Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient.This is the first gastric denocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation,heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

Influência do FK-506 sobre a expressão de RANKL e OPG na doença periodontal induzida: estudo in vivo e in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Impacto de drogas antiplaquetárias na doença e no reparo periodontal experimental em ratos

Pós-graduação em Odontologia - FOAR

Efeito antimicrobiano e modulador da resposta imune dos peptídeos hBD-3 e LL-37 e dos polifenóis o chá verde e do cranberry

Pós-graduação em Odontologia - FOAR

Host responses induced by different animal models of periodontal disease: a literature review

Periodontitis is an infectious disease characterized by chronic inflammation of the periodontium, and it is mediated and modulated by the host immune system. In the presence of microorganisms or other antigens, immune cells (macrophages/monocytes, dendritic cells, lymphocytes, neutrophils), endothelial cells and fibroblasts secrete cytokines and trigger immune and inflammatory reactions. However, when synthesized at high levels, cytokines modify the pattern of cellular response, participating substantially in the development of chronic inflammatory pathologies, such as periodontal disease. Understanding the origin and progression of bone resorption is one of the primary goals of the field of periodontics, aiming to arrest the disease progression and to optimize future treatments. For this purpose, the development of experimental models is an important and necessary step before entering into clinical trials with new therapies. The purpose of this study is to characterize/evaluate the tissue changes induced by various models of experimental periodontitis through a literature review.

Treatment of periodontal disease with an Er,Cr:YSGG laser in rats exposed to cigarette smoke inhalation

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

Influence of nicotine and cotinine impregnation on the first step of periodontal regeneration: clot stabilization

This study analyzes the clot stabilization on root surfaces of teeth impregnated with cotinine and nicotine and the influence of the scaling in the adhesion of blood components, observing the influence of new exposition to nicotine and/or cotinine after scaling. Fifteen human teeth extracted due to periodontal disease of non-smokers patients were selected and manually scaled. Four dentin blocks were obtained from each tooth (n = 60). Samples received blood application or reimpregnation with nicotine and/or cotinine, depending on the groups. Group 1: PBS immersion + root scaling + blood; group 2: nicotine + root scaling + blood; group 3: nicotine + root scaling + nicotine reapplication + blood; group 4: cotinine + root scaling + blood; group 5: cotinine + root scaling + cotinine reapplication+ blood; group 6: nicotine and cotinine + root scaling + nicotine and cotinine + blood. Samples were kept in 2 ml of each substance for 24 hours. Each group received a blood drop and was analyzed by SEM. The higher amount of blood components was present in teeth exposed to cotinine and the groups submitted to scaling and blood application in comparison with groups that received reapplication of toxic substances after scaling. The greater toxic effect on root dentin surface was after the exposure to nicotine and cotinine. Results suggest that periodontal healing may be delayed in smokers due to the direct inhibition of clot stabilization on the root surface when nicotine and cotinine are present concomitantly.

Effect of LPS treatment on the viability and chemokine synthesis by epithelial cells and gingival fibroblasts

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

The effect of blue light on periodontal biofilm growth in vitro

We have previously shown that blue light eliminates the black-pigmented oral bacteria Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, and Prevotella melaninogenica. In the present study, the in vitro photosensitivity of the above black-pigmented microorganisms and four Fusobacteria species (Fusobacterium nucleatum ss. nucleatum, F. nucleatum ss. vincentii, F. nucleatum ss. polymorphum, Fusobacterium periodonticum) was investigated in pure cultures and human dental plaque suspensions. We also tested the hypothesis that phototargeting the above eight key periodontopathogens in plaque-derived biofilms in vitro would control growth within the dental biofilm environment. Cultures of the eight bacteria were exposed to blue light at 455 nm with power density of 80 mW/cm(2) and energy fluence of 4.8 J/cm(2). High-performance liquid chromatography (HPLC) analysis of bacteria was performed to demonstrate the presence and amounts of porphyrin molecules within microorganisms. Suspensions of human dental plaque bacteria were also exposed once to blue light at 455 nm with power density of 50 mW/cm(2) and energy fluence of 12 J/cm(2). Microbial biofilms developed from the same plaque were exposed to 455 nm blue light at 50 mW/cm(2) once daily for 4 min (12 J/cm(2)) over a period of 3 days (4 exposures) in order to investigate the cumulative action of phototherapy on the eight photosensitive pathogens as well as on biofilm growth. Bacterial growth was evaluated using the colony-forming unit (CFU) assay. The selective phototargeting of pathogens was studied using whole genomic probes in the checkerboard DNA-DNA format. In cultures, all eight species showed significant growth reduction (p < 0.05). HPLC demonstrated various porphyrin patterns and amounts of porphyrins in bacteria. Following phototherapy, the mean survival fractions were reduced by 28.5 and 48.2 % in plaque suspensions and biofilms, respectively, (p < 0.05). DNA probe analysis showed significant reduction in relative abundances of the eight bacteria as a group in plaque suspensions and biofilms. The cumulative blue light treatment suppressed biofilm growth in vitro. This may introduce a new avenue of prophylactic treatment for periodontal diseases.

Antimicrobial activity of plants infusions on oral fusobacteria and their adherence to human erythrocytes

Periodontal disease is the result of the interrelationship between microbiotic aggression and the host’s organic defence. Amongst the microorganisms involved in periodontopathies, Fusobacterium nucleatum is conspicuous by establishing a link between the initial and final colonizers, besides producing toxic compounds and adhering to the host’s cells. Control of bacterial biofilm can be achieved by use of chemical agents, many of which extracted from plants. Thus the object of this study was to evaluate the inhibitory activity in vitro of some teas, generally taken in a normal diet, on Fusobacterium nucleatum and your adherence to host’s cells. Minimum inhibitory and bactericidal concentrations were established and haemagglutinative test in microplaques was effected. It was ascertained that all plant extracts have inhibitory activity and that infusions of Camellia sinensis (black tea and green tea), Mentha piperita (mint) and Pimpinella anixem (aniseed) added to the bacteria/erythrocyte compound reduced significantly the adherence of microorganisms.

Avaliação da sensibilidade à insulina e concentração do cortisol salivar em gestantes com doença periodontal

Pós-graduação em Odontologia Preventiva e Social - FOA

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Cyto and genotoxicity of gold nanoparticles in human hepatocellular carcinoma and peripheral blood mononuclear cells

Engineered nanomaterials have been extensively applied as active materials for technological applications. Since the impact of these nanomaterials on health and environment remains undefined, research on their possible toxic effects has attracted considerable attention. It is known that in humans, for example, the primary site of gold nanoparticles (AuNps) accumulation is the liver. The latter has motivated research regarding the use of AuNps for cancer therapy, since specific organs can be target upon appropriate functionalization of specific nanoparticles. In this study, we investigate the geno and cytotoxicity of two types of AuNps against human hepatocellular carcinoma cells (HepG2) and peripheral blood mononuclear cells (PBMC) from healthy human volunteers. The cells were incubated in the presence of different concentrations of AuNps capped with either sodium citrate or polyamidoamine dendrimers (PAMAM). Our results suggest that both types of AuNps interact with HepG2 cells and PBMC and may exhibit in vitro geno and cytotoxicity even at very low concentrations. In addition, the PBMC were less sensitive to DNA damage toxicity effects than cancer HepG2 cells upon exposure to AuNps. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FHR127H) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-gamma were able to synthesize FHR127H, the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-gamma secrete FH without retention in the ER. Moreover, the retention of FHR127H provoked enlargement of ER cisterns after treatment with IFN-gamma. A similar ER retention was observed in Cos-7 cells expressing the mutant FHR127H protein. Despite this deficiency in secretion, we show that the FHR127H mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein. The Journal of Immunology, 2012, 189: 3242-3248.

ANATOMICAL AND FUNCTIONAL STUDY OF THE MEDIAL COLLATERAL LIGAMENT COMPLEX OF THE ELBOW

Objective: To carry out an anatomical study of the medial collateral ligament, an important elbow stabilizer in different degrees of elbow flexion-extension. Methods: Forty elbows were dissected in order to analyze the functional behavior of the anterior, posterior and transverse ligament bands during valgus stress maneuvers of the elbow in different degrees of flexion and extension. Two groups were determined; in the group GPA the posterior band of the ligament was sectioned initially, then the articular capsule and finally the anterior band; in group GAP this order was reversed. Results: Instability was observed in GPA only in the third stage, when there was a greater mean elbow's opening during the flexion (between 50 degrees and 70 degrees); in GAP the instability was present since the first stage; the degrees of flexion with greater instability were the same as in group GPA. Conclusion: The anterior band of the medial collateral ligament of the elbow is the most important stabilizer of the elbow valgus instability, and its principal action occurs between 50 degrees and 70 degrees of elbow flexion. Level of Evidence III, Diagnostic Studies Investigating a diagnostic test.