Roman Janas

Endothelin-1 inactivating peptidase in the human kidney and urine.

Objective Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology. Methods Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37°C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [125I]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography. The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft. Results ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155–1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (Rf 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (KM180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (KM11.5 μmol/l), endothelin 11–21 fragment (KM15.3 μmol/l), endothelin antagonist Trp- Leu-Asp-Ile-Ile-Trp (KM3.1 μmol/l), gastrin (KM2.2 μmol/l) and cholecystokinin (KM4.0 μmol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme. The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 μmol/l), cyclosporin (IC50 400 μmol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l). Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft. Conclusions Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system.

Interactions between the growth hormone and cytokines – A review

Numerous reports on the interactions between the immune and endocrine systems, especially growth hormone axis, can be found in the literature. Growth hormone acts mainly indirectly through insulin-like growth factor-1, which stimulates the growth and development processes, metabolism of lipids, proteins, and carbohydrates, and it also has a modulating effect on the cells of the immune system. Several studies have been conducted on the influence of growth hormone therapy on the immunological parameters in children and adults with and without growth hormone deficiency. However, there have been no definite results and some of them have been even contradictory. Some studies have suggested that administration of growth hormone increases the production of tumor necrosis factor and certain pro- and anti-inflammatory cytokines; whereas other studies have demonstrated the lack of correlation between growth hormone and interleukins. The aim of this paper was to evaluate the available literature on the interaction between growth hormone and TNF-α, pro-inflammatory (IL-1β, IL-2, IL-6) and anti-inflammatory (IL-4, IL-10) interleukins.

Evaluation of the immunoradiometric and electrochemiluminescence method for the measurement of serum insulin in children

Human insulin is a polypeptide hormone produced, stored, and secreted by the ß-cells in the pancreatic islets of Langerhans. Its secretion is stimulated by an increase of the glucose concentration in circulation. Non-radioactive assays are frequently used in many laboratories to measure hormone concentrations, as an alternative to the traditional “gold standard” radioimmuno- and immunoradiometric assays. The precise and reliable determination of the insulin concentration is an important concern in numerous diagnostic procedures. The aim of this study was to compare two commercially available assays (manual and automated) for measurement of serum insulin concentrations. Aliquots of the 86 randomly selected serum samples were measured by Elecsys Insulin Assay (cobas e411 immunoassay analyzer, Roche Diagnostics GmbH, Mannheim, Germany) and DIAsource INS-IRMA Kit (DIAsource ImmunoAssays S.A., Louvain-la-Neuve, Belgium). Compared assays exhibit good correlation (r = 0.996). Insulin concentrations were on average 4.2 μIU/mL lower (p < 0.05) with the cobas e411 immunoassay analyzer when compared to those measured with DIAsouce Immunoassay. Our findings suggest that electrochemiluminescence method on the cobas e411 analyzer and manual IRMA method offered by the DIAsource for the serum insulin determination could be considered interchangeable.

Generation and Identification of Thymic Epithelial Progenitor Cells pTEC by In-Vitro Processing of Human Thymic Fragments for Allotransplantation

The procedure of generation and identification of stromal progenitor cells derived from human thymic fragments (PL patent 378431) has been described in this article. Our aim was to prepare material for transplantation in elderly people. The method is based on in-vitro processing of thymic fragments to get rid of all immunogenic elements of lymphocytes, endothelial cells, macrophages, and fibroblasts. In the thymic culture process, this organ dies out in the incubation medium and epithelial cells emerge out of the organ. After about 4 weeks from the start of the culture, the population of various developmental forms of epithelial cells was generated, namely CK AE1/AE3+, SDF-1 alpha+ and a weak expression of FGF+ S-100+. Finally, we obtained approximately 3 million cells as a monolayer. The progenitor cells were experimentally transplanted into a 72-year-old volunteer in order to prove that they do not induce neither a local nor a systemic rejection response.

Serum carnitine and acyl-carnitine in patients with meningitis due to tick-borne encephalitis virus infection

BACKGROUND Hard ticks are the main vectors of tick-borne encephalitis virus (TBEV). Free carnitine (FC) and acylcarnitines (AC) have the basic role in β-oxidation as well as the modulation of immune and nervous system. Homeostasis of carnitines in the TBE patients was not studied so far. OBJECTIVES This study aimed to evaluate FC and AC serum concentrations in patients with meningitis due to TBEV infection before and after 14 ± 3 days of treatment. MATERIAL AND METHODS The study was performed in 14 patients aged 48 ± 29 years that were divided a posteriori (based on their FC level before and after treatment) into 2 subgroups: 1-8 and 9-14. Diagnosis was based on the neurological, serological and pleocytosis evaluation. RESULTS The FC level in patients 1-8 before treatment (24.1 ± 8.1) was significantly lower than in patients post-treatment (34.4 ± 8.3), lower than in the control group (40.5 ± 7.6), and lower than in patients 9-14 before treatment (40.0 ± 13.5) but not lower than in the patients 9-14 after treatment (24.7 ± 7.3 μmol/L), respectively, p < 0.05. AC concentration in the patients 1-8 before treatment (4.7 ± 2.2) was apparently lower than in patients post-treatment (9.5 ± 3.9 μmol/L) but the values were not significantly different. In patients 9-14 before treatment the AC concentration (16.3 ± 12.6) was higher than in patients after treatment (5.3 ± 4.0 μmol/L), but the difference was not statistically significant. CONCLUSIONS FC and AC homeostasis in circulation was disturbed in the patients with meningitis due to TBEV infection patients. The mean levels of FC and AC in 60% of the patients were below the normal range but normalized after treatment whereas in 40% of the patients they were near or at a normal range and significantly decreased after treatment. Explanation of this intriguing finding and its clinical significance is not easy without further studies.

Serum carnitine concentration is decreased in patients with Lyme borreliosis.

BACKGROUND Lyme borreliosis (LB) is a serious infectious disease. Carnitine plays a crucial role in metabolism and inflammatory responses. Carnitine may be important in improving neuronal dysfunction and loss of neurons. AIM To evaluate serum carnitine concentration in adult patients with various clinical types of LB. MATERIAL/METHODS Groups: 1) patients with erythema migrans (EM, n=16), 2) neuroborreliosis (NB, n=10), 3) post-Lyme disease (PLD, n=22) and healthy controls (HC, n=32). Total (TC) and free (FC) carnitine were determined with the spectrophotometric method. RESULTS TC levels (44.9±10.4, 28.0±8.4, 35.9±15.6 μmol/L) in the EM, NB and PLD patients were lower than in HC (54.0±11.4 μmol/L), p < 0.001. FC levels (32.7±7.7, 23.6±6.8, 26.3±11.2 μmol/L) in the EM, NB and PLD patients were lower than in HC (40.5±7.6 μmol/L), p < 0.001. AC levels (12.2±5.2, 4.4±2.6, 9.6±7.4 μmol/L) in the EM, NB and PLD patients were lower in the NB and PLD patients than in HC (13.5±8.40 μmol/L), p <0.001. AC/FC ratio was 0.31±0.14, 0.18±0.09, 0.39±0.33 in the EM, NB and PLD patients. CONCLUSIONS LB patients exhibit a significant decrease of their serum carnitine concentrations. The largest changes were in the NB and PLD patients. To prevent late complications of the disease a possibility of early supplementation with carnitine should be considered. Further studies are required to explain the pathophysiological significance of our findings.

DETERMINANTS OF HYPERTENSION SEVERITY AND TARGET ORGAN DAMAGE IN CHILDREN WITH ESSENTIAL HYPERTENSION: PP.35.462

In 86 children (14.1 ± 2.4 years) with newly diagnosed essential hypertension (EH) the determinants of stage of hypertension (HT) and target organ damage (TOD) were assessed. Results: 58.1% of pts had stage 1 of HT and 41.9% stage 2. Pts with stage 2 of HT in comparison with pts with stage 1 of HT had significantly lower birth weight (3182 ± 751 vs 3469 ± 555, p < 0.05), greater left ventricular mass (LVM) (42.3 ± 12.1 vs 35.7 ± 8.7 g/m2.7, p < 0.01), higher LDL concentration (125.2 ± 29 vs 109 ± 34.2 mg/dl, p < 0.05). Pts with stage 2 in comparison with pts with stage 1 trended to be younger (13.6 ± 3 vs 14.5 ± 1.7 yrs, p = 0.08), visceral obese (waist –hip ratio (WHR): 0.87 ± 0.07 vs 0.84 ± 0.07, p = 0.08) and had tendency to greater albuminuria (32.2 ± 49.0 vs 20.1 ± 19.4 mg/24 h, p = 0.1). Birth weight negatively correlated with 24 h SBP (p < 0.05, r = -0.27), 24 h DBP (p < 0.05, r = -0.25), albuminuria (p < 0.05, r = -0.23), 120 min-glucose concentration after oral load (p < 0.05, r = -0.26) and positively correlated with insulin sensitivity index (ISI[0,120]: p < 0.05, r = 0.23). Step-wise regression analysis revealed that the main predictor of HT stage was WHR (R2 = 0.195, beta = 0.442, p = 0.009). The main predictor of LVM was waist circumference (R2 = 0.279, beta = 0.561, p = 0.008) and of carotid intima-media thickness – fasting insulin concentration (R2 = 0.208, beta = 0.510, p = 0.04). Conclusions: Lower birth weight and visceral obesity are main factors differentiating stage of HT and development of TOD in children with EH. Main predictors of TOD in children with EH are related with visceral obesity and hyperinsulinemia.

Role of the rat gastrointestinal mucosa in catabolism of endothelin peptides.

Endothelin-1 is involved in physiology and pathophysiology of the alimentary tract. The peptide modulates blood flow in the gastrointestinal microvasculature and regulates contractility of smooth muscles and, when present in excess, may be an important factor contributing to pathogenesis of various forms of mucosal injury and peristaltic disorders. Mechanisms that regulate endothelin concentration in the gastrointestinal tissues are unknown. Therefore, the aim of our study was to identify and characterize endothelin inactivating peptidases in the rat gastrointestinal mucosa and smooth muscle cells. We have found three high affinity and efficient endothelin-1 inactivating peptidases. The acidic (pH optimum 5.5), membrane-bound, thiorphan- (ED(50) 1.2+/-0.2 nM) and phosphoramidon (ED(50) 150+/-25 pM) sensitive, endothelin-1 inactivating peptidase (K(M) 0.12+/-0.03 microM) was present in the mucosal cells of duodenum and small intestine. The enzyme exhibited high molecular weight (>100 kDa) and characteristics similar to that of the rat and human kidney, acidic metalloendopeptidase that was recently described. Two forms of the unique, low molecular weight (100>MW>30 kDa), alkaline (pH optimum 8.5), specific (K(M) 0.5+/-0.2 microM), thiorphan- and phosphoramidon insensitive, 1,10 phenanthroline inhibitable (ED(50) 0.65+/-0.20 mM, mean+/-S.E.M.) endothelin-1 inactivating peptidase were present exclusively in the duodenal mucosal cells; soluble form in cytosol and membrane-bound form exhibiting an abundance ratio 5:1, respectively. Mucosa of the stomach and large intestine, and gastrointestinal smooth muscle cells do not contain the specific endothelin-1 inactivating peptidases. The enzymes may play a crucial role in regulation of endothelin concentration in the gastrointestinal tissues. Whether impairment of activity of the mucosal endothelin inactivating peptidases, resulting in the increase of concentration of endothelin peptides in gastrointestinal tissues, occurs in various pathological conditions is actually studied in our laboratory.