randomly selected clones were tested for the presence of anti-Sj-MAg ScFvs, 6 clones showed positive. The specificity of these 6 clones was confirmed by binding them to antigens of other four trematodes. Two clones (B04, C24) were found to bind to Sj-MAg but not to any of the antigens of other four trematodes and their expression products were about 31 kDa in size. CONCLUSION: ScFv antibodies against the circulating antigens from Schistosoma japonicum Sj-MAg can be selected and manufactured from the antibody library.

Stimuli-responsive blue fluorescent supramolecular polymers based on a pillar[5]arene tetramer.

A tetraphenylethene-bridged pillarene tetramer with aggregation-induced emission properties forms an A4/B2-type supramolecular polymer and a gel with a symmetric neutral guest linker, showing a remarkable fluorescence emission enhancement in solution and the solid state and a good responsiveness to temperature and solvent composition.