Interferon-λ3 as a Predictor of Survival in Hemodialysis Patients.

BACKGROUND: IFNL4 polymorphisms are associated with circulating IFN-λ3, and higher plasma IFN-λ3 are associated with higher production of antibodies to HBV surface antigen (anti-HBs). The IFNL4 rs8099917 T allele and anti-HBs development in response to HBV vaccine are associated with better survival in hemodialysis (HD) patients. OBJECTIVE: To show whether plasma IFN-λ3 is also a predictor of survival in HD patients. METHODS: Plasma IFN-λ3 was measured in 135 HD patients who were followed-up for 2.6 years. Survival probability was tested using the Kaplan-Meier method and the Cox proportional hazard model. RESULTS: Plasma IFN-λ3 (ng/L) was 116.8 (20.4-227.5) in survivors on HD (n=89, 65.9%), 75.1 (36.0-228.8) in deceased patients (n=37, 27.4%), and 109.0 (40.0-232.7) in subjects submitted to kidney transplantation (n=9, 6.7%). IFN-λ3 was lower in deceased patients than that in all remaining patients (P=0.039) and patients who continued HD without transplantation (P=0.028). IFN-λ3 and anti-HBs titers were correlated (r=0.587, P<0.000001). Patients showing IFN-λ3 >126.1 ng/L (3rd tertile) presented better survival compared with patients with IFN-λ3 in the 1st (<73.8 ng/L, P=0.005) and 2nd (≥73.8 - <126.1 ng/L, P=0.013) tertiles. Each decrease in IFN-λ3 per 10 ng/L was associated with a hazard ratio equal to 1.076 (95%CI 1.015-1.140, P=0.013). In multivariate analysis, the independent predictors of survival were age (P=0.008), dialysis modality (P=0.038), circulating IFN-λ3 (P=0.044), and diabetic nephropathy (P=0.047), but not gender, dialysis duration prior to the study, mean arterial pressure, BMI, CRP, albumin, 25(OH)D, or anti-HBs. CONCLUSION: Circulating IFN-λ3 is a promising predictor of HD patient survival.

Photodynamic diagnosis (PDD) using 5-aminolevulinic acid-supplemented cultures of human endometrial epithelial cells.

PURPOSE: The studies were aimed at monitoring 5-aminolevulinic acid (5-ALA)-dependent accumulation of endogenous protoporphyrin IX (PpIX) in epithelial cells originating from normal endometrium or endometriotic foci, as related to steroid treatment. MATERIAL AND METHODS: Epithelial cells were cultured in presence of estradiol-17 beta (E2) and progesterone (P) in concentrations typical for the follicular stage (E2 alone, 220 pg/ml) or the luteal stage (E2 100 pg/ml and P 2 ng/ml) or in presence of progesterone alone (2 ng/ml) for a period of 24, 48 or 72 h. Effect of 5-ALA concentration on the accumulation of PpIX was defined in the cells incubated with 2.0 mmol/l 5-ALA for 2 h. PpIX fluorescence was detected using a confocal microscope. RESULTS: After hormonal stimulation, intensity of PpIX-specific fluorescence was only slightly increased in epithelial cells originating from normal endometrium. Cultures of epithelial cells from endometriosis foci showed higher concentration of PpIX than did the cells originating from normal endometrium. The highest peak of PpIX fluorescence was noted in epithelial endometriotic cells after 48h incubation with progesterone. CONCLUSIONS: The data on PpIX accumulation in epithelial cells in the presence of estradiol-17 beta or progesterone may provide indications as to the menstrual cycle phase(s) in which photodynamic therapy for endometriosis should be performed. It is concluded that hormonal condition of female body must be taken into account for diagnosis and treatment of endometriosis.

Effect of sperm subpopulation's kinetics on human fertilization in vitro.

The present study was carried out to investigate the relationship between sperm subpopulation kinetics on in vitro fertilization rate. The ability of human sperm to achieve fertilization oocytes was investigated in relation to particular motility parameters obtained on a computer aided sperm analysis system base. Analysis covers velocity straight linear (VSL), cross beat frequency (CBF), lateral head displacement (LHD) and homogeneity of progressive motility velocity (HPMV) of fresh semen and semen after density gradient selection. Investigation was based on sperm samples from 82 infertile couples undergoing IVF. Two subpopulations were extracted from each sample using the clustering method with respect to VSL parameter: a slow and rapid one. Comparison of obtained results before and after selection shows no significant change of subpopulations percentage. However, this method of selection strongly influences motility parameters of both subpopulations. There was found a positive correlation for VSL, LHD and HPMV and a negative correlation for CBF parameters found in slow fraction of fresh semen and percentage of fertilized oocytes. On the other hand, rapid subpopulation parameters for fresh semen and parameters found for both subpopulations in semen after selection did not correlate with one. This means that information of slow sperm subpopulation kinetics carries important prognostic value of IVF success. Since the current prognosis factors ignore motility parameters of slow sperms, our results show the importance of such an analysis.

Predictive value of selected sperm parameters for classical in vitro fertilization procedure of oocyte fertilization.

A proportion of fertilized oocytes during classical in vitro fertilization (IVF) procedure was analysed depending on the following factors: number of mature oocytes, seminological criteria such as sperm morphology in raw semen and after its selection in a density gradient (six structural defects of a male gamete were taken into consideration), sperm concentration, motility parameters according to World Health Organization criteria and the functional tests: hypo-osmotic swelling assay and acrosomal reaction induced by calcium ionophore. Evaluation of DNA content in sperm by image cytometry and determination of malonyldialdehydes in seminal plasma were also performed. Seventy-nine semen samples from patients undergoing IVF were assessed. Apart from significant correlations obtained for selected semen parameters and proportion of fertilized eggs, logistic regression analysis showed that the best predictive factors for oocyte fertilization were normal morphology of sperm before and after gradient selection, grade B and C of sperm movement in raw semen, and DNA content after density gradient centrifugation, which all accounted for 76.7% of fertilization predictive value.

Dielectric properties of a protein-water system in selected animal tissues.

Dielectric spectroscopy has been applied to study aspects of the organization of water in selected animal tissues (tendon, bone and horn). The measurements of the relative permittivity epsilon' and the dielectric loss epsilon'' were carried over the frequency range of 10-100 kHz and at temperatures from 22 to 240 degrees C. The water content was 10% for bone and horn, and 22% for tendon by mass at room temperature at a relative humidity of 70%. The temperature dependencies of epsilon' and epsilon'' reveal distinctively the temperature ranges corresponding to the release of water in temperatures up to about 200 degrees C for all tissues and the melting of the crystalline structure only for tendon and horn, above this temperature. The frequency dependencies of epsilon' and epsilon'' show a remarkable dispersion in the low-frequency at selected temperatures up to 200 degrees C for all tissues due to the release of the loosely and strongly bound water. The results were discussed in terms of the interfacial (Maxwell-Wagner) polarization and polarization mechanism involving hopping charge carriers interacting with the bound water molecules. The information on the effect of temperature, water content and frequency of the electromagnetic field on the dielectric behaviour of the tissues studied is of importance in the design and construction of medical diagnostic or therapeutic instruments based on the use of electric signals.

Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells.

5-aminolevulinic acid (ALA) is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX), which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX.

Effect of 5-aminolevulinic acid (ALA) doses and oestrogen/progesterone on protoporphyrin IX (ppIX) accumulation in human endometrial epithelial cells.

Endometriosis represents one of the most frequent causes of restricted fecundity. Despite the progress in medicine, appropriate diagnosis and treatment pose significant problems. The aim of this study was to evaluate ALA-induced PpIX fluorescence of normal endometrial epithelial cells for the diagnosis of endometriosis. PpIX-fluorescence was measured after stimulation with estradiol-17 beta (E2) or with estradiol-17 beta (E2) and progesterone (P) and after incubation with ALA under a confocal microscope. The epithelial cells showed a significantly higher fluorescence of PpIX in the course of 24 and 48h incubation with hormones, than the cells without stimulation. After 72h, a significant decrease in cellular PpIX concentration was noted. The results suggested that E2 and P were required to convert ALA to PpIX in epithelial cells and increased PpIX concentration in a time-dependent fashion.

Temperature dependent hysteresis-like changes of motility parameters in human spermatozoa.

We studied the impact of temperature changes on selected parameters of normozoospermic spermatozoa motility. The examinations were carried out in the temperature range of 11 degrees C to 21 degrees C both during cooling and heating. We found that a phenomenon of hysteresis, i.e. alternate means of changes in velocity straight linear and lateral head displacement was obtained both at cooling and at heating. This phenomenon was not found for other parameters examined.

Cell cycle and apoptosis in tumours of oral cavity estimated by image cytometry.

The aim of investigations was to examine whether diploid and aneuploid tumours of oral cavity differ in frequencies of occurrence of individual phases of cell cycle and of apoptosis. Investigations were carried out on 53 tumours using techniques of image cytometry. The studies were performed on properly isolated cells of tumours, which were stained using Feulgen method and analysed by image cytometry. Using a suitable computer programme, the percentages of cells present in individual phases of cell cycle and in apoptosis were estimated. No respective differences could be disclosed between diploid and aneuploid tumours. In addition, the ratios of the fraction of cells in S phase of the cell cycle and those in apoptosis were calculated and were found to carry certain prognostic significance in patients with squamous cell carcinomas of oral cavity.

[Compensatory mechanisms of food transmission and lower respiratory tract protection after classical and extended supraglottic laryngectomies. Computerized topokinetic analysis of roentgen-cinematographic images]. / Mechanizmy kompensacyjne przewodzenia pokarmów i ochrony dolnych dróg oddechowych po klasycznych i poszerzonych laryngektomiach nadglosniowych. Komputerowa analiza topokinetyczna obrazów rentgenokinematograficznych.

Studies were performed on 22 patients, aged from 17 to 78 years, in whom, owing to laryngeal cancers, partly classical or extended supraglottic laryngectomy was carried out. The evaluation involving the pharyngeal deglutition course was accomplished by resorting to computerized topokinetic analysis of the roentgen-cinematographic images. The completed observation revealed good mobility of the anatomical structures participating in deglutition, small volume and number of glossolaryngeal recesses. There were few patients in whom the contrast medium passed to the trachea. Better passage of food as well as protection of lower respiratory tract were associated with the improvement of parameters facilitating the deglutition.

Neurofilament phosphorylation and axon diameter in the squid giant fibre system.

Electron energy loss spectroscopic analysis of squid giant axons in a phosphorus energy window yielded bright signals, which were shown to originate from highly phosphorylated neurofilaments. The frequency and distribution of these signals were analysed at defined intervals in cross-sections of the giant axon, starting from its origin in the stellate ganglion and extending distally along the stellar nerve. The analysis revealed a proximodistal gradient of increasing neurofilament phosphorylation. Within the stellate ganglion and for some distance beyond, the increase in frequency of signals correlated with the widening of the neurofilament meshwork and the radial growth of the axon. This agrees with the hypothesis that neurofilament phosphorylation regulates axon calibre by affecting interfilament spacing. In distal axon domains where the axon diameter diminished, contrary to expectations, the spacing of signals increased and the signals were significantly larger. Hyperphosphorylation apparently compensated for a diminishing supply of neurofilament protein. Contrary to predictions, the presynaptic terminal of the giant synapse contained a distinct and highly phosphorylated neurofilament meshwork. We conclude that the growth of the axon diameter is a function of neurofilament phosphorylation, interfilament spacing and neurofilament density. A mature and highly phosphorylated neurofilament cytoskeleton completely filled the presynaptic terminal of the giant synapse.

[Studies on DNA content in sperm of men with oligozoospermia]. / Badania nad zawartoscia DNA w plemnikach mezczyzn z oligozoospermia.

The aim of our study was to analysis the ploidy of spermatozoa from oligozoospermic individuals. The study was performed in 48 patients with oligozoospermia. Spermatozoal DNA was analysed using MagiCal imaging cytometry. The percentage of aneuploid cells was determined using CYT-2 program. The results indicated aneuploidy in 73% of the examined patients. The amount of cells with abnormal DNA content ranged from 30 to 100%. Monitoring of the DNA in human spermatozoa may have practical applications in establishing the reasons for infertility as well as for screening of the semen for assisted reproduction techniques.

[Computer-assisted analysis of sperm motility]. / Analiza ruchu plemników przy zastosowaniu komputerowej analizy obrazu.

The fully automatic method for reconstruction of spermatozoa trajectories was presented. The method is based on computer image analysis. It allow to process pure semen without any treatment like diluting or purifying. Since no human interacting is available, this method lead to objective results. The method was used to analyse semen taken from healthy men (WHO 92 standard). The result shows absence of different populations in single sample.

[The course of the oropharyngeal stage of deglutition in physiological condition. Computer topokinetic analysis of videofluoroscopic images]. / Przebieg fazy gardlowej aktu polykania w warunkach fizjologicznych. Komputerowa analiza topokinetyczna obrazów rentgenokinematograficznych.

The performed analysis covers the physiology of the pharyngeal phase of deglutition in 11 patients aged 45-65 years. The studies were carried out with the aid of roentgenocinematographic examinations (RTGC) and after preparing an adequate computer program, a computer topokinetic analysis was accomplished (CTA). The measurement of parameters established in CTA make it possible to obtain the image of the pathway passed by the anatomical structures, the dynamics, the shape and the relative distances of two anatomical structures, with the duration of the deglutition act being taken into consideration. The use of RTGC images in CTA allows for better understanding of the mechanism acting in the pharyngeal phase of deglutition act in physiological conditions.

[Disorders of the pharyngeal stage of deglutition after classical and extended supraglottic laryngectomies. Computerized tomographic analysis of x-ray images]. / Zaburzenia fazy gardlowej aktu polykania po laryngektomiach nadglosniowych klasycznych i poszerzonych. Komputerowa analiza topokinetyczna obrazów rentgenokinematograficznych.

In 20 patients with laryngeal cancers, of both sexes and aged from 17 to 76 years, a partly classical or extended supraglottic laryngectomy was performed. The accomplished roentgenocinematographic analysis dealt with the disorders of the pharyngeal phase of deglutition act (RTGC). Next, by selecting characteristic schemas of frames for individual phases of deglutition, a computerized topokinetic analysis was carried out (CTA). The deglutition disorders were estimated in relation to the spared anatomical structures, formation of glosso-laryngeal recesses after this type of operations as well as leakage of contrast medium into the trachea.

Analysis of spermatozoa movement using a video imaging technique.

The aim of this paper is to present methods for a fully computerised analysis of spermatocyte movement. The technique were designed for processing pure images of spermatozoa captured from a light microscope. The techniques described allowed for the reduction of background light inhomogeneity and for the correct detection of moving cells and involved densitometric equalization of background inhomogeneity and implementation of the dynamic threshold, adopted for the recognition of objects. The method for relating cells (found in each frame processed) to movement trajectories used heuristic rules, describing the behaviour of moving cells. This permitted samples containing high numbers of spermatocytes within the observed area to be processed with maximum accuracy.

The human insulin analog insulin lispro improves insulin binding on circulating monocytes of intensively treated insulin-dependent diabetes mellitus patients.

The rapidly absorbed analog of human insulin, insulin lispro (LP), is characterized by a faster onset of action, a higher peak insulin level, and a shorter duration of action compared with regular insulin (RI). The aim of this study was to investigate whether intensified treatment with either LP or RI influences insulin receptor status. Twelve patients with insulin-dependent diabetes mellitus (IDDM) participating in a multicenter randomized cross-over trial were allocated to this study. Four patients began with LP, whereas eight patients started with RI. Each patient was switched to the other insulin after a 3-month treatment period. Competitive [125I]A-14-insulin binding studies were performed with isolated monocytes. Treatment with insulin lispro increased the total number of insulin binding sites from 9,400 +/- 2,200 (RI) to 20,300 +/- 3,000 (LP)/monocyte (P < 0.001). The insulin concentration required for a 50% competition of [125I]insulin binding (IC50) decreased from 0.6 +/- 0.2 (RI) to 0.1 +/- 0.03 (LP) nmol/L, indicating significantly higher affinity of insulin binding sites during LP treatment (P < 0.001). In additional experiments, the time course of insulin binding was determined after an oral meal. In LP-treated IDDM patients, the affinity and capacity of insulin binding showed a nadir 1 h after insulin injection and a regained binding affinity and capacity 5 h later. These changes observed after LP treatment were comparable to the effect of endogenous insulin secretion in healthy control subjects. In contrast, the IDDM patients who injected RI showed a decreasing insulin binding affinity and capacity, most markedly expressed after 5 h. The corresponding serum levels of insulin were inversely correlated with the affinity and capacity of insulin-binding sites. Pretreatment of cultured human IM-9 lymphoblasts with LP or RI yielded no difference in the down-regulation of insulin binding. In summary, intensified conventional insulin therapy with LP increased the number and affinity of insulin receptors on circulating monocytes to a level similar to that observed in healthy subjects. We conclude that the improved insulin receptor status observed during LP treatment is caused by its more physiological pharmacokinetic profile.

An inexpensive computer-aided morphometric device for the analysis of dissociated brain cell cultures.

Morphometric analysis of brain cells growing in dissociated cell culture is a promising approach in neuroscience research. Here we present new software written in Turbo PASCAL which has the advantage of requiring only a minimum in equipment accessible in most neuroscience labs, i.e., a microscope with a camera lucida attachment and a personal computer. The camera lucida is used to superimpose the pictures from the microscope and the computer screen. With the aid of a mouse, cells are drawn to the computer screen. The program counts pixels and calculates morphometric data including the area occupied by the cell soma as well as number, length and arborization of the processes. The program may be obtained from the authors upon request.