Different antigen presentation tendencies of granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived macrophages and peritoneal macrophages

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced bone marrow-derived cells (BMCs) and primary peritoneal exudate cells (PECs) are usually used for antigen presentation in in vitro experiments. In order to expound their tendency for uptake and antigen presentation, we compared differences in the degree of phagocytosis, the expression of co-stimulatory molecules, and the activation of T lymphocytes between these two cell types. These assays used the F4/80 marker expression, as it is the general marker for macrophages. The BMC population was found to contain both F4/80(bright) and F4/80(dim) subtypes, while PECs were mainly composed of the F4/80(bright) subtype. Expression levels of cell surface co-stimulatory molecules, CD80, CD86, CD54, and CD40, were significantly higher for F4/80(+)BMCs than F4/80(+)PECs. Their expressions were further upregulated for F4/80(+)BMCs than for F4/80(+)PECs after stimulation with flagellin. F4/80(+)BMCs had a weaker ability to phagocytize microbeads than F4/80(+)PECs (P?相似文献   

Reciprocal regulatory interaction between human herpesvirus 8 and human immunodeficiency virus type 1

Human herpesvirus 8 (HHV8) is the primary viral etiologic agent in Kaposi's sarcoma (KS). However, individuals dually infected with both HHV8 and human immunodeficiency virus type 1 (HIV-1) show an enhanced prevalence of KS when compared with those singularly infected with HHV8. Host immune suppression conferred by HIV infection cannot wholly explain this increased presentation of KS. To better understand how HHV8 and HIV-1 might interact directly in the pathogenesis of KS, we queried for potential regulatory interactions between the two viruses. Here, we report that HHV8 and HIV-1 reciprocally up-regulate the gene expression of each other. We found that the KIE2 immediate-early gene product of HHV8 interacted synergistically with Tat in activating expression from the HIV-1 long terminal repeat. On the other hand, HIV-1 encoded Tat and Vpr proteins increased intracellular HHV8-specific expression. These results provide molecular insights correlating coinfection with HHV8 and HIV-1 with an unusually high incidence of KS.     

Rhodococcus kronopolitis sp. nov., a novel actinobacterium isolated from a millipede (Kronopolites svenhedind Verhoeff)

A novel actinobacterium, designated strain NEAU-ML12^T, was isolated from a millipede (Kronopolites svenhedind Verhoeff), which was collected from Fenghuang Mountain in Wuchang, Heilongjiang Province, north China. The strain was characterized using a polyphasic approach. Strain NEAU-ML12^T was found to have morphological and chemotaxonomic characteristics typical of the members of the genus Rhodococcus. 16S rRNA gene sequence similarity analysis showed that the strain NEAU-ML12^T belongs to the genus Rhodococcus, and was most closely related to Rhodococcus tukisamuensis Mb8^T (98.9 %) and Rhodococcus koreensis DNP505^T (97.7 %). Phylogenetic analysis based on 16S rRNA gene sequences also demonstrated that strain NEAU-ML12^T should be classified in the genus Rhodococcus, forming a distinct clade with R. tukisamuensis Mb8^T supported by a 99 % bootstrap value. However, the DNA–DNA relatedness between strain NEAU-ML12^T and R. tukisamuensis Mb8^T was found to be 41.9 ± 0.7 %. Furthermore, strain NEAU-ML12^T could also be differentiated from R. tukisamuensis Mb8^T and other closely related strains (R. koreensis DNP505^T and Rhodococcus maanshanensis M712^T) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-ML12^T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kronopolitis sp. nov. is proposed. The type strain is NEAU-ML12^T (=CGMCC 4.7145^T = DSM 46702^T).     

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.     

Molecular cloning, expression profile, polymorphism and the genetic effects of the dopamine D1 receptor gene on duck reproductive traits

The dopamine D1 receptor (DRD1), a member of the dopamine receptor (DR) gene family, participates in the regulation of reproductive behaviors in birds. In this study, a 1,390 bp fragment covering the complete coding region (CDS) of duck DRD1 gene was obtained. The cDNA (GenBank: JQ346726) contains a 1,353 bp CDS and a 37 bp 3'- UTR including a TGA termination codon (nucleotides 1,354-1,356 bp). The duck DRD1 shares about 76-96 % nucleic acid identity and 82-98 % amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences displays that duck DRD1 protein is closely related with those of chicken and zebra finch. The quantitative real-time PCR analysis indicates that the DRD1 mRNA is widely expressed in all examined tissues. Five single nucleotide polymorphisms (SNPs) (c.189A > T, c.507C > T, c.681C > T, c.765A > T, c.1044A > G) in the CDS of duck DRD1 gene were indentified, c.681C > T and c.765A > T were genotyped and analyzed in a two generations duck population by using of PCR-RFLP. Association analysis demonstrated that the c.681C > T genotypes were significantly associated with body weight at sexual maturity (when laying their first egg) (P < 0.01), egg production within 360 days (P < 0.05) and 420 days (P < 0.01); the c.765A > T genotypes were significantly associated with egg shape index and egg shell strength (P < 0.05). Those results suggest that the DRD1 gene may be a potential genetic marker to improve some reproductive traits in ducks.     

Quercetin supplementation suppresses the secretion of pro-inflammatory cytokines in the lungs of Mongolian gerbils and in A549 cells exposed to benzo[a]pyrene alone or in combination with β-carotene: in vivo and ex vivo studies

In vitro studies have shown that quercetin modulates the effects of β-carotene induced by stimulants. Whether these reactions happen in vivo, however, is unclear. Thus, we investigated whether quercetin supplementation suppresses the harmful effects of benzo[a]pyrene (BaP) alone or combined with β-carotene in the lungs of Mongolian gerbils. The gerbils were given quercetin (100 mg/kg body wt, 3 times/week), β-carotene (10 mg/kg body wt, 3 times/week), and BaP (8 mmol, 2 times/week) alone or in combination by gavage for 6 months. β-Carotene supplementation enhanced the pro-inflammatory effects of BaP in the lungs of gerbils. In contrast, quercetin supplementation significantly decreased the infiltration of inflammatory cells as well as the levels of TNF-α and IL-1β in the bronchoalveolar lavage fluid and plasma of gerbils exposed to BaP or BaP+β-carotene (P<.05). Such effects of quercetin supplementation were accompanied by a down-regulation of the expression of phospho-c-Jun and phospho-JNK induced by BaP or BaP+β-carotene in the lungs of gerbils. Furthermore, in the ex vivo study, we found that quercetin-metabolite-enriched plasma (QP) obtained from gerbils acted like a JNK inhibitor to significantly suppress the secretion of pro-inflammatory cytokines induced by BaP or BaP+β-carotene in A549 cells (P<.05). QP also suppressed the activation of the JNK pathway in the A549 cells. These results suggest that supplemental quercetin suppress the pro-inflammatory effect of β-carotene induced by BaP in vivo and ex vivo. The regulation of the JNK pathway by the metabolites of quercetin contributes, at least in part, to such effects of quercetin in vivo.     

Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.     

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.     

Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. In this study, four B-class Phalaenopsis DEF-like MADS-box genes were identified and characterized, including PeMADS2, PeMADS3, PeMADS4 and PeMADS5. Differential expression profiles of these genes were detected in the floral organs of P. equestris, suggesting distinctive roles in the floral morphogenesis of orchids. Furthermore, expressions of these genes were varied to different extents in the peloric mutants with lip-like petals. Expression of PeMADS4 was in lips and columns of wild type, and it extended to the lip-like petals in the peloric mutant. Expression of PeMADS5 was mainly in petals and to a lesser extent in columns in the wild type, whereas it was completely eliminated in the peloric mutant. Disruption of the PeMADS5 promoter region of the peloric mutant was detected at nucleotide +312 relative to the upstream of translational start codon, suggesting that a DNA rearrangement has occurred in the peloric mutant. Genomic structure analysis of the PeMADS5 showed that the exon length was conserved in exons 1-6, similar to DEF-like genes of other plants. Collectively, this is the first report that four DEF-like MADS genes were identified in a single monocotyledonous species and that they may play distinctive morphogenetic roles in the floral development of an orchid.