Effect of Short Peptides of Pregnancy-Specific ß1-Glycoprotein on Differentiation of Human Regulatory T Cells In Vitro and on Their Cytokine Profile.

Pregnancy-specific ß1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor ß (TGFß) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1ß, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1ß, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences.

Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic ß1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

[The molecular marker of the preconditioning phenomenon HIF1α is a new pathway for early detection of visceral hypoxic conditions].

Improvement and development of technologies for laboratory and instrumental examination of patients in recent years have greatly facilitated the diagnosis of ischemic myocardial damage. However, a decrease in the rating of cardiovascular diseases is not expected in the short term. This is due to an increase in the life expectancy of the population, general aging of the population and improving diagnostic capabilities and the provision of medical care. The time for verification of the diagnosis of ischemic disease, simplified the decision on treatment tactics were significantly reduced by introduction of X-ray contrast visualization examination methods such as angiography, quantitative and qualitative laboratory tests, development of diagnostic criteria based on the results of ultrasound and electrophysiological examination methods. Unfortunately, all these techniques are secondary in nature and are applied, when organ damage is for the most part already irreversible. Full restoration of organs is possible only if the patient is successfully evacuated to the hospital and there are specialists of the appropriate level of experiebce, X-ray surgical equipment with suitable supplies or pharmacological agents, usefull for quickly restoration the patency of the great vessels and normal blood flow. A large number of studies appear on the phenomenon of preconditioning at the present stage of development of fundamental medical science. The purpose of this article is to reveal the possibilities of using molecular markers of the phenomenon of preconditioning in the framework of the early detection of hypoxic conditions, the assessment of their diagnostic use in the clinic and the prevention of hypoxia-associated diseases.

[The use of stalevo in the treatment of patients with Parkinson's disease].

OBJECTIVE: To assess the efficacy and tolerability of the three component drug stalevo in the treatment of patients with Parkinson's disease (PD). MATERIAL AND METHODS: We analyzed the experience of using the antiparkinsonian drug stalevo (levodopa/carbidopa/entacapone) in the treatment of acute stages of PD and elderly patients with restricted possibilities of using other antiparkinsonian drugs. The study included 47 patients. A clinical example is presented. RESULTS AND СONCLUSION: Stalevo increased motor and daily activities, reduced the severity of basic symptoms of PD and improved quality of life of the patients.

Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs.

Standardized green tea extract was evaluated for exposure and toxicity in Beagle dogs following oral dosing by capsules. The main component (-)-epigallocatechin gallate (EGCG) accounted for 56-72% of the material. A 9-month chronic study (0, 200, 500, and 1000 mg/kg/day) was done in fasted dogs to take advantage of the reported improved catechin bioavailability with fasting. Extensive morbidity, mortality, and pathology of many major organs led to its early termination at 6.5 months and prevented identification of the toxicity mechanisms. A follow-up 13-week study examined the exposure to and toxicity of the extract. In general, toxicities were less severe than in the chronic study during the same interval. Dosing in a fed state resulted in considerably lower and less variable exposure than found under fasted conditions. Toxicity was less frequent and of lesser severity with lower exposure but limited sample size and large variability prevented reaching that definitive conclusion. Differences in mortality and morbidity between the preliminary terminated chronic and follow-up subchronic studies with the same dose of the same drug lot and similar exposure were not fully resolved as there may be other as yet unclear confounding factors.

Direct determination of selenium in rat blood plasma by Zeeman atomic absorption spectrometry.

The method was developed to be applied for direct determination of selenium in rat plasma by graphite-furnace atomic absorption spectrometry with Zeeman background correction. Blood was obtained from CD rats of both sexes 2h after dosing in weeks 7 and 13 in order to acquire data on the levels of selenium in these animals during 13-week gavage administration of l-seleno-methylselenocysteine (SeMC), a new candidate chemopreventive agent under development. Application of the commonly used method of standard addition was found to be unsuitable to calculate the selenium content in rat plasma (within-run and between-run accuracy and precision parameters were less than 85%). Therefore, a new analytical method was developed. In this method, samples of rat plasma (50 microL) were diluted 10-fold with a reducing agent containing l-ascorbic acid, a modifier solution containing palladium chloride and Triton X-100. Samples were atomized in pyrolytically coated graphite tubes and peak height signals were measured. Selenium concentrations were determined by linear least squares regression analysis based on the standard curve generated in pooled rat blank plasma. Since selenium is normally present in plasma, a three-step approach was used to calculate selenium plasma levels. Initially selenium levels were determined based on the standard curve with selenium-spiked pool plasma. In the second step, background selenium levels in the pooled plasma were determined based on the same standard curve. In the third step, background level was added to the previously derived number. The relative errors were in the range from -4.6 to 11.4% (intra-day assay) and from -0.4 to 8.8% (inter-day assay) which proved good accuracy. The relative standard deviations were in the range from 1.88 to 4.70% (intra-day precision) and from 3.28 to 5.38% (inter-day precision). In rat plasma, the following dose-dependent selenium levels (mean+/-S.D.) in males and females, respectively, were observed at 13 weeks: 655.5+/-48.8 and 595.8+/-43.9 ng/mL (control group), 927.9+/-85.3 and 859.3+/-164.3 ng/mL (0.4 mg/kg per day dose group), 1238.9+/-182.4 and 1169.9+/-112.6 ng/mL (0.8 mg/kg per day dose group), and 1476.5+/-138.1 and 1320.1+/-228.6 ng/mL (2.0mg/kg per day dose group). No significant sex differences in selenium plasma levels were seen in the SeMC-treated groups. No significant differences in selenium plasma levels were seen between mean plasma levels at 7 and 13 weeks. The described method is simple, rapid, accurate, precise and can be easily applied in other laboratories for a large number of samples.

Comparing pharmacokinetic and pharmacodynamic profiles in female rats orally exposed to lovastatin by gavage versus diet.

The objective of this study was to assess how the dosing method (i.e., gavage versus diet) affects the absorption and disposition of lovastatin, as well as its effect on two biological markers of exposure, such as serum levels of cholesterol and triglycerides. In preclinical safety studies the test agent is normally administered by gavage, but in chemoprevention efficacy studies the test agent is usually administered with the diet. Therefore, extrapolation of safety and efficacy data from laboratory animals to humans should consider the influence of the method of administration on the absorption, disposition and effect of the drug. Lovastatin, a blood cholesterol-lowering drug with a short elimination half-life in humans, was used to assess the influence of two different dosing methods on the drug pharmacokinetics and pharmacodynamics. Plasma and liver concentrations of lovastatin and its active metabolite lovastatin-Na were measured in female rats at sequential times after administration. Serum concentrations of triglycerides and cholesterol were measured at similar times and used as biomarkers of effect. Significant differences in pharmacokinetics and pharmacodynamics were observed after administration of lovastatin by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of lovastatin and lovastatin-Na in plasma and liver, lower area under the concentration-time curve of lovastatin-Na in plasma and liver, and less of an effect on the serum concentrations of triglycerides and cholesterol than the corresponding diet dosing. Although no inverse linear relationship was observed between pharmacokinetic and pharmacodynamic markers, in the case of serum cholesterol a visual trend could be observed which might have proven significant had data from a larger number of dose levels been available. As in our previous study with sulindac, this study illustrates potential limitations in trying to extrapolate from data obtained using different dosing schemes to potential safety and efficacy in humans.

Effects of oral dosing paradigms (gavage versus diet) on pharmacokinetics and pharmacodynamics.

In cancer chemopreventive studies, test agents are typically administered via diet, while the preclinical safety studies normally employ oral gavage dosing. Correspondence in pharmacokinetic and pharmacodynamic profiles between the two dosing approaches cannot be assumed a priori. Sulindac, a non-steroidal anti-inflammatory agent with potential chemopreventive activity, was used to assess effects of the two oral dosing paradigms on its pharmacokinetics and pharmacodynamics. Time-dependent concentrations of sulindac and its sulfone metabolite were determined in plasma and potential target organ, mammary gland. Prostaglandin E(2) was used as a pharmacodynamic biomarker and measured in mammary gland. An inverse linear relationship was detected between pharmacodynamic and pharmacokinetic markers, area under the curve for prostaglandin E(2) levels and sulindac sulfone concentrations, respectively, in the mammary tissue. Marked differences in pharmacokinetics and pharmacodynamics were observed after administration of sulindac by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of sulindac in plasma and mammary tissue, higher area under concentration-time curve in plasma and mammary tissue, and greater effect on prostaglandin E(2) levels than the corresponding diet dosing. This study illustrates potential pitfalls and limitations in trying to generalize based on data obtained with different oral dosing schemes and their extrapolation to potential efficacy and health risks in humans.

Quantitative analysis of ES-285, an investigational marine anticancer drug, in human, mouse, rat, and dog plasma using coupled liquid chromatography and tandem mass spectrometry.

A method was developed for the quantitative analysis of the novel anticancer agent ES-285 (spisulosine; free base) in human, mouse, rat, and dog plasma using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in order to support pre-clinical and clinical studies with the drug. Sample preparation was carried out by protein precipitation with acetonitrile, containing isotopically labeled (d(3)) ES-285 as internal standard. Aliquots of 10 micro l of the supernatant were injected directly on to an Inertsil ODS-3 column (50 x 2.0 mm i.d., 5 micro m). Elution was carried out using methanol-10 mM ammonium formate (pH 4) in water (80 : 20, v/v) pumped at a flow-rate of 0.2 ml min(-1) with a run time of 8 min. Multiple reaction monitoring chromatograms obtained on an API365 triple-quadrupole mass spectrometer were used for quantification. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in human, mouse, rat, and dog plasma and the linear dynamic range extended to 500 ng ml(-1). A full validation of the method was performed in human plasma, and partial validations were performed in mouse, rat and dog plasma. Accuracies and precisions were <20% at the LLOQ concentration and <15% for all other concentrations in all matrices. ES-285 was stable during all steps of the assay. Thus far this method has been used successfully to analyze over 500 samples in pre-clinical trials, and will be implemented in the planned clinical phase I studies.

Simplified urinary immunoassay for 2,4-D: validation and exposure assessment.

Urinary monitoring of exposed workers by either analytic chemical methods or radioimmunoassay suggests that urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) exceeding 30 ppb are indicative of occupational exposure. However, the current methods do not lend themselves to clinical laboratory use in the rural medical setting. The major goal of this project was to provide medical practitioners who care for members of the agricultural community with a cost-efficient way to conduct exposure assessment. This project used a direct 2,4-D enzyme immunoassay (EIA) and measurement of the ratio between 2,4-D-spiked and non-spiked samples of the same urine to quantify 2,4-D levels. This simplified approach minimizes the effects of non-specific interfering substances in urine and eliminates the need for sample extraction and clean-up. Possible urine co-contaminants (2,4-dichlorophenol and 2,5-dichlorophenol) do not significantly interfere with this immunoassay. Twenty-two forest pesticide applicators who apply and use chlorophenoxy herbicides in their work and 14 comparable control subjects were studied to validate the assay in the occupational setting. Coded urine specimens were examined for levels of 2,4-D by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and compared with immunoassay results from the same specimens. A correlation coefficient of r = 0.982 with a P value of .0001 for a plot of HPLC-MS/MS versus immunoassay demonstrated that the results from these methods were comparable over urinary dose levels ranging from not detectable (<19 ppb) to 1700 ppb 2,4-D, as determined by immunoassay.

Developmental neurotoxicity and immunotoxicity of 2,4,6-tribromophenol in Wistar rats.

Pregnant Wistar rats were exposed to 2,4,6-Tribromophenol (TBP) by whole body inhalation (0, 0.03, 0.1, 0.3, 1.0 mg/m3, 24 hr/day, 7 days/weeks, from day 1 to 21 of gestation). Significant decreases in orientation reactions were noted at concentrations of 1.0 mg/m3 (p < 0.05) in the open field test. Nonsignificant trends (p > 0.05) toward decreased horizontal movement and emotionality in the open field and increased electrical impulse skin pain threshold (SPT) were observed. No significant exposure-related differences in the nonspecific immunological status (phagocytosis and blood anti-microbe activity) of pregnant rats were seen after the exposure. Preimplantation and postimplantation embryo losses were significantly increased in a dose-dependent manner and were seen in all treated groups except the lowest concentration (0.03 mg/m3) group. Signs of retarded fetal skeletal development and increased frequencies of visceral abnormalities were found at concentrations of 0.1 and 1.0 mg/m3. Significant effects were found for lower incisor eruption and ear unfolding at a concentration of 0.3 mg/m3. The grooming behavior of 30-day old male progeny was significantly less than control in all experimental groups. Grooming behavior in female subjects exposed to a concentration of 0.3 mg/m3 and emotionality in subjects exposed to a concentration of 1 mg/m3 were decreased significantly. At 60 days of age emotional reactions were significantly decreased in female subjects from the 0.03, 0.3 and 1.0 mg/m3 groups. SPT was significantly increased in the 1 mg/m3 group for both male and female pups. Thus, evidence of CNS depression influence of TBP both in maternal and offspring groups was found. The NOEL (No Observed Effect Level) for developmental neurotoxicity is thus < 0.03 mg/m3, and the NOEL for maternal neurotoxicity is 0.3 mg/m3. These results suggest that exposure to TBP for 24 hr/day throughout gestation may cause developmental neurotoxicity, embryotoxicity and fetotoxicity, but not immunotoxicity.