Safety and In Situ Antierosive Effect of CaneCPI-5 on Dental Enamel.

The sugarcane cystatin (CaneCPI-5) was recently cloned and showed strong binding force to dental enamel and protection against initial erosion. However, evaluations on its safety and efficacy in a situation closer to the clinical condition are necessary. In the present study we analyzed 1) the cytotoxicity of CaneCPI-5 on human gingival fibroblasts (HGFs); 2) the ability of CaneCPI-5 to reduce enamel erosion and erosion+abrasion in situ. In part 1, HGFs were treated with CaneCPI-5 (0.025, 0.05, 0.1, 0.5 or 1.0 mg/mL) or no treatment (control). The cytotoxicity was assessed after 60 s and 24 h by mitochondrial activity (MTT), confocal microscopy, and hematoxylin/eosin staining. In part 2, 15 volunteers participated in a double-blind crossover protocol consisting of 3 phases, according to the following treatments: 1) 0.1 mg/mL CaneCPI-5; 2) SnCl2/NaF/AmF (Elmex; positive control); 3) water (negative control). The volunteers wore an appliance containing 4 bovine enamel specimens for 5 d. Each day, the specimens were individually treated with 50 µL of the tested solutions per 60 s and then subjected to erosive challenges (0.1% citric acid, pH 2.5, for 90 s, 4 times per day). After the first and last erosive challenge each day, 2 samples were abraded (toothbrushing, 15 s). Enamel wear was measured by contact profilometry. One or two-way analysis of variance (ANOVA)/Tukey's or Sidak's tests (P < 0.05) were applied. Regardless of the concentration and the experimental time, CaneCPI-5 did not decrease the cell viability compared to the negative control (P < 0.05). Erosion+abrasion led to significantly greater wear compared to erosion only. For both conditions, the lowest wear was found for SnCl2 and CaneCPI-5, which did not differ significantly from each other, but showed significant protection when compared to the negative control. In conclusion, CaneCPI-5 is safe on HGFs and reduces enamel erosive wear to the same extent as a commercial solution used to control erosive tooth wear (ETW).

Molecular detection of Wolbachia pipientis in natural populations of sandfly vectors of Leishmania infantum in endemic areas: first detection in Lutzomyia longipalpis.

A polymerase chain reaction-based method was used to screen sandflies for infection with Wolbachia (Rickettsiales: Rickettsiaceae), an intracellular bacterial endosymbiont found in many arthropods and filarial hosts. Positive results were obtained in five of 200 field-collected sandflies and were confirmed by sequencing. All sandflies were Lutzomyia longipalpis (Diptera: Psychodidae) captured in a region endemic for visceral leishmaniasis in Brazil. This is the first study to identify Wolbachia infection in this Lutzomyia species, which is the main vector of leishmaniasis in the study area. The low infection rate found in this study (2.5%), together with the lack of detection of Wolbachia in previous studies and the diversity found in the sequences analysed, suggests horizontal transmission to these sandflies.

Response of carious enamel to TiF4 varnish treatment under diverse cariogenic activities in situ.

OBJECTIVE: To compare the effect of TiF4 and NaF varnishes on demineralized bovine enamel under different cariogenic activities in situ. METHODS: Twenty subjects participated of this in situ study with 3 crossover phases (14days each), in which they wore palatal appliances containing demineralized bovine enamel samples (8 samples/appliance for phase) treated with TiF4, NaF (all with 2.45% F) or placebo varnish. The samples were subjected to different cariogenic conditions (1. absence of biofilm accumulation and sucrose exposure; 2. presence of biofilm and absence of sucrose exposure; 3. presence of biofilm and 20% sucrose exposure 4×/day; 4. presence of biofilm and 20% sucrose exposure 8×/day). All were exposed to fluoride dentifrice (2×/day). The mineral content and lesion depth were evaluated using transverse microradiography (TMR) and the data were subjected to RM two-way ANOVA/Bonferroni tests (p<0.05). RESULTS: TiF4 varnish significantly increased the remineralization of artificial carious lesions compared to placebo, regardless of the cariogenic activity. On the other hand, the remineralizing effect of NaF varnish was dependent on the cariogenic activity. For NaF, remineralization happened only in conditions 1 and 3 compared to placebo varnish (p<0.0001). NaF was unable to prevent further demineralization under biofilm accumulation and sucrose exposure 8×/day (condition 4). In the absence of fluoride treatment, demineralization happened in all conditions, except in the condition 1. CONCLUSION: Therefore, 4% TiF4 varnish was the only treatment able to improve enamel remineralization regardless of the cariogenic activity, while NaF varnish failed in preventing further demineralization under high cariogenic activity in situ. CLINICAL SIGNIFICANCE: 4% TiF4 varnish showed better remineralizing effect compared to NaF varnish, which was seen regardless of the cariogenic activity. This is a promising finding to support the indication of TiF4 in the clinic.

Lactococcus lactis carrying a DNA vaccine coding for the ESAT-6 antigen increases IL-17 cytokine secretion and boosts the BCG vaccine immune response.

AIMS: A regimen utilizing Bacille Calmette-Guerin (BCG) and another vaccine system as a booster may represent a promising strategy for the development of an efficient tuberculosis vaccine for adults. In a previous work, we confirmed the ability of Lactococcus lactis fibronectin-binding protein A (FnBPA+) (pValac:ESAT-6), a live mucosal DNA vaccine, to produce a specific immune response in mice after oral immunization. In this study, we examined the immunogenicity of this strain as a booster for the BCG vaccine in mice. METHODS AND RESULTS: After immunization, cytokine and immunoglobulin profiles were measured. The BCG prime L. lactis FnBPA+ (pValac:ESAT-6) boost group was the most responsive group, with a significant increase in splenic pro-inflammatory cytokines IL-17, IFN-γ, IL-6 and TNF-α compared with the negative control. CONCLUSIONS: Based on the results obtained here, we demonstrated that L. lactis FnBPA+ (pValac:ESAT-6) was able to increase the BCG vaccine general immune response. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is of great scientific and social importance because it represents the first step towards the development of a booster to the BCG vaccine using L. lactis as a DNA delivery system.

Comparação do teor de taninos entre duas espécies de espinheira-santa (Maytenus aquifolium Mart.e Maytenus ilicifolia Mart. ex Reissek)cultivadas no Horto Medicinal do Refúgio Biológico Bela Vista - RBBV da Itaipu Binacional - Foz do Iguaçu, PR - Brasil / Comparison of the tannin doses between two species of thorn-santa (Maytenus ilicifolia Mart. and Maytenus aquifolium Mart. ex Reissek) grown in the medicinal garden of Bela Vista Biological Refuge - RBBV of Itaipu Binational - Foz do Iguacu, PR - Brazil

RESUMO As duas espécies de espinheira-santa Maytenus aquifolium Mart. e Maytenus ilicifolia Mart. ex Reissek pertencentes à família Celastraceae e têm sido intensamente exploradas nas populações nativas devido seu alto valor medicinal. O grande interesse pela espinheira-santa é para o tratamento de gastrites, úlceras gástricas e duodenais. O efeito antiulcerogênico está relacionado com a presença de polifenóis totais, mais especificamente com os taninos. Este trabalho teve como objetivo comparar o teor de taninos entre essas duas espécies cultivadas no Horto Medicinal do Refúgio Biológico Bela Vista - RBBV da Itaipu Binacional no município de Foz do Iguaçu, PR - Brasil. Foram realizadas duas coletas com intervalo de um mês para cada espécie e para a quantificação foi realizada a análise por espectrofotometria segundo a Farmacopeia Brasileira V. Os resultados foram analisados através do teste de variância (ANOVA) e a diferença no teor de taninos foi evidenciada pelo teste Tukey, a 5% de nível de significância empregando-se o software SISVAR. Foram obtidos em média 0,61% para o lote 1 e 2 de Maytenus aquifolium e (3,90%) para Maytenus ilicifolia, resultando em uma média de 84,35% de taninos a mais para Maytenus ilicifolia em comparação com Maytenus aquifolium concluindo assim que as espécies não devem ser intercambiáveis.

ABSTRACT The two species of (espinheira-santa) Maytenus aquifolium and Maytenus ilicifolia, which belong to the Celastraceae family, have been intensively explored in native populations, due to its medicinal potential. The great interest in the espinheira-santa is due to its effectiveness on the treatment of gastritis, gastric and duodenal ulcers. The antiulcerogenic effect is related to the presence of phenolic compounds, more specifically the tannins. This study aimed to compare the tannin doses between these two species grown in the Medicinal Garden of Bela Vista Biological Refuge - RBBV of Itaipu in the city of Foz do Iguaçu, PR - Brazil. Two trials were conducted with one month interval, for the quantification of the tannins by a spectrophotometry, according to the Brazilian Pharmacopoeia V. The results were analyzed through the analysis of variance (ANOVA) and the difference in the tannins doses was demonstrated in a Tukey test at 5% level of significance employing the SISVAR software. It were found 0.61% of tannins for lot 1 and 2 of Maytenus aquifolium, and 3.90% for Maytenus ilicifolia, which represents 84.35% more tannin at Maytenus ilicifolia than at Maytenus aquifolium. Therefore, the species should not be interchanged.

Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom.

Honeybee stings are a severe public health problem. Bee venom contains a series of active components, including enzymes, peptides, and biogenic amines. The local reactions observed after envenoming include a typical inflammatory response and pain. Honeybee venom contains some well-known polycationic peptides, such as Melittin, Apamin, MCD peptide, Cardiopep, and Tertiapin. Secapin in honeybee venom was described 38 years ago, yet almost nothing is known about its action. A novel, variant form of this peptide was isolated from the venom of Africanized honeybees (Apis mellifera). This novel peptide, named Secapin-2, is 25 amino acid residues long. Conformational analyses using circular dichroism and molecular dynamics simulations revealed a secondary structure rich in strands and turns, stabilized by an intramolecular disulfide bridge. Biological assays indicated that Secapin-2 did not induce hemolysis, mast cell degranulation or chemotactic activities. However, Secapin-2 caused potent dose-related hyperalgesic and edematogenic responses in experimental animals. To evaluate the roles of prostanoids and lipid mediators in the hyperalgesia and edema induced by this peptide, Indomethacin and Zileuton were used to inhibit the cyclooxygenase and lipoxygenase pathways, respectively. The results showed that Zileuton partially blocked the hyperalgesia induced by Secapin-2 and decreased the edematogenic response. In contrast, Indomethacin did not interfere with these phenomena. Zafirlukast, a leukotriene receptor antagonist, blocked the Secapin-2 induced hyperalgesia and edematogenic response. These results indicate that Secapin-2 induces inflammation and pain through the lipoxygenase pathway in both phenomena.

Effect of experimental mouthrinses containing the combination of NaF and TiF4 on enamel erosive wear in vitro.

OBJECTIVE: This study analysed the anti-erosive effect of experimental solutions containing TiF4 and NaF. METHODS: Bovine enamel samples (n=15) were treated with: (1) commercial solution with SnCl2/NaF (Erosion Protection(®)); (2) experimental solution with 0.0815% TiF4; (3) 0.105% NaF; (4) 0.042% NaF + 0.049% TiF4; (5) 0.063% NaF + 0.036% TiF4 or (6) control. The samples were submitted to pH cycles and daily fluoride applications for seven days. The enamel wear was measured using a contact profilometer and analysed by ANOVA (p < 0.05). RESULTS: The best anti-erosive effect was found for experimental solution with 0.0815% TiF4 (99% reduction in enamel wear), followed by SnCl2/NaF (78%) and 0.049% TiF4 + 0.042% NaF solution (41%). CONCLUSIONS: The experimental solution containing a specific combination of TiF4 + NaF has the ability to partially reduce enamel erosion.

Risk factors associated with Chlamydia psittaci infection in psittacine birds.

Chlamydia psittaci is the aetiological agent of chlamydiosis in birds, especially Psittaciformes. The objective of the present study was to detect C. psittaci by means of semi-nested PCR among psittacine birds sold at pet markets and kept as pet birds in Salvador, Bahia, Brazil. Questionnaires were used to identify risk factors involved in the epidemiology of the disease. In addition, the management of birds and cages was observed at each location studied. The frequency of C. psittaci infection was 10.6% (33/311) in the psittacine birds studied. Birds kept in households were less frequently positive (3.4%; 5/148) than those at pet markets (17.2%; 28/163). Among the several factors analysed in the epidemiology of the disease, only population density (P = 0.001) and cage hygiene (P = 0.041) in birds at pet markets were significantly associated with C. psittaci infection. These results demonstrate the presence of C. psittaci infection in Psittaciformes kept as pets and held at pet markets in Salvador, Bahia, showing that this micro-organism is a public health concern. Control measures should be encouraged to prevent the spread of the agent among birds, as well as among employees and customers.

Inhibition of tooth erosion by milk containing different fluoride concentrations: an in vitro study.

OBJECTIVES: This in vitro study assessed the effect of milk containing different fluoride concentrations on tooth erosion. METHODS: Bovine enamel and root dentine specimens were treated with: (1) bovine whole milk with 0ppm F; (2) 2.5ppm F; (3) 5ppm F; (4) 10ppm F (all after erosion); (5) whole milk with 0ppm F (before erosion); (6) NaF (0.05% F, positive control, after erosion) or (7) 0.9% NaCl (negative control, after erosion). The specimens were submitted to pH cycles (4× 90s in soft drink) and treatments for 5 days. The specimens were immersed in the treatment solutions for 1min (only at the first cycle each day) with further exposition to 1:1 milk:saliva slurry for 10min. The tooth loss was measured using a contact profilometer and statistically analysed (p<0.05). RESULTS: Rinsing with milk before erosive challenge significantly reduced tooth loss compared to negative control (67% and 24% reduction in dentine and enamel loss, respectively) and to milk after erosive challenge, only for dentine. The addition of fluoride to milk also reduced tooth loss compared to negative control, but with no significant differences among fluoride concentrations for enamel and dentine (µm), respectively: 0ppm (3.63±0.04 and 2.51±0.53), 2.5ppm F (2.86±0.42 and 1.96±0.47), 5ppm F (2.81±0.27 and 1.77±0.44), 10ppm F (2.03±0.49 and 1.68±0.59). There was a negative and significant correlation between [F] and the tooth loss. CONCLUSIONS: Daily rinse with milk containing F is able to reduce both enamel and dentine erosion in vitro. CLINICAL SIGNIFICANCE: Since the prevalence of dental erosion is steadily increasing, rinse with milk or its derivate might be an important strategy to reduce the progression of tooth erosion.

Progesterone increases resistance of ophthalmic and central retinal arteries in climacteric women.

OBJECTIVE: To evaluate the effect of a synthetic progestin on the vascular resistance of the ophthalmic and central retinal arteries in climacteric women, compared to placebo, using transorbital ultrasound with Doppler velocimetry. METHODS: We performed a prospective, randomized, double-blinded, placebo-controlled study with 216 climacteric women. Subjects were randomly allocated to one of two groups: either the group receiving placebo (one pill/day for 30 days) (n = 108) or the group receiving progestin (5 mg medroxyprogesterone acetate/day for 30 days) (n = 108). Transorbital Doppler velocimetric ultrasound was performed, before and after treatment; we measured the pulsatility index, resistance index and systole/diastole ratio. RESULTS: The mean ages of the participants in the study group and the control group were 54 ± 6.2 years (range 48-59 years) and 55 ± 6.8 years (range 46-60 years), respectively. When we compared the effect of the progestin on the central retinal artery before and after treatment, we observed a significant increase after the treatment in all Doppler indices. The same was observed when we compared the effect of the progestin on the ophthalmic artery. In the group of women receiving placebo, the Doppler indices were similar before and after treatment. CONCLUSIONS: Our results demonstrate the existence of a progestogenic vasoconstrictive effect in the ophthalmic and central retinal arteries. As this study provides new data, the observed effect needs further investigations to better elucidate its extent. Moreover, our findings may be particularly useful to others interested in understanding the vascular dynamics of the cerebral vessels and to researchers running clinical trials related to hormone replacement therapy.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae).

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH2 and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH2, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom.

Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.

Nitric oxide synthesized from L-arginine regulates vascular tone in the coronary circulation of the rabbit.

1. The role of nitric oxide (NO) in the regulation of the vascular tone of the coronary circulation of the Langendorff-perfused rabbit heart was investigated. 2. NG-monomethyl-L-arginine (L-NMMA; 10-100 microM), a specific inhibitor of NO formation from L-arginine (L-Arg), but not its D-enantiomer (D-NMMA; 100 microM) produced a dose-related, sustained increase in the coronary perfusion pressure (CPP). In addition, L-NMMA inhibited the vasodilator responses of acetylcholine (ACh), unmasking in some instances its direct vasoconstrictor effect. These effects of L-NMMA were attenuated by L-Arg. 3. L-NMMA (10 and 30 microM), but not D-NMMA (30 microM), caused a long-lasting inhibition of NO formation which was reversed by L-Arg (30 and 100 microM), but not by D-Arg (100 microM). 4. This study indicates that the formation of NO from L-Arg in the coronary circulation of the rabbit plays a role both as a regulator of vascular tone and as a mediator of the vasodilatation induced by ACh.