Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

OKN-007 decreases free radical levels in a preclinical F98 rat glioma model

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin–Gd-DTPA–albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.     

The use of a mirror reduces isolation stress in horses being transported by trailer

Horse trailers are a common form of transportation for horses and ponies and often require the animal to travel alone or with a single companion. The current study investigated the effect of transporting horses alone, in company or with an acrylic safety mirror (measuring 81 cm × 61.5 cm) that provided surrogate companionship. The behavioural and physiological responses of 12 mature horses during a 30-min journey by trailer under the three treatments were compared. Behaviours (vocalisation, eating, head-tossing, pawing, and head-turning) were recorded. In order to assess circulatory changes that occur as part of the response to transport, heart rate (HR), rectal (T_r) and ear-pinna (T_p) temperatures were recorded. When travelling with a live companion significantly less time was spent vocalising (p < 0.001), head-turning (p < 0.001), head-tossing (p < 0.01) and pawing (p < 0.01); eating behaviour increased (p < 0.05). Physiological responses (increases in HR and T_r and decreases in T_p) were also significantly reduced when travelling with a live companion (p < 0.01). Travelling with the mirror did not significantly affect physiological responses compared with travelling alone, but the rise in T_r and fall in T_p was reduced (p = 0.052 and p = 0.051, respectively) and can be considered a trend. When travelling with a mirror significantly less time was spent turning the head (p < 0.01), vocalising (p < 0.05) and head-tossing (p < 0.05); eating behaviour increased (p < 0.05). The only significant difference between travelling with a live companion and a mirror was that the time spent turning the head round was less with a live companion (p < 0.05). The provision of surrogate companionship in the form of a mirror was found to be preferable to travel alone, but where possible a live companion is recommended. Isolation during transportation was found to suppress feeding behaviour. Although peripheral blood flow (T_p) has been used to assess transport stress in other species it has not previously been used in the horse. Further evaluation of this non-invasive measure is now required.     

Aluminum chloride impacts dentate gyrus structure in male adult albino Wistar rats

To get better insights into the aluminum neurotoxicity, rats were treated with AlCl₃ for increasing doses and periods. Body and brain weights, plasma and brain AlCl₃ levels were assayed. Light microscopy observation of brain was performed. AlCl₃ exposure showed a significant decrease (p < 0.05) on body and brain weight with the highest dose at 18 months. Statistical analysis confirms no significant interaction during 6 months (ρ = 0.357; p > 0.05) while, significant correlation was observed during 12 (ρ = 0.836; p < 0.001) and 18 months (ρ = 0.769; p < 0.001) between body and brain weight. Plasma and brain AlCl₃ concentration increased significantly (p < 0.05) with dose and period dependent manner. Statistical analysis confirms significant interaction between brain concentrations of AlCl₃ and administrated doses during 6 (ρ = 0.969; p < 0.001), 12 (ρ = 0.971; p < 0.001) and 18 months (ρ = 0.965; p < 0.001). Similar relation was established between plasma AlCl₃ concentration and administrated doses during 6 (ρ = 0.970; p < 0.001), 12 (ρ = 0.971; p < 0.001) and 18 months (ρ = 0.964; p < 0.001). Significant relation was confirmed between plasma and brain AlCl₃ concentration during 6 (ρ = 0.926; p < 0.001), 12 (ρ = 0.983; p < 0.001) and 18 months (ρ = 0.906; p < 0.001). Morphological alterations mainly targeted the subgranular layer with modulation of the dentate gyrus appearance. This study highlights the toxic effect of AlCl₃ on the brain which may affects learning and memory and seems to be different according to dose and duration of exposure.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Neutrophil CD64 expression and serum IL-8: Sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24 h of septic onset, Interleukins (IL)-8, -1β, -6, -10, and -12p70, tumor necrosis factor-α (TNF-α), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p < 0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p < 0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p < 0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR = 1.3, p = 0.01 for CD64 and OR = 1.26, p = 0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

Antioxidant activity of blueberry fruit is impaired by association with milk

The antioxidant properties of dietary phenolics are believed to be reduced in vivo because of their affinity for proteins. In this study we assessed the bioavailability of phenolics and the in vivo plasma antioxidant capacity after the consumption of blueberries (Vaccinium corymbosum L.) with and without milk. In a crossover design, 11 healthy human volunteers consumed either (a) 200 g of blueberries plus 200 ml of water or (b) 200 g of blueberries plus 200 ml of whole milk. Venous samples were collected at baseline and at 1, 2, and 5 h postconsumption. Ingestion of blueberries increased plasma levels of reducing and chain-breaking potential (+ 6.1%, p < 0.001; + 11.1%, p < 0.05) and enhanced plasma concentrations of caffeic and ferulic acid. When blueberries and milk were ingested there was no increase in plasma antioxidant capacity. There was a reduction in the peak plasma concentrations of caffeic and ferulic acid (? 49.7%, p < 0.001, and ? 19.8%, p < 0.05, respectively) as well as the overall absorption (AUC) of caffeic acid (p < 0.001). The ingestion of blueberries in association with milk, thus, impairs the in vivo antioxidant properties of blueberries and reduces the absorption of caffeic acid.     

Effects of dietary whole cottonseed and crude protein level on rumen protozoal population and fermentation parameters

In this investigation in vitro and in vivo trials were performed to determine the efficacy of a cottonseed to limit protozoal population and fermentation parameters. The composition of diets given to the different treatments were as follow: (1) control (without whole cottonseed), 16% crude protein (CP), 3.2% ether extract (E.E.); (2) 20% whole cottonseed, 16% CP, 6.5% E.E.; (3) 20% whole cottonseed, 13% CP, 6.4% E.E. and (4) 20% crushed whole cottonseed, 13% CP, 6.4% E.E. DM disappearance (DMD) and fermentation characteristics of the treatments were determined by in vitro incubation studies. In the in vivo trial, ruminal fluid was taken by rumenocentesis (3 h after feeding) on days 1, 3, 5, 7, 9, 11, 14, 21 and 28 from four sheep fed about treatment diets. The pH and protozoal counts were determined in each sample, while ammonia nitrogen and volatile fatty acid (VFA) were determined in samples taken on days 7, 14, 21 and 28. The in vitro DMD after 24 h incubation decreased (p < 0.01) with the addition of cottonseed in diets 3 and 4 and DMD after 72 h incubation was highest (p < 0.01) for the control diet. The fractional rate of gas production (c) for the control and diet 2 was higher (p < 0.05) than for the diets 3 and 4. Feeding crushed whole cottonseed decreased molar proportion of propionate (p < 0.05) and increased molar proportion of butyrate (p < 0.01). Low crude protein level increased the molar proportion of propionate (p < 0.05) and decreased molar proportion of butyrate (p < 0.05) and cellolytic protozoa population (p < 0.05). Feeding cottonseed decreased (p < 0.05) the total protozoa population from approximately 500,000 to 250,000 ml⁻¹ and Holotrich and cellulolytic protozoa disappeared from the rumen of sheep and only Entodinium sp., remained. This was associated with lower concentration of ammonia nitrogen in rumen fluid of sheep fed diets 4 (p < 0.05) and 2 (p < 0.01). It was concluded that cottonseed reduced rumen fauna and ammonia nitrogen, but had no effect on ruminal VFA while the crushed whole cottonseed decreased molar proportion of isovalerate only. In vivo molar proportion of propionate and butyrate and valerate were increase and decrease, respectively, by decreasing CP percentage in treatment diets.     

Effect of 1,25 dihydroxyvitamin D3 on intracellular IFN-γ and TNF-α positive T cell subsets in pulmonary tuberculosis

We studied the immunomodulatory effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n = 22) and normal healthy subjects (n = 22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)₂D₃ (10^?7 M) for 48 h. T cell subsets positive for IFN-γ and TNF-α were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-γ and TNF-α expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)₂D₃ compared to cultures without 1,25(OH)₂D₃ (NHS; CD3+ IFN-γ+: p < 0.0001; CD3+TNF-α +: p = 0.0292 and PTB; CD3+ IFN-γ+: p = 0.0292; CD3+ TNF-α +: p = 0.0028). The levels of IFN-γ and TNF-α in the culture supernatants of 1,25(OH)₂D₃ treated cultures were also found to be significantly decreased in both groups (NHS; IFN-γ: p = 0.0001; TNF-α: p < 0.0001) and (PTB; IFN-γ: p < 0.0001; TNF-α: p < 0.0001). A positive correlation was observed between IFN-γ and TNF-α expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)₂D₃ in NHS (p = 0.0001; p = 0.001, respectively) and PTB patients (p = 0.002; p = 0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)₂D₃ on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.     

Interaction between vitamin B6 and source of selenium on the response of the selenium-dependent glutathione peroxidase system to oxidative stress induced by oestrus in pubertal pig

This study aimed to assess the interaction between vitamin B₆ and selenium (Se) for the flow of Se towards the Se-dependent glutathione peroxidase (GPX) system in response to oxidative stress naturally induced by oestrus in a pubertal pig model. At first oestrus, forty-five gilts were randomly assigned to the experimental diets (n = 9/group): basal diet (CONT); CONT + 0.3 mg/kg of Na-selenite (MSeB₆0); MSeB₆0 + 10 mg/kg of HCl-B₆ (MSeB₆10); CONT + 0.3 mg/kg of Se-enriched yeast (OSeB₆0); and OSeB₆0 + 10 mg/kg of HCl-B₆ (OSeB₆10). Blood samples were collected at each oestrus (long-term profiles), and daily from day −4 to +3 (slaughter) of the fourth oestrus (peri-oestrus profiles) after which liver, kidneys, and ovaries were collected. For long-term profiles, CONT had lower blood Se than Se-supplemented gilts (p < 0.01) and OSe was higher than MSe (p < 0.01). Lower erythrocyte pyridoxal-5-phosphate was found in B₆0 than B₆10 (p < 0.01). No treatment effect was observed on GPX activity. For peri-oestrus profiles, treatment effects were similar to long-term profiles. Treatment effects on liver Se were similar to those for long-term blood Se profiles and OSe had higher renal Se concentrations than MSe gilts (p < 0.01). Gene expressions of GPX1, GPX3, GPX4, and selenocysteine lyase in liver and kidney were greatest in OSeB₆10 gilts (p < 0.05). These results suggest that dietary B₆ modulate the metabolic pathway of OSe towards the GPX system during the peri-oestrus period in pubertal pigs.     

Dosimetric comparison of RapidPlan and manually optimized plans in volumetric modulated arc therapy for prostate cancer

PurposeThis study evaluated whether RapidPlan based plans (RP plans) created by a single optimization, are usable in volumetric modulated arc therapy (VMAT) for patients with prostate cancer.MethodsWe used 51 previously administered VMAT plans to train a RP model. Thirty RP plans were created by a single optimization without planner intervention during optimization. Differences between RP plans and clinical manual optimization (CMO) plans created by an experienced planner for the same patients were analyzed (Wilcoxon tests) in terms of homogeneity index (HI), conformation number (CN), D_95%, and D_2% to planning target volume (PTV), mean dose, V_50Gy, V_70Gy, V_75Gy, and V_78Gy to rectum and bladder, monitor unit (MU), and multi-leaf collimator (MLC) sequence complexity.ResultsRP and CMO values for PTV D_95%, PTV D_2%, HI, and CN were significantly similar (p < 0.05 for all). RP mean dose, V_50Gy, and V_70Gy to rectum were superior or comparable to CMO values; RP V_75Gy and V_78Gy were higher than in CMO plans (p < 0.05). RP bladder dose-volume parameter values (except V_78Gy) were lower than in CMO plans (p < 0.05). MU values were RP: 730 ± 55 MU and CMO: 580 ± 37 MU (p < 0.05); and MLC sequence complexity scores were RP: 0.25 ± 0.02 and CMO: 0.35 ± 0.03 (p < 0.05).ConclusionsRP plans created by a single optimization were clinically acceptable in VMAT for patient with prostate cancer. Our simple model could reduce optimization time, independently of planner’s skill and knowledge.     

Nutrient composition,digestion and rumen fermentation in sheep of wheat straw treated with calcium oxide,sodium hydroxide and alkaline hydrogen peroxide

This study tested the effect of calcium oxide (CaO), sodium hydroxide (NaOH) and NaOH plus hydrogen peroxide (H₂O₂; AHP) on cell wall composition, digestion and fermentation of wheat straw (straw) in sheep. Treated straws were prepared by mixing straw either with water followed by dusting with CaO at 160 g kg⁻¹ DM or with a NaOH solution alone at 3 l kg⁻¹ DM to supply 80 g NaOH kg⁻¹ DM (Na) or pre-soaked with Na exactly as in the previous treatment for 27 h followed by mixing with 130 g H₂O₂ kg⁻¹ DM (AHP) for 6 h. After 14 days of storage, the treated straws and an untreated straw (U) were fed automatically every 2 h to four individually housed sheep together with a supplement in a 4×4 latin square experiment. Each kilogram supplement DM contained 422 g CP and 10.8 MJ ME. NDF (p<0.001) and hemicellulose (p<0.01) contents were significantly reduced whereas cellulose was increased (p<0.001) in treated compared to untreated straw. ADL was reduced in Ca (p<0.05) but increased (p<0.05) in Na and AHP compared with U. The rumen and total tract digestibility were significantly (p<0.001) greater in sheep fed treated compared with untreated straw. Significant differences (p<0.05) between treatments for pH, NH₃ and VFA were also observed. All treatments improved the nutritive value of straws compared with untreated through modification of cell wall with a subsequent increase in digestibility by sheep. Although the digestibility for Ca was lower than that for Na despite reduction in cell wall, its use to treat straws may be more safe and cost effective than Na. AHP was the most effective and could also improve the energy value of other low quality forages for ruminants. However, the need of AHP for a large amount of NaOH to achieve highly alkaline pH limits its farm scale application. Therefore, further studies should either consider reducing the amount of NaOH or finding alternative alkalis that are cost effective and user-friendly.     

The effects of feeding on the swimming performance and metabolic response of juvenile southern catfish,Silurus meridionalis,acclimated at different temperatures

To test whether the effects of feeding on swimming performance vary with acclimation temperature in juvenile southern catfish (Silurus meridionalis), we investigated the specific dynamic action (SDA) and swimming performance of fasting and feeding fish at acclimation temperatures of 15, 21, 27, and 33 °C. Feeding had no effect on the critical swimming speeding (U_crit) of fish acclimated at 15 °C (p = 0.66), whereas it elicited a 12.04, 18.70, and 20.98% decrease in U_crit for fish acclimated at 21, 27 and 33 °C, respectively (p < 0.05). Both the maximal postprandial oxygen consumption rate (VO_2peak) and the active metabolic rate (VO_2active, maximal aerobic sustainable metabolic rate of fasting fish) increased significantly with temperature (p < 0.05). The postprandial maximum oxygen consumption rates during swimming (VO_2max) were higher than the VO_2active of fasting fish at all temperature groups (p < 0.05). The VO_2max increased with increasing temperature, but the relative residual metabolic scope (VO_2max? VO_2peak) during swimming decreased with increasing in temperature. The present study showed that the impairment of postprandial swimming performance increased with increasing temperature due to the unparalleled changes in the catfish's central cardio-respiratory, peripheral digestive and locomotory capacities. The different metabolic strategies of juvenile southern catfish at different temperatures may relate to changes in oxygen demand, imbalances in ion fluxes and dissolved oxygen levels with changes in temperature.     

Effects of considering the rate of comminution of particles and microbial contamination on accuracy of in situ studies of feed protein degradability in ruminants

Ruminal degradation of dry matter (DM) and crude protein (CP) in samples of soybean meal (SBM), barley grain (BG) and lucerne hay (LH) were measured by an in situ ruminal technique considering either only the outflow rate of particles from the rumen (k_p) or, both k_p and rate of particle comminution (k_c). The effect of correction for the microbial contamination using an ¹⁵N technique was also determined for LH. Degradation and transit studies were completed on three wethers cannulated in the rumen and duodenum and fed a mixed diet of LH and concentrate (2:1 on DM), at an intake level of 50 g DM/kg BW^0.75. Transit studies of concentrates and forages were completed with SBM and LH samples marked with Yb and Eu, respectively. Mean values of k_c were higher in SBM than in LH (0.357 versus 0.204 h, P < 0.05). The same trend was observed for k_p (0.0587 versus 0.0452/h; P < 0.1). Apparent estimates of the proportion of rumen undegraded CP obtained using both rates were 0.884, 0.745 and 0.825 of those obtained using only k_p (0.535, 0.247 and 0.303 for SBM, BG and LH, respectively). Differences occurred for SBM and LH (P < 0.01) and for BG (P < 0.05). The pattern of microbial contamination of LH, expressed as a proportion of residual DM or CP, fitted exponential curves with asymptotic values of 0.076 and 0.586, respectively. Correction for microbial contamination reduced (P < 0.05) estimates of undegraded CP from 0.303 to 0.238, which was further reduced to 0.178 (P < 0.01) when the effect of k_c was also considered.     

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n = 46) by EIA. TXB₂ levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB₂ levels were correlated with VEGF (p < 0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p < 0.0001) and microvessel density (CD-31; p < 0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p < 0.0001) and female patients (p < 0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p < 0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.     

Elements of margin of safety,toxicity and action of sodium selenite in a lipopolysaccharide rat model

ProjectBoth septic shock and sodium selenite (Na₂SeO₃) lead to multiple organ failure through oxidation. Na₂SeO₃ has direct oxidant effects above the nutritional level and indirect anti-oxidant properties.In a lipopolysaccharide (LPS) rat model we assessed margin of safety, toxicity and beneficial effect of pentahydrate Na₂SeO₃ (5H₂O·Na₂SeO₃) at oxidant doses.ProcedureIn a three-step study on 204 rats we: (i) observed toxic effects of Na₂SeO₃ injected intraperitoneously (IP) and determined its Minimum Dose Without Toxic effect (MDWT) 0.25–0.35 mg/kg selenium (Se) content; (ii) injected IP LPS at 70% lethal dose (LD) followed, or not, one hour later by IP Na₂SeO₃ at MDWT and (iii) by doses > MDWT. At 48 h, in survivors, we measured plasma creatinine, lactate, aspartate and alanine aminotransferase (AST, ALT), nitric oxide (NO) and Se concentrations.Results(i) Na₂SeO₃ alone did not increase NO and lactate. Encephalopathy appeared at 1 mg Se/kg. Creatinine increased at 1–1.75 mg Se/kg, AST, ALT at 3–4.5 mg Se/kg, and the minimum LD was 3 mg Se/kg. (ii) Mortality after LPS was 37/50 (74%, [62–86%]) vs. 20/30 (67%, [50–84%]) when followed by Na₂SeO₃ at MDWT (p = 0.483) with a decreased in NO (−31%, p = 0.038) a trend for lactate decrease (−19%, p = 0.068) and an increased Se in plasma of survivals. (iii) All rats died at doses ≥0.6 mg/kg (p < 0.001).ConclusionMechanisms of LPS and Na₂SeO₃ toxicity differ (i.e. NO, lactate). In septic shock 5H₂O·Na₂SeO₃ toxicity increased, margin of safety decrease, but IP administration of dose considered as oxidant of 5H₂O·Na₂SeO₃ showed beneficial effects.     

Differential pattern of cell-surface and soluble TREM-1 between sepsis and SIRS

ObjectiveTriggering receptor expressed on myeloid cells-1 (TREM-1) was reported to play a key roll in amplification of production of inflammatory cytokines. TREM-1 is suggested to be a specific biomarker for sepsis for this reason, but the clinical significance of TREM-1 has not been elucidated. We investigated TREM-1 expression on the cell-surface, and plasma levels of soluble TREM-1 (sTREM-1) in patients with non-infectious systemic inflammatory response syndrome (SIRS) and sepsis admitted to the ICU.MethodsThirty-five patients with SIRS and 21 patients with sepsis admitted to ICU were subjected to the study. TREM-1 expressions on the surfaces of monocytes and neutrophils were measured by flow cytometry. Plasma sTREM-1 level and serum interleukin (IL)-6 level were measured.ResultsSeptic patients had decreased TREM-1 expression, clearly on neutrophils or to a lesser extent on monocyte compared to SIRS patients on ICU admission (neutrophils p < 0.001, monocyte p < 0.05). TREM-1 expression on neutrophils had a significant inverse correlation with serum IL-6 level (r = ?0.64, p < 0.0001). Plasma sTREM-1 level in septic patients was significantly higher than that in SIRS patients (p < 0.05). Plasma sTREM-1 level positively correlated with severity score and non-survivors had increased plasma sTREM-1 level compared to survivors in all SIRS/sepsis patients (p < 0.05).ConclusionsPatients with sepsis had increased soluble TREM-1 and decreased TREM-1 expression on neutrophil compared to SIRS patients. sTREM-1 may be useful to evaluate disease severity and outcome of patients with SIRS or sepsis.     

Relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to Sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs

The main purpose of this study was to determine if temporal relationships exist between serum concentrations of free fractions of thyroxin (fT₄) and triiodothyronine (fT₃), follicle-stimulating hormone (FSH) levels, and Sertoli cell differentiation in euthyroid ram lamb testes. Additionally, testicular thyroid hormone (TH) receptors (TRs) were identified using immunohistochemistry and Western blot analysis. Weekly testicular biopsies and jugular blood samples were collected from 12 ram lambs over the 9 weeks of study. Hormone concentrations and the numbers of dividing Sertoli cells per seminiferous tubule (ST) area were analyzed relative to chronological age of animals and the two distinctive stages of Sertoli cell differentiation: (a) tight junction/ST lumen formation and (b) the onset of support mechanisms for the development of multiple germ cell types (presence of primary spermatocytes in >95% STs). Circulating FSH concentrations increased (p < 0.05) immediately after first detection of ST lumen and reached a nadir (p < 0.05) just prior to the end of the first wave of spermatogenesis. A decline in both fT₄ and fT₃ levels (p < 0.05) occurred after Sertoli cells had formed the ST lumen and began supporting germ cell differentiation. There was a positive correlation between the numbers of proliferating Sertoli cells and serum fT₄ (r = 0.51, p < 0.001) and fT₃ (r = 0.52, p < 0.001) concentrations. TRs were expressed throughout the study period; however, prior to the formation of ST lumen, two isoforms were detected while only one TR isoform was present by the end of the first wave of spermatogenesis. Overall, the exit of Sertoli cells from the cell cycle that presages their final differentiation begins when THs and FSH levels are high, suggesting a permissive role of these hormones in the maturation of STs in prepubertal ram lambs.     

Synthesis and pharmacological evaluation of new arylpiperazines N-{4-[4-(aryl) piperazine-1-yl]-phenyl}-amine derivatives: Putative role of 5-HT1A receptors

In an attempt to design novel 5-HT_1A agonists/partial agonists, based on an arylpiperazine nucleus, a series of N-{4-[4-(aryl)piperazine-1-yl]-phenyl}-amine derivatives were synthesized and biologically tested. The anxiolytic effect of the compounds was investigated employing the Elevated plus Maze (EPM) task. On the basis of in vivo functional test, compound 1c (3 mg/kg) and 4c (3 mg/kg) induced significant increments in open arm entries and time on EPM as compared to Buspirone. The anxiolytic effects of compounds 1c and 4c were effectively antagonized by WAY-100635, a 5-HT_1A receptor antagonist (0.5 mg/kg). Furthermore, we have also evaluated the concentration of 5-HT in the brain tissue using HPLC with fluorescent detection. Our result showed that serotonin levels were significantly decreased by ～38% (p < 0.001) and ～32% (p < 0.001) after acute administration of compounds 1c and 4c, respectively. These findings suggest that the anxiolytic like activity of these new arylpiperazines is mediated via 5-HT_1A receptors in the brain.