Synergistic Antifungal Interactions between Antibiotic Amphotericin B and Selected 1,3,4-thiadiazole Derivatives, Determined by Microbiological, Cytochemical, and Molecular Spectroscopic Studies.

In recent years, drug-resistant and multidrug-resistant fungal strains have been more frequently isolated in clinical practice. This phenomenon is responsible for difficulties in the treatment of infections. Therefore, the development of new antifungal drugs is an extremely important challenge. Combinations of selected 1,3,4-thiadiazole derivatives with amphotericin B showing strong synergic antifungal interactions are promising candidates for such formulas. In the study, microbiological, cytochemical, and molecular spectroscopy methods were used to investigate the antifungal synergy mechanisms associated with the aforementioned combinations. The present results indicate that two derivatives, i.e., C1 and NTBD, demonstrate strong synergistic interactions with AmB against some Candida species. The ATR-FTIR analysis showed that yeasts treated with the C1 + AmB and NTBD + AmB compositions, compared with those treated with single compounds, exhibited more pronounced abnormalities in the biomolecular content, suggesting that the main mechanism of the synergistic antifungal activity of the compounds is related to a disturbance in cell wall integrity. The analysis of the electron absorption and fluorescence spectra revealed that the biophysical mechanism underlying the observed synergy is associated with disaggregation of AmB molecules induced by the 1,3,4-thiadiazole derivatives. Such observations suggest the possibility of the successful application of thiadiazole derivatives combined with AmB in the therapy of fungal infections.

Influence of measurement mode on the results of glioblastoma multiforme analysis with the FTIR microspectroscopy.

Fourier Transform Infrared (FTIR) microspectroscopy is well known for its effectiveness in spectral and biochemical analyses of various materials. It enables to determine the sample biochemical composition by assigning detected frequencies, characteristic for functional groups of main biological macromolecules. In analysis of tissue sections one of two measurement modes, namely transmission and transflection, is usually applied. The first one has relatively straightforward geometry, hence it is considered to be more precise and accurate. However, IR-transparent media are very fragile and expensive. Transflection does not require expensive substrates, but is more prone to disruptive influence of Mie scattering as well as electric field standing wave effect. The excessive comparison of spectra' characteristics, obtained via both measurement modes, was performed in this paper. By the means of Mann-Whitney non-parametrical U test and PCA, the comparison of results obtained with both modes and assessment of usefulness of IR spectra obtained with transmission and transflection modes to differentiate between healthy and GBM-affected tissue, were performed. The main objective of the presented research is to compare the results of FTIR analysis of unfixed biological samples performed with transflection and transmission mode. In frame of the study we demonstrated the discrepancies between results of biochemical analysis performed based on data obtained with transmission and transflection. Such observation suggests that caution should be taken in drawing conclusions from the results obtained with transflection geometry, as its more prone to disruptive effects. Despite that, IR spectra developed with both modes allowed to distinguish GBM area from healthy tissue, which proves their diagnostic potential. Especially, application of the ME-EMSC correction of spectra before PCA enhances the performance of both methods to distinguish the analysed tissue areas.

Effect of Antibiotic Amphotericin B Combinations with Selected 1,3,4-Thiadiazole Derivatives on RPTECs in an In Vitro Model.

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections.

Biochemical changes of macrophages and U87MG cells occurring as a result of the exposure to iron oxide nanoparticles detected with the Raman microspectroscopy.

The core size of iron oxide nanoparticles (IONPs) is a crucial factor defining not only their magnetic properties but also toxicological profile and biocompatibility. On the other hand, particular IONPs may induce different biological response depending on the dose, exposure time, but mainly depending on the examined system. New light on this problem may be shed by the information concerning biomolecular anomalies appearing in various cell lines in response to the action of IONPs with different core diameters and this was accomplished in the present study. Using Raman microscopy we studied the abnormalities in the accumulation of proteins, lipids and organic matter within the nucleus, cytoplasm and cellular membrane of macrophages, HEK293T and U87MG cell line occurring as a result of 24-hour long exposure to PEG-coated magnetite IONPs. The examined nanoparticles had 5, 10 and 30 nm cores and were administered in doses 5 and 25 µg Fe/ml. The obtained results showed significant anomalies in biochemical composition of macrophages and the U87MG cells, but not the HEK293T cells, occurring as a result of exposure to all of the examined nanoparticles. However, IONPs with 10 nm core diminished the accumulation of biomolecules in cells only when they were administered at a larger dose. The Raman spectra recorded for the macrophages subjected to 30 nm IONPs and for the U87MG cells exposed to 5 and 10 nm showed the presence of additional bands in the wavenumber range 1700-2400 cm-1, probably resulting from the appearance of Fe adducts within cells. Our results indicate, moreover, that smaller IONPs may be effectively internalized into the U87MG cells, which points at their diagnostic/therapeutic potential in the case of glioblastoma multiforme.

The influence of IONPs core size on their biocompatibility and activity in in vitro cellular models.

Although the key factor affecting the biocompatibility of IONPs is the core size, there is a lack of regular investigation concerning the impact of the parameter on the toxicity of these nanomaterials. Therefore, such studies were carried out in this paper. Their purpose was to compare the influence of PEG-coated-magnetite NPs with the core of 5, 10 and 30 nm on six carefully selected cell lines. The proliferation rate, viability, metabolic activity, migration activity, ROS levels and cytoskeleton architecture of cells have been evaluated for specified incubation periods. These were 24 and 72-h long incubations with IONPs administered in two doses: 5 and 25 µg Fe/ml. A decrease in viability was observed after exposure to the tested NPs for all the analyzed cell lines. This effect was not connected with core diameter but depended on the exposure time to the nanomaterials. IONPs increased not only the proliferation rate of macrophages-being phagocytic cells-but also, under certain conditions stimulated tumor cell divisions. Most likely, the increase in proliferation rate of macrophages contributed to the changes in the architecture of their cytoskeleton. The growth in the level of ROS in cells had been induced mainly by the smallest NPs. This effect was observed for HEK293T cells and two cancerous lines: U87MG (at both doses tested) and T98G (only for the higher dose). This requires further study concerning both potential toxicity of such IONPs to the kidneys and assessing their therapeutic potential in the treatment of glioblastoma multiforme.

The Use of Fourier Transform Infrared Microspectroscopy for the Determination of Biochemical Anomalies of the Hippocampal Formation Characteristic for the Kindling Model of Seizures.

The animal models of seizures and/or epilepsy are widely used to identify the pathomechanisms of the disease as well as to look for and test the new antiseizure therapies. The understanding of the mechanisms of action of new drugs and evaluation of their safety in animals require previous knowledge concerning the biomolecular anomalies characteristic for the particular model. Among different models of seizures, one of the most widely used is the kindling model that was also applied in our study. To examine the influence of multiple transauricular electroshocks on the biochemical composition of rat hippocampal formation, Fourier transform infrared (FT-IR) microspectrosopy was utilized. The chemical mapping of the main absorption bands and their ratios allowed us to detect significant anomalies in both the distribution and structure of main biomolecules for electrically stimulated rats. They included an increased relative content of proteins with ß-sheet conformation (an increased ratio of the absorbance at the wavenumbers of 1635 and 1658 cm-1), a decreased level of cholesterol and/or its esters and compounds containing phosphate groups (a diminished intensity of the massif of 1360-1480 cm-1 and the band at 1240 cm-1), as well as increased accumulation of carbohydrates and the compounds containing carbonyl groups (increased intensity of the bands at 1080 and 1740 cm-1, respectively). The observed biomolecular abnormalities seem to be the consequence of lipid peroxidation promoted by reactive oxygen species as well as the mobilization of glucose that resulted from the increased demand to energy during postelectroshock seizures.

The methods of vibrational microspectroscopy reveals long-term biochemical anomalies within the region of mechanical injury within the rat brain.

Traumatic brain injury (TBI), meaning functional or structural brain damage which appear as a result of the application of the external physical force, constitutes the main cause of death and disability of individuals and a great socioeconomic problem. To search for the new therapeutic strategies for TBI, better knowledge about posttraumatic pathological changes occurring in the brain is necessary. Therefore in the present paper the Fourier transform infrared microspectroscopy and Raman microscopy were used to examine local and remote biochemical changes occurring in the rat brain as a result of focal cortex injury. The site of the injury and the dorsal part of the hippocampal formation together with the above situated cortex and white matter were the subject of the study. The topographic and quantitative biochemical analysis followed with the statistical study using principal component analysis showed significant biomolecular anomalies within the lesion site but not in the area of the dorsal hippocampal formation and in the above situated white matter and cortex. The observed intralesional anomalies included significantly decreased accumulation of lipids and their structural changes within the place of injury. Also the levels of compounds containing phosphate and carbonyl groups were lower within the lesion site comparing to the surrounding cortex. The opposite relation was, in turn, found for the bands characteristic to proteins and cholesterol/cholesterol esters.

The assessment of the usability of selected instrumental techniques for the elemental analysis of biomedical samples.

The fundamental role of major, minor and trace elements in different physiological and pathological processes occurring in living organism makes that elemental analysis of biomedical samples becomes more and more popular issue. The most often used tools for analysis of the elemental composition of biological samples include Flame and Graphite Furnace Atomic Absorption Spectroscopy (F-AAS and GF-AAS), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Each of these techniques has many advantages and limitations that should be considered in the first stage of planning the measurement procedure. Their reliability can be checked in the validation process and the precision, trueness and detection limits of elements belong to the most frequently determined validation parameters. The main purpose of this paper was the discussion of selected instrumental techniques (F-AAS, GF-AAS, ICP-OES and ICP-MS) in term of the achieved validation parameters and the usefulness in the analysis of biological samples. The focus in the detailed literature studies was also put on the methods of preparation of the biomedical samples. What is more based on the own data the usefulness of the total reflection X-ray fluorescence spectroscopy for the elemental analysis of animal tissues was examined. The detection limits of elements, precision and trueness for the technique were determined and compared with the literature data concerning other of the discussed techniques of elemental analysis. Reassuming, the following paper is to serve as a guide and comprehensive source of information concerning the validation parameters achievable in different instrumental techniques used for the elemental analysis of biomedical samples.

Verification of electron beam parameters in an intraoperative linear accelerator using dosimetric and radiobiological response methods.

BACKGROUND: The availability of linear accelerators (linac) for research purposes is often limited and therefore alternative radiation sources are needed to conduct radiobiological research. The National Centre for Radiation Research in Poland recently developed an intraoperative mobile linac that enables electron irradiation at energies ranging from 4 to 12 MeV and dose rates of 5 or 10 Gy/min. The present study was conducted to evaluate the electron beam parameters of this intraoperative linac and to verify the set-up to evaluate out-of-field doses in a water phantom, which were determined through dosimetric and biological response measurements. MATERIALS AND METHODS: The distribution of radiation doses along and across the radiation beam were measured in a water phantom using a semiconductor detector and absolute doses using an ionisation chamber. Two luminal breast cancer cell lines (T-47D and HER2 positive SK-BR-3) were placed in the phantom to study radiation response at doses ranging from 2 to 10 Gy. Cell response was measured by clonogenic assays. RESULTS AND CONCLUSION: The electron beam properties, including depth doses and profiles, were within expected range for the stated energies. These results confirm the viability of this device and set-up as a source of megavoltage electrons to evaluate the radiobiological response of tumour cells.

Comparison of Elemental Anomalies Following Implantation of Different Cell Lines of Glioblastoma Multiforme in the Rat Brain: A Total Reflection X-ray Fluorescence Spectroscopy Study.

Glioblastoma multiforme (GBM) is a primary brain tumor with a very high degree of malignancy and is classified by WHO as a glioma IV. At present, the treatment of patients suffering from GBM is based on surgical resection of the tumor with maximal protection of surrounding tissues followed by radio- and pharmacological therapy using temozolomide as the most frequently recommended drug. This strategy, however, does not guarantee success and has devastating consequences. Testing of new substances or therapies having potential in the treatment of GBM as well as detection of their side effects cannot be done on humans. Animal models of the disease are usually used for these purposes, and one possibility is the implantation of human tumor cells into rodent brains. Such a solution was used in the present study the purpose of which was comparison of elemental anomalies appearing in the brain as a result of implantation of different glioblastoma cell lines. These were two commercially available cell lines (U87MG and T98G), as well as tumor cells taken directly from a patient diagnosed with GBM. Using total reflection X-ray fluorescence we determined the contents of P, S, K, Ca, Fe, Cu, Zn, and Se in implanted-left and intact-right brain hemispheres. The number of elemental anomalies registered for both hemispheres was positively correlated with the invasiveness of GBM cells and was the highest for animals subjected to U87MG cell implantation, which presented significant decrease of P, K, and Cu levels and an increase of Se concentration within the left hemisphere. The abnormality common for all three groups of animals subjected to glioma cell implantation was increased Fe level in the brain, which may result from higher blood supply or the presence of hemorrhaging regions. In the case of the intact hemisphere, elevated Fe concentration may also indicate higher neuronal activity caused by taking over some functions of the left hemisphere impaired as a result of tumor growth.

Intravenously administered d-mannitol-coated maghemite nanoparticles cause elemental anomalies in selected rat organs.

In this study novel d-mannitol coated maghemite nanoparticles (MIONPs) are presented in terms of their influence on elemental homeostasis of living organisms and for this purpose highly sensitive total reflection X-ray fluorescence was used. Because of the biological indifference of d-mannitol and presumed lower toxicity of maghemite, compared to the most commonly used magnetite in nanomedicine, such nanoparticles seem to be promising candidates for biomedical applications. The examined dose of MIONPs was comparable with one of the lowest doses used in medical diagnostics. However, it should be emphasized that the amount of iron injected in this form is still significant compared to its total content in organs, especially in kidneys or the heart, and may easily disrupt their elemental homeostasis. The aim of the present study was to evaluate the elemental changes occurring in selected rat organs after injecting a low dose of MIONPs. The results were compared with those obtained for previously examined PEG-coated nanoparticles with magnetite cores. In the light of our findings the elemental changes observed after exposure to MIONPs were less extensive than those following PEG-coated magnetite nanoparticle administration.

Comparison of ultrasmall IONPs and Fe salts biocompatibility and activity in multi-cellular in vitro models.

In the paper, the results of the first regular studies of ultra-small iron oxide nanoparticles (IONPs) toxicity in vitro were presented. The influence of PEG-coated NPs with 5 nm magnetite core on six different cell lines was examined. These were: human bronchial fibroblasts, human embryonic kidney cells (HEK293T), two glioblastoma multiforme (GBM) cell lines as well as GBM cells isolated from a brain tumor of patient. Additionally, mouse macrophages were included in the study. The influence of IONPs in three different doses (1, 5 and 25 µg Fe/ml) on the viability, proliferation and migration activity of cells was assessed. Moreover, quantifying the intracellular ROS production, we determined the level of oxidative stress in cells exposed to IONPs. In the paper, for the first time, the effect of Fe in the form of IONPs was compared with the analogical data obtained for iron salts solutions containing the same amount of Fe, on the similar oxidation state. Our results clearly showed that the influence of iron on the living cells strongly depends not only on the used cell line, dose and exposure time but also on the form in which this element was administered to the culture. Notably, nanoparticles can stimulate the proliferation of some cell lines, including glioblastoma multiforme. Compared to Fe salts, they have a stronger negative impact on the viability of the cells tested. Ultra-small NPs, also, more often positively affect cell motility which seem to differ them from the NPs with larger core diameters.

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like γ-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that γ-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

FTIR microspectroscopy revealed biochemical changes in liver and kidneys as a result of exposure to low dose of iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) have biomedical and biotechnological applications in magnetic imaging, drug-delivery, magnetic separation and purification. The biocompatibility of such particles may be improved by covering them with coating. In presented paper the biochemical anomalies of liver and kidney occurring in animals exposed to d-mannitol-coated iron(III) oxide nanoparticles (M-IONPs) were examined with Fourier transform infrared (FTIR) microspectroscopy. The dose of IONPs used in the study was significantly lower than those used so far in other research. Liver and kidney tissue sections were analysed by chemical mapping of infrared absorption bands originating from proteins, lipids, compounds containing phosphate groups, cholesterol and cholesterol esters. Changes in content and/or structure of the selected biomolecules were evaluated by comparison of the results obtained for animals treated with M-IONPs with those from control group. Biochemical analysis of liver samples demonstrated a few M-IONPs induced anomalies in the organ, mostly concerning the relative content of the selected compounds. The biomolecular changes, following exposition to nanoparticles, were much more intense within the kidney tissue. Biochemical aberrations found in the organ samples indicated at increase of tissue density, anomalies in fatty acids structure as well as changes in relative content of lipids and proteins. The simultaneous accumulation of lipids, phosphate groups as well as cholesterol and cholesterol esters in kidneys of rats exposed to IONPs may indicate that the particles stimulated formation of lipid droplets within the organ.

Organ Metallome Processed with Chemometric Methods Enable the Determination of Elements that May Serve as Markers of Exposure to Iron Oxide Nanoparticles in Male Rats.

The systemic influence of iron oxide nanoparticles on the elemental homeostasis of key organs was examined in male rats. In tissues taken at different intervals from nanoparticles injection, the dynamics of elemental changes was analyzed. The organ metallome was studied using total reflection X-ray fluorescence. The obtained data were processed with advanced cluster and discriminant analyses-to classify the tissues according to their organs of origin and to distinguish accurately the nanoparticle-treated and normal rats. Additionally, in the case of liver and heart, it was possible to determine the elements of highest significance for different treatments, which may serve as markers of exposure to iron oxide nanoparticles.

Chemo-enzymatic Baeyer-Villiger oxidation of 4-methylcyclohexanone via kinetic resolution of racemic carboxylic acids: direct access to enantioenriched lactone.

A new method for the asymmetric chemo-enzymatic Baeyer-Villiger oxidation of prochiral 4-methylcyclohexanone to (R)-4-methylcaprolactone in the presence of (±)-4-methyloctanoic acid, Candida Antarctica lipase B and 30% aq. H2O2 has been developed. A mechanism for the asymmetric induction based on kinetic resolution of racemic carboxylic acids is proposed.

[Nocturnal and diurnal oxygen saturation in patients with chronic pulmonary embolism]. / Dzienne i nocne wysycenie krwi tetniczej tlenem u chorych na przewlekla, zatorowosc plucna.

UNLABELLED: Little is known on oxygen saturation in patients with chronic pulmonary embolism. AIM OF THE STUDY WAS: Tto assess the occurrence and importance of oxygen desaturations (D) in these patients. MATERIAL AND METHODS: The study involved 58 normotensive patients with chronic pulmonary embolism (18 males, 40 females, mean age 60 years, range 22-87 years) and was carried out 6 weeks to 2 years after an acute pulmonary embolic event. During 24-hour pulse oximetry mean oxygen saturation (SpO2), and number and duration of desaturations (D), defined as at least a 6% drop of pSO2, below 88%, lasting a minimum of 8 s, were recorded. Simultaneously echocardiographic study and blood gases analysis was performed. RESULTS: Desaturations were found in 39 (67.2%) patients, whereas 79% patients had pSO2 <95% in gasometry. 27 patients had both diurnal (06(00)-22(00)) and nocturnal (22(00)-06(00)) D, 9 patients only nocturnal D, and 3 patients only diurnal D. The number (14.7 vs 36.1) and duration (733.9 vs 1528 s) of D episodes were approximately 2 times greater at night than day. There were 18 (75%) desaturators among patients with pulmonary hypertension (defined as an echocardiographic tricuspid gradient >30 mmHg), and 21 (61.8%) desaturators among patients with chronic pulmonary embolism and without pulmonary hypertension. The patients with pulmonary hypertension had a lower mean SpO2 (p=0.005) and a lower number and duration of nocturnal (p=0.008, 0.03) and diurnal (p=0.008, 0.035) D. CONCLUSION: A large number of D episodes were found in the patients with chronic pulmonary embolism, mostly at night and in the patients with pulmonary hypertension. It is unclear whether D merely reflects pulmonary vasculature embolization or whether they are capable of impacting development of chronic thromboembolic pulmonary hypertension on the basis of vicious circle. In the latter case the need for a long term oxygen therapy, or at least nighttime oxygenation, should be taken into account to slow down progression of the disease.

[Results of one year pulse oxymetry in patients with chronic thromboembolic pulmonary hypertension]. / Roczna obserwacja przezskórnego wysycenia krwi tetniczej tlenem u chorych na przewlekle zakrzepowo-zatorowe nadcisnienie plucne.

UNLABELLED: It is little known on oxygen saturation in patients with chronic thromboembolic pulmonary hypertension (CTEPH). AIM OF THE STUDY: To assess the occurrence, evolution and clinical significance of oxygen desaturations (D) during 1-year observation in CTEPH patients. MATERIAL AND METHOD: The study involved 24 consecutive patients with CTEPH (6 males, 18 females, mean age 63, range 22-75 years). During 24-hour pulse oxymetry mean oxygen saturation (SpO2), number and duration of desaturations, defined as at least a 6% drop of SO2, below 88%, lasting a minimum of 8 s, were recorded at baseline and after 6 and 12 months of follow-up. Simultaneously echocardiographic study and blood gases analysis was performed. RESULTS: At baseline 18 of 24 (75%) CTEPH patients had desaturations. During follow-up none of nondesaturators had desaturations episodes and all baseline desaturators had desaturations episodes after 6 and 12 months. In desaturators there was trend to aggravate the number and duration of diurnal but not nocturnal desaturations episodes after 6 and 12 months. It was accompanied by trend (p = 0.05) to increase of pulmonary pressure as assessed by echocardiographic study, whereas the opposite trend to decrease of pulmonary pressure was seen in nondesaturators. CONCLUSIONS: Results of the study show that desaturations occur in most patients with CTEPH and desaturators may have worse clinical course than nondesaturators. It may suggest the need for long oxygen therapy in desaturating CTEPH patients to slow down progression of the disease.

Ghrelin inhibits vascular superoxide production in spontaneously hypertensive rats.

BACKGROUND: Ghrelin is a novel peptide involved in the control of appetite, but its role in vascular pathologies remains to be elucidated. Ghrelin was shown to decrease blood pressure (BP) and improve endothelial function. Its plasma levels are correlated with BP in humans. Mechanisms of these effects are unknown. Because oxidative stress and increased superoxide production by NAD(P)H oxidases (Nox) are critical in the pathogenesis of hypertension, we aimed to study the effects of ghrelin on vascular superoxide production and NAD(P)H oxidase activity in spontaneously hypertensive rats (SHR). METHODS: Aortic superoxide production and NAD(P)H oxidase activity were measured using lucigenin (5 micromol/L) chemiluminescence. Aortas from Wistar-Kyoto rats (WKY) were used as control. Direct superoxide scavenging properties of ghrelin were tested using xanthine-xanthine oxidase system. RESULTS: Both basal superoxide production and vascular NADPH oxidase activity were significantly higher in aortas from SHR, than from WKY. Preincubation of aortic segments from SHR or WKY with ghrelin caused concentration-dependent (from 50 pg/mL to 5 ng/mL) decrease of basal superoxide production. Vascular NAD(P)H oxidase activity was inhibited by ghrelin, abolishing the difference between SHR and basal WKY. Ghrelin did not affect superoxide release from the in vitro xanthine-xanthine oxidase system, indicating lack of direct superoxide scavenging properties or inhibitory effects on xanthine oxidase in vitro. Nitric oxide synthase (NOS) inhibition, using N(omega)-nitro-L-arginine methyl ester (L-NAME), partially blunted the effects of ghrelin on NADPH oxidase activity indicating potential role of nitric oxide. CONCLUSIONS: Ghrelin inhibits vascular oxidative stress in SHR. This effect is likely related to the inhibition of vascular NAD(P)H oxidases.