Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro‐inflammatory cytokines that up‐regulate iNOS contribute to pre‐eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre‐eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham‐operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N‐[3‐(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8‐isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ~30 mmHg in RUPP rats, and 1400 W attenuated this increase by ~50% (P < 0.05). While RUPP increased plasma 8‐isoprostane levels, aortic ROS levels, and NADPH‐dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre‐eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.     

Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken

Background

Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax?, or probiotic, Dairyman's Choice? paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.

Findings

Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillus flavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax? removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice? paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax? removed the cytotoxicity in vitro. Celmanax? also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice? paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.

Conclusion

The current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax?, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.     

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.     

Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability

Vascular dysfunction associated with two-kidney, one-clip (2K-1C) hypertension may result from both altered matrix metalloproteinase (MMP) activity and higher concentrations of reactive oxygen species (ROS). Doxycycline is considering the most potent MMP inhibitor of tetracyclines and attenuates 2K-1C hypertension-induced high blood pressure and chronic vascular remodeling. Doxycycline might also act as a ROS scavenger and this may contribute to the amelioration of some cardiovascular diseases associated with increased concentrations of ROS. We hypothesized that in addition to its MMP inhibitory effect, doxycycline attenuates oxidative stress and improves nitric oxide (NO) bioavailability in 2K-1C hypertension, thus improving hypertension-induced arterial endothelial dysfunction. Sham operated or 2K-1C hypertensive rats were treated with doxycycline 30 mg/kg/day (or vehicle). After 8 weeks of treatment, aortic rings were isolated to assess endothelium dependent vasorelaxation to A23187. Arterial and systemic levels of ROS were respectively measured using dihydroethidine (DHE) and thiobarbituric acid reactive substances (TBARS). Neutrophils-derived ROS were tested in vitro using the fluoroprobe Carboxy-H(2)DCFDA and human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). NO levels were assessed in rat aortic endothelial cells by confocal microscopy. Aortic MMP activity was determined by in situ zymography. Doxycycline attenuated 2K-1C hypertension (169 ± 17.3 versus 209 ± 10.9mm Hg in hypertensive controls, p<0.05) and protected against hypertension-induced reduction in endothelium-dependent vasorelaxation to A23187 (p<0.05). Doxycycline also decreased hypertension-induced oxidative stress (p<0.05), higher MMP activity (p<0.01) and improved NO levels in aortic endothelial cells (p<0.01). Therefore, doxycycline ameliorates 2K-1C hypertension-induced endothelial dysfunction in aortas by inhibiting oxidative stress generation and improving NO bioavailability, in addition to its inhibitory effects on MMP activity.     

Effect of Regular Circus Physical Exercises on Lymphocytes in Overweight Children

Obesity associated with a sedentary lifestyle can lead to changes in the immune system balance resulting in the development of inflammatory diseases. The aim of this study was to compare lymphocyte activation mechanisms between overweight children practicing regular circus physical exercises with non-exercised children. The study comprised 60 pubescent children randomly divided into 4 groups: Overweight Children (OWC) (10.67 ± 0.22 years old), Overweight Exercised Children (OWE) (10.00 ± 0.41 years old), Eutrophic Children (EC) (11.00 ± 0.29 years old) and Eutrophic Exercised Children (EE) (10.60 ± 0.29 years old). OWE and EE groups practiced circus activities twice a week, for 4.3 ± 0.5 and 4.4 ± 0.5 months, respectively. Percentage of T regulatory cells (Treg) and the expression of CD95 and CD25 in CD4⁺ lymphocytes were evaluated by flow cytometry. Lymphocyte proliferation capacity was measured by [¹⁴C]-thymidine incorporation and mRNA expression of IL-35, TGF-beta, IL-2 and IL-10 by real-time PCR. Lymphocyte proliferation was higher in OWC and OWE groups compared with the EC (3509 ± 887; 2694 ± 560, and 1768 ± 208 cpm, respectively) and EE (2313 ± 111 cpm) groups. CD95 expression on lymphocytes was augmented in the EC (953.9 ± 101.2) and EE groups (736.7 ± 194.6) compared with the OWC (522.1 ± 125.2) and OWE groups (551.6 ± 144.5). CTLA-4 expression was also lower in the OWC and OWE groups compared with the EC and EE groups. Percentage of Treg, IL-35, and IL-10 mRNA expression were lower in the OWC and OWE groups compared with the EC and EE groups. In conclusion, overweight children present altered immune system balance characterized by elevated lymphocyte proliferation due to a decrease in T regulatory cell percentage. These effects were partially reverted by moderate physical exercise, as demonstrated by decreased lymphocyte proliferation.     

Peroxisomes,lipid droplets,and endoplasmic reticulum “hitchhike” on motile early endosomes

Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.     

CD14 C-159T and early infection with Pseudomonas aeruginosa in children with cystic fibrosis

Early acquisition of Pseudomonas aeruginosa is associated with a poorer prognosis in patients with cystic fibrosis. We investigated whether polymorphisms in CD14, the lipopolysaccharide receptor, increase the risk of early infection. Forty-five children with cystic fibrosis were investigated with annual bronchoalveolar lavage (BAL) and plasma sCD14 levels. Plasma sCD14 levels were significantly lower in children from whom P.aeruginosa was subsequently isolated (492.75 μg/ml vs. 1339.43 μg/ml, p = 0.018). Those with the CD14 -159CC genotype had a significantly increased risk of early infection with P.aeruginosa suggesting that CD14 C-159T plays a role in determining the risk of early infection with P.aeruginosa.     

Individual mRNA expression profiles reveal the effects of specific microRNAs

Background  

MicroRNAs (miRNAs) are oligoribonucleotides with an important role in regulation of gene expression at the level of translation. Despite imperfect target complementarity, they can also significantly reduce mRNA levels. The validity of miRNA target gene predictions is difficult to assess at the protein level. We sought, therefore, to determine whether a general lowering of predicted target gene mRNA expression by endogenous miRNAs was detectable within microarray gene expression profiles.     

Isoform specificity of P2X2 purinergic receptor C-terminus binding to tubulin

Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X2 is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X2 binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X2 with a naturally occurring splicing variant named P2X2b. These isoforms differ in their C-terminal regions which contain the proposed beta-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X2 binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X2 C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X2 interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles.     

Effects of age and leg length upon central loop of the Gastrocnemius-soleus H-reflex latency

Background  

central loop of the gastrocnemius-soleus H-reflex latency (T_c) that looks promising in the diagnosis of S1 radiculopathy; has been investigated in a few studies and only two of them have focused on the constitutional factors affecting it. Although leg length has been shown to contribute to the T_c, the role of age is controversial. More confusing, none of the previously performed studies have used strict criteria to rule out subclinical neuropathy, so the results could be misleading. This study has been performed to determine the influence of leg length and age on T_c among a carefully selected group of healthy volunteers.