Effects of Astragalus Polysaccharide on Immune Responses of Porcine PBMC Stimulated with PRRSV or CSFV

BACKGROUND: Astragalus polysaccharide (APS) has been used as an immunomodulator that can enhance immune responses, whereas the immunomodulatory effects of APS on porcine peripheral blood mononuclear cells (PBMCs) exposed to porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) have not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: Porcine PBMCs were cultured in complete RPMI media in the presence of the R98-strain of PRRSV (5×10(4) TCID(50)/ml) or C-strain of CSFV (10(3) TCID(50)/ml) with or without APS. The expression of mRNA for CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), transforming growth factor-β (TGF-β), interleukin 2 (IL-2) and IL-10 was assayed by TaqMan real-time RT-PCR. The expression of mRNA for CD28 and CTLA-4 increased at 24 h after stimulation of PBMCs with CSFV and the increased production of CTLA-4 was confirmed by western blot analysis, whereas the increases were inhibited by the addition of APS. In addition, APS alone upregulated IL-2 and TGF-β mRNA expression in PBMCs and the addition of APS had the capacity to prevent a further increase in IL-2 mRNA expression in PBMCs during CSFV or PRRSV infection, but had no effect on TGF-β mRNA expression. The production of tumor necrosis factor-alpha (TNF-α) increased at 12 h after stimulation with PRRSV or CSFV, but not with PRRSV plus APS or CSFV plus APS, whereas the addition of APS to PBMCs infected with PRRSV or CSFV promoted IL-10 mRNA expression. CONCLUSIONS: We suggested that APS had immunomodulatory effects on cells exposed to PRRSV or CSFV. It might be that APS via different mechanisms affects the activities of immune cells during either PRRSV or CSFV infection. This possibility warrants further studies to evaluate whether APS would be an effective adjuvant in vaccines against PRRSV or CSFV

Regional Variability in Microphysical Characteristics of Precipitation Features with Lightning across China: Observations from GPM

The statistical characteristics of precipitation microphysics in lightning clouds are not yet fully understood, as a result of the limitations of traditional observational methods. Using the latest observations from the dual-frequency radar and microwave imager onboard the Global Precipitation Mission (GPM) and ground-based lightning observations, the precipitation microphysics of precipitation features with and without lightning (LPFs and NLPFs) was investigated across four typical regions of China in summer during the time period of 2014–2021. The statistical results show that the LPFs are characterized by smaller concentration and larger mass-weighted mean diameter (Dm) for rain and ice hydrometeors than those of NLPFs. Below the melting layer, the radar reflectivity (Ze) for both the LPFs and NLPFs generally decreases toward the surface, indicating the evaporation or strong break-up of rain hydrometeors. Above the melting layer, the Ze values mainly increase as the altitudes decrease for both LPFs and NLPFs, indicating the rimming, aggregation, or deposition processes. However, the change in slope is much smaller for the LPFs than for the NLPFs, which suggests a more uniform distribution of large ice hydrometeors at high altitudes, probably as a result of the stronger updrafts within the LPFs. The microphysical structures of the LPFs show great regional differences among the four regions of China, which is characterized by the low concentration of large-sized rain hydrometeors over Northeast China, and a high concentration of small-sized rain hydrometeors near the surface over the Yangtze-Huaihe River basin

Identification of ireA, 0007, 0008, and 2235 as TonB-dependent receptors in the avian pathogenic Escherichia coli strain DE205B

International audienceAbstractAvian pathogenic Escherichia coli (APEC), a pathotype of extraintestinal pathogenic E. coli, causes one of the most serious infectious diseases of poultry and shares some common virulence genes with neonatal meningitis-associated E. coli. TonB-dependent receptors (TBDRs) are ubiquitous outer membrane β-barrel proteins; they play an important role in the recognition of siderophores during iron uptake. Here, in the APEC strain DE205B, we investigated the role of four putative TBDRs—ireA, 0007, 0008, and 2235—in iron uptake. Glutathione-S-transferase pulldown assays indicated that the proteins encoded by these genes directly interact with TonB. Moreover, the expression levels of all four genes were significantly upregulated under iron-depleted conditions compared with iron-rich conditions. The expression levels of several iron uptake-related genes were significantly increased in the ireA, 0007, 0008, and 2235 deletion strains, with the upregulation being the most prominent in the ireA deletion mutant. Furthermore, iron uptake by the ireA deletion strain was significantly increased compared to that by the wild-type strain. Moreover, a tonB mutant strain was constructed to study the effect of tonB deletion on the TBDRs. We found that regardless of the presence of tonB, the expression levels of the genes encoding the four TBDRs were regulated by fur. In conclusion, our findings indicated that ireA, 0007, 0008, and 2235 indeed encode TBDRs, with ireA having the most important role in iron uptake. These results should help future studies explore the mechanisms underlying the TonB-dependent iron uptake pathway

Enzyme-linked immunosorbent assay of TNF-α in cell culture supernatants.

<p>Porcine PBMCs were cultured with medium alone, APS, PRRSV, PRRSV plus APS, CSFV, or CSFV plus APS. Data are presented as means ± SEM of three independent experiments. Within the same time: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

The cell proliferation determined by MTT assay.

<p>Porcine PBMCs were cultured with medium alone, APS, PRRSV, PRRSV plus APS, CSFV, or CSFV plus APS. Cell proliferation was assessed after 24, 48 and 72 h of cultivation. The results are presented as a percentage relative to the OD570 values of nonstimulated PBMC (100%) at the same day. Data are presented as means ± SEM of three independent experiments. Within the same time: *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Relative mRNA expression of CD28 (A), CTLA-4 (B), IL-2 (C), TGF-β (D) and IL-10 (E) in porcine PBMCs.

<p>Relative mRNA expression in porcine PBMCs cultured with medium alone, APS, PRRSV, PRRSV plus APS, CSFV, or CSFV plus APS. Gene expression was analysed by Taqman real-time RT-PCR. Data are presented as means ± SEM of three independent experiments. Within the same time: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Sequences of oligonucleotide primers and probes used for TaqMan real-time RT-PCR, the length of the PCR products and accession numbers.

<p>IL-10, interleukin 10; TGF-β1, transforming growth factor-beta1; HPRT, hypoxanthine phosphoribosyl-transferase; CTLA4, cytotoxic T-lymphocyte antigen 4; F, forward primer; R, reverse primer. 5′and 3′modifications for the probes were as followed: FAM, 6-carboxyfluorescein; TAMRA, tetramethylrhodamine;</p>a<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029320#pone.0029320-Zhu1" target="_blank">[48]</a>.</p>b<p>Taqman Gene Expression Assays (Applied Biosystems, Foster City, CA).</p