Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others^1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition⁴, and it can be difficult to manage clinically⁵. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria^6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota⁸. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal^9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV^11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform¹³. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample¹⁴. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.Download video file.^{(68M, mov)}     

Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway

Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.     

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.     

Comparative sequence and genetic analyses of asparagus BACs reveal no microsynteny with onion or rice

The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e < −20) to expressed sequences on different rice chromosomes, revealing no evidence for microsynteny between asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e < −20) expressed sequence tags (ESTs) of onion. These onion ESTs mapped to different onion chromosomes and no relationship was observed between physical or genetic linkages in asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome model for plants in the Asparagales with enormous nuclear genomes.     

Microglia recognize double-stranded RNA via TLR3

Microglia are CNS resident innate immune cells of myeloid origin that become activated and produce innate proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors for pathogen-associated molecular patterns that drive innate immune responses. We have recently shown that mice deficient in TLR3 (TLR3(-/-) mice) are resistant to lethal encephalitis and have reduced microglial activation after infection with West Nile virus, a retrovirus that produces dsRNA. We wished to determine whether microglia recognize dsRNA through the TLR3 pathway. In vitro, murine wild-type primary cultured microglia responded to synthetic dsRNA polyinosinic-polycytidylic acid (poly(I:C)) by increasing TLR3 and IFN-beta mRNA and by morphologic activation. Furthermore, wild-type microglia dose dependently secreted TNF-alpha and IL-6 after poly(I:C) challenge, whereas TLR3(-/-) microglia produced diminished cytokines. Activation of MAPK occurred in a time-dependent fashion following poly(I:C) treatment of wild-type microglia, but happened with delayed kinetics in TLR3(-/-) microglia. As an in vivo model of encephalitis, wild-type or TLR3(-/-) mice were injected intracerebroventricularly with poly(I:C) or LPS, and microglial activation was assessed by cell surface marker or phospho-MAPK immunofluorescence. After intracerebroventricular injection of poly(I:C), microgliosis was clearly evident in wild-type mice but was nearly absent in TLR3(-/-) animals. When taken together, our results demonstrate that microglia recognize dsRNA through TLR3 and associated signaling molecules and suggest that these cells are key sensors of dsRNA-producing viruses that may invade the CNS.     

Blocking TGF-beta-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology

Alzheimer's disease is the most common dementia and is pathologically characterized by deposition of amyloid-beta peptide (Abeta) into beta-amyloid plaques, neuronal injury and low-level, chronic activation of brain immunity. Transforming growth factor-betas (TGF-betas) are pleiotropic cytokines that have key roles in immune cell activation, inflammation and repair after injury. We genetically interrupted TGF-beta and downstream Smad2/3 signaling (TGF-beta-Smad2/3) in innate immune cells by inducing expression of CD11c promoter-driven dominant-negative TGF-beta receptor type II in C57BL/6 mice (CD11c-DNR), crossed these mice with mice overexpressing mutant human amyloid precursor protein, the Tg2576 Alzheimer's disease mouse model, and evaluated Alzheimer's disease-like pathology. Aged double-transgenic mice showed complete mitigation of Tg2576-associated hyperactivity and partial mitigation of defective spatial working memory. Brain parenchymal and cerebrovascular beta-amyloid deposits and Abeta abundance were markedly (up to 90%) attenuated in Tg2576-CD11c-DNR mice. This was associated with increased infiltration of Abeta-containing peripheral macrophages around cerebral vessels and beta-amyloid plaques. In vitro, cultures of peripheral macrophages, but not microglia, from CD11c-DNR mice showed blockade of classical TGF-beta-activated Smad2/3 but also showed hyperactivation of alternative bone morphogenic protein-activated Smad1/5/8 signaling and increased Abeta phagocytosis. Similar effects were noted after pharmacological inhibition of activin-like kinase-5, a type I TGF-beta receptor. Taken together, our results suggest that blockade of TGF-beta-Smad2/3 signaling in peripheral macrophages represents a new therapeutic target for Alzheimer's disease.     

The implementation of the consensus on the management of Helicobacter pylori and barriers to consensus

Background

A consensus on the management of Helicobacter pylori has been developed. We aimed to assess whether dissemination through continuing medical education (CME) could enhance the adoption of this consensus among clinicians and to explore potential barriers to acceptance.

Materials and methods

Four CME courses were held to disseminate the consensus. Adoption surveys were performed to evaluate participants’ behavior in the past and their commitment to adopt the consensus in future clinical practice after CME. The gaps and barriers to adoption were also surveyed.

Results

A total of 240 physicians had attended the CME courses and received surveys with the 22 statements/substatements of the consensus. Before CME, adoption was good in six, fair in ten, and poor in six. After CME, 21 statements had either an initial >90% adoption or improvement to good or fair (P < 0.001), but one still had poor even though it showed improvement (P = 0.02). Although commitment was good or fair after CME, there was a >20% gap between “commitment” and “no barrier” to adoption for 11 statements, ten of which had a main barrier of financial incentives. Among the statements with fair or poor commitment after CME, less commitment to adoption and more barriers related to financial incentives were pronounced in clinicians serving in regional/district hospitals or clinics compared to those serving in medical centers.

Conclusions

Continuing medical education may improve the adoption of the H. pylori consensus. The financial incentives were shown to be a main barrier to adoption of the consensus and should be improved.     

CD40 signaling and Alzheimer's disease pathogenesis

The interaction between CD40 and its cognate ligand, CD40 ligand, is a primary regulator of the peripheral immune response, including modulation of T lymphocyte activation, B lymphocyte differentiation and antibody secretion, and innate immune cell activation, maturation, and survival. Recently, we and others have identified CD40 expression on a variety of CNS cells, including endothelial cells, smooth muscle cells, astroglia and microglia, and have found that, on many of these cells, CD40 expression is enhanced by pro-inflammatory stimuli. Importantly, the CD40–CD40 ligand interaction on microglia triggers a series of intracellular signaling events that are discussed, beginning with Src-family kinase activation and culminating in microglial activation as evidenced by tumor necrosis factor- secretion. Based on the involvement of microglial activation and brain inflammation in Alzheimer's disease pathogenesis, we have investigated co-stimulation of microglia, smooth muscle, and endothelial cells with CD40 ligand in the presence of low doses of freshly solubilized amyloid-β peptides. Data reviewed herein show that CD40 ligand and amyloid-β act synergistically to promote pro-inflammatory responses by these cells, including secretion of interleukin-1β by endothelial cells and tumor necrosis factor- by microglia. As these cytokines have been implicated in neuronal injury, a comprehensive model of pro-inflammatory CD40 ligand and amyloid-β initiated Alzheimer's disease pathogenesis (mediated by multiple CNS cells) is proposed.     

Doppler optical coherence tomography for measuring flow in engineered tissue

The engineering of human tissue represents a major paradigm shift in clinical medicine. Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures. However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required. This in turn will require the development of reliable on-line monitoring and control systems. This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship. Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo. Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro. As a base line, the current status of DOCT in vivo is therefore reviewed. This paper also reports the first preliminary use of DOCT in tissue engineering. The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ.