Quercetin protects cardiomyoblasts against hypertonic cytotoxicity by abolishing intracellular Ca2+ elevations and mitochondrial depolarisation.

BACKGROUND AND AIM: Osmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. EXPERIMENTAL PROCEDURE: Protective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. KEY RESULTS: Quercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. CONCLUSIONS AND IMPLICATIONS: Quercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.

Deterministic proton dose calculation from first principles.

The purpose of this paper is to find a deterministic pencil beam algorithm that computes, from first principles, the dose in some region of interest when a known heterogeneous terrain is irradiated by known proton beams. The terrain is discretized into slabs perpendicular to the nominal beam direction. Pencil beams (PBs) are transported through each slab using generalized Fermi-Eyges theory with the correct stopping and scattering powers for each material (no water-like assumption). At transverse heterogeneities PBs are divided into smaller ones using either redefinition (all PBs divided) or dynamic splitting (PBs divided one at a time as needed). Dynamic splitting recurs until the transverse heterogeneity is sufficiently well resolved. After a PB is transported, its dose contribution at the slab exit (if designated as a measuring plane) is added to each point of interest. The calculation depends solely on accurate descriptions of the terrain and of the incident beams. It does not require measurements such as effective source size and position. These follow from the beam line specifications. The calculation ignores nuclear reactions (hard scatters), as do other PB algorithms. Our first comparison with experiment confronts the widely used 'Hong' algorithm. The second is a simple study of collimator scatter and the third studies collimator scatter under more extreme conditions. All agree well with the high dose region. The latter two agree substantially with collimator scatter and thickness effects, ignored by conventional algorithms. The algorithm gives a good account of the high dose region, including realistic collimator effects. It is equally amenable to passive beam spreading or pencil beam scanning (PBS). It dispenses with measurements (commissioning) of beam line properties. In PBS it promises to be faster than conventional algorithms since virtual PBs are generated only as needed. These results justify further work towards a full-fledged therapy dose engine.

Water equivalent path length measurement in proton radiotherapy using time resolved diode dosimetry.

PURPOSE: To verify water equivalent path length (WEPL) before treatment in proton radiotherapy using time resolved in vivo diode dosimetry. METHODS: Using a passively scattered range modulated proton beam, the output of a diode driving a fast current-to-voltage amplifier is recorded at a number of depths in a water tank. At each depth, a burst of overlapping single proton pulses is observed. The rms duration of the burst is computed and the resulting data set is fitted with a cubic polynomial. RESULTS: When the diode is subsequently set to an arbitrary depth and the polynomial is used as a calibration curve, the "unknown" depth is determined within 0.3 mm rms. CONCLUSIONS: A diode or a diode array, placed (for instance) in the rectum in conjunction with a rectal balloon, can potentially determine the WEPL at that point, just prior to treatment, with submillimeter accuracy, allowing the beam energy to be adjusted. The associated unwanted dose is about 0.2% of a typical single fraction treatment dose.

Test of GEANT3 and GEANT4 nuclear models for 160 MeV protons stopping in CH2.

Monte Carlo simulations are used for many problems in proton radiation therapy, some of which are sensitive to the nuclear interaction model. The available models have been little tested in the regime of interest, namely in their ability to predict the secondary particle yield, including their angle and energy, when 70-250 MeV protons stop in various materials. The present study provides one such test in carbon, complementing a previous one in copper. Using a multilayer Faraday cup we have measured the projected range distribution of charged nuclear secondaries from 160 MeV protons stopping in polyethylene (CH2). To test the popular GEANT Monte Carlo we have simulated the experiment with GEANT3 using the "Gheisha" (default) and "Fluka" models and with GEANT4.5 using the "low-energy" and "precompound" models. The GEANT3/Fluka and GEANT4/precompound simulations agree moderately well with the observed range distribution. The data are given in a convenient form for testing other Monte Carlo programs.

Identification and characterization of a novel secreted immunoglobulin binding protein from group A streptococcus.

Immunoglobulin binding proteins are one of several pathogenicity factors which have been associated with invasive disease caused by group A streptococci. The surface-bound M and M-like proteins of Streptococcus pyogenes are the most characterized of these immunoglobulin binding proteins, and in most cases they bind only a single antibody class. Here we report the identification of a novel non-M-type secreted protein, designated SibA (for secreted immunoglobulin binding protein from group A streptococcus), which binds all immunoglobulin G (IgG) subclasses, the Fc and Fab fragments, and also IgA and IgM. SibA has no significant sequence homology to any M-related proteins, is not found in the vir regulon, and contains none of the characteristic M-protein regions, such as the A or C repeats. Like M proteins, however, SibA does have relatively high levels of alanine, lysine, glutamic acid, leucine, and glycine. SibA and M proteins also share an alpha-helical N-terminal secondary structure which has been previously implicated in immunoglobulin binding in M proteins. Evidence presented here indicates that this is also the case for SibA. SibA also has regions of local similarity with other coiled-coil proteins such as Listeria monocytogenes P45 autolysin, human myosin heavy chain, macrogolgin, and Schistoma mansoni paramyosin, some of which are of potential significance since cross-reactive antibodies between myosin proteins and M proteins have been implicated in the development of the autoimmune sequelae of streptococcal disease.

Identification and molecular analysis of PcsB, a protein required for cell wall separation of group B streptococcus.

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated pcsB, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (M(r), 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from Listeria monocytogenes. Northern blot analysis revealed a monocistronic transcriptional organization for pcsB in GBS. Insertional inactivation of pcsB in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.

Nuclear interactions of 160 MeV protons stopping in copper: a test of Monte Carlo nuclear models.

To estimate the influence of nuclear interactions on dose or biological effect one uses Monte Carlo programs which include nuclear models. We introduce an experimental method to check these models at proton therapy energies. We have measured the distribution of charge deposited by 160 MeV protons stopping in a stack of insulated copper plates. A buildup region ahead of the main peak contains approximately 20% of the total charge and is entirely due to charged secondaries from inelastic nuclear interactions. The acceptance for charged secondaries is 100%. Therefore the data are a good benchmark for nuclear models. We have simulated the stack using GEANT with two nuclear models. FLUKA agrees fairly well with the measurement but GHEISHA, designed for much higher energies, does not. The experimental method will work for many other materials, including insulators. Therefore it can also be used for light nuclei.

A pencil beam algorithm for proton dose calculations.

The sharp lateral penumbra and the rapid fall-off of dose at the end of range of a proton beam are among the major advantages of proton radiation therapy. These beam characteristics depend on the position and characteristics of upstream beam-modifying devices such as apertures and compensating boluses. The extent of separation, if any, between these beam-modifying devices and the patient is particularly critical in this respect. We have developed a pencil beam algorithm for proton dose calculations which takes accurate account of the effects of materials upstream of the patient and of the air gap between them and the patient. The model includes a new approach to picking the locations of the pencil beams so as to more accurately model the penumbra and to more effectively account for the multiple-scattering effects of the media around the point of interest. We also present a faster broad-beam version of the algorithm which gives a reasonably accurate penumbra. Predictions of the algorithm and results from experiments performed in a large-field proton beam are presented. In general the algorithm agrees well with the measurements.

Physical specifications of clinical proton beams from a synchrotron.

Tumor treatment with charged particle beams is a quickly developing field aimed to translate the potential advantages offered by the superior physical dose distribution and relative biological effectiveness of heavy charged particles into a real improvement of tumor therapy. To this purpose the new proton and light-ion radiation therapy facilities must be designed according to strict clinical specifications to provide a reliable and effective tool against cancer. This paper provides the performance specifications of the accelerator and of the beam transport and delivery systems of the Italian Hadrontherapy Centre, which should be satisfied to meet the clinical specifications. A discussion is given on the requirements on energy range, energy variability, beam intensity, lateral penumbra, distal dose falloff, source-to-surface distance, time structure of the extracted beam, raster scanning system specifications, and beam abort time. Though the physical specifications are given for a particular accelerator, they can be used as a general guideline for the design of future biomedical particle accelerator facilities.

Subtle speech and motor deficits of children with congenital hypothyroid treated early.

This study surveyed the development and functioning of a group of 16 children with congenital hypothyroidism who had been followed closely since treatment was instituted at an average age of 15.6 days. This group of early-treated young children had no deficits in cognitive or adaptive functioning. Some isolated motor deficits were found, although results of the Finger-tapping and Marching subtests of the Reitan-Indiana battery did not replicate the New England Congenital Hypothyroid Collaborative (1985) finding of impaired performance. Speech deficits were documented in some. Congenitally hypothyroid children with delayed neonatal bone-age performed more poorly on most measures than those whose bone-age had been normal at birth.

[Ultrasound changes of the pancreas in patients with mucoviscidosis]. / Sonographische Befunde am Pankreas bei Patienten mit Mukoviszidose.

Pancreas sonography was performed on 171 patients with cystic fibrosis. The pancreas was visualized in 138 patients (81%) and could not be visualized in 33. 125 patients (90%) had abnormal echogenicity of the pancreas. Cysts were found in 5 patients. Two patients showed a dilatation of the pancreatic duct. The pancreas was abnormal in 98% of all patients aged more than 6 years.

Increased efficacy of radiation therapy by use of proton beam.

Proton beam treatment techniques provide a powerful approach to improving dose distribution (decrease treatment volume towards target volume) and hence increasing dose to target with resultant higher tumor control rates and lesser morbity. To achieve these dose distributions in patients requires use of modern imaging techniques, rigid immobilization systems, confirmation of target position vis a vis the proton beam at each treatment session, treatment planning which feature beam's eye view, displays of uncertainty, dose at each anatomic point, boli based on accurate assessment of density along each pixel, etc. Experience at MGH/MEEI/HCL has yielded a disease-free survival of 78% for patients with chordoma/chondrosarcoma of base of skull. Local control is achieved by 98% of patients treated for choroidal melanoma.

[Comparison of 2 pancreatic enzyme preparations in the treatment of digestive insufficiency in mucoviscidosis (cystic fibrosis)]. / Vergleich von zwei Pankreasenzym-Präparaten zur Behandlung der Verdauungsinsuffizienz bei Mukoviszidose (zystische Fibrose).

In an open trial 10 patients with cystic fibrosis were treated with two acid-protected pancreatic enzyme preparations formed as microtablets or pellets. The difference between Panzytrat 20,000 and Kreon is that with only 225 mg pancreatin, the former has twice the lipase activity of the latter. Therefore, the patients who had been taken Kreon were given only half the number of Panzytrat 20,000 capsules. There were no significant differences seen between the amounts of fecal fat nor in the fecal weight. In our study the fat absorption coefficient was somewhat too low with 67.4% for Kreon and 71.3% for Panzytrat 20,000 because of too low enzyme dosage, which was based only on an improvement of the clinical symptoms. Therefore, we would recommend a higher dose of 1000-1500 units of lipase/l g of dietary fat ingested. This requires the use of a preparation with high enzyme activity.

[Antibiotic pharmacokinetics in patients with mucoviscidosis]. / Antibiotika-Pharmakokinetik bei Patienten mit Mukoviszidose.

In this review we summarize the available literature on the pharmacokinetics of antibacterials in cystic fibrosis. A special impact is given on the results of our group which will be put in perspective with the results of other authors. The homogeneity of our patient population allows a valid comparison between patient and volunteer data. We do not confirm the previously suggested strongly enhanced elimination of antibacterials in CF. Our findings have recently been confirmed by other investigators. However, since in the clinical situation a more heterogeneous group of patients is treated it seems rational to increase the dose of the antibacterials by about 20-30%.

[Sonographic findings in the gallbladder in mucoviscidosis patients]. / Sonographische Befunde der Gallenblase bei Patienten mit Mukoviszidose.

The improved survival time of patients with mucoviscidosis has revealed some new complications. 137 patients were studied by sonography. The findings in the gall bladder have been compared with liver echogenicity. A micro-gall bladder was found in 37 patients (27%). 74 patients had a normal gall bladder (54%). Concretions were found in 22 (16%) of patients. 10 out of the 22 patients with concretions showed sonographic changes in the liver. In view of the frequent abdominal symptoms in patients with mucoviscidosis, sonography should be used routinely as a non-invasive method of investigation.

High dose treatment with antibiotics in cystic fibrosis--a reappraisal with special reference to the pharmacokinetics of beta-lactams and new fluoroquinolones in adult CF-patients.

In this review we analyzed the pharmacokinetic basis for high dose treatment with antibiotics of patients with cystic fibrosis. Both our results and those from other well designed pharmacokinetic studies do not support the view that low blood levels of antibacterials are a common feature of CF. We were unable to detect a decrease in absorption, nor could we find evidence for enhanced elimination of antibacterials in CF. Both these factors have been considered responsible for reducing the plasma (and tissue) levels of antibiotics. Most recent studies on kidney function are in agreement with these findings, since neither inulin nor creatinine clearance differ between CF-patients and healthy volunteers. In contrast to previous discussion, the volume of distribution (Vdss) was not elevated for any compound. The rational of weight correction of volume terms like Vdss or total clearance has never been clearly demonstrated and should therefore not be used without prior proof of relevance. Since the variability of pharmacokinetic parameters of antibiotics in CF-patients may be considerable, we suggest that a dose increase of 20-30% may be justified, but cannot agree with two to fourfold increases in dosage as previously proposed and applied in many CF-centers. Until more findings become available for non-adult CF-patients, these conclusions are only valid for adult CF-patients.

Bronchial secretions and bronchial mucosa in children with cystic fibrosis: comparison of bronchoscopic, biochemical, bacteriological, microscopic and ultrastructural findings.

In children (mean age 12.1 +/- 2.9 years) with cystic fibrosis, 44 bronchoscopic examinations were done under general anaesthesia with muscle relaxation using a Friedel type ventilation bronchoscope. The endoscopic picture of the mucous membranes was compared with the state of the bronchial secretions, its bacteriologic findings and content of acid mucopolysaccharides and DNA fibres (semiquantitative estimations). In all patients biopsy of the mucous membrane (central part of the bronchial tree) was performed for light and electron microscopy. The degree of reddening, swelling of the mucous membrane and hypersecretion was in some agreement with the intensity of the cellular infiltration and the production of pus (microscopic investigation). Secondary ultrastructural changes were detected in nearly all children, consisting of cellular oedema, swelling of mitochondria, dilatation of the endoplasmatic reticulum, protrusion of cells and fusion of cilia, enlarged intercellular spaces, thickening of the epithelial basal membrane, increased number of goblet cells, microtubular abnormalities of the cilia, lesions of the apical cell membrane with loss of cilia and microvilli. These ultrastructural changes were not correlated with the above-mentioned signs of inflammation.