Thermal Characterization and Cytotoxicity of Complexes Formed by Papain and Cyclodextrin

Papain is a proteolytic enzyme with restricted applications due to its limited stability. Cyclodextrins are widely used in pharmaceutical formulations once they are able to form complexes with other molecules, improving their stability and bioavailability. The purpose of the present paper was to analyze complexes formed by papain/hydroxypropyl-beta-cyclodextrin and papain/beta-cyclodextrin by thermal analysis and cytotoxicity tests to verify their possible interactions and toxicological behavior. Complex solutions were prepared at different molar ratios and collected as a function of time to be lyophilized and analyzed. Results showed changes in endothermic events and cytotoxicity profiles. A complex formation for both complexes is observed; nevertheless, beta-cyclodextrin was able to change the enzyme characteristics more efficiently.

Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase

Matrix metal loprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

Type 2 heat-labile enterotoxin (LT-II)-producing Escherichia coli isolated from ostriches with diarrhea

The culture supernatant of Escherichia coli, isolated from ostriches with diarrhea in Brazil, caused elongation in Vero cell, rounding in Chinese hamster ovary (CHO) cells and a cytoplasmic vacuolation in ostrich embryo fibroblasts (OEF), but it was not cytotoxic for chicken embryo fibroblasts (CEF). These effects were not neutralized by antiserum to cholera toxin. Polymerase chain reaction assays showed that the ostrich E.coli contained the gene encoding (eltII-A), but not those for type 1 heat-labile enterotoxin (eltA), heat-stable enterotoxins (estA, estB), verocytotoxins (stx-I, stx-II), or cytotoxic necrotizing factors (cnf 1, cnf 2). All isolates belonged to serotype O15:H8. The enteropathogenic relevance of LT-II in ostrich diarrhea remains undetermined. (C) 2004 Elsevier B.V. All rights reserved.

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD 147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Evaluation of the Effects of Endodontic Materials on Fibroblast Viability and Cytokine Production

Introduction: Recently, a new sealer composed of Portland cement named Endo-CPM-Sealer was developed. The aim of this study was to investigate the effects of Endo-CPM-Sealer (EGEO SRL, Buenos Aires, Argentina), Sealapex (Sybron Endo, Glendora, CA), and Angelus MTA (Angelus, Londrina, Brazil) on cell viability and cytokine (interleukin [IL]-1 beta and IL-6) production by mouse fibroblasts. Methods: Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts. Cells cultured with only empty polyethylene tubes were used as the control. After 24 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate the cell viability. For cytokine assay, mouse fibroblasts were incubated in 24-well flat-bottom plates with set material disks at the bottom. Cells cultured without the material disks served as the negative control. After 24 hours of incubation, culture media were collected for cytokine evaluation by using an enzyme-linked immunosorbent assay. The data were statistically analyzed by analysis of variance and Bonferroni correction. Results: Endo-CPM-Sealer, Sealapex, and Angelus MTA did not inhibit the cell viability. All materials induced IL-6 releasing, but the amount was not statistically significant compared with the control group. Angelus MTA induced IL-1 beta releasing significantly more than the control. Conclusions: All materials were not considered cytotoxic in fibroblast culture. (J Endod 2009;35:1577-1579)

Myenteric Denervation Downregulates Galectin-1 and-3 Expression in Gastric Carcinogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fresh autogenous rib cartilage graft to the malar process of rats with and without removal of the perichondrium: a histological study.

A comparison was made of autogenous grafts of rib cartilage with and without removal of the perichondrium, applied to the malar process of rats. Seventy-two male albino rats were divided into two groups according to the kind of graft received by each animal. The experimental periods were 5, 10, 20, 30, 60 and 120 postoperative days. The results showed that, in the control group, the grafts maintained their vitality for the whole experimental period and the perichondrium was biologically integrated into the host bed. Appositional growth was also observed. The treated animals showed intense resorption of the grafts and more intense bone neoformation. The newly formed bone was in intimate contact with the graft in both groups.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Biocompatibility of two current adhesive resins

The purpose of the study was to evaluate the biocompatibility of two current adhesive resins and a calcium hydroxide cement. Fifty-four polyethylene tubes were filled with these dental materials, which were hand-mixed or light-cured according to the manufacturer's directions: group 1-Clearfill Liner Bond 2 (Kuraray); group 2-Single Bond (3M); and group 3-calcium hydroxide cement (Dycal-Dentsply). The materials were implanted into dorsal connective tissue of rats, which were killed 7, 30, and 60 days after the implantation procedure. The implant sites were excised, immersed in buffered Karnovsky's fixative, and processed using routine histological techniques. Sections of 6 μm thickness were stained with hematoxylin and eosin and assessed under light microscopy. Both adhesive resins at 7 days elicited a moderate/intense inflammatory reaction that decreased over time. Fibrous capsules surrounding the tubes were observed at 30 days. Half of the samples in groups 1 and 2 showed thin fibrous capsule formation containing macrophages, capillaries, lymphocytes, fibroblasts, and collagen fibers. Connective tissue healing was observed even though many specimens exhibited a persistent inflammatory reaction mediated by macrophages and giant cells at the 60-day evaluation. Dycal allowed complete healing at 30 days with only a thin fibrous capsule. In conclusion, all experimental materials were successfully walled off by the connective tissue of the rat. However the adhesive resins may release particulates that may, in turn, induce a persistent local inflammatory reaction. Consequently, in this specific condition, these materials cannot be regarded as biocompatible. Dycal was less irritating than the adhesive resins and was better tolerated by the connective tissue. Copyright © 2000 by The American Association of Endodontists.

Homogenous implant in rat tibias of matrix preserved in 98% glycerin: histomorphologic study.

As a chemical medium for preservation of tissues, glycerin has shown good results because it maintains the cellular integrity despite the tissue dehydration it causes. Taking advantage of the osteoinducing properties of the osseous matrix and glycerin as a proper medium for tissue preservation, osseous matrix was implanted in rat tibias. Twenty-four rats were used, each receiving two surgical wounds. In one of the wounds an osseous matrix preserved in 98% glycerin was implanted and the other received a matrix without preservatives. Six animals were sacrificed on days 10, 20, 30 and 60 post-implant. After routine histological processing, the specimens were stained in hematoxylin-eosin and Masson's trichrome. The results showed that the matrixes preserved in glycerin presented faster resorption with replacement by newly formed tissue.

Reaction of rat connective tissue to implanted dentin tube filled with mineral trioxide aggregate, Portland cement or calcium hydroxide.

The subject of this study was to observe the rat subcutaneous connective tissue reaction to implanted dentin tubes filled with mineral trioxide aggregate, Portland cement or calcium hydroxide. The animals were sacrificed after 7 or 30 days and the undecalcified specimens were prepared for histological analysis with polarized light and Von Kossa technique for mineralized tissues. The results were similar for the studied materials. At the tube openings, there were Von Kossa-positive granules that were birefringent to polarized light. Next to these granulations, there was an irregular tissue like a bridge that was Von Kossa-positive. The dentin walls of the tubes exhibited in the tubules a structure highly birefringent to polarized light, usually like a layer and at different depths. The mechanism of action of the studied materials has some similarity.

Hydroxylapatite implants with or without collagen in the zygomatic arch of rats. Histological study.

The authors studied the behavior of calcium phosphate materials used as inlay implants into bone cavities prepared in the zygomatic arch of rats. Fifty male albino rats were divided into four groups as follows: group I-preparation of bone cavities which did not receive any implant material as controls; group II-implants of Interpore 200; group III-implants of experimental hydroxylapatite; group IV-implants of experimental hydroxylapatite combined with collagen. The animals were sacrificed after 5, 15, 30, 60 and 120 days and the specimens were submitted to histological analysis. Results showed that the experimental hydroxylapatite used in group III presented better osteogenic properties compared to the other materials. All tested materials were biocompatible, although group IV presented a more intense inflammatory response.

Morphometric evaluation of gingival overgrowth and regression caused by cyclosporin in rats

Cyclosporin A is a selective immunosuppressant, used in organ transplants to prevent graft rejection. Cyclosporin A can cause various side effects including gingival overgrowth. The aim of this work was to evaluate gingival overgrowth of rats treated daily with 10 mg/kg body weight of Cyclosporin A for 60 days, as well as the regression after the interruption of treatment. All rats treated with Cyclosporin A developed gingival overgrowth, with increased thickness of the epithelium, height and width of the connective tissue. The density of fibroblasts and collagen fibers also increased. Five to 90 days after the interruption of treatment with Cyclosporin A, there was a progressive reduction of the gingival volume and of collagen fibers and fibroblast densities. The reduction was more pronounced in the initial periods and after 90 days did not return to the normal values.

In vitro cytotoxicity of some natural and semi-synthetic isocoumarins from Paepalanthus bromelioides

Numerous natural compounds have a potential for therapeutic applications, but may have to be chemically modified to alter toxic side effects. We investigated structural parameters that could affect the cytotoxicity of isocoumarins similar to 9,10-dihydroxy-5,7-dimethoxy-1H-naphtho(2,3c)pyran-1-one (paepalantine 1). Paepalantine 1 has antimicrobial activity, as well as significant in vitro cytotoxic effects in the McCoy cell line. Two other natural and two semi-synthetic isocoumarins with similar structures obtained from the capitula of Paepalanthus bromelioides were tested on the same cell line by the neutral red assay. Substitution of the 9 and/or 10-OH group made these compounds less cytotoxic.

Morphological evaluation of combined effects of cyclosporin and nifedipine on gingival overgrowth in rats

It has previously been shown that, while cyclosporin A (CsA) and nifedipine both cause gingival overgrowth in the rat. the combined use of these drugs increases the severity of overgrowth. The aim of this study was to describe the histometry and densities of fibroblasts, collagen fibers and vessels in the gingival tissue of rats that were treated with CsA and nifedipine, either alone or in combination. Rats were treated for 60 days with a daily subcutaneous injection of 10 mg/kg body weight of CsA and/or with 50 mg/kg body weight of nifedipine added to the chow. The results confirmed that CsA causes a more severe overgrowth than nifedipine, and that the combined use of these drugs increases the overgrowth severity. All the rat groups that were studied showed that, as the severity of overgrowth increased, there was a parallel increase in fibroblasts and collagen, and a decrease in vessel content. Therefore, independently of whether the gingival overgrowth was caused by CsA alone, nifedipine alone, or both treatments in combination, the fibroblast and collagen density increased in parallel with the severity of the overgrowth. © Blackwell Munksgaard, 2002.