Novel Sol-Gel Route to Prepare Eu3+-Doped 80SiO2-20NaGdF4 Oxyfluoride Glass-Ceramic for Photonic Device Applications.

Oxyfluoride glass-ceramics (OxGCs) with the molar composition 80SiO2-20(1.5Eu3+: NaGdF4) were prepared with sol-gel following the "pre-crystallised nanoparticles route" with promising optical results. The preparation of 1.5 mol % Eu3+-doped NaGdF4 nanoparticles, named 1.5Eu3+: NaGdF4, was optimised and characterised using XRD, FTIR and HRTEM. The structural characterisation of 80SiO2-20(1.5Eu3+: NaGdF4) OxGCs prepared from these nanoparticles' suspension was performed by XRD and FTIR revealing the presence of hexagonal and orthorhombic NaGdF4 crystalline phases. The optical properties of both nanoparticles' phases and the related OxGCs were studied by measuring the emission and excitation spectra together with the lifetimes of the 5D0 state. The emission spectra obtained by exciting the Eu3+-O2- charge transfer band showed similar features in both cases corresponding the higher emission intensity to the 5D0→7F2 transition that indicates a non-centrosymmetric site for Eu3+ ions. Moreover, time-resolved fluorescence line-narrowed emission spectra were performed at a low temperature in OxGCs to obtain information about the site symmetry of Eu3+ in this matrix. The results show that this processing method is promising for preparing transparent OxGCs coatings for photonic applications.

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.

Liposomes as Tools to Improve Therapeutic Enzyme Performance.

The drugs concept has changed during the last few decades, meaning the acceptance of not only low molecular weight entities but also macromolecules as bioagent constituents of pharmaceutics. This has opened a new era for a different class of molecules, namely proteins in general and enzymes in particular. The use of enzymes as therapeutics has posed new challenges in terms of delivery and the need for appropriate carrier systems. In this review, we will focus on enzymes with therapeutic properties and their applications, listing some that reached the pharmaceutical market. Problems associated with their clinical use and nanotechnological strategies to solve some of their drawbacks (i.e., immunogenic reactions and low circulation time) will be addressed. Drug delivery systems will be discussed, with special attention being paid to liposomes, the most well-studied and suitable nanosystem for enzyme delivery in vivo. Examples of liposomal enzymatic formulations under development will be described and successful pre-clinical results of two enzymes, L-Asparaginase and Superoxide dismutase, following their association with liposomes will be extensively discussed.

Antagonist G-targeted liposomes for improved delivery of anticancer drugs in small cell lung carcinoma.

Ligand-mediated targeted liposomes have the potential to increase therapeutic efficacy of anticancer drugs. This work aimed to evaluate the ability of antagonist G, a peptide targeting agent capable of blocking the action of multiple neuropeptides, to selectivity improve targeting and internalization of liposomal formulations (long circulating liposomes, LCL, and stabilized antisense lipid particles containing ionizable amino lipid, SALP) to H69 and H82 small cell lung carcinoma (SCLC) cell lines. Antagonist G-targeted LCL and SALP were prepared by two different methods (either by direct covalent linkage at activated PEG grafted onto the liposomal surface or by post-insertion of DSPE-PEG-antagonist-G-conjugates into pre-formed liposomes). Association of the liposomal formulations with target SCLC cells was studied by fluorescence microscopy using fluorescence-labelled liposomes and confirmed quantitatively with [3H]-CHE-labelled liposomes. An antisense oligodeoxynucleotide against the overexpressed oncogene c-myc(as(c-myc)) was efficiently loaded into SALP formulations, the encapsulation efficiency decreased due to the inclusion of the targeting ligand. Also, liposome size was affected by as(c-myc) physical chemical properties. The amount of antagonist G linked to the surface of the liposomal formulations was dependent on the coupling method and lipid composition used. Covalent attachment of antagonist G increased liposomes cellular association and internalization via receptor-mediated and clathrin-dependent endocytosis, as assessed in SCLC cell lines. Biodistribution studies in healthy mice revealed a preferential lung accumulation of antagonist G-targeted SALP as compared to the non-targeted counterpart. Lung levels of the former were up to 3-fold higher 24 h after administration, highlighting their potential to be used as delivery vectors for SCLC treatment.

Animal models of acute gastric mucosal injury: Macroscopic and microscopic evaluation.

Acute gastric mucosal injury is a common disorder of the gastrointestinal tract and the search for new therapeutics is ongoing. The aim of this study is to update and expand the information related to the most widely used rat models of acute gastric ulcer, the ethanol-induced ulcer and the indomethacin-induced ulcer. These two models are compared in terms of macroscopic and microscopic features. Experimentally, ethanol was given orally in a single dose and indomethacin was subcutaneously injected into male Wistar rats. After ulcerative challenges, the stomachs were removed and visually inspected. Anti-ulcerative drugs were used to validate the models. Histological analysis of the stomachs determined the microscopic score. The methodology used for model evaluation applied to macroscopic and microscopic gastric lesions. With these methods it was possible to induce lesions in the gastric mucosa. Microscopic evaluation permitted assessment of the inflammatory and apoptotic impact in the mucosa not observable by macroscopic evaluation. Groups of animals were treated with two standard drugs: sulcralfate suspension or lansoprazole solution. Both drugs reduced macroscopic and microscopic lesions, particularly the hemorrhagic ones. Both models induced acute gastric mucosal injury and no single evaluation method can address all the aspects of the pathology of gastric lesions. As a complement to macroscopic evaluation, microscopy appears to be a relevant tool to selectively identify specific aspects of the development of mucosal injury, quantify the extent of lesions, and contribute to an appropriate interpretation of results. The score systems established here offer a reliable method for testing antiulcer drugs.

Targeted delivery of paromomycin in murine infectious diseases through association to nano lipid systems.

Treatment of intracellular infections such as those caused by Mycobacterium spp. and Leishmania spp. is often hampered by limited access of drugs to infected cells. This is the case of paromomycin (PRM), an antibiotic with broad spectrum in vitro activity against protozoa and mycobacteria. Association of chemotherapeutics to liposomes is a worthy strategy to circumvent poor drug accessibility. Six different PRM liposomal formulations were produced, physicochemically characterized and biologically evaluated in a macrophagic cell line confirming their adequacy for in vivo studies. Biodistribution profiles of PRM liposomes revealed preferential targeting of the antibiotic to the liver, spleen and lungs, relative to free PRM, which translated into an enhanced therapeutic effect in murine models infected with Mycobacterium avium and Leishmania infantum and an absence of toxic effects. Our findings demonstrate the advantages of associating PRM to liposomes indicating their potential as an alternative therapeutic strategy for mycobacterial and parasite infections. FROM THE CLINICAL EDITOR: Infections caused by intracellular organisms such as Mycobacterium and Leishmania remain a significant problem worldwide. Although effective drugs are available, their actions are limited by access into the intracellular compartment. In this article, the authors developed different liposomal formulations as drug carriers of paromomycin and investigated their efficacy in a mouse model. The positive should provide another treatment option for these organisms in the near future.

Utilização de compostos bioativos de plantas medicinais na pós-colheita de tomate / Use of bioactive compounds from medicinal plants in post-harvest tomato

Vários métodos são usados para controle de doenças em pós-colheita de frutos de tomate. Com a crescente restrição ao uso de fungicidas, por questões de segurança alimentar e impacto ambiental, tem-se estimulado o uso de métodos alternativos para controle de doenças pós-colheita. O objetivo deste trabalho foi verificar o efeito de produtos alternativos na conservação e no controle de doenças pós-colheita de tomate cv. Delta e Débora, obtidos de sistemas de cultivo convencional (SC) e em transição ao orgânico (SO). Os frutos foram tratados por imersão (1 min) com acibenzolar-S-metil (ASM), extrato bruto de Azadirachta indica e extrato de biomassa cítrica, e por fumigação com os óleos essenciais (OE) de Syzygium aromaticum e de Citrus aurantium ssp. bergamia. Após 14 dias de armazenamento em temperatura ambiente avaliou-se a perda de massa, pH, sólidos solúveis (SS), a acidez total titulável (ATT) e a incidência de patógenos. Os resultados mostraram que os tratamentos não influenciaram nos parâmetros analisados nos frutos oriundos do SC, para ambas as cultivares. Para os frutos produzidos no SO, observou-se menor perda de massa e maiores valores de SS e ATT para a cv Débora, tratados com os OEs e ASM. Os frutos da cv Delta, apresentaram os maiores valores de pH quando tratados com OE de S. aromaticum. Nenhum dos tratamentos reduziu a incidência de doenças pós-colheita.

Aspectos clínicos y epidemiológicos en pacientes con ERGE que acuden al Servicio de Gastroentrología del Hospital “Dr. Jesús Yerena”: enero-junio 2009 / Clinical and epidemiological aspects of gerd patients going to Hospital

La enfermedad de reflujo gastroesofágico (ERGE) es uno de los trastornos gastrointestinales con mayor prevalencia que afecta a la población en general constituyendo una de las causas frecuentes de consulta. Está relacionada con el flujo retrógrado de contenido gástrico al Esófago siendo la pirosis y la regurgitación los síntomas típicos de esta patología. Las comorbilidades, tales como Obesidad, Diabetes Mellitus, Asma, Hipertensión Arterial y Patologías de ORL se asocian con la disfunción de los mecanismos que normalmente previenen el reflujo gastroesofágico. Describir la relación existente entre los aspectos clínicos y epidemiológicos y la enfermedad de reflujo gastroesofágico en pacientes que consultan al Servicio de Gastroenterología del Hospital “Dr. Jesús Yerena”. Enero- Junio 2009. Estudio prospectivo de corte longitudinal, realizado en la población general adulta que acudió a la consulta del Servicio de Gastroenterología del Hospital General Dr. Jesús Yerena, desde Enero hasta Junio del 2009. Se utilizó una encuesta basada en criterios clínicos, epidemiológicos, de acuerdo a consensos latinoamericanos y europeos sobre ERGE. De 80 sujetos encuestados, 51 fueron femeninos y 29 masculinos. El promedio de edad fue de 52,2 años. La prevalencia de síntomas cardinales determinó que la pirosis presentó un 50,9% con respecto a la regurgitación 49%. La Hipertensión Arterial representó el 50% de las comorbilidades con respecto al resto de las mismas. Encontramos que el 25,563% consumen café, y el 28,571% ingerían alcohol al menos una vez al mes y 24,06% referían hábitos tabáquicos. Así mismo se evaluó la presencia de síntomas atípicos, describiéndose entre ellos la tos (28,03%), dolor Retroesternal (16,6%), ronquera (14,39%) y disnea (12,87%), entre otras. Nuestro estudio determinó la mayor prevalencia de ERGE en el sexo femenino, los síntomas típicos gástricos como pirosis y regurgitación no solo son predominantes también en la ERGE sino que síntomas...

The Gastroesophageal Reflux Disease (GERD) is one of the higher- prevalence gastroenterological disorders affecting the general population, placing itself as one of the most frequent causes of consult. It is related to the gastric contents’ backward flow to the esophagus; heartburn and regurgitation are the typical symptoms of this pathology. Comorbidities such as obesity, diabetes mellitus, asthma, high blood pressure, and ENT pathologies are associated with the disorder of mechanisms that regularly prevent the gastroesophageal reflux. Description of the relationship existing between the clinical and epidemiologic aspects and the gastroesophageal reflux disease on patients going to the Hospital Jesus Yerena’s Gastroenterology Service, between January and June 2009. Longitudinal section prospective study performed on a consult-attending general adult population at Hospital Jesus Yerena’s Gastroenterology Service between January and June 2009. A survey based on clinical and epidemiological criteria was used, as per Latin American and European consensus on GERD. 80 surveyed subjects: 51 female and 29 male. The average age was 52.2. The main symptoms prevalence determined that heartburn showed 50.9% regarding regurgitation that was 49%. 50% of comorbilities was represented by High Blood Pressure regarding the others. Other findings: 25.563% were coffee consumers and 28.571% were at least once a month alcohol consumers, and 24.06% were tobacco users. Likewise, the presence of atypical symptoms was assessed, and the following were described: coughing (28.03%), retrosternal pain (16.6%), hoarseness (14.39%) y dyspnea (12.87%), among others. The study determined that GERD had higher prevalence on females; that not only the typical gastric symptoms, such as heartburn and regurgitation, prevail on GERD but extraesophageal symptoms too, such as coughing, retrosternal pain and hoarseness, were ...

Efeito dos compostos naturais bioativos na conservação pós-colheita de frutos de mangueira cv. Tommy Atkins / Effects of bioactive natural compounds on the postharvest conservation of mango fruits cv. Tommy Atkins

A antracnose causa severos danos na pós-colheita de manga. Como o controle químico tem demonstrado desvantagens, o potencial uso de óleos essenciais para o controle de fitopatógenos tem sido uma alternativa. Neste trabalho, objetvou-se avaliar a eficácia de produtos alternativos no controle da antracnose e na conservação de mangas (Mangifera indica) cv. Tommy Atkins tratado com bicarbonato de sódio diluído em água destilada, na concentração de 3 por cento (v/v), via imersão. Como tratamento, foram utilizados os óleos essenciais de Origanum majorana, Citrus sinensis, Cymbopogon citratus e Eucalyptus citriodora, via fumigação, na dosagem de 1000 μL e água destilada via fumigação; todos por 3 minutos. Frutos da Unidade CEASA-Maringá/PR, selecionados, pesados em balança semianalítica, desinfetados em imersão por 3 minutos em solução de hipoclorito de sódio a 0,5 por cento (v/v); foram submetidos aos tratamentos, permanecendo sob condições ambientes (26 ± 2ºC e UR 90 ± 5 por cento). Exceto o tratamento com Citrus sinensis, que proporcionou um pH maior do que a testemunha, diferindo significativamente dos demais tratamentos que elevaram a variável. A redução da acidez em todos os tratamentos e aumentos dos sólidos solúveis totais nos frutos, sendo que o tratamento com bicarbonato de sódio proporcionou 14,42 ºBrix. O número de frutos com antracnose foi menor no tratamento com Citrus sinensis e este diferiu dos demais comprovando o efeito de espécies do gênero Citrus como antifúngicos. O uso de óleos essenciais como o de Citrus sinensis controlou a antracnose e manteve a qualidade dos frutos.

The anthracnose causes severe damages to the mango post-harvest. As chemical control has shown disadvantages, the potential of use of essential oils has been an alternative for the phytopathogen control. The objective of the present work was to evaluate the efficacy of alternative products in the anthracnose control and in the conservation of mangos (Mangífera mandica) cv. Tommy Atkins treated with sodium bicarbonate diluted in distilled water, concentration of 3 percent (v/v), through immersion. For the treatment, the essential oils of Origanum majorana, Citrus sinensis, Cymbopogon citratus and Eucalyptus citriodora were used through fumigation, in the dosage of 1000 μL and distilled water through fumigation; all for 3 minutes. Mango fruits of the Unity CEASA-Maringá/PR, selected, weighed in a semi-analytical scale, disinfected by immersion of 3 minutes in a solution of sodium hypochlorite to 0.5 percent (v/v); rinsed in running water and dried. They were submitted to the treatments under ambient conditions (26 ± 2ºC and RH 90 ± 5 percent). Except for the treatment of Citrus sinensis, that resulted in a pH larger than the witness, significantly differing from the treatments that elevate the variable. There was reduction of the acidity in all of the treatments and increment of TSS in the fruits and with sodium bicarbonate provided 14.42 º Brix. The number of fruits with anthracnose was smaller in those treated with Citrus sinensis and this differed from the others, proving the effect of Citrus as fungitoxic. The use of essential oils such as that obtained from Citrus sinensis controlled the anthracnose and maintained the quality of the fruits.

Anti-inflammatory effects of locally applied enzyme-loaded ultradeformable vesicles on an acute cutaneous model.

Superoxide dismutase (SOD) and catalase (CAT) are active scavengers of reactive oxygen species and were incorporated into ultradeformable vesicles with the aim of increasing enzyme bioavailability (skin delivery). These special very adaptable vesicles have been formulated and optimized for enzyme transport in order to penetrate into or across the intact skin barrier. Anti-inflammatory activity of SOD-loaded, CAT-loaded and of SOD- and CAT-loaded ultradeformable vesicles applied epicutaneously was measured using different protein doses on the skin, on an arachidonic acid-induced mouse ear oedema. The biological anti-oedema activity is a measurement of drug-targeting potentiation in the organ. Delivery by means of deformable vesicles was compared to conventional vesicles or the absence of an enzyme carrier mediated transport. This was done at various times following prophylactic application of the test formulations. Positive reference groups were treated epicutaneously with several low molecular weight non-steroidal anti-inflammatory drugs (NSAIDs). The latter included indomethacin (3 mg kg(-1)), etofenamate (30 mg kg(-1)) and piroxicam (1 mg kg(-1)) and reduced the oedema by 94 +/- 4%, 81 +/- 4% and 42 +/- 5%, respectively, if measured 30 min after ear treatment with a NSAID. Of the enzyme-loaded carriers tested, only the enzyme-loaded ultradeformable vesicles reduced the swelling of ears significantly: SOD (90 microg kg(-1)), CAT (250 microg kg(-1)) and SOD (90 microg kg(-1)) plus CAT (250 microg kg(-1)) reduced the oedema by 70 +/- 12%, 65 +/- 10% and 61 +/- 19%, respectively, at t = 30 min. Aqueous enzyme solutions and empty carriers had no such effect. The combination of two enzymes resulted in no increased therapeutic effect, but the results are inconclusive since only two dose combinations were tested. The results presented in this study suggest that antioxidant enzymes delivered by means of ultradeformable lipid vesicles can serve as a novel region-specific treatment of inflammation.

Atividades antibacteriana, antifúngica e indutora de fitoalexinas de hidrolatos de plantas medicinais / Antibacterial, antifungal and phytoalexins induction activities of hydrolates of medicinal plants

(O objetivo do trabalho foi avaliar a atividade como antifúngico, antibacteriano e indutor da produção de fitoalexinas dos hidrolatos de Helietta apiculata (canela-de-veado) (HA), Conyza canadensis (buva) (CC) e Cymbopogon nardus (citronela) (CN) nas concentrações de 1, 5, 10, 15, 20 e 25%, buscando seu uso no controle alternativo de doenças em plantas. Para o ensaio de fitoalexinas foram utilizados mesocótilos estiolados de sorgo. Para o efeito antibacteriano foi avaliado o crescimento da bactéria Xanthomonas campestris pv. campestris em meio caldo nutriente suplementado com os tratamentos, tendo como testemunha mistura de antibióticos (22,5 mg/L de oxitetraciclina + 225 mg/L de estreptomicina). O efeito antifúngico foi avaliado através da mensuração do crescimento vegetativo, esporulação, germinação de esporos e desenvolvimento do tubo germinativo de Alternaria brassicae, sendo o fungicida azoxystrobin (0,08 g i.a./L) usado como controle. Foi observado acréscimo na síntese de fitoalexinas com o aumento na concentração dos hidrolatos, sendo o melhor resultado obtido com CN, o qual promoveu aumento de cerca de 4,3 vezes mais, seguido de HA (2,5 vezes) e CC (2,1 vezes), em relação a testemunha água. Resultados semelhantes foram obtidos para crescimento bacteriano com os seguintes resultados de inibição (em %) no desenvolvimento da bactéria: CN: 29,8, HA: 14,9, CC: 14,6 e antibióticos: 97,7. Com relação à ação antifúngica foi observada inibição do desenvolvimento dos tubos germinativos, sendo que os hidrolatos de CC e HA inibiram 69,2 e 56,2%, respectivamente, resultado semelhante ao fungicida. O hidrolato de CN não apresentou efeito antifúngico. Estes resultados indicam a presença de compostos indutores de fitoalexinas, antibacterianos e antifúngicos nesses hidrolatos, porém em baixas concentrações.

The aim of this work was to evaluate the antifungical, antibacterial and phytoalexins elicitors activities ofhydrolates, for using in the alternative control of plant diseases. The hydrolates of Helietta apiculata(HA), Conyza canadensis (CC) and Cymbopogon nardus (CN) were used in the concentrations of 1, 5,10, 15, 20 and 25%. In the phytoalexins assay, sorghum etiolated mesocotyls were used. The antibacterialeffect was evaluated on the growth of the bacterium Xanthomonas campestris pv. campestris in liquidnutrient medium and antibiotic (oxytetraciclin 22.5 mg/L + streptomycin 225 mg/L) was used as controltreatment. The antifungical effect was evaluated on the micelial growth, esporulation, conidia germinationand development of germinative tubes of Alternaria brassicae. The fungicide azoxystrobin (0.08 g a.i./L) was used as control treatment. There was increment in the phytoalexins synthesis with the increase inhydrolates concentration. The better result was obtained with CN that promoted increase 4.3 times largerin relation to the value of the control treatment with water, followed by HA (2.5 times) and CC (2.1 times).Similar results were obtained to the antibacterial activity with the following inhibition results (%) in the development of the bacterium: CN: 29.8, HA: 14.9, CC: 14.6 and antibiotic: 97.7. The main antifungicaleffect was observed on the development of the germinative tubes. Hydrolates of CC and HA showedinhibition of up to 69.2 and 56.2%, respectively, being similar to the fungicide azoxystrobin. The hydrolateof CN did not show antifungical effect. These results indicate the presence of phytoalexins elicitors, and antibacterial and antifungical compounds in those hydrolates, however in low concentrations

Enzymosomes with surface-exposed superoxide dismutase: in vivo behaviour and therapeutic activity in a model of adjuvant arthritis.

Acylated Superoxide Dismutase (Ac-SOD) enzymosomes, liposomal enzymatic systems expressing catalytic activity in the intact form, were previously characterized. The main scope of the present work was to investigate the biological behaviour of Ac-SOD inserted in the lipid bilayer of liposomes, in comparison with SOD located in the aqueous compartment of liposomes. Two types of liposomes were used: conventional liposomes presenting an unmodified external surface and long circulating liposomes coated with poly (ethylene glycol) (PEG). Liposomal formulations of Ac-SOD and SOD were prepared and labelled with indium-111 and their in vivo fate compared. Data obtained led us to the conclusion that, for liposomes coated with PEG the in vivo fate was not influenced by the insertion of Ac-SOD in the lipid bilayers. The potential therapeutic effect of Ac-SOD enzymosomes was compared with SOD liposomes in a rat model of adjuvant arthritis. A faster anti-inflammatory effect was observed for Ac-SOD enzymosomes by monitoring the volume of the inflamed paws. The present results allowed us to conclude that Ac-SOD enzymosomes are nano-carriers combining the advantages of expressing enzymatic activity in intact form and thus being able to exert therapeutic effect even before liposomes disruption, as well as acting as a sustained release of the enzyme.

Estudo experimental sobre o tratamento de cáries incipientes manchas brancas / Experimental study about the incipients caries

No presente trabalho fizemos uma extensiva revisão da literatura sobre remineralização de cáries incipientes - manchas brancas - para dar apoio à parte experimental que constitui na avaliação da eficácia de remineralização de 6 tratamentos testados em dentes permanentes jovens que apresentavam lesões naturais de cáries incipientes. Na parte metodológica, foram estabelecidos detalhadamente critérios para a caracterização das cáries incipientes, tanto para o diagnóstico (início do tratamento) como para a avaliação (final do tratamento). Foi utilizada, como método de avaliação, unicamente a observação clínica (macroscópica). A fase experimental teve uma duração de 8 semanas. Para cada tratamento seguimos uma técnica padronizada. Os resultados foram analisados estatisticamente e apresentados em tabelas e gráficos, com fins de ilustrar o trabalho. A análise dos resultados comprovou que todos os tratamentos mostram-se eficazes, quando considerados em conjunto, como também os tratamento II (Profilaxia + Flúor) e IV (Profilaxia + Ácido Cítrico) mostram-se mais eficazes, quando considerados os tipos de lesões independentemente. Por outro lado, foi surpreendente o comportamento regressivo das lesões para os tipos menos severos e até deu total desaparecimento, já que das 363 manchas brancas tratadas, 76,74 por cento foram remineralizadas, sendo que estas, 38,367 por cento adquiriram uma aparência de completa normalidade (sadia/sem lesão) e em 38,07 por cento ainda permaneceu leve coloração, de extensão menor do que a inicial. Acreditamos que, com um tempo mais logo de aplicação dos medicamentos propostos, estas lesões possam regredir completamente, sendo, portanto, interessante a tentativa de remineralização artificial.