Clinical adjuvant combinations stimulate potent B-cell responses in vitro by activating dermal dendritic cells.

CD14(+) dermal DCs (CD14(+) DDCs) have a natural capacity to activate naïve B-cells. Targeting CD14(+) DDCs is therefore a rational approach for vaccination strategies aimed at improving humoral responses towards poorly immunogenic antigens, for example, HIV-1 envelope glycoproteins (Env). Here, we show that two clinically relevant TLR ligand combinations, Hiltonol plus Resiquimod and Glucopyranosyl lipid A plus Resiquimod, potently activate CD14(+) DDCs, as shown by enhanced expression of multiple cytokines (IL-6, IL-10, IL-12p40 and TNF-α). Furthermore, the responses of CD14(+) DDCs to these TLR ligands were not compromised by the presence of HIV-1 gp120, which can drive immunosuppressive effects in vitro and in vivo. The above TLR ligand pairs were better than the individual agents at boosting the inherent capacity of CD14(+) DDCs to induce naïve B-cells to proliferate and differentiate into CD27(+) CD38(+) B-cells that secrete high levels of immunoglobulins. CD14(+) DDCs stimulated by these TLR ligand combinations also promoted the differentiation of Th1 (IFN-γ-secreting), but not Th17, CD4(+) T-cells. These observations may help to identify adjuvant strategies aimed at inducing better antibody responses to vaccine antigens, including, but not limited to HIV-1 Env.

Characterization of European forage maize lines for stover composition and associations with polymorphisms within O-methyltransferase genes.

Cell wall components, such as lignin, cellulose, and hemicelluloses, play an important role in the conversion efficiency of corn stover into ethanol. Understanding the molecular basis of cell wall formation is fundamental for marker assisted selection to develop lines more suitable for ethanol production. In this study, we evaluated a set of 40 European forage maize lines for cellulose, lignin, total hemicellulose, glucuronoarabinoxylan (GAX), and monosaccharides, such as arabinose (ara), xylose (xyl), and glucuronic acid (GlcA). The most significant correlations were observed between hemicelluloses and GAX (0.9), and hemicelluloses and cellulose (-0.81). Cell wall digestibility (CWD, estimated by digestible neutral detergent fiber, DNDF) was negatively correlated with Xyl (-0.34). The association analysis between the evaluated traits and polymorphisms within ten "lignin" genes revealed significant associations between polymorphisms within CCoAOMT1, CCoAOMT2, 4CL2 and C4H, and cellulose/xyl, cellulose, cellulose, and GclA, respectively. None of the QTPs identified in this study corresponded to previously reported CWD QTPs.

Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay.

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5' noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus.

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

Combined immunomagnetic separation-molecular beacon-reverse transcription-PCR assay for detection of hepatitis A virus from environmental samples.

In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.