Applicability of Gene Expression in Saliva as an Alternative to Blood for Biodosimetry and Prediction of Radiation-induced Health Effects.

As the great majority of gene expression (GE) biodosimetry studies have been performed using blood as the preferred source of tissue, searching for simple and less-invasive sampling methods is important when considering biodosimetry approaches. Knowing that whole saliva contains an ultrafiltrate of blood and white blood cells, it is expected that the findings in blood can also be found in saliva. This human in vivo study aims to examine radiation-induced GE changes in saliva for biodosimetry purposes and to predict radiation-induced disease, which is yet poorly characterized. Furthermore, we examined whether transcriptional biomarkers in blood can also be found equivalently in saliva. Saliva and blood samples were collected in parallel from radiotherapy (RT) treated patients who suffered from head and neck cancer (n = 8) undergoing fractioned partial-body irradiations (1.8 Gy/fraction and 50-70 Gy total dose). Samples were taken 12-24 h before first irradiation and ideally 24 and 48 h, as well as 5 weeks after radiotherapy onset. Due to the low quality and quantity of isolated RNA samples from one patient, they had to be excluded from further analysis, leaving a total of 24 saliva and 24 blood samples from 7 patients eligible for analysis. Using qRT-PCR, 18S rRNA and 16S rRNA (the ratio being a surrogate for the relative human RNA/bacterial burden), four housekeeping genes and nine mRNAs previously identified as radiation responsive in blood-based studies were detected. Significant GE associations with absorbed dose were found for five genes and after the 2nd radiotherapy fraction, shown by, e.g., the increase of CDKN1A (2.0 fold, P = 0.017) and FDXR (1.9 fold increased, P = 0.002). After the 25th radiotherapy fraction, however, all four genes (FDXR, DDB2, POU2AF1, WNT3) predicting ARS (acute radiation syndrome) severity, as well as further genes (including CCNG1 [median-fold change (FC) = 0.3, P = 0.013], and GADD45A (median-FC = 0.3, P = 0.031)) appeared significantly downregulated (FC = 0.3, P = 0.01-0.03). A significant association of CCNG1, POU2AF1, HPRT1, and WNT3 (P = 0.006-0.04) with acute or late radiotoxicity could be shown before the onset of these clinical outcomes. In an established set of four genes predicting acute health effects in blood, the response in saliva samples was similar to the expected up- (FDXR, DDB2) or downregulation (POU2AF1, WNT3) in blood for up to 71% of the measurements. Comparing GE responses (PHPT1, CCNG1, CDKN1A, GADD45A, SESN1) in saliva and blood samples, there was a significant linear association between saliva and blood response of CDKN1A (R2 = 0.60, P = 0.0004). However, the GE pattern of other genes differed between saliva and blood. In summary, the current human in vivo study, (I) reveals significant radiation-induced GE associations of five transcriptional biomarkers in salivary samples, (II) suggests genes predicting diverse clinical outcomes such as acute and late radiotoxicity as well as ARS severity, and (III) supports the view that blood-based GE response can be reflected in saliva samples, indicating that saliva is a "mirror of the body" for certain but not all genes and, thus, studies for each gene of interest in blood are required for saliva.

Increasing prevalence of celiac disease - where to look for answers?

Celiac disease or gluten-sensitive enteropathy is a relevant health concern in today's world. Three prerequisites need to be met to trigger the disease, namely a genetic predisposition, gluten consumption, and environmental factors. Retrospective studies conducted across all age groups have ruled out the possibility that improved diagnostic methods were behind the increased prevalence. Since the genetic predisposition is more or less constant in the population, it is assumed that external factors may play a major role in this increase. Although it is generally believed that modern wheat varieties are to be blamed for the increase in gluten intolerance-related diseases, this assumption is refuted based on the analysis of the current and 100-year-old varieties. However, the increased prevalence could be related to modern lifestyles, changes in food preparation technology or composition, disruption of the intestinal barrier in viral disease, and other factors leading to intestinal dysbiosis. A possible preventive strategy in predisposed individuals could be the avoidance of gluten from the diet when ill, especially with a viral infection. This article openup a new perspective on the currently common autoimmune disease.

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes.

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

Polydispersity of liposome preparations as a likely source of peak width in capillary zone electrophoresis.

Negatively charged liposomes consisting of phosphatidylcholine/phosphatidylglycerol/cholesterol in various ratios when subjected to capillary zone electrophoresis (CZE) in Tris-HCl (pH 8) buffer of different concentrations have been shown previously to exhibit a size-dependent migration rate at low ionic strength. The present study, focusing on the peak width under those conditions, shows that the polydispersity of liposomes correlated with, and appears to be a dominant source of, the peak width of the liposomes in CZE in a buffer of low ionic strength (2 to 5 mM Tris-HCl buffer, pH 8) at moderate electric field strengths (200 V cm(-1) or less). This finding, beyond allowing for the analysis of liposome polydispersity by CZE, suggests that the size-dependent fractionation of liposome preparations by a preparative electrophoretic technique such as free-flow electrophoresis is potentially feasible.

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Rapid capillary coating by epoxy-poly-(dimethylacrylamide): performance in capillary zone electrophoresis of protein and polystyrene carboxylate.

A fast and simple method for the internal coating of capillaries in capillary zone electrophoresis (CZE) is that with epoxy-poly(dimethylacrylamide) (EPDMA). Duration of coating by that method is 30 min, compared with that of 24 h when using uncross-linked polyacrylamide (PA) under otherwise identical conditions. Under the conditions used for the CZE of proteins (pH 9.0, 2% polyethylene glycol), the capillary coating with EPDMA is stable for at least 50 consecutive runs as judged by the constancy of low electroosmotic flow, equalling the stability of coating achieved by PA. Protein mobilities and protein peak asymmetry (suggestive of reversible interaction with the capillary wall) are also found to be the same in EPDMA and PA coated capillaries. Differences between EPDMA and PA coating also exist: The former is unstable upon lowering the ionic strength of the buffer to 0.003, upon the addition of sodium dodecyl sulfate (SDS) to the buffer and in application to the hydrophobic analyte, polystyrene carboxylate.

Discrimination between peak spreading in capillary zone electrophoresis of proteins due to interaction with the capillary wall and due to protein microheterogeneity.

Our study attempts to find an approach to distinguishing between the contribution to peak spreading in capillary zone electrophoresis (CZE) due to protein microheterogeneity and that due to interaction with the capillary wall, by analyzing correlations between observed peak spreading and peak asymmetry. The peak asymmetry was measured as ln[(tm-t1)/(t2-tm)] where tm, t1, and t2 are migration times at the mode of the peak and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively. Two isoforms of recombinant green fluorescent protein (GFP-1 and GFP-2, 27 kDa molecular mass), glucose-6-phosphate dehydrogenase (GPD, 104 kDa), and the naturally fluorescent protein R-phycoerythrin (PHYCO, 240 kDa) were subjected to CZE in polyacrylamide-coated fused-silica capillaries of 50 and 100 microns diameters under varying conditions of protein concentration, field strength, and the initial zone length. Under conditions such that contributions to peak spreading from axial diffusion, thermal effects, and electrophoretic dispersion are negligible, the analysis of the interrelations between peak width and peak asymmetry was found to allow a conclusion as to the cause of peak spreading in CZE of protein. It appears that the peak width of GFP-2 originates mostly in protein microheterogeneity while that of GFP-1 is due to protein-capillary wall interactions. For PHYCO, both microheterogeneity and protein-capillary wall interactions contribute to peak spreading. GPD exhibits relatively little microheterogeneity or interaction with capillary walls. Thus, its peak width appears to be mostly affected by an extracolumn source of spreading such as the initial zone length.

A survey of different successful microsurgical vascular prostheses in animal experiments.

The authors summarize historical findings from the literature on the success of microsurgical vascular prostheses in experimental practice with regard to different production and construction technologies. They reference authors and surgeons using Gore-Tex, polyurethane, polyactide, silicone and woven vascular microprostheses.

Woven vascular microsurgical prostheses in our experimental practice.

The authors have been concerned for six years with the development of woven microsurgical prostheses in experiments on laboratory rats, beagle dogs and domestic white pigs. In rats, in 224 experiments employing 38 types of prostheses with an inner diameter of 2 mm, a reliable prosthesis was selected, described as No. 36, with a 100% patency after insertion of a 1 cm long portion into a defect in the abdominal aorta in 40 experiments. The follow-up of this type of prosthesis lasted 12 months. In large animals this type of prosthesis was implanted into a defect of the radial artery and cephalic vein in dogs and into the femoral artery and the artery of a vascular pedicle of groin and lateral thigh flap. These experiments on large animals are not yet complete and will be the subject of a separate paper.

Size-dependent electrophoretic migration and separation of liposomes by capillary zone electrophoresis in electrolyte solutions of various ionic strengths.

The size-dependent electrophoretic migration and separation of liposomes was demonstrated and studied in capillary zone electrophoresis (CZE). The liposomes were extruded and nonextruded preparations consisting of phosphatidylcholine/phosphatidylglycerol/cholesterol in various ratios and ranging from 125 to 488 nm in mean diameter. When liposomes of identical surface charge density were subjected to CZE in Tris-HCl (pH 8) buffers of various ionic strengths (0.001-0.027), they migrated in order of their size. Size-dependent electrophoretic migration and separation of liposomes in CZE can be enhanced or brought about by decreasing the ionic strength of the buffer. It was shown that size-dependent migration is primarily a function of kappaR, where kappa(-1) is the thickness of the electric double layer (which can be derived from the ionic strength, I, of the buffer) and R, the liposome radius. Liposome mobility depends on kappaR and surface charge density in a manner consistent with that expected from the Overbeek-Booth electrokinetic theory. Thus, the relaxation effect appears to be the physical mechanism underlying the size-dependent electrophoretic separation of liposomes.

Capillary zone electrophoresis of sub-microm-sized particles in electrolyte solutions of various ionic strengths: size-dependent electrophoretic migration and separation efficiency.

To gain insight into the mechanisms of size-dependent separation of microparticles in capillary zone electrophoresis (CZE), sulfated polystyrene latex microspheres of 139, 189, 268, and 381 nm radius were subjected to CZE in Tris-borate buffers of various ionic strengths ranging from 0.0003 to 0.005, at electric field strengths of 100-500 V cm(-1). Size-dependent electrophoretic migration of polystyrene particles in CZE was shown to be an explicit function of kappaR, where kappa(-1) and rare the thickness of electric double layer (which can be derived from the ionic strength of the buffer) and particle radius, respectively. Particle mobility depends on kappaR in a manner consistent with that expected from the Overbeek-Booth electrokinetic theory, though a charged hairy layer on the surface of polystyrene latex particles complicates the quantitative prediction and optimization of size-dependent separation of such particles in CZE. However, the Overbeek-Booth theory remains a useful general guide for size-dependent separation of microparticles in CZE. In accordance with it, it could be shown that, for a given pair of polystyrene particles of different sizes, there exists an ionic strength which provides the optimal separation selectivity. Peak spreading was promoted by both an increasing electric field strength and a decreasing ionic strength. When the capillary is efficiently thermostated, the electrophoretic heterogeneity of polystyrene microspheres appears to be the major contributor to peak spreading. Yet, at both elevated electric field strengths (500 V/cm) and the highest ionic strength used (0.005), thermal effects in a capillary appear to contribute significantly to peak spreading or can even dominate it.

Capillary zone electrophoresis of proteins in semidilute polymer solutions: inter- and intra-polymer predictability of size-dependent retardation.

The retardation of three "spherical" proteins with Stokes' radii of 2.0, 2.4, and 3.0 nm (35-104 kDa) was studied in capillary zone electrophoresis (CZE), using semidilute solutions of polyethylene glycol (PEG), linear polyacrylamide (PA), and polyvinyl alcohol (PVA). The purpose was to test the models predicting that the ratio of particle radius, R, to the mesh size of polymer network (the correlation or screening length of a semidilute polymer solution), xi, directly governs the size-dependent retardation in the form: mu/muo = exp (-R/xi). Here xi = kc-0.75, where c is polymer concentration and the numerical factor kcan be calculated based on polymer molecular weight. In application to polymers in a "good solvent" (PA and PEG in the aqueous buffer) and to proteins of 2.4 and 3.0 nm radius, that relation between relative mobility and R/xi was found to be obeyed for PA, while for PEG the value of k derived from retardation experiments significantly exceeded that which was theoretically calculated. Thus, the retardation appears to be polymer-specific, rather than universal, even for polymers in a "good solvent". It is suggested that, in that case, retardation of proteins of R > 2 nm be quantitatively described in the form mu/muo = exp[-p(R/xi], where p is a parameter depending on monomer type and/or polymer polydispersity. For PVA, the logarithm of mu/muo was found to be linearly related to c (in line with the prediction that the aqueous buffer is a "poor solvent" for this polymer) and to be near-independent of R.

[Do we need predictions for health care expenditures?]. / Potrebujeme prognózu výdaju na zdravotnictví?

The unfavourable health status of our population is generally known, its improvement being one of the basic prerequisites for the further development of our society. To a certain extent improvement may be promoted by high standard health services. The standard of these services depends, in addition to effective use of available financial means, on the increase of the total volume of financial resources for the health services. For a qualified decision on the actual volume of financial resources it will be necessary to have a well elaborated prognosis of expenditure on health services which among others will justify the financial demands of the branch. The prognosis of expenditure on health services will be a socio-economic prognosis which may be started in the near future, if suitable conditions will be created.

[The present status and trends in health care expenditures throughout the world and in Czechoslovakia]. / Soucasný stav a tendence v oblasti výdaju na zdravotnictví ve svete a u nás.

Expenditure on health services on a world-wide scale have an ever increasing trend. The trends in advanced countries where nowadays the expenditure on health services is cca 10% of the national gross product is to restrict a further rise and to make maximum use of available resources. In Czechoslovakia the position is different, as expenditure on health services is roughly half as compared with advanced countries. Provisions thus will be specific and funds from other sources than the stage budget will be increased and there will be efforts to achieve a maximum effectiveness as regards the use of available resources. At the same time it will be, however, necessary to make an effort to increase the total volume of financial resources for the health services. The importance of these efforts ensues from the present health status of the population.

[Factors affecting higher expenditures in health care]. / Nekteré faktory ovlivnující výsi výdaju na zdravotnictví.

The funds available for the health services at present are not sufficient. When assessing their desirable volume, we must consider a number of factors which influence the needs of financial means. This comprises direct and indirect action, in different spheres and direction and of different intensity. Different factors are not quantifiable to the same extent; some can be expressed only verbally. The resultant of their action which may have the same trend (then the effects add) or have an opposite trend (then the effects cancel out), is then the concrete pressure which in a concrete situation affects the concrete need of financial means. A team of specialists will have to study the identification of factors and assess the assumed forces of their action.

[Problems in expressing performance in relation to costs in the health services]. / Nekteré problémy vyjadrování výkonu ve vztahu k nákladum ve zdravotnictví.

Although in our country considerable financial means are spent on the health services, they do not suffice for the health services to meet their tasks under adequate conditions and to contribute to the favourable health status of the population. Under conditions of the reconstruction of the economic mechanism the authorities accept the necessity to increase the financial means for the health services, they ask, however, for concrete justification of these demands. This involves considerable efforts in conjunction with attempts to express performed work in the health services in relation to their costs. This means that the contents of different tasks have to be defined, to create a uniform classification, define the sources of information, the method of calculation and perform other essential tasks associated with the financial assessment of health activities. Certain provisions in this respect were already made. It is essential to proceed with this work or take over the results of work performed in other countries. Optimation of expenditure on the health services which ought to be the result of this work will be a valuable contribution in relation to social resources justifying the financial demands of the health services, as well as in relation to different health facilities and their staff who will get a better grasp of the medical and economic relations.

[The present share of health services financing provided by state enterprises]. / Soucasný podíl podnikové sféry na financování zdravotnických sluzeb.

At the moment none of the managing departments is concerned with investigations of financing of factory health facilities by enterprises and organizations. A pilot study revealed that hitherto valid methods of investigation of these costs, as regards investments, were not very successful and thus it is very difficult to follow up these data. As regards operational costs, considerable differences were revealed between different departments in their contribution to the costs of factory health facilities. It was revealed that during the 7th Five-year Plan all financial means spent by enterprises on health services were cca 3.2% of the expenditure for health services (i. e. the total expenditure of the state health administration and enterprises). From the fund of cultural and social needs 0.1% to 1% are spent on the health services. The paper discusses also possibilities of the further development of factory health services.