An optimized method for the functional analysis of human regulatory T cells.

Naturally occurring regulatory T cells (Treg) suppress the activation of antigen-responsive T cells in a cell contact-dependent manner. In order to investigate the impact of soluble mediators and receptor-ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen-presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti-CD25 monoclonal antibody (MoAb)-coated Dynabeads followed by a detachment step. The remaining CD4+ CD25- responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T-cell Activation/Expansion Beads from Miltenyi Biotec pre-coated with anti-CD3 plus anti-CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 microg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti-CD3/anti-CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg-mediated T-cell suppression by some cytokines (IL-2, IL-15), soluble IL-6 receptor/IL-6 fusion protein and recombinant GITR-ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25- responder T cells.

Physiological aging of an all-ceramic restorative material.

Ceramic materials exposed to a liquid environment may be subject to stress corrosion and/or time-delayed failure. The intent of this project was to evaluate the susceptibility of a magnesia alumina spinel (Cerestore) to stress corrosion and degradation. Bars 2.5 x 2.5 x 30.0 mm were prepared according to manufacturer's instructions. Specimens were aged in distilled water or air at 37 degrees C. The modulus of rupture was evaluated at zero, six, and 12 months in four-point loading at loading rates of 0.05, 0.5, and 5.0 mm/min. The modulus of rupture of each specimen was tested in its respective aging medium. The data were analyzed by one-way analysis of variance with a multiple-means comparison test, linear regression analysis, and Weibull statistics. The pooled data sets of specimens aged in water vs. those aged in air showed a significant difference in the respective modulus of rupture (air, 108.50 +/- 16.11; water 96.94 +/- 15.04 MPa). The one-way analysis of variance showed no significant difference between the aging times zero, six, and 12 months in each respective aging medium. The Weibull analysis also showed no difference between the Weibull constants, 7.66 air vs. 7.64 water, but a significant difference between the characteristic strengths, 115.22 air vs. 103.02 water. This study indicates that distilled water has a significant degradative effect on a magnesia alumina spinel, more likely affecting the mode of fracture rather than the stress corrosion characteristics.

Determinants controlling iatrogenic external root resorption and repair during and after palatal expansion.

The mechanisms controlling iatrogenic external root resorption (ERR) and repair were studied on 8 Macaca fascicularis monkeys. The animals were divided into short-term and long-term groups, and were treated with jackscrew, magnetic and sham palatal expansion appliances. Scanning electron microscopy morphometric analysis found major evidence of ERR in the tooth-borne jackscrew appliance, in the long-term group, in the maxillary premolars, on the buccal and furcation root surfaces, on the mesiobuccal root, and in the apical zone. Correspondingly, the ERR mechanism is controlled by the impulse (F. delta t) and the critical barrier of the periodontal ligament as primary determinants and by the environmental density as a secondary determinant. ERR is initially regulated by the force component of the impulse and, with increased duration, by the time component of the impulse. The impairment/repair dynamics were found to be regulated by three principles: ERR level of irreversibility, delayed resorption response and jiggling.

Surface analysis of tarnished dental alloys.

Six crown and bridge alloys ranging in nobility between 25-63 wt % (18-45 at %) were analyzed by optical microscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and secondary ion mass spectroscopy (SIMS), as well as by L*a*b* colorimetry before and after in vitro tarnishing in artificial saliva with and without additions of 0.00016, 0.016, and 1.6 % Na2S with a rotating wheel apparatus. All alloys except the lowest of 18 at % changed colors to about the same degree after 72 h of tarnishing. All alloys decreased in L*, while increased in both a* and b*, thus appearing darker and with increased redness and yellowness. This was due to localized darkening and to other products. For all alloys except one, saliva without sulfide promoted color changes more severe than for saliva with 0.016 % Na2S. For the most part, analysis by EDS was unable to detect differences between the tarnished films and the as-polished surfaces. SIMS analysis, however, showed changes in the substrate ion (Cu, Ag, Pd, and In) peak intensities. In most cases the intensities decreased and with the decrease greater with the sulfide-free saliva than with sulfide-containing. This indicated that sulfide promoted insoluble deposition of products. Changes in the Ag, Pd, and In peak intensities followed much the same pattern as with Cu. The as-polished surfaces, even though carefully prepared, showed much contamination in the form of organics, namely C, CH, N, NH, O, CHN, CN, as well as from Na, K, Ca, Si, S, Cl, and others. Most tarnished surfaces showed large increases in Na, K, and Ca, and with the sulfide-free saliva being more severe in this regard. The mass spectrum also showed peaks with atomic mass units in the range 55-58 related to only some of the tarnished surfaces.

Shear testing of the porcelain-metal bond.

A simple shear test was employed to determine the bonding strength of porcelain to a gold metal substrate and to a non-precious metal substrate after they had been aged in double-distilled water at 37 degrees C for four and 12 months. A 7.4% and 18.1% decrease in bond strength after four months and 12 months, respectively, was observed for the porcelain-gold system, and a decrease of 21.2% and 21.4% after four and 12 months, respectively, for the non-precious-porcelain system. A significant difference was observed between the bond strengths of the porcelain-metal composites: 51.17 +/- 11.29 MPa for the porcelain-gold system and 31.83 +/- 3.65 MPa for the porcelain-non-precious system.