Influence of Different Etching Modes on Bond Strength to Enamel using Universal Adhesive Systems.

INTRODUCTION: The adhesive systems and the techniques currently used are designed to provide a more effective adhesion with reduction of the protocol application. The objective of this study was to evaluate the bond strength of universal adhesive systems on enamel in different etching modes (self-etch and total etch). MATERIALS AND METHODS: The mesial and distal halves of 52 bovine incisors, healthy, freshly extracted, were used and divided into seven experimental groups (n = 13). The enamel was treated in accordance with the following experimental conditions: FUE-Universal System - Futurabond U (VOCO) with etching; FUWE - Futurabond U (VOCO) without etching; SB-Total Etch System - Single Bond 2 (3M); SBUE-Universal System - Single Bond Universal (3M ESPE) with etching; SBUWE - Single Bond Universal (3M ESPE) without etching; CLE-Self-etch System - Clearfil SE Bond (Kuraray) was applied with etching; CLWE - Clearfil SE Bond (Kuraray) without etching. The specimens were made using the composite spectrum TPH (Dentsply) and stored in distilled water (37 ± 1°C) for 1 month. The microshear test was performed using the universal testing machine EMIC DL 2000 with the crosshead speed of 0.5 mm/minute. The bond strength values were analyzed using statistical tests (Kruskal-Wallis test and Mann-Whitney test) with Bonferroni correction. RESULTS: There was no statistically significant difference between groups (p < 0.05), where FUE (36.83 ± 4.9 MPa) showed the highest bond strength values and SBUWE (18.40 ± 2.2 MPa) showed the lowest bond strength values. The analysis of adhesive interface revealed that most failures occurred between the interface composite resin and adhesive. CONCLUSION: The universal adhesive system used in dental enamel varies according to the trademark, and the previous enamel etching for universal systems and the self-etch both induced greater bond strength values. CLINICAL SIGNIFICANCE: Selective enamel etching prior to the application of a universal adhesive system is a relevant strategy for better performance bonding.

Enzymatic synthesis of melatonin in avian pineal body: extraretinal response to light.

In the chick pineal body, activity of the melatonin-forming enzyme hydroxyindole-O-methyl transferase is greater in the light than in darkness. Neither bilateral enucleation of the eyes nor sympathetic denervation prevented this light-induced elevation of enzyme activity. This fact indicates that in the bird, in contrast to mammals, neither the retinas nor sympathetic innervation of the pineal body are essential for environmental control of melatonin formation.

Fingerprints of anergic T cells.

Peripheral T cell tolerance may result from activation-induced cell death [1], anergy [1], and/or immune response modulation by regulatory T cells [2]. In mice that express a transgenic receptor specific for peptide 111-119 of influenza hemagglutinin presented by E(d) class II MHC molecules as well as hemagglutinin under control of the immunoglobulin-kappa promoter, we have found that anergic T cells [3] can also have immunoregulatory function and secrete IL-10 [4]. In order to obtain information on molecular mechanisms involved in anergy and immunoregulation, we have compared expression levels of 1176 genes in anergic, naive, and recently activated CD4+ T cells of the same specificity by gene array analysis. The results provide a plausible explanation for the anergic phenotype in terms of proliferation, provide new information on the surface phenotype of in vivo-generated anergic CD4+ T cells, and yield clues with regard to new candidate genes that may be responsible for the restricted cytokine production of in vivo-anergized CD4+ T cells. The molecular fingerprints of such T cells should enable the tracking of this small population in the normal organism and the study of their role in immunoregulation.

Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells.

Although multiple sclerosis (MS) patients and healthy individuals have similar frequencies of myelin basic protein (MBP)-specific T cells, the activation state of these cells has not been well characterized. Therefore, we investigated the dependence of MBP-reactive T cells on CD28-mediated costimulation in MS patients, healthy controls, and stroke patients. MBP-reactive T cells from healthy controls and stroke patients failed to proliferate efficiently when costimulation was blocked using anti-CD28, consistent with a naive T cell response. In contrast, MBP-specific T cell proliferation was not inhibited, or was only partially inhibited when CD28-mediated costimulation was blocked in MS patients. Blockade of CD28 failed to inhibit tetanus toxoid-specific T cell proliferation in both the controls and MS patients, demonstrating that memory cells are not dependent on CD28-mediated costimulation. Limiting dilution analysis indicated that the frequency of MBP-reactive T cells was significantly decreased in healthy controls compared with MS patients when CD28-mediated costimulation was blocked. These data suggest that MBP-reactive T cells are more likely to have been activated in vivo and/or differentiated into memory T cells in MS patients compared with controls, indicating that these cells may be participating in the pathogenesis of MS.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

Detection of small sequence differences using competitive PCR: molecular monitoring of genetically improved, mercury-reducing bacteria.

A quantitative PCR approach is presented to detect small genomic sequence differences for molecular quantification of recombinant DNA. The only unique genetic feature of the mercury-reducing, genetically improved Pseudomonas putida KT2442::mer73 available to distinguish it from its native mercury-resistant relatives is the DNA sequence crossing the border of the insertion site of the introduced DNA fragment. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively co-amplified DNA standard constructed by point mutation PCR. After computing the denaturation behavior of the target DNA stretch, a single base difference was introduced to achieve maximum migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to detect small sequence differences and to monitor recombinant DNA in effluxes of biotechnological plants.

Lid suture myopia in chicks.

Domestic chicks reared under continuous light developed light-induced avian glaucoma (LIAG), including eye enlargement and flat cornea. Chicks reared under dim light developed dim light buphthalmos (DLB), a somewhat similar eye enlargement, superimposed on the LIAG effect when, as in one subgroup of the present experiments, the dim light was also continuous. Unilateral lid suture elicited experimental myopia on the operated side, additive to the LIAG and/or DLB effects. When the rearing conditions were diurnal (ie, not such as to induce LIAG eye enlargement), and the light of at least "normal" brightness, lid suture still caused unilateral myopia, accompanied by a pronounced bulging of the cornea. We discuss the probable effects of these changes on refraction and accommodation during the chick's development.

Phenylethylamine-induced stereotypies in the rat: a behavioral test system for assessment of MAO-B inhibitors.

Stereotyped sniffing behavior together with forepaw padding--defined as the beta-phenylethylamine (PEA) syndrome--is induced by MAO-B inhibitors in rats injected with 30 mg/kg IP PEA. The comparison of the abilities of the MAO-B inhibitors to induce the syndrome and to inhibit MAO-B in rat brain homogenates indicated that at least 75% of MAO-B activity in rat brain had to be inhibited to induce the PEA syndrome. A good correlation was found between the abilities of MAO-B inhibitors to induce the behavioral syndrome and to increase levels of PEA in rat brain. Specific MAO-A inhibitors potentiated the behavioral effect of the MAO-B inhibitor deprenyl, while they did not induce the syndrome themselves or only at very high doses. Inhibitors of the reuptake of 5-HT or noradrenaline were inactive under the described experimental conditions. This behavioral test system seems to be a useful in vivo screening test in rats for detecting compounds with strong MAO-B inhibiting activity.

Increased frequency of recognition of delipidated versus intact CNS myelin in multiple sclerosis and control subjects.

The proliferative response of mononuclear cells from MS patients and normal control subjects to intact and delipidated myelin membranes was examined. The mean frequency of recognition in both groups of human subjects was greater for delipidated myelin than for intact myelin. Human T cell lines established using intact or delipidated myelin as the antigen were highly heterogeneous in response, and were each able to recognize myelin basic protein and myelin proteolipid protein peptides. However, there was no difference in the frequency of recognition of either form of myelin membrane when MS patients were compared to control subjects. Our results suggest that the presentation of delipidated forms of membrane proteins might enhance the response to myelin antigens in vivo, and be relevant to demyelinating diseases.

Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors.

We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R(-)-baclofen are mediated by the GABAB receptor, whereas only the induction of SWD by GHB is dependent on GABAB receptor mediation, the decrease in cGMP being only partially so. Taken together with the observations of Marescaux et al. (1992), these results indicate that GABAB receptors are of primary importance in experimental absence epilepsy and that GABAB receptor antagonists may represent a new class of anti-absence drugs.

Hand rejuvenation. The state of the art.

Intrinsic aging and photoaging, two distinct biologic processes, contribute to making hands appear aged. Restoration to a more youthful appearance for aged-appearing hands often requires a multitherapeutic approach individualized to the patient's pathology. This article reviews the role and technique of microlipoinjection for the rejuvenation of aged-appearing hands.

Retinal pigment epithelium: ring mitochondria and lesions induced by continuous light.

Electron micrographs of avian retinal pigment epithelium (RPE) reveal a basal zone of abundant mitochondria, approximately 15% of which present ring-shaped profiles. We infer that these ring profiles represent cross sections of "donut-shaped" bodies, randomly deployed in the RPE mitochondrial zone. The per cent of all RPE mitochondria which are in the ring form increases with age, and is further increased in chicks reared under continuous light, an environmental condition leading also to light-induced avian glaucoma (LIAG). We calculate that a given mitochondrion, on assuming the ring form, would more than double its surface area. Thus the RPE mitochondria of chicks developing LIAG may present to the surrounding cytoplasm a total surface area more than fourfold greater than in normal chicks.

Diurnal mitochondrial changes in avian retinal pigment epithelium: a search for correlation with thyroid state.

The avian retinal pigment epithelium (RPE) is well endowed with mitochondria, 10-15% of which appear to be "donut-shaped". The number of such mitochondria presenting ring profiles varies with the photoperiod, with a peak early in the dark period and a low early in the light period. This cycle is inversely correlated with the diurnal rhythm of plasma thyroxine (T4), but is in phase with the T3 cycle. We could detect no effect on the numbers of ring mitochondria when exogenous TSH, T4 or T3 was injected. Numbers and appearance of ring mitochondria were likewise unaltered in chicks breathing pure oxygen or spent air for 15 min. before sacrifice. We speculate that this aberrant mitochondrial form may have some relationship to the processing of shed outer segment discs, also a photoperiod-driven phenomenon. It has been suggested that the daily manufacture of hydrolytic enzymes for breakdown of disc proteins is probably highly energy-expensive. We also note that the peroxidation of the lipid components of these membranes must be another aspect of disc disposal. Ascorbic acid, whose photooxidation in the eye generates hydrogen peroxide, may play a role in this process.

Chicks blinded with formoguanamine do not develop lid suture myopia.

The involvement of the retina in avian lid suture myopia was investigated by use of formoguanamine (FG), which induces blindness in chicks, characterized by damage to the retinal pigment epithelium and photoreceptor layers. In control chicks reared under a diurnal lighting schedule of 12L/12D, unilateral eyelid suture at 3 days of age induced a typical lid suture myopia response on the operated side (eye enlargement and bulging cornea) by 4 wks. of age. On the other hand, in FG-treated blind chicks also reared under 12L/12D, there was no difference in eye parameters, between sutured and non-sutured eyes. All globe parameters measured were very similar in FG-treated and control non-sutured chicks. Corneal diameter and anterior chamber depth in the eyes of FG-treated chicks were significantly smaller than in non-sutured control eyes.

Ocular responses of genetically blind chicks to the light environment and to lid suture.

Blind rc chicks were examined as to growth characteristics of the developing eye. We also explored the relationship between hereditary blindness and two environmental interventions affecting eye development, light-induced avian glaucoma (LIAG) and the axial lengthening associated with lid-suture. Genetically blind rc chicks could be identified by their behavior even during the first hours after hatching. However, they retained the pupillary reflex, at least to 5 weeks of age. Blind chicks reared under a diurnal lighting schedule had eyes 18% larger (by eye weight) than sighted hatchmates, with corresponding increases in globe diameters. Of the anterior segment parameters, neither corneal diameter, anterior chamber depth nor corneal curvature was demonstrably different from sighted controls. When rc chicks were reared under continuous light, the environmental condition which brings on LIAG, their already enlarged eyes showed no superimposed increases in either eye weight or globe dimensions. The corneas showed some characteristic LIAG effects: small anterior chamber depth and increased corneal radius of curvature (flat cornea), as compared with diurnal rc birds. Some birds were subjected to unilateral eyelid-suture, a protocol which usually induces axial lengthening and corneal bulging. Blind rc birds did not respond to this surgical intervention: in comparison by paired t-test between sutured and contralateral eye, there was little or no difference in eye weight, globe diameters or anterior segment parameters. Thus vision, or at least an intact retina/RPE, seems to be necessary for development of the characteristic axial lengthening in response to eyelid suture. The globe enlargement of LIAG also appears to be vision-dependent, although the anterior segment manifestations of LIAG are systemic effects.

Automated sample-preparation technologies in genome sequencing projects.

A robotic workstation system (BioRobot 96OO, QIAGEN) and a 96-well UV spectrophotometer (Spectramax 250, Molecular Devices) were integrated in to the process of high-throughput automated sequencing of double-stranded plasmid DNA templates. An automated 96-well miniprep kit protocol (QIAprep Turbo, QIAGEN) provided high-quality plasmid DNA from shotgun clones. The DNA prepared by this procedure was used to generate more than two mega bases of final sequence data for two genomic projects (Arabidopsis thaliana and Schizosaccharomyces pombe), three thousand expressed sequence tags (ESTs) plus half a mega base of human full-length cDNA clones, and approximately 53,000 single reads for a whole genome shotgun project (Pseudomonas putida).

Eye enlargement in birds induced by dim light.

We here report changes in eye weight and other measurements, in chickens reared in dim light. These effects are distinguished from those seen in light-induced avian glaucoma (brought on by exposure to continuous light). We confirm and extend the results of Berkovitz and Harrison on the dim light effect, but we differ from Axmith and Morin, who were apparently unable to reproduce light-induced avian glaucoma. We cannot yet offer an explanation as to the mechanism(s) of eye damage in either light-induced avian glaucoma or dim light-induced macrophthalmos.

The water provocative test in preglaucomatous chickens.

Our ability to induce glaucoma in birds provides an experimental system for exploring the mechanisms involved in the provocative water test. Waters loads of a level comparable to those used clinically produced larger intraocular pressure (IOP) changes in preglaucomatous and glaucomatous birds than in control birds. The magnitude of the response corresponded to the level of IOP before testing, that is, to the severity of the glaucoma. Changes in systemic blood pressure were not involved in the response. While hyotonicity of plasma may be the systemic determinant, the normal range of variation in this factor does not allow us at present to consider this a causal relationship.

Timolol and pilocarpine are hypotensive in light-induced avian glaucoma.

Two antiglaucoma drugs were tested on Hubbard chicks with light-induced avian glaucoma (LIAG). This animal model for open-angle glaucoma was further assessed by a novel method for monitoring aqueous inflow and outflow simultaneously. When the chicks were 8 to 9 weeks of age the preglaucomatous eyes were significantly enlarged (p less than 0.001), weighing a mean of 3.37 g, compared with 2.59 g for a group of normal eyes, but the intraocular pressure (IOP) was slightly lower in the former (13.79 v. 16.46 mm Hg; p less than 0.05). At this age the aqueous outflow was markedly reduced (to 0.70 v. 2.47 microL/min; p less than 0.05), but no change in aqueous inflow could be demonstrated. By 18 to 20 weeks the glaucomatous eyes were further enlarged (mean weight 4.67 v. 3.76 g; p less than 0.001) and hypertensive (mean IOP 29.85 v. 22.27 mm Hg; p less than 0.05). Timolol elicited a 29% to 33% reduction in the IOP at 8 to 9 weeks in both groups of eyes. At 18 to 20 weeks it reduced the IOP of the glaucomatous eyes to normal values but caused a less marked reduction in the normal eyes. Pilocarpine elicited an initial sharp rise in the IOP but was ultimately hypotensive, causing about a 33% decrease from the preinfusion IOP, in both young and adult birds.

Herpes simplex virus infection complicating amniotic band syndrome in the newborn.

Amniotic band syndrome, which is frequently seen in association with prolonged rupture of membranes, is due to the amnion forming fibrous strands that encircle fetal parts. This causes compression and necrosis of the underlying skin. We report a case of herpes simplex virus infection localized to these lesions.