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Introduction: Postoperative paresis of the laryngeal nerves may disrupt the protection function of the larynx during swallowing. Material and method: Stage 1: Retrospective analysis of FEES reports in patients with unilateral laryngeal paralysis (n = 99) in terms of the occurrence of penetration or aspiration of saliva and liquid and solid food. Stage 2: Prospective analysis of the results of the EAT-10 screening questionnaire and reports (n = 12) of FEES examination performed in the early postoperative period (up to 7 days after the surgery) in patients with iatrogenic palsy of the X cranial nerve. Assessment of: sensory of the larynx, effectiveness for cough and pressure tests, presence of "white out", PAS scale for a regular diet according to IDDSI (ST 0; ST 7). Results: Stage 1: Penetration or aspiration was found in 65% of patients, in 29% of patients it were silent symptoms. Salivary penetration or aspiration concerned 60.60% of patients. Among people who developed silent disorders, 20 had isolated n. X paralysis, and 79% - polyneuric palsy. The PAS level between 2-8 was found in 65% of patients. Stage 2: 11/12 patients obtained the result of the EAT 10 questionnaire > = 3. "White out" elongation and sensory loss were found in 75% of patients, ineffective cough in 33%. In oral trials, residue (50% ST0), premature swallowing (8% ST0), penetration (33% ST0, 8% ST7) and aspiration (16% ST0, 8% ST7) were found. Adaptive and compensatory techniques were the most frequently combined (66%), and the dominant technique was turning the head towards the affected side. Conclusions: In every sixth patient with iatrogenic laryngeal paralysis, aspiration of non-condensed fluids occurs early after the surgery, and the penetration of these contents into the larynx occurs in every third patient. It is therefore advisable to improve the comfort associated with the quality of life dependent on swallowing and to prevent complications of dysphagia in the study group. Therefore, it is justified to extend diagnostics to include clinical assessment of swallowing and qualify patients for FEES assessment. Key words: dysphagia, penetration, aspiration, laryngeal paralysis, FEES, vocal fold paresis, oropharyngeal dysphagia

Dysphagia concerns 10–89% patients after total laryngectomy; to a greater extent, it concerns patients receiving complementary radiotherapy. The disease mechanism is associated with anatomical changes after surgery (scope of surgery) or complications of adjuvant therapy (xerostomia, neuropathy, swelling of tissue, etc.). The above changes lead to: decreased mobility of the lateral walls of the pharynx and tongue retraction, the occurrence of lingual pumping, decreased swallowing reflex, weakening of the upper esophageal sphincter opening, contraction of the cricopharyngeal muscle, tissue fibrosis, formation of pharyngeal pseudodiverticulum, etc. As a result: regurgitation of food through the nose and oral cavity, food sticking in middle and lower pharynx, prolongation of bolus transit time. Upon the formation of tracheoesophageal fistula, there may be aspiration of gastric contents. The above changes considerably reduce patients’ quality of life after surgery. The diagnostic protocol includes: medical interview (questionnaires can be helpful such as: EAT 10, SSQ, MDADI, DHI), clinical swallowing assessment and instrumental examinations: primarily videofluoroscopy but also endoscopic evaluation of swallowing. In selected cases, multifrequency manometry is necessary. The treatment options include: surgical methods (e.g. balloon dilatation of the upper esophageal sphincter, cricopharyngeal myotomy, pharyngeal plexus neurectomy, removal of the pharyngeal pseudodiverticulum), conservative methods (e.g. botulinum toxin injection of the upper esophageal sphincter, speech therapy, nutritional treatment) and supportive methods such as consultation with a psychologis physiotherapist, clinical dietitian. The selection of a specific treatment method should be preceded by a diagnostic process in which the mechanism of functional disorders related to voice formation and swallowing will be established.

Celem pracy była ocena funkcjonowania chorych z zaburzeniami połykania po operacji radykalnego usunięcia krtani. Do badania wstępnie zakwalifikowano 20 pacjentów, będących dłużej niż 6 miesięcy po całkowitym usunięciu krtani, wymagających przeprowadzenia badania wideofluoroskopowego (VFSS) ze względu na zgłaszane zaburzenia połykania. Grupę ostateczną stanowiło 10 osób (8 mężczyzn i 2 kobiety). Procedura badania obejmowała: zebranie wywiadu, wykonania pełnego badania VFSS oraz wypełnienie przez pacjenta kwestionariusza MDADI. Najczęściej stwierdzanymi problemami były: brak kontaktu nasady języka z tylną ścianą gardła, zaburzenia obróbki oralnej kęsa oraz ruchy pompujące języka. Chorzy oceniali nisko swoją jakość życia. Obecność lub brak regurgitacji treści pokarmowych do nosa znacznie wpływał na funkcjonowanie pacjentów. Zgłaszanie przez chorych zaburzeń połykania oraz problemów z wytworzeniem głosu i mowy zastępczej jest wskazaniem do dalszej diagnostyki (np. VFSS). Dobór skutecznej metody terapeutycznej może wpłynąć na poprawę jakości życia tych osób.

Wines produced from grapes cultivated in cool climate areas are characterized by high levels of organic acids. One method to correct this is malolactic fermentation (MLF). The aim of this study was to determine the effectiveness of different strains of Oenococcus oeni bacteria (Viniflora CH11, Viniflora CH16, Viniflora CH35, Viniflora Oenos, SIHA LACT Oeno) during the biological acidity reduction process. Red wine from Rondo and Regent cultivars was obtained by ethanol fermentation of the pulp, at 20 °C for 14 days. The profile of organic acids was examined with a particular focus on changes in the content of l-malic and l-lactic acids. Additionally, the impact on profile and quantity of phenolic compounds and antioxidant capacity was measured. The results showed that MLF had a positive influence on content of organic acids through the reduction of l-malic acid content with a simultaneous increase of the amount of l-lactic acid. The best effect was obtained with the CH11 and CH35 bacterial strains. The biological acidity reduction process had no significant (P > 0.05) impact on phenolic content or antioxidant capacity. However, the wine making process (ethanol fermentation, maturation) contributed to the reduction of polyphenols and in consequence lower antioxidant capacity of the final tested wines. PRACTICAL APPLICATION: The present study provides useful information on the impact of different Oenococcus oeni bacterial strains on MLF in red wines, reduction of l-malic to l-lactic acid, and stability of phenolic compounds during MLF and the maturation period. Also, this article provides information about phenolic compounds and antioxidant capacity during malolactic fermentation and maturity of red wines made from hybrids of Vitis vinifera such as Rondo and Regent cultivars.

Antimicrobial resistance is a major and growing global problem and new approaches to combat infections caused by antibiotic resistant bacterial strains are needed. In recent years, increasing attention has been paid to nanomedicine, which has great potential in the development of controlled systems for delivering drugs to specific sites and targeting specific cells, such as pathogenic microbes. There is continued interest in metallic nanoparticles and nanosystems based on metallic nanoparticles containing antimicrobial agents attached to their surface (core shell nanosystems), which offer unique properties, such as the ability to overcome microbial resistance, enhancing antimicrobial activity against both planktonic and biofilm embedded microorganisms, reducing cell toxicity and the possibility of reducing the dosage of antimicrobials. The current review presents the synergistic interactions within metallic nanoparticles by functionalizing their surface with appropriate agents, defining the core structure of metallic nanoparticles and their use in combination therapy to fight infections. Various approaches to modulate the biocompatibility of metallic nanoparticles to control their toxicity in future medical applications are also discussed, as well as their ability to induce resistance and their effects on the host microbiome.

Background: The aim of the study was to determine the prevalence of dysphagia in patients with chronic cough and its relationship with the long-term persistence of these symptoms. Methods: Thirty consecutive patients. All patients underwent physical examination, ENT assessment, videolaryngoscopy, functional phoniatric assessment at rest and speech, Water-Swallow Test, and Fiberoptic Endoscopic Evaluation of Swallowing disorders with Reflux Finding Score. Reflux Symptom Index questionnaire was performed. The study was approved by the local Ethics Committee Review Board (KB/39/A/2016). Results: The results of the RFS and the RSI questionnaire showed the risk of reflux in participating patients. The patients presented episodes of spillage, double swallows, penetration, aspiration and residue of food at the hypopharynx. The results of functional assessment correlated with the Water-Swallow Test. The correlation between Fiberoptic Endoscopic Evaluation of Swallowing disorders and Water-Swallow Test results was found for aspiration risk, spillage, and retention of saliva. Conclusion: The results of the study showed prevalence of dysphagia in most patients with chronic chough. It seems that phoniatric assessment in those cases should be expanded and the following tests should be performed: assessment of the laryngeal elevation, Water-Swallow Test, and Fiberoptic Endoscopic Evaluation of Swallowing disorders.

Improved cost-effective bioethanol production using inexpensive enzymes preparation was investigated. Three types of waste lignocellulosic materials were converted&mdash, for the production of enzyme preparation, a mixture of sugar beet pulp and wheat bran, while the source of sugars in hydrolysates was sweet sorghum biomass. A novel enzyme cocktail of Trichoderma citrinoviride C1 is presented. The one-step ultrafiltration process of crude enzyme extract resulted in a threefold increase of cellulolytic and xylanolytic activities. The effectiveness of enzyme preparation, compared to Cellic&reg, CTec2, was tested in an optimized enzymatic hydrolysis process. Depending on the test conditions, hydrolysates with different glucose concentrations were obtained&mdash, from 6.3 g L&minus, 1 to 14.6 g L&minus, 1 (representing from 90% to 79% of the CTec2 enzyme yield, respectively). Furthermore, ethanol production by Saccharomyces cerevisiae SIHA Active Yeast 6 strain DF 639 in optimal conditions reached about 120 mL kg d.m.&minus, 1 (75% compared with the CTec2 process). The achieved yields suggested that the produced enzyme cocktail C1 could be potentially used to reduce the cost of bioethanol production from sweet sorghum biomass.

Background and Purpose Treatment of infections caused by NDM-1 carbapenemase-producing Enterobacteriaceae (CPE) represents one of the major challenges of modern medicine. In order to address this issue, we tested ceragenins (CSAs – cationic steroid antimicrobials) as promising agents to eradicate various NDM-1-producing Gram-negative enteric rods. Materials and Methods Susceptibility to CSA-13, CSA-44, and CSA-131 of four reference NDM-1 carbapenemase-producing strains, ie, Escherichia coli BAA-2471, Enterobacter cloacae BAA-2468, Klebsiella pneumoniae subsp. pneumoniae BAA-2472, and K. pneumoniae BAA-2473 was assessed by MIC/MBC testing of planktonic cells as well as biofilm formation/disruption assays. To define the mechanism of CSAs bactericidal activity, their ability to induce generation of reactive oxygen species (ROS), permeabilization of the inner and outer membranes, and their mechanical and adhesive properties upon CSA addition were examined. Additionally, hemolytic assays were performed to assess CSAs hemocompatibility. Results All tested CSAs exert substantial bactericidal activity against NDM-1-producing bacteria. Moreover, CSAs significantly prevent biofilm formation as well as reduce the mass of developed biofilms. The mechanism of CSA action comprises both increased permeability of the outer and inner membrane, which is associated with an extensive ROS generation. Additionally, atomic force microscopy (AFM) analysis has shown morphological alterations in bacterial cells and the reduction of stiffness and adhesion properties. Importantly, CSAs are characterized by low hemolytic activity at concentrations that are bactericidal. Conclusion Development of ceragenins should be viewed as one of the valid strategies to provide new treatment options against infections associated with CPE. The studies presented herein demonstrate that NDM-1-positive bacteria are more susceptible to ceragenins than to conventional antibiotics. In effect, CSA-13, CSA-44, and CSA-131 may be favorable for prevention and decrease of global burden of CPE.

Sea buckthorn (Hippophae rhamnoides L.) berries have high biological value as a rich source of phenolic compounds, fatty acids and vitamins A, C, E. Due to the high organic acid content and sour taste, the fruits are rarely used in juice production. Therefore, the study aimed to determine the metabolic activity of Lactobacillus plantarum, Lactobacillus plantarum subsp. argentoratensis and Oenococcus oeni strains along with the dynamics of changes in organic acids, sugars, phenolic compounds, and antioxidant activity during 72-h fermentation of 100% sea buckthorn and mixed with apple (1:1) juices. The strongest malolactic conversion was in mixed juices (to 75.0%). The most efficient strains were L. plantarum DSM 10492, 20174 and 6872. L. plantarum strains caused an increase in flavonols and antioxidant activity of sea buckthorn-apple juices. The results can be used to select conditions and strains in industrial-scale fermentation, to produce novel sea buckthorn products and increase their consumption.

Aim of study: Evaluation of the speech therapy on voice quality in patients with unilateral vocal fold palsy. Material and methods: The study group included 11 patients, 8 women and 3 men, in age between 16 to 72 years, with unilateral vocal fold palsy, diagnosed in ENT Department of Warsaw Medical University between 2017-2018. Each person completed questionnaires: the voice disability self-assessment scale (VHI), the voice-based quality of life (VRQoL) scale, the vocal tract discomfort scale (VTD). All questionnaires were completed twice, before and after the voice therapy. In addition, the acoustic analysis of the voice, the assessment of the maximum phonation time and the breathing tract were performed twice in each patient. Each of the patients had a voice rehabilitation consisting of a series of 10 meetings. Results: Statistical analysis of the results of maximum phonation time, the self-assessment of voice disability, the quality of life depending on the voice, discomfort of the vocal tract voice acoustic analysis showed statistically significant differences in the results before and after rehabilitation (p

It is commonly known that dysphagia is associated with primary (malnutrition, dehydration, aspiration pneumonia) as well as secondary consequences (longer hospital stay, increased treatment cost, higher risk of mortality). Therefore screening tests in swallowing disorders, especially at risk groups are essential. The aim of screening is identification of patients at risk of dysphagia and referring patient to further instrumental methods. Test should be non invasive, quick, easy to perform by medical staff, with highest sensitivity and specifity. One of the test is Viscosity – Volume Screening Test (VVST) with 3 different consistencies at 3 volumes (5, 10 and 20 ml), what wider possibilities of these tool in safe consistency and volume indication.

Dysphagia is an interdisciplinary problem. Doctors of different specialties (phoniatrician, ENT, gastrologist, neurologist), speech language therapists, physiotherapists and dieticians should be involved in the therapy of patients. The task of the first group is instrumental evaluation of swallowing, defining the safe consistency of fluids and foods, showing effective therapeutic maneuvers (restitutive and compensatory maneuvers), and finally - adapting the optimal diet to patient's eating ability (adaptive methods). The purpose of the study is to present a modification of the consistency of fluids and foods according to the recommendations of the International Dietary Standardization Initiative (IDDSI). The work shows the basic consistencies and tests that allow for them to be checked.

The study investigated 15 variants of wine differing in their must pre-treatment (maceration by enzymes: Siha Pectinase or SihazymUni) and fermentation by different yeast strains (S.cerevisiae as ICV - D-47; SIHA- Cryaroma; Challenge Aroma White; and S. bayanus as SIHA Active Yeast). The effects of experimental conditions on chemical composition (pH, acidity, sugar), content of phenolic (LC–PDA-QTOF/MS) and volatile compounds (GC-MS), antioxidant activity (ABTS and FRAP assay), color property (CIEL*a*b*), and ethanol content were measured. For all production processes, significant changes in basic parameters and in the amount of polyphenolic compounds and antioxidant activity in wine were observed as compared to must. The wines obtained with S. cerevisiae were characterized by higher polyphenol content, smaller reduction of antioxidant activity and the strongest aroma. Challenge Aroma White yeast strain particularly contributed to good quality of wine. Enzymatic pre-treatment did not significantly affect the studied parameters (expect pH and the content of phenolic acids).

The aim of this study was to investigate the influence of various Saccharomyces cerevisiae and Saccharomyces bayanus on the content of phenolic compounds (UPLC-PDA-FL), antioxidant activity (ABTS and FRAP), colour (CIEL*a*b*) and sensory attributes of chokeberry wine. The pH, content of sugar, polyphenols, total acidity, colour, and antioxidant activity were evaluated in chokeberry must, wine after fermentation, and wine after 10 months of storage at 4 and 25 °C. High contents of polyphenol compounds and antioxidant activity were obtained in chokeberry wine after storage at 4 °C. The colour and sensory attributes of these wines were intensively red, attractive and accepted by the consumer. The temperature during storage significantly influenced increase in the quality of the products. However, the different types of yeasts used to ferment chokeberry wine did not significantly affect the bioactive compounds, antioxidant activity or colour of chokeberry wine.

In this article, we proposed a questionnaire for assessment of videofluorscopic examination in patients with swallowing disorders. It was developed during years 2015 - 2016 by the Department of Otolaryngology at the Medical University of Warsaw. The main body of the form consists of 3 parts assessing the oral phase, the pharyngeal phase and the esophageal phase. The main column contains a list of specific symptoms with yes/no answers. There is a four-point scale assessing the severity of the disorders from 0 to 3, where 0 corresponds to clinically insignificant findings, 1- mild impairment, 2 - moderate impairment, and 3 – severe impairment. Other elements of the form include: basic demographic and nutritional data, assessment of the consistency and texture of ingested food, food texture recommended based on the evaluation acc. to the recommendations of the International Dysphagia Diet Standardization Initiative (www.iddsi.org), and therapeutic assessment before implementation of speech therapy. The questionnaire was prepared on the basis of Trinity College survey, own experience and literature data.

This study present the effects of different pre-treatment maceration techniques (microwave, thermo-maceration, and enzymatic treatment) on the content of phenolic compounds (by UPLC-PDA) and their redox potential by cyclic voltammetry (CV), antioxidant capacity (ABTS radical cation decolorization assay and FRAP as Ferric Reducing Ability of Plasma), and other basic chemical properties of red wine made from cv. Dornfelder. The pre-treatment maceration technique significantly (p 0.05) affected the content of total phenolics in Dornfelder must (freshly crushed grapes), with particularly high amounts in the must pre-treated by microwaves (4344.0 mg/100 mL), and the lowest in the must pre-treatment by thermo-maceration (2979.8 mg/100 mL). A positive correlation was found between the content of total phenolics and antioxidant activity (R = 0.69 and 0.52 by ABTS and FRAP assay, respectively) and individual groups of polyphenols (for ABTS assay with anthocyanins, flavonols, flavan-3-ols, respectively R = 0.60, 0.64 and 0.66, while for FRAP method only for anthocyanins R = 0.79). The highest antioxidant activity was also determined for the variant with microwave pre-treatment (must 3.31 mM and 3.05 mM Trolox/100 mL for ABTS and FRAP assay, respectively). Following the fermentation and maturation, the amount of polyphenols decreased. No thermal pre-treatment maceration methods gave lighter or redder must. After winemaking process all the samples were characterized by less red and more yellow shade.

The yeast strain and storage time is an important factor affecting the development of phytochemicals and sensory attributes in ciders. Therefore, the aim of this study was to determine the influence of yeast strains (Saccharomyces bayanus and Saccharomyces cerevisiae), &beta, cyclodextrin (BCD), and storage time on physicochemical parameters, contents of phenolic compounds (ultra-performance liquid chromatography with photodiode array detector coupled to quadrupole time-of-flight tandem mass spectrometry (UPLC&ndash, PDA&ndash, QToF-MS/MS)), antioxidative activity (free radical-scavenging ability (ABTS) and ferric reducing antioxidative power (FRAP) assay), and sensory attributes of new cider from the &ldquo, Bella Marii&rdquo, cultivar of red apple. The pH value, acidity, concentrations of alcohol, organic acids, and polyphenols, and the color and antioxidative properties were evaluated in red apple ciders immediately after fermentation and after three months of storage at 4 &deg, C. S. cerevisiae SIHAFERM Finesse Red with BCD and SIHAFERM Finesse Red yeast strain especially contributed to obtaining ciders with a high content of the tested compounds. The use of BCD during fermentation significantly influenced the protection of bioactive compounds, by as much as 18%. Storage time had an impact on concentrations of the tested components (mainly on the total flavan-3-ols and phenolic acids). Based on the achieved values of parameters analyzed in red apple ciders and results of the consumer acceptance test, it may be concluded that red apple offers vast potential for the production of ciders with a high content of polyphenolic compounds.

The aim of this study was to identify and compare phenolic acids, flavan-3-ols, flavonols, and anthocyanins in two the most popular interspecific hybrids of red grapes, Rondo and Regent, nowadays very popular in red wine production in Poland. The phenolic profiles of these hybrids have not yet been reported. Thirty-three phenolic compounds, including 2 flavan-3-ols, 3 phenolic acids, 5 flavonols, and 23 anthocyanins, were determined in the examined samples using the ultra-performance liquid chromatography photodiode detector-quadrupole/time of flight-mass spectrometry (UPLC-PDA-Q/TOF-MS) method. Major differences were found in the phenolic profiles of investigated cultivars. The Regent hybrid exhibited the highest total phenolics content (27029.75 mg/kg dry matter) but Rondo was characterized by the highest concentration of anthocyanins (19342.36 mg/kg dm). The dominant fraction was anthocyanin compounds, especially acetylated > diglucosylated forms than glucosylated ones. This data represents valuable information that may be useful for oenological practices and to valorise these varieties as sources of bioactive compounds.

This study investigated the energy outputs from methane and ethanol production from sweet sorghum (Sorghum bicolor (L.) Moench). Different methods of sorghum biomass management were compared. The effect of bio-based waste products – sewage sludge and digestate replacing urea – on the energy output of biofuels produced from two different hybrids of sweet sorghum was evaluated. Conversion of fresh sorghum biomass into methane generated 76.6–179.5 GJ ha−1, while the gross energy output from ethanol was only 22.6–70.5 GJ ha−1. Application of digestate allowed the highest energy efficiency ratio to be obtained in terms of ethanol production for both tested hybrids and in terms of methane for Rona 1. Sorghum should be used as biogas feedstock in the temperate climate of Central Europe. The application of waste – sewage sludge and digestate – in feedstock management increased the energy efficiency of biofuel production.

Static and dynamic electric dipole polarisabilities and first hyperpolarisabilities of test molecules are evaluated within the CCSD response approach using medium-size basis sets: the large polarised LPol-n (n = ds, dl, fs, fl) sets, the aug-pc-n (n = 1, 2) basis sets of Jensen, and the SVPD and TZVPD basis sets of Rappoport and Furche. Results are compared to reference values obtained using the x-aug-cc-pVXZ (x= -, d, t; X = D, T, Q, 5) basis sets of Dunning. The aug-pc-2 basis set of Jensen proves to be an attractive choice in the case of polarisability calculations, and among the smaller sets, the LPol-ds and the TZVPD basis sets can be recommended. For the first hyperpolarisability calculations, we recommend in particular the use of the LPol-ds basis set. Other attractive choices here are the LPol-dl, the LPol-fs and the aug-pc-2 sets.

Introduction: The Focal Infection Theory, originally presented at the beginning of the 20 th century, postulates that systemic diseases can be caused by microorganisms that arise from the focus of infection. Foci of infections have been described as sinuses, adenoids, tonsils, teeth, genitourinary tract, gall bladder and kidneys. A focus of infec - tion is defined as the area that can occur in any part of the body, contains a pathogen (microorganism) and is usu - ally asymptomatic. There are discordant opinions about the role of focal infections in the pathogenesis of psoria - sis and urticaria. Aim: To establish whether there is a higher incidence of focal infections in patients with chronic urticaria and pso - riasis. Material and methods: We retrospectively reviewed 129 patients with a history of psoriasis and chronic urticaria: 58 women and 71 men treated in the Department of Dermatology of the Jagiellonian University Medical College in Krakow. Results:In the analyzed group, 11 patients had a dental consultation, 58 - laryngological consultation and 29 women had a gynecological consultation. The most common examples of focal infection were tonsillitis, upper respiratory tract infections, sinusitis, dental caries and genitourinary tract infections. Aggravating factors were similar to pre - viously described. Conclusions: A high incidence of focal infections in patients with psoriasis and urticaria suggests that infections may play a significant role in the pathogenesis of these skin disorders. Treatment of infection foci may play the key role in the remission of skin changes.

IGF-I is regarded as the most sensitive marker of growth hormone (GH) secretion in both GH deficient individuals and in individuals with excessive GH production. Studies on the effect of inhibitors of GH action in normal experimental animals are difficult to evaluate due to the complex relationship and feed back mechanisms of the GH/IGF-I system and the hypothalamo-pituitary axis. To circumvent the GH/IGF-I feedback mechanisms, we have used hypophysectomized (HX) rats treated with GH to assess the potential of a new low molecular weight compound, BVT-A ((N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2-furamide), to act as a GH receptor antagonist in vivo. GH treatment of HX rats induced serum IGF-I, body weight and hepatic mRNA levels of IGF-I, IGFBP-3, ALS and the IGF-I and GH receptors. Co-treatment with BVT-A suppressed all the GH-induced effects. We conclude that the GH substituted HX rat is a useful model for studies on GH receptor antagonists, and for the first time, a small molecule GH receptor antagonist with in vivo activity has been revealed. This opens up for development of new drugs for diseases in which lowering of GH receptor activity would be beneficial.

The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor-1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human alpha1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds.

In the present study, the influence of chromium(III) complexes (acetate, chloride, glycinate, histidinate, lactate and propionate) on insulin binding and signal transduction [phosphorylation of tyrosine and serine in the insulin receptor substrate (IRS)-1] was investigated in vitro using three experimental models: isolated rat liver membranes and cultured mouse C2C12 myoblasts or 3T3-L1 preadipocytes. The examined complexes did not elevate the binding of insulin to the liver membranes. Moreover, chromium histidinate, lactate, acetate and propionate complexes diminished the specific binding of insulin. Simultaneously, chromium chloride, which did not significantly elevate insulin binding, increased the number of membrane accessible particles of the insulin receptors. However, it was accompanied by slightly diminished affinity of the receptor to the hormone. Chromium acetate and propionate significantly diminished the binding capacity of the low-affinity insulin receptor class. Investigations with the myoblast cell line C2C12 and preadipocyte cell line 3T3-L1 did not allow differentiation of the influence of the examined complexes on insulin binding. Immunodetection of phosphorylated forms of IRS-1 showed that the chromium compounds modulated the transduction of the insulin signal. Chromium glycinate, acetate and propionate decreased the amount of IRS-1 phosphorylated at serine. Since it is generally thought that phosphorylation of serine in IRS-1 may moderate insulin action, the above mentioned chromium complexes may, in this way, enhance insulin effects inside target cells. Phosphorylation of tyrosine in IRS-1, which acts as a stimulatory signal for further steps of insulin action, was elevated after the incubation of 3T3-L1 cells with insulin. Chromium supplementation did not additionally intensify this process. However, in the absence of insulin, chromium glycinate and acetate slightly elevated the level of IRS-1 phosphorylated at tyrosine. This fact may be important in vivo at low levels of insulin in blood. The results indicate that the action of chromium(III) complexes involves a direct effect on the number of receptors accessible to insulin, their affinity to the hormone and the modulation of the signal multiplying proteins by their phosphorylation.

An improved assay method for tissue plasminogen activator (t-PA) in plasma samples is described, which allows the plasma t-PA activity to be determined within 3 hours. The method minimizes the action of possible inhibitors by immediate acidification of blood samples prior to separation of the blood cells. In almost all plasma samples variable amounts of a rapid t-PA inhibitor could be demonstrated. The inhibitor was present in plasma and seemed not to be associated with the blood cells. A quantitative method for determination of the inhibitor content in plasma was developed. In this investigation 1 unit of inhibitor is defined as the amount that neutralized 1 unit (0.06 pmol) of t-PA in 10 minutes. The content of t-PA inhibitor in plasma samples from healthy individuals was determined as 0.6 U/ml +/- 0.5 U/ml (range 0-3.2, median 0.4). In a group of patients (n = 75) with suspected disturbance of the haemostatic system, elevated levels were found (3.8 +/- 2.7, U/ml median 3.6 U/ml, range 0 - 15 U/ml). Kinetic analysis with plasma from three patients with elevated inhibitor content showed very similar rate of t-PA inhibition and assuming the formation of a 1:1 t-PA-inhibitor complex a second order rate constant of 10(7) M(-1) s(-1) was calculated. A regulatory function may be anticipated for this inhibitor, but its exact patophysiological role has still to be established.

The production of PA-inhibitor by the human cell-lines Hep G2 and HT 1080 have been studied by immunochemical and functional methods. The data suggest that the PA-inhibitor is released from the cells in an active form and that it is subsequently transformed into an inactive form. The active form seems to be partially protected by foetal calf serum and can be separated from the inactive form by gel-filtration. The inactive form behaves as a low M r (about 50 000) component in the absence and in the presence of sodium dodecylsulphate. In contrast, the active form of PA-inhibitor behaves as a high M r (>300000) compound in the absence of sodium dodecylsulphate but as a low M r compound in its presence. The low Mr inactive PA-inhibitor form has been purified to homogeneity from HT 1080 conditioned medium, collected in the absence of foetal calf serum. This was achieved by chromatography on Concanavalin A-Sepharose, gel-filtration on Sephacryl S-300 and affinity chromatography on insolubilized monoclonal antibody against PA-inhibitor. On treatment of this form of the inhibitor with 4 mol/L guanidinium chloride, the activity was regained, but its gel-filtration behaviour was unchanged ( M r about 50 000). The high M r spontaneously active form of the PA-inhibitor neither changed its activity nor its gel-filtration behaviour by this treatment.

SummaryPolyclonal antibodies have been raised against the inhibitor moiety in the purified complex between tissue plasminogen activator and its fast inhibitor (PA-inhibitor) in human plasma/ serum. A radioimmunoassay for quantitation of PA-inhibitor antigen was developed. The polyclonal antiserum and a previously described monoclonal antibody against the PA-inhibitor (14) have been used to study the immunological relationship between PA-inhibitors from plasma, serum, platelets, placenta extract and conditioned media from Hep G2 and HT 1080 cells. It was demonstrated that the ratio between PA-inhibitor activity and antigen varied considerably between the different sources. In the plasma samples studied, similar activity and antigen concentrations were found, suggesting that the PA-inhibitor in these samples mainly was in an active form. On the other hand the other sources seemed to contain variable amounts of inactive PA-inhibitor forms. Immunoadsorption experiments revealed that the PA-inhibitor (activity and antigen) from all the sources were specifically bound to the insolubilized antibodies (polyclonal and monoclonal). In no case, however, could active PA-inhibitor be eluted from the immunoadsorption columns. Also the competitive radioimmunoassays suggested that the PA-inhibitors from the different sources studied, were closely immunologically related.

Summary. A comparison has been made between the prothrombin time test using British Comparative Thromboplastin (BCT) and a chromogenic substrate assay for factor VII in the assessment of laboratory control of oral anticoagulants in short-term and long-term patients. Opportunity was also taken to compare the findings with parallel results obtained with the venous Thrombotest technique and a specific clotting assay for factor VII. There was good agreement between the amidolytic factor VII assay, using a method modified from Seligsohn et al (1978) with the Quick test using BCT and Thrombotest in 60 long-term patients. Tests in 53 patients within the first 3 weeks of starting oral anticoagulant administration gave less satisfactory agreement between the above amidolytic method and the conventional tests. In contrast, there was a good correlation between the two conventional tests in both groups and also between the clotting and amidolytic factor VII method. Although the results are an improvement on previous, less satisfactory correlations between the BCT prothrombin time method and amidolytic assays for factor II and X, the present study indicates the limitations of a specific clotting assay versus a broad spectrum extrinsic clotting test in oral anticoagulant control. While not warranting the routine use of the chromogenic assay for factor VII in place of the prothrombin time using BCT, the factor VII amidolytic assay offers a limited but dependable guide to dosage in long-term patients. The complexity of the technique in its present form militates against its adoption for routine anticoagulant control in hospital laboratories.

SummaryHybridoma cells were produced by fusing mouse myeloma cells (SP 2/0 - Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MCI, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MCI) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was devloped with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).

Pleural fluid from an early, active phase of BCG-induced pleurisy was compared with fluid from late, healing phase, characterized by fibrinous adhesions. Exudates were tested for proteolytic activity on chromogenic peptide substrates designed for plasmin, tissue plasminogen activator, factor Xa, thrombin and plasma kallikrein. Considerable activity of active-phase pleural fluid was found on all of these substrates, and significantly lower values in the healing phase. Most exudates from both stages had very low fibrinogen concentration. Fibrinopeptide A, fibrinolytic products and antiplasmin were found in all exudates. Little or no fibrinolytic effect of pleural fluid was demonstrable on plasminogen free fibrin plates, despite the high activities on the low molecular weight substrates. Occurrence of α 2 -macroglobulin-enzyme complexes is suggested as an explanation. The experimental results indicate that protease of the fibrinolytic and coagulation systems are active in the chronic inflammation of pleurisy, with higher levels of activity in active pleurisy phase.

This study investigated the effects of a flash release versus traditional maceration expansion on the content of phenolic compounds (by LC–MS, UPLC–PDA), antioxidant activity (ABTS and FRAP), and other physical and chemical properties of wine from Pinot noir cultivar. The content of polyphenols in a must obtained by the flash release technology was higher than when using a traditional method. Flash release expansion improved the extraction of polyphenol compounds, especially anthocyanins, as well as color properties and antioxidant activity. However, during maturation and storage, the content of polyphenols significantly decreased, especially in the wine exposed to the flash release expansion. Despite lower total polyphenols content (1435 vs. 1686 mg/L in control), the final flash release wine contained more anthocyanins which resulted in approximately 2 times higher antioxidant activity. High correlation between antioxidant activity (r 2 = 0.84 and 0.79 for ABTS and FRAP assay, respectively) and the content of anthocyanins was determined in both types of examined wines. Changes in color were also observed in the must prepared by flash release versus traditional maceration. During fermentation, L* and a* parameters, the variables associated with more light and red color, increased, and during maturation, a rise in b* parameter, associated with yellow color, was observed.

Chronic malignant pleural exudation is generally characterized by little or no fibrin deposition. However, during induced inflammation, fibrin deposition concomitant with cessation of exudation is seen. To judge the involvement of the fibrinolytic system in this process, concentrations of fibrinolytic factors were followed in 10 patients during treatment by quinacrine instillation and tube drainage. Plasminogen and alpha-2-antiplasmin were found in low concentrations and did not show significant changes during treatment. The plasminogen activator inhibitor PAI-1, which plays an important role in the regulation of fibrinolysis, was also studied during treatment. Before treatment the concentration of PAI-1 was 21.7 +/- 12.0 (mean +/- SD) AU/ml and it increased to 86.9 +/- 25.9 AU/ml 6 h after quinacrine instillation. D-dimer, a product of the lysis of fibrin, was found in high concentrations before treatment (62.7 +/- 25.5 micrograms/ml) and in low concentrations 6 h after treatment (12.2 +/- 7.9 micrograms/ml). Thus, it was possible to demonstrate that the fibrinolytic system is activated during chronic malignant pleural exudation and, furthermore, that the activity decreases during induced inflammation.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a ‘second-site’ interaction, in addition to that between the enzyme active site and the inhibitor ‘bait’ peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the ‘free’ concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.

Neuropeptide B/W receptor 1 (NPBWR1) and neuropeptide B/W receptor 2 (NPBWR2) are two structurally related orphan receptors linked to protein G. In rodents NPBWR2 is absent, and its counterpart is described as being similar to neuropeptide B/W receptor 2. Endogenous ligands of these receptors have been identified. One of them is 29 amino acid residues long, uniquely modified with bromine and, thus, termed neuropeptide B (NPB). The other, neuropeptide W (NPW), has been identified in two molecular forms of 23 and 30 amino acids (NPW23 and NPW30), respectively. Both NPB and NPW affect food intake and energy expenditure. Since leptin, a potent anti-obesity hormone, and insulin are involved in the control of energy homeostasis, the present study aimed to investigate whether NPB and NPW affect leptin and insulin secretion in the rat. RT-PCR technique revealed the presence of ppNPB, ppNPW, NPBWR1 and NPBWR2-like mRNAs in isolated pancreatic islets of the rat. NPB and NPW immunoreactivities were observed in all of the cells of the pancreatic islets. Only when a higher dose was administered (3 nmol/100 g body weight) did NPW transiently lower blood insulin levels whereas NPB injection did not alter insulinaemia in the studied rats. At 30 min, but not 60, of the experiment, NPW notably lowered blood leptin concentrations at both tested doses. On the contrary, NPB injections had no effect on blood leptin and insulin concentrations. Thus, the results suggest that NPW but not NPB exerts a potent suppressive effect on blood leptin concentrations in the rat, and this mechanism may be involved in NPW regulation of energy homeostasis.