Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection.

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

Electrochemically generated acid and its containment to 100 micron reaction areas for the production of DNA microarrays.

An addressable electrode array was used for the production of acid at sufficient concentration to allow deprotection of the dimethoxytrityl (DMT) protecting group from an overlaying substrate bound to a porous reaction layer. Containment of the generated acid to an active electrode of 100 micron diameter was achieved by the presence of an organic base. This procedure was then used for the production of a DNA array, in which synthesis was directed by the electrochemical removal of the DMT group during synthesis. The product array was found to have a detection sensitivity to as low as 0.5 pM DNA in a complex background sample.

Use of semiconductor-based oligonucleotide microarrays for influenza a virus subtype identification and sequencing.

In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.

A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease.

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-alpha release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.

Synthetic toll-like receptor 4 agonists stimulate innate resistance to infectious challenge.

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

Structure-activity relationship of synthetic toll-like receptor 4 agonists.

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators.