Ping Shen

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM(+)CD138(hi)TACI(+)CXCR4(+)CD1d(int)Tim1(int) plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138(+) plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

B cells have paradoxical roles in autoimmunity, exerting both pathogenic and protective effects. Pathogenesis may be antibody independent, as B cell depletion therapy (BCDT) leads to amelioration of disease irrespective of autoantibody ablation. However, the mechanisms of pathogenesis are poorly understood. We demonstrate that BCDT alleviates central nervous system autoimmunity through ablation of IL-6-secreting pathogenic B cells. B cells from mice with experimental autoimmune encephalomyelitis (EAE) secreted elevated levels of IL-6 compared with B cells from naive controls, and mice with a B cell-specific IL-6 deficiency showed less severe disease than mice with wild-type B cells. Moreover, BCDT ameliorated EAE only in mice with IL-6-sufficient B cells. This mechanism of pathogenesis may also operate in multiple sclerosis (MS) because B cells from MS patients produced more IL-6 than B cells from healthy controls, and this abnormality was normalized with B cell reconstitution after Rituximab treatment. This suggests that BCDT improved disease progression, at least partly, by eliminating IL-6-producing B cells in MS patients. Taking these data together, we conclude that IL-6 secretion is a major mechanism of B cell-driven pathogenesis in T cell-mediated autoimmune disease such as EAE and MS.

ABSTRACT We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC - cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

GM-CSF–producing B cells contribute to multiple sclerosis pathogenesis and the therapeutic action of B cell depletion.

The myeloid differentiation primary response gene 88 (Myd88) is critical for protection against pathogens. However, we demonstrate here that MyD88 expression in B cells inhibits resistance of mice to Salmonella typhimurium infection. Selective deficiency of Myd88 in B cells improved control of bacterial replication and prolonged survival of the infected mice. The B cell-mediated suppressive pathway was even more striking after secondary challenge. Upon vaccination, mice lacking Myd88 in B cells became completely resistant against this otherwise lethal infection, whereas control mice were only partially protected. Analysis of immune defenses revealed that MyD88 signaling in B cells suppressed three crucial arms of protective immunity: neutrophils, natural killer cells, and inflammatory T cells. We further show that interleukin-10 is an essential mediator of these inhibitory functions of B cells. Collectively, our data identify a role for MyD88 and B cells in regulation of cellular mechanisms of protective immunity during infection.

Compounds that target the peroxisome proliferator-activated receptors PPARalpha and PPARgamma are used to correct dyslipidemia and to restore glycemic balance, respectively. Because the majority of diabetic patients suffer from atherogenic lipid abnormalities, in addition to insulin resistance, ligands are required that can activate both PPARalpha and PPARgamma. In this study, we used chimeric PPARalpha/gamma reporter-gene bioassays to screen herbal extracts with purported antidiabetic properties. Extracts of Astragalus membranaceus and Pueraria thomsonii significantly activated PPARalpha and PPARgamma. Bioassay-guided fractionation resulted in the isolation of the isoflavones, formononetin, and calycosin from Astragalus membranaceus, and daidzein from Pueraria thomsonii as the PPAR-activating compounds. We investigated the effects of these and 2 common isoflavones, genistein and biochanin A, using chimeric and full-length PPAR constructs in vitro. Biochanin A and formononectin were potent activators of both PPAR receptors (EC50 = 1-4 micromol/L) with PPARalpha/PPARgamma activity ratios of 1:3 in the chimeric and almost 1:1 in the full-length assay, comparable to those observed for synthetic dual PPAR-activating compounds under pharmaceutical development. There was a subtle hierarchy of PPARalpha/gamma activities, indicating that biochanin A, formononetin, and genistein were more potent than calycosin and daidzein in chimeric as well as full-length receptor assays. At low doses, only biochanin A and formononetin, but not genistein, calycosin, or daidzein, activated PPARgamma-driven reporter-gene activity and induced differentiation of 3T3-L1 preadipocytes. Our data suggest the potential value of isoflavones, especially biochanin A and their parent botanicals, as antidiabetic agents and for use in regulating lipid metabolism.

B lymphocytes have a unique role as antibody-producing cells. Antibodies are key mediators of humoral immunity against infections, and are thought to account for the protection afforded by successful vaccines. B cells can also secrete cytokines and subsequently regulate immune responses mediated by T and innate cells. Remarkably, recent studies identified plasma blasts/plasma cells as the main types of activated B cells producing the cytokines interleukin (IL)-10, IL-35, tumor necrosis factor (TNF)-α, IL-17, and GM-CSF in various contexts in mice. Here, we discuss these observations, which suggest the existence of various subsets of plasma blast/plasma cells distinguishable through their cytokine expression pattern.

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack.

Abstract Successful isolation of human endometrial stem cells from menstrual blood, namely menstrual blood‐derived endometrial stem cells (Men SC s), has provided enticing alternative seed cells for stem cell‐based therapy. Men SC s are enriched in the self‐regenerative tissue, endometrium, which shed along the periodic menstrual blood and thus their acquisition involves no physical invasiveness. However, the impact of the storage duration of menstrual blood prior to stem cell isolation, the age of the donor, the number of passages on the self‐renewing of Men SC s, the paracrine production of biological factors in Men SC s and expression of adhesion molecules on Men SC s remain elusive. In this study, we confirmed that Men SC s reside in shedding endometrium, and documented that up to 3 days of storage at 4°C has little impact on Men SC s, while the age of the donor and the number of passages are negatively associated with proliferation capacity of Men SC s. Moreover, we found that Men SC s were actually immune‐privileged and projected no risk of tumour formation. Also, we documented a lung‐ and liver‐dominated, spleen‐ and kidney‐involved organic distribution profile of Men SC 3 days after intravenous transfer into mice. At last, we suggested that Men SC s may have potentially therapeutic effects on diseases through paracrine effect and immunomodulation.

Rice tungro disease is caused by a combination of two viruses: rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (Jones et al. (1991) J. Gen. Virol. 72, 757-761.). The genome of RTSV is a single-stranded polyadenylated RNA. We present here the 12,433-nucleotide complete sequence of RTSV genomic RNA and its deduced coding regions. This sequence contains a large open reading frame (ORF) which initiates following a 514-nucleotide 5' leader sequence and is capable of encoding a viral polyprotein of 390.3 kDa. Two viral subgenomic RNAs of ca. 1.2 and 1.4 kb, respectively, were detected in RTSV-infected leaf tissues and mapped by S1 nuclease protection assay. These RNAs were determined to be congruent with the genomic RNA sequence proximal to the 3' terminus and could contain up to two small ORFs in their 5' to 3' orientation. There are at least three capsid protein subunit cistrons near the N-terminus of the large ORF. A computer-aided search of the C-terminal half of the large ORF revealed conserved protein sequence motifs for a viral RNA polymerase, proteinase, and a putative NTP-binding protein. These sequence motifs are arranged in a manner that resembles those of picorna-like viruses. Taken together, these data indicate that RTSV is a distinct type of positive-strand RNA virus. The evolutionary relationships between RTSV and other picorna-like plant viruses are discussed.

B lymphocytes contribute to immunity through production of antibodies, antigen presentation to T cells, and secretion of cytokines. B cells are generally considered in autoimmune diseases as drivers of pathogenesis. This view is certainly justified, given the successful utilization of the B cell-depleting reagent rituximab in patients with rheumatoid arthritis or other autoimmune pathologies. In a number of cases, however, the depletion of B cells led to an exacerbation of symptoms in patients with autoimmune disorders. In a similar manner, mice lacking B cells can develop an aggravated course of disease in several autoimmune models. These paradoxical observations are now explained by the concept that activated B cells can suppress immune responses through the production of cytokines, especially interleukin-10. Here, we review the stimulatory signals that induce interleukin-10 secretion and suppressive functions in B cells and the phenotype of the B cells with such characteristics. Finally, we formulate a model explaining how this process of immune regulation by activated B cells can confer advantageous properties to the immune system in its combat with pathogens. Altogether, this review proposes that B-cell-mediated regulation is a fundamental property of the immune system, with features of great interest for the development of new cell-based therapies for autoimmune diseases.

We present evidence that rice tungro spherical virus (RTSV) has a genome of polyadenylated single-stranded RNA of about 10 kb whereas rice tungro bacilliform virus (RTBV) contains double-stranded circular DNA. RTBV DNA has been mapped and shown to have two discontinuities, one in each strand, at specific sites; it thus resembles that of the caulimo-viruses. Gel electrophoresis of RTSV preparations revealed two protein bands (M r 35K and 26K). RTBV yielded two major protein bands of 37K and 33K together with several minor species of higher and lower M r which react with antiviral serum.

To understand the factors contributing to estrogenic properties of extracts from the genus Epimedium L. (Berberidaceae), we performed taxonomic, genetic and chemical characterization on 37 specimens from 18 species and related these to estrogen receptor (ERα and ERβ) bioactivity, as measured by reporter genes in stable human cells. Boot strap values derived from amplified fragment length polymorphisms indicated that specimens of E. koreanum, E. brevicornum, E. myrianthum, E. leishanense, and E. membranaceum were genetically distinct and this was supported by their very similar ERα activities. In contrast, specimens from E. pubescens and E. sagittatum were diverse both genetically, chemically and in terms of ERα and ERβ bioactivities. Strikingly, a genetic cluster comprising six rare Epimedium species exhibited strongest ERα and ERβ activity, and this bioactivity was positively correlated with content of trace flavonoid aglycones (kaempferol, apigenin, quercetin, luteolin and breviflavone B). In contrast, there was no association between estrogenic activity and the major flavonol glycoside constituents (icariin and epimedin A–C). Although they exhibited equally strong ERα and ERβ activity, E. koreanum can be clearly differentiated from E. pubescens and E. brevicornum by genetic distance and its significantly lower content of epimedin C. Our morphologic, genetic, chemical and bioactivity profiling provide the basis for the production of extracts with reproducible estrogenic properties. Such reproducibility will be critical for the standardization of Epimedium-based products.

To explore pharmacokinetic properties of prenylflavonoids from the Traditional Chinese Medicinal plant Epimedium, three doses of a standardized extract (100, 300 and 600 mg/kg body weight), were administered to ovariectomized rats and serial blood samples were obtained. Serum concentrations of the Epimedium prenylflavonoids icariin, icariside I, icariside II, icaritin and desmethylicaritin were determined by LC-MS/MS. Aliquots of sera were also applied to human cell lines that permanently express ERalpha and ERbeta proteins for the ex vivo measurement of estrogenic activity. All five prenylflavonoids exhibit non-linear dose-dependent increases in the area under concentration versus time curves. Two distinct pharmacokinetic patterns were evident, an early phase wherein icariin and icariside II reached t(max) 0.5-1 h, and a late phase wherein icariside I, icaritin and desmethylicaritin peaked at t(max) 8h. Total concentrations of icaritin and desmethylicaritin reached C(max) approximately 2 microM and approximately 0.25 microM respectively. Estrogenic activity in Epimedium-treated rat sera lagged by several hours compared to animals treated with control drug estradiol benzoate, corresponding to the appearance of bioactive metabolites desmethylicaritin, icaritin and icariside I. Following glucuronidase/sulphatase treatment, prolonged estrogenic activity at higher Epimedium doses (300 and 600 mg/kg of body weight) was evident, and correlated with the persistence of micromolar levels of icaritin at the 48-72 h sampling period. The depot effect resulted in time-concentration bioactivity profiles at the three Epimedium doses (area under curve 374, 543, and 771pME2 h(-1)) that exceeded that observed for estradiol benzoate (148pME2 h(-1)). Our study correlated the pharmacokinetics of prenylflavonoids with the dynamics of their estrogenic effects and reveals the potential estrogenicity of this Epimedium extract. This study may aid the development of prenylflavonoids as drugs for menopause and other conditions requiring estrogenic action.

Surface modification of mesoporous silica nanoparticles (MSNs) is a promising way to enhance therapeutic efficacy and minimize side effects of anticancer drugs. In this work, MSNs with reduced particle size and optimum pore diameter were obtained and catalyzed by ammonia/triethanolamine. In view of the negatively charged carboxymethyl chitosan (CMC) and positively charged chitosan oligosaccharide (CS), the pH-triggered charge-reversal CS/CMC bilayer was designed as a stimuli-responsive switch for MSNs via the protonation and deprotonation effect. The results showed that MSNs-CS/CMC were core-shell and mesoporous in structure. Surface charge conversion and pH dependence were clearly observed in the doxorubicin hydrochloride (DOX) delivery. The intracellular uptake indicated that DOX@MSNs-CS/CMC could be distributed in the cytoplasm of MCF-7 cells and exhibited lower toxicity, which would improve the stability and prolong the retention time compared to free DOX and unmodified DOX@MSNs at pH 7.4. Moreover, the cellular uptake and internalization of DOX@MSNs-CS/CMC were enhanced to promote drug delivery into the cell nucleus at pH 6.5. The biocompatible and surface-charge-reversible MSNs-CS/CMC have the potential to prolong the retention time in the bloodstream, facilitate the endosome escape, and enrich the targeted antitumor strategy, providing an alternative platform for efficient drug delivery in breast cancer therapy.

The major promoter region for the transcription of the genome of rice tungro bacilliform virus (RTBV), a newly described badnavirus, has been identified. Fragments of the RTBV genome upstream of the site of transcription initiation were isolated and tested for promoter activity using a β‐glucuronidase reporter gene ( gusA ). Assays of transient gusA expression were performed following introduction of the chimeric gene into protoplasts via electroporation. The chimeric RTBV‐promoter: gusA gene was more active in rice protoplasts than in maize or tobacco protoplasts, but was weaker than gusA controlled by an enhanced 35S promoter from cauliflower mosaic virus. Analysis of gusA gene expression following introduction of chimeric reporter genes into intact leaves via micro‐projectile bombardment indicated that the GUS activity is present primarily in vascular tissues. Transgenic rice plants carrying the chimeric gusA gene had GUS activity only in the phloem of the vascular bundles in the leaf. Tissue printing studies demonstrated that RTBV accumulates in the vascular bundles of infected rice leaves. The results of our study indicate that phloem‐specific expression from the RTBV promoter is an intrinsic property of the viral promoter.

Osteoarthritis (OA) is a joint disease featuring cartilage breakdown and chronic pain. Although age and joint trauma are prominently associated with OA occurrence, the trigger and signaling pathways propagating their pathogenic aspects are ill defined. Following long-term catabolic activity and traumatic cartilage breakdown, debris accumulates and can trigger Toll-like receptors (TLRs). Here we show that TLR2 stimulation suppressed the expression of matrix proteins and induced an inflammatory phenotype in human chondrocytes. Further, TLR2 stimulation impaired chondrocyte mitochondrial function, resulting in severely reduced adenosine triphosphate (ATP) production. RNA-sequencing analysis revealed that TLR2 stimulation upregulated nitric oxide synthase 2 ( NOS2 ) expression and downregulated mitochondria function-associated genes. NOS inhibition partially restored the expression of these genes, and rescued mitochondrial function and ATP production. Correspondingly, Nos2 −/− mice were protected from age-related OA development. Taken together, the TLR2–NOS axis promotes human chondrocyte dysfunction and murine OA development, and targeted interventions may provide therapeutic and preventive approaches in OA.

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.

Estrogens maintain female sexual health. The hormone also drives the growth of estrogen receptor (ER) positive breast tumors, and ER modulators, like tamoxifen, are used to reduce tumor recurrence. To identify phytoestrogens with possible health benefits, we screened several Traditional Chinese Medicines and encountered an extract from the leaves of Epimedium brevicornum (EB), with strong (EC50: 1.3 microg/mL) and specific ER-stimulatory activity. It increased estrogen-responsive human breast cancer cell proliferation at low doses, but paradoxically caused profound inhibition of growth at higher doses. Using bioassay-guided fractionation, we isolated and characterized a new prenylflavone, breviflavone B, which exerted biphasic stimulatory and inhibitory effects on breast cancer cell proliferation, mimicking the effects of EB. In contrast to estradiol and genistein, high doses (> 2 microM) of breviflavone B almost eliminated ERalpha protein; a process that may be mediated through increased proteasome degradation. Pre-clinical studies are needed to explore whether these prenylflavones are of value in estrogen-deficiency states and for prophylaxis of breast cancer.

The aim of this study was to investigate the effects of Lycium barbarum polysaccharide (LBP) on irradiation- or chemotherapy-induced myelosuppressive mice and cultured peripheral blood mononuclear cells (PBMCs).In an in vivo experiment, mice were irradiated with a sublethal dose of 550 cGy X-ray or intraperitoneally (i.p.) injected with carboplatin (CB) 125 mg/kg to produce severe myelosuppression. Four to 6 hours after the irradiation or injection, mice were subcutaneously (s.c.) injected with LBP (50, 100, and 200 mg/kg) daily from day 0 to day 6. Blood samples were collected from the tail veins of mice at different time points, and peripheral white blood cells (WBC), red blood cells (RBC), and platelet (PLT) counts were monitored. In an in vitro experiment, human PBMCs were incubated with LBP at different concentrations in combination with phytohemagglutinin (PHA), and the production of granulocyte colony-stimulating factor (G-CSF) was tested.Compared to the control, 50 mg/kg LBP (LBP-L) significantly ameliorated the decrease of peripheral WBC of irradiated myelosuppressive mice on day 13, and 100 mg/kg LBP (LBP-M) did the same on days 17 and 21. All dosages of LBP significantly ameliorated the decrease of peripheral RBC of irradiated myelosuppressive mice on days 17 and 25. Two-hundred mg/kg LBP (LBP-H) and LBP-M significantly enhanced peripheral PLT counts of irradiated myelosuppressive mice on days 10, 13, 17, and 21, as did LBP-L on days 13 and 17. All dosages of LBP increased peripheral WBC counts of chemotherapy-induced myelosuppressive mice to some extent, but there was no statistic difference when compared to the control. LBP-H significantly ameliorated the decrease of peripheral RBC of chemotherapy-induced myelosuppressive mice on days 13, 15, 17, and 20, and LBP-M and LBP-L did the same on days 15 and 17. All dosages of LBP significantly enhanced peripheral PLT counts of chemotherapy-induced myelosuppressive mice on days 7 and 10, as did LBP-H on days 13, 15, and 17, and LBP-M on days 13 and 15. Also, LBP could obviously stimulate human PBMCs to produce G-CSF.LBP promoted the peripheral blood recovery of irradiation or chemotherapy-induced myelosuppressive mice, and the effects may be the result of the stimulation of PBMCs to produce G-CSF.

Five dimeric phthalides were isolated from rhizomes of Ligusticum chuanxiong and their structures deduced based on spectral data. All compounds and their parent extracts were assessed for progesterone-like activity using a progesterone receptor driven reporter-gene bioassay. Among all the compounds, riligustilide, displayed weak progesterone-like activity (EC50 approximately 81 microM), whereas, (3Z')-(3a'R,6'R,3R,6R,7R)-3,8-dihydro-6.6',7.3a'-diligustilide (Mr: 382, EC50 approximately 90 nM), was found to be a potent and specific activator of the progesterone receptor. Levistolide A, although having a very similar plenary structure, was inactive indicating the importance of stereochemistry of chiral centers and flexibility of butylidene side chain for progestogenic activity. These bioactive phthalides and their parent extracts (EC50 approximately 8 microg/ml) may have utility for treatment of conditions requiring progesterone action.

Abstract Activated B cells can regulate immunity and have been envisaged as a potential cell‐based therapy for treating autoimmune diseases. However, activated human B cells can also propagate immune responses, and the effects resulting from their infusion into patients cannot be predicted. This led us to consider resting B cells, which in contrast are poorly immunogenic, as an alternative cellular platform for the suppression of unwanted immunity. Here, we report that resting B cells can be directly engineered with lentiviral vectors to express antigens in a remarkably simple, rapid, and effective way. Notably, this neither required nor induced activation of the B cells. With this approach we were able to produce reprogrammed resting B cells that inhibited antigen‐specific CD4 + T cells, CD8 + T cells, and B cells upon adoptive transfer in mice. Furthermore, resting B cells engineered to ectopically express myelin oligodendrocyte glycoprotein antigen protected recipient mice from severe disability and demyelination in EAE, and even induced complete remission from disease in mice lacking functional natural Tregs, which otherwise developed chronic paralysis. In conclusion, our study introduces reprogrammed quiescent B cells as a novel tool for suppressing undesirable immunity.

B cells and regulatory T (Treg) cells can both facilitate remission from experimental auto immune encephalomyelitis (EAE), a disease of the central nervous system (CNS) used as a model for multiple sclerosis (MS). Considering that B-cell-depletion therapy (BCDT) is used to treat MS patients, we asked whether Treg-cell activation depended on B cells during EAE. Treg-cell proliferation, accumulation in CNS, and augmentation of suppressive activity in the CNS were normal in B-cell-deficient mice, indicating that B cells are not essential for activation of the protective Treg-cell response and thus provide an independent layer of regulation. This function of B cells involved early suppression of the encephalitogenic CD4(+) T-cell response, which was enhanced in B-cell-deficient mice. CD4(+) T-cell depletion was sufficient to intercept the transition from acute-to-chronic EAE when applied to B-cell-deficient animals that just reached the peak of disease severity. Intriguingly, this treatment did not improve disease when applied later, implying that chronic disability was ultimately maintained independently of pathogenic CD4(+) T cells. Collectively, our data indicate that BCDT is unlikely to impair Treg-cell function, yet it might produce undesirable effects on T-cell-mediated autoimmune pathogenesis.

B-cell depletion can improve disease in some patients with rheumatoid arthritis or multiple sclerosis, indicating the pathogenic contribution of B cells to autoimmunity. However, studies in mice have demonstrated that B cells have immunosuppressive functions as well, with IL-10 being a critical mediator of B-cell-mediated suppression. IL-10-secreting B cells have been shown to promote disease remission in some mouse models of autoimmune disorders. Human B cells also produce IL-10, and evidence is accumulating that human IL-10-producing B cells might inhibit immunity. There is considerable interest in identifying the phenotype of B cells providing IL-10 in a suppressive manner, which would facilitate the analysis of the molecular mechanisms controlling this B-cell property. Here, we review current knowledge on the B-cell subpopulations found to provide suppressive functions in mice, considering both the pathological context in which they were identified and the signals that control their induction. We discuss the phenotype of B cells that have IL-10-dependent regulatory activities in mice, which leads us to propose that antibody-secreting cells are, in some cases at least, the major source of B-cell-derived regulatory IL-10 in vivo. Anti-inflammatory cytokine production by antibody-secreting cells offers a novel mechanism for the coordination of innate and humoral immune responses.

B lymphocytes are often essential to successfully control invading pathogens and play a primary role in the protection afforded by successful vaccines through the production of specific antibodies. However, recent studies have highlighted the complex roles of B cells in infectious diseases, showing unexpectedly that some activated B cells limited host defense towards pathogens. This B-cell function involves production of regulatory cytokines including IL-10 and IL-35 and is reminiscent of the regulatory functions of B cells initially defined in autoimmune diseases. It is now known that various types of microbes including bacteria, helminths and viruses can induce IL-10-expressing B cells with inhibitory functions, indicating that this response is a general component of anti-microbial immunity. Interestingly, IL-10-producing B cells induced in the course of some microbial infections can inhibit concurrent immune responses directed towards unrelated antigens in a bystander manner and as a consequence ameliorate the course of autoimmune or allergic diseases. This could explain how some micro-organisms might provide protection from these pathologies, as formulated in the 'hygiene hypothesis'. In this review, we discuss the regulatory functions of B cells in bacterial, parasitic and viral infections, taking into account the phenotype of the B cells implicated, the signals controlling their induction and the cell types targeted by their suppressive activities.

B cells are usually considered primarily for their unique capacity to produce antibodies after differentiation into plasma cells. In addition to their roles as antibody-producing cells, it has become apparent during the last 10 years that B cells also perform important functions in immunity through the production of cytokines. In particular, it was shown that B cells could negatively regulate immunity through provision of interleukin (IL)-10 during autoimmune and infectious diseases in mice. Here, we review data on the suppressive functions of B cells in mice with particular emphasis on the signals controlling the acquisition of such suppressive functions by B cells, the phenotype of the B cells involved in the negative regulation of immunity, and the processes targeted by this inhibitory circuit. Finally, we discuss the possibility that human B cells might also perform similar inhibitory functions through the provision of IL-10, and review data suggesting that such B cell-mediated regulatory activities might be impaired in patients with autoimmune diseases.

The prenyl-flavones, icaritin and desmethylicaritin, are bioactive compounds from the traditional Chinese medicinal herb, Epimedium, extracts of which can enhance bone health in animal models. In order to examine their bioavailability in humans, we have developed and validated a sensitive method to quantify icaritin and desmethylicaritin in human sera, using gas chromatography-mass spectrometry. The serum samples were extracted with ethyl acetate and then derivatized with BSTFA in pyridine (4:1). With genistein as internal standard, calibration curves with good linearity (R(2)>0.99) within the concentration range of 0.15-10nM in the selective ion monitoring mode were obtained. The limits of detection and quantization were 11 and 33 pM for icaritin, and 23 and 70 pM for desmethylicaritin, respectively; inter- and intra-assay variabilities were <15%, and accuracies were between 89 and 110%. Icaritin, but not desmethylicaritin, was detected from 1h, increasing to a peak at 8h (1.51+/-1.6 nM) in sera of human volunteers after ingestion of an aqueous decoction of Epimedium. This sensitive method can be used to quantify serum levels of icaritin and desmethylicaritin for pharmacokinetic studies.

The Chinese medicinal herb, Epimedium, used traditionally for bone health exerts estrogenic activity (EA) in vitro. A genetically characterized Epimedium brevicornum (EB) extract induced biphasic responses in the mRNA and protein expression of the estrogen-regulated progesterone receptor gene in breast cancer (MCF-7) cells. These changes were mirrored changes in estrogenic receptor (ERα) content. In male Sprague–Dawley rats, administration of the estrogenic prodrug, estradiol valerate increased area-under-curve of serum effects for ERα (AUC difference: 18,900EA(ERα) min; 95% CI: 0–37,800; p = 0.05) and breast cancer cell (MCF-7) growth (AUC difference: 30,200EA(MCF-7) min; 95% CI: 24,200–36,200; p < 0.001), compared to placebo. Oral administration of Epimedium brevicornum increased ERα activity (1320EA(ERα) min, p < 0.01). Our data indicate that estrogen-responsive bioassays can measure the pharmacokinetic/pharmacodynamics of estrogenic activity in serum. Epimedium brevicornum extract increases estrogenic activity in serum and human studies are required to evaluate whether Epimedium extracts have utility for estrogen replacement therapy.

A rapid and sensitive method to separate and quantify icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera was developed using liquid chromatography–tandem mass spectrometry. Serum samples were extracted with ethyl acetate without further derivatization. Using coumestrol as an internal standard, calibration curves with good linearity (r2 > 0.99) within the concentration range of 0.78–12.5 nM for icariin, icaritin and desmethylicaritin, and 0.78–100 nM for icariside I and II, were obtained in the multiple reaction monitoring mode. For all analytes, the limits of detection and quantification were <1 nM and 1–2 nM, respectively. Inter- and intra-assay variabilities were <15% and accuracies were between 94% and 114%, respectively. This method was successfully applied to quantify levels of icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera after oral administration of an Epimedium preparation.

Accumulation of advanced glycation end products (AGEs) has been implicated in the development of diabetic nephropathy. We investigated the effects of Pu-erh tea on AGE accumulation associated with diabetic nephropathy. Although it did not affect blood glucose levels and insulin sensitivy, Pu-erh tea treatment for 8 weeks attenuated the increases in urinary albumin, serum creatinine, and mesangial matrix in db/db mice. We found that Pu-erh tea prevented diabetes-induced accumulation of AGEs and led to a decreased level of receptor for AGE expression in glomeruli. Both production and clearance of carbonyl compounds, the main precursor of AGE formation, were probably attenuated by Pu-erh tea in vivo independent of glyoxalase I expression. In vitro, HPLC assay demonstrated Pu-erh tea could trap methylglyoxal in a dose-dependent manner. Our study raises the possibility that inhibition of AGE formation by carbonyl trapping is a promising approach to prevent or arrest the progression of diabetic complications.

Tumor cells often produce high levels of reactive oxygen species (ROS) and display an increased ROS scavenging system. However, the molecular mechanism that balances antioxidative and oxidative stress in cancer cells is unclear. Here, we determined that oncogenic multiple copies in T-cell malignancy 1 (MCT-1) activity promotes the generation of intracellular ROS and mitochondrial superoxide. Overexpression of MCT-1 suppresses p53 accumulation but elevates the manganese-dependent superoxide dismutase (MnSOD) level via the YY1-EGFR signaling cascade, which protects cells against oxidative damage. Conversely, restricting ROS generation and/or targeting YY1 in lung cancer cells effectively inhibits the EGFR-MnSOD signaling pathway and cell invasiveness induced by MCT-1. Significantly, MCT-1 overexpression in lung cancer cells promotes tumor progression, necrosis and angiogenesis, and increases the number of tumor-promoting M2 macrophages and cancer-associated fibroblasts in the microenvironment. Clinical evidence further confirms that high expression of MCT-1 is associated with an increase in YY1, EGFR and MnSOD expression, accompanied by tumor recurrence, poor overall survival and EGFR mutation status in patients with lung cancers. Together, these data indicate that the MCT-1 oncogenic pathway is implicated in oxidative metabolism and lung carcinogenesis.

In contrast to men, women have experienced a rapid increase in lung cancer mortality. Numerous studies have found that the sex differences in lung cancer are due to reproductive hormones. Experiments in female mice with and without ovariectomy were performed to explore the possible mechanism by which sex hormones (and their receptors) influence lung cancer.Twenty-four female C57BL/6 mice aged 56-62 days were randomly divided into the ovariectomized group and the control group. In the ovariectomized group, the bilateral ovaries were removed via the dorsal approach, while the control group underwent a sham operation with bilateral ovarian fat resection at the same sites. After 3 weeks of recovery, Lewis lung cancer cells were transplanted into these mice by subcutaneous inoculation of a tumour cell suspension to establish the ovariectomized lung cancer model. Beginning on the 6th day after subcutaneous inoculation, mouse weight and transplanted tumour volume were measured every 3 days. After 3 weeks, all the mice were killed by cervical dislocation, and we measured the tumour weight. Mouse serum and tumour tissues were removed. Then, the serum levels of E2 (oestradiol) and T (testosterone) were detected by ELISA; the protein expression levels of AR (androgen receptor), ERα (oestrogen receptor α) and ERβ (oestrogen receptor β) were detected by Western Blot and IHC (immunohistochemistry); and the mRNA expression levels of AR, ERα and ERβ were detected by qRT-PCR (quantitative real-time polymerase chain reaction) in the ovariectomized and control groups.Compared with the control group, both mouse weight and transplanted tumour volume increased rapidly in the ovariectomized group, and the transplanted tumour weight was significantly heavier in the ovariectomized group (1.83±0.40 and 3.13±0.43, P<0.05). E2 and T serum levels decreased exponentially in the ovariectomized group, while the E2/T ratio increased compared with the control group (E2: 55.88±11.45 and 78.21±9.37; T: 0.82±0.14 and 1.46±0.16; ratio: 69.62±14.43±29.81 and 52.22±5.42; all P<0.05). The Western blot and IHC results indicated that AR, ERα and ERβ protein expression levels were obviously higher in transplanted tumour and lung tissues from the ovariectomized group, with particular increases in ERβ in transplanted tumour tissue and in ERα in lung tissue. The PCR results also showed markedly higher mRNA expression levels of AR, ERα and ERβ in the ovariectomized group, and in particular, ERβ in transplanted tumour tissue and ERα in lung tissue were significantly increased in the ovariectomized group.Ovariectomy decreased E2 and T serum levels and increased the E2/T ratio in mice, and this imbalance in the internal environment promoted the growth of transplanted tumours. Sex hormone disorder not only promoted transplanted tumour growth but also significantly reduced the protein and mRNA expression levels of sex hormone receptors. The metabolism of E2 and T may affect the growth, proliferation and metabolism of lung cancer cells, and the mechanism by which sex hormones and their receptors influence lung cancer is worthy of further research.

The global climate change made the heavy rainfall happen more frequently, and the non-point source pollution caused by it would exacerbate the risk to the water ecological environment. In this study, we took a reservoir (Shahe reservoir, Beijing, China) supplied with reclaimed water as an exapmle to investigate how spatiotemporal changes in the quantity and diversity of prokaryotic, eukaryotic, and algal communities respond to heavy rainfall. Results showed that heavy rainfall could directly impact the composition of the prokaryotic community by introducing amounts of runoff closely associated bacterium especially for the human potential pathogens such as Aliarcobacter, Aeromonas and Pseudomonas in the Shahe reservoir area. While the eukaryotic community was rather stable, and the development and changes in algal communities occurred in the last few days after heavy rainfall. The microbial source tracking through FEAST indicated that Nansha river (S) was the major contributor to the development of all the three concerned communities in the reservoir. The co-occurrence analysis showed that the modules with the highest cumulative abundance in each community were all strongly and positively connected with Chl-a, pH, turbidity, COD and TOC, but negatively correlated with NO3-N (p < 0.01). The network analysis showed that the eukaryotes played a key role in the interaction network among the three communities, and were more likely to interact with algae and prokaryotes. It was suggested that the controlling of human potential pathogens associated with prokaryotic community should be emphasized at the beginning of the heavy rainfall, but the prevention of the eutrophication bloom should be another focus after the heavy rainfall. This study provided valuable information concerning the role of heavy rainfall on the water ecological environment from the perspective of microbial community.

Purpose Sphingosine-1-phosphate (S1P) is a signaling lipid involved in many biological processes, including inflammatory and immune regulatory responses. The study aimed to determine whether admission S1P levels are associated with disease severity and prognosis after spontaneous intracerebral hemorrhage (ICH). Methods Data of 134 patients with spontaneous ICH and 120 healthy controls were obtained from Biological Resource Sample Database of Intracerebral Hemorrhage at the First Affiliated Hospital of Zhengzhou University. Plasma S1P levels were measured. Regression analyses were used to analyze the association between S1P levels and admission and 90-day modified Rankin scale (mRS) scores. Receiver operating characteristic (ROC) curves assessed the predictive value of S1P levels for ICH severity and prognosis. Results Patients with ICH exhibited elevated plasma S1P levels compared to the control group (median 286.95 vs. 239.80 ng/mL, p &amp;lt; 0.001). When divided patients into mild-to-moderate and severe groups according to their mRS scores both at admission and discharge, S1P levels were significantly elevated in the severe group compared to the mild-to-moderate group (admission 259.30 vs. 300.54, p &amp;lt; 0.001; 90-day 275.24 vs. 303.25, p &amp;lt; 0.001). The patients were divided into three groups with different concentration gradients, which showed significant statistical differences in admission mRS scores (3 vs. 4 vs. 5, p &amp;lt; 0.001), 90-day mRS scores (2.5 vs. 3 vs. 4, p &amp;lt; 0.001), consciousness disorders (45.5% vs. 68.2% vs. 69.6%, p = 0.033), ICU admission (29.5% vs. 59.1% vs. 89.1%, p &amp;lt; 0.001), surgery (15.9% vs. 47.7% vs. 82.6%, p &amp;lt; 0.001), intraventricular hemorrhages (27.3% vs. 61.4% vs. 65.2%, p &amp;lt; 0.001) and pulmonary infection (25% vs. 47.7% vs. 84.8%, p &amp;lt; 0.001). Multivariate analysis displayed that S1P level was an independent risk factor for disease severity (OR = 1.037, 95% CI = 1.020–1.054, p &amp;lt; 0.001) and prognosis (OR = 1.018, 95% CI = 1.006–1.030, p = 0.003). ROC curves revealed a predictive value of S1P levels with an area under the curve of 0.7952 (95% CI = 0.7144–0.8759, p &amp;lt; 0.001) for disease severity and 0.7105 (95% CI = 0.6227–0.7983, p &amp;lt; 0.001) for prognosis. Conclusion Higher admission S1P is associated with worse initial disease severity and 90-day functional outcomes in intracerebral hemorrhage.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a central regulator of lipid metabolism. Fibrate drugs act on PPARalpha to modulate dyslipidemias. A natural variant (V227A) affecting the PPARalpha hinge region was associated with perturbations in blood lipid levels in Asian populations. In this study, we investigated the functional significance of the V227A substitution. The variant significantly attenuated PPARalpha-mediated transactivation of the cytochrome P450 4A6 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) genes in the presence of fibrate ligands. Screening of a panel of PPARalpha coregulators revealed that V227A enhanced recruitment of the nuclear corepressor NCoR. Transactivation activity of V227A could be restored by silencing NCoR or by inhibition of its histone deacetylase activity. Deletion studies indicated that PPARalpha interacted with NCoR receptor-interacting domain 1 (ID1) but not ID2 or ID3. These interactions were dependent on the intact consensus nonapeptide nuclear receptor interaction motif in NCoR ID1 and were enhanced by the adjacent 24 N-terminal residues. Novel corepressor interaction determinants involving PPARalpha helices 1 and 2 were identified. In hepatic cells, the V227A substitution stabilized PPARalpha/NCoR interactions and caused defective release of NCoR in the presence of agonists on the HMGCS2 promoter. These results provide the first indication that defective function of a natural PPARalpha variant was due, at least partially, to increased corepressor binding. Our data suggest that the PPARalpha/NCoR interaction is physiologically relevant and can produce a discernable phenotype when the magnitude of the interaction is altered by a naturally occurring variation.

Abstract Background Immunosuppressive properties grant mesenchymal stromal cells (MSCs) promising potential for treating autoimmune diseases. As autologous MSCs suffer from limited availability, the readily available allogeneic MSCs isolated from menstrual blood (MB-MSCs) donated by young, healthy individuals offer great potential. Here, we evaluate the therapeutic potential of MB-MSCs as ready-to-use allo-MSCs in multiple sclerosis, an autoimmune disease developed by the activation of myelin sheath-reactive Th1 and Th17 cells, by application in its animal model experimental autoimmune encephalomyelitis (EAE). Methods We assessed the therapeutic effect of MB-MSCs transplanted via either intravenous (i.v.) or intraperitoneal (i.p.) route in EAE in comparison with umbilical cord-derived MSCs (UC-MSCs). We used histology to assess myelin sheath integrity and infiltrated immune cells in CNS and flow cytometry to evaluate EAE-associated inflammatory T cells and antigen-presenting cells in lymphoid organs. Results We observed disease-ameliorating effects of MB-MSCs when transplanted at various stages of EAE (day − 1, 6, 10, and 19), via either i.v. or i.p. route, with a potency comparable to UC-MSCs. We observed reduced Th1 and Th17 cell responses in mice that had received MB-MSCs via either i.v. or i.p. injection. The repressed Th1 and Th17 cell responses were associated with a reduced frequency of plasmacytoid dendritic cells (pDCs) and a suppressed co-stimulatory capacity of pDCs, cDCs, and B cells. Conclusions Our data demonstrate that the readily available MB-MSCs significantly reduced the disease severity of EAE upon transplantation. Thus, they have the potential to be developed as ready-to-use allo-MSCs in MS-related inflammation. Graphical abstract

The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules of the 90-kDa heat shock protein (hsp90) and one molecule of the 56-kDa heat shock protein (hsp56). In the absence of hormone, this complex is found in the cytosolic fraction of cells, and upon hormone-binding the complex dissociates and the GR is recovered in the nuclear pellet fraction. Given the association of heat shock proteins with the cytosolic form of the GR, we have examined the effects of heat shock on GR subcellular localization and transcription enhancement activity in a series of Chinese hamster ovary (CHO) cells which express either low levels of endogenous GR (CHOd cells), high levels of the mouse GR (WCL2 cells), or high levels of mutated mouse GR unable to bind DNA (NB cells). It was found that heat shock treatment of WCL2 cells results in wild-type mouse GR that is recovered almost entirely within the nuclear pellet fraction, a response similar to that seen in hormone-treated cells. In contrast, heat shock treatment of NB cells results in complete loss of GR from the cytosolic fraction, but almost no shift of GR to the nuclear pellet. These results indicate that heat shock-mediated conversion to high-affinity nuclear binding by the wild-type GR requires a functional DNA-binding domain, and that heat shock will result in loss of GR to proteolysis in the absence of nuclear sequestration. Analysis of MMTV-CAT reporter gene expression in these cells revealed that heat or chemical shock, in comparison to hormone-treatment, results in a small induction of MMTV-CAT expression in the WCL2 cells, but not in the CHOd or NB cells. These results indicate that cellular stress can cause at least a partial induction of hormone-independent GR-mediated gene expression.

Prepubertal testicular dysfunction and the subsequent development of hypogonadism affects an estimated one in 200 children worldwide.As the testosterone levels are dynamic during development and puberty, traditional hormone treatment regimens are often inadequate, thereby leaving associated physiological conditions unresolved.Therefore, we have investigated the potential therapeutic effect of mature Leydig cell transplantation for the treatment of prepubertal primary hypogonadism through the use of a surgically induced hypogonadistic rat model system.In the experiment, Leydig cells were surgically isolated from mature Sprague-Dawley rats and transplanted into prepubertal recipients.Serum testosterone levels and microscopic analysis of the stained testicular interstitium were compared with sham-treated controls, as well as with castrated and intact rats during sexual development.At 4 weeks post-implantation, serum testosterone was detectable in Leydig cell recipients, but not in surgical controls, and progressively increased as a function of time until reaching levels comparable with sexually mature males at 12 weeks post-implantation.Histological analysis revealed a high rate of Leydig cell survival as well as steroidogenic secretory activity.Therefore, we conclude that mature Leydig cell transplantation in prepubertal hypogonadism recipients has therapeutic potential in rats and merits further investigation for clinical application.

At present, cardiovascular disease is the global leading cause of mortality. Total paeony glycoside (TPG) is a traditional Chinese medicine, which serves a pivotal role in the cardiovascular system. In the present study, the effects and underlying mechanisms of TPG on ischemia/reperfusion (I/R) injury‑induced apoptosis of cardiomyocytes were investigated in vitro. Cell Counting kit‑8 and flow cytometry were used to assess the viability, reactive oxygen species (ROS) content and apoptosis of H9C2 cells. The activities of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were analyzed by commercial detection kits. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were conducted to evaluate the expression levels of various factors. The results demonstrated that the viability of H9C2 cells was not significantly altered in response to various concentrations of TPG. However, following I/R injury, TPG markedly enhanced cell viability in a time‑ and dose‑dependent manner. Furthermore, TPG decreased the rate of apoptosis and ROS levels, and reduced the activities of MDA and LDH. Conversely, TPG increased SOD and GPX activities. In addition, TPG upregulated the expression levels of pro‑caspase‑3 and B‑cell lymphoma2 (Bcl‑2), whereas it downregulated cleaved‑caspase‑3, poly (ADP‑ribose) polymerase 1, Bcl‑2‑associated X protein, phosphorylated (p)‑phosphatidylinositol 3 kinase (PI3K) and p‑protein kinase B (Akt) expression. Treatment with insulin‑like growth factor‑1 increased the apoptosis of H9C2 cells, thus suggesting that activation of the PI3K/Akt signaling pathway reversed the protective effects of TPG. Taken together, TPG may suppress I/R‑induced apoptosis and oxidative stress of H9C2 cells possibly by inhibiting the PI3K/Akt signaling pathway; such a phenomenon may have a therapeutic effect on cardiovascular disease.

Chemical examination of the fresh rhizomes of Tupistra wattii HOOK. f. led to the isolation of three new steroidal saponins, wattoside G (1), H (2), and I (3), together with one known steroidal saponin, (25S)-1β,3β,4β-trihydroxyspirotan-5β-yl-O-β-D-glucopyranoside (4). The structures of 1—3 were established to be (25R)-1β,2β,3β,5β-tetrahydroxyspirostan-4β-yl-O-β-D-xylopyranoside (1), (24S,25S)-24-[(β-D-glucopyranosyl)oxy]-1β,2β,3β,4β,5β,7β-hexahydroxyspirostan-6-one (2), and (24S,25S)-1β,3β-dihydroxy-5β-spirostan-24-yl-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (3) on the basis of detailed analyses of physical, chemical, and spectral data. The isolated compounds were evaluated for cytotoxic activity against the cancer cell line K562 in vitro.

Flavonoids present in food, botanicals, and body fluids occur as complex mixtures, and data on their combinatorial estrogenic effects are sparse. Human cell lines that permanently express estrogen receptor (ER) alpha and ERbeta proteins were developed for the measurement of the global estrogenicity of flavonoids in such complex mixtures. The presence of estrogenic ligands, known and unknown, in these mixtures can be detected by activation of an ER-driven luciferase reporter gene. We also examined the effect of hydroxylation on the estrogenic activities of four common flavonoids-apigenin, kaempferol, luteolin, and quercetin, alone and in combination. An inverse relationship was observed between the number of hydroxyl groups in flavonoids and ERalpha bioactivity. When submaximal doses of apigenin, luteolin, kaempferol, genistein, and estradiol were combined in binary and higher order mixtures, the experimental estrogenic effects matched those obtained by summing effects extrapolated from dose-response curves of individual compounds. The estrogenic activities of mixtures containing quercetin were observed to deviate from additivity, suggesting that it was a partial agonist/antagonist. Our assay reveals superagonistic, additive, and antagonistic ERalpha or ERbeta actions of flavonoids and adds to our understanding of the estrogenic effects of phytoestrogens in complex mixtures.

Standard estrogenic prodrugs such as estradiol valerate (E2V) and increasingly popular phytoestrogen formulations are commonly prescribed to improve menopausal health. These drugs are metabolized to numerous bioactive compounds, known or unknown, which may exert combinatorial estrogenic effects in vivo. The aim of this study is to develop and validate estrogen receptor (ER) α/ERβ reporter gene and MCF-7 breast cancer cell proliferation bioassays to quantify serum estrogenic activities in a clinical trial setting. We measured changes in serum estrogenicity following ingestion of E2V and compared this to mass spectrometric measurements of its bioactive metabolites, estrone and 17β-stradiol. ERα bioactivity of the 192 serum samples correlated well (R = 79%) with 17β-estradiol levels, and adding estrone improved R to 0.83 (likelihood ratio test, P < 0.0001), suggesting that the ERα assay reflects summated activity of compounds in serum. ERβ correlated moderately (R = 0.52) with estrone and 17β-estradiol, with an estrone/17β-estradiol coefficient ratio that was twice that of ERα, indicating estrone was more active on a molar basis in the ERβ assay. Unlike the ERα and ERβ bioassays, MCF-7 cell proliferation was driven by 17β-estradiol, and addition of estrone did not increase the predictive value of the model, suggesting that the driver or drivers for breast cancer cell proliferation were not the same as for ERα and ERβ transactivation. In contrast, a decoction of the traditional Chinese medicinal herb Epimedium pubescens did not induce significant changes in estrogenic bioactivity over baseline. These data indicate that ERα/ERβ reporter gene and MCF-7 breast cancer cell proliferation bioassays reflect different aspects of estrogenic activity and that these assays suggest that the Epimedium formulation tested is unlikely to exert significant estrogenic effects in humans.

A novel hydrogen sulfide-activatable block copolymer prodrug with high tumor specificity was developed for cancer chemotherapy.

Abstract The RE‐1 silencing transcription factor (REST) is a master transcription factor that plays a critical role in embryo development, especially during the process of neurogenesis and neural plasticity. However, the mechanism of REST gene transcription regulation is still an open question. Here, by combining bioinformatics analysis and experimental studies, we report that the transcription factor Yin Yang 1 (YY1) bound to a conserved YY1 binding site in the promoter of the mouse REST gene and positively regulated activity of this promoter in SH‐SY5Y cells. Furthermore, analysis of microarray data revealed a significant correlation between the expression of YY1 and REST genes. Overall, this study suggests that YY1 directly regulates expression of the REST gene. © 2007 Wiley‐Liss, Inc.

Phase 2 detoxification enzymes protect against carcinogenesis and oxidative stress. Ginseng ( PANAX spp.) extracts and components were assayed for inducer activity of NQO1 (quinone reductase), a phase 2 enzyme, in Hepa1c1c7 cells. Ginseng extracts were analyzed for ginsenosides and panaxytriol. Korean red PANAX GINSENG extracts demonstrated the most potent phase 2 enzyme induction activity (76,900 U/g dried rhizome powder and 27,800 U/g for two similar preparations). The ginsenoside-enriched HT-1001 American ginseng ( PANAX QUINQUEFOLIUS) extract was the next most potent inducer, with activity of 15,900 U/g, followed by raw American ginseng root with activity of 8700 U/g. Neither a polysaccharide-enriched extract of American ginseng nor a commercial white PANAX GINSENG preparation showed any inducer activity. Pure ginsenosides showed no inducer activity. Protopanaxadiol and protopanaxatriol, deglycosylated ginsenoside metabolic derivatives, showed potent induction activity (approximately 500,000 U/g each). Synthetic panaxytriol was over 10-fold more potent (induction potency 5,760,000 U/g). There was no correlation between ginsenoside content and phase 2 enzyme induction. The most potent inducing red ginseng extract also had the highest panaxytriol content, 120.8 microg/g. We found that ginseng induced NQO1 and that polyacetylenes are the most active components.

Rheumatoid arthritis (RA) is associated with abnormal B cell-functions implicating antibody-dependent and -independent mechanisms. B cells have emerged as important cytokine-producing cells, and cytokines are well-known drivers of RA pathogenesis. To identify novel cytokine-mediated B-cell functions in RA, we comprehensively analysed the capacity of B cells from RA patients with an inadequate response to disease modifying anti-rheumatic drugs to produce cytokines in comparison with healthy donors (HD). RA B cells displayed a constitutively higher production of the pathogenic factors interleukin (IL)-8 and Gro-α, while their production of several cytokines upon activation via the B cell receptor for antigen (BCR) was broadly suppressed, including a loss of the expression of the protective factor TRAIL, compared to HD B cells. These defects were partly erased after treatment with the IL-6-signalling inhibitor tocilizumab, indicating that abnormal IL-6 signalling contributed to these abnormalities. Noteworthy, the clinical response of individual patients to tocilizumab therapy could be predicted using the amounts of MIP-1β and β-NGF produced by these patients' B cells before treatment. Taken together, our study highlights hitherto unknown abnormal B-cell functions in RA patients, which are related to the unbalanced cytokine network, and are potentially relevant for RA pathogenesis and treatment.

We have examined the effects of heat shock on glucocorticoid receptor (GR)-mediated gene transcription in an L929 cell line derivative (LMCAT2) stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid. Exposure of the LMCAT2 cells to heat or chemical shock resulted in a large increase in dexamethasone (Dex)-induced expression of CAT enzyme activity. This potentiation of hormone-induced MMTV-CAT expression was dependent on the magnitude of the stress event and on the Dex concentration, with maximal increases observed for 1 microM Dex after 2 h at 43 C or 2 h at 200 microM sodium arsenite. Heat shock potentiation of MMTV-CAT expression was not seen in an L929 cell derivative devoid of GR or in LMCAT2 cells treated with RU486 antagonist, suggesting that this effect of stress on CAT gene expression was mediated by the GR. Using a quantitative Western blot procedure, the amount of GR protein in the nucleus of cells subjected to combined heat shock and Dex treatment was no greater than the amount of nuclear GR in cells treated with hormone alone, indicating that the stress potentiation effect was not the result of increased nuclear translocation or retention by the GR. In addition, equally strong potentiations of MMTV-CAT expression were observed for cells subjected to heat shock either before or after Dex-mediated translocation of the GR to the nucleus. Thus, the major effect of stress on GR transcription enhancement activity appears to occur after the GR is bound to its high affinity nuclear acceptor sites. We have used a series of MMTV-CAT reporter constructs containing varying portions of the long terminal repeat regulatory region to show that a putative heat shock transcription factor-binding sequence at position -437 of the long terminal repeat is not required for this effect of heat shock on MMTV-CAT expression. A stress-induced increase in hormone-mediated CAT gene expression was observed for a minimal CAT reporter controlled by two synthetic glucocorticoid response elements and a TATA box sequence. Thus, it is unlikely that any DNA-binding transcription factor, other than GR, is required for this effect of stress on transcription by the hormone-bound GR. Based on these results, a model of heat shock enhancement of GR-mediated gene expression is developed in which stress acts on the DNA-bound GR, on a putative heat shock-activated adaptor, or on components of the RNA-polymerase-II complex.

B cells can contribute to immunity through production of antibodies, presentation of antigen to T cells, and secretion of cytokines. B cell activation can result in various outcomes for the host. In general B cell responses are beneficial during infections, and deleterious during autoimmune diseases. However, B cells can also limit host defence against pathogens, and protect from autoimmune pathologies. B cells can therefore act both as drivers and as regulators of immunity. Understanding how these opposite functions are mediated shall stimulate the elaboration of novel approaches for manipulating the immune system. B cells might acquire distinct functional properties depending on their mode of activation. Antigen-specific B cell responses require triggering of B cell receptor (BCR) by antigen, and provision of helper signals by T cells. B cells also express various innate immune receptors, and can directly respond to microbial products. Here, we discuss how intrinsic signalling via Toll-like receptors contributes to the suppressive functions of B cells during autoimmune and infectious diseases.

To determine whether the RNA of bacterial viruses is polyadenylated like bacterial mRNAs, pulse‐labelled as well as the steady‐state population of bacteriophage T7‐specific transcripts were examined for the presence of poly(A) tracts by binding to oligo(dT) cellulose followed by hybridization with specific gene probes. Representatives of all classes of bacteriophage‐specific mRNA — early, middle and late — were found to be polyadenylated. This conclusion was confirmed by screening the products of oligo(dT)‐dependent cDNA synthesis. A cDNA library was prepared from RNA synthesized after bacteriophage T7 infection and the sequence of bacteriophage‐specific clones was determined to define the sites of polyadenylation. About half of the clones were polyadenylated near the end of a protein‐coding region, one of them at the site of post‐transcriptional processing by RNase III. Other clones were polyadenylated within protein‐coding regions. These observations suggest that polyadenylation occurs after the nucleolytic processing of primary transcripts and in some cases also after mRNA degradation has already begun.

Many women are using botanical alternatives for menopausal hormone replacement therapy (HRT) because current progestins, compounds with progesterone activity, have adverse risk profiles. However the development of phyto-progestins for HRT is hampered by the absence of basic pharmacokinetic/pharmacodynamic (PK/PD) data due to the lack of methods to capture summated effects of the numerous compounds that contribute to bioactivity in vivo. In this study, we explored the utility of progesterone receptor (PR)-driven bioassays to track changes in serum progestogenic activity following administration of traditional Chinese medicinal herb, Ligusticum chuanxiong, with potent progestogenic activity. Sensitive and specific (> 300-fold) increases in progestogenic activity were observed when HeLa cells transfected with PR and a PR-driven promoter were exposed to the progestogenic drug, medroxy-progesterone acetate (MPA), suggesting the utility of the bioassay to measure progestogenic effects for PK/PD studies. Progestogens were administered to male Sprague–Dawley rats and serum extracted for measurement of progestogenic activity. Effect–time studies indicate that injection of MPA and L. chuanxiong extract raised area-under-curve of progestogenic activity in sera by 8.2-fold (p < 0.001) and 4.5-fold (p < 0.01) respectively, compared to sera from rats administered vehicle only. Administration of MPA and L. chuanxiong extract by the oral route resulted in a 5.4 (p < 0.001) and 2.3-fold (p = 0.07) increase respectively. Our data suggest that PR-responsive reporter gene bioassays can measure bioavailability of compounds, known and unknown, of complex botanicals for hormone replacement therapy. L. chuanxiong extracts exert progestogenic activity in vivo, and may have utility for progesterone-replacement therapy.

Botanical extracts differ from conventional supplements in that they are complicated mixtures of many bioactive compounds. Here we describe our experience with a traditional Chinese medicinal plant Epimedium sp. to illustrate the scientific challenges of firstly, obtaining a standardized product from a complex mixture and secondly, evaluating that product for preclinical and clinical efficacy. In contrast, to its colloquial name 'Horny goat weed' and Internet advertisements as a herbal 'Viagra' for men, extracts of Epimedium are strongly oestrogenic due to the presence of novel potent phytoestrogens of the prenyl-flavone family. Since Epimedium is not cultivated, it was necessary to source for taxonomically identified samples and to authenticate their species by phylogenetic, chemical and bioresponse profiling. The feasibility of using a panel of oestrogen-responsive cell-based bioassays to measure summated oestrogenic effects at close time points for pharmacokinetic/pharmacodynamic (PK/PD) modelling was evaluated. We document proportionate oestrogenic responses in sera of animals fed oestrogenic drugs and botanical extracts, indicating that these target molecule responsive cell-based bioassays may have utility to capture the global effects of the myriad bioactive compounds in botanical extracts, informing the design of rigorous clinical trials for safety and efficacy.

The efflux transporter MRP1 actively transports antiretrovirals and reduces intracellular accumulation in peripheral blood mononuclear cells (PBMCs). We studied MRP1 expression and function in healthy volunteers treated with darunavir/ritonavir and efavirenz.Seven healthy HIV-negative volunteers were recruited. PBMCs were collected at baseline, 9 days after administration of darunavir (900 mg) and ritonavir (100 mg) once daily, 9 days after coadministration of darunavir/ritonavir and efavirenz (600 mg) once daily and 13 days after administration of efavirenz alone. MRP1 expression was measured in PBMCs using flow cytometry with fluorescein isothiocyanate-conjugated antibody against MRP1m6. MRP1 expression was also measured in CD4(+) T-cells with a phycoerythrin-conjugated antibody against CD4. MRP1 efflux function was assessed by incubating PBMCs with carboxyfluorescein diacetate (CFDA) and comparing CFDA fluorescence with and without the modulators MK571 and probenecid.MRP1 expression was reduced after darunavir/ritonavir administration (geometric mean ratio [GMR] 0.58, 95% confidence interval [95% CI] 0.51-0.65; P<0.001) and darunavir/ritonavir plus efavirenz coadministration (GMR 0.74, 95% CI 0.64-0.84; P=0.001), but not after efavirenz administration alone (GMR 0.82, 95% CI 0.64-1.06; P=0.10). MRP1 protein expression was 41% higher in CD4(+) T-cells. MRP1 efflux function was increased after efavirenz administration (GMR 3.13, 95% CI 2.73-3.59; P<0.001) and darunavir/ritonavir plus efavirenz coadministration (GMR 4.35, 95% CI 3.35-5.68; P<0.001), but not after darunavir/ritonavir administration (GMR 1.06, 95% CI 0.80-1.42; P=0.42).Darunavir/ritonavir and efavirenz treatment exerted differential effects on MRP1 expression and function. These effects could potentially alter antiviral activity, especially in CD4(+) T-cells.

Ritonavir-boosted darunavir with efavirenz may be considered a nucleoside-sparing regimen for treatment-naïve HIV-infected patients. However, the pharmacokinetics of this combination administered once daily have not been studied. We conducted a three-period interaction study with healthy volunteers. The subjects were given darunavir at 900 mg with ritonavir at 100 mg once daily for 10 days. Efavirenz at 600 mg once daily was added for 14 days. Darunavir-ritonavir was then stopped and efavirenz alone was given for 14 days. At the end of each period, blood was taken predosing and for up to 24 h postdosing to measure the drug concentrations. We recruited seven males and five females ages 24 to 49 years and weighing 50 to 83 kg. The darunavir trough concentrations were reduced after efavirenz administration (geometric mean ratio [GMR], 0.43; 90% confidence interval [CI], 0.32 to 0.57]; P < 0.001). The mean darunavir trough concentrations were 1,180 ng/ml (standard deviation, 1,138 ng/ml) after efavirenz administration, but all darunavir trough concentrations were above the 50% effective concentration (EC(50)) of 55 ng/ml for the wild-type virus. For darunavir, the area under the concentration-time curve from 0 to 24 h (AUC(0-24)) (GMR, 0.86; 90% CI, 0.75 to 0.97; P = 0.05) and the half-life (GMR, 0.56; 90% CI, 0.49 to 0.65; P < 0.001) were also significantly reduced. The darunavir peak concentrations were not significantly changed (GMR, 0.92; 90% CI, 0.82 to 1.03; P = 0.23). The ritonavir trough concentrations (GMR, 0.46; 90% CI, 0.33 to 0.63; P = 0.001), AUC(0-24) (GMR, 0.74; 90% CI, 0.64 to 0.86; P = 0.004), and half-life (GMR, 0.80; 90% CI, 0.75 to 0.86; P < 0.001) were also significantly reduced. The efavirenz half-life was significantly longer when it was coadministered with darunavir-ritonavir than when it was given alone (GMR, 1.66; 90% CI, 1.24 to 2.23; P = 0.01), but there were no differences in the efavirenz trough or peak concentration or AUC(0-24) when it was coadministered with darunavir-ritonavir. Efavirenz reduced the trough concentrations of darunavir significantly, but the concentrations remained above the EC(50) for the wild-type virus. This regimen should be evaluated with treatment-naïve patients with no preexisting resistance.

From the fresh rhizomes of Dioscorea deltoidea Wall var. orbiculata, a novel ergostanol saponin, orbiculatoside A (1), was isolated and identified as 3-O-beta-D-glucopyranosyl-ergost-5-ene-3beta, 26-diol-26-O-beta-D-glucopyranosyl(1-->3)-[beta-D-glucopyranosyl(1-->2)-beta-D-glucupyranosyl(1-->6)]-beta-D-glucopyranoside by various NMR techniques in combination with chemical methods. The new saponin showed strong activity against Pyricularia oryzae, with a MMDC (minimum morphological deformation concentration) value of 28.04 micromol/l and was cytotoxic to cancer cell line K562, HCT-15, A549, HT1080, and A2780a in vitro.

Breast cancer is one of the most serious diseases, posing threats to women's physical and mental health. Gene therapy has been gradually regarded as an important part of tumor therapeutics. In the present study, the breast cancer‑specific gene 1‑small interference RNA (BCSG1‑siRNA) plasmid was designed, then encapsulated by chitosan‑silicon dioxide nanometer carriers. The results demonstrated a successful encapsulation of BCSG1‑siRNA in chitosan‑silicon dioxide nanoparticles (encapsulation efficiency exceeded 90%). BCSG1‑siRNA was released slowly (the release rate was almost 30% after 24 h). The cytotoxic effect on MCF‑7 cells was enhanced by increasing the concentration of nanoparticle (the proliferation rate was reduced to 13.4±5.3% and apoptosis rate was increased to 71.5±6.8%). Therefore, the materials presented in the current study acted as successful gene carriers and exhibited significant antitumor effects in breast cancer cells.

Gene therapy for cancer treatment has been an attractive option in the last 2-3 decades; however, the development of efficient gene delivery systems is still underway. Low molecular weight of polyethyleneimines (LMW PEIs), especially 1.8K PEI, have received much attention due to their low toxicity. Hydrophobic modification is a versatile method to address the poor transfection efficiency of LMW PEIs. In this study, a series of mole ratios of folic acid were grafted to 1.8K PEI for hydrophobic modification and targeting purposes. All PEI-FAX conjugates were characterized by UV absorption, 1H nuclear magnetic resonance spectroscopy (1HNMR), gel electrophoresis, dynamic light scattering (DLS), zeta potential, luminometry, flow cytometry and confocal microscopy. As expected, all PEI-FAX conjugates maintained the low toxicity of 1.8K PEI. With the optimal FA substitution, PEI-FA0.65 conjugate achieved the highest transfection efficiency on several cell lines, nearly 100-fold higher than that of the 1.8K PEI, which could be attributed to the better pDNA binding ability and favourable pH buffering capacity. Furthermore, folate receptor-mediated specific cell uptake of the PEI-FA0.65/pDNA polyplex also played an important role in its high transfection efficiency. With low cytotoxicity and high transfection efficiency, PEI-FA0.65 holds potential for in vivo study.

BACKGROUND Overexpression of breast cancer-specific gene 1 (SNCG) is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer cells by using small hairpin RNA (shRNA). METHODS Four different SNCG shRNA oligonucleotides were designed and chemically synthesized to construct mammalian expression vectors. These vectors were then stably transfected into a breast cancer MCF-7 cell line to knockdown SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively in both the stable cell lines and xenografts. RESULTS All four SNCG shRNA constructs significantly reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the unrelated sequence control shRNA and the liposome control mice (P < 0.05). SNCG-knockdown MCF-7 cells formed significantly smaller tumor masses than cells expressing the unrelated sequence control or the liposome control mice (P < 0.05). CONCLUSION SNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer.

To study the prevalence, treatment policy and control of hypertension in patients with maintenance hemodialysis, and to analyze the influencing factors of hypertension control.We studied the current status of 1382 patients with maintenance hemodialysis in 11 dialysis centers in Shanghai, among them 809 were male, and 573 were female. Hypertension was defined as systolic blood pressure (SBP)≥140 and/or diastolic blood pressure (DBP)≥90 mm Hg (1 mm Hg=0.133 kPa). Those who had a history of hypertension and requiring antihypertensive therapy were also diagnosed as hypertension though their blood pressure was within normal range during the survey. Hypertension control was defined as blood pressure<140/90 mm Hg before each dialysis session.The prevalence of hypertension in the hemodialysis patients was 86.3%. The treatment rate and control rate in those patients were 96.8% and 25.5% respectively. More than half (50.4%) of patients were treated with only one kind of anti-hypertensive drug, and 34.4% with 2 kinds, 14.2% with 3 kinds, 1.0% with 4 kinds or more. Calcium channel blocker (CCB) was the most frequently prescribed drug (61.0%), followed by angiotensin II receptor blockers (56.4%), centrally acting anti-hypertensive agent (26.4%), beta blockers and alpha, beta-blockers (14.0%). The control rate of hypertension in those hemodialysis people was aggravated by the existence of coronary artery disease. The patients who need more kinds of antihypertensive agents have a poorer control rate of hypertension. The hypertension control rate elevated significantly with the adequate hemodialysis.There is a very high prevalence of hypertension in maintenance hemodialysis patients. Although the treatment rate is high, the control rate is unsatisfactory. So the control of hypertension in hemodialysis patient is still a clinical challenge. Appropriate dialysis adequacy, reasonable use of erythropoietin, treatment of heart disease and judicious use of antihypertensive drugs may be helpful to improve the clinical outcome.

The immune system is composed of multiple cell types, which together improve the resistance of the organism against infections. The unfolding of a successful host response ensuring effective protection against pathogens requires an appropriate coordination of the different players of the immune system. Innate cells and T cells extensively communicate during immune reactions, providing multiple opportunities for the mutual coordination of these two defense pathways. Little is known about the functional interactions between B and innate cells, and it is generally assumed that they influence each other indirectly through effects on T cells. However, recent studies highlighted important roles for innate cells in initial presentation of antigen to B cells after immunization, and in long-term maintenance of antibody-producing cells in bone marrow after resolution of immune responses. Furthermore, it was found that activated B cells could regulate the activity of innate cells through production of cytokines. Here, we review how direct interactions between innate and B cells can contribute to orchestration of humoral and cellular immunity.

The aim of the present study was to investigate the effects of synuclein‑γ (SNCG) downregulation by RNA interference (RNAi) on the clonogenicity and invasiveness of MCF‑7 breast cancer cells. This study used four pairs of SNCG‑specific siRNAs which were designed and cloned into the pGPU6 plasmid for introduction into an MCF‑7 cell line. The SNCG knockdown efficacies of the four siRNAs were compared using the reverse transcription polymerase chain reaction (RT‑PCR) and immunocytochemistry. The cells' clonogenic and invasive phenotypes were examined with clonogenic and Boyden chamber assays. In comparison with the non‑specific siRNA and empty vector controls, all four SNCG siRNAs were observed to significantly inhibit SNCG expression at the mRNA and protein levels (F=481.06, P<0.001; F=147.42, P<0.0001). SNCG suppression mediated by RNAi successfully inhibited the clonogenicity (P=0.002) and invasiveness (P<0.001) of transfected MCF‑7 cells. According to the results of the present study, we concluded that SNCG suppression mediated by RNAi significantly suppressed SNCG expression at the mRNA and protein levels, suggesting that SNCG suppression mediated by an RNAi strategy may become a novel approach for treating advanced breast cancer.

Objective: The aim of this study was to determine whether the methylation patterns of the breast cancer-specific gene 1 (BCSG1) and the breast cancer susceptibility gene 1 (BRCA1) can be used as biomarkers for predicting the occurrence and development of breast cancer. Methods: Methylation-specific polymerase chain reaction (PCR) was used to detect the methylation status of the BCSG1 and BRCA1 genes in ductal infiltrating carcinomas of the breast; carcinoma in situ of the breast; fibroadenoma of the breast and adjacent normal tissues. Quantitative real-time PCR and immunohistochemistry were used to detect the expression levels of BCSG1 and BRCA1. The BCSG1 and BRCA1 genes were knocked down by siRNA to study their effect of BCSG1 and BRCA1 on the behaviour of breast cancer cell lines. Results: The BCSG1 gene was hypomethylated in breast cancer tissues, and its mRNA as well as its protein levels showed elevated expression compared to normal adjacent tissues. In contrast, the BRCA1 gene was hypermethylated in breast cancer tissues and showed correspondingly decreased mRNA and protein expression levels. In vitro experiments demonstrated that BCSG1 could promote the proliferation and migration of breast cancer cells. After inhibiting the methylation, the expression of both the BCSG1 and BRCA1 genes were increased. Conclusion: Abnormal methylation patterns of the BCSG1 and BRCA1 genes are associated with the development of breast cancer. Thus, methylatedion analyses of these genes have biomarker potential for breast cancer prognoses.

To investigate the therapeutic effects of Lycium barbarum polysaccharide (LBP) on mitomycin C (MMC)-induced myelosuppressive mice.Mice were intravenously injected with MMC 150 mg/kg for two consecutive days from day -1 to day 0 to produce severe myelosuppression, and then treated by s.c. injections of LBP (100 or 200 mg/kg/day) from days 0 to 6. Blood samples were collected from the tail veins of mice on days 7, 10, 12, 14, 17, 19, 21, 24 and 27, and peripheral white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB) and platelet counts (PLT) etc. were monitored.LBP at 100 mg/kg (LBP-L) on day 14 and LBP at 200 mg/kg (LBP-H) on days 10, 14, 17, 19 and 21, significantly ameliorated the decrease of peripheral RBC, HGB and hematocrit (HCT) of myelosuppressive mice compared to the control. LBP-L on days 12 and days 14 and LBP-H on days 10, 12, 14, 17, 19 and 21, significantly enhanced peripheral PLT recovery of myelosuppressive mice compared to the control. LBP-H on days 12, 17, 19 and 21, significantly inhibited the increase of mean platelet volume (MPV) of myelosuppressive mice compared to the control. LBP showed no obviously effect on neutropenia induced in mice by MMC.LBP is effective on peripheral RBC and PLT recovery of MMC-induced myelosuppressive mice.

In recent years, the carcinoma of the breast threatens to women's health heavily. Invasion and metastasis is the main reason that results in the patients death.A prospective study is made in the center hospital of zhengzhou, in order to approach the expression of nm23, MMP-2 (Matrix metallo-proteinase-2), TIMP-2 (it's tissue-inhibitor of the metalloproteinase-2) in the breast neoplasm and the relationship with invasion and metastasis.This study applied the immunohistochemistry technique SP methodIn fibroadenoma, ductal carcinoma in situ and invasive ductal carcinoma of the breast 4 groups, the positive immunostaining rate of nm23, MMP-2 and TIMP-2 have significant difference among 4 groups (p < 0.05). In the breast invasive ductal carcinoma, the expression of nm23 and TIMP-2 decreased or the expression of MMP-2 increasedThis suggested that invasion and metastasis is ability of the neoplasm. MMP-2 in the breast ductal carcinoma in situ appears of high expression and this suggested that the positive expression of this onco-proteins was the early incident in the genetic course of the breast cancer The unite detection of nm23, MMP-2 and TIMP-2 expression would contribute to the early diagnosis and prognostic assessment of the carcinoma of the breast.

Abstract Background and Aim The role of STMN1 in the development and progression of esophageal carcinoma is not yet determined. The present study aimed to systematically evaluate the correlation between STMN1 and prognosis of patients with esophageal carcinoma. Methods Electronic databases including PubMed, Embase, the Cochrane library, and Chinese Biomedical Literature Database (CBM) were searched to identify studies evaluating the impact of STMN1 on the survival of esophageal cancer patients, without the language limitation. Two investigators screened the literature according to the inclusion and exclusion criteria and evaluated the quality of the included studies. The combined analysis was performed using RevMan 5.3 software. Results A total of eight studies, involving 1240 esophageal carcinoma patients, were included in this retrospective design. Meta‐analysis showed that esophageal carcinoma patients with low STMN1 had a superior overall survival and disease‐free survival than those with high expression of STMN1. Compared with the high expression of STMN1, the 5‐year survival rate was significantly higher in patients with low level of STMN1. Patients with high STMN1 expression had a higher risk of experiencing clinical grade III–IV disease, lymph node metastasis, and tumor invasion than those with low STMN1. Conclusion STMN1 is an indicator for the prognosis of esophageal carcinoma patients.

To investigate the role of microRNA-337-5p in osteosarcoma (OS) and its underlying mechanism.The microRNA (microRNA-337-5p) that may be related to OS development was screened out by GEO (Gene Expression Omnibus) database. Survival analysis and ROC curve were performed according to microRNA-337-5p expressions in OA patients. Besides, the correlation between microRNA-337-5p expression and clinical parameters was evaluated by Chi-square analysis. Cox regression analysis was performed to detect the relationship between the overall survival and clinical parameters of OA patients. Subsequently, enriched functions and pathways of microRNA-337-5p were predicted by GESA (gene enrichment sets analysis). MicroRNA-337-5p expression was detected in 65 OS tissue samples and 30 normal tissue samples by qRT-PCR (quantitative Real-Time Polymerase Chain Reaction). For in vitro experiments, after microRNA-337-5p mimics or microRNA-337-5p inhibitor was transfected into OS cells, proliferative and invasive abilities were detected by CCK-8 (Cell Counting Kit-8) and transwell assay, respectively. Finally, Western blot was used to explore the underlying mechanism of microRNA-337-5p in regulating OS.MicroRNA-337-5p was overexpressed in serum and tissue samples of OS patients, which was valuable in diagnosing OS. Besides, microRNA-337-5p expression was correlated with the overall survival and necrosis range of OA patients, whereas not correlated with age and sex. GESA indicated that microRNA-337-5p was enriched in ERBB, MAPK, and VEGF pathways. In vitro experiments indicated elevated proliferative and invasive abilities in MG63 and U2OS cells after microRNA-337-5p overexpression. Furthermore, increased expressions of ERBB2, Erk1/2, and VEGF121 were observed in OS cells after microRNA-337-5p overexpression.MicroRNA-337-5p is upregulated in OS tissues, which is an independent prognostic factor in OS. Overexpressed microRNA-337-5p can promote proliferative and invasive abilities of OS cells via activating ERBB, MAPK, and VEGF pathways.

To investigate the effects of antipyretic and purgative herbs on intestinal mucosal barrier and inflammatory response in the treatment of acute cholangitis.Sixty SD rats were randomly divided into group A (untreated group, acute cholangitis was induced, n=20), group B (treatment group, acute cholangitis was induced and treated with antipyretic and purgative herbs, n=20) and group C (sham operation group, n=20). At the third or fifth day after operation, the rats were sacrificed and sampled. The serum endotoxin, cytokines and inflammatory mediators were tested and the numbers of labeled bacteria in the liver, spleen and mesenteric lymph nodes translocated from the gut were assayed.As compared with group A, the serum content of endotoxin, IL-6, IL-8, TNF-alpha, CRP and NO was significantly lower and that of IL-2 was significantly higher, and the translocated numbers of labeled bacteria from gut were reduced in both group B and group C (P<0.01).Antipyretic and purgative herbs can play therapeutic roles in the treatment of acute biliary tract infections, including the protection of intestinal mucosal barrier from bacterial translocation, reduction of serum endotoxin content and regulation of inflammatory response.

Curcumin (Cur), an antioxidant molecule, has multiple medicinal properties and displays therapeutic values. However, in some functional food applications, the incorporation of Cur is limited because of its poor solubility and inherent physicochemical instability. Herein, to address these issues, PEGylated poly (trimethylene carbonate) (MPEG-PTMC) was used as the polymeric carrier for fabricating the Cur loaded nanoparticles (MPEG-PTMC@Cur NPs). The MPEG-PTMC@Cur NPs had an average size of ∼130 nm. Importantly, after being incorporated in MPEG-PTMC@Cur NPs, the photochemical stability of Cur was significantly enhanced. The degradation rate of Cur was only about 15%. Furthermore, the MPEG-PTMC@Cur NPs exhibited improved DPPH scavenging ability (SC25 = 14.40 μg/mL) compared with that of the free Cur (SC25 > 30 μg/mL). Importantly, the pharmacokinetic study demonstrated that the oral relative bioavailability of MPEG-PTMC@Cur NPs was significantly improved. All these results support that MPEG-PTMC@Cur NPs could be an effective delivery system for Cur.

Objective: To evaluate the accommodative response of patients with intermittent exotropia (IXT) objectively, and study the changes of accommodative response of intermittent exotropia patients when maintaining binocular fusion. Methods: The prospective cohort study was used in this study. Twenty-four patients diagnosed with basic intermittent exotropia who visited the eye hospital of Wenzhou Medical University during October 2016 through January 2017 together with 24 normal volunteers were included, the 48 participants aged from 10 to 27 years old. The participants were divided into the case group and the control group. There were 11 males and 13 females in the case group, and 7 males and 17 females in the control group. The Open-filed autorefractor WAM-5500 (Grand Seiko, Japan) was used to measure the accommodative response of each eye under binocular and monocular viewing conditions at 5 m and 40 cm respectively. During the measurement, patients wore full correction spectacles to achieve distant best-corrected visual acuity of both eyes. The accommodative responses of each eye under binocular and monocular viewing conditions at distance or near between fellow eyes and groups were compared. Results: Under near fixation (40cm) binocular viewing conditions, the accommodative response of the fixating eye (-1.915±0.301)D was different from the deviating eye -1.649(-2.020, -0.304)D in the case group (Z=-3.714, P<0.001). Under near fixation monocular viewing conditions, the accommodative response of the fixating eye (-1.653±0.271)D was also different from the deviating eye -1.565 (-2.031, -0.667)D in the case group (Z=-2.971, P=0.003). During binocular viewing, the asymmetric value of the accommodative response between both eyes of the case group was 0.389(0.102, 1.458)D which was more significant than the normal controls' 0.155(0.009, 0.573)D (Z=-3.505, P<0.001), but during monocular viewing, there was no significant difference between the groups (Z=-1.908, P=0.056). Under near viewing conditions, the variation value of the fixating eyes of the case group was -0.228(-0.796, 0.382)D, which was greater than the variation value -0.086(-0.606, 0.628)D of the right eye of the normal controls, such difference is of statistical significance (Z=-2.279, P=0.023). Under distance viewing conditions, there was no significant difference in the accommodative response between fixating eyes and deviating eyes in case group neither during monocular viewing nor binocular viewing (t=-1.525, -1.729, P>0.05). Besides, the asymmetric values of accommodative response between groups were not significantly different (Z=-1.433, P=0.152. Z=-0.938, P=0.348). Under distance viewing conditions, the changes in accommodative response of each eye during both monocular viewing and binocular viewing were not significantly different between case group and normal controls (Z=-0.041, P=0.967. Z=-1.433, P=0.152). Conclusions: The accommodative responses of the fixating eye and deviating eye of patients with intermittent exotropia were asymmetric under near fixation binocular viewing conditions, and the accommodative response of the deviating eye tends to decrease. Besides, the change of accommodative response of the patients with intermittent exotropia when maintaining binocular fusion is more significant than that of the normal controls. (Chin J Ophthalmol, 2018, 54: 55-61).目的： 探讨间歇性外斜视患者注视眼和偏斜眼的调节反应特点及其保持双眼融像时的调节反应变化。 方法： 前瞻性队列研究。纳入2016年10月至2017年1月在温州医科大学附属眼视光医院就诊的基本型间歇性外斜视患者24例以及健康志愿者24名，年龄10~27岁，分为病例组和对照组，其中病例组男性11例，女性13例；对照组男性7名，女性17名。采用开放视野式自动验光仪分别测量受试者在双眼注视和单眼注视条件下视近（40 cm）和远距（5 m）视标时右眼和左眼的调节反应，测量过程中患者配戴视远全矫框架眼镜。采用配对t检验或配对资料的Wilcoxon符号秩和检验比较双眼注视和单眼注视条件下病例组患者注视眼及偏斜眼的调节反应及对照组右眼和左眼的调节反应，并采用独立样本t检验或两样本比较的Wilcoxon秩和检验比较病例组和对照组之间调节反应、调节不对称性和调节负荷量变化值。 结果： 视近（40 cm）时，在双眼注视条件下，病例组注视眼的调节反应为（-1.915±0.301）D，偏斜眼调节反应为-1.649（-2.020，-0.304）D，两者之间差异有统计学意义（Z=-3.714，P<0.001）；单眼注视条件下，病例组注视眼的调节反应为（-1.653±0.271）D，偏斜眼调节反应为-1.565（-2.031，-0.667）D，两者之间差异有统计学意义（Z=-2.971，P=0.003）；双眼注视时病例组患者两眼之间调节反应不对称性值为0.389（0.102，1.458）D与对照组0.155（0.009，0.573）D相比差异有统计学意义（Z=-3.505，P<0.001），而在单眼注视条件下两组患者的两眼调节反应不对称性的差异无统计学意义（Z=-1.908，P=0.056）。病例组患者的注视眼的调节负荷量变化值为-0.228（-0.796，0.382）D大于对照组右眼相应的变化值-0.086（-0.606，0.628）D，差异有统计学意义（Z=-2.279，P=0.023）。视远（5 m）时，病例组患者在双眼注视及单眼注视条件下注视眼与偏斜眼之间的调节反应差异无统计学意义（t=-1.525，-1.729；P>0.05），病例组和对照组的调节反应不对称性在双眼注视和单眼注视条件下差异均无统计学意义（Z=-1.433，-0.938；P>0.05），病例组患者注视眼与对照组右眼以及偏斜眼与左眼的调节负荷量变化值相比差异亦无统计学意义（Z=-0.041，-1.433；P>0.05）。 结论： 间歇性外斜视患者双眼视近目标时注视眼和偏斜眼之间的调节反应不对称，偏斜眼表现出调节反应降低。间歇性外斜视患者保持双眼融像时所增加的调节反应比健康人群明显增多。（中华眼科杂志，2018，54：55-61）.

Breast cancer is the most common cancer diagnosed in women worldwide. The current treatments for breast cancer, including surgery, radiotherapy and chemotherapy aim to destroy cancer cells, whereas they also cause damage to normal tissues and cells. Thus, an effective, safe and specific breast cancer treatment is urgently needed. The breast cancer-specific gene 1 (BCSG1) has been shown to be specific for the development of breast cancer and is a target for breast cancer diagnosis and treatment. It is expected to silence the expression of BCSG1 at the gene level for the purpose of treating breast cancer. The effect of RNAi technology on silencing target genes is comparable to gene knockout and has been widely used in animal experiments and plant genetic research. In the field of cancer therapy, numerous investigators have used siRNAs to specifically inhibit target genes, demonstrating that siRNAs can treat cancers at the molecular level. However, the delivery of siRNAs into humans needs to overcome multiple physiological barriers, limiting the clinical applications of siRNAs. This review focuses on the application of BCSG1 gene, siRNAs in cancer treatments, and the nanocarrier delivery system of siRNAs. The potential application and research value of BCSG1-specific siRNA in the treatment of breast cancer are discussed.

Two novel polyhydroxylated steroidal sapogenins, wattigenin B ((25R)-spirost-1beta, 2beta, 3beta, 4beta, 5beta, 6beta, 7alpha-heptol, 1) and wattigenin C ((25S)-spirost-1beta, 2beta, 3beta, 4beta, 5beta, 7alpha-hexalrydroxyl-6-one, 2), together with two known sapogenins, kitigenin (3) and convallagenin B (4), were isolated froth the fresh rhizomes of Tupistra wattii Hook. f. The structures of the compounds were determined on the basis of spectroscopic analysis. The four sapogenins were evaluated for the cytotoxicities on the cancer cell line K562 and A2780a in vitro. Compounds 1 - 4 were obtained from the plant for the first time.

The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo . Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome. Cartilage tissues were transferred into a hypoxia chamber (4% O 2 ) immediately after being removed from OA patients and minced finely. Collagenase II at concentrations of 0.02%, 0.1%, 0.25%, 0.5%, 1%, and 2% was applied for 0.5, 1, 2, 4, and 18 h to digest the minced tissue. Actinomycin D (ActD) was added to test its capacity in stabilizing the transcriptome. Cell yield, viability, cell size, and transcriptome were determined using counter chamber, flow cytometry, and RNA sequencing (RNA-seq). Collagenase II at 2% concentration released small chondrocytes from cartilage matrix during the first digestion hour and started to release large cells thereafter, reaching a complete release at 4 h. During 4-h digestions, collagenase II at 2% and 1% but not at lower concentrations yielded maximal release also of the large chondrocyte population. RNA-seq analysis revealed that a 4-h digestion period with 1% or 2% collagenase II plus Actinomycin D optimally preserved the transcriptome. Thus, this study provides an isolation protocol for single chondrocytes from human articular cartilage optimized for transcriptome preservation and RNA-seq analysis.

Pu-erh tea, one of the most popular beverages, is reported to have many healthy beneficial effects of lowering the plasma levels of lipids and lipoproteins, which may be relevant with its oxidation activity. However, the possible mechanisms of affecting the cholesterol biosynthesis and metabolism of LDL-C remain unknown. In this study, we examined the effects of different doses of Pu-erh tea on weight gain, plasma levels of lipids and lipoprotein, protein expression and activity of Squalene synthase, and transcription levels of LDLR, in a rat hyperlipidemia model. The inhibition ratio of cholesterol biosynthesis is determined by the method of liquid scintillation counting in HepG2 cell line. We found that the plasma TC and LDL-C levels were significantly reduced compared with the value of hyperlipidemic control group after 4 weeks treatment of different doses of Pu-erh tea. The protein expression of squalene synthase in rat liver was significantly decreased in Pu-erh tea-treated groups while activity of this rate-limiting enzyme was inhibited in the same groups. Pu-erh tea also inhibited the cholesterol biosynthesis in a dose-dependent manner from 3 H-Mevalonate in HepG2 cell line. Our results suggest that Pu-erh tea exerts lipid-lowering effects by inhibiting the expression and activity of squalene synthase, as well as evaluating the transcription level and protein expression of LDLR.

To observe the changes of intracellular free calcium level ([Ca(2+)]i) in gallbladder cells of guinea pigs with gallstones so as to study the mechanisms of gallstone formation and the prevention and treatment function of traditional Chinese herbs for nourishing the liver.Eighty guinea pigs were randomly divided into four groups, which were normal control group, untreated group, nourishing-liver Chinese drug (NLCD) group and ursodeoxycholic acid (UDCA) group, with 20 guinea pigs in each group. Gallstones were induced in the guinea pigs of the latter 3 groups by the feed of diet inducing cholelithiasis with high cholesterol, while the corresponding medicines were used in NLCD group and UDCA group for prevention and treatment for 7 weeks. Then the state of the guinea pigs, the formation of gallstones, and the changes of [Ca(2+)]i in gallbladder cells were observed.The [Ca(2+)]i in gallbladder cells of guinea pigs in the untreated group was decreased significantly. NLCD improved the behavioral signs of the guinea pigs, significantly decreased the formative rate of gallstones and increased the [Ca(2+)]i in gallbladder cells.The [Ca(2+)]i in gallbladder cells is the important factor for contractile function of gallbladder and the information of gallstones. Traditional Chinese herbs for nourishing the liver may significantly increase the [Ca(2+)]i in gallbladder cells to facilitate contraction of the smooth muscle cells of gallbladder and relieve the cholestatis. It may be one of the mechanisms of traditional Chinese herbs for nourishing the liver in preventing and treating cholelithiasis.

Abstract Background The role of STMN1 in the development and progression of esophageal carcinoma is not yet determined. The present study aimed to systematically evaluate the correlation between STMN1 and prognosis of patients with esophageal carcinoma. Methods Electronic databases including PubMed, Embase, the Cochrane library, and Chinese Biomedical Literature Database (CBM) were searched to identify studies evaluating the impact of STMN1 on the survival of esophageal cancer patients, without the language limitation. Two investigators screened the literature according to the inclusion and exclusion criteria and evaluated the quality of the included studies. The combined analysis was performed using RevMan5.3 software. Results A total of 8 studies, involving 1240 esophageal carcinoma patients, were included in this retrospective design. Meta-analysis showed that esophageal carcinoma patients with low STMN1 had a superior overall survival (OS) and disease-free survival (DFS) than those with high expression of STMN1. Compared to the high expression of STMN1, the 5-year survival rate was significantly higher in patients with low level of STMN1. Patients with high STMN1 expression had a higher risk of experiencing clinical grade III-IV disease, lymph node metastasis, and tumor invasion than those with low STMN1. Conclusion STMN1 is an indicator for the prognosis of esophageal carcinoma patients.

To identify the functional contributions of cooperations among transcription factors on regulatory DNA is critical for understanding transcription activation. But so far there is a great lack of effective identifying methods. Here we describe a novel strategy, based on comprehensively perturbed experiments and a computational model, to identify the cooperations among NF‐κB (p65), CREB, and AP‐1 in transcription activation of human cytomegalovirus major IE1 promoter/enhancer (MIEP). In this strategy, functional profiles of protein–MIEP association and RNA synthesis are achieved through comprehensively perturbing the association of p65, CREB or AP‐1 with MIEP and then subjected to the computational model. Consequently, the ‘real’ cooperations contributing to MIEP activation are found to comprise five but not seven types of potential cooperations. Thus, our research provides a facile systematic approach to identifying the DNA‐organized cooperations among transcription factors and understanding transcription activation.

Magnetic resonance cholangiopancreatography (MRCP) is an emerging imaging technique for direct visualization of biliary and pancreatic ducts without the need for an invasive procedure, ionizing radiation, or iodine contrast media administration. The purpose of this study was to evaluate the efficacy of non-breath-hold MRCP in depicting normal and diseased biliary and pancreatic ducts. A retrospective analysis of 162 patients who underwent MRCP was performed, and a comparison between MRCP and direct cholangiography was made. The overall accuracy of MRCP in diagnosing malignant and benign pancreaticobiliary diseases was also calculated. MRCP depicted more than three hepatic segments in 99% of patients with dilated intrahepatic ducts and in 63% of patients with nondilated intrahepatic ducts. MRCP demonstrated the main hepatic duct, gallbladder, and cystic duct in 100%, 89%, and 72% of patients, respectively. The dilated extrahepatic duct, nondilated extrahepatic duct, dilated pancreatic duct, and nondilated pancreatic duct were visualized in 100%, 98%, 95%, and 77% of patients, respectively. The accuracy of MRCP in diagnosing hepatolithiasis, cholecystolithiasis, and choledocholithiasis was 96%, 97%, and 96%, respectively. The obstruction levels and characteristics determined by MRCP were in agreement with those determined from direct cholangiography in 98% of malignant obstructions and 89% of benign obstructions. The overall accuracy of a combination of MRCP and conventional magnetic resonance imaging in diagnosing pancreaticobiliary diseases was 81% for malignant diseases, 86% for benign diseases, and 82% for stone diseases. We conclude that non-breath-hold MRCP can reliably depict normal and diseased pancreaticobiliary ducts except for cystic ducts and nondilated pancreatic ducts.

The exact mechanisms of defect closure in patients with perimembranous ventricular septal defect (PMVSD) remain unknown. We hypothesized that the expression of urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) may mediate extracellular matrix (ECM) remodeling in aneurysms.Seven normal heart tricuspid septal leaflet and 33 aneurysms were collected in Shanghai Renji Hospital and Shanghai Children's Medical Center from January 2008 to June 2010. Immunohistochemical expression of uPA and PAI-1 in 4 normal heart valvular tissues and 15 aneurysms was detected with immunohistochemical methods. The expression of uPA and PAI-1 mRNA in 3 normal heart valvular tissues and 7 aneurysms was studied by real time fluorescent PCR; the protein expression of uPA and PAI-1 in 4 normal heart valvular tissues and 11 aneurysms was tested with Western blotting.The surface of the aneurysms were completely covered by endothelial cells. Two types of granulation tissue, myxoid and fibrous, were associated with the aneurismal formation. uPA were recognized predominantly in valvar interstitial cells (VICs) which located mainly in regions adjacent to the endothelium and smooth muscle cells of blood vessels. PAI-1 was found in both VICs which located mainly in granulation tissue and endothelial cells. Nine aneurysms expressed a higher uPA activity than 4 normal valvular tissues ((74.6±11.8)% vs. (49.5±7.4)%; t = 3.87, P = 0.003) and six aneurysms expressed a low uPA activity ((10.3±3.1)% vs. (49.5±7.4)%; t=11.78, P=0.000) and a high PAI-1 activity ((55.2±1.7)% vs. (50.8±3.8)%; t=2.55, P=0.034) using immunohistochemical methods. uPA / PAI-1 ratio of protein expression tested by Western blot was 0.88±0.22 in four normal heart vavular tissues; five aneurysms expressed high uPA activity and low PAI-1 activity and uPA/PAI-1 ratio was 4.26±2.04; while the other 6 cases expressed low uPA activity and high PAI-1 activity and uPA/PAI-1 ratio was 0.30±0.07; the difference among the three groups was statistically significant (P<0.05). The rate of uPA/PAI-1 in relative copy of mRNA expression among normal heart valvular tissue, high uPA expressed aneurysms and low uPA expressed aneurysms are also significantly different (2.14±0.17 vs. 0.45±0.04; 2.14±0.17 vs. 4.38±1.41, P<0.05) respectively.The expression of uPA and PAI-1 in VICs suggests that interactions among these molecules contribute to the aneurysm formation and development. This provides a potential mechanism for defect closure in patients with PMVSD.

B lymphocytes have a unique capacity to produce antibodies and are important antigen-presenting cells. In addition to these primary functions, they can secrete cytokines and subsequently influence immunity in a non-antigen-restricted manner. Through these activities, B cells can act as drivers and regulators of immune responses. B cell-mediated regulatory activities primarily involve their production of antiinflammatory cytokines such as interleukin (IL)-10 and IL-35. Through production of these factors, B cells can protect from autoimmune disease and limit host resistance to pathogens. Here, we review current data on the suppressive activities of B cells, using several examples of autoimmune or inflammatory disorders, and infectious diseases in mice. We also discuss available data on the phenotype and function of human IL-10-producing B cells. Finally, we detail throughout the text the recently identified relationship between IL-10-producing B cells and antibody-secreting cells, which demonstrates that plasmablasts/plasma cells can have antibody-independent functions.

目的 探讨食管良性肿瘤临床病理特征。 方法1 058例食管良性肿瘤均来自1973年1月至2015年1月郑州大学第一附属医院河南省食管癌重点开放实验室50万例食管和贲门癌生物样本和临床信息数据库。采用SPSS 21.0软件进行统计学分析。 结果本组50万例数据库中，临床病理信息完整的食管肿瘤患者共249 246例，食管良性肿瘤占0.42%（1058/249 246），其中男性544例，年龄（50±11）岁；女性514例，年龄（52±11）岁。在组织病理学诊断的10种良性肿瘤中食管平滑肌瘤最常见为84.50%（894/1 058），其次为乳头状瘤6.90%（73/1 058），腺瘤最少为0.38%（4/1 058）。平滑肌瘤、间质瘤、神经纤维瘤以男性为主；脂肪瘤、颗粒细胞瘤、神经鞘瘤和血管瘤以女性为主。此外，本组发现的5例错构瘤全部发生于女性。以发生率≥50%为易发标准，青年男性易发食管良性瘤依次为平滑肌瘤和间质瘤，而青年女性依次为颗粒细胞瘤和脂肪瘤；老年男性依次为乳头状瘤、间质瘤和平滑肌瘤；老年女性依次为神经鞘瘤、乳头状瘤、平滑肌瘤、间质瘤和错构瘤；此外，男性脂肪瘤、血管瘤、神经纤维瘤和腺瘤，女性神经纤维瘤均发生在老年；不同年龄良性肿瘤总体发生率（P=0.034）和平滑肌瘤发生率（P=0.004）差异均具有统计学意义。本组平滑肌瘤、乳头状瘤、间质瘤和神经鞘瘤好发部位以中段为主，脂肪瘤以下段为主。本组良性肿瘤治疗以单纯手术为主为57.54%（492/855），其次为内镜下切除38.01%（325/855）和其他为4.45%（38/855）。 结论食管良性肿瘤发生率低，组织类型较多，且不同性别和年龄患者易发类型明显不同，肿瘤组织类型不同，好发部位不同，手术及内镜治疗为主要治疗方式。

Objective:To establish a method with Gas Chromatography-Mass Spectrometry also detection of various common poisons in food in same conditions,including 18 kinds of organophosphorus,8 kinds of organochlorine pesticides,killing rat poisons fluoroacetamide and tetramine,and optimize the sample pretreatment of extraction and purification conditions.Methords: Extracted samples were classified pesticide category,containing fat fewer extracted using acetonitrile sample after nearly dry,concentrated extract by acetone and methylene chloride mixture dissolve,over Na2SO4 dehydration without water,had Carb/NH2 column and silicon magnesium column purification,concentrated to determine;Containing fat more samples,first with petroleum ether extract the fat content,using mixture of acetonitrile and petroleum ether liquid-liquid allocation methods will adipose the pesticide residue separation,over Na2SO4 dehydration without water,with different volume percentage of methylene chloride,acetonitrile and are hexane as eluent,over florisil column and Carb/NH2 column,samples to determination after purification and concentration.Containing killed of rat poison samples by ethyl acetate extracted,over Na2SO4 dehydration without water,with ethyl acetate as eluent,had silicon magnesium column purification then concentrated determination.Results: The results showed that 18 kinds of organophosphorus detection of limiting was 0.01 mg/L~0.05 mg/L,8 kinds of organochlorines in the 0.001 mg/L~0.01 mg/L,tetramine was 0.03 mg/L,Fluoroacetamide was 0.05 mg/l,the method recoveries were 67.7%~83.2%,68.6%~85.3%,89.6%~92.7%,81.5%~83.6%,and the RSD were 1.3%~7.9%,1.8%~3.7%,4.5%,2.5 %.Conclusion: This method is able to determinate various poisons in food for good separation and determination,accurate qualitative and quantitative,experimental results show that the method is sensitive and reproducibility,and high accuracy and precision,and can be used in the actual testing analysis.

Objective To observe the clinical effect of Dingtong decoction in gouty arthritis. Methods 39 patients with gouty arthritis were investigated and were treated by basic treatment, included psychological nursing and keep on a diet and with Dingtong decoction taken by mouth. After a course of treatment, the biochemical index and symptom index were investigated. Results After treatment, clinical recovery was 15 cases, produce effects was 13 cases, validness was 7 cases, invalidation was 4 cases, the total effective rate was 89.74% after the treatment, the clinical symptom of the patients was to improve,and there was a remarkable difference (P 0.05) in biochemical index before and after treatment. Conclusion The clinical effect of Dingtong decoction in gouty arthritis is significantly and can improve the concomitant symptom and shorten the course, and the safety is higher.

Objective: To study the effect of Zhengqinfengtongning injection iontophoresis on rheumatoid arthritis. Methods: Seventy-eight patients of RA were chosen and devided randomly into two groups for investigating their changes of tenderness joint number and degree, number of swollen joints and severity, duration of morning stiffness, RF,ESR,CRP before and after treatmenting. Results: The treatment group was significantly better than the control group, the difference was statistically significant. Conclusion: Effect of Zhengqinfengtongning injection iontophoresis in the treatment of RA is good.

A perimembranous ventricular septal defect (PMVSD) may be partially or completely occluded by aneurysms that originate from the tricuspid valve leaflets, though the exact mechanisms of closure remain unknown. It is hypothesized that valvar interstitial cells (VICs) mediate extracellular matrix (ECM) remodeling in aneurysms via the secretion of a serine proteinase and its inhibitor.The functional characteristics of VICs in 15 aneurysms and in four normal tricuspid valve leaflets obtained at autopsy were evaluated by detecting the expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and alpha-smooth muscle actin (alpha-SMA) in the specimens, using immunohistochemical methods.uPA and alpha-SMA were recognized predominantly in VICs located mainly in regions adjacent to the endothelium and smooth muscle cells of blood vessels. PAI-1 was identified in VICs found mainly in granulation tissues, and in endothelial cells. Two types of granulation tissue (myxoid and fibrous tissue) were associated with aneurysms. Nine aneurysms expressed a high uPA activity and a low PAI-1 activity (uPA/PAI-1 ratio 1.78), while six aneurysms expressed a low uPA activity and a high PAI-1 activity (uPA/PAI-1 ratio 0.14).The expression of uPA, PAI-1 and alpha-SMA in VICs suggests that interactions among these molecules contribute to ECM remodeling during aneurysm formation and development. This provides a potential mechanism for defect closure in patients with PMVSD.

Bioactivity-guided fractionation led to the isolation of a new steroidal saponin, orbiculatoside B, together with a pair of furostanol saponins, protobioside and methyl protobioside, from the fresh rhizomes of Dioscorea deltoidea Wall var. orbiculata. The new Compound was identified to be isonarthogenin 3 - O - alpha - L - rhamnopyranosyl (1-->2) - [alpha - L - rhamnopyranosyl - (1 --> 4)] - beta - D - glucopyranoside by various NMR techniques in combination with chemical methods. The three saponins showed strong activity against Pyricularia oryzae, and were cytotoxic to cancer cell line K562, HCT-15, A549, and A2780a in vitro.

Objective To investigate the inhibitory effects of siRNA targeting BCSG1 gene expression in tumor transplants of human breast cancer cell line in nude mice. Methods Four-pairs of small interfering RNA sequences of BCSG1 were chemically synthesized and inserted into the plasmid expression vectors, and were then transfected into human breast carcinoma cell line MCF7 by liposome method. Plasmid vector with unrelated sequence was used as the vector control. Cells transfected with 4siRNA sequences, control vector and naive FCF7 cells were transplanted into the nude mice. The tumor inhibition was analysised. Immunohistochemical SP method and semi-quantitative RT-PCR were adopted to detect the BCSG1 mRNA and protein expression, respectively. Breast tissue samples of human infiltrating ductal carcinoma, ductal hyperplasia and fibroadenoma were also used as the controls. Results The inhibition rates of tumor growth in four BCSG1-siRNA transfected groups were remarkably higher than those of the vector control group and naive MCF7 cells (P ＜0. 01 ). Compared with that of the vector control and na(i)ve MCF7 cell group, there was a significant decrease of BCSG-1 protein expression in the four experimental groups by immuno-histochemistry staining (P ＜0. 01 ). In addition, BCSG1 mRNA expression in the four groups transfected with BCSG1-siRNA were significantly less than that of the control vector group,naive MCF7 cell control group and human breast IDC (P ＜ 0. 01 ). Conclusion BCSG1-siRNA downregulates the expression of BCSG1 and inhibits effectively growth of the transplaned human breast cancer cell line in nude mice. Key words: Breast neoplasms; Proto-oncogenes; RNA interference; Tumor cells,cultured