Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions.

Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes involved in remodeling of extracellular matrix. Although proteolytic enzymes are produced by many cell types, mast cells seem to be more important than other types in remodeling of pulmonary arteries during hypoxia. Therefore, we tested in vitro production of MMPs and serine proteases in four cell types (mast cells, fibroblasts, vascular smooth muscle cells and endothelial cells) cultivated for 48 h under normoxic or hypoxic (3% O2) conditions. MMP-13 was visualized by immunohistochemistry, MMP-2 and MMP-9 were detected by zymography in cell lysates. Enzymatic activities (MMPs, tryptase and chymase) were estimated in the cultivation media. Hypoxia had a minimal effect on total MMP activity in the cultivation media of all types of cells, but immunofluorescence revealed higher intensity of MMP-13 in the cells exposed to hypoxia except of fibroblasts. Tryptase activity was three times higher and chymase activity twice higher in mast cells cultivated in hypoxia than in those cultured in normoxia. Among all cell types studied here, mast cells are the most abundant source of proteolytic enzymes under normoxic and hypoxic conditions. Moreover, in these cells hypoxia increases the production of both specific serine proteases tryptase and chymase, which can act as MMPs activators.

In vitro hypoxia increases production of matrix metalloproteinases and tryptase in isolated rat lung mast cells.

Chronic hypoxia results in hypoxic pulmonary hypertension characterized by fibrotization and muscularization of the walls of peripheral pulmonary arteries. This vessel remodeling is accompanied by an increase in the amount of lung mast cells (LMC) and the presence of small collagen cleavage products in the vessel walls. We hypothesize that hypoxia activates LMC, which release matrix metalloproteinases (MMPs) cleaving collagen and starting increased turnover of connective tissue proteins. This study was designed to determine whether in vitro hypoxia stimulates production of MMPs in rat LMC and increases their collagenolytic activity. The LMC were separated on the Percoll gradient and then were divided into two groups and cultivated for 24 h in 21 % O(2) + 5 % CO(2) or in 10 % O(2) + 5 % CO(2). Presence of the rat interstitial tissue collagenase (MMP-13) in LMC was visualized by immunohistological staining and confirmed by Western blot analysis. Total MMPs activity and tryptase activity were measured in both cultivation media and cellular extracts. Exposure to hypoxia in vitro increased the amount of cells positively labeled by anti-MMP-13 antibody as well as activities of all measured enzymes. The results therefore support the concept that LMC are an important source of increased collagenolytic activity in chronic hypoxia.

Inhalation trauma in burns, cytology and biochemical findings.

We have followed in a pilot study a group of patients for cytological and biochemical changes of lavage in the upper and lower respiratory system. Into the study patients with respiratory burns confirmed by bronchoscopy and skiagraphy were included. We divided patients according to the Lung Injury Scores (LIS). We obtained the values in our sample group between intubation and last swab (before extubation). Listed are the risk factors, probability of survival, and lung histology results are listed for patients who died.

[Findings in bronchial lavage in patients with burns of the upper respiratory tract]. / Nálezy v bronchiálních lavázích u pacientu s popálením horních dýchacích cest.

The study is focused on cytologic and biochemical changes in bronchoalveolar lavage (BAL) at the group of patients from the Clinic of Burns Medicine at the University Hospital Královské Vinohrady in Prague. Only patients with inhalation trauma were involved in the study. The obtained data are from BAL measurements collected between intubation and extubation. There was the greater number of polymorphonuclears in the first two weeks in all patients. After 2-3 weeks there was a gradual increase in the number of macrophages at four patients and three patients had still the greater number of polymorphonuclears. There was a positive findings of PCNA at one patient and other markers (bcl-2, p53) were negative. The cytoflowmetry proved the increase in number of CD3 cells by 20% in one patient. The amount of proteins in the last BAL just before extubation was lower than 22 g/l. Serin proteinase activity was lower than 50 ncat/l at five patients and the ratio of MMP-8/TIMP-2 was higher than one in all analysed samples. There was no development of lung inflammation into chronic stadium. Two patients died.

[The exogenous surfactant and its effect on the respiratory distress syndrome (RDS)]. / Exogenní surfaktant a jeho vliv na syndrom respiracní tísne (RDS).

In this article the pathophysiology and diagnosis of neonatal respiratory distress syndrome as a primarily form of surfactant deficiency is disclosed. Attention is paid to surfactant composition, productive and secretion in the alveoli and metabolic turnover as well. From clinical point the view basic principles concerning the surfactant replacement is discussed. Surfactant minimizes the surface tension and friction between the gas molecules and lung tissues during breathing. It also helps keep lung tissues dry and alveoli patent. At 26 to 28 weeks of gestation alveolar ducts and bronchioles are present but pulmonary capillaries are not in close contact with them. Alveoli may not even be distinguishable. Surfactant develops all through gestation but does not surge until about the 34th week. Infants born before 35 weeks of gestation are at risk for respiratory distress syndrome which is at first characterised by decrease of lung compliance. It leads to progressive respiratory failure.

[Changes in bone metabolism in girls with Turner's syndrome]. / Zmeny kostního metabolismu u dívek s Turnerovým syndromem.

BACKGROUND: Data of the prevalence of osteoporosis in girls with Turner's syndrome are not uniform, and its causes have not been fully elucidated. Information on the mineralization of osseous tissue is controversial. The objective of the present work was to examine some more recent indicators of bone metabolism in a group of girls with Turner's syndrome. METHODS AND RESULTS: A group of girls aged 4-20 years was examined where genetic examination had revealed karyotype 45 X (7 patients) or mosaic 45 X (46 XX) (9 patients). On X-ray examination osteoporosis was found in 71%, densitometric evidence of reduced bone density was provided in 2% of the examined patients. As to biochemical osteologically oriented examinations, a significantly reduced osteocalcin value was found (1.18 +/- 0.42 mug/l, as compared with 11.38 +/- 0.03 in controls, p < 0.001), reduced values of alkaline phosphatase (2.67 mukat/l as compared with 8.46 +/- 4.16 mukat/l in controls, p < 0.005) and reduced values of the bone isoenzyme of alkaline phosphatase (1.47 mukat/l as compared with 6.04 +/- 0.27 in controls, p < 0.001). The values of calcemia (2.63 / 0.13 mmol/l) and phosphataemia (1.36 +/- 0.25 mmol/l) did not differ significantly from values recorded in controls. To six patients the authors administered for a period of three months 1,25(OH)2D3, 0.25 mg on alternate days. The osteocalcin values rose to 5.27 +/- 3.14 micrograms/l, similarly as alkaline phosphatase (6.32 +/- 1.83 mukat/l) and the bone isoenzyme of alkaline phosphatase (2.42 +/- 1.95 mukat/l), not evaluated statistically because of the small number of patients. CONCLUSIONS: In girl with Turner's syndrome a reduced bone density was revealed in 25%, reduced values of osteocalcin, alkaline phosphatase and its bone isoenzyme indicate a reduced osteoblast activity. It appears that administration of 1,25(OH)2D3 can have a favourable effect.

[Growth in children with the exudative enteropathy syndrome due to a congenital heart defect--cor triatriatum dextrum]. / Rust u dítete se syndromem exsudativní enteropatie na podklade vrozené srdecní vady--cor triatriatum dextrum.

Retarded growth in a child can be the sign of serious chronic disease. The authors present an account of a six-year-old boy where growth retardation persisted at least from the age of three. During this period his height dropped from the zone between the 25th and 50th percentile into the zone between the 3rd and 10th percentile. From the clinical point of view a large abdomen, loose stools and hypocalcaemia with tetany were striking, as they were moreover refractory to vitamin D2, calcitriol and calcium administration by the oral route. The authors revealed severe hypoproteinaemia, a 150 times increased value of alpha-1-antitrypsin in faeces, and exudative enteropathy syndrome was diagnosed. The cause was venous congestion due to a rare heart disease--cor triatriatum dextrum. The septum in the right atrium was resected. Immediately after surgery the consistency and frequency of stool decreased. Calcaemia and plasma protein levels reached normal levels within two months. A growth spurt of 11 cm/year followed. Fifteen months after operation the patient's height reached almost the 50th percentile.

[Functional determination of C1-inhibitor in plasma with a low level of prekallikrein]. / Funkcní stanovení C1-inhibitoru v plazmách se snízenou hladinou prekalikreinu.

The method of assessment of the C1-inhibitor (C1-INH) based on activation of plasma precallicrein (PC) by the fragment of Hageman's factor (HFf) was described in a previous publication (6). It was recommended as an alternative of already described methods used for assessment of C1-INH. To define the reliability of the recommended method in plasma with a reduced PC level the authors used plasma of mothers where during childbirth as a rule a steep drop of this proenzyme occurs. In a group of 23 parturient women the plasma PC levels were assessed and were within the range from 0.067-0.397 U/ml incl. only 6 plasma specimens where the PC level varied within the range of normal donors, i.e. 0.319-0.438 U/ml (mean value mean = 0.369 +/- 0.035). The mean value in plasma of the mothers was 0.237 +/- 0.100. In both groups the C1-INH was assessed by the recommended method and by the method of callicrein inactivation. In the group of mothers the results of C1-INH correlated with estimations in plasma where the PC level was within the range from 42.5 to 107.6% of the mean PC value of donors (r = 0.82). In plasmas of mothers whose levels varied from 18.2 to 50.2% of the mean value of PC donors the assessment of C1-INH by the method of HF fragment activation gave lower results in all instances. The dependence of C1-INH estimation on the amount of PC was demonstrated on deficient plasma with a defined PC level.(ABSTRACT TRUNCATED AT 250 WORDS)

[C1-inhibitor plasma levels in neonates and mothers during labor]. / Hladiny C1-inhibitoru v plazme novorozencu a matek pri porodu.

The authors investigated C1-inhibitor (C1-INH) plasma levels in blood collected from mothers during delivery: mothers with a normal pregnancy and a group of mothers who displayed during the perinatal period direct or indirect signs of infectious disease. Both groups, as compared with a group of healthy donors, had low C1-INH levels. The neonates were divided into two groups (normal children and children with perinatal risk of infection) with sub-groups of mature full-term neonates (38-41 weeks of gestation) and premature infants (29-36 weeks of gestation). In the group of normal infants the neonates had on average somewhat lower C1-INH levels, as compared with healthy donors, in premature neonates of this group the lowest mean C1-INH level was recorded. In this group the authors observed a correlation between the inhibitor level and the gestation period. The results of C1-INH assessment in the group of neonates with a perinatal risk of infection were different. In premature neonates a higher average C1-INH level was observed than in mature neonates and the relationship between the C1-INH level and the gestation period was a linear negative regression. The postnatal increase of C1-INH levels on the 1st to 5th day was more rapid in premature neonates of both groups.

[Alkaline phosphatase activity and bone isoenzymes in children treated with lithium]. / Aktivita alkalické fosfatázy a kostního izoenzymu u detí lécených lithiem.

The acute stage of manic depressive psychosis before the onset of lithiotherapy is characterized, similarly as the group of patients with the diagnosis of schizoaffective psychosis, by low levels of bone isoenzyme of alkaline phosphatase and normal values of alkaline phosphatase which in the course of lithium treatment decline markedly, similarly as in patients with unipolar manic depressive psychosis. The mentioned changes are very probably associated with changes of the extracellular calcium homeostasis. Schizophrenic patients do not have the described changes and the activities of AP and bone isoenzyme are the same before and after lithiotherapy.

[Classification of acute pancreatitis in correlation with serum protease levels]. / Klasifikace akutní pankreatitidy v korelaci s hladinou sérových proteáz.

Based on previous work of their own and data in the literature the authors outlined diagnostic and classification criteria of acute pancreatitis. The latter were applied in a prospective study. Thus a group of 71 patients with acute pancreatitis was obtained. In 10 patients of this group at the same time intervals and while the same therapeutic procedures were used, the serum protease levels were followed up. The authors assumed that it will be possible to express thus objectively the severity of the disease and to diagnose in time necrotizing forms of acute pancreatitis. The results re demonstrated the feasibility and suitability of this procedure only for assessment of the severity of the disease.

[The fatty acid spectrum in the serum of mothers and infants after premature delivery]. / Spektrum mastných kyselin v séru matek a detí po predcasném porodu.

Using the method of gas chromatography on a Carlo Erba 2531 apparatus in Base's modification, the authors identified serum fatty acids of mothers (n = 23) and premature infants (17 girls and 6 boys). The mean birth weight was 2224.3 (800-2650 g), the mean gestation age 34 weeks (28-37 weeks). The fatty acid levels were compared with fatty acid levels of control mothers (n = 7) and control healthy neonates born at term (n = 15). Individual fatty acids were expressed as the ratio in the total volume of fatty acids (100%). The neonates and their mothers were divided into three groups. The most numerous group (n = 12) are mothers who lost the amniotic fluid prematurely (6 hours-12 days), mothers under the influence of Partusisten (n = 10); the third group is formed by mothers treated with ATB (n = 8). In these groups the authors assessed, as compared with controls, the rise of the saturated fatty acid ratio, in particular short-chain fatty acids. The greatest increase was recorded in mothers and children under the influence of Partusisten. The ratio of mono- and polyunsaturated n-6 fatty acids was not altered in the infants, there was, however, a marked reduction of the n-3 fatty acid ratio which are known to play an important role in the regulation of immune responses and to have an anti-inflammatory effect; they are also important for the development of the CNS and retina.(ABSTRACT TRUNCATED AT 250 WORDS)

[Determination of prekallikrein in the plasma of neonates]. / Stanovení prekallikreinu v plazme novorozencu.

The authors assessed plasma prekallikrein (PK) after its conversion into the active enzyme kallikrein from the rate of breakdown of the specific chromogenic substrate NO-Pro-Pre-Arg-pNA. Activation of PK was achieved by the parallel contact method, using dextran sulphate (DS) and by direct activation by the Hageman's factor fragment (HFf). Contact activation assumes the presence of Hageman's factor (HF) and high molecular kininogen (HMW-K) in plasma. Both methods of activation and x = 0,451 +/- 0.146 U/ml in HFf activation). The mean PK value in neonates assessed by activation of DS was lower [mean = 0.105 +/- 0.068 U/ml] than the value assessed by activation of HFf [mean = 0.175 +/- 0.072 U/ml]. In adults the PK values varied at a significantly higher level, the results being higher when HFf was used as activator [mean = 0.346 +/- 0.087 U/ml in DS activation and mean = 0.451 +/- 0.146 U/ml in HFf activation]. The increase of mean values of PK assessed in umbilical blood and on the 3rd and 5th day after delivery was more marked in direct HFf activation. The correlation coefficient of both ways of assessment in neonates was r = 0.79, in the group of adults there was a close correlation r = 0.92. The lower correlation in neonates can be explained by the low level of co-factors, in particular HMW-K, necessary for complete activation of PK by the contact method. In some neonates the PK value assessed by DS expresses only the total activating plasma capacity and not the actual PK level.

[Strategies in prenatal diagnosis of cystic fibrosis after the introduction of DNA analysis. Initial experience]. / Strategie prenatální diagnostiky cystické fibrózy po zavedení analýzy DNA. Prvé zkusenosti.

The authors describe their experience with the prenatal genetic diagnosis of cystic fibrosis (CF), using DNA analysis in the first trimester of pregnancy in three families with a 25% risk of CF. The authors examined polymorphisms of probes J3.11, met D, met H, KM-19 and XV-2c. All families were fully informative when one or two probes were used. In two families the development of unaffected children--carriers of the gene for CF was proved. In one of these foetuses in the 17th and 21st week false pathological values of microvilillous enzymes were assessed. With regard to this possibility the authors do not recommend to supplement the DNA analysis in the first trimester by biochemical examination of amniotic fluid. The results were confirmed by delivery of unaffected children. In one family DNA analysis revealed the development of an unaffected homozygote, the pregnancy was, however, terminated by a miscarriage. In women with an increased risk of abortion the authors recommend therefore to make the molecular genetic examination during the second trimester from amniotic fluid cells.

The experience with the foetal diagnosis of the cystic fibrosis in the second and first trimester.

The amniotic fluid activity of gamma glutamyl transpeptidase (GGT), leucine aminopeptidase (LAP) and alcaline phosphatase (AP) and disacharidases was examined in 66 pregnancies with the risk of cystic fibrosis (CF) in the 17th-21st weeks of gestation. So far 28 pregnancies continue. The prenatal diagnosis was confirmed in all so far delivered children or aborted foetuses if the GGT activity was higher than 400 U/1 (10th percentile) or lower than 190 U/1 (3rd percentile) in the 17th-18th weeks. The results of other microvillar and ultrasound examinations were consistent with it. From 3 pregnancies with GGT activity in the range of 3-5 percentiles and abnormal activities of other microvillar enzymes, the CF was confirmed only in one aborted foetus with meconium ileus and with abnormal ultrasound examination. In other 2 pregnancies with normal ultrasound, healthy children were delivered. In 3 pregnancies with the GGT in the range of 5-10 percentiles and abnormal other microvillar enzymes, one false negative GGT and ultrasound examination was disclosed. The other 2 aborted foetuses did not exhibit the signs of CF in necropsy examinations. The meconium ileus was found in 2/4 of aborted foetuses with GGT lower than 3 percentiles, abnormal activities of other microvillar enzymes and abnormal ultrasound examination. The ultrasound examination was correct in 2/10 of pregnancies with GGT lower than 3 percentiles or abnormal activities of other microvillar enzymes. The GGT examination in 19th-21st weeks provided similarly reliable diagnostic results. The importance of fetal karyotyping and ultrasound elimination of other severe congenital anomalies is pointed out for critical interpretation of microvillar enzyme activities testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of intravenously administered intralipid on free fatty acid concentration and albumin binding capacity to bilirubin in newborn infants.

During a 24-hour interval, examination was carried out of 5 full-term and 4 preterm newborns in whom no disturbance of postnatal adaptation was found. The newborns received parenteral nutrition using infusates containing 20% Intralipid during 7 hours on the average. The mean daily dose of Intralipid was 1.2 g/kg, infusion rate was 0.17 g/kg/h. The concentration of FFA (n = 9) and ABC/Bi was examined three times in the course of 24 hours, and that prior to Intralipid administration, after one-hour infusion, and approximately 17 hours after the end of infusion. Before infusion and 24 hours after the first collection, blood bilirubin and glucose concentrations, and the values of pH, pCO2 and BE (= 9) were examined. One hour after the start of Intralipid infusion the mean FFA concentration was reduced and decreased again during the following 23 hours. The differences were mostly insignificant. One hour after the start of infusion the mean value of the ABC/Bi ratio decreased insignificantly, but returned to the initial value by the end of the follow-up period (24 hours). The mean concentration of bilirubin and blood glucose and the values of pH, pCO2 and BE did not change significantly. It may be reasonably assumed, that in newborns showing normal postnatal adaptation in the first days of life, the administration of 20% Intralipid (as parenteral nutrition) exerts no substantial effect on the plasma FFA concentration and ABC/Bi ratio in the course of 24 hours. General principles for the indication of parenteral Intralipid administration to the newborn are given.

Postnatal development of plasma FFA concentration and its influence of the separation of bilirubin from albumin binding in newborns.

The first part of the study gives data on the FFA concentrations, glycaemia and energy quotient in newborns with uncomplicated postnatal adaptation in the first 2 weeks of life. The study was carried out in a group of 69 full-term and 69 pre-term infants at the age of 24, 48, 72, 96 hours and 7 and 14 days. Although it is not the case of consecutive values of FFA concentrations in plasma, a certain developmental trend can be suggested. This is analogous in both groups with the only difference that in pre-term infants FFA culminate and decrease earlier (pre-term 48-72 hours, full-term infants 72-96 hours). The study shows that, in pre-term and full-term infants with satisfactory postnatal adaptation and above nutrition, stabilization of glucose-lipid metabolism occurs on the 3rd and 4th day after birth, respectively. In pre-term infants supplementary nutrition by the glucose system (glucose and amino acids) had a favourable effect. The second part of the study assesses the potential risk of increasing plasma FFA concentration for the separation of bilirubin from albumin binding. The means plasma FFA concentrations reached 25-48 and 49-72 hours after birth had no significant effect on the BCA/Bi ratio and, therefore, they are not dangerous as regards the possibility of displacing bilirubin from albumin binding.