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1.  Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay 
Archives of Oral Biology  2011;57(2):197-204.
The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9 mg/ml; SPLUNC1 concentrations ranged from 34.7 ng/ml to 13.8 μg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7 ng/ml to 5.3 μg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies.
PMCID: PMC3260398  PMID: 21925642
SPLUNC; SPLUNC1; Luminex; xMAP; saliva; innate immunity
2.  Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis 
The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects.
Materials and Methods
Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and 2 healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analyzed utilizing a multiplexed immunoassay (Luminex®). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests.
Compared to healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of IL-1α, IL-1β, IL-6, IL-12 (p40) (pro-inflammatory cytokines); IL-8, MCP-1, MIP-1α, RANTES (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 (regulator of T-cells and NK cells). Smokers displayed decreased amounts of pro-inflammatory cytokines (IL-1α, IL-6, IL-12 (p40)), chemokines (IL-8, MCP-1, MIP-1, RANTES) and regulators of T-cells and NK cells (IL-7, IL-15).
Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.
PMCID: PMC3038432  PMID: 21198766
Smoking; periodontal disease; immune response; periodontitis/etiology; chronic periodontitis; gingival crevicular fluid
3.  Targeted antimicrobial activity of a specific IgG–SMAP28 conjugate against Porphyromonas gingivalis in a mixed culture 
Antimicrobial peptides coupled to a ligand, receptor or antibody for a specific pathogenic bacteria could be used to develop narrow-spectrum pharmaceuticals with ‘targeted’ antimicrobial activity void of adverse reactions often associated with the use of broad-spectrum antibiotics. To assess the feasibility of this approach, in this study sheep myeloid antimicrobial peptide (SMAP) 28 was linked to affinity- and protein G-purified rabbit immunoglobulin G (IgG) antibodies specific to the outer surface of Porphyromonas gingivalis strain 381. The selective activity of the P. gingivalis IgG–SMAP28 conjugate was then assessed by adding it to an artificially generated microbial community containing P. gingivalis, Aggregatibacter actinomycetemcomitans and Peptostreptococcus micros. The specificity of the P. gingivalis IgG–SMAP28 conjugate in this mixed culture was concentration-dependent. The conjugate at 50 μg protein/mL lacked specificity and killed P. gingivalis, A. actinomycetemcomitans and P. micros. The conjugate at 20 μg protein/mL was more specific and killed P. gingivalis. This is an initial step to develop a selective antimicrobial agent that can eliminate a specific periodontal pathogen, such as P. gingivalis, from patients with periodontal disease without harming the normal commensal flora.
PMCID: PMC3169388  PMID: 18778918
Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans; Peptostreptococcus micros; Cathelicidins; Targeted antimicrobial activity; SMAP28

Results 1-3 (3)