Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in placenta growth factor (PlGF) deficiency

Placenta growth factor (PlGF) deficient mice are fertile at a Mendelian ratio. Interestingly, low maternal plasma levels of PlGF are strongly associated with early onset of preeclampsia, a pregnancy hypertensive disorder characterised by high blood pressure, proteinuria and fetal growth restriction. PlGF is increasingly being recognised as an early diagnostic biomarker, but the physiological importance of PlGF in the pathogenesis of preeclampsia is unknown. We investigated whether the decreased levels of PlGF in pregnancy exacerbate the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1 and the potential of hydrogen sulphide to ameliorate these effects. Pregnant PlGF−/− mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at 1 × 109 pfu/ml at E10.5 and mean arterial blood pressure (MAP), biochemical and histological analysis of maternal kidney, placenta and embryos were assessed at the end of pregnancy. Ad-sFlt-1 significantly increased MAP and induced severe glomerular endotheliosis in PlGF−/− mice compared to wild-type animals. Soluble Flt-1 also significantly elevated albumin–creatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury. Furthermore, sFlt-1 over expression increased fetal resorption rate in the PlGF−/− mice and promoted abnormal placental vascularisation. To determine whether placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/− placentas and embryos in dams and exposed to high sFlt-1 environment. These mothers showed reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF−/− mice. Furthermore, treatment with hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria observed in Ad-sFlt-1 treated pregnant PlGF−/− mice. Our study shows that placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 associated with preeclampsia and activation of the hydrogen sulphide pathway may rescue preeclampsia phenotypes even under low PlGF environment.

Dysregulation of placental cystathionine-β-synthase promotes fetal growth restriction

Fetal growth restriction (FGR) is characterized by the birth weight and body mass below the tenth percentile for gestational age. FGR is a major cause of perinatal morbidity and mortality and babies born with FGR are prone to develop cardiovascular diseases later in life. The underlying pathology of FGR is inadequate placental transfer of nutrients from mother to fetus, which can be caused by placental insufficiency. Hydrogen sulfide (H2S), a gaseous messenger is produced endogenously by cystathionine-lyase (Cth), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), which are present in human placenta. Recently, we demonstrated that the dysregulation of H2S/Cth pathway is associated with preeclampsia and blockade of CSE activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in H2S pathways promote FGR and H2S donor restores fetal growth in mice where CBS or CSE activity has been compromised. Western blotting and qPCR revealed that placental CBS expressions were significantly reduced in women with FGR. ELISA analysis showed reduced placental growth factor production (PlGF) from first trimester (8–12 weeks gestation) human placental explants following inhibition of CBS activity by aminooxyacetic acid (AOA). Administration of AOA to pregnant mice had no effects on blood pressure, but caused fetal growth restriction. This was associated with reduced PlGF production. Histological analysis revealed a reduction in the placental junction zone, within which trophoblast giant cells and glycogen cells were less prominent in CBS inhibitor treated mice. These results imply that placental CBS is required for placental development and that dysregulation of CBS activity may contribute to the pathogenesis of FGR but not preeclampsia.

Dysregulation of placental cystathionine-β-synthase impact on fetal growth restriction

INTRODUCTION: Fetal growth restriction (FGR), which causes perinatal morbidity and mortality, is characterized by birth weight and body mass being below 10th percentile for gestational age. FGR babies are prone to develop cardiovascular diseases later in life. Inadequate placental transfer of nutrients from mother to fetus due to placental insufficiency is considered the underlying cause of FGR. Recently, we demonstrated that blockade of cystathionine-γ-lyase (CSE) activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in cystathionine-β-synthase (CBS) / H2S pathway may promote FGR. METHODS: Placental CBS expressions were determined in women with FGR (n=9) and normal controls (n=14) by Western blotting and real-time qPCR. ELISA was used to determine angiogenic factors levels in plasma and first-trimester (8–12 weeks gestation) human placental explants. Time pregnant mice were treated with CBS inhibitor, aminooxyacetic acid (AOA). Mean arterial blood pressure (MBP), histological assessments of placenta and embryos were performed. RESULTS: Placental CBS expressions were significantly reduced in women with FGR. Inhibition of CBS activity by AOA reduced PlGF production from first-trimester human placental explants, Administration of AOA to pregnant mice had no effects on blood pressure, but caused fetal growth restriction, which was associated with reduced placental PlGF production. Histological analysis revealed a reduction in the placental junction zone, within which trophoblast giant cells and glycogen cells were less prominent in CBS inhibitor-treated animals. Furthermore, H2S donor GYY4137 treatment restored fetal growth in pregnant mice exposed to high level of sFlt-1. CONCLUSIONS: These results imply that placental CBS is required for placental development and that dysregulation of CBS activity may contribute to the pathogenesis of FGR but not preeclampsia opening up the therapeutic potentials of H2S therapy in this condition.

Microbial community of black band disease on infection, healthy and dead part of scleractinian Montipora sp. colony at Seribu islands, Indonesia

It is crucial to understand the microbial community associated with the host when attempting to discern the pathogen responsible for disease outbreaks in scleractinian corals. This study determines changes in the bacterial community associated with Montipora sp. in response to black band disease in Indonesian waters. Healthy, diseased, and dead Montipora sp. (n = 3 for each sample type per location) were collected from three different locations (Pari Island, Pramuka Island, and Peteloran Island). DGGE (Denaturing Gradient Gel Electrophoresis) was carried out to identify the bacterial community associated with each sample type and histological analysis was conducted to identify pathogens associated with specific tissues. Various Desulfovibrio species were found as novelty to be associated with infection samples, including Desulfovibrio desulfuricans, Desulfovibrio magneticus, and Desulfovibrio gigas, Bacillus benzoevorans, Bacillus farraginis in genus which previously associated with pathogenicity in corals. Various bacterial species associated with uninfected corals were lost in diseased and dead samples. Unlike healthy samples, coral tissues such as the epidermis, endodermis, zooxanthellae were not present on dead samples under histological observation. Liberated zooxanthellae and cyanobacteria were found in black band diseased Montipora sp. samples.

Simvastatin improves the healing of infected skin wounds of rats

This study explores the potential of the simvastatin to ameliorate inflammation and infection in open infected skin wounds of rats. Methods: Fourteen Wistar rats weighing 285±12g were used. The study was done in a group whose open infected skin wounds were treated with topical application of sinvastatina microemulsion (SIM, n=7) and a second group with wounds treated with saline 0.9 % (SAL, n=7). A bacteriological exam of the wounds fluid for gram positive and gram negative bacteria, the tecidual expression of TNFá and IL-1â by imunohistochemical technique, and histological analysis by HE stain were performed. Results: The expression of TNFa could be clearly demonstrated in lower degree in skin wounds treated with simvastatin (668.6 ± 74.7 ìm2) than in saline (2120.0 ± 327.1 ìm2). In comparison, wound tissue from SIM group displayed leukocyte infiltration significantly lower than that observed in SAL group (p<0.05). Culture results of the samples taken from wound fluid on fourth post treatment day revealed wound infection in only one rat of group simvastatin (SIM), where Proteus mirabilis, Escherchia coli and Enterobacter sp were isolated. In the rats whose wounds were treated with saline (SAL), polymicrobial infection with more than 100,000 CFU/g was detected in all the wounds. Conclusion: In addition to its antiinflammatory properties, the protective effects of simvastatin in infected open skin wounds is able to reduce infection and probably has antibacterial action. The potential to treat these wounds with statins to ameliorate inflammation and infection is promising

Efeito de agentes de controle sobre a qualidade reprodutiva de rainhas de Apis mellifera L. (Hymenoptera: Apidae) africanizadas

The agriculture with the use of products with less environmental impact is expanding. In it, the producers offer their products without the use of synthetic chemical inputs, solving the phytosanitary problems with the use of biological or alternative control agents such as parasites, predators, entomopathogenic, alternative products, plant extracts and essential oils. These products can be considered safe to non-target organisms, but studies are needed to find these features on natural enemies and on the beneficial insects such as bees, common frequenter of cultures. In this sense, this study aims to evaluate the effects of control over reproductive quality queens of Apis mellifera L. (Hymenoptera: Apidae) Africanized. For this, it tested the action of control products on the production of A. mellifera queens, using the commercial entomopathogenic fungus Boveril® 1,0x108 (Beauveria bassiana) and aqueous extract of pomegranate (Punica granatum) at a concentration of 5% sterile distilled water with Tween (0.01%) and sterile distilled water (controls). The treatments were incorporated into a tissue type gauze, wrapped in an acrylic plate and packed inside minirrecrias type colonies for the production of queens on the day before the transfer of larvae. The next day were introduced battens with 30 domes with larvae to produce queens, so the workers have contacted the agent tested. From the emergence of all the queens, they were monitored to determine the measures of body weight (mg), length and width of wing and abdomen, length, width and height of the chest (mm) as well as the time of emergence of queens. The next step was evaluated the influence of the control agents in production creates, performing measurements of creating areas in cm2 for six straight weeks. It was found that the area creates Queens did not differ among the treatments. Histological analysis of hipofaringeanas of workers glands that came into contact with the control agents and the midgut of virgin queens were also held. Histological analysis differences were observed in the tissues when the treatments were compared with the respective controls.

The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.

Simvastatin improves the healing of infected skin wounds of rats

This study explores the potential of the simvastatin to ameliorate inflammation and infection in open infected skin wounds of rats. Methods: Fourteen Wistar rats weighing 285±12g were used. The study was done in a group whose open infected skin wounds were treated with topical application of sinvastatina microemulsion (SIM, n=7) and a second group with wounds treated with saline 0.9 % (SAL, n=7). A bacteriological exam of the wounds fluid for gram positive and gram negative bacteria, the tecidual expression of TNFá and IL-1â by imunohistochemical technique, and histological analysis by HE stain were performed. Results: The expression of TNFa could be clearly demonstrated in lower degree in skin wounds treated with simvastatin (668.6 ± 74.7 ìm2) than in saline (2120.0 ± 327.1 ìm2). In comparison, wound tissue from SIM group displayed leukocyte infiltration significantly lower than that observed in SAL group (p<0.05). Culture results of the samples taken from wound fluid on fourth post treatment day revealed wound infection in only one rat of group simvastatin (SIM), where Proteus mirabilis, Escherchia coli and Enterobacter sp were isolated. In the rats whose wounds were treated with saline (SAL), polymicrobial infection with more than 100,000 CFU/g was detected in all the wounds. Conclusion: In addition to its antiinflammatory properties, the protective effects of simvastatin in infected open skin wounds is able to reduce infection and probably has antibacterial action. The potential to treat these wounds with statins to ameliorate inflammation and infection is promising

Efeitos da exposição ao chumbo no sistema reprodutivo de Chrysomus ruficapillus (AVES: Icteridae)

O chumbo é um metal pesado que constitui um dos grandes problemas ambientais em termos de poluição atmosférica, aquática e terrestre. O impacto da exposição ao chumbo tem consequências nas características morfológicas e bioquímicas deem aves, porém são escassos os estudos sobre os efeitos no sistema reprodutivo das aves. O objetivo deste trabalho é avaliar os efeitos do acetato de chumbo em parâmetros de integridade, histopatologia e bioquímica emnas células espermáticas. Foram coletadas 36 aves silvestres (Chrysomus ruficapillus) adultos, machos e expostas em gaiolas. Foi administrada uma dose única de 50 e 100 mg/kg de acetato de chumbo através de uma injeção intraperitoneal e o grupo controle recebeu uma injeção de solução salina. Após sete dias da administração das doses, foi realizada a coleta dos ductos deferentes e testículos para as análises nas células espermáticas. Os resultados mostraram que houve deterioração na integridade da membrana e DNA, e diminuição da funcionalidade mitocondrial nos testículos das aves expostas ao acetato de chumbo nas duas doses do estudo (P<0,05). Na histopatologia foi observada diminuição na quantidade de células dos estágios de desenvolvimento da espermatogênese, além de patologias nas mesmas. Observou-se danos oxidativos nas aves tratadas com 100mg/kg e um aumento da peroxidação lipídica nos testículos. Portanto, o acetato de chumbo causou efeitos negativos no aparelho reprodutivo de Chrysomus ruficapillius

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatatos da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Superparamagnetic Liposomes for MRI Monitoring and External Magnetic Field-Induced Selective Targeting of Malignant Brain Tumors

International audience

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatados da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Utilização da berberina na identificação dos mastócitos do molusco Anomalocardia brasiliana

Berberine is an alkaloid used as a fluorochrome in the identification of heparin and DNA. Enerback, 1974, described the technique used until today to study granules rich in heparin of vertebrate mast cells. Santos et al., 2003, studied mast cells of the mollusk Anomalocardia brasiliana using biochemical and histological analysis. This work used the fluorescent dye berberine technique to improve characterization of these cells. Mollusk organs (ctenidium and mantle) were processed with routine histological techniques. Tissue sections were treated with berberine 0,02% in redistilled water acidified to pH 4, by the addition of citric acid for 20 minutes. The visualization was made through fluorescence microscopy with ultraviolet region emission. The mast cell fluorescence had a strong yellow color, where cell nuclei appeared more greenish. This result was very similar to the ones reported before. Mast cells are location at the epithelium surface is the same in both organs, mantle and ctenidium. The fluorescence was easily observed in the granules. Therefore, this technique showed to be good and sensitive to study mast cell of invertebrates

Endoluminal calprotectin measurement in assessment of pouchitis and a new index of disease activity: a pilot study

Pouchitis is the most common complication following proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis (UC). To provide a standardized definition of pouchitis clinical, endoscopic and histological markers were grouped and weighted in the pouch disease activity index (PDAI). However, the delay in the assessment of the final score due to the time requested for histological analysis remains the main obstacle to the index implementation in clinical practice so that the use of modified-PDAI (mPDAI) with exclusion of histologic subscore has been proposed. We tested the ability of calprotectin measurement in the pouch endoluminal content to mimic the histologic score as defined in the PDAI, the index that we adopted as gold standard for pouchitis diagnosis. Calprotectin was measured by ELISA in the pouch endoluminal content collected during endoscopy in 40 consecutive patients with J-pouch. In each patient PDAI and mPDAI were calculated and 15% of patients were erroneously classified by mPDAI. ROC analysis of calprotectin values vs. acute histological subscore ≥ 3 identified different calprotectin cut-off values with corresponding sensitivity and specificity allowing the definition and scoring of different range of calprotectin subscores. We incorporated the calprotectin score in the mPDAI obtaining a new score that shows the same specificity as PDAI for diagnosis of pouchitis and higher sensitivity when compared with mPDAI. The use of the proposed new score, once validated in a larger series of patients, might be useful in the early management of patients with symptoms of pouchitis.

Stability and Local Toxicity Evaluation of a Liposomal Prilocaine Formulation

This study reports a physicochemical stability evaluation of a previously reported liposomal prilocaine (PLC(LUV)) formulation (Cereda el al. J. Pharm. Pharmaceut. Sci. 7:235, 2004) before and after steam sterilization as well as its local toxicity evaluation. Prilocaine (PLC) was encapsulated into extruded unilamellar liposomes (LUVs) composed by egg phosphatidylcholine:cholesterol:alfa-tocopherol (4:3:0.07, mole %). Laser light-scattering analysis (p > 0.05) and thiobarbituric acid reaction (p > 0.05) were used to evaluate the liposomes physical (size) and chemical (oxidation) stability, respectively. The prilocaine chemical stability was followed by (1)H-nuclear magnetic resonance. These tests detected no differences on the physicochemical stability of PLC or PLCLUV, sterilized or not, up to 30 days after preparation (p > 0.05). Finally, the paw edema test and histological analysis of rat oral mucosa were used to assess the possible inflammatory effects of PLC(LUV). PLC(LUV) did not evoke rat paw edema (p > 0.05), and no significant differences were found in histological analysis, when compared to the control groups (p > 0.05). The present work shows that PLC(LUV) is stable for a 30-day period and did not induce significant inflammatory effects both in the paw edema test and in histological analysis, giving supporting evidence for its safely and possible clinical use in dentistry.