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.

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.

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.

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.

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.

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.

Suloctidil therapy in migraine: an open pilot study.

An open pilot study was carried out in 27 patients with severe and frequent migraine attacks who had failed to respond adequately to other drug therapy, to assess the effectiveness and tolerance of prophylactic treatment with suloctidil. Patients received 600 mg suloctidil daily for 6 weeks. Response, assessed on the reduction in frequency of attacks, was considered as good in 7 patients and as significant improvement in 11 patients. Treatment was rated as of doubtful value or as ineffective in the other 9 patients. One patient stopped treatment because of adverse reactions and 10 others reported mild side-effects.

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.

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.

[Thyrotropic and thyroid hormone (T3 and T4) content in Horton' s headache patients]. / Stezenie hormonu tyreotropowego i hormonów tarczycy (T3 I T4) u chorych z bólem glowy Hortona.

In 18 men with Horton's headaches the levels of the following hormones were determined in the serum: TSH, T3 and T4. The determinations were performed during disease relapse and before starting treatment. The control group included 18 blood donors. In the control group the mean TSH level was 3.97 +/- 0.88 microunits/ml, T4 level was 109.06 +/- 26.88 nmol/l, and T3 1.48 +/- 0.14 nmol/l. In the group of patients the mean values were: 2.58 +/- 2.09 microunits/ml, 97.62 +/- 21.81 nmol/l, 1.07 +/- 0.21 nmol/l respectively. The analysis of the results showed statistically significant differences in the concentrations of T3 between the compared groups. In the light of these results the authors believe that there is not sufficient support of the hypothesis on a hypothalamic pathogenesis of the disease. The causes of reduced T3 concentration in these patients are discussed.

[Platelet aggregation in migraine and other headaches]. / Agregacja plytek krwi w migrenie i innych bólach glowy.

In 43 patients with migraine, 20 with Horton's headaches, 13 with trigeminal neuralgia and 50 healthy subjects the circulating platelet aggregates were determined by the method of Wu and Hoak. Moreover, in 23 patients with migraine, 8 with Horton's headache and 21 healthy subjects spontaneous platelet aggregation was determined in platelet-rich plasma with a transmission aggregometer. It was found that the aggregation coefficient was much lower in patients with migraine and Horton's headache than in the control group. In the group with migraine the per cent of spontaneous aggregation in 8 patients differed significantly from the mean value which was similar to that in the control group or in the group with Horton's headache. In the light of these results the authors conclude that significant changes of platelet aggregation occur in migraine and Horton's headache.

[Treatment of amyotrophic lateral sclerosis with TRH]. / Leczenie stwardnienia zanikowegò bocznego przy pomocy TRH.

In view of earlier reports in the literature it was tried to use TRH (thyrotropin releasing hormone) in cases of amyotrophic lateral sclerosis. TRH was given during 3 weeks once daily intravenously in drip infusions in a 0.4 mg dose. For objectivization of the results the muscle power was assessed using a five-grade scale of Lovette. TRH effect on EMG was analysed also. It was found that in only 3 out of 14 patients with moderately progressed disease no improvement was achieved, while in 11 cases the improvement was from 10 to 20%. However, the improvement was transient, and TRH treatment failed to stop the progression of the disease.

[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.

[Neurological assessment of health status in dentists]. / Próba oceny neurologicznej stanu zdrowia lekarzy stomatologów.

A thorough neurological examination supplemented with electroneurographic and radiological examinations was performed in 31 dentists. A special attention was focused on the condition of the peripheral nervous system, particularly cervical roots, and peripheral nerves of upper limbs. All persons under study complained of lumbar pains. In 80.6% of subjects these pains indicated radicular neuralgia. Lesion of ulnar nerve was found in 22.6% and lesion of median nerve in 35.5% of dentists examined. The studies performed also allowed to detect some micro-symptoms.

[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.

[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.

[Congenital syndromes of oculomotor disturbances--diagnosis and results of surgical treatment]. / Wrodzone zespoly zaburzen narzadu ruchowego oczu--rozpoznawanie i wyniki operacyjnego leczenia.

During the period of 30 years (1964-1994), 363 children with different congenital syndromes of oculomotor disturbances were treated: 300 with Duane's retraction syndrome, 29 with Moebius syndrome, 34 with the superior oblique tendon sheath syndrome of Brown. Symptoms of these syndromes are presented. The choice of appropriate therapy of oculomotor disturbances are described. For aesthetic indications surgical treatment was carried out in 142 (47.3%) patients with retraction syndrome, in 20 (68.7%) with Moebius syndrome and in 23 (67.75%) with Brown's syndrome. Marked reduction of deviation in primary position and reduction of anomalous head posture were obtained.

[Selected problems with ocular accommodation in children and youth]. / Wybrane zagadnienia zaburzen akomodacji u dzieci i mlodziezy.

This paper intends to present some accommodative disturbances which cause difficulties in reading and should be differentiated from dyslexia. The work describes the way of evaluation of accommodative convergence to accommodation rate (AC/A ratio). Clinical forms of two types of nonrefractive accommodative convergence excess connected with high AC/A ratio, namely hyperkinetic and hypoaccommodative, are presented. In hyperkinetic type, disturbances of ocular movements coordination during reading prevail. Patients with hypoaccommodation disorders suffer from youth presbyopia. The paper describes the ways of treatment of these disorders.