Contribution of local regeneration of glucocorticoids to tissue steroid pools.

11ß-Hydroxysteroid dehydrogenase 1 (11ßHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11ßHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution vs glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11ßHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) vs adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2H3]-cortisol (d3F) from [9,12,12-2H3]-cortisone (d3E), measuring 11ßHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix-assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh-/- mice, showing the importance for whole-body 11ßHSD1 reductase activity. Disruption of liver 11ßHSD1 reduced the amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11ßHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regenerated of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11ßHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.

Dual-drugs delivery in solid lipid nanoparticles for the treatment of Candida albicans mycosis.

Nowadays, a combinatorial drug delivery system that simultaneously transports two or more drugs to the targeted site in a human body, also recognized as a dual-drugs delivery system, represents a promising strategy to overcome drug resistance. Solid lipid nanoparticles loaded with clotrimazole (CLZ) and alphalipolic acid (ALA), considered as an effective agent in the reduction of reactive oxygen species, can enhance anti-infective immunity being proposed as a non-toxic and mainly non-allergic dual-drugs delivery system. In this study, uncoated and cationic CLZ-ALA-loaded SLN were prepared and compared. Suspensions with a narrow size distribution of particles of mean size below 150 nm were obtained, having slight negative or highly positive zeta potential values, due to the presence of the cationic lipid, which also increased nanoparticles stability, as confirmed by Turbiscan® results. Calorimetric studies confirmed the rationale of separately delivering the two drugs in a dual-delivery system. Furthermore, they confirmed the formation of SLN, without significant variation in presence of the cationic lipid. In vitro release studies showed a prolonged drug release without the occurrence of any burst effect. In vitro studies performed on 25 strains of Candida albicans showed the antimicrobial drug activity was not altered when it was loaded into lipid nanoparticles. The study has proved the successfully encapsulation of CLZ and ALA in solid lipid nanoparticles that may represent a promising strategy to combine ALA protective effect in the treatment with CLZ.

Evaluation of reference genes for quantitative real-time PCR in Wharton's Jelly-derived mesenchymal stem cells after lentiviral transduction and differentiation.

Quantitative real time reverse transcription PCR, qRT-PCR, is one of the most important techniques for assessing the level of gene expression. Selecting the correct reference gene to normalize the results is a key step in this method. Inaccurate data can be generated if the correct reference gene is not selected. The level of the expression of reference genes is tissue-variable, and in the case of mesenchymal stem cells (MSC), it can be different depending on the source of their origin. The aim of this study was to select the reference gene for Wharton's Jelly-derived MSC (WJ- MSC) that were undergoing transduction and differentiation. In this work, the expression of 32 genes was analyzed, of which two (RPS17 and 18S rRNA), which had the most stable expression level, were selected. A comparative analysis of the expression stability of the selected genes was then performed with the genes that are most commonly used in the literature, i.e. ß-actin and GAPDH. Next, it was determined that a false picture of the expression level of the studied genes can be obtained when a reference gene with variable expression level is used for normalization. RPS17 and 18S rRNA proved to be the most stable reference genes for the WJ-MSC that had been subjected to the lentiviral transfection procedure followed by differentiation. The expression of ß-actin and GAPDH was highly unstable and therefore these genes are not suitable for use as reference genes in studies involving WJ- MSC.

3D printing in the design of pharmaceutical dosage forms.

Three-dimensional (3D) printing technologies are manufacturing approaches with widespread use in industry (e.g. automotive, automobile, pharmaceutical industries). With regard to its use in pharmaceutical industry, 3D printing is demonstrating to be of added value attributed to the possibility of printing tailored pharmaceutical products, namely personalized medical devices, such as implants and other dosage forms. However, with the approval of the first 3D-printed drug-product in 2015, a new perspective has arisen, i.e. the use of this technology to produce solid oral dosage forms exhibiting complex drug release profiles and allowing for individual dosing. Technological hurdles and regulatory issues still have to be overcome before this technology can truly find its place in the healthcare sector, where it can certainly contribute to a personalized and patient-centered healthcare. This manuscript offers a comprehensive analysis of the most extensively used methods of 3D printing in the pharmaceutical field, with examples of solid oral dosage forms and other medical devices currently under development or already marketed.

Uveal melanoma: physiopathology and new in situ-specific therapies.

Uveal melanoma is the most common primary intraocular tumor in adults. It can arise from melanocytes in the anterior (iris) or posterior uveal tract (choroid and ciliary body). Uveal melanoma has a particular molecular pathogenesis, being characterized by specific chromosome alterations and gene mutations (e.g., GNAQ/GNA11; BAP1), which are considered promising targets for molecular therapy. Primary treatment of uveal melanoma includes radiotherapy (brachytherapy and charged-particle therapy), phototherapy (photocoagulation, transpupillary thermal therapy, and photodynamic therapy) and surgery (local resection, enucleation and exenteration). Approximately half of patients with uveal melanoma will, however, develop metastasis, especially in the liver. The treatment of metastatic uveal melanoma includes systemic chemotherapy, immunotherapy and molecular targeted therapy. Liver-directed therapies, such as resection, chemoembolization, immunoembolization, radioembolization, isolated hepatic perfusion and percutaneous hepatic perfusion, are also available to treat metastatic uveal melanoma. Several clinical trials are being developed to study new therapeutic options to treat uveal melanoma, mainly for those with identified liver metastases. The present work discusses the physiopathology and new in situ-specific therapies for the treatment of uveal melanoma.

Levels of betatrophin decrease during pregnancy despite increased insulin resistance, beta-cell function and triglyceride levels.

AIM: Evidence in support of an association between betatrophin and insulin resistance (IR) is mounting, with studies demonstrating that betatrophin is elevated in patients with type 2 diabetes, obesity and gestational diabetes. The aim of this study was to evaluate the role of betatrophin in IR and physiological proliferation of beta cells during pregnancy in healthy women. METHODS: Eighty healthy pregnant women were examined at each trimester [T1 (first), T2 (second), T3 (third)], with a subgroup (n=45) that was also examined at 3 months postpartum (3MPP). The controls comprised 30 non-pregnant healthy women (HW) of reproductive age. Also measured were levels of betatrophin (ELISA), glucose (enzymatic method with hexokinase), insulin (IRMA), C-peptide (EASIA) and HbA1c (HPLC), while HOMA-IR and HOMA-ß scores were calculated. RESULTS: Betatrophin concentration was highest at T1, and differed significantly from T2 and T3 (1.84 [Q1=1.16, Q3=2.67]ng/mL vs 1.46 [Q1=0.96, Q3=2.21]ng/mL; P<0.05 and 1.23 [Q1=0.85, Q3=2.14]ng/mL; P<0.01, respectively). The T3 median concentration of betatrophin was the lowest of all trimesters, and significantly lower than at 3MPP (1.23 [Q1=0.85, Q3=2.14]ng/mL vs 1.49 [Q1=1.06, Q3=2.60]ng/mL; P<0.01, respectively). At 3MPP, the level of betatrophin was similar to that of HW (1.47 [Q1=0.89, Q3=2.67]ng/mL). HOMA-IR and HOMA-%ß index scores increased during gestation, peaking at T3 (2.3 [Q1=1.66, Q3=2.72] and 227.7 [Q1=185.49, Q3=326.31], respectively) and returning to levels similar to those of HW at 3MPP (1.53 [Q1=1.12, Q3=2.41] and 88.86 [Q1=62.73, Q3=130.45] vs 1.35 [Q1=1.02, Q3=1.62] and 92.5 [Q1=74.20, Q3=111.47], respectively). CONCLUSION: Concentrations of betatrophin decrease during pregnancy, suggesting that the hormone does not play a significant role in the expansion of beta-cell mass and IR during pregnancy.

Stability determination of the formulations containing hyaluronic acid.

OBJECTIVES: The aim of this work was to investigate the long-term stability of four emulsions (creams and ointments) developed for carrying hyaluronic acid (HA) for dermal penetration. METHODS: The stability of obtained formulations was analysed by multiple light scattering and laser diffraction methods. RESULTS: The experimental results showed that the moisturizing creams were more stable than the ointments containing HA. The migration phenomenon of particles was observed in soft ointment with HA and the flocculation phenomenon was detected in ointment based on lanolin. The larger the water content, the more stable formulation can be, due to hygroscopic properties of HA. CONCLUSIONS: The identification of instability phenomena was shortened dramatically using optical method such as multiple light scattering. Even small changes in the stability of emulsion were determined very early. We suggest that the moisturizing creams may be more suitable than ointments to carry HA for dermal absorption. These studies are a significant first step towards further exploration into what form of HA would assure maximum effect, give consumers satisfaction and guarantee safety during application.

Melatonin-induced glycosaminoglycans augmentation in myocardium remote to infarction.

Elevated levels of collagen as well as transient increases of glycosaminoglycans (GAG) have been shown in the myocardium remote to the infarction. The aim of the study is to observe the effect of melatonin on the accumulation of collagen and GAG in the left ventricle wall, remote to the infarction. A second aim is to determine whether the effect of the pineal indole is mediated by the membrane melatonin receptors of heart fibroblasts. Rats with myocardial infarction induced by ligation of the left coronary artery were treated with melatonin at a dose of 60 µg/100 g b.w. or vehicle (2% ethanol in 0.9% NaCl). The results were compared with an untreated control. In the second part of the study, the fibroblasts from the non-infarcted part of myocardium were isolated and cultured. Melatonin at a range of concentrations from 10(-8) M to 10(-6) M was applied to the fibroblast cultures. In the final part of the study, the influence of luzindole (10(-6) M), the melatonin membrane receptor inhibitor, on melatonin-induced GAG augmentation was investigated. Both collagen and GAG content were measured in the experiment. Melatonin elevated GAG content in the myocardium remote to the infarcted heart. Collagen level was not changed by pineal indoleamine. Fibroblasts isolated from the myocardium varied in shape from fusiform to spindle-shaped. Moreover, the pineal hormone (10(-7)M and 10(-6)M) increased GAG accumulation in the fibroblast culture. Luzindole inhibited melatonin-induced elevation of GAG content at 10(-6)M. Melatonin increased GAG content in the myocardium remote to infarction. This effect was dependent on the direct influence of the pineal indole on the heart fibroblasts. The melatonin-induced GAG elevation is blocked by luzindole, the melatonin membrane receptors inhibitor, indicating a direct effect of this indole.

Vitamin D analogs decrease in vitro secretion of RANTES and enhance the effect of budesonide.

PURPOSE: Eosinophils appear to be central inflammatory cells in the pathogenesis of rhinosinusitis with nasal polyps (NP). One of the most predominantly recognized eosinophil chemoattractants is RANTES. The aim of this study was to assess the influence of vitamin D (VD) derivates on RANTES expression in the culture of nasal polyp fibroblasts. MATERIAL AND METHODS: NP fibroblast cell cultures derived from 16 patients with NP were first stimulated with bacterial LPS and than incubated in increasing concentrations (from 10(-7)M to 10(-4)M) of calcitriol, tacalcitol or budesonide and in combination with one of VD derivate with budesonide in 1:1, 1:3 and 3:1 ratios. Quantitative analysis of RANTES level was conducted in culture supernatants using an ELISA method. RESULTS: The highest calcitriol concentration (10(-4)M) as well as tacalcitol at 10(-5)M and 10(-4)M reduced RANTES production significantly compared to the control (201.1pg/ml, 338.7pg/ml, 211.3pg/ml v 571.78pg/ml; p<0.05). Budesonide and calcitriol administered in 1:3 ratio and budesonide and tacalcitol in 1:1 and 1:3 reduced RANTES concentration significantly better than each of the drug used in monotherapy (p<0.05). Budesonide and tacalcitol in 1:1 and 1:3 ratios suppressed RANTES production to the lowest level (171.8±97.6pg/ml and 178.7±105.22pg/ml, respectively). CONCLUSION: Active VD compounds via downregulation of RANTES production exert a potential role as a complementary element in the therapy of chronic rhinosinusitis with NP. Compounds consisting of budesonide and VD derivate have an advantage over both drugs used in monotherapy.

Effect of macroalgae enriched with microelements on egg quality parameters and mineral content of eggs, eggshell, blood, feathers and droppings.

This study presents results obtained from feeding experiment on laying hens, which were fed with the diet supplemented with two marine macroalgae: Enteromorpha prolifera and Cladophora sp., enriched with microelements [Cu(II), Zn(II), Co(II), Mn(II), Cr(III)]. The applicability of the preparation was tested on five experimental groups of laying hens and one control group. In the control group, microelements were supplemented in the inorganic form, whereas in experimental groups, Cu, Zn, Co, Mn and Cr were replaced by macroalgae enriched with a given microelement ion. During feeding experiment, weight of laying hens, weight of eggs, eggshell thickness and mineral content of blood, feathers, droppings, eggs content (separately yolk and egg white) and eggshell were measured. Also egg number was counted and microclimate (temperature and relative humidity) was monitored. Supplementing bio-metallic feed additives to the diet of laying hens resulted in higher microelement transfer to eggs and enhanced the colour of yolk. It was also found that the presence of Enteromorpha prolifera and Cladophora sp. in laying hens diet influenced advantageously eggs weight, eggshell thickness as well as body weight of hens. On the basis of these results, it could be concluded that Enteromorpha prolifera and Cladophora sp. enriched with microelement ions could be potentially used as mineral feed additives in laying hens feeding.

Youth's knowledge and attitude to epilepsy.

PURPOSE: The aim of this research is to assess youth's knowledge and attitude towards their epileptic peers. MATERIAL AND METHODS: The present study concerned 181 students of two secondary schools in Poznan, including 107 girls and 74 boys. The tool used was a self-made questionnaire. The criterion of including a questionnaire into data was the answer to the following question: "Have you heard about epilepsy?" RESULTS: The questionnaire has shown that 46% of secondary school students had already known a person with epilepsy. 91% of students knew that epilepsy was not a contagious disease, but 12% of pupils thought that epilepsy was a mental illness. Almost 23% of respondents claimed they knew what could cause epilepsy. Only 2% of the students knew how to provide first aid during an epileptic fit. 92% of the respondents accepted a person with epilepsy among their friends. CONCLUSIONS: The level of the knowledge about epilepsy among pupils is insufficient. The youth do not know the causes of epilepsy, provocative factors of convulsions, and symptoms and the rules of giving first aid during an epileptic fit. The main source of information about the disease were television programs. The majority of the school - graduates expressed willingness to make friends with epileptic people and acceptance of the ill.

Peripheral blood lymphocyte population in children infected with Helicobacter pylori.

PURPOSE: Helicobacter pylori infection in children is associated with a chronic inflammatory process of gastric and duodenal mucosa, which may have a various clinical course ranging from asymptomatic and chronic inflammatory condition to gastric ulceration. The immune system may contribute especially to chronic gastric mucosa inflammation. The aim of our study was to assess the levels of peripheral blood T (CD3+, CD4+, CD8+) and B lymphocyte subpopulation (CD19+) in children with Helicobacter pylori infection and to evaluate their relation to degree of antrum mucosa inflammation. MATERIAL AND METHODS: The study was performed in 32 children aged 7-18 years, hospitalized due to dyspeptic symptoms. The endoscopic examination of upper gastrointestinal tract was performed and gastric and duodenal mucosa was estimated in all patients. The endoscopic and histological evaluation of gastric mucosa was performed according to the Sydney System [4]. The urease test (CLO-test-H. pylori) was made to estimate the severity of the infection. RESULTS: Moderate antrum mucosa inflammation was found in 41.2% of the examined. The highest percentage of children (58.8%) presented marked inflammation. No mild inflammation was found in children examined. CONCLUSIONS: No correlation was found between lymphocyte levels and the degree of the inflammatory changes in antrum mucosa. The evaluation of peripheral blood lymphocytes performed in children with Helicobacter pylori infection suggests that T lymphocytes may play a predominant role in this infection.

Nitrogen-15 fractionation in the thermal decomposition of nitrous oxide of natural isotopic composition.

The 15N fractionation in the thermal decomposition of nitrous oxide (N2O) of natural isotopic composition has been investigated in quartz reaction vessel in the temperature interval 888-1073 K. The formulas relating the observed experimentally 15N fractionations with the primary 15N kinetic isotope effect, (k14/k15)p for 14N15N16O, and secondary 15N kinetic isotope effect, (k14/k15)s for 15N14N16O, have been derived. The experimentally estimated 15N kinetic isotope effects have been compared with the primary and secondary 15N kinetic isotope effects calculated with the absolute rate theory formulations applied to linear three atom molecules. A good agreement was found for the primary 15N kinetic isotope effect, (k14/k15)p, in the temperature interval 888-1007 K. But at 1073 K the decompositions of N2O, accompanied by NO (nitric oxide) formation proceed with a twice times smaller primary kinetic isotope effect, (k14/k15)p of 1.0251 +/- 0.0009, only, suggesting the nonlinear transition state structures with participation of the fourth external atom at high temperature decompositions of nitrous oxide. The nitrogen isotope effects determined in this study correlate well with nitrogen isotope fractionations observed in the natural biological, earth and atmospheric processes.

[Endoscopic intranasal microsurgery in the management of cerebrospinal fluid rhinorrhea]. / Zastosowanie endoskopowej mikrochirurgii wewnatrznosowej w leczeniu plynotoku nosowego.

The surgical management of cerebrospinal fluid rhinorrhoea has changed significantly after the introduction of functional endoscopic sinus surgery in the management of chronic rhinosinusitis. It gave the excellent visualization and atraumatic surgical technique in the management of eight cases of cerebrospinal fluid leaks treated in Department of Otolaryngology Medical University of Warsaw. Five of them were grafted with a pedicled middle turbinate mucosal flap. In the next three cases cerebrospinal fluid leaks came from sphenoid sinus and were stopped by free autograft of temporal fascia or abdominal fat. The indications and the advantages of the endoscopic management of cerebrospinal fluid leaks were discussed.

Carbon isotope fractionation in the decarboxylation of phenylpropiolic acid in hydrogen donating media.

13C kinetic isotope effect (KIE) in the decarboxylation of phenylpropiolic acid (PPA) in tetralin medium (Tn) has been determined at 409-432 K and found to be of magnitude similar to the 13C KIE observed in the decarboxylation of malonic acid where the rupture of the C-C bond is the rate determining step. 13C KIE equals 1.0318/at 136 degrees C in the decarboxylation of PPA in Tn medium. Intramolecular 13C KIE in the decarboxylation of malonic acid equals 1.0316 at this temperature. Thus it has been shown that the nearly "full" 13C KIE can be achieved by providing the excess hydrogen to Calpha of PPA (or to triple acetylene bond) using not only strong mineral acids as the source of protons but also by carrying out the decarboxylation in organic medium like tetralin. A mechanism of decarboxylation of PPA in Tn is suggested.

Anatomic variations of the lateral nasal wall in the computed tomography scans of patients with chronic rhinosinusitis.

The development of computed tomography (CT) technique results in the advance in visualization of the anatomic structures of the lateral nasal wall. It allows the anatomic variations of this region to be identified precisely, which is important in surgical treatment of chronic rhinosinusitis. The aim of this study was to determine the incidence of anatomic variations of the lateral nasal wall in a group of patients with chronic rhinosinusitis. The preoperative, frontal plane CT scans of the study group were evaluated using the method of the four-zone interpretation. The incidence of the anatomic variations of the lateral nasal wall was consistent with the results reported by other authors.

[Strabismus and binocular vision disorders in premature infants with retinopathy treated with cryotherapy]. / Wystepowanie zeza i zaburzen obuocznego widzenia u wczesniaków z retinopatia leczona krioterapia.

PURPOSE: To assess frequency of refraction errors, abnormal eye position and binocular vision disorders in children with retinopathy treated with cryotherapy. MATERIAL AND METHODS: Subjects of the study were 67 children, aged 2-7, treated with cryotherapy for active phase retinopathy. RESULTS: The largest group consisted of children with good visual acuity (48%). Refraction error was found in 94% of the examined eyes. Hypermetropia was more common (54%) than myopia (40%). Majority of the examined children had squint, and esotropia was much more frequent (52%). The deviation in 37% of children was over 10 degrees. In the group with birth weight under 1000 grams as well as in those with birth weight 1000-1500 grams the squint occurred in 48%, and in children weighing over 1500 grams in 4.0%. Only 54% of the studied children had normal binocular vision. CONCLUSIONS: Retinopathy of prematurity is a risk factor for development of refraction errors, strabismus and amblyopia. In infants with retinopathy of prematurity treated with cryotherapy it is necessary to have vision check-ups during their early childhood years.

[Classification of rhinosinusitis]. / W sprawie klasyfikacji zapalen zatok przynosowych.

Rhinosinusitis is a multifactorial disease which has been variously defined by far. There is a strong need among all physicians to create a commonly accepted classification of rhinosinusitis, providing indications for an appropriate evaluation, diagnosis and treatment of the disease. The goal of this review is to present leading definitions of different forms of rhinosinusitis as well as classifications based on clinical findings, temporal considerations and the patophysiology of rhinosinusitis.

[Occupational N-hexane neuropathy: clinical and neurophysiological investigation]. / Zawodowa toksyczna polineuropatia po narazeniu na n-heksan--obserwacja kliniczna i elektrofizjologiczna.

A group of 37 patients (26 men and 11 women), aged 18-49 (mean: 29.7) with subacute toxic polyneuropathy was investigated clinically and electrophysiologically. In all cases the disease was induced by occupational exposure to n-hexane in a small enterprise of purse makers. Toxicological investigations revealed a highly increased concentration of n-hexane in glue used in the manufacture process. The clinical course, including delayed worsening of symptoms after cessation of exposure, was typical when compared with previously reported cases of n-hexan-induced neuropathy after glue sniffing and industrial exposure. Electroneurographic (ENeG) studies showed a predominately decreased motor nerve conduction velocity (MNCV) with focal conduction block followed by dramatically diminished CMAPs. Recovery of EneG abnormalities paralled the clinical improvement.