Sérgio Aparecido Torres

Activity of endodontic antibacterial agents against selected anaerobic bacteria

The antimicrobial activity of substances used as antibacterial agents (solutions of 10% calcium hydroxide, camphorated paramonochlorophenol - PMCC, 2% chlorhexidine digluconate and 10% castor oil plant detergent) on anaerobic bacteria (Fusobacterium nucleatum ATCC 25586, Prevotella nigrescens ATCC 33563, Clostridium perfringens ATCC 13124 and Bacteroidesfragilis ATCC 25285), using a broth dilution technique, was evaluated in vitro. For determination of minimum inhibitory and minimum bactericide concentrations (MIC and MBC), two culture broths, Reinforced Clostridial Medium (RCM) and supplemented Brucella, standardized inoculum and serially diluted solutions were used. All antibacterial agents presented antimicrobial activity that varied for different bacteria. There were no differences in the performance of the two broths. Chlorhexidine digluconate was the most effective, with the lowest MICs, followed by castor oil detergent, PMCC and calcium hydroxide. C. perfringens and B. fragilis were the most resistant bacteria to all agents.

The essential role of toll like receptor-4 in the control of Aggregatibacter actinomycetemcomitans infection in mice

OBJECTIVE Aggregatibacter actinomycetemcomitans is an oral Gram-negative bacterium that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs). In this study, we evaluated the role of TLR4 in the control of A. actinomycetemcomitans infection. MATERIAL AND METHODS We examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4(-/-) mice. The production of cytokines was evaluated by ELISA. The bacterial load was determined by counting the number of colony-forming units per gram of tissue. RESULTS The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. However, the absence of TLR4 facilitated the A. actinomycetemcomitans dissemination. Myeloperoxidase activity was diminished in the periodontal tissue of TLR4(-/-) mice. We observed a significant reduction in the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in the periodontal tissue of TLR4(-/-) mice. CONCLUSION The results of this study highlighted the role of TLR4 in controlling A. actinomycetemcomitans infection.

Absence of TLR2 influences survival of neutrophils after infection with Candida albicans

Candida albicans is an opportunistic pathogen, which causes local and/or disseminated diseases in immunosuppressed humans. Phagocytic cells play a critical role in the immune response against C. albicans. Toll like receptors (TLR) are important in the identification of invading microorganisms and in the regulation of neutrophil survival. TLR2 has been shown to participate in the response against pathogenic yeasts and to increase the functional life span of neutrophils. In view of these observations, we studied the involvement of TLR2 in neutrophil function after C. albicans infection. The absence of TLR2 resulted in lower chemotaxis of neutrophils to the site of infection. This in turn was associated with lower levels of chemokines from neutrophils, facilitating the dissemination of the pathogen to the lymph nodes and spleen. A high frequency of apoptotic neutrophils and macrophages in the inflammatory exudates from TLR2(-/-) mice was found. In addition, the phagocytic activity of neutrophils and macrophages, nitric oxide production and myeloperoxidase activity were diminished in cells from TLR2(-/-) mice. Together, these data demonstrate the importance of TLR2 signals for neutrophils activation and survival after C. albicans infection.

Absence of functional TLR4 impairs response of macrophages after Candida albicans infection.

Candida albicans is recognized by phagocytic cells through a set of recognition receptors patterns. Recently, we showed the importance of TLR2 in the regulation of neutrophil survival after C. albicans infection. In the present work, we analyzed the involvement of TLR4 in the recognition of C. albicans by neutrophils and macrophages. Our results show that the absence of functional TLR4 resulted in lower chemotaxis of neutrophils to the site of infection, lower levels of TNF-α, CXCL1 and nitric oxide, and dissemination and persistence of the pathogen in lymph nodes and spleen. In vitro, the phagocytic activity, nitric oxide production and myeloperoxidase activity, CXCL1, IL-1β production by neutrophils from TLR4-defective mice were not changed. In contrast, macrophages from TLR4-defective mice demonstrated lower phagocytosis and lower levels of CXCL1, IL-1β and TNF-α. Together, these data demonstrate that TLR4 signals are important for the recognition of C. albicans by macrophages and their absence allows persistence of the infection.

PD-1 blockage delays murine squamous cell carcinoma development

Engagement of programmed death-1 (PD-1) with its two ligands [programmed death ligand-1 (PD-L1) and PD-L2] has been associated with the suppression of tumor-reactive T cells; however, the underlying mechanism for this T-cell dysfunction is not clear. We hypothesized that PD-1 and PD-L1 signals are, in part, responsible for squamous cell carcinoma (SCC) escape from immune antitumor regulation by modulation of the tumor environment. In the present study, we used a multistage model of SCC to examine the role of PD-1/PD-L1 activation during tumor development. Tumor sites presented an increased percentage of CD4(+) and CD8(+) T cells expressing PD-1 when compared with non-tumorigenic control mice, whereas the expression of PD-L1 was particularly increased in F4/80(+) macrophages in tumor sites. Further, the systemic immune neutralization of PD-1 resulted in a decreased number and delayed incidence rate of papillomas followed by a differential expression of cytokeratins, suggesting that the PD-1-PD-L1 interaction contributes to the progression of SCC by downregulation of antitumor responses. In fact, blocking PD-1 increased the percentage of CD8(+) and CD4(+) T cells, and the levels of interferon-γ in the tumor sites. Our results indicated involvement of PD-1(+) T cells in SCC development and in the modulation of the inflammatory immune response.

Antimicrobial activity of calcium hydroxide and chlorhexidine on intratubular Candida albicans

This study investigated the efficacy of calcium hydroxide and chlorhexidine gel for the elimination of intratubular Candida albicans (C. albicans). Human single-rooted teeth contaminated with C. albicans were treated with calcium hydroxide, 2% chlorhexidine gel, calcium hydroxide plus 2% chlorhexidine gel, or saline (0.9% sodium chloride) as a positive control. The samples obtained at depths of 0–100 and 100–200 µm from the root canal system were analyzed for C. albicans load by counting the number of colony forming units and for the percentage of viable C. albicans using fluorescence microscopy. First, the antimicrobial activity of calcium hydroxide and the 2% chlorhexidine gel was evaluated by counting the number of colony forming units. After 14 days of intracanal medication, there was a significant decrease in the number of C. albicans colony forming units at a depth of 0–100 µm with chlorhexidine treatment either with or without calcium hydroxide compared with the calcium hydroxide only treatment. However, there were no differences in the number of colony forming units at the 100–200 µm depth for any of the medications investigated. C. albicans viability was also evaluated by vital staining techniques and fluorescence microscopy analysis. Antifungal activity against C. albicans significantly increased at both depths in the chlorhexidine groups with and without calcium hydroxide compared with the groups treated with calcium hydroxide only. Treatments with only chlorhexidine or chlorhexidine in combination with calcium hydroxide were effective for elimination of C. albicans.

Níveis de infecção de estreptococos do grupo mutans em gestantes

The CFU number of mutans streptococci in saliva has been used to indicate dental caries risk, as well as to assess the effectiveness or the need for preventive measures. Considering that the acquisition in early childhood of mutans streptococci is a risk factor for developing dental caries in children, and that the transmission of these bacteria is dependent of the mothers salivary levels, the oral status of 50 pregnant women aged between 16 to 37 years (mean of 22.8 years) and of low socio-economic class, that attended a public medical center in Bauru-SP- Brazil for prenatal check up was studied. The dental examination was analysed through the DMF-T and DMF-S indexes using a mirror and an explorer, without radiographs. Paraffin-stimulated saliva was collected and handled according to the instructions of the Caritest-SM manufacturer (HERPO) for researching mutans groups. Of the 50 examined women, 8 (16%) had all teeth present, 39 (78%) had lost from 1 to 10 teeth and 4 (8%) had lost from 13 to 22 teeth. The DMF-S index registered a range from 5 to 114 (mean 37.1). Mutans streptococci were detected in 48 (96%) saliva samples, 4 (8%) of which had an infection level of 1x104 CFU/mL saliva; 10 (20%), 5x104 CFU/mL; and 4 (8%), 1x105 CFU/mL. Levels of infection considered more favorable to transmission were exhibited by 30 (60%) pregnant women 10 (20%) of which showed 2,5x105 CFU/mL, 12 (24%) 5x105 CFU/mL and 8 (16%) 1x106 CFU/mL. The results indicated that there are many pregnant women harbouring high levels of mutans streptococci, prompting the need of a preventive program directed to them in order to reduce or delay the intrafamilial transmission of these microorganisms.

In vitro effect of intracanal medicaments on strict anaerobes by means of the broth dilution method

The determination of bacterial susceptibility to intracanal medicaments is a necessity. Nevertheless, few studies utilize the proper methodology to carry out that evaluation with anaerobes. In this study, the steps of a broth dilution method, carried out in microplates (microdilution) and tubes (macrodilution), to test the effect of traditional intracanal medicaments on anaerobic bacteria are described. The results are presented as values of minimal inhibitory and bactericidal concentrations (MIC and MBC). Standardized inocula of the anaerobic bacteria Prevotella nigrescens (ATCC 33563), Fusobacterium nucleatum (ATCC 25586) and Clostridium perfringens (ATCC 13124), in reinforced Clostridium medium (RCM) and supplemented Brucella broth, were submitted to different concentrations of calcium hydroxide, chlorhexidine digluconate, camphorated paramonochlorophenol and formocresol solutions. The drugs were diluted in the same culture broths, in microplates and tubes, and were then incubated in anaerobiosis jars at 37 degrees C for 48 or 96 hours. The determination of MICs was carried out through visual and spectrophotometric readings, and the determination of MBCs, through the plating of aliquots on RCM-blood agar. For that kind of study, the macromethod with spectrophotometric reading should be the natural choice. MICs and MBCs obtained with the macromethod were compatible with the known clinical performance of the studied medications, and the values varied according to the bacteria and culture media employed. RCM was the most effective medium and C. perfringens, the most resistant microorganism.

Efeito da profilaxia profissional com jato de bicarbonato de sódio sobre a microbiota cariogênica

Avaliou-se o efeito da profilaxia dentaria profissional com o jato de bicarbonato de sodio na contagem salivar de estreptococos do grupo mutans e de lactobacilos em 32 criancas entre 7-10 anos. Coletou-se saliva total estimulada antes e 60 minutos apos o procedimento, e decorridos 30 dias, sendo o numero de UFC/mL saliva detectado atraves do sistema Caritest. Constatou-se uma reducao imediata, estatisticamente significativa, nos niveis salivares de ambos os microrganismos, sendo de 50% para estreptococos do grupo mutans e de 27% para lactobacilos. Para os estreptococos do grupo mutans, esta reducao persistiu pelo periodo de 30 dias, o mesmo nao ocorrendo para os lactobacilos, que retornaram aos seus valores iniciais.

Inhibitory Signals Mediated by Programmed Death-1 Are Involved With T-Cell Function in Chronic Periodontitis

BACKGROUND Inhibitory signals mediated via molecules such as programmed death-1 (PD-1) play a critical role in downmodulating immune responses and maintaining peripheral tolerance. We investigated the involvement of cytokines and PD-1 engagement in mediating the T-cell unresponsiveness to bacterial and ubiquitous antigens in periodontal diseases. METHODS Gingival and peripheral blood samples from healthy individuals and patients with chronic periodontitis were collected and used for the subsequent assays. Leukocytes in the lesion site and blood were evaluated using flow cytometry. The production of interferon-gamma, interleukin-10, and transforming growth factor-beta proteins was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of PD-1+ cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and PD-1 colocalization. RESULTS T cells from patients with chronic periodontitis proliferated poorly in response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) antigen. T-cell unresponsiveness was not associated with imbalanced cytokine production. However, T cells from patients with chronic periodontitis expressed significantly higher levels of PD-1 either upon isolation or after culture with antigens. Moreover, PD-1 blocking did not result in significant T-cell proliferation in cells cultured with phytohemagglutinin or bacterial antigens. The blockade of PD-1 resulted in the increased production of IFN-gamma. In addition, CD4+ and CD8+ T cells expressing PD-1 accumulated in lesions with chronic periodontitis. CONCLUSION These data show that PD-1 engagement could be involved in the modulation of IFN-gamma production by T cells in patients with chronic periodontitis.