Fernanda Miriane Bruni

Effect of mangiferin on the development of periodontal disease: Involvement of lipoxin A4, anti-chemotaxic action in leukocyte rolling

UNLABELLED Mangiferin is a polyphenol compound obtained from mango and has been reported to possess antioxidant and anti-inflammatory properties. AIM We propose to evaluate the influence of mangiferin in preventing and treating experimental periodontitis induced in Wistar rats. MAIN METHODS Periodontitis was induced in rats by applying a ligature around the lower right first molar. After ligature, groups of animals were submitted orally to the following treatments: saline 10 mL/kg, piroxicam 20 mg/kg or mangiferin 100 mg/kg. On days 1, 4 or 7 after ligature application the alveolar bone loss (ABL) was determined. We evaluated the effect of mangiferin on ABL by histological techniques (alveolar bone loss and cellularity), enzyme immunoassay (lipoxin A(4)), intravital microscopy (rolling leukocytes and endothelial-leukocyte adhesion), zymographic analyses (metalloproteinases, MMPs 2 and 9), immunohistochemistry (PCNA, COX-2 and CXCR4) and toxicology. KEY FINDINGS Oral administration of mangiferin significantly reduced ABL. We also observed the reduction of cellularity in mangiferin-treated rats. Treatment with mangiferin inhibited COX-2 expression and the rolling and adhesion of leukocytes, while maintaining normal lipoxin A(4) levels. The mangiferin did not interfere in the activity of MMP-2 or -9. The mangiferin-treated rats presented an earlier peak of cell proliferation and augmented angiogenesis in the injured region. SIGNIFICANCE Our results have demonstrated promising therapeutic potential of mangiferin both in the prevention and treatment of periodontitis.

Activation of leukocyte rolling by the cysteine‐rich domain and the hyper‐variable region of HF3, a snake venom hemorrhagic metalloproteinase

The functionality of the disintegrin‐like/cysteine‐rich domains of snake venom metalloproteinases (SVMPs) has been shown to reside in the cysteine‐rich region, which can interact with VWA‐containing proteins. Recently, the hyper‐variable region (HVR) of the cysteine‐rich domain was suggested to constitute a potential protein–protein adhesive interface. Here we show that recombinant proteins of HF3, a hemorrhagic P‐III SVMP, containing the cysteine‐rich domain (disintegrin‐like/cysteine‐rich and cysteine‐rich proteins) but not the disintegrin‐like protein were able to significantly increase leukocyte rolling in the microcirculation. Peptides from the HVR also promoted leukocyte rolling and this activity was inhibited by anti‐alphaM/beta2 antibodies. These results show, for the first time, that the cysteine‐rich domain and its HVR play a role in triggering pro‐inflammatory effects mediated by integrins.

Characterization of a new bioactive peptide from Potamotrygon gr. orbignyi freshwater stingray venom

Brazilian freshwater stingrays, Potamotrygon gr. orbigyni, are relatively common in the middle-western regions of Brazil, where they are considered an important public health threat. In order to identify some of their naturally occurring toxin peptides available in very low amounts, we combine analytical protocols such as reversed-phase high-performance liquid chromatography (RP-HPLC), followed by a biological microcirculatory screening and mass spectrometry analysis. Using this approach, one bioactive peptide was identified and characterized, and two analogues were synthesized. The natural peptide named Porflan has the primary structure ESIVRPPPVEAKVEETPE (MW 2006.09 Da) and has no similarity with any bioactive peptide or protein found in public data banks. Bioassay protocols characterized peptides as presenting potent activity in a microcirculatory environment. The primary sequences and bioassay results, including interactions with the membrane phospholipids, suggest that these toxins are a new class of fish toxins, directly involved in the inflammatory processes of a stingray sting.

Inflammatory effects of the toxic cyanobacterium Geitlerinema amphibium.

Toxic cyanobacteria in public water reservoirs may cause severe health issues for livestock and human beings. Geitlerinema amphibium, which is frequently found in São Paulo Citys drinking water supplies, showed toxicity in the standard mouse bioassay, while displaying signs of intoxication and post-mortem findings different from those showed by animals intoxicated by known cyanotoxins. We report here the alterations caused by G. amphibium methanolic extract on mouse microcirculatory network, as seen by in vivo intravital microscopy, besides observations on leukocyte migration, cytokine quantitation, and results of toxicological essays. Our data showed that G. amphibium methanolic extract displayed time- and dose-dependent pro-inflammatory activity, and that at lower doses [125 and 250 mg/kg body weight (b.w.)] increased the leukocyte rolling, caused partial venular stasis, as well as induced an increase in leukocyte counts in the peripheral blood and peritoneal washings. At higher doses (500 and 1000 mg/kg b.w.), the extract caused ischemic injury leading to animal death. As confirmed by mass spectrometric studies and polymyxin B test, the G. amphibium methanolic extract did not contain lipopolysaccharides.

Identification of bradykinin: related peptides from Phyllomedusa nordestina skin secretion using electrospray ionization tandem mass spectrometry after a single-step liquid chromatography

Amphibian skin secretions are a source of potential new drugs with medical and biotechnological applications. Rich in peptides produced by holocrine-type serous glands in the integument, these secretions play different roles, either in the regulation of physiological skin functions or in the defense against predators or microorganisms. The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of Phyllomedusa nordestina. In order to achieve this goal, the crude skin secretion of this frog was pre-fractionated by solid phase extraction and separated by reversed-phase chromatography. The fractions were screened for low-molecular-mass peptides and sequenced by mass spectrometry. It was possible to identify three novel bradykinin-related peptides, namely: KPLWRL-NH2 (Pnor 3), RPLSWLPK (Pnor 5) and VPPKGVSM (Pnor 7) presenting vascular activities as assessed by intravital microscopy. Pnor 3 and Pnor 7 were able to induce vasodilation. On the other hand, Pnor 5 was a potent vasoconstrictor. These effects were reproduced by their synthetic analogues.

Delayed polymorphonuclear leukocyte infiltration is an important component of Thalassophryne maculosa venom pathogenesis

Thalassophryne maculosa fish envenomation is characterized by severe pain, dizziness, fever, edema and necrosis. Here, the dynamic of cellular influx, activation status of phagocytic cells, and inflammatory modulator production in the acute inflammatory response to T. maculosa venom was studied using an experimental model. Leukocyte counting was performed (2 h to 21 days) after venom injection in BALB/c mice footpads. Our results showed an uncommon leukocyte migration kinetic after venom injection, with early mononuclear cell recruitment followed by elevated and delayed neutrophil influx. The pattern of chemokine expression is consistent with the delay in neutrophil recruitment to the footpad: T. maculosa venom stimulated an early production of IL-1beta, IL-6, and MCP-1, but was unable to induce an effective early TNF-alpha and KC release. Complementary to these observations, we detected a marked increase in soluble KC and TNF-alpha in footpad at 7 days post-venom injection when a prominent influx of neutrophils was also detected. In addition, we demonstrated that bone marrow-derived macrophages and dendritic cells were strongly stimulated by the venom, showing up-regulated ability to capture FITC-dextran. Thus, the reduced levels of KC and TNF-alpha in footpad of mice concomitant with a defective accumulation of neutrophils at earlier times provide an important clue to uncovering the mechanism by which T. maculosa venom regulates neutrophil movement.

The action of fish peptide Orpotrin analogs on microcirculation

In order to investigate the relationship between the primary structure of Orpotrin, a vasoactive peptide previously isolated from the freshwater stingray Potamotrygon gr. orbignyi, and its microcirculatory effects, three Orpotrin analogs were synthesized. The analogs have a truncated N‐terminal with a His residue deletion and two substituted amino acid residues, where one Nle is substituted for one internal Lys residue and the third analog has a substitution of a Pro for an Ala (Orp‐desH1, Orp‐Nle and Orp‐Pro/Ala, respectively). Only Orp‐desH1 could induce a lower vasoconstriction effect compared with the natural Orpotrin, indicating that besides the N‐terminal, the positive charge of Lys and the Pro residues located at the center of the amino acid chain is crucial for this vasoconstriction effect. Importantly, the suggestions made with bioactive peptides were based on the molecular modeling and dynamics of peptides, the presence of key amino acids and shared activity in microcirculation, characterized by intravital microscopy. Moreover, this study has demonstrated that even subtle changes in the primary structure of Orpotrin alter the biological effects of this native peptide significantly, which could be of interest for biotechnological applications. Copyright