Prognostic significance of clinical parameters and Epstein-Barr virus infection in non-endemic undifferentiated carcinoma of nasopharyngeal type: a Serbian report.

Undifferentiated carcinoma of nasopharyngeal type (UCNT) is very rare tumour in Serbia, like in most of the countries of Europe, with incidence less than 0.5 per 100,000 people per year. The aim of this study was to assess the presence of Epstein-Barr virus (EBV) in the UCNT of a non-endemic population in Serbia and identify the main clinical parameters that interfere with patients' survival rate. This study included 102 patients with UCNT who were diagnosed between 1996 and 2003. Biopsies were analysed for EBV-encoded RNA (EBER) by in situ hybridization of tumour tissue microarray. Of 102 patients, 76 were men and 26 were women with ages ranging between 18 and 82 years (median 52.5, mean 53.0±14.1). Survival rates were 80, 39 and 31% for one, three and five years, respectively. Ninety-three of 102 cases were EBER positive (92%). Factors with unfavourable prognostic values were age over 50 years at the time of diagnosis, advanced clinical stage, therapy other than chemoradiotherapy and EBER negative status. In regard to the clinical data, EBER expression in UCNT was shown to be a strong independent predictor of overall and progression-free survival. To our knowledge, the current report constitutes the largest European non-endemic series of UCNT samples from a single institution with correlation between survival and clinical parameters/EBER status.

Dental identification after two mass disasters in Croatia.

AIM: To determine the usefulness of dental methods in the identification of victims in the railway accident in Zagreb (August 30, 1974) and midair collision of a British and a Slovenian airplane near Vrbovec (September 10, 1976). METHODS: There were 152 people killed in the railway accident, and 176 fatalities in the plane crash (63 in the British and 113 in the Slovenian plane). Individual victim identification and autopsy forms, and group identification reports were analyzed. RESULTS: Of the railway accident victims, 111 were identified. Dental characteristics, along with clothes, personal descriptions, personal documents, fingerprints, and jewelry, proved to be decisive in 5% of the cases. All 63 passengers and crew members from the British plane were identified; in 33% of the victims dental features, along with other characteristics, were decisive. From the Slovenian plane 103 victims were identified, 14% exclusively by teeth and 16% by teeth in combination with other characteristics. Ten bodies remained unidentified. CONCLUSION: The reasons for the small number of dental identifications in the victims of the railway accident were incomplete or unavailable antemortem data provided by relatives and friends of the deceased, and the predominant orientation toward other forensic identification methods. The significant number of dental identifications in the plane crash is explained by the provision of complete and accurate antemortem odontological data. Dental characteristics proved to be particularly valuable in the identification of carbonized victims.

Functional profile of the giant metacerebral neuron of Helix aspersa: temporal and spatial dynamics of electrical activity in situ.

1. Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. However, action potential initiation and the preceding integration of the synaptic signals in neuronal processes of individual cells are complex and difficult to understand in the absence of detailed, spatially resolved measurements. Multi-site optical recording with voltage-sensitive dyes from individual neurons in situ was used to provide these kinds of measurements. We analysed in detail the pattern of initiation and propagation of spikes evoked synaptically in an identified snail (Helix aspersa) neuron in situ. 2. Two main spike trigger zones were identified. The trigger zones were activated selectively by different sets of synaptic inputs which also produced different spike propagation patterns. 3. Synaptically evoked action potentials did not always invade all parts of the neuron. The conduction of the axonal spike was regularly blocked at particular locations on neuronal processes. 4. The propagating spikes in some axonal branches consistently reversed direction at certain branch points, a phenomenon known as reflection. 5. These experimental results, when linked to a computer model, could allow a new level of analysis of the electrical structure of single neurons.

Imaging membrane potential with voltage-sensitive dyes.

Membrane potential can be measured optically using a variety of molecular probes. These measurements can be useful in studying function at the level of an individual cell, for determining how groups of neurons generate a behavior, and for studying the correlated behavior of populations of neurons. Examples of the three kinds of measurements are presented. The signals obtained from these measurements are generally small. Methodological considerations necessary to optimize the resulting signal-to-noise ratio are discussed.

Fast optical recordings of membrane potential changes from dendrites of pyramidal neurons.

Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment; 2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons; 6) the dye spread approximately 500 micron into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites.

[Hypertension as a risk factor in acute infarct in young people]. / Hipertenzija kao rizik u akutnom infarktu mladih.

INTRODUCTION: Hypertension is one of the most frequent risk factors for acute myocardial infarction. The aim of this paper was to establish both the incidence of hypertension as a risk factor for myocardial infarction in young people and its characteristics. METHODS: The investigation involved patients younger than 40 years of age with acute myocardial infarction treated at the Institute of Cardiovascular Diseases in Sremska Kamenica from 1989 to 1994. RESULTS: The investigation involved 80 patients with acute myocardial infarction younger than 40 years--40 male and 40 female. Hypertension as a risk factor was found in 22 (55%) women and in 14 (35%) men and it was a moderate hypertension of systolic-diastolic type or unstable hypertension. In 10 (27.77%) investigated patients hypertension was detected during hospitalization (before that, it had not been controlled) while in 22 (61%) it was not seriously taken into account either by the patients, or their physicians and therefore it was not treated. As a single risk factor hypertension was found in 4 (10%) women, while it was not found in men. DISCUSSION: Hypertension is found in 10-15% of population, and in 10% young men and women. The existence of hypertension as the only risk factor for the occurrence of acute myocardial infarction was sufficient in these women, although it was considered that women in their generative period are protected from ishaemic heart diseases by their hormonal status. All that points to hypertension as a secret and mysterious killer. CONCLUSION: Hypertension as a risk factor was found in 22 (55%) women, and in 14 (35%) men. In 10% of women hypertension was the only risk factor, while it was not found as such in men. It was quite sufficient in these women for the occurrence of acute myocardial infarction, although it was considered that women in their generative period are protected from ishaemia in heart diseases by their hormonal status. Detection and adequate treatment of hypertension are of great importance as well as elimination of other risk factors.

[Coronary score in patients with low ejection fraction after myocardial infarct]. / Koronarni skor u bolesnika sa niskom ejekcionom frakcijom nakon prelezanog infarkta miokarda.

INTRODUCTION: The most frequent cause of heart failure is ischemic heart disease (1). This paper was aimed at comparing the coronary score of patients with low ejection fraction whose ejection fraction was not significantly changed after sustained myocardial infarction. MATERIAL AND METHODS: The study involved patients after sustained myocardial infarction treated at the Institute of Cardiovascular Diseases in Sremska Kamenica. Total coronary score and score of each individual coronary artery were emphasized. RESULTS: The investigation study comprised 56 patients aged 33-83 years of various occupations. Patients were divided into two groups: the first--A group consisted of 28 (50%) patients with ejection fraction 35% or lower; the second--B group also consisted of 28 (50%) patients with ejection fraction higher that 35%. Table 1. shows the dominant coronary artery in investigated groups of patients. Table 2. shows values of total and scores of each coronary artery. The right coronary artery was dominant in 75% of patients from the A group and in 82.1% of patients from the B group. A significantly higher individual score of coronary arteries, as well as the total score, was established in the group of patients with low ejection fraction, and especially the score of the anterior descendent artery which is almost twice higher in regard to the second group of examined patients. Table 3. describes the analyzed score in male and female patients. DISCUSSION: Patients with low ejection fraction after sustained myocardial infarction have more changes of coronary arteries than patients with better ejection fraction. The total score, score of the right coronary artery (ACD), circumflex artery (RCX) and especially anterior descendent artery (LAD) are significantly higher in patients with ejection fraction lower than 35%. There are no differences in the dominant coronary artery in investigated patients. In both investigated groups women had a smaller score of ACD and RCX and a higher score of LAD, but the difference is not significant. In regard to total score there were no differences in men and women. Numerous investigations also point to the fact that patients with lower ejection fraction and ischemic heart disease have more changes on coronary artery than patients with better ejection fraction. CONCLUSION: 1. Patients with low ejection fraction after sustained myocardial infarction have a higher total score and scores of ACD. LAD and RCX. 2. There are no differences in coronary score of men and women within the same investigated groups. 3. There are no differences in dominant coronary artery in investigated groups of patients.

Fast optical measurement of membrane potential changes at multiple sites on an individual nerve cell.

In the past 15 years, there has been renewed interest in the detailed spatial analyses of signalling in individual neurons. The behaviour of many nerve cells is difficult to understand on the basis of microelectrode measurements from the soma. Regional electrical properties of neurons have been studied using sharp microelectrode and patch-electrode recordings from neuronal processes, high-resolution multisite optical recordings of Ca2+ concentration changes and by using models to predict the distribution of membrane potential in the entire neuronal arborization. Additional, direct evidence about electrical signalling in neuronal processes of individual cells in situ can now be obtained by recording of membrane potential changes using voltage-sensitive dyes. A number of recent studies have shown that active regional electrical properties of individual neurons are extraordinarily complex, dynamic and, in the general case, impossible to predict by present models. This places a great significance on measuring capabilities in experiments studying the detailed functional organization of individual neurons. The main difficulty in obtaining a more accurate description was that experimental techniques for studying regional electrical properties of neurons were not available. With this motivation, we worked on the development of multisite voltage-sensitive dye recording as a potentially powerful approach. The results described here demonstrate that the sensitivity of voltage-sensitive dye recording from branches of individual neurons was brought to a level at which it can be used routinely in physiologically relevant experiments. The crucial figure-of-merit in this approach, the signal-to-noise ratio from neuronal processes in intact ganglia, has been improved by a factor of roughly 150 over previously available signals. The improvement in the sensitivity allowed, for the first time, direct investigation of several important aspects of the functional organization of an individual neuron: (1) the direction and the velocity of action potential propagation in different neuronal processes in the neuropile was determined; and (2) the interaction of two independent action potentials (spike collision) was monitored directly in a neurite in the neuropile; (3) it was demonstrated that several action potentials are initiated in the same neuron at different sites (multiple spike trigger zones) by a single stimulus; (4) the exact location and the size of one of the remote spike trigger zones was determined; (5) the spread of passive subthreshold signals was followed in the neurites in the neuropile. This kind of information was not previously available. Preliminary experiments on vertebrate neurons indicate partial success in the effort to use intracellularly applied voltage-sensitive dyes to record from neurons in a mammalian brain slice preparation. The results suggest that, with further improvements, it may be possible to follow optically synaptic integration and spike conduction in the dendrites of vertebrate nerve cells. The main impact of these results is a demonstration of a new way of analysing how individual neurons are functionally organized. Limitations and prospects for the further refinement of the technique are discussed mostly in terms of the signal-to-noise ratio; both improvements in the apparatus and design of more sensitive dyes are addressed.

Dental identification of war victims from Petrinja in Croatia.

In this paper the authors report their experiences and problems encountered in the identification of war victims from Petrinja in Croatia. Soon after Croatian forces regained Petrinja in 1995, four mass graves were discovered from which the bodies of 46 civilians, 38 males and 8 females, were recovered. Identification of the victims was performed at the Department of Forensic Medicine and Criminology at the School of Medicine in Zagreb. A forensic odonto-stomatologist from the Department of Dental Anthropology of the School of Dental Medicine at the University of Zagreb participated in the identification process by carrying out dental identifications. A total of 27 victims (59%) were identified, while 19 (41%) are at present still unidentified. Identification by supportive and anthropological evidence (e.g. sex, age, height, personal documents, dress, jewellery) was achieved in 43% of cases, while identification based only on dental records was achieved in 16%. The most useful dental characteristics for the purpose of identification were fixed and removable prosthetic appliances for oral rehabilitation. The reason for the low number of dental identifications was the lack of antemortem dental data which could be compared with postmortem dental records.

Short tandem repeat polymorphism at the HUMCD4 and HUMF13B loci in a Croatian population.

Population studies were carried out on unrelated individuals of Croatian ancestry. Genomic DNA was amplified by the polymerase chain reaction (PCR) at the polymorphic microsatellite loci HUMCD4 (n = 105 individuals) and HUMF13B (n = 108 individuals). After horizontal polyacrylamide gel electrophoresis followed by silver staining 6 alleles and 12 genotypes were observed for HUMCD4 and 6 alleles and 13 genotypes could be identified for HUMF13B. Data obtained were in concordance with the prediction of Hardy-Weinberg equilibrium. The allele frequency data were compared with Austrian and Italian population samples and no significant deviations between these populations were observed.

Multiple spike-initiation zones in single neurons revealed by voltage-sensitive dyes.

The primary function of the nerve cell is to process electrical signals. Over the past fifteen years there has been renewed interest in the detailed spatial analysis of signalling in individual neurons owing to experimental evidence that the regional electrical properties of neurons are complex. Thus the behaviour of many nerve cells cannot be understood on the basis of microelectrode measurements from the soma. Regional electrical properties of neurons have been studied using sharp microelectrode and patch-electrode recordings from neuronal processes, high-resolution multisite optical recordings of Ca2+ concentration changes and by using models to predict the distribution of membrane potential in the entire neuronal arborization. Additional direct evidence about electrical signalling in neuronal processes in situ can now be obtained by recording membrane potential changes using voltage-sensitive dyes. Here I demonstrate the existence of multiple action potential trigger zones in separate regions of the neuronal arborization of an identified molluscan neuron.

Optical recording from cerebellar Purkinje cells using intracellularly injected voltage-sensitive dyes.

We evaluated several techniques for their ability to record membrane potential changes with voltage-sensitive dyes introduced into CNS neurons in the brain slice preparation. Using a probe designed for intracellular application, JPW1114, we found that iontophoresis or pressure pulses could not push the lipophilic dye through electrodes whose resistance was sufficiently high to produce good electrical recordings in cerebellar Purkinje neurons. However, properly selected patch electrodes could introduce the dye into the cell and still give good electrical records. Using this technique we recorded depolarizing and hyperpolarizing transients and climbing fiber responses using either a single photodiode or a fast, cooled CCD camera. While these results are promising, there are still problems due to the slow diffusion of the dye in the dendrites and a low sensitivity which requires signal averaging to acquire traces with a good signal to noise ratio.

Optical signals from neurons with internally applied voltage-sensitive dyes.

We carried out experiments to monitor optically the generation and spread of action potentials and subthreshold potentials in the processes of individual neurons in ganglia of the snail, Helix aspersa. The neurons were selectively stained by intracellular pressure injection of voltage-sensitive dyes. Optical signals were detected by a system for fast, multiple-site optical monitoring, utilizing a silicon photodiode array. After testing 30 voltage-sensitive dyes using absorption, we concluded that this mode was probably not sensitive enough to allow monitoring neuronal signals from distal processes. Satisfactory signals were obtained in fluorescence measurements using a newly synthesized styryl dye, JPW1114, specifically designed for intracellular application. There was an improvement in sensitivity (as defined by the signal-to-noise ratio) by a factor of about 50 over previously reported absorption and fluorescence signals from neuronal processes stained by either intra- or extracellular application of dyes. Recordings with good signal-to-noise ratio and adequate spatial and temporal resolution were obtained simultaneously from the cell body and long axonal branches. From this data, the site of action potential initiation was determined. Also, the propagation velocity of the action potential was calculated for different axonal segments; the results suggest that different regions have different velocities ranging from 0.53 m/sec to 0.07 m/sec. The present sensitivity was adequate to allow the recording of a 10 mV hyperpolarizing electrotonic response along axonal branches and to observe directly the decline of this passive response with distance from the site of stimulation. Relatively modest improvements in sensitivity will allow systematic analyses of the spread and summation of synaptic potentials in individual neurons.

[Ischemic heart disease in young women. Its importance and possibilities of prevention]. / Ishemijska bolest srca u mladih zena. Vaznost i mogucnost prevencije.

Ischemic heart disease in young women is a rare disease, but nowadays it is more frequently detected. The investigation involved 83 women up to the age of 19 with ischemic heart disease. The most frequent risk factors were as follows: hypertension, hyperlipoproteinaemia, smoking, stress and obesity. The association of three factors was most often observed. Electrocardiogram at rest pointed to the changes as follows: anteroseptal region, the whole anterior wall, postero-inferior region and subendocardial localization. Out of 11 exercise tests 36 (81.81%) of them were positive. Coronary arteriography was performed in 72 (86.71%), with positive changes in 61 (81.72%) patients. Occlusive changes were most frequently found on anterior descendent artery. Since a great number of risk factors in women may be easily corrected by an adequate diet and healthier way of life, their detection and implementation of measures of primary and secondary prevention have been attached a great importance.

Myocardial perfusion imaging using 99mTc-MIBI in patients with dilated cardiomyopathy--methodology and its clinical use.

The aim of the study was to demonstrate the methodology of investigation of myocardial perfusion and viability using 99mTc-MIBI in patients with dilated cardiomyopathy and to estimate its clinical use. Myocardial perfusion and viability were investigated by using the planar and SPECT scintigraphy and by estimating the wall motion of the myocardium. The foci of decreased myocardial viability were found in 7 patients. One patient had perfusion defect only at stress. Analyzing the patients data, by using the gated studies, we found various degrees of wall motion myocardial abnormalities.

Optical measurement of action potential activity in invertebrate ganglia.

With a voltage-sensitive dye and an array of silicon photodiodes we monitored the action potential activity of a large fraction of the cells in the Aplysia abdominal ganglion during the gill-withdrawal reflex. This capability allowed a direct determination of the spread of sensory information within the 1,000 neurons of the ganglion. Surprisingly, approximately 30% of the neurons are activated by a light touch to a small area of siphon skin. It is likely that many other neurons also receive large synaptic potentials, either excitatory or inhibitory, and thus even a very mild and restricted stimulus will have widely distributed effects. It seems to us that these results will force a more pessimistic view of the present understanding of the neuronal basis of apparently simple behaviors.