Comparison of short-term and medium-term swimming training on cardiodynamics and coronary flow in high salt-induced hypertensive and normotensive rats.

The aim of present study was to evaluate the effects of 3- and 6-week swimming exercise on cardiodynamics and coronary flow in high salt-induced hypertensive and normotensive rats. 80 male Wistar albino rats (6 weeks old) were divided into 8 groups: hypertensive animals that swam for 3 weeks; hypertensive animals that swam for 6 weeks and their respective sedentary controls; normotensive animals that swam for 3 weeks; normotensive animals that swam for 6 weeks and their respective sedentary controls. Hypertensive animals were on high sodium (8% NaCl solution) diet for 4 weeks, and these animals did not drink tap water during the experimental protocol. After sacrificing, hearts were isolated and perfused according to Langendorff technique at gradually increased coronary perfusion pressure (40-120 cmH2O). The following parameters of cardiac function were continuously recorded: maximum and minimum rate of pressure development in LV, systolic, and diastolic left ventricular pressure, and heart rate. Coronary flow was measured flowmetrically. Findings of the present study may help in better understanding of short- to medium-term exercise-induced direct effects on cardiac function and perfusion. Generally viewed, swimming of both durations did not change myocardial function and perfusion in hypertensive and normotensive conditions.

Effects of atorvastatin and simvastatin on oxidative stress in diet-induced hyperhomocysteinemia in Wistar albino rats: a comparative study.

Considering the well-known antioxidant properties of statins, it seems important to assess their impact on major markers of oxidative stress (superoxide anion radical, nitric oxide, and index of lipid peroxidation) to compare the antioxidative potentials of atorvastatin and simvastatin during the different degrees of hyperhomocysteinemia (HHcy) in rats. This study was conducted on adult male Wistar albino rats (n = 90; 4 weeks old; 100 ± 15 g body mass) in which HHcy was achieved by dietary manipulation. For 4 weeks, the animals were fed with one of the following diets: standard rodent chow, diet enriched in methionine with no deficiency in B vitamins (folic acid, B6, and B12), or diet enriched in methionine and deficient in B vitamins (folic acid, B6, and B12). At the same time, animals were treated with atorvastatin at doses of 3 mg/kg/day i.p. or simvastatin at doses of 5 mg/kg/day i.p. Levels of superoxide anion radical and TBARS were significantly decreased by administration of simvastatin in normal and high-homocysteine (Hcy) groups (p < 0.05). At 4 weeks after feeding with purified diets, the concentrations of the GSH, CAT, and SOD antioxidants were significantly affected among all groups (p < 0.05). Our results indicated that statin therapy had variable effects on the redox status in hyperhomocysteinemic rats, and simvastatin demonstrated stronger antioxidant effects than did atorvastatin.

Differential transcriptome of tolerogenic versus inflammatory dendritic cells points to modulated T1D genetic risk and enriched immune regulation.

Tolerogenic dendritic cells (tolDCs) are assessed as immunomodulatory adjuvants to regulate autoimmunity. The underlying gene expression endorsing their regulatory features remains ill-defined. Using deep mRNA sequencing, we compared transcriptomes of 1,25-dihydroxyvitaminD3/dexametasone-modulated tolDCs with that of non-modulated mature inflammatory DCs (mDCs). Differentially expressed genes controlled cellular interactions, metabolic pathways and endorse tolDCs with the capacity to regulate cell activation through nutrient and signal deprivation, collectively gearing tolDCs into tolerogenic immune regulators. Gene expression differences correlated with protein expression, designating low CD86 and high CD52 on the cell surface as superior discriminators between tolDCs and mDCs. Of 37 candidate genes conferring risk to developing type 1 diabetes (T1D), 11 genes differentially expressed in tolDCs and mDCs regulated immune response and antigen-presenting activity. Differential-expressed transcripts of candidate risk loci for T1D suggest a role of these 'risk genes' in immune regulation, which targeting may modulate the genetic contribution to autoimmunity.

Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model.

Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery.

8-Iso-prostaglandin F2α as a potential biomarker in patients with unipolar and bipolar depression.

OBJECTIVE: Previous studies have shown that the disturbance of redox homeostasis plays a role in the pathogenesis of mood disorders. It is currently unclear whether oxidative stress parameters can be used as biomarkers (state vs. trait). The aim of the present study was to investigate oxidative stress markers in patients with major depressive disorder (MDD) and bipolar disorder (BP) in acute depressive episodes and remission, and healthy individuals. PATIENTS AND METHODS: Thirty-two patients with a diagnosis of MDD, 32 patients with a diagnosis of BP and 32 matched healthy controls were included in the study. We measured the serum levels of markers of oxidative damage, including 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-Iso-prostaglandin F2α (8-iso-PGF2α; 8-isoprostane), and malondialdehyde (MDA), and also serum activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) in both acute and remission phase, and in control group. RESULTS: After controlling for the effects of age, sex, body mass index, and smoking status, serum 8-iso-PGF2α levels were significantly higher in both patient groups compared to controls, regardless of disease phase. The activities of GPX and GR were significantly lower in the acute phase in MDD patients compared to controls. Serum GR activity was lower in both acute and remission phase in MDD compared to BP. CONCLUSIONS: Our results suggest that both MDD and BP are associated with a disturbed redox balance with a particularly pronounced increase in serum 8-iso-PGF2α levels in both groups and the presence of glutathione metabolism disorders in MDD patients. Further research is needed to confirm the importance of oxidative stress parameters as potential biomarkers of MDD and BP.

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.

Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit.

We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.

Spatio-temporal patterns in pollination of deceptive Aristolochia rotunda L. (Aristolochiaceae).

Pollination success of highly specialised flowers is susceptible to fluctuations of the pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers exhibiting a highly specialised pollination system. The sole pollinators are kleptoparasitic flies in search of food. This study investigates these pollinators on a spatio-temporal scale and the impact of weather conditions on their availability. Two potential strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and an increased floral life span, were tested. A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian populations. Availability of the main pollinator was correlated to meteorological data. Artificial pollination experiments were conducted and the floral life span was recorded in two populations according to pollinator availability. Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollinator compositions varied among populations. Weather conditions 15-30 days before pollination had a significant effect on availability of the main pollinator. Flowers were not autonomously selfing, and the floral life span exhibited considerable plasticity depending on pollinator availability. A. rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of kleptoparasitic flies, rather than on a single species. Pollinator variability may result in differing selection pressures among populations. The availability/abundance of pollinators depends on weather conditions during their larval development. Flowers show a prolonged trapping flower stage that likely increases outcrossing success during periods of pollinator limitation.

Central versus peripheral site of action of the tachykinin NK1-antagonist RP 67580 in inhibiting chemonociception.

1. Many studies indicate an involvement of substance P in the transmission of nociceptive stimuli, without, however, presenting any conclusive evidence as to its exact site and mode of action. The present experiments tested the involvement of substance P in the mediation of chemical nociception using the non-peptidic specific tachykinin NK1-receptor antagonist, RP 67580 (2-[1-imino-2-(2-methoxyphenyl-ethyl]-7,7diphenyl-4-perhydroiso indolone (3aR, 7aR)). 2. Mean arterial pressure (MAP) and intragastric pressure (IGP) were measured in anaesthetized rats. The reflex changes of these parameters in response to i.p. or s.c. injections of hydrochloric acid or capsaicin were taken to indicate nociception. 3. Intravenous administration of RP 67580 up to 5 mg kg-1 had little influence on the reflex changes in MAP or IGP in response to hydrochloric acid or capsaicin. In contrast, the sensitization of rats to i.p. capsaicin by preinjection of prostaglandin E2 was significantly reduced by 1 mg kg-1 RP 67580. 4. Intrathecal injection of 5 micrograms RP 67580 inhibited the reflex changes of MAP and IGP in response to i.p. or s.c. capsaicin whereas the inactive enantiomer RP 68651 was ineffective. 5. The results indicate that spinal NK1-receptors are involved in the acute transmission of chemically induced pain, while such receptors in the periphery take part in the sensitization by prostaglandin E2. The rather minor ability of i.v. RP 67580 to inhibit the acute nociceptive reflex is attributed to an insufficient penetration of the blood-brain-barrier.

Treatment protocol determines the efficacy of clindamycin in acute murine toxoplasmosis.

To determine the contribution to the efficacy of clindamycin in acute murine toxoplasmosis of treatment protocol variables, groups of Swiss Webster mice inoculated intraperitoneally with 10(2) RH strain Toxoplasma gondii tachyzoites were treated with peroral clindamycin at 25, 50 and 400 mg/kgBM per day for 1, 2 and 3 weeks. While the lowest drug dose applied for a single week prolonged survival time as compared to untreated animals, not even the highest dose applied for 1 or 2 weeks completely prevented mortality. Conversely, 100% protection was achieved with 3-week treatment courses at both 50 and 400 mg/kg per day. While both survival rates and survival times increased in parallel with the drug dose and treatment duration, the latter was shown to be critical to the outcome, suggesting the use of clindamycin as an antitoxoplasmic agent should be as a prolonged course.

Increased activity of lymph node cells in experimental thermal injury: changes in accessory cells in injured area-draining lymph nodes.

Accessory cell content and some of their functional characteristics were determined in regional lymph nodes which drain burn injury (DLN) in rats. Increase in percentages of non-specific esterase-positive cells and NBT+ macrophages and in numbers of dendritic cells were noted in cytospin preparations of draining lymph node cells (DLC) 24 and 72 h following thermal injury. An accumulation of B cells was also noted in the DLN paracortex region at these time points. Enrichment of ED1+ (rat macrophage marker) cells was noted in the adherent DLC population. Increased activity of interleukin-1 (IL-1) in conditioned medium from adherent DLC population and the increased stimulatory capacity of whole DLC or dendritic cell enriched-DLC fraction were noted in functional assays. Enrichment in accessory cells and an increase in their functional activity could contribute to the endogenous activity of regional lymph nodes which drain burned areas.

Study description and baseline characteristics of the population enrolled in a multinational observational study of extended teriparatide use (ExFOS).

OBJECTIVE: To better characterize patients who are currently being prescribed teriparatide in Europe, this article describes the study design and baseline characteristics of participants of the Extended Forsteo * Observational Study (ExFOS). RESEARCH DESIGN AND METHODS: ExFOS is a noninterventional, multicenter, prospective, observational study in men and women with osteoporosis treated with teriparatide during the course of normal clinical practice for up to 24 months and with a post-treatment follow-up of at least 18 months. MAIN OUTCOME MEASURES: Baseline characteristics, including history of fracture and back pain, and health-related quality of life (HRQoL, assessed using the EuroQol-5 Dimension [EQ-5D]). RESULTS: Of 1607 patients enrolled, 90.9% were women. At baseline, mean (standard deviation [SD]) age was 70.3 (9.8) years, and 85.8% of patients had a history of fracture (64.7% with ≥2 fragility fractures). Of those with historic fractures, 90.8% had vertebral fractures (67.8% had thoracic fractures). The mean (SD) of reported bone mineral density T-scores were -3.0 (1.2), -2.4 (1.0), and -2.5 (0.9) for lumbar spine, total hip (left), and femoral neck (left), respectively. Overall, 39.3% of patients had experienced ≥1 fall during the 12 months before enrollment. At baseline, 11.4% of patients were osteoporosis-treatment naïve and 15% were currently using glucocorticoids. The mean (SD) visual analog scale score for back pain during the last month was 50.7 (26.9), and 62.1% of patients experienced daily or almost daily back pain. The median EQ-5D health state value at baseline was 0.62 (first and third quartiles: 0.19, 0.74). CONCLUSIONS: Baseline characteristics of the ExFOS study cohort indicate that patients prescribed teriparatide in Europe have severe osteoporosis with highly prevalent vertebral fractures, frequent and disabling back pain, and a poor HRQoL, despite previous pharmacotherapy for osteoporosis. Limitations include non-randomization, lack of a comparator group, and patient self-report for data on prior medication and fracture history.

Synergistic effect of clindamycin and atovaquone in acute murine toxoplasmosis.

The effect of clindamycin (CLI) combined with autovaquone (ATO) was examined in a murine model of acute toxoplasmosis. Swiss Webster mice intraperitoneally infected with 10(2) or 10(4) tachyzoites of the RH strain of Toxoplasma gondii were perorally treated with either drug alone (for ATO, 5, 25, 50, or 100 mg/kg of body weight/day; for CLI, 25, 50, or 400 mg/kg/day) or both combined (for ATO plus CLI, respectively, 5 plus 25, 25 plus 25, 25 plus 50, 50 plus 50, or 100 plus 400 mg/kg/day) starting with day 1 for 14 days. Survival was monitored during 7 weeks. Residual infection was assessed by a bioassay of representative 4-week survivors and by parasite DNA detection by PCR for representative 7-week survivors. An effect of treatment was shown in all treatment groups compared to untreated control mice (P = 0.0000). Among mice infected with 10(2) parasites, ATO and CLI at any dose combination protected significantly more animals than ATO alone (P = 0.0000), but compared to CLI alone, given its good effect, the combined drugs were no more effective (P > 0.05). For mice infected with 10(4) parasites, the drugs combined at the lowest and highest doses (5 plus 25 and 100 plus 400 mg/kg/day) were, similarly, more effective than ATO alone (P = 0.035 and 0.000, respectively) but not than CLI alone (P > 0. 05). However, treatment with ATO plus CLI at 25 plus 25, 25 plus 50, and 50 plus 50 mg/kg/day protected 20, 33, and 78% of mice, respectively, compared to virtually no survivals among those treated with either drug alone (P < 0.0005), thus demonstrating a significant synergistic effect of ATO and CLI against T. gondii. Furthermore, the dose of ATO at a given dose of CLI was shown to be critical to the effect. Moreover, the absence of residual infection in some survivors shows the potential of this drug combination to eliminate the parasite.

Regulation of phosphatidylinositol 3'-kinase by tyrosyl phosphoproteins. Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit.

Phosphatidylinositol 3'-kinase (PI 3'-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3'-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3'-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3'-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3'-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3'-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3'-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3'-kinase by tyrosylphosphorylated proteins requires occupancy of both SH2 domains in p85.