Optimization of cardiac resynchronization therapy based on speckle tracking.

OBJECTIVES: The aim of this study was to evaluate the correlation between the change in heart strains and the success rate of Cardiac Resynchronization Therapy (CRT) optimization. We further explored the benefit of speckle tracking for CRT. METHODS: In this prospective cohort study, CRT-Ds were implanted to 60 patients. 3 months later, the response was evaluated. In the non-responders, optimization based on speckle tracking was performed. The AV interval was optimized with respect to the quality of left ventricle filling and the VV interval was optimized with respect to heart strains. After a further three months, the optimization success was evaluated. RESULTS: Thirty-nine patients responded well to the initial CRT. The response was independent of etiology; the subsequent optimization was however more successful in dilated cardiomyopathy (DCM) (8 out of 9) than in ischemic heart disease (IHD) patients (3 out of 10 responded). The ejection fraction increase and area strain were the best predictors of NYHA improvement. CONCLUSION: AV and VV optimization in patients who do not respond well to initial CRT seems to have better results in patients suffering from DCM. Speckle tracking (specifically A-strain) may be used to guide CRT optimization (Tab. 2, Fig. 3, Ref. 22).

Ankle-brachial index in diabetic patients - which upper cut-off value is to be used?

OBJECTIVES: In diabetic patients, there is a discrepancy in guidelines for ankle-brachial index (ABI) screening for peripheral arterial disease (PAD). While diabetes organizations suggest the value of upper limit of normal ABI to be 1.3, cardiologists recommend 1.4. Also, guidelines recommend using the higher value of ankle pressure (HAP) but multiple recent studies propose the opposite (LAP). METHODS: In this prospective study, we performed ABI measurements in 62 diabetic patients. Results were calculated by comparing higher and lower values of ankle pressure to those of duplex ultrasound (stenosis ≥ 50 % was considered PAD). Special attention was paid to patients with high and non-measurable ABI. RESULTS: LAP ABI appears to be a preferable method for PAD screening in diabetics. The upper cut-off value of 1.4 yielded better results with sensitivity of 93 % and negative predictive value of 91 %. No limbs with ABI between 1.3 and 1.4 with significant stenosis were found. However, using HAP for the upper cut-off captured additional PAD patients. PAD was abundant among patients with high or non-measurable ABI. CONCLUSIONS: LAP should be used for assessing low ABI (cut-off 0.9) while HAP for detecting the abnormally high ABI. The preferable high ABI cut-off is 1.4. Condition with abnormally high or non-measurable ABI should be considered as PAD (Tab. 3, Ref. 22).

[An effect of continuous and intermittent renal replacement therapy on antibiotic treatment in critically ill patients with sepsis - a practice-based perspective of vancomycin and gentamycin therapies]. / Vliv kontinuální a intermitentní náhrady renálních funkcí na antibiotickou lécbu u kriticky nemocných v sepsi - praktický pohled na lécbu vankomycinem a gentamicinem.

Sepsis and septic shock are common cause of hospitalisation in intensive care unit. Acute kidney injury is an accompanying manifestation of sepsis/septic shock leading to worsening of morbidity and also mortality and requiring use of intermittent or continual renal replacement therapy. Life saving effect is attributed to early and effective antibiotic therapy. Therapeutic drug monitoring and do-sage adjustment is important for successful treatment. Despite therapeutic drug monitoring of both antibiotic agents vankomycin and gentamicin the treatment still rises many questions about the convenient use in septic patients due to their nephrotoxicity.

[Primary hepatic neuroendocrine carcinoma]. / Primární neuroendokrinní karcinom jater.

UNLABELLED: Primary neuroendocrine carcinoma of the liver is a rare tumour, probably arising from scattered neuroendocrine cells of the bile duct. We present the case of a 72-year-old male who experienced gradual weight loss and diarrhoea. Given the fact that he had stayed in the Dominican Republic, a parasitic disease was initially suspected. However, this was not confirmed. Further examination showed tumour infiltration of the liver. Fine needle aspiration cytology of the tumour site was performed. The diagnostic procedure revealed neuroendocrine carcinoma. The tumour cells expressed the following neuroendocrine markers (chromogranin, synaptophysin, CD56 and NSE) as well as the epithelial marker AE1-AE3. The tumour was considered metastasis of the primary tumour located in the gastrointestinal tract. A thorough clinical examination was performed including gastroscopy, colonoscopy, In-111 Octreoscan scintigraphy, computed tomography and magnetic resonance imaging. These methods revealed metastases in the vertebrae, pelvis, long bones and skull. No other tumour sites were found in the lungs, gastrointestinal tract or pancreas. The patient became increasingly cachexic and later died. An autopsy showed massive multicentric tumour infiltration of the liver. Histological examination revealed well differentiated neuroendocrine carcinoma which transformed into intermediate and small cells. The autopsy found no tumour sites in the gastrointestinal tract, lungs or pancreas. The results were suggestive of primary neuroendocrine carcinoma of the liver. KEYWORDS: neuroendocrine carcinoma - liver - primary tumour.

[Diagnostic algorithm of syncope: integrative approach]. / Diagnostický algoritmus synkop: integrativní prístup.

Syncope is a symptom, defined as transient loss of consciousness and postural tone with spontaneous and mostly prompt recovery. At first it is necessary to differentiate other non-syncopal transient loss of consciousness and simple falls, where thorough history taking is pivotal. EGSYS and OESIL risk scores seem to be contributional in initial risk stratification, however they are neither widely accepted nor a part of national guidelines. They are part of the European society of cardiology guidelines, though. Next it is essential expert ECG evaluation, thorough physical status examination, supine and standing blood pressure measurement and carotid sinus massage, if not contraindicated. Successively one has to decide if hospitalization or outpatient management is more suitable. Recently it has been shown, that so-called syncope management units (aimed for short-term hospitalization or fast outpatient examination, including vital function monitoring, echocardiography and facile cathlab access) are effective in fast syncope evaluation. Echocardiography, ECG monitoring and head-up tilt test should be a part of complex diagnostic evaluation. If syncope is not clarified by upon stated methods moreover syncope is recurrent, electrophysiological study, ILR implantation or both are justified. Despite of entire health practitioner's effort, more than 1/3 of syncopes remain unexplained.

[Primary hepatic carcinoid]. / Primární jaterní karcinoid.

Primary hepatic carcinoid (PHC) is considered to be particularly sporadic diagnosis; in current world literature about 60 cases have been reported. Most of the patients present with abdominal pain, diarrhea, icterus, flush, weight loss or respiratory disease; even though the course of the disease might stay asymptomatic for a long time. An illuminating case of at presentation oligosymptomatic 72-year-old patient, which was after extensive examination based on OctreoScan and histological verification diagnosed to have generalized PHC, is reported. Paliative therapy with somatostatine analogues followed. At autopsy 8 months later the clinical diagnosis of PHC was confirmed. PHC is difficult to diagnose both due to radiological similarity to other hepatic lesions and demanding exclusion of other primary foci. The diagnosis of PHC is based on negative imaging techniques result in other possible localizations. Minimal symptomatology in stage of generalization, atypical primary localization and rapid progression is of interest in current case.

[Head-up tilt test--do we really know all about it?]. / Polohový test--víme o nem opravdu vsechno?

Head-up tilt test (HUTT) falls within the competence of functional cardiological examination. Most often it is being used for the diagnostical approach to possibly neurally-mediated syncope, less is known about the possibility of using tilt table test in the treatment of vasovagal syncope. The main goal of this article is to make clear the principles, appropriate indication, performance and subsumption of HUTT in the algorithm of syncope investigation. Moreover it will be marginally mentioned how HUTT can be used in predicting cardiovascular risk of patients with coronary artery disease.

Flow-cytometric monitoring of mitochondrial depolarisation: from fluorescence intensities to millivolts.

Redistribution potentiometric dyes represent a powerful tool for monitoring membrane potential of mitochondria, especially when these dyes are used with flow cytometry. In particular, tetramethylrhodamine methyl ester proved to be suitable for the screening of mitochondrial membrane potential in cultured human skin fibroblasts from patients suffering from different defects of oxidative phosphorylation. We have developed a method that makes it possible to measure the changes in mitochondrial membrane potential, or to assess the differences between respective mitochondrial membrane potentials in investigated cells and controls in the absolute scale of millivolts. Our approach employs the fact that a logarithmic transformation of Nernst equation-controlled intensity of fluorescence from potentiometric dyes accumulated in mitochondria leads to a linear scale for mitochondrial membrane potentials.

Pseudo real-time method for monitoring of the limiting anisotropy in membranes.

Data acquisition and analysis of the time-resolved fluorescence anisotropy is typically a time consuming process preventing usage of this experimental method for monitoring of time-dependent phenomena. We describe a method for pseudo real-time monitoring of the limiting fluorescence anisotropy r(infinity) allowing to track changes of the membrane order occurring on the time scale of minutes. Principle and performance of the method is demonstrated in the time domain with the time-correlated single photon counting detection. DMPC liposomes stained with 1,6-diphenyl-1,3,5-hexatriene (DPH) have been used to test influence of the diffusion membrane potential on the membrane order during the temperature-induced phase transition in DMPC membranes. It has been found that the transmembrane field of the order of -70 mV increases the phase transition temperature by about 1.5 degrees C-2 degrees C. It is proposed that the full advantage of the method can be utilized with a gated detection, which besides a faster data acquisition brings additional advantage of excitation light suppression. The method can be also used for imaging.

Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase.

An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.

Limitations in linearized analyses of binding equilibria: binding of TNP-ATP to the H4-H5 loop of Na/K-ATPase.

Binding of TNP-ATP [2',3'- O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, a fluorescent analogue of ATP] to the K605 protein was studied. This is an isolated N-domain in the cytoplasmic loop of the Na/K-ATPase alpha-subunit, lying between membrane-spanning segments 4 and 5 (sequence L(354)-I(604)). A titration equation is derived that explicitly makes it possible to relate the fluorescence of TNP-ATP and K605 solutions to total probe concentration in the sample. Using this, it is possible to obtain the value of the dissociation constant from the titration experiment without resorting to the Scatchard plot, which is far from optimal from the statistical point of view. Using the new formula with non-linear regression analysis, a value of the dissociation constant K(D) for TNP-ATP binding to the K605 protein of 3.03 +/- 0.28 microM at 22 degrees C was obtained. A series of fits to simulated data with added noise demonstrated clearly the advantage of non-linear regression using the new formula over the commonly employed linear regression using the Scatchard plot. The procedure presented is generally applicable to binding assays using changes in the fluorescence of ligands on binding.

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C(3)(3) in yeast.

The redistribution fluorescent dye diS-C(3)(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by Delta psi-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambda(max) and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Delta psi-independent fluorescence responses that may mimic Delta psi-dependent blue or red shift in lambda(max). These lambda(max) shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced Delta psi collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambda(max)) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the Delta psi collapse causes a blue lambda(max) shift mimicking an apparent depolarisation.

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells.

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts.

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.

Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae.

Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

Measurement of membrane potential in Saccharomyces cerevisiae by the electrochromic probe di-4-ANEPPS: effect of intracellular probe distribution.

Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane. The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time. The presence and properties of the cell wall affected the extent of cell staining. The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy. The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min. Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

[Is homeopathic therapy more effective that placebos?]. / Je homeopatické lécení úcinnejsí nez placebo?

A critical report is presented on the reliability of two clinical trials by Reilly et al. (Lancet, 1986ii, pp. 881-886 and 1994ii, pp. 1601-1606) who has claimed that the effect of homeopathy differs significantly from placebo. Biophysical hypothesis dealing with the intriguing problem of the mechanism of action of homeopathic remedies are also reviewed in this paper.

Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis.

Basic tenets related to the use of three main classes of potentiometric redistribution fluorescent dyes (carbocyanines, oxonols, and rhodamines) are discussed in detail. They include the structure/function relationship, formation of nonfluorescent (H-type) and fluorescent (J-type) dimers and higher aggregates, probe partitioning between membranes and medium and binding to membranes and intracellular components (with attendant changes in absorption and emission spectra, fluorescence quantum yield and lifetime). The crucial importance of suitable probe-to-cell concentration ratio and selection of optimum monitored fluorescence wavelength is illustrated in schematic diagrams and possible artifacts or puzzling results stemming from faulty experimental protocol are pointed out. Special attention is paid to procedures used for probe-response calibration (potential clamping by potassium in the presence of valinomycin, use of gramicidin D in combination with N-methylglucamine, activation of Ca-dependent K-channels by A23187, the null-point technique). Among other problems treated are dye toxicity, interaction with mitochondria and other organelles, and possible effects of intracellular pH and the quantity of cytosolic proteins and/or RNA on probe response. Individual techniques using redistribution dyes (fluorescence measurements in cuvettes, flow cytometry and microfluorimetry of individual cells including fluorescence confocal microscopy) are discussed in terms of reliability, limitations and drawbacks, and selection of suitable probes. Up-to-date examples of application of slow dyes illustrate the broad range of problems in which these probes can be used.

Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.

The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.