Laser confocal endomicroscopy as a novel technique for fluorescence diagnostic imaging of the oral cavity

Malignancies of the oral cavity are conventionally diagnosed by white light endoscopy, biopsy, and histopathology. However, it is often difficult to distinguish between benign and premalignant or early lesions. A laser confocal endomicroscope (LCE) offers noninvasive, in vivo surface and subsurface fluorescence imaging of tissue. We investigate the use of an LCE with a rigid probe for diagnostic imaging of the oral cavity. Fluorescein and 5-aminolevulinic acid (ALA) were used to carry out fluorescence imaging in vivo and on resected tissue samples of the oral cavity. In human subjects, ALA-induced protoporphyrin IX (PpIX) fluorescence images from the normal tongue were compared to images obtained from patients with squamous cell carcinoma (SCC) of the tongue. Using rat models, images from normal rat tongues were compared to those from carcinogen-induced models of SCC. Good structural images of the oral cavity were obtained using ALA and fluorescein, and morphological differences between normal and lesion tissue can be distinguished. The use of a pharmaceutical-grade solvent Pharmasolve enhanced the subsurface depth from which images can be obtained. Our initial results show that laser confocal fluorescence endomicroscopy has potential as a noninvasive optical imaging method for the diagnosis of oral cavity malignancies.     

Mechanoenzymatics of titin kinase

Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.     

The effect of low and ultra-low dosages Thymoglobulin on peripheral T, B and NK cells in kidney transplant recipients

IntroductionRabbit Anti-Thymocyte Globulin (r-ATG) is a polyclonal antibody preparation, used to prevent and treat acute rejection episodes after organ transplantation. However, despite more than 40 years of clinical use, the optimal dose of r-ATG is still not defined. To find a better balance between efficacy and infectious complications, we embarked on a controlled study and monitored the effect of low and ultra-low dosages Thymoglobulin (Genzyme) on peripheral T, B, and NK cells.Patients and methodsKidney transplant recipients received either 0.5 mg/kg, 1.0 mg/kg or 2.0 mg/kg on the first 3 consecutive days post-transplantation. Thus, total doses were 1.5 mg/kg, 3.0 mg/kg and 6.0 mg/kg. A total of 40 patients were enrolled, including 11 controls. All patients were treated with Prednisolon, Advagraf (Astellas) and Mycophenolate Mofetil (Roche). T (CD3 +), B (CD19 +) and NK (CD3-CD16 + 56 +) cells were analyzed by flow cytometry. Baseline cell counts were compared to forty age and sex matched healthy persons. Post-transplantation cell counts of the 3 Thymoglobulin groups were compared to the 11 control patients, who received no induction therapy.ResultsAbsolute numbers of T, B, and NK cells were comparable in all patients pre-transplantation, but T and B cells were lower than in healthy persons (p = 0.007 and p = 0.0003, Mann Whitney test). In the first week, T cells and NK cells were significantly lower in all Thymoglobulin groups compared to controls. B cells were not affected. One month after Thymoglobulin NK cells had returned to control numbers in all groups, while T cells had already recovered to control counts in the 1.5 mg/kg group. During follow-up, T cells in the 3.0 mg/kg group also returned to control values, but at one year the patients in the 6.0 mg/kg group still had significantly lower T cells (p = 0.03). Patient and graft survival, rejection and infection incidence and renal function did not differ between groups.ConclusionPatients with end stage renal disease have significantly lower peripheral T and B cell counts than healthy persons. (Ultra-) low Thymoglobulin schedules deplete peripheral lymphocytes in a dose dependent way. Knowledge of the duration of this depletion contributes to finding the optimal immunosuppressive strategy for kidney transplant recipients.     

Systematic review of physical activity and health in the early years (aged 0-4 years)

The early years represent a critical period for promoting physical activity. However, the amount of physical activity needed for healthy growth and development is not clear. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework, we aimed to present the best available evidence to determine the relationship between physical activity and measures of adiposity, bone and skeletal health, motor skill development, psychosocial health, cognitive development, and cardiometabolic health indicators in infants (1 month - 1 year), toddlers (1.1-3.0 years), and preschoolers (3.1-4.9 years). Online databases, personal libraries, and government documents were searched for relevant studies. Twenty-two articles, representing 18 unique studies and 12?742 enrolled participants, met inclusion criteria. The health indicators of interest were adiposity (n = 11), bone and skeletal health (n = 2), motor development (n = 4), psychosocial health (n = 3), cognitive development (n = 1), and cardiometabolic health indicators (n = 3); these indicators were pre-specified by an expert panel. Five unique studies involved infants, 2 involved toddlers, and 11 involved preschoolers. In infants, there was low- to moderate-quality evidence to suggest that increased or higher physical activity was positively associated with improved measures of adiposity, motor skill development, and cognitive development. In toddlers, there was moderate-quality evidence to suggest that increased or higher physical activity was positively associated with bone and skeletal health. In preschoolers, there was low- to high-quality evidence on the relationship between increased or higher physical activity and improved measures of adiposity, motor skill development, psychosocial health, and cardiometabolic health indicators. There was no serious inconsistency in any of the studies reviewed. This evidence can help to inform public health guidelines. (PROSPERO registration: CRD42011001243).     

The Global Rating Scale complements the AGREE II in advancing the quality of practice guidelines

ObjectiveTo explore the role of a four-item Global Rating Scale (GRS) that could be used in place of the Appraisal of Guidelines, Research and Evaluation II (AGREE II).Study Design and SettingA mixed four-factor design was used (User Type, Evaluation Type, Clinical Topic, Guideline). Participants were asked to read and evaluate a guideline using both the AGREE II draft and GRS or GRS only and to complete a series of questions regarding overall guideline quality, adoption, utility, and acceptability.ResultsOne GRS item varied as a function of User Type. Each item was a significant predictor of participants' outcome measures. All items were rated as useful by stakeholders. The GRS rating scores, outcome measures, and usefulness scores did not vary between the two Evaluation Type conditions. Correlations between the GRS and the outcome measures were stronger compared with those between the AGREE II draft and these measures.ConclusionAlthough the GRS is less sensitive than the AGREE II in detecting differences in guideline quality as a function of User Type, its items did predict important outcome measures related to guideline adoption. The GRS may play a role in guideline evaluation, although further study is warranted.     

Calmodulin antagonists inhibit and phorbol esters enhance transferrin endocytosis and iron uptake by immature erythroid cells

Seven antagonists of the calcium-binding protein calmodulin were found to inhibit iron and transferrin uptake by reticulocytes. This inhibition could be completely accounted for by inhibition of the endocytosis and exocytosis of transferrin. When four of the antagonists were tested with the nucleated erythroid cells from the liver of the fetal rat, inhibition of iron uptake was also observed but at higher concentrations than required for the same degree of inhibition with reticulocytes. The tumor promoters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) were shown to increase the rates of iron and transferrin uptake by reticulocytes and fetal liver erythroid cells by accelerating the rates of transferrin endocytosis and exocytosis. Since these substances are known to stimulate the calcium-activated enzyme protein kinase C while calmodulin antagonists are inhibitory, it is concluded that this enzyme plays an important role in the endocytosis and intracellular cycling of transferrin, and iron uptake by immature erythroid cells. However, the possibilities that calmodulin is also involved or that the inhibitory effects of the calmodulin antagonists are due to nonspecific actions on the cell membrane cannot be excluded.     

Diffusion capacity in heart transplant recipients.

Preoperative diffusion capacity per liter alveolar volume (Kco) in cardiac transplant recipients with an intrinsic normal lung is within the normal range. In the first postoperative year, Kco showed a significant mean decrease of 12 percent (p less than 0.004). Lung function (TLC, VC, FEV1) tended to normalize after heart transplantation. Ventilation distribution remained stable before and after heart transplantation. Preoperatively, weak correlations were found between Kco and diastolic pulmonary arterial pressure (dPAP) and mean pulmonary capillary wedge pressure (PCWP). Postoperatively, correlation between Kco and PCWP was weak, and between Kco and dPAP it was not significant at all. These pressures determine the capillary blood volume before and after transplantation. Probably these weak correlations indicate that intrapulmonary factors, not cardiac factors, are of primary importance in the regulation of blood distribution. The percentage of decrease in Kco in the first postoperative year correlated with the change in dPAP and PCWP, but also with the cyclosporine level in the first posttransplant year. No correlation was found between cyclosporine level and pulmonary vascular resistance. It is suggested that higher levels of cyclosporine influence the alveolar capillary membrane, so that Kco decreases. The percentage of decrease in Kco was significantly more outspoken in patients who had rales on auscultation preoperatively. Using multiple regression analysis, we found that the factors most strongly related to the percentage of change in Kco in the first posttransplant year were the preoperative Kco, the cyclosporine level in the first postoperative year, and the change in dPAP in that year.