Caspase-9 inhibition decreases expression of Mmp9 during chondrogenesis.

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Recent approaches in tooth engineering research.

Tooth absence and defects caused by various reasons are frequent events in humans. They are not life threatening but may bring about social consequences. Recent dentistry provides solutions in the form of prosthetics or dental implants; however, several complications and distinct limitations favour bioengineering of dental and periodontal structures. At least two types of cells (epithelial and mesenchymal) have to be recombined to produce a new functional tooth. Moreover, the tooth must be vascularized, innervated and properly anchored in the bone. To study these issues, different approaches have been established in both basic and applied research. In this review, recent strategies and techniques of tooth engineering are comprehensively summarized and discussed, particularly regarding manipulation using stem cells.

Properties of neural crest-like cells differentiated from human embryonic stem cells.

Neural crest cells (NCCs) derive early in vertebrate ontogenesis from neural tube as a population of migratory cells with exquisite differentiation potential. Abnormalities in NCC behaviour are cause of debilitating diseases including cancers and a spectrum of neurocristopathies. Thanks to their multilineage differentiation capacity NCCs offer a cell source for regenerative medicine. Both these aspects make NCC biology an important issue to study, which can currently be addressed using methodologies based on pluripotent stem cells. Here we contributed to understanding the biology of human NCCs by refining the protocol for differentiation/propagation of NCClike cells from human embryonic stem cells and by characterizing the molecular and functional phenotype of such cells. Most importantly, we improved formulation of media for NCC culture, we found that poly-L-ornithine combined with fibronectin provide good support for NCC growth, we unravelled the tendency of cultured NCCs to maintain heterogeneity of CD271 expression, and we showed that NCCs derived here possess the capacity to react to BMP4 signals by dramatically up-regulating MSX1, which is linked to odontogenesis.

Coupling activation of pro-apoptotic caspases with autophagy in the Meckel´s cartilage.

Mammalian Meckel´s cartilage is a temporary structure associated with mandible development. Notably, its elimination is not executed by apoptosis, and autophagy was suggested as the major mechanism. Simultaneous reports point to pro-apoptotic caspases as novel participants in autophagic pathways in general. The aim of this research was to find out whether activation of pro-apoptotic caspases (-2, -3, -6, -7, -8 and -9) was associated with autophagy of the Meckel´s cartilage chondrocytes. Active caspases were examined in serial histological sections of mouse mandible using immunodetection and were correlated with incidence of autophagy based on Beclin-1 expression. Caspase-2 and caspase-8 were found in Beclin-1 positive regions, whereas caspase-3, -6, -7 and -9 were not present. Caspase-8 was further correlated with Fas/FasL and HIF-1alpha, potential triggers for its activation. Some Fas and FasL positivity was observed in the chondrocytes but caspase-8 activation was found also in FasL deficient cartilage. HIF-1alpha was abundantly present in the hypertrophic chondrocytes. Taken together, caspase-8 activation in the Meckel´s cartilage was demonstrated for the first time. Caspase-8 and caspase-2 were the only pro-apoptotic caspases detected in the Beclin-1 positive segment of the cartilage. Activation of caspase-8 appears FasL/Fas independent but may be switched on by HIF-1alpha.

The effect of fluvastatin on cICAM-1 as a biomarker of endothelial dysfunction in patients with dyslipidemia.

AIM: Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression has been suggested to play an important role in the pathogenesis of atherosclerosis. Lipid-lowering therapy with 3-hydroxy-methylglutaryl-coenzyme A reductase inhibitors reduces the incidence of atherosclerosis-related cardiovascular events. The aim of this study was to establish the effect of a 3-month treatment of fluvastatin on circulating (c)ICAM-1 and standard lipid parameters. METHODS: A total of 14 patients (6 men and 8 women), 59.7+/-11 years old, with mean systolic and diastolic blood pressure 135+/-16.3 and 81+/-9.3 mmHg, respectively, fasting plasma cholesterol 6.2+/-1 mmol/L and plasma triglycerides 2.6+/-1 mmol/L, took part in the study. The observed parameters were measured before and after 3 months of therapy. RESULTS: When comparing total cholesterolemia, low-density lipoprotein (LDL) cholesterol, and apolipoprotein B, there were significant decreases after a 3-month treatment. By contrast, no significant difference was observed when we compared the values of triglycerides, high-density lipoprotein (HDL) cholesterol, apolipoprotein A1 and, especially, of cICAM-1. Our results demonstrate the well-known capacity of fluvastatin to lower LDL- and total cholesterol; however, it failed to reduce cICAM after a 3-month treatment. CONCLUSIONS: It can be concluded that a 3-month therapy with fluvastatin does not decrease cICAM-1 levels, despite normalization of cholesterol levels. The implication is that cholesterol may not induce endothelial activation by the initial upregulation of this adhesive molecule.

Mouse Incisor Stem Cell Niche and Myb Transcription Factors.

Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B-Myb is often linked to the stem cell phenotype, and c-Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo-spatial expression of B-Myb and c-Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B-Myb expression was localised mostly in the zone of transit-amplifying cells, and c-Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B-Myb nor c-Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c-Myb in differentiation of hard tissue cells.

Diagnosis of pleural effusions. Experience with clinical studies, 1986 to 1990.

OBJECTIVES: To identify in patients with pleural effusion which procedures are most useful in separating malignant from nonmalignant pleural effusions and to identify which procedures most commonly lead to a definitive diagnosis. DESIGN: Prospective consecutive case series. SETTING: Pulmonary referral hospital in Prague, Czech Republic. PATIENTS: One hundred seventy-one adults between ages 18 and 70 years with a pleural effusion and a Karnofsky score of 70 or above. INTERVENTIONS: All patients underwent history, physical, pleural fluid cytologic study, laboratory evaluation of serum and pleural fluid, pleural biopsy, bronchoscopy, and lung scan and/or pulmonary arteriogram. RESULTS: In this series in which 45% of the patients had malignant effusions, 19% had paramalignant effusions, and 36% had benign diseases, the pleural fluid cytologic study was the best for establishing a diagnosis. The pleural fluid carcinoembryonic antigen (CEA) levels above 10 had a high specificity (90%) for malignancy but had low sensitivity (37%). The pleural fluid CEA level was increased only in 19% of patients with paramalignant effusions. Although there were statistically significant differences in the mean results on several biochemical tests of pleural fluid, none were very accurate in separating malignant from benign disease. CONCLUSION: From this study, we conclude that patients with an undiagnosed pleural effusion should be evaluated in an individualized stepwise manner. If malignancy is strongly considered, the initial three steps should be relatively noninvasive and include clinical evaluation and cytologic study.

Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style.

The original purpose of our study was to determine if the detection of chromosomal aberrations in peripheral lymphocytes of children might be used as a biomarker of environmental pollution and life style. We compared the results of cytogenetic analyses performed in children and adolescents in the periods 1984-1993 and 1994-1999, in a total of 3402 subjects. The frequency of aberrant cells (AB.C.) markedly decreased in the period 1994-1999 compared with the period 1984-1993. The decreases in AB.C. were significant in the age groups 7-15 and 16-19 years: 1.63% AB.C. versus 1.14% AB.C. and 2.02% AB.C. versus 1.08% AB.C., respectively (P<0.01). No difference in the frequency of AB.C. was observed in newborns. Based on our experience, we believe that monitoring the spontaneous level of chromosomal aberrations in children over 5 year periods may be used to examine the general changes in environmental pollution in larger geographic areas.

Spontaneous level of chromosomal aberrations in peripheral blood lymphocytes of control individuals of the Czech Republic population.

In order to assess the potential of cytogenetic determinations on peripheral blood lymphocytes as a mean of monitoring human population subjects to occupational and environmental exposures to genotoxins, accurate baseline data are required. During the past 20 years many results of the cytogenetic studies on peripheral blood lymphocytes from monitored occupationally exposed and non-exposed groups were obtained. At the time of blood drawing a questionnaire was administered. The questions covered a brief medical and family history including age, sex, medication, infectious diseases, smoking habits, X-ray examinations, alcohol consumption etc. Cytogenetic analysis from whole blood was carried out in short-term cultures. The cultivation time was 52 hours with all cells being in the first mitosis. A total of 100 well-spread metaphases containing 46 +/- 1 centromere were examined per donor on coded slides. Four categories of chromosome aberrations were evaluated: Chromatid and chromosome breaks, chromatid and chromosome exchanges. Cells bearing breaks or exchanges were classified as aberrant cells. Gaps were recorded but not scored as aberrations. Results of the cytogenetic analysis from control individuals (N = 5,430) indicated elevation of spontaneous frequency of aberrant cells (AB.C.) with age. We found 1.10% AB.C. (N = 551) in newborns; 0.71% AB.C. (N = 105) in the group 5-6 yr; 1.20% (N = 1,734) in the group 7-15 yr; 1.25% AB.C. (N = 239) in the group 16-19 yr and 1.59% (N = 2,801) in the group 20-63 yr.

Maganese retention in rat brain.

Manganese retention was observed in brains and in several other tissues of female Wistar rats after the intratracheal instillation of an inorganic manganese compound: manganese dioxide. Two categories of rats, younger (180 to 200g) and older (330 to 350g), were divided into a control group, in which animals received vehicle only (0.5 mL physiological saline), and an experimental group, in which rats received a dose of 0.48 mg of Mn/kg body weight (in 0.5 mL saline), twice a week for 3 months, for a total dosage of 11.80 mg of Mn/kg body weight. At the end of the exposure period, manganese retention in selected rat organs, brain, liver, kidney, and lung, was analyzed using atomic absorption spectrophotometry. At the end of the 6-wk or 12-wk manganese dioxide exposure period, analysis of variance of the manganese retention results revealed significant differences between Mn-exposed and unexposed rats in brain, kidney, and lung tissues (p<0.01) for both experimental age categories. Moreover, at the end of the 12-wk exposure period, significant results (p<0.05) between younger and older rats were obtained for both brain and kidneys. In both types of tissue, the manganese retention in the younger group was higher than that in older animals.

The effect of an acute fat load on endothelial function after different dietary regimens in young healthy volunteers.

Attention has recently been focused on endothelial function after a single high-fat meal, i.e. on the anticipated direct atherogenic effect of triglyceride-rich lipoproteins. Our study was designed to investigate the effect of a low-fat diet given for four weeks followed by a high-fat diet for another four weeks. At the end of each dietary period, a noninvasive ultrasound investigation of endothelial function of the brachial artery was performed along with laboratory tests. Endothelial function was measured immediately before the dietary load and after three and six hours in 11 healthy volunteers. The results were expressed as percentage of the changes in artery diameter at rest and during hyperemia; the data were processed using computer technology. When compared to the low-fat regimen, the total cholesterol content rose after the high-fat diet from 4.28 mmol/l to 5.15 mmol/l (p<0.05) in the whole group of volunteers. There was no difference between both dietary regimens in baseline triglycerides. The brachial artery dilatation under basal conditions was 5.26+/-2.88 mm after the high-fat diet compared with the value of 3.13+/-3.01 mm (p<0.05) after the low-fat diet. When measured individually endothelial function in the whole group of volunteers in the course of the day, the degree of arterial dilatation after one month on low-fat diet was 3.13+/-3.0%, 3.88+/-2.5% and 5.23+/-3.3% at single measurement. When comparing arterial dilatation at two closest measurements, a non-significant trend, p>0.05 was seen in either case. The following values were obtained after one month on the high-fat diet: 5.26+/-2.9%, 4.47+/-1.7%, and 6.2+/-3.6%; again showing a non-significant trend of p>0.05. In this study, a single high-fat meal at the different dietary regimen did not significantly influence the vasoreactivity of the brachial artery in young volunteers.

Characterization of immunomodulatory polysaccharides from Salvia officinalis L.

Crude polysaccharide fractions, rich mainly in arabinogalactans (A), pectin (B) and glucuronoxylan-related polymers (D), have been obtained from aerial parts of sage (Salvia officinalis L.) by sequential extraction with various reagents. Arabinogalactans displayed on HPLC a dominance of lower molecular-mass polymers (MW < 10,000), while pectin and glucuronoxylan-related polysaccharides showed predominance of polymers with MW > 50,000. Individual polysaccharide fractions were examined for their immunomodulatory activity in the in vitro comitogenic thymocyte test. The polysaccharide fractions tested possessed the capacity to induce rat thymocyte proliferation in the order D>B>A. Besides, fraction D possessed a significant comitogenic effect, and the SIcomit/SImit ratio 3-4 indicates potential adjuvant properties of this glucuronoxylan-rich material.

Interaction of bromine with iodine in the rat thyroid gland at enhanced bromide intake.

In experiments with rats, we have found that at enhanced intake of bromide, bromine does not replace chlorine in the thyroid; it replaces iodine. Under our experimental conditions, more than one-third of the iodine content in the thyroid was replaced by bromine. In the thyroid, bromine probably remained in the form of bromide and, in proportional to its increased concentration, the production of iodinated thyronines decreased, with the sum of the iodine and bromine concentrations being constant at the value of 20.51 +/- 1.16 mumol/g dry wt of the thyroid. In contrast to other organs, the biological behavior of bromine in the thyroid is not similar to the biological behavior of chlorine but resembles more that of iodine.

Bromide kinetics and distribution in the rat. I. Biokinetics of 82Br-bromide.

Biological half-lives of bromine in 15 different organs and tissues of the rat, in addition to the whole-body half-life, were determined by measuring the radioactive concentration of 82Br-bromide in samples of tissues collected at the time intervals of 12-396 h from animals that continuously (up to 17 d) received 82Br-labeled bromide in their drinking water. The half-life values, calculated from the experimental data by the method of gradual estimates of the parameters in question with the SPSS statistical program, ranged from 94.3+/-14.6 h in the thyroid gland to 235.0+/-88.9 h in liver. In most of the studied tissues, the biological half-lives of bromine were shorter than in the whole body, in which it equaled 197.8+/-22.2 h. Significant correlation between the values of the steady-state concentration of bromide and of the biological half-life was found for most tissues (except for liver). The steady-state concentrations of 82Br in tissues are probably proportional to the magnitude of bromide space, and, consequently, of chloride space.

In vitro studies of cell-mediated immunity in rabbits sensitized with Mycobacterium kansasii and Mycobacterium tuberculosis.

Rabbits, sensitized with M. kansasii, responded by a profound inhibition of migration of macrophages elicited by both antigens: the migration index for homologous antigen was 0.51 in the direct test and 0.65 in the indirect test; for heterologous antigen the indexes were 0.53 and 0.67. However, significant differences in reactivity were found in rabbits sensitized with M. tuberculosis. In the homologous system, high reactivity was maintained and the migration index reached the value of 0.53 in the direct and 0.63 in the indirect test. On the other hand, the heterologous antigen M. kansasii influenced the migration in both direct and indirect assays significantly less, the migration indexes being 0.62 and 0.72. The differences were statistically significant at 1% and 5% levels.

Detection of respiratory syncytial virus serum antibodies by an ELISA system.

An ELISA test for respiratory syncytial (RS) virus assay was adapted and standardized; it gave 10-15 times higher antibody titres than complement fixation (CF) but was not a more sensitive test for detecting recent RS virus infection in persons above 1 year of age. In testing normal-population sers, ELISA revealed twice more positive sera than the CF test. Owing to its high sensitivity and apparent ability to detect long-persisting antibodies, ELISA is the test of choice for sero-epidemiological surveys on RS virus infections.

A laboratory study of age-related varicella incidence and prevalence in the Czech Socialist Republic.

During 1972-1981, ten representative population samples totalling 2,916 individuals were tested for antibodies to varicella-zoster virus (VZV) by an indirect-haemagglutination assay (IHA). Statistical analysis of the results provided estimates of age-related varicella prevalence and incidence rates. It transpired that 45% of the child population had encountered varicella at preschool age and another 45% during the attendance of school. Adult seropositivity rates amounted to 97.5-100%. The highest varicella incidence was observed in the 4-year age interval of 2-6 years. For control, additional 324 and 297 individuals were tested by RIA and ELISA IgG, respectively; there was good correlation of results. For the period 1970-1985 varicella morbidity was also studied from notification data. As far as the authors are aware, the present investigation performed on a total of 3,537 subjects furnishes the most comprehensive information on nation-wide varicella incidence and prevalence obtained by laboratory methods.

Antibodies to human coronaviruses 229E and OC43 in the population of C.R.

Of 1200 human sera tested by enzyme-linked immunosorbent assay (ELISA), 53% contained antibodies to human coronavirus (HCV) 229E and 88% to HCV OC43. The sera were from persons aged 13 months to 80 years, both males and females, and were collected in four different regions in 1986. The percentage of positives increased with increasing age and tended to vary according to the geographic area. Additional paired human sera from 218 patients with acute respiratory diseases (ARD) were collected between October 1986 and June 1987 in Prague. Significant antibody rises to HCV strain 229E were detected in 7 (3.2%) patients 9 months to 17 years old, to HCV strain OC43 in 4 (1.8%) patients under 2 years of age.

Cell-mediated immunity to varicella-zoster virus as assessed by the migration inhibition test.

The migration inhibition method was used to test cell-mediated immunity to varicella-zoster (VZ) virus in 10 varicella and 11 herpes zoster patients. Control groups consisted of eight children susceptible to VZ infection on serological evidence and 49 normal persons of different age categories. Depending on the positivity criterion adopted, positive results during disease were obtained in 43% or 90% or all tests performed in varicella patients and 47% or 74% in herpes zoster patients. Irrespective of which criterion of positivity was applied, a high rate of positive results was obtained in the normal control groups; in the age range from 20--44 years it was comparable to that for patients. This finding would suggest a high activity of VZ virus among the human population. Since a positive result in migration inhibition test offers evidence of recent contact with antigen, either exogeneous contact with VZ infection or endogenous contact with latent VZ virus must have been involved.