Aerosolized clindamycin is superior to aerosolized dexamethasone or clindamycin-dexamethasone combination in the treatment of severe Porphyromonas gingivalis aspiration pneumonia in an experimental murine model

Adjunctive corticosteroid treatment to reduce excessive local inflammatory response in pneumonia is controversial. To study the effects of an early local adjunct dexamethasone treatment on the course of pneumonia and inflammatory/cytokine response, mice were intratracheally inoculated with live Porphyromonas gingivalis and treated with either clindamycin (C), dexamethasone (D), C+D combination, or were not treated (Pg). Six mice from each group were euthanized at 6, 24, 72, and 168 hours after inoculation. Levels of tumor necrosis factor (TNF)-α, soluble TNF-α receptors (sTNFR1 and sTNFR2), interleukin (IL)-1β, and IL-6 in the serum and lung-homogenate supernatant were determined. Lung samples were histopathologically assessed and all findings compared to those found in 24 sham-inoculated mice (phosphate-buffered saline [PBS]). Severe P. gingivalis-induced bronchopneumonia progressed from 24 hours, peaked at 72 hours, and resolved after 168 hours with changes in local and systemic cytokine levels. Clindamycin-treated mice developed only mild bronchopneumonia that resolved fast (72 hours) with an early (6-24 hours) normalization of local and systemic cytokine levels. Similar course of pneumonia and cytokine level changes were observed in mice treated with C+D, but later. Early (6-24 hours) local elevation of sTNFRs was observed in C and C+D groups of mice, whereas nontreated (Pg) mice had increased systemic sTNFRs. Severe bronchopneumonia with delayed resolution was observed in D-group mice, with an early local and systemic decrease in sTNFR1 and persistent elevation of local TNF-α. Clindamycin or a clindamycin-dexamethasone combination treatment significantly improves the course of P. gingivalis-aspiration pneumonia, but more so if clindamycin alone is used. A favorable course of pneumonia seems to be associated with an early elevation of sTNFRs and normalization of TNF-α.     

Comparison of paper-based and electronic data collection process in clinical trials: Costs simulation study

An alternative to clinical trial paper-based data collection (PDC) is internet based electronic data collection (EDC), where the investigators over the internet enter data directly in the electronic database by themselves. In our study we considered clinical trial as a business process. Our objective was to model PDC and EDC process and to estimate the difference of the costs of PDC and EDC process for a sample clinical trial based on these models.We used Extended Event-driven Process Chains (eEPC) modeling technique to model PDC and EDC process. In order to evaluate the costs of the processes we assigned costs functions to each process function which appears in the model. The parameters which appear in these functions include efforts, staff prices and data quality parameters. We estimated the values of all these parameters and performed costs calculations for a sample clinical trial.Through an analysis and modeling efforts we identified sub-processes which contain main differences affecting duration and costs of the PDC and EDC process: data gathering at the research center; monitoring; and data management. The most significant model difference between PDC and EDC process appeared in data management sub-process. For the sample clinical trial considered in our simulation study and our parameters estimations the EDC process decreased data collection costs for 55%. For different scenarios of parameters variations we show that the EDC process may bring from 49% to 62% of savings when compared to PDC process.     

Perturbation of blood flow as a mechanism of anti-tumour action of direct current electrotherapy

Anti-tumour effects of direct current electrotherapy are attributed to different mechanisms depending on the electrode configuration and on the parameters of electric current. The effects mostly arise from the electrochemical products of electrolysis. Direct toxicity of these products to tumour tissue is, however, not a plausible explanation for the observed tumour growth retardation in the case when the electrodes are placed into healthy tissue surrounding the tumour and not into the tumour itself. The hypothesis that the anti-tumour effectiveness of electrotherapy could result from disturbed blood flow in tumours was tested by the measurement of changes in blood perfusion and oxygenation in tumours with three different methods (in vivo tissue staining with Patent Blue Violet dye, polarographic oximetry, near-infrared spectroscopy). The effects induced by electrotherapy were evaluated in two experimental tumour models: Sa-1 fibrosarcoma in A/J mice and LPB fibrosarcoma in C57B1/6 mice. We found that perfusion and oxygenation were significantly decreased after electrotherapy. Good agreement between the results of different methods was observed. The effect of electrotherapy on local perfusion of tumours is probably the prevalent mechanism of anti-tumour action for the particular type of electrotherapy used in the study. The importance of this effect should be considered for the optimization of electrotherapy protocols in experimental and clinical trials. The non-invasive technique of near-infrared spectroscopy proved to be a reliable method for detecting perfusion and oxygenation changes in small solid tumours.     

Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

Background

Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer.

Materials and methods

A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment.

Results

Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected.

Conclusions

We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation.     

Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE)

Background

Irreversible electroporation (IRE) is a tissue ablation method, which relies on the phenomenon of electroporation. When cells are exposed to a sufficiently electric field, the plasma membrane is disrupted and cells undergo an apoptotic or necrotic cell death. Although heating effects are known IRE is considered as non-thermal ablation technique and is currently applied to treat tumors in locations where thermal ablation techniques are contraindicated.

Materials and methods.

The manufacturer of the only commercially available pulse generator for IRE recommends a voltage-to-distance ratio of 1500 to 1700 V/cm for treating tumors in the liver. However, major blood vessels can influence the electric field distribution. We present a method for treatment planning of IRE which takes the influence of blood vessels on the electric field into account; this is illustrated on a treatment of 48-year-old patient with a metastasis near the remaining hepatic vein after a right side hemi-hepatectomy.

Results

Output of the numerical treatment planning method shows that a 19.9 cm3 irreversible electroporation lesion was generated and the whole tumor was covered with at least 900 V/cm. This compares well with the volume of the hypodense lesion seen in contrast enhanced CT images taken after the IRE treatment. A significant temperature raise occurs near the electrodes. However, the hepatic vein remains open after the treatment without evidence of tumor recurrence after 6 months.

Conclusions

Treatment planning using accurate computer models was recognized as important for electrochemotherapy and irreversible electroporation. An important finding of this study was, that the surface of the electrodes heat up significantly. Therefore the clinical user should generally avoid placing the electrodes less than 4 mm away from risk structures when following recommendations of the manufacturer.     

Nanosecond Electric Pulses are Equally Effective in Electrochemotherapy with Cisplatin as Microsecond Pulses

BackgroundNanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 μs pulses and to investigate the effects of electric pulses on cisplatin structure.Materials and methodsChinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.ResultsNanosecond pulses equivalent to 8 × 100 μs pulses were established in vitro based on membrane permeabilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and accumulating cisplatin intracellularly as 8 × 100 μs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin.ConclusionsEquivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 μs pulses.Key words: electroporation, electrochemotherapy, nanosecond pulses, cisplatin     

Laser Welding of Ti6Al4V Titanium Alloy in Air and a Water Medium

Ti6Al4V titanium alloys are widely used in a variety of scientific and industrial fields. Laser beam welding is one of the most effective techniques for the joining of titanium plates. The main objective of this study was to investigate the influence of the most important laser parameters on welding performance of titanium alloy in two different physical environments such as air and water (i.e., serum) media. Specifically, the laser beam welding of 2 mm thick Ti6Al4V samples was applied using an Nd:YAG laser in open-air welding using argon as a shielding gas, and in wet welding using a serum environment. The deepest penetration was achieved at −3 mm focal position with 11 J of laser energy in both investigated media (i.e., air and serum). The maximum hardness (1130 HV) was achieved for the focal position of −4 mm in serum medium while it was 795 HV for a focal position of −5 mm in air medium. The minimum (1200 μm and 800 μm) and maximum (1960 μm and 1900 μm) weld widths were observed for air and serum medium, respectively. After the welding process, martensite, massif martensite, and transformed martensite were observed in the microstructure of Ti6Al4V. To the best of our knowledge, the underwater wet welding of titanium alloy was carried out and reported for the first time in this study.     

Prognostic factors in the prediction of chronic wound healing by electrical stimulation

The aim of the study is to determine the effects of wound, patient and treatment attributes on the wound healing rate and to propose a system for wound healing rate prediction. Predicting the wound healing rate from the initial wound, patient and treatment data collected in a database of 300 chronic wounds is not possible. After considering weekly follow-ups, it was determined that the best prognostic factors are weekly follow-ups of the wound healing process, which alone were found to predict accurately the wound healing rate after a minimum follow-up period of four weeks (at least five measurements of wound area). After combining the follow-ups with wound, patient and treatment attributes, the minimum follow-up period was reduced to two weeks (at least three measurements of wound area). After a follow-up period of two weeks, it was possible to predict the wound healing rate of an independent test set of chronic wounds with a relative squared error of 0.347, and after three weeks, with a relative squared error of 0.181 (using regression trees with linear equations in its leaves). Regression trees with a relative squared error close to 0 produce better prediction than with an error closer to 1. Results show that the type of treatment is just one of many prognostic factors. Arranged in order of decreasing prediction capability, prognostic factors are: wound size, patient's age, elapsed time from wound appearance to the beginning of the treatment, width-to-length ratio, location and type of treatment. The data collected support former findings that the biphasic- and direct-current stimulation contributes to faster healing of chronic wounds. The model of wound healing dynamics aids the prediction of chronic wound healing rate, and hence helps with the formulation of appropriate treatment decisions.     

Hereditary medullary thyroid cancer in Slovenia – genotype-phenotype correlations

BACKGROUND: Medullary thyroid cancer (MTC) is a rare endocrine tumor that may be sporadic or inherited in settings of MEN2A, MEN2B and FMTC. Germline point mutations in the RET proto-oncogene are responsible for tumor occurrence, inheritance and great clinical variability. The aim of this study was to correlate the genotype and phenotype of patients with hereditary MTC (age at diagnosis, sex, TNM classification and clinical features). PATIENTS: Between 1997 and 2003 genetic testing was performed in 69 out of 98 patients with "sporadic" MTC. Carriage of mutation was found in 14 (20.2%) patients (index patients) and in 16 out of 31 (51.6%) of their relatives. One patient with MEN2B and codon 918 mutation was excluded from further analysis. METHODS: Genomic DNA was isolated from peripheral blood leukocytes. Exons 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene were amplified in polymerase chain reactions. Point mutations of the RET gene were detected with single-strand conformation analysis and DNA sequencing. Detected mutations were confirmed with restriction enzyme analysis. RESULTS: Codon 634 mutations were detected in 15 patients (50%; aged 18-76 years; 6 families), codon 618 in nine patients (30%; aged 12-65 years; 4 families) and codon 790 in five patients (16.6%; aged 16-74 years; 3 families). The median age at diagnosis was 31 +/- 17.3, 33 +/- 15.9 and 36 +/- 23.8 years for patients with codon 618, 634 and 790 mutations. Selected by sex, females with codon mutations 618 and 634 versus 790 had median age at diagnosis of 34.5 +/- 15.6 years and 43.5 +/- 22.9 years, whereas the inverse result was observed in males (26.5 +/- 18.0 versus 16 years). The male/female ratio was 1:2 for patients with codon 618 and 634 mutations and 1:4 for patients with codon 790 mutations. Some of the data suggested correlation between specific genotypes, tumor size, stage of MTC and age at diagnosis. Pheochromocytoma (12 out of 15 patients) and primary hyperparathyroidism (6 out of 15 patients) were diagnosed solely in patients with codon 634 mutations. One patient with FMTC and Hirschprung disease was found in a family with codon 618 mutations. CONCLUSION: Correlation between tumor size, stage of MTC at diagnosis in view of patient's age, and specific genotype were indicated in our limited series and were more evident in female patients with codon 790 mutations. Later onset and a probably less aggressive course of MTC in these patients than in patients with other mutations should be considered in planning prophylactic thyroid surgery. MEN2A syndrome was related solely to codon 634 mutations.