An analytical model for elucidating tendon tissue structure and biomechanical function from in vivo cellular confocal microscopy images.

Fibered confocal laser scanning microscopes have given us the ability to image fluorescently labeled biological structures in vivo and at exceptionally high spatial resolutions. By coupling this powerful imaging modality with classic optical elastography methods, we have developed novel techniques that allow us to assess functional mechanical integrity of soft biological tissues by measuring the movements of cells in response to externally applied mechanical loads. Using these methods we can identify minute structural defects, monitor the progression of certain skeletal tissue disease states, and track subsequent healing following therapeutic intervention in the living animal. Development of these methods using a murine Achilles tendon model has revealed that the hierarchical and composite anatomical structure of the tendon presents various technical challenges that can confound a mechanical analysis of local material properties. Specifically, interfascicle gliding can yield complex cellular motions that must be interpreted within the context of an appropriate anatomical model. In this study, we explore the various classes of cellular images that may result from fibered confocal microscopy of the murine Achilles tendon, and introduce a simple two-fascicle model to interpret the images in terms of mechanical strains within the fascicles, as well as the relative gliding between fascicles.

Ultrasound-based nonviral gene delivery induces bone formation in vivo.

Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.

The effect of verapamil, lanthanum and local anesthetics of serotonin release from rabbit platelets.

The effect of calcium blockers (verapamil, local anesthetics and lanthanum chloride) on serotonin release from rabbit platelets was studied. The following results were obtained: (1) Verapamil and tetracaine (but not lanthanum) caused a time- and dose-dependent release of serotonin. The curves describing the time-course and those describing the concentration dependence of the release were sigmoid, suggesting cooperativity. (2) Thrombin-induced release from the platelets was dependent upon extracellular sodium ions, while no dependence was observed for the drug-induced release. (3) The release by verapamil was partially inhibited by prostaglandin E1 and theophylline which are known to raise intracellular cAMP levels, but was unaffected by the prostaglandin-synthesis inhibitor, indomethacin. (4) Verapamil, tetracaine and lanthanum inhibited thrombin-induced release of serotonin. The curve of dose dependence of the inhibition by verapamil and tetracaine was not sigmoid. The inhibition by verapamil and tetracaine was reversed by extracellular calcium ions, but no effect of this ion on the drug-induced release reaction was observed. It is concluded that the serotonin release induced by some calcium blockers and the inhibition of the thrombin-induced release by the same drugs are two separate phenomena. It is suggested that verapamil and tetracaine-induced release are mediated by exocytotic processes brought about by the interference of the drugs with calcium distribution between the cytosol and storage compartments within the platelet.

High oligomycin concentrations augment 6-keto-PGF1 alpha production in ventricular cardiomyocytes.

Incubation of cultured ventricular cardiomyocytes with high oligomycin concentrations (100 micrograms/ml), either alone or combined with 2-deoxyglucose (20 mM), led to the rapid depletion of cellular ATP. Inositol (poly)phosphate production decreased, and 6-keto PGF1 alpha production was increased. In cells depleted of ATP, either by low oligomycin concentrations or by sodium azide, 6-keto PGF1 alpha was not appreciably increased. There was a 25% rise in the release of fatty acids from the sn-2 position in glycerophospholipids. We suggest that oligomycin at high concentrations causes the release of free arachidonic acid from phospholipids either by non-PIP2-specific PLC and DG lipase or by phospholipase D, phosphatidic acid phosphatase and DG lipase. The effect is unrelated to decreased cellular ATP content.

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

UNLABELLED: Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. IN CONCLUSION: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent.

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells.

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.

Dissociation between inositol polyphosphate production and mitogenesis in mouse thymocytes.

Cortical and medullary thymocytes can be separated from each other by virtue of the fact that only cortical thymocytes bear peanut agglutinin (PNA) receptors. The mitogenic responses of subpopulations of thymocytes were studied. We have confirmed the results of Conlon et al. [(1982) J. Immun. 128, 797-801], that lectin-induced stimulation of unseparated cells, and PNA- but not PNA+ thymocytes, results in DNA synthesis. In contrast, both subpopulations, as well as unseparated cells, synthesize DNA in response to the calcium ionophore A23187 in the presence of the phorbol ester TPA, suggesting an impairment of signal transduction in PNA+ cells. However, comparable amounts of inositol phosphates were accumulated in PNA- and PNA+ thymocytes in response to Concanavalin A (Con A). We suggest that mitogenic lectins generate a third signal in addition to elevation of intracellular free calcium concentration and activation of protein kinase C. This signal is generated in PNA- but not in PNA+ thymocytes and is obligatory for lectin-induced stimulation.

Mitogenic activation of phosphoinositide turnover and DNA synthesis in murine CD4+8+ thymocytes.

Eighty percent of the lymphoid cells in the murine thymus are premature CD4+8+ (double positive) thymocytes. The vast majority of the double-positive cells do not maturate and die in the thymus. Although these cells are subjected to thymic selection processes, their activation competence has been an enigma. We have separated out CD4+8+ cells and studied their early and late responses to several mitogens. Concanavalin A, anti-CD3 (145-2C11) or anti-Thy1 (G7) monoclonal antibodies enhanced phosphoinositide turnover in double-positive thymocytes. However, DNA synthesis in the mitogen-stimulated cells was only accomplished if IL-2 or IL-4 was added. Alternatively, DNA synthesis could be induced by the calcium ionophore A23187 and phorbol myristate acetate (PMA). The latter mode of activation did not require the addition of exogenous lymphokines. CD4+8+ thymocytes did not secrete IL-2 or IL-4 following activation by either mitogens or A23187 and PMA. These findings demonstrate that CD4+8+ thymocytes resemble mature T cells in their ability to respond with DNA synthesis when activated by T-cell mitogens and IL-4 as well as IL-2. The results also delineate the difference between receptor mediated mitogenesis and pharmacological stimulation.

Apoptosis of thymic lymphoma clones by thymic epithelial cells: a putative model for 'death by neglect'.

We have previously described an in vitro system in which thymic epithelial cells induce apoptosis in CD4+ 8+ thymocytes or thymic lymphoma cells, in the absence of an exogenous antigen. A thymic epithelial cell line (TEC) recapitulated the response, by inducing apoptosis in CD4+ 8+ thymocytes of the thymic lymphoma clone, PD1.6. The present study pursues the involvement of the T-cell receptor (TcR) in the response of PD1.6 to TEC. TcR cross-linking did not cause apoptosis of PD1.6, although it induced tyrosine phosphorylation of p95vav. In contrast, TEC did not induce phosphorylation of p95vav but induced apoptosis of PD1.6 cells. These results suggest that TcR-evoked signals are not involved in TEC-induced apoptosis of PD1.6. Intracellular calcium chelation, using BAPTA-loaded PD1.6 cells, diminished TEC-induced apoptosis. Protein kinase C depletion in PD1.6 cells augmented their apoptotic response to TEC. Thus, the response of PD1.6 to TEC is calcium-dependent and inhibited by PKC. Likewise, the apoptotic response of PD1.6 to A23187 was abrogated by PKC activation. PD1.6 cells may represent an immature double positive thymocyte population, which does not undergo negative selection. The interaction of PD1.6 with TEC may thus serve as a model for the TcR-independent 'Death by Neglect', which takes place in the thymus during thymocyte development.

Multiple effects of staurosporine, a kinase inhibitor, on thymocyte functions. Comparison with the effect of tyrosine kinase inhibitors.

The effects of staurosporine, a protein kinase inhibitor, on the signal transduction and proliferation of thymocytes were studied. Signal transduction in response to Concanavalin A (Con A) as well as Concanavalin A (Con A)-induced augmentation of [3H]inositol incorporation into phospholipids were inhibited by staurosporine (> or = 10(-8) M). Staurosporine inhibited thymocyte proliferation in response to Con A in the presence or absence of the phorbol ester, phorbol myristate acetate (TPA) (10 nM). This inhibition was observed regardless of whether staurosporine was added together with Con A or 3 hr later. High concentrations of staurosporine (> 10(-8) M) inhibited thymocyte proliferation induced by the calcium ionophore A23187 and the phorbol ester TPA, whereas lower concentrations of the inhibitor (< or = 10(-8) M) enhanced thymidine incorporation in response to these activators. This dual effect of staurosporine was also observed in the presence of the staurosporine-related kinase inhibitor, K252a. In contrast, the tyrosine kinase inhibitor, tyrphostin AG490, inhibited the response to A23187 and TPA at all concentrations of the inhibitor and no augmentation was seen. Interleukin 2 (IL-2)-driven mitogenesis in IL-2-dependent cells was also inhibited by staurosporine. We suggest that the inhibition of thymocyte proliferation by staurosporine results from inhibition of both protein kinase C and tyrosine kinase: the augmentation of the response to A23187 and TPA results from inhibition of protein kinase C. Inhibition of signal transduction as well as inhibition of IL-2-driven mitogenesis result from inhibition of tyrosine kinase.

The effect of topical application with an organic and inorganic fluoride compound on the inhibition of dental plaque in humans.

Accumulation of dental plaque for 3 and 7 days respectively was determined by calculating the difference between the postcollection and precollection weights of enamel slabs attached to orthodontic bands cemented to the upper first molars of 3 subjects. Pretreatment of the enamel slabs with amine-fluoride or -chloride respectively-at an equivalent amine concentration-prevented plaque deposition on the slabs, whereas pretreatment with sodium monofluorophosphate was less effective. The fluoride concentrations of all fluoride-pretreated enamel slabs were similar. No convincing demonstration of significant differences between the treatments could be predicted in view of the discrepancy in results at the 3- and 7-day test periods.

Estimation of mesiodistal width of permanent canines and premolars in early mixed dentition.

The accuracy for predicting mesiodistal widths of unerupted permanent canines and premolars from x-rays and their estimations based on the already erupted permanent teeth were tested in a group of Israeli children. The observed posteruptive widths related more closely to predicted values obtained from x-ray measurements than from tabulated estimations.

The use of phospholipid liposomes for lithium administration. Polydipsic effect and tissue distribution of lithium.

Administration of lithium entrapped in phospholipid liposomes increased the lithium-induced polydipsia, but did not accelerate the onset of this effect. It also resulted in a larger accumulation of lithium in the liver, kidney and spleen, but not in the brain. The time course of polydipsia suggests that it depends on the intracellular lithium concentration. However, the rate of development of this effect depends on some additional factor.

Effect of trauma to the primary incisors on the alignment of their permanent successors in Israelis.

117 children who had experienced trauma to their primary incisors were re-examined in their transitional or permanent dentition stage. The control group consisted of 174 children with a corresponding dental developmental age. All the children were examined clinically and the intra- and interarch relations of the anterior segments were recorded. The prevalence of patients with at least one malposed incisor was higher in the trauma group. Trauma to the primary dentition was found to be a contributing factor affecting the alignment of the permanent successors. Early loss of the primary incisors did not cause loss of space in most of the cases; however, it was associated with malposition of their permanent successors. Lack of eruption guidance or the direct effect of the injury on the position of the developing bud could be considered as contributing etiologic factors. There was a very low prevalence of more serious malocclusion features, like impaction, in the trauma group.

Iatrogenic exfoliation of teeth by the incorrect use of orthodontic elastic bands.

Four cases of improper use of elastic bands for minor orthodontic treatment are described. For all the patients, the treated teeth were damaged and local surgical intervention was necessary. To prevent complications when elastic bands are being used, the dentist must provide stabilization of the elastic bands by brackets, bands, or bonded attachments, inform the patient about the possibility of losing the rubber elastic band, and mark the elastic rubber by radiopaque material. Every effort must be made to preserve the damaged anterior teeth by fixation and surgical treatment.

Simultaneous Analog Stimulation (SAS)--Continuous Interleaved Sampler (CIS) pilot comparison study in Europe.

The recent availability of the enhanced bipolar electrode array in the CLARION Multi-Strategy Cochlear Implant has permitted clinicians to fit patients successfully with the Simultaneous Analog Stimulation (SAS) strategy by means of bipolar coupling. Out of 22 consecutively implanted subjects, 20 subjects could be fitted with both Continuous Interleaved Sampler (CIS) and SAS strategies. Their performance was evaluated up to 3 months postoperatively. Speech perception results, as well as the patient's preference for either CIS or SAS, were examined. It was found that half of the subjects selected SAS as their preferred strategy and that the use of a preferred strategy resulted in higher overall patient performance. Further analyses showed that those subjects preferring SAS had higher electrode impedance values and lower threshold and most comfortable loudness levels than subjects preferring CIS. We presume that these psychophysical findings are, in part, related to the position of the intracochlear electrode array. Specifically, SAS users may have a more modiolus-hugging electrode array position.

Orthodontic-prosthetic treatment to replace maxillary incisors exfoliated because of improper use of orthodontic elastics: a case report.

This article describes the iatrogenic exfoliation of maxillary central incisors following the improper use of orthodontic elastic bands. The unsecured rubber band had migrated apically and caused an almost "bloodless extraction" of both maxillary central incisors. A combined orthodontic-prosthetic solution was used to replace the lost incisors.

Functional and esthetic rehabilitation of an adolescent cleft lip and palate patient.

Many patients with clefts that also affect the alveolar ridge present with congenital absence of the permanent maxillary lateral incisors. This paper describes the treatment of an adolescent cleft lip and palate patient whose missing and unesthetic maxillary incisors were replaced by a combination of fixed and removable partial dentures.

Root length of lateral incisors adjacent to palatally-displaced maxillary cuspids.

An evaluation of 70 palatally-displaced cuspids and 106 controls finds significantly shorter lateral incisor adjacent to the palatal cuspids.