Tetramer-Guided Epitope Mapping: A Rapid Approach to Identify HLA-Restricted T-Cell Epitopes from Composite Allergens.

Tetramer-guided epitope mapping (TGEM) is a technique in immunology that permits the rapid identification of allergenic epitopes through peptide screening procedures utilizing human lymphocyte antigen (HLA) class II tetramers as staining reagents for detection. The identification of allergenic epitopes is a prerequisite for the accurate characterization of allergen-specific CD4+ T cells without in vitro stimulation. Additionally, these MHC-II/peptide complexes that interact with T-cell receptors (TCR) of pathogenic CD4+ T cells are compatible with a different number of assays like Intracelullar Cytokine Staining (ICS), and Carboxyfluorescein succinimidyl ester (CFSE) making it a robust technology to study the functionality of allergen-specific CD4+ T cells.

Chronic cat allergen exposure induces a TH2 cell-dependent IgG4 response related to low sensitization.

BACKGROUND: In human subjects, allergen tolerance has been observed after high-dose allergen exposure or after completed allergen immunotherapy, which is related to the accumulation of anti-inflammatory IgG4. However, the specific T-cell response that leads to IgG4 induction during chronic allergen exposure remains poorly understood. OBJECTIVE: We sought to evaluate the relationship between cat allergen-specific T-cell frequency, cat allergen-specific IgE and IgG4 titers, and clinical status in adults with cat allergy with and without cat ownership and the cellular mechanism by which IgG4 is produced. METHODS: Fel d 1-, Fel d 4-, Fel d 7-, and Fel d 8-specific T-cell responses were characterized by CD154 expression after antigen stimulation. RESULTS: In allergic subjects without cat ownership, the frequency of cat allergen (Fel d 1 and Fel d 4)-specific TH2 (sTH2) cells correlates with higher IgE levels and is linked to asthma. Paradoxically, we observed that subjects with cat allergy and chronic cat exposure maintain a high frequency of sTH2 cells, which correlates with higher IgG4 levels and low sensitization. B cells from allergic, but not nonallergic subjects, are able to produce IgG4 after cognate interactions with sTH2 clones and Fel d 1 peptide or the Fel d 1 recombinant protein. CONCLUSION: These experiments suggest that (1) allergen-experienced B cells with the capacity to produce IgG4 are present in allergic subjects and (2) cat allergen exposure induces an IgG4 response in a TH2 cell-dependent manner. Thus IgG4 accumulation could be mediated by chronic activation of the TH2 response, which in turn drives desensitization.

Stain Removal Assessment of Two Manual Toothbrushes with an Interproximal Tooth Stain Index.

OBJECTIVE: To assess a newly developed index to measure interproximal stain and evaluate the stain removal efficacy of two commercially available manual toothbrushes. METHODS: This was a randomized, examiner-blind, parallel-group, two-treatment clinical trial of two weeks' duration. Subjects qualified for the study if they had an average Modified Lobene Stain Index of ≥ 1.5 from two anterior teeth. At baseline, subjects brushed in front of a mirror for one minute under supervision. All subjects were provided with a standard 0.243% sodium fluoride dentifrice and were randomly assigned either an Oral-B Pulsar manual brush (OBP) or a Colgate Whitening manual brush (CW) to use for two weeks. Stain was reassessed after two weeks of product use. Stain measurements were conducted using the Modified Lobene Stain Index and the new Interproximal Modified Lobene Stain Index, which allows for assessment of stain in hard-to-reach areas using the same area and intensity scales as the Modified Lobene Stain Index. RESULTS: Use of the two manual brushes resulted in statistically significant reductions in surface stain relative to baseline after two weeks of use. Median stain reductions were 78% and 60% for the OBP and CW, respectively, as measured by the Modified Lobene Stain Index. The mean changes in the composite scores from baseline to week two were 1.85 and 1.57 for the two treatment groups, respectively. Statistically significant reductions from baseline were also found for the intensity and extent of stain measures (p < 0.001). Similar trends were found using the new Interproximal Modified Lobene Index. Composite median stain removal percentages versus baseline were 88% and 73% for the OBP and CW groups, respectively (p < 0.001). For the interproximal sites, a median stain removal of 92% was observed with the OBP brush and 83% reduction with the CW brush. For the gingival sites, the median stain removal percentages were 83% and 50%, respectively For the body region, a median stain removal of 100% was found for both treatment groups. No statistically significant differences were found between the two groups for the mean composite scores for either index. CONCLUSION: Both manual brushes showed effective stain removal, including interproximal hard-to-reach sites. The Interproximal Modified Lobene Stain Index gave clinically relevant results consistent with the traditional Modified Lobene Stain Index, while allowing assessment of interproximal regions.

Jug r 2-reactive CD4(+) T cells have a dominant immune role in walnut allergy.

BACKGROUND: Allergic reactions to walnut can be life-threatening. Although IgE epitopes of walnut have been studied, CD4(+) T cell-specific epitopes for walnut remain uncharacterized. In particular, the relationship of both phenotype and frequency of walnut-specific T cells to the disease have not been examined. OBJECTIVES: We sought to provide a thorough phenotypic analysis for walnut-reactive T cells in allergic and nonallergic subjects, particularly the relationship of phenotypes and frequencies of walnut-specific T cells with the disease. METHODS: The CD154 upregulation assay was used to examine CD4(+) T-cell reactivity toward the walnut allergens Jug r 1, Jug r 2, and Jug r 3. A tetramer-guided epitope mapping approach was used to identify HLA-restricted CD4(+) T-cell epitopes in Jug r 2. Direct ex vivo staining with peptide-major histocompatibility complex class II tetramers enabled comparison of the frequency and phenotype of Jug r 2-specific CD4(+) T cells between allergic and nonallergic subjects. Jug r 2-specific T-cell clones were also generated, and mRNA transcription factor levels were assessed by using quantitative RT-PCR. Intracellular cytokine staining assays were performed for further phenotypic analyses. RESULTS: Jug r 2 was identified as the major allergen that elicited CD4(+) T-cell responses. Multiple Jug r 2 T-cell epitopes were identified. The majority of these T cells in allergic subjects have a CCR4(+) phenotype. A subset of these T cells express CCR4(+)CCR6(+) irrespective of the asthmatic status of the allergic subjects. Intracellular cytokine staining confirmed these TH2-, TH2/TH17-, and TH17-like heterogenic profiles. Jug r 2-specific T-cell clones from allergic subjects mainly expressed GATA3, nonetheless, a portion of T-cell clones both GATA3 and RAR-related orphan receptor C (RORC) or RORC alone, confirming the presence of TH2, TH2/TH17, and TH17 cells. CONCLUSIONS: Jug r 2-specific responses dominate walnut T-cell responses in patients with walnut allergy. Jug r 2 central memory CD4(+) cells and terminal effector T cells were detected in peripheral blood, with the central memory phenotype as the most prevalent phenotype. In addition to conventional TH2 cells, TH2/TH17 and TH17 cells were also detected in nonasthmatic and asthmatic patients with walnut allergy. Understanding this T-cell heterogeneity might render better understanding of the disease manifestation.

Antigingivitis efficacy of a stabilized stannous fluoride/sodium hexametaphosphate dentifrice in subjects previously nonresponsive to a triclosan/copolymer dentifrice.

Antimicrobial agents, such as stannous fluoride and triclosan, have been incorporated into dentifrice formulations and shown to be effective in the prevention and reduction of gingivitis. However, not all subjects respond to treatment. The objective of this study was to assess the antigingivitis efficacy of a 0.454% stannous fluoride/sodium hexametaphosphate dentifrice among subjects who were identified as nonresponsive or minimally responsive to a sodium fluoride/triclosan/copolymer dentifrice. This was a 12-week, examiner-blind, single-center study. Forty-one subjects with no or minimal gingivitis response to 6 months of use of a sodium fluoride/triclosan/copolymer dentifrice were enrolled in this study and 38 of them provided complete data. All subjects were provided with a 0.454% stannous fluoride/sodium hexametaphosphate dentifrice to use over the next 3 months (12 weeks). Subjects were instructed to brush twice daily for 60 seconds using their assigned product. Toothbrushing was supervised for 3 days of each week. Clinical examinations using the Löe-Silness Gingival Index were performed at 6 and 12 weeks posttreatment. The 6-month scores after the use of the sodium fluoride/triclosan/copolymer dentifrice served as the baseline. After 12 weeks of product use, participants had statistically significantly less gingivitis by 54% and statistically significantly less bleeding by 55% on average compared to baseline. No adverse oral soft tissue effects were reported in the study. This study demonstrated that the use of an experimental 0.454% stannous fluoride/sodium hexametaphosphate dentifrice over a 12-week period provides a statistically significant and clinically relevant effect in the control of gingivitis among subjects who previously did not respond to 6 months of use of a sodium fluoride/triclosan/copolymer dentifrice.

The comparative efficacy of stabilized stannous fluoride/sodium hexametaphosphate dentifrice and sodium fluoride/triclosan/copolymer dentifrice for the control of gingivitis: a 6-month randomized clinical study.

BACKGROUND: Antimicrobial agents such as stannous fluoride and triclosan have been incorporated into dentifrice formulations and have been shown to be effective in reducing gingivitis. The objective of this study was to compare the anti-gingivitis efficacy of a 0.454% stannous fluoride/sodium hexametaphosphate dentifrice to a positive control triclosan/copolymer dentifrice. METHODS: This was a 6-month, randomized, double-masked, parallel-group study conducted according to the American Dental Association guidelines for evaluating chemotherapeutic products for the control of gingivitis. A 0.454% stannous fluoride/sodium hexametaphosphate dentifrice was tested against a commercially available positive control dentifrice (0.30% triclosan/2.0% Gantrez copolymer). Following baseline measurements, subjects received a dental prophylaxis. Subjects were then instructed to brush twice daily for 60 seconds using their assigned product. Tooth brushing was supervised for 3 days of each week. Clinical examinations using a gingival index were performed at baseline and at 3 and 6 months. RESULTS: A total of 199 subjects were enrolled and 186 completed the 6-month study. Average baseline gingivitis and bleeding scores were similar for the two treatment groups. After 6 months, the experimental group had statistically significantly less gingivitis (25.8%) and statistically significantly less bleeding (27.4%) on average compared to the control group. Neither adverse oral soft tissue effects nor tooth staining were reported. CONCLUSION: Within the limits of the study protocol, the results demonstrated superior therapeutic benefits for the stabilized 0.454% stannous fluoride/sodium hexametaphosphate dentifrice in reducing gingivitis compared to the triclosan/copolymer control in this partially supervised study.

Dose response efficacy of sodium fluoride dentifrice at 9 and 21 months with supervised brushing.

PURPOSE: To assess whether a fluoride dose response could be detected with a relatively small sample size in a short duration using a novel randomized, double-blind study design. METHODS: Subjects (N = 644, with approximately 215 per group) with a mean age of 10.4 years old (9-12 years old) used a placebo dentifrice, an 1100 ppm F dentifrice or a 2800 ppm F dentifrice for the first 9 months of the study. Subjects in the placebo group were then switched to either 1100 ppm or 2800 ppm F dentifrice for the remainder of the study, while subjects in the fluoride groups continued with their original treatment assignments. Three calibrated examiners measured visual-tactile caries as DMFS that was supplemented with a radiographic examination at baseline, 9 months and 21 months for each subject. RESULTS: The results of this study are consistent with the previous results reported for sodium fluoride dentifrices. For all examiners, the 1100 ppm and 2800 ppm fluoride dentifrices delivered statistically significantly (P < 0.05) lower DMFS scores than the placebo control dentifrice at 9 months, while at 21 months the 1100 ppm and 2800 ppm fluoride dentifrices delivered statistically significantly lower DMFS scores compared to the both the placebo/1100 ppm and the placebo/2800 ppm dentifrice groups. In addition, one of the three examiners observed a directional (P = 0.11) dose response (2800 ppm F < 1100 ppm F) at 9 months, while at 21 months all three examiners observed evidence of a dose response, with one examiner observing a statistically significant difference between 1100 ppm and 2800 ppm F. Caries scores on occlusal surfaces provided the strongest evidence of an 1100 ppm F vs. 2800 ppm F difference.

Dental caries in school-age children residing in five Guatemalan communities.

OBJECTIVE: The caries epidemiology within the country of Guatemala is poorly understood. This work reports the cross-sectional prevalence of caries in the permanent teeth of children between the ages of 6 and 12 years in five different communities within Guatemala, and relates the caries experience to the fluoride levels in the communal drinking water in each of these cities. METHODOLOGY: These cities were selected because they represent geographically distinct regions of Guatemala, where there is no fluoridation of public drinking water. A total of 1,145 children were enrolled and examined in this study, with 227, 230, 231, 228 and 229 enrolled at Coban, Solola, Guatemala City, Chiquimula, and Estanzuela, respectively. Caries examinations were performed by a calibrated examiner using a visual tactile examination method performed with the aid of an artificial light, mouth mirror, compressed air, and a dental explorer employing a modified Radike criteria. Fluoride concentrations from drinking water, collected from the primary drinking water spigot at each elementary school in each city, were determined using ion chromatography. RESULTS: The cross-sectional mean DMFS in children 6-8 years old was 4.61, 4.34, 3.75, 3.52 and 2.71 for Coban, Solola, Guatemala City, Chiquimula, and Estanzuela, respectively. The cross-sectional mean DMFS in children 9-12 years old was 10.96, 10.09, 8.67, 8.71 and 6.02 for Coban, Solola, Guatemala City, Chiquimula, and Estanzuela, respectively. The prevalence of caries in children between the ages of 9 and 12 years was greater than 90% in all five cities. The natural fluoride concentration in water was 0.05 ppm, 0.23 ppm, 0.14 ppm, 0.50 ppm, and 0.60 ppm for Coban, Solola, Guatemala City, Chiquimula, and Estanzuela, respectively. CONCLUSION: Collectively, these data demonstrate that the high prevalence of caries in Guatemala appears to be directly correlated to levels of fluoride in the community drinking water.

Effect of three concentrations of sodium fluoride dentifrices on clinical caries.

PURPOSE: To assess whether the anti-caries effectiveness of three concentrations of fluoride dentifrice (placebo, 500 ppm F- and 1450 ppm F-) could be differentiated with small sample sizes in short time frames. MATERIALS AND METHODS: A controlled fluoride (F) dose-response study was conducted in concurrence with a supervised school oral hygiene regimen to assess whether dentifrices with increasing fluoride levels could be differentiated with small sample sizes in short time frames. The study was a randomized, double-blind study conducted for a period of 21 months. Subjects (N = 657 with approximately 219 per group) were randomized to placebo dentifrice, 500 ppm F- dentifrice or 1450 ppm F- dentifrice treatments for the first 9 months of the study. Subjects in the placebo group were then switched to either 500 ppm F- or 1450 ppm F- dentifrice for the remainder of the study, while subjects in the fluoride groups continued with their original treatment assignments. A calibrated examiner measured visual-tactile caries as DMFS that was supplemented with a radiographic examination at baseline, 9 months and 21 months for each subject. RESULTS: The mean caries increments at 9 months were 0.35 surfaces, 0.34 surfaces, and 1.28 surfaces for the 1450 ppm F-, 500 ppm F-, and placebo groups, respectively. The mean caries increments at 21 months were 0.21 surfaces, 0.26 surfaces, 1.75 surfaces and 1.90 surfaces for the 1450 ppm F-, 500 ppm F-, placebo/1450 ppm F-, and placebo/500 ppm F- groups, respectively. The 500 ppm F- and 1450 ppm F- fluoride dentifrices delivered statistically significantly (P< 0.05) lower DMFS scores than the placebo control dentifrice at 9 months, while at 21 months the 500 ppm F- and 1450 ppm F- fluoride dentifrices delivered statistically significantly lower DMFS scores as compared to the both the placebo/500 ppm F- and the placebo/1450 ppm F- dentifrice groups. There was no evidence of a dose response (1450 ppm F- < 500 ppm F-) at 9 months or at 21 months. In this study, the effectiveness of the two fluoride dentifrices was observed at 9 months and these outcomes were still present at 21 months, confirming that caries benefits can be observed in time frames as short as 9 months with approximately 200 subjects per treatment group.

The effect of shared genetic and environmental factors on periodontal disease parameters in untreated adult siblings in Guatemala.

BACKGROUND: Increasing evidence supports the role of genetic factors in susceptibility to infectious diseases, including chronic periodontitis. The role of genetic factors in phenotypic expression can be estimated from the degree of resemblance between relatives, as compared with that of unrelated members of a population. Heritability is an estimate of the proportion of total phenotypic variation of a quantitative trait, which is attributable to genetic factors, and is based on the variance within versus between family members. The aim of this study was to determine whether there is a familial basis for periodontal disease status in an untreated population in Guatemala using heritability estimates as a measure of familial clustering of disease. METHODS: One-hundred and thirteen adult subjects (including both siblings and spouse pairs), age range 35 to 60 years, participated in this study. Full-mouth periodontal examinations were performed and heritability estimates were calculated for mean plaque score, mean gingival index (GI), probing depth (PD), and clinical attachment level (CAL). Intraclass correlation coefficients (ICCs) were calculated using the same parameters for spouses to determine whether a common family environment in adulthood plays a role in disease expression. RESULTS: Only in the case of mean plaque score and mean recession score were heritability estimates significantly above zero at alpha = 0.05. For spouse pairs, mean GI score, mean PD, and percentage of sites of PD > or = 5 mm showed a statistically significant ICC. CONCLUSIONS: These results lead us to reject the hypothesis that there is substantial heritability for periodontal disease expression in this population. This may be due to an underlying lack of genetic variation within this sample or may indicate that, compared with the role of environmental factors, the genetic contribution to periodontal disease phenotypes is relatively minor.