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.

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.

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.

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.

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.

Non-apoptotic functions of caspase-7 during osteogenesis.

Caspase-3 and -7 are generally known for their central role in the execution of apoptosis. However, their function is not limited to apoptosis and under specific conditions activation has been linked to proliferation or differentiation of specialised cell types. In the present study, we followed the localisation of the activated form of caspase-7 during intramembranous (alveolar and mandibular bones) and endochondral (long bones of limbs) ossification in mice. In both bone types, the activated form of caspase-7 was detected from the beginning of ossification during embryonic development and persisted postnatally. The bone status was investigated by microCT in both wild-type and caspase-7-deficient adult mice. Intramembranous bone in mutant mice displayed a statistically significant decrease in volume while the mineral density was not altered. Conversely, endochondral bone showed constant volume but a significant decrease in mineral density in caspase-7 knock-out mice. Cleaved caspase-7 was present in a number of cells that did not show signs of apoptosis. PCR array analysis of the mandibular bone of caspase-7-deficient versus wild-type mice pointed to a significant decrease in mRNA levels for Msx1 and Smad1 in early bone formation. These observations might explain the decrease in the alveolar bone volume of adult knock-out mice. In conclusion, this study is the first to report a non-apoptotic function of caspase-7 in osteogenesis and also demonstrates further specificities in endochondral versus intramembranous ossification.

Mybs in mouse hair follicle development.

The Myb transcription factors are involved in essential cellular processes, such as cell proliferation, differentiation and cell death. Biological functions carried out by specific Myb proteins are distinct. Hair follicles are ectodermal-derived organs with cycling character of the growth resulting from the presence of somatic stem cells. In this study, we followed the expression of the Myb proteins in developing hair follicles and in the hair follicle stem cell niche by immunofluorescence staining. During hair follicle development, B-Myb was present in a few cells located in the area of cell division; c-Myb was abundant postanally in dividing cells but also in keratinizing zone. In addition, c-Myb was also detected in cells under the hair follicle bulge. These findings indicate possible involvement of c-Myb in regulation of activated stem cells leaving the niche.

Caspase-7 in molar tooth development.

OBJECTIVES: The primary enamel knot (PEK) is a population of cells that shows spatio-temporal restricted apoptosis during tooth development. It has been shown that caspase-9 and Apaf-1 are essential for apoptosis in the PEK as well as the central caspase-3. Caspase-7, as another executioner member in the caspase machinery, is considered to have caspase-3 like properties. DESIGN: The aim of this study was to detect caspase-7 activation during molar tooth development with a special focus on the cells of the PEK and to correlate the expression with the pattern of apoptosis and caspase-3 activation. Apoptosis in the PEK was investigated in caspase-7 deficient mice to examine the functional consequence of loss of this specific caspase. In addition, odontoblasts and ameloblasts, which are known to undergo cell death during their secretory and maturation stages, were investigated. RESULTS: Cleaved caspase-7 was found in the apoptotic region of the PEK, however, caspase-7-deficient mice still possessed apoptotic cells in the PEK in a similar distribution to the wild type. Caspase-7 is therefore not essential for apoptosis in the PEK. Notably, cleaved caspase-7-positive cells were found at later stages in odontoblasts and ameloblasts, but expression did not correlate with apoptosis in these tissues. CONCLUSIONS: The results indicate a non-essential apoptotic role of caspase-7 in the PEK apoptosis but suggest also possible non-apoptotic functions for caspase-7 in tooth development.

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.

[Results of treatment of malignant stenoses of the respiratory tract in a long term study]. / Výsledky lécby maligních stenóz dýchacích cest v dlouhodobém sledování.

BACKGROUND: :The malignant stenoses complicate the course of the disease in about 50% of patients with lung tumors. Management of them can influence the quality of life of the afflicted. There is no general agreement about optimal methods of their therapy, of selection of the best methods and also there is only few data about survival of these patients. METHODS AND RESULTS: 130 patients with tumors stenoses of the airways were prospectively followed in the 1998-2003 period, 93 of them suffered from lung cancer. Besides other treatment of the tumor they underwent also interventional bronchoscopy (Nd YAG laser, electrocautery i.e.). The airways were opened completely in 62% of the set of patients. The interventional bronchoscopy was complicated in 9% of patients, 32 patients lived at the 31st December of 2003, 98 of them had died. One year survival was 23.1%, median survival of the whole set of patients was 7 months. The patients with lung cancer had significantly worse survival than patients with other types of lung tumors, 6 months versus 10 months. There were no significant survival differences of the whole set according to the achieved grade of recanalisation, or according the inclusion of the brachytherapy to the set of interventional methods. The patients with inserted stent had significantly worse prognosis than the those without stenting. To homogenize the study group, the subset of patients with epidermoid lung cancer in the stage IIIB were selected (n = 51). Significantly longer survival of patients with complete recanalisation, insignificant longer survival in patients who underwent also brachytherapy and significantly shorter survival in patients with stenting were proved in this subgroup of patients. CONCLUSIONS: The interventional bronchoscopy has a low rate of complications and in most cases it has a palliative effect. The survival of patients is more influenced by the extension and the type of the tumor, their general status, polymorbidity, age etc. than by effect or selection of interventional bronchoscopical methods.

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.

The 2001 serological survey in the Czech Republic--measles.

In total, 3,013 sera from a representative population sample of the Czech Republic aged from 1 to 64 years were tested according to the requirements of the ESEN 2 project using a commercial kit Measles IgG (II) EIA manufactured by Denka Seiken, Japan. The highest seropositivity rate and GMT values were found in the population group aged over 35 years coming into natural contact with the causative agent. Among the vaccinee population, the best outcomes were achieved in 2-9-year-olds (seropositivity rate: 94-100%, GMT: 2500-4000 EIA) and 10-14-year-olds (seropositivity rate: 93-97.1%, GMT almost 2000 EIA). The lowest seropositivity rate was found in the age group 20 to 24 years, more precisely in the 20-year-olds (78.4%), vaccinated with only one dose before the end of the first year of life. The seropositivity rates and GMT values vary with the vaccination strategies adopted in different years. The morbidity trend is supportive of the appropriateness of the vaccination strategy selected.

The 2001 serological survey in the Czech Republic--rubella.

In total, 3,009 sera from a representative population sample of the Czech Republic, 1508 females and 1,501 males, were tested by EIA using a commercial kit ETI-RUBEK-G Plus manufactured by Dia Sorin, Italy. The vaccination strategy was as follows: vaccination of 12-year-old girls since 1982 and all 2-year-olds vaccinated since 1986. Currently, all women under 31 years of age and all men under 17 years of age have been vaccinated. The results of the serological survey confirmed lower seropositivity rates in young men compared to vaccinated women of the same age as expected. An epidemic outbreak of rubeola among young men is another evidence of that The seropositivity rate of 2- 9-year-old vaccinee children reaches almost 95%. The long-term morbidity trend in the Czech Republic is downward sloping, with a marked differentiation between the male and female populations.

The 2001 serological survey in the Czech Republic--mumps.

In total, 3,010 sera from a representative population sample of the Czech Republic aged from 1 to 64 years were tested according to the requirements of the ESEN 2 project using a commercial kit Mumps IgG (II) EIA manufactured by Denka Seiken, Japan. To date, the regular programme of vaccination against mumps has covered the population of children under 15 years of age. The vaccination coverage achieved (97-100%) does not correspond to the antibody prevalence rates of 70.2 to 86.4% as found. After implementation of regular vaccination in 1987, the morbidity rates have fallen dramatically. Nevertheless, the collective immunity achieved is not sufficient to prevent epidemics of mumps.

The 2001 serological survey in the Czech Republic--viral hepatitis.

Within serological surveys 2001, prevalence of markers of hepatitis viruses A (anti-HAV), B (anti-HBc, HBsAg, anti-HBs) and for the first time also C (anti-HCV) was investigated. Sera were collected in 2001 and tested by respective kits AxSYM, Abbott. HAV: 2,623 sera were tested for the presence of anti-HAV antibodies. Comparison with serological surveys of 1984 and 1996 revealed again shifts of the age prevalence curve for anti-HAV antibodies towards higher age groups corresponding to time intervals between epidemiological surveys. High prevalence rates of anti-HAV antibodies (more than 20%) were only found for the population age groups who lived in the period of high incidence of VHA, i.e. up to 1965. The prevalence of anti-HAV antibodies increased by about 5-10% in the population under 20 years of age, the increase being significant and assumingly attributable to vaccination against VHA, and remained the same as in 1996 in the age group 20-29 years. HBV: 2,568 sera were tested for the presence of anti-HBc antibodies and 76 reactive specimens were further tested for the presence of HBsAg and anti-HBs antibodies. The prevalence of anti-HBc antibodies continuously increases with age. The total prevalence of anti-HBc antibodies calculated for the Czech population is 5.59% compared to 6.95% recorded in 1996. The calculated prevalence rate of HBsAg is 0.56% and that of anti-HBs antibodies is 3.99% for the non-vaccinee population. HCV: The prevalence rate of anti-HCV antibodies was 0.2% with 6 out of 2,950 sera testing positive. Age dependence could not be assessed because of the small number of positive persons. HCV infection is known to afflict high-risk groups, likely to escape a general serological survey, rather than the normal population.

Effect of acrylate chemistry on human health.

OBJECTIVES: The prospective cohort study of 1992-1999 describes the effect of occupational exposure to chemical substances in the production of acrylic acid, acrylic acid esters and acrylate dispersions at the various workplaces of one chemical plant. METHODS: Exposure to selected chemicals (acrylonitrile, n-butanol, butyl acrylate, ethyl acrylate, methyl acrylate, methyl methacrylate, toluene, and styrene) was determined by personal passive dosimetry (GC/MS method). The annual examinations included general health, by guided interview, a general medical examination, hematological and biochemical examinations, examination of the parameters of serum immunity and selected tumor markers, and spirometry. The authors also repeatedly performed cytogenetic analysis of human peripheral lymphocytes. RESULTS: The authors followed a group of 120 employees (60 exposed, 60 controls), mean age 40+/-8 years in both groups, with average period of exposure to chemicals (exposed group) 13+/-5 years. The measured concentrations of chemicals in the working atmosphere were generally low; maximum allowable concentrations (MAC) values or suggested limits of certain chemicals were occasionally exceeded (most frequently for butyl acrylate). The results of the examination of the workers over the 8 years have not revealed any marked differences between the exposed and control groups that could be attributable solely to the acrylate exposure. CONCLUSIONS: Exposure to chemical substances at the workplace was relatively low, the limits being exceeded only sporadically (each such case was investigated at the workplace), and the level of exposure continues to decrease gradually over the years. Considering the fact that the exposed individuals are expected to work for 23 additional years on average, we feel that long-term monitoring of selected health-related parameters, not including tumor markers, appears desirable. The examination of tumor markers has not contributed to the problem evaluation for a number of false-positive results.

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.

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.

[A statistical method for long-term monitoring of selected causes of death]. / Statistická metodika dlouhodobého sledování úmrtí na vybrané príciny smrti.

The death rate statistics belongs to the essential health parameters and it is therefore frequently analysed. Authors suggest reviewing problems, which could be met, and discussing statistical methods in order to make the comparison and analysis of the development as much corresponding to the actual health state of the population as possible. Authors also discuss methods of the "International classification of diseases" and describe methods of direct data standardisation, including estimation of the variability. Using respiratory diseases as an example, authors illustrate the problems of revision of the code system in the "International classification of diseases" and the give examples of numerical evaluation.