The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization

Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, Src kinases are involved in regulating the cell architecture. In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening. Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase. Its predicted size of 71 kDa was confirmed by Western blot analysis. Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development. Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female. Its enzymatic activity was confirmed by functional analyses. In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein. Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms. These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.     

Photodynamic therapy of prostate cancer by means of 5-aminolevulinic acid-induced protoporphyrin IX - in vivo experiments on the dunning rat tumor model

OBJECTIVE: In order to expand the use of photodynamic therapy (PDT) in the treatment of prostate carcinoma (PCA), the aim of this study was to evaluate PDT by means of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) in an in vivo tumor model. METHODS: The model used was the Dunning R3327 tumor. First of all, the pharmacokinetics and the localization of PPIX were obtained using fluorescence measurement techniques. Thereafter, PDT using 150 mg 5-ALA/kg b.w. i.v. was performed by homogenous irradiation of the photosensitized tumor (diode laser lambda = 633 nm). The tumors were resected 2 days post-PDT and the extent of the necrosis was determined histopathologically. RESULTS: The kinetics of PPIX fluorescence revealed a maximum intensity in the tumor tissue within 3 and 4.5 h post-application of 5-ALA. At this time, specific PPIX fluorescence could be localized selectively in the tumor cells. The PDT-induced necrosis (n = 18) was determined to be 94 +/- 12% (range 60-100%), while the necrosis of the controls (n = 12) differs significantly (p < 0.01), being less than 10%. CONCLUSION: These first in vivo results demonstrate the effective potential of 5-ALA-mediated PDT on PCA in an animal model.     

Feasibility of using interpolated contours of targets and organs at risk in intensity‐modulated radiation therapy treatment planning for advanced‐stage nasopharyngeal carcinoma

To assess the dosimetric effect of using interpolated contours in planning intensity‐modulated radiation therapy (IMRT) for advanced T‐stage nasopharyngeal carcinoma. The present study focused on T3–T4 tumours where the proximity of targets to neurological organs poses a stringent test on the feasibility of such an approach. Contours of targets and organs were delineated on CT images of 2.5‐mm interval and a reference IMRT plan was generated. An investigative (INV) IMRT plan was then generated with the same planning protocol, but based on interpolated contours that replaced deleted contours on alternate slices. The reference and INV plans were compared. Regarding target coverage, all targets in the INV plans met the acceptance criteria except for the PTV in one case. Regarding organs, the mean dose to 1% volume of the brainstem and spinal cord in the INV plans were kept below their dose limits. No significant differences in the mean doses to others organs were found. Satisfactory target coverage and protection of critical organs to a degree similar to full‐scale contouring could be achieved with use of interpolated contours. The saving in manpower time for contouring is expected to significantly improve the throughput of the IMRT planning process.     

Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model

Objective Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) may represent a treatment option for malignant brain tumors. We used a three-dimensional cell culture system, the C6 glioma spheroid model, to study acute effects of PDT and how they might be influenced by treatment conditions. Methods Spheroids were incubated for 4 h in 100 μg/ml ALA in 5% CO₂ in room air or 95% O₂ with subsequent irradiation using a diode laser (λ = 635 nm, 40 mW/cm², total fluence 25 J/cm²). Control groups were “laser only”, “ALA only”, and “no drug no light”. Annexin V-FITC, a marker used for detection of apoptosis, propidium iodide (PI), a marker for necrotic cells and H 33342, a chromatin stain, were used for morphological characterization of PDT effects by confocal laser scanning and fluorescence microscopy. Hematoxylin–eosin staining and TdT-FragEL (TUNEL) assay were used on cryosections. Growth kinetics were followed for 8 days after PDT. Results PDT after incubation in 5% CO₂ provided incomplete cell death and growth delay in spheroids of >350 μm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 μm). Incubation in 95% O₂ with subsequent PDT resulted in complete cell death and growth arrest regardless of spheroid size. In incompletely damaged spheroids viable cells were restricted to spheroid centers. The rate of cell death in all control groups was negligible. Cell death was accompanied by annexin/PI costaining, but there was also evidence for annexin V-FITC staining without PI uptake. Conclusions PDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O₂ concentration and spheroid size.     

KATP-channels in beta-cells in tissue slices are directly modulated by millimolar ATP

In pancreatic beta-cells, inhibition of K_ATP-channels plays a pivotal role in signal transduction of glucose-induced insulin release. However, the extreme sensitivity of K_ATP-channels to its ligand ATP as found in inside-out patches is not directly compatible with modulation of these channels at physiological [ATP]_i. We studied K_ATP-channel sensitivity to ATP in beta-cells in dispersed culture and in fresh pancreatic tissue slices. Physiological [ATP]_i blocks more than 99% of K_ATP-channels in cultured beta-cells, while only 90% in beta-cells in slices, indicating reduced sensitivity to ATP in the fresh slices. Applying cytosolic factors like ADP, phosphatidylinositol-4,5-bisphosphate (PIP₂) or oleoyl-CoA did not restore the K_ATP-channel sensitivity in cultured beta-cells. Our data suggest that interaction between SUR1 and Kir6.2 subunit of the K_ATP-channel could be a factor in sensitivity modulation. Tissue slices are the first beta-cell preparation to study direct K_ATP-channel modulation by physiological [ATP]_i.     

The Waterlow score for risk assessment in surgical patients

Introduction

Perioperative scoring systems aim to predict outcome following surgery and are used in preoperative counselling to guide management and to facilitate internal or external audit. The Waterlow score is used prospectively in many UK hospitals to stratify the risk of decubitus ulcer development. The primary aim of this study was to assess the potential value of this existing scoring system in the prediction of mortality and morbidity in a general surgical and vascular cohort.

Methods

A total of 101 consecutive moderate to high risk emergency and elective surgical patients were identified through a single institution database. The preoperative Waterlow score and outcome data pertaining to that admission were collected. The discriminatory power of the Waterlow score was compared against that of the American Society of Anesthesiologists (ASA) grade and the Portsmouth Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity (P-POSSUM).

Results

The inpatient mortality rate was 17% and the 30-day morbidity rate was 29%. A statistically significant association was demonstrated between the preoperative Waterlow score and inpatient mortality (p<0.0001) and 30-day morbidity (p=0.0002). Using a threshold Waterlow score of 20 to dichotomise risk, accuracies of 0.84 and 0.76 for prediction of mortality and morbidity were demonstrated. In comparison with P-POSSUM, the preoperative Waterlow score performed well on receiver operating characteristic analysis. With respect to mortality, the area under the curve was 0.81 (0.80–0.85) and for morbidity it was 0.72 (0.69–0.76). The ASA grade achieved a similar level of discrimination.

Conclusions

The Waterlow score is collected routinely by nursing staff in many hospitals and might therefore be an attractive means of predicting postoperative morbidity and mortality. It might also function to stratify perioperative risk for comparison of surgical outcome data. A prospective study comparing these risk prediction scores is required to support these findings.     

Update of endovenous laser therapy and the latest application studies

Endovenous laser therapy (ELT) has been applied in clinical practice as a therapy for truncal vein incompetence for about 10 years. One characteristic of ELT is the broad spectrum of different treatment protocols using a variety of laser systems and different forms of endovenous application. The principles behind the way ELT is carried out and the clinical results are described in detail in the following article.Despite good clinical results with effective, relatively pain-free occlusion of incompetent truncal veins, undesired side effects have been observed such as ecchymosis, phlebitis and recanalization. These can mainly be traced to thermal lesions in the vein wall concentrated in certain spots with transmural ablations of the tissue and perforations. In recent years systematic experimental investigations and the analysis of clinical results have increased understanding of the connection between endovenous laser application and clinical results. This has led to a continuous development and optimization of ELT. Particularly the use of longer wavelengths and radially irradiating optical fibers, together with endovenous laser irradiation with continuous pull-back of the optical fiber seem to have had a positive influence on the side effects. As a result ELT treatment is coming closer to the goal of standardizing an effective method for the treatment of varicose veins. Further controlled studies are required to compare optimized ELT treatment protocols with not only other endothermal modes of treatment but also with conventional open surgery.     

Hutchinson-Gilford progeria syndrome: oral and craniofacial phenotypes

Objective:  Hutchinson-Gilford progeria syndrome (HGPS) is a rare early-onset accelerated senescence syndrome. In HGPS, a recently identified de novo dominant mutation of the lamin A gene ( LMNA ) produces abnormal lamin A, resulting in compromised nuclear membrane integrity. Clinical features include sclerotic skin, cardiovascular and bone abnormalities, and marked growth retardation. Craniofacial features include 'bird-like' facies, alopecia, craniofacial disproportion, and dental crowding. Our prospective study describes dental, oral soft tissue, and craniofacial bone features in HGPS.
Methods:  Fifteen patients with confirmed p.G608G LMNA mutation (1–17 years, seven males, eight females) received comprehensive oral evaluations. Anomalies of oral soft tissue, gnathic bones, and dentition were identified.
Results:  Radiographic findings included hypodontia ( n  = 7), dysmorphic teeth ( n  = 5), steep mandibular angles ( n  = 11), and thin basal bone ( n  = 11). Soft tissue findings included ogival palatal arch ( n  = 8), median sagittal palatal fissure ( n  = 7), and ankyloglossia ( n  = 7). Calculated dental ages (9 months to 11 years 2 months) were significantly lower than chronological ages (1 year 6 months to 17 years 8 months) ( P  = 0.002). Eleven children manifested a shorter mandibular body, anterior/posterior cranial base and ramus, but a larger gonial angle, compared to age/gender/race norms.
Conclusion:  Novel oral-craniofacial phenotypes and quantification of previously reported features are presented. Our findings expand the HGPS phenotype and provide additional insight into the complex pathogenesis of HGPS.     

DLA-identical bone marrow grafts after low-dose total body irradiation: the effect of canine recombinant hematopoietic growth factors

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.