Katia Sabrina Paludo

B1 and B2 kinin receptor participation in hyperproliferative and inflammatory skin processes in mice

BACKGROUND Kinins are released during dermal injury and inflammation and seem to contribute to the pathogenesis of cutaneous diseases. OBJECTIVE Participation of kinins in skin inflammatory process was evaluated using knockout mice and non-peptide kinin receptor antagonists. METHODS Chronic skin inflammation was induced by multiple applications of TPA in mice ear. RESULTS The B(2) knockout mice (B(2)(-/-)) showed a significant increase of ear weight (23 ± 10%) and epidermal cellular hyperproliferation and acanthosis formation upon histological analysis when compared with wildtype mice. Also, evaluation of PCNA levels by Western blot and immunohistochemistry confirmed the increase in the epidermis hyperproliferation in the ear skin of B(2)(-/-) mice. In contrast, no modification in these parameters was detected in B(1) knockout mice (B(1)(-/-)). However, mice lacking both kinin receptors (B(1)B(2)(-/-)) presented a considerable reduction of epidermis thickness and in PCNA levels. Following the establishment of skin inflammation (5th day of TPA application) treatment with the non-peptide antagonists SSR 240612 (B(1) receptor antagonist), FR 173657 (B(2) receptor antagonist), or SSR 240612 plus FR 173657 topically applied, caused a significant inhibition of ear weight (20 ± 5%, 34 ± 4% and 32 ± 6%, respectively). In the histological analysis, the antagonists produced a reduction in epidermal hyperplasia and acanthosis formation; but the treatment with a combination of the two antagonists did not increase efficacy. CONCLUSION Kinin receptors seem to be involved in the control of the keratinocyte hyperproliferative process, and non-peptide kinin receptor antagonists may be useful tools in the treatment of hyperproliferative skin disorders.

Glycosaminoglycan chains from alpha5beta1 integrin are involved in fibronectin-dependent cell migration.

Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha5beta1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in alpha5beta1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha5beta1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha5beta1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha5beta1 integrin are involved in cellular migration on fibronectin.

Characterization of a specific interaction between ADAM23 and cellular prion protein.

ADAMs are transmembrane proteins implicated in several biological functions, including cytokine and growth factor shedding, fertilization, muscle and nervous system development. Here, we show for the first time that ADAM23, which is predominantly expressed in the central nervous system, co-localizes with cellular prion protein (PrP(C)) at plasma membrane of mouse hippocampal neurons and neuroblastoma cells. Co-immunoprecipitation and pull-down assay showed a physical interaction between ADAM23 and both recombinant and endogenous PrP(C). Glycosylation seems to be not relevant to the observed interaction since both ADAM23 and PrP(C) recombinant proteins expressed in bacteria or extracted from eukaryotic cells treated with tunicamycin are still able to bind each other. In vitro binding assays also suggested that the disintegrin domain of ADAM23 is able to interact directly with PrP(C). Taken together, these findings point out PrP(C) as a novel molecular partner for ADAM23 in the nervous systems.

Effects of the venom and the dermonecrotic toxin LiRecDT1 of Loxosceles intermedia in the rat liver

Brown spider bites cause dermonecrotic lesions and systemic manifestations known as loxoscelism. The Loxosceles intermedia venom contains many active proteins, as phospholipase D. There are reports of increased levels of hepatic transaminases in humans with loxoscelism, but detailed studies about the action of the Loxosceles intermedia venom on the liver functions are lacking. The aim of this study was to investigate the effects of the venom and the dermonecrotic recombinant toxin 1 (LiRecDT1) in the liver of Wistar rats injected subcutaneously with venom (80 microg) or toxin (80 microg). After 6 and 12h the liver immunofluorescence was positive for venom and toxin. Hepatocytes from the venom group were tumefacted and apoptotic. There was leucocyte infiltration in the portal region combined with a high degree of steatosis in 12h. In the toxin group the histological alterations were less severe. Plasma levels of alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl-transferase were significantly elevated only in the venom group in 6h. Hepatic metabolism was modified: the venom, but not LiRecDT1, reduced gluconeogenesis and ureagenesis from alanine and glycogen accumulation. These results show that the venom is hepatotoxic and that the dermonecrotic toxin is only partly responsible.

Cytotoxic biomonitored study of Euphorbia umbellata (Pax) Bruyns

ETHNOPHARMACOLOGICAL RELEVANCE Euphorbia umbellata latex (sap) has normally been used in folk medicine in southern Brazil to treat different types of cancers. AIM OF STUDY To carry out a biomonitored investigation of partitioned latex using in vitro assay, to identify the main mechanisms related with the action of the most active fraction as well as to develop a phytochemical study with this material. MATERIALS AND METHODS Biological screening was performed with hexane, chloroform, ethyl acetate and methanol fractions from the latex of E. umbellata using MTT, trypan blue, and neutral red assays to determine the cytotoxicity against HRT-18, HeLa and Jurkat cells and flow cytometry, DNA quantification, acridine orange and Hoechst 33342 staining to investigate mechanisms of action for the hexane extract. The phytochemical study of the hexane fraction was performed by chromatographic procedures and the substances were identified by NMR analysis. The isolated terpenes were evaluated using MTT to determine the cytotoxicity against Jurkat cells. RESULTS All the fractions presented concentration and time dependent cytotoxicity. The hexane fraction showed the highest cytotoxicity; whereas the Jurkat cell was the lineage with the highest sensitivity (IC50 1.87µg/mL). Fragmentation of DNA and apoptosis are two mechanisms related with the toxicity of hexane fraction. The hexane fraction arrested the cell cycle in the G0/G1 phase, and the selectivity index was 4.30. Phytochemical study of the hexane fraction led to isolation of euphol (main compound) and germanicol acetate. Both substances demonstrated some slight cytotoxic activity against Jurkat cells after 72h; however the activity was minimal compared to vincristine (anticancer standard drug). CONCLUSION The current research proves that the fractions of the latex from E. umbellata have a cytotoxic effect against three different cancer cells lines. The hexane fraction showed high in vitro cytotoxic effects against Jurkat cells demonstrating that the effect may be due to non-polar constituents. The two isolated terpenes (euphol and germanicol acetate) showed poor cytotoxic activity indicating that the anticancer properties of the extract may be caused by other substances present in the hexane fraction.

Detection of Diarrheagenic Escherichia coli Using a Two‐System Multiplex‐PCR Protocol

Diarrheagenic Escherichia coli (DEC) strains are important causes of diarrhea. However, they cannot be distinguished from E. coli of the intestinal microbiota by conventional microbiological tests.

Brown spider (Loxosceles intermedia) venom triggers endothelial cells death by anoikis

Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α₅β₁ integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.

Standardized extract of Dicksonia sellowiana Presl. Hook (Dicksoniaceae) decreases oxidative damage in cultured endothelial cells and in rats

AIMS Aging and a variety of pathologies, including cancer, diabetes, cardiovascular and inflammatory diseases have been associated with reactive oxygen species (ROS), such as superoxide anion (O₂·⁻), hydroxyl radical (·OH) and hydrogen peroxide (H₂O₂) generation. Plant polyphenols bear radical scavenging/antioxidant activity. A phytomedicinal preparation obtained from aerial parts of Dicksonia sellowiana (Dicksoniaceae), a native plant from Central and South America, has been widely used in Brazil against asthma and presents beneficial effects in several other diseases, including cardiovascular disturbance. In this work, we investigated whether Dicksonia sellowiana, which is also known to contain high levels of polyphenols, presents antioxidant activity. METHODS The antioxidant activity of the hydroalcoholic extract obtained from Dicksonia sellowiana leaves (HEDS) was investigated by in vitro and in vivo tests. RESULTS HEDS (0.1-100 μg/mL) exhibited a strong scavenging activity against all reactive species tested (DPPH, O₂·⁻,·OH and H₂O₂; IC₅₀=6.83±2.05, 11.6±5.4, 2.03±0.4, and 4.8±0.4 μg/mL, respectively). HEDS strongly protected endothelial cells against H₂O₂-induced oxidative stress by mechanisms other than increasing catalase activity. In addition, HEDS protected cell membrane from oxidative damage. HEDS, (20 and 40 mg/kg) inhibited lipid peroxidation in vivo (29.8% and 24.5%, respectively). CONCLUSIONS According to our results, we can speculate that the traditional uses of Dicksonia sellowiana for cardiovascular diseases, asthma and skin diseases could be, at least in part, related to the potent antioxidant and endothelial protective activities of the plant.

Investigation of anti-inflammatory and anti-proliferative activities promoted by photoactivated cationic porphyrin

BACKGROUND Photodynamic therapy (PDT) is a technique that uses light and a photosensitizer, converting local molecular oxygen into singlet oxygen, which eliminates a target unhealthy tissue. It has been increasingly used for the treatment of several diseases including skin disorders. Psoriasis is a chronic inflammatory skin disease expressing immune and hyperproliferative features. OBJECTIVE This study aimed to evaluate the effect of the photosensitizer 5,10-diphenyl-15,20-di(N-methylpyridinium-4-yl)porphyrin (Di-cis-Py+) in in vivo models whereby some psoriasis-like parameters could be investigated. METHODS The antiinflammation and antiproliferative activities of Di-cis-Py+ photoactivated was measured by myeloperoxidase (MPO) and N-acetyl-β-d-glucosaminidase (NAG) enzyme activity assay, measurement of IL-6, IL-1β and TNF-α levels, evaluation of proliferating cell nuclear antigen (PCNA) levels by immunohistochemistry and by Western blot. RESULTS Treatment involving PDT and Di-cis-Py+ resulted in reduction of edema, cellular infiltration, proinflammatory cytokines, as well as reduced hyperproliferation of the epidermis. All the evaluated parameters were promoted by topical application of phlogistic agents and are similar to that observed in lesions of psoriatic skin. CONCLUSION The results shows the advantage of topical application, do not cause apparently photosensitivity and have effects comparable to dexamethasone, a first-line drug for the treatment of the disease.

Biochemical Characteristics, Adhesion, and Cytotoxicity of Environmental and Clinical Isolates of Herbaspirillum spp.

ABSTRACT Herbaspirillum bacteria are best known as plant growth-promoting rhizobacteria but have also been recovered from clinical samples. Here, biochemical tests, matrix-assisted laser deionization–time of flight (MALDI-TOF) mass spectrometry, adherence, and cytotoxicity to eukaryotic cells were used to compare clinical and environmental isolates of Herbaspirillum spp. Discrete biochemical differences were observed between human and environmental strains. All strains adhered to HeLa cells at low densities, and cytotoxic effects were discrete, supporting the view that Herbaspirillum bacteria are opportunists with low virulence potential.