Evidence for altered levels of IgD in the nasal airway mucosa of patients with chronic rhinosinusitis.

BACKGROUND: IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD+IgM- B-cell populations have been described in the human upper respiratory mucosa. OBJECTIVE: We assessed whether levels of sIgD and IgD+ B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD+ B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels. METHODS: sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD+ cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records. RESULTS: sIgD levels and numbers of IgD+ cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P < .05). IgD+ cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD+CD19+CD38bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues (P < .05). Among numerous factors tested, IL-2 levels were increased in UT from patients with CRSsNP and were positively correlated with tissue IgD levels. Additionally, supernatants of IL-2-stimulated dissociated tissue from patients with CRSsNP had significantly increased sIgD levels compared with those in IL-2-stimulated dissociated control tissue ex vivo (P < .05). Tissue from patients with CRS with preoperative antibiotic use or those with pathogenic bacteria showed higher IgD levels compared with tissue from patients without these variables (P < .05). CONCLUSION: sIgD levels and IgD+CD19+CD38bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP.

Use of nanobarcodes particles in bioassays.

We have developed striped metal nanoparticles, Nanobarcodes particles, which can act as encoded substrates in multiplexed assays. These particles are metallic, encodeable, machine-readable, durable, submicron-sized tags. The power of this technology is that the particles are intrinsically encoded by virtue of the difference in reflectivity of adjacent metal stripes. This chapter describes protocols for the attachment of biological molecules, and the subsequent use of the Nanobarcodes particles in bioassays.

Development of a cost-effective assay for genotyping of HIV-1 non-B subtype for drug resistance.

Highly Active Antiretroviral Therapy (HAART) is the most effective way to control HIV-1 replication in infected patients. Prior to the start of therapy, genotyping of HIV-1 for mutations that confer resistance to potential drug candidates is crucial for it allows formulating an effective regimen. Ineffective drugs are excluded and potentially effective ones are included. A number of diagnostic kits are commercially available for this purpose but are tailored for HIV-1 subtype-B, a strain chiefly found in AIDS patients of Europe and America. However, AIDS patients of South-East Asia including Thailand are predominant infected with HIV-1 subtype non-B. In this study, an inexpensive assay was developed that genotypes HIV-1 non-B for drug resistance and tested it on 99 Thai AIDS patients. Results showed that 98 were infected with HIV-1 subtype non-B (or CRF01_AE) and one with subtype-B. Within the HIV-1 polymerase (pol), reverse transcriptase (RT) gene, the assay identified 18 codon mutations associated with resistance to Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs) and 17 Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). Employing a commercially available kit, parallel genotyping of patient samples confirmed results providing validation of the assay. This method approximately costs 100 US dollars compared to $300 for a commercially available test. In Thailand, the burden of cost for treating HIV-infections is high not only for the average citizen but the country's health care systems. Therefore the low cost and yet effective genotyping test for HIV-1 subtype non-B is a practical and viable solution to expensive genotyping platforms.

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles.

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16+/-0.27 (mean+/-S.D.), 1.35+/-0.285 and 2.72+/-0.47 per 1000 microm(2) resulted in percent deaths of 3.96+/-5.22, 45.91+/-10.89, 90.52+/-12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death.

Particles for multiplexed analysis in solution: detection and identification of striped metallic particles using optical microscopy.

In this report, we present data demonstrating that cylindrical metallic particles, with various submicrometer striping patterns, may be readily distinguished in an optical microscope. Accurate particle identification is discussed relative to synthesis reproducibility and the limitations of optical microscopes. Results from a library of these particles, of which over 100 different striping patterns have been produced, are presented. For these particles, made with Au and Ag stripes, more than 70 patterns may be identified with greater than 90% accuracy. The ability to chemically modify the surface of these particles, making them useful for bioanalytical measurements, is also demonstrated. Finally, we discuss improvements in our manufacturing and identification processes that will lead to both larger numbers of striping patterns and improved identification accuracy.