Chez l’homme, la primo-infection par le vírus Herpès simplex de type 1 (HSV-1) a lieu au niveau de la muqueuse oro-pharyngée. Après une phase de réplication, les virions pénètrent les terminaisons axonales et migrent vers le ganglion trijumeau ipsilatéral (TG), puis vers le TG controlatéral. Le virus entre alors dans un état de latence dans les 2 TG. Les réactivations d’HSV-1 dans ces neurones sont responsables des kératites herpétiques (KH), unilatérales et survenant toujours du même côté chez un patient donné.En utilisant un modèle murin oro-oculaire, qui reproduit les aspects essentiels de la maladie chez l’homme, nous avons précédemment démontré que l’inoculation virale latéralisée d’un côté de la bouche entraine une réplication virale dans la lèvre, suivie d’une KH ipsilatérale à l’inoculation 6 jours plus tard. De manière concomitante, le virus se propage aux 2 TG, mais les réactivations ne surviennent que du côté ipsilatéral à l’inoculation. Nous avons également observé qu’après une primo-infection dans une lèvre avec une souche d’HSV-1 non-neurovirulente, les souris étaient protégées contre les signes de la maladie et contre la réactivation d’une souche sauvage pleinement virulente inoculée secondairement dans l’autre lèvre (souche ré-infectante). Afin de comprendre les mécanismes immunitaires en jeu dans cet état de protection, nous avons combiné une analyse en cytométrie en flux multiparamétrique à des tests immunologiques, pour quantifier et définir l’infiltrat immunitaire hématopoïétique et les chémokines inflammatoires au site d’inoculation et dans les TG. Nous avons démontré qu’après une inoculation unique avec la souche sauvage virulente, un infiltrat immun riche en cellules pro-inflammatoires, survenaient de manière retardée dans les lèvres inoculées, et persistaient dans les TG. A l’opposé, l’infiltrat immunitaire était plus précoce dans les tissus réinfectés (après une primo-infection par la souche non neurovirulente), plus riche en cellules de l’immunité adaptative, et associé à des concentrations moindres de chémokines inflammatoires. En outre, cet infiltrat s’estompait plus rapidement, avec une disparition concomitante des chémokines inflammatoires. Ces données permettent de mieux comprendre la nature et la cinétique de la réponse immunitaire anti-HSV-1, et pourront être utiles pour le développement futur de stratégies vaccinales anti-HSV-1. / In humans, Herpes simplex virus type 1 (HSV-1), primary infection occurs in the oral mucocutaneous tissues. Virions replicated here penetrate sensitive neuronal axons, migrate to both trigeminal ganglion (TG) where it established a lifelong latency. Reactivations of HSV-1 in the TG neurons induce clinical recurrences in the connected peripheral tissues. This process is involved in herpes simplex keratitis (HSK), a condition that, strikingly, occurs almost exclusively in the same eye for a given patient. Based on an experimental oro-ocular (OO) model of HSV-1 infection, that recapitulates most of these human clinical features, we previously demonstrated that a virus inoculation on one side of the mouth, leads to viral replication in the lip, followed by HSK. Virus concomitantly disseminates to both TG, but reactivation only occurs in the TG ipsilateral to the inoculation site. We also observed that after a primary inoculation with a non-neurovirulent strain of HSV-1 in one lip, mice are protected against both acute phase disease and reactivation after a superinfection with a fully virulent wild-type strain of HSV-1 in the contralateral lip.In order to understand the underlying mechanisms involved in this state of protection, we combined high resolution flow cytometry and bead-based immunoassays, to quantify hematopoietic subsets and inflammatory chemokines in the site of inoculation and in the TG. We demonstrated that after a single inoculation with the wild-type strain, a delayed immune infiltrate, boasting more proinflammatory subsets, occurred in the lip and persisted in the TG. In contrast, the immune infiltrate occurred earlier in the superinfected lip and ipsilateral TG, with less inflammatory chemokines but more adaptive immune subsets. Moreover, cellular infiltrate resolved faster, correlating with nullification of inflammatory chemokines locally. These data show that immune response kinetics influence the development of natural immunity to HSV-1, and can be harnessed to protect against disease and reactivations.

Cerca de 38% das árvores cítricas do Estado de São Paulo são afetadas pela Clorose Variegada dos Citros - CVC, causando perdas anuais de cerca de 100 milhões de dólares para as indústrias citrícolas. O agente causal da CVC é uma bactéria gram-negativa, fastidiosa e habitante dos vasos de xilema denominada Xylella fastidiosa, transmitida por cigarrinhas pertencentes à família Cicadellidae, subfamília Cicadellinae. Já foi observado que laranjas doces jovens, na faixa de 3 a 4 anos de idade, são mais sensíveis à CVC do que as plantas adultas. A doença é mais severa em regiões onde estresses adicionais ocorrem, como alta demanda atmosférica por água (decorrente do aumento da temperatura e do déficit de pressão de vapor) e déficit hídrico no solo. Os mecanismos fisiológicos e químicos que caracterizam o estabelecimento da CVC ainda não foram totalmente compreendidos, especialmente sob condições de estresse. Sendo assim, estudos que consideram a interação entre época do ano, regime hídrico e o estabelecimento da CVC em plantas jovens são necessários para auxiliar na elaboração de programas de manejo da doença. O presente trabalho avaliou a sobrevivência de infecções iniciais de X. fastidiosa em plantas cítricas jovens no campo, mantidas com e sem irrigação, 6, 12 e 18 meses após inoculações mecânicas realizadas no verão (mês de janeiro) e inverno (mês de julho). Visando analisar a influência do regime hídrico isoladamente foram realizadas, na época do outono (final de maio e início de junho), inoculações mecânicas e inoculações com as cigarrinhas vetoras Bucephalogonia xanthophis e Dilobopterus costalimai em plantas cítricas jovens mantidas em capacidade de campo e déficit hídrico em casa-de-vegetação; as plantas inoculadas mecanicamente foram avaliadas 5 meses após a data de inoculação, enquanto que as plantas inoculadas com insetos foram avaliadas 10 meses após a data de inoculação. Em condições naturais, foram observadas, 6 meses da data de inoculação, maiores taxas de sobrevivência de infecções iniciais de X. fastidiosa em plantas jovens inoculadas na época de inverno na ausência de estresse hídrico; no entanto, 12 meses da data de inoculação, as plantas inoculadas mantidas com e sem irrigação apresentavam médias populacionais bacterianas estatisticamente iguais. As plantas mantidas em casa-devegetação e inoculadas mecanicamente apresentaram resultados semelhantes àqueles obtidos 6 meses da data de inoculação em condições naturais; no caso da inoculação com cigarrinhas vetoras, foi detectada a presença da bactéria apenas em plantas em capacidade de campo. Estes resultados indicam que o déficit hídrico é um fator desfavorável para o estabelecimento de infecções primárias de X. fastidiosa em plantas cítricas jovens. Os resultados obtidos 12 meses após a inoculação mecânica em condições naturais, logo após a estação de chuva, indicam que, uma vez retirado o estresse hídrico, tal influência negativa deixa de existir e a bactéria readquire sua capacidade de infecção e colonização do hospedeiro. / In Brazil, about 38% of the citric trees of the São Paulo State are affected by the Citrus Variegated Chlorosis - CVC, causing annual losses of about 100 million dollars for the citrus industries. The causal agent of CVC is a gram-negative, fastidious bacterium inhabitant of the xylem vessels called Xylella fastidiosa. It is vectored by sharpshooter leafhoppers pertaining to family Cicadellidae and subfamily Cicadellinae. It was observed that young citrus plants, between 3 and 4 years of age, are more susceptible to CVC than adult plants. The disease is more severe in regions where additional stresses occur, such as high atmospheric demand (due to the increase of temperature and vapor pressure deficit) and water deficit in the soil. The physiological and chemical mechanisms that characterize the establishment of CVC within the host have not yet been totally understood, especially under conditions of stress. Therefore, studies that consider the interaction between seasons, water regime and the establishment of CVC in young plants are necessary in the elaboration of more efficient management programs. The survival of initial infections of X. fastidiosa in young citrus plants mechanically inoculated during the summer and winter, and kept in the field with and without irrigation, were evaluated 6, 12 and 18 months after mechanical inoculations. To analyze the influence of water regime separately, an experiment was carried through during the autumn (final of May and beginning of June) in a greenhouse. Young citrus plants kept in field capacity and water deficit were mechanically inoculated and inoculated with sharpshooter leafhoppers Bucephalogonia xanthophis and Dilobopterus costalimai. The mechanically inoculated plants were evaluated 5 months after the date of inoculation, whereas the plants inoculated with insect vectors were evaluated 10 months after the date of inoculation. In natural conditions, 6 months of the date of inoculation, higher survival rates of initial infections of of X. fastidiosa were detected in inoculated young plants during winter in the absence of water deficit; however, 12 months after inoculation, the young citrus plants kept with and without irrigation presented statistically equal bacterial population averages. The mechanically inoculated plants kept in the greenhouse presented similar results to those kept in natural conditions and analyzed 6 months after mechanical inoculation. In the case of inoculation with sharpshooter leafhoppers, the presence of the bacterium was detected only in plants kept in field capacity. The results demonstrate that the water deficit is unfavorable for the establishment of primary infections of X. fastidiosa in young citrus plants. The results obtained 12 months after mechanical inoculation in natural conditions, right after the rainy season, indicate that, once the water deficit is removed, its negative influence ceases to exist and the bacterium reacquires its capacity of infection and colonization of the host.

Cerca de 38% das árvores cítricas do Estado de São Paulo são afetadas pela Clorose Variegada dos Citros - CVC, causando perdas anuais de cerca de 100 milhões de dólares para as indústrias citrícolas. O agente causal da CVC é uma bactéria gram-negativa, fastidiosa e habitante dos vasos de xilema denominada Xylella fastidiosa, transmitida por cigarrinhas pertencentes à família Cicadellidae, subfamília Cicadellinae. Já foi observado que laranjas doces jovens, na faixa de 3 a 4 anos de idade, são mais sensíveis à CVC do que as plantas adultas. A doença é mais severa em regiões onde estresses adicionais ocorrem, como alta demanda atmosférica por água (decorrente do aumento da temperatura e do déficit de pressão de vapor) e déficit hídrico no solo. Os mecanismos fisiológicos e químicos que caracterizam o estabelecimento da CVC ainda não foram totalmente compreendidos, especialmente sob condições de estresse. Sendo assim, estudos que consideram a interação entre época do ano, regime hídrico e o estabelecimento da CVC em plantas jovens são necessários para auxiliar na elaboração de programas de manejo da doença. O presente trabalho avaliou a sobrevivência de infecções iniciais de X. fastidiosa em plantas cítricas jovens no campo, mantidas com e sem irrigação, 6, 12 e 18 meses após inoculações mecânicas realizadas no verão (mês de janeiro) e inverno (mês de julho). Visando analisar a influência do regime hídrico isoladamente foram realizadas, na época do outono (final de maio e início de junho), inoculações mecânicas e inoculações com as cigarrinhas vetoras Bucephalogonia xanthophis e Dilobopterus costalimai em plantas cítricas jovens mantidas em capacidade de campo e déficit hídrico em casa-de-vegetação; as plantas inoculadas mecanicamente foram avaliadas 5 meses após a data de inoculação, enquanto que as plantas inoculadas com insetos foram avaliadas 10 meses após a data de inoculação. Em condições naturais, foram observadas, 6 meses da data de inoculação, maiores taxas de sobrevivência de infecções iniciais de X. fastidiosa em plantas jovens inoculadas na época de inverno na ausência de estresse hídrico; no entanto, 12 meses da data de inoculação, as plantas inoculadas mantidas com e sem irrigação apresentavam médias populacionais bacterianas estatisticamente iguais. As plantas mantidas em casa-devegetação e inoculadas mecanicamente apresentaram resultados semelhantes àqueles obtidos 6 meses da data de inoculação em condições naturais; no caso da inoculação com cigarrinhas vetoras, foi detectada a presença da bactéria apenas em plantas em capacidade de campo. Estes resultados indicam que o déficit hídrico é um fator desfavorável para o estabelecimento de infecções primárias de X. fastidiosa em plantas cítricas jovens. Os resultados obtidos 12 meses após a inoculação mecânica em condições naturais, logo após a estação de chuva, indicam que, uma vez retirado o estresse hídrico, tal influência negativa deixa de existir e a bactéria readquire sua capacidade de infecção e colonização do hospedeiro. / In Brazil, about 38% of the citric trees of the São Paulo State are affected by the Citrus Variegated Chlorosis - CVC, causing annual losses of about 100 million dollars for the citrus industries. The causal agent of CVC is a gram-negative, fastidious bacterium inhabitant of the xylem vessels called Xylella fastidiosa. It is vectored by sharpshooter leafhoppers pertaining to family Cicadellidae and subfamily Cicadellinae. It was observed that young citrus plants, between 3 and 4 years of age, are more susceptible to CVC than adult plants. The disease is more severe in regions where additional stresses occur, such as high atmospheric demand (due to the increase of temperature and vapor pressure deficit) and water deficit in the soil. The physiological and chemical mechanisms that characterize the establishment of CVC within the host have not yet been totally understood, especially under conditions of stress. Therefore, studies that consider the interaction between seasons, water regime and the establishment of CVC in young plants are necessary in the elaboration of more efficient management programs. The survival of initial infections of X. fastidiosa in young citrus plants mechanically inoculated during the summer and winter, and kept in the field with and without irrigation, were evaluated 6, 12 and 18 months after mechanical inoculations. To analyze the influence of water regime separately, an experiment was carried through during the autumn (final of May and beginning of June) in a greenhouse. Young citrus plants kept in field capacity and water deficit were mechanically inoculated and inoculated with sharpshooter leafhoppers Bucephalogonia xanthophis and Dilobopterus costalimai. The mechanically inoculated plants were evaluated 5 months after the date of inoculation, whereas the plants inoculated with insect vectors were evaluated 10 months after the date of inoculation. In natural conditions, 6 months of the date of inoculation, higher survival rates of initial infections of of X. fastidiosa were detected in inoculated young plants during winter in the absence of water deficit; however, 12 months after inoculation, the young citrus plants kept with and without irrigation presented statistically equal bacterial population averages. The mechanically inoculated plants kept in the greenhouse presented similar results to those kept in natural conditions and analyzed 6 months after mechanical inoculation. In the case of inoculation with sharpshooter leafhoppers, the presence of the bacterium was detected only in plants kept in field capacity. The results demonstrate that the water deficit is unfavorable for the establishment of primary infections of X. fastidiosa in young citrus plants. The results obtained 12 months after mechanical inoculation in natural conditions, right after the rainy season, indicate that, once the water deficit is removed, its negative influence ceases to exist and the bacterium reacquires its capacity of infection and colonization of the host.

La primo-infection est un moment critique de l’établissement du réservoir justifiant de l’initiation d’un traitement précoce. Nous avons initié un essai randomisé évaluant l’impact de deux ans d’un traitement antirétroviral intense (essai ANRS147 OPTIPRIM, trithérapie versus pentathérapie) sur le réservoir et avons initié des études physiopathologiques au cours de cet essai. Nous montrons ainsi la faible diversité génétique des virus en primo-infection dans les compartiments sanguins et rectaux. Le réservoir s’établit dès le premier mois de l’infection par diffusion d’un cluster viral homogène au sein des lymphocytaires T CD4 naïfs (TN) et mémoires centrales (TCM), transitionnelles (TTM), effectrices (TEM) quiescents. Il en résulte une perturbation de l’homéostasie lymphocytaire associée à une faible contribution au réservoir des cellules peu différenciées à longue demi-vie, TN et TCM. Par ailleurs nous montrons que la majorité des patients au moment de leur primo-infection n’ont pas la capacité de développer des réponses T CD8 à même de supprimer la réplication virale comme chez les patients HIV Controllers. Après deux ans de traitement, nous observons que la diversité virale n’a pas évolué, par contre la taille du réservoir est fortement réduite. Les anomalies de l’homéostasie lymphocytaire T CD4 persistent, par contre le traitement très précoce a permis de protéger les TN et TCM. Il n’y a pas de bénéfice additionnel d’une pentathérapie mais nous avons validé le concept qu’un traitement précoce permet d’induire un contrôle virologique au long cours après arrêt de traitement. Nos résultats indiquent qu’un traitement plus long que deux ans permettrait de renforcer la diminution du réservoir. Ces résultats seront à prendre en compte pour l’élaboration de futurs essais en primo-infection visant à réduire le réservoir pour une rémission au long cours. / HIV primary infection is a critical period in the establishment of the reservoirs that justifies the initiation of an early treatment. We started a randomised trial to assess the impact of a two-year intense HAART (ANRS147 OPTIPRIM trial: five-drug therapy versus. three-drug therapy) on the blood reservoir; within this this trial, we included some pathophysiological studies. Thus, we show that during the primary infection, viruses have a low genetic diversity in blood and rectal compartments. The reservoir establishes itself as early as the first month of the infection by spreading a homogeneous viral cluster in CD4 T cells subsets, naive T cells (TN), central memories (TCM), transitional memories (TTM), effector memories (TEM), and resting T cells. This results in a disruption of the lymphocyte homeostasis, linked to the low contribution to the reservoir of TN and TCM, which are little differentiated cells with long half-lives. Moreover, we show that, at the time of the primary infection, the majority of patients do not have the ability to develop CD8 T cells responses that could suppress the viral replication, as HIV Controllers patients do. After two years of treatment, we observe that there is no evolution of the viral diversity, but the size of the reservoir is significantly reduced. The abnormalities of the CD4 T cells lymphocyte homeostasis remain, but the very early treatment was able to protect the TN and TCM. The five-drug therapy does not have any additional benefit, but we confirm the idea that early treatment can induce long-term virological control after the discontinuation of the treatment. Our results show that a treatment lasting more than two years would be able to reinforce the reduction of the reservoir. These results should be taken into account in the development of future trials aiming to reduce the reservoir in patients treated at the time of primary infection for a sustainable remission.

O objetivo deste estudo foi avaliar os efeitos da irradiação do canal radicular pelo laser de diodo, quando associado à terapêutica endodôntica tradicional: no aumento da temperatura na superfície radicular externa; nas alterações morfológicas da dentina do canal radicular; no selamento marginal apical após obturação do canal radicular e no potencial de redução microbiana. O aumento de temperatura foi monitorado durante a irradiação do canal radicular de incisivos inferiores por câmera termográfica. Os parâmetros de irradiação foram: grupo de emissão contínua (CW): P=2,5 W e grupo de emissão pulsada ?P=1,25 W. A avaliação das alterações morfológicas por MEV das amostras irradiadas nas condições acima citadas foram comparadas com amostras não irradiadas. O grau de infiltração marginal apical foi mensurado em amostras irradiadas (CW; P = 2,5 W) ou não irradiadas, e obturadas por um dos três cimentos endodônticos: N-Rickert, AH Plus ou Apexit. O grau de penetração linear foi medido após o período de imersão das amostras na solução de azul de metileno. O potencial de redução microbiana promovido pela associação do laser à terapêutica endodôntica tradicional foi avaliado em dentes portadores de infecção endodôntica primária por técnicas de cultura e PCR. A irrigação de NaOCl 0,5 % e uso de hidróxido de cálcio (grupo controle) foram associados à irradiação no grupo laser (CW; P=2,5 W). Foram avaliadas: a redução do número de UFC dos anaeróbios viáveis, a quantidade de E. faecalis e de lactobacilos/amostra, e a proporção de E. faecalis e de lactobacilos/microrganismos viáveis. O aumento de temperatura na superfície radicular externa durante a irradiação do canal radicular nos regimes de emissão contínuo e pulsado não excedeu o limite crítico de 10 ºC. A variação de 10 temperatura mediana máxima na região apical usando o método de Wilcoxon (p < 0,05) foi de 8,6 °C na irradiação contínua e de 3,3 °C na emissão pulsada. O tempo de resfriamento de 20 segundos foi determinado entre os 5 ciclos de irradiação. As alterações morfológicas promovidas pela irradiação do canal revelaram fusão da dentina e obliteração dos túbulos dentinários especialmente no terço apical, em ambas as condições de irradiação. A irradiação do canal radicular pelo laser de diodo reduziu significativamente o grau de infiltração marginal apical nos canais obturados pelo cimento Apexit (Kruskal Wallis p < 0,05). A prevalência dos anaeróbios viáveis ao final do tratamento foi menor no grupo laser do que no grupo controle, embora não tivesse sido constatada diferença significante entre os grupos (Mann-Whitney p > 0,05). O porcentual de lactobacilos/microrganismos viáveis no início do tratamento foi 22 % reduzindo a zero ao final, no grupo controle. Esse microrganismo não foi detectado no grupo laser. A proporção de Enterococcus/microrganismos viáveis variou de 36 %-50 % no grupo controle, e 22 %-0 % no grupo laser, no início e final do tratamento, respectivamente. Nas condições testadas, os resultados sugerem que o laser de diodo mostrouse uma ferramenta possível de ser incorporada à Endodontia, contudo os efeitos biológicos são similares à terapêutica endodôntica tradicional bem executada. / The aim of this study was to evaluate the effects of diode laser irradiation associated with endodontic therapy. The aspects analyzed were: temperature rise at the external root surface, morphological changes at the dentine root walls, apical marginal leakage after root sealing, and microbiological reduction after laser irradiation. The temperature rise in inferior incisor teeth was monitored using a thermographic camera. Two laser treatment parameters were investigated: continuous emission mode (CW) with P = 2.5 W and pulsed emission mode (PL) with average power?P = 1.25 W. The morphological changes of irradiated samples were analyzed by SEM for both treatment modes and compared with a non-irradiated control group. The apical marginal leakage was measured after the immersion in methylene blue solution for irradiated (CW; P = 2.5 W) and non-irradiated samples sealed by one of the following endodontic sealers: N-Rickert, AH Plus or Apexit. The reduction in microbiological activity after conventional endodontic therapy and after laser irradiation was evaluated in primary endodontic infection using culture and PCR techniques, where the control group used conventional 0.5 % NaOCl solution and calcium hydroxide treatment was compared with laser irradiation (CW; P = 2.5 W). The reduction of viable anaerobic microorganisms, the quantity of E. faecalis and Lactobacillus per samples, and the rate of E. faecalis and Lactobacillus per viable microorganism were analyzed. The temperature rise at external root surface during root canal irradiation in both continuous and pulsed emission modes was demonstrated not exceed the safety limit of 10 ºC. The maximum median temperature variation was 8.6 °C (CW) at the continuous emission mode and 1.6 °C at pulsed mode (PL) (Wilcoxon, p < 0.05). The optimal ?resting time? between each of five irradiation cycles was 12 determined to be 20 seconds to allow for tissue cooling and hence to prevent potential dangerous rises in the tissue temperature. SEM analysis revealed melting of dentine and closure of dentinal tubules especially at apical third for both irradiation conditions. The apical marginal leakage was significant reduced in the irradiated samples sealed with Apexit (Kruskal Wallis, p < 0.05). The prevalence of the viable anaerobic microorganisms exhibited reduced with time in both the control and laser groups with no statistical difference (Mann-Whitney p > 0.05). The Lactobacillus rate per viable microorganisms in the control group was 22 % at the beginning of the treatment and decreasing to zero at the end. This microorganism was not detected in laser group. The rate of Enterococcus per viable microorganism ranged 36 % to 50 % in the control group and 22 % to 0 % in laser group at the begin and the end of treatments, respectively. The conclusion is that diode laser technology is suitable for applications in endodontic therapy under the conditions tested in this study, however, the biological effects are similar to well done conventional endodontic therapy.

O objetivo deste estudo foi avaliar os efeitos da irradiação do canal radicular pelo laser de diodo, quando associado à terapêutica endodôntica tradicional: no aumento da temperatura na superfície radicular externa; nas alterações morfológicas da dentina do canal radicular; no selamento marginal apical após obturação do canal radicular e no potencial de redução microbiana. O aumento de temperatura foi monitorado durante a irradiação do canal radicular de incisivos inferiores por câmera termográfica. Os parâmetros de irradiação foram: grupo de emissão contínua (CW): P=2,5 W e grupo de emissão pulsada ?P=1,25 W. A avaliação das alterações morfológicas por MEV das amostras irradiadas nas condições acima citadas foram comparadas com amostras não irradiadas. O grau de infiltração marginal apical foi mensurado em amostras irradiadas (CW; P = 2,5 W) ou não irradiadas, e obturadas por um dos três cimentos endodônticos: N-Rickert, AH Plus ou Apexit. O grau de penetração linear foi medido após o período de imersão das amostras na solução de azul de metileno. O potencial de redução microbiana promovido pela associação do laser à terapêutica endodôntica tradicional foi avaliado em dentes portadores de infecção endodôntica primária por técnicas de cultura e PCR. A irrigação de NaOCl 0,5 % e uso de hidróxido de cálcio (grupo controle) foram associados à irradiação no grupo laser (CW; P=2,5 W). Foram avaliadas: a redução do número de UFC dos anaeróbios viáveis, a quantidade de E. faecalis e de lactobacilos/amostra, e a proporção de E. faecalis e de lactobacilos/microrganismos viáveis. O aumento de temperatura na superfície radicular externa durante a irradiação do canal radicular nos regimes de emissão contínuo e pulsado não excedeu o limite crítico de 10 ºC. A variação de 10 temperatura mediana máxima na região apical usando o método de Wilcoxon (p < 0,05) foi de 8,6 °C na irradiação contínua e de 3,3 °C na emissão pulsada. O tempo de resfriamento de 20 segundos foi determinado entre os 5 ciclos de irradiação. As alterações morfológicas promovidas pela irradiação do canal revelaram fusão da dentina e obliteração dos túbulos dentinários especialmente no terço apical, em ambas as condições de irradiação. A irradiação do canal radicular pelo laser de diodo reduziu significativamente o grau de infiltração marginal apical nos canais obturados pelo cimento Apexit (Kruskal Wallis p < 0,05). A prevalência dos anaeróbios viáveis ao final do tratamento foi menor no grupo laser do que no grupo controle, embora não tivesse sido constatada diferença significante entre os grupos (Mann-Whitney p > 0,05). O porcentual de lactobacilos/microrganismos viáveis no início do tratamento foi 22 % reduzindo a zero ao final, no grupo controle. Esse microrganismo não foi detectado no grupo laser. A proporção de Enterococcus/microrganismos viáveis variou de 36 %-50 % no grupo controle, e 22 %-0 % no grupo laser, no início e final do tratamento, respectivamente. Nas condições testadas, os resultados sugerem que o laser de diodo mostrouse uma ferramenta possível de ser incorporada à Endodontia, contudo os efeitos biológicos são similares à terapêutica endodôntica tradicional bem executada. / The aim of this study was to evaluate the effects of diode laser irradiation associated with endodontic therapy. The aspects analyzed were: temperature rise at the external root surface, morphological changes at the dentine root walls, apical marginal leakage after root sealing, and microbiological reduction after laser irradiation. The temperature rise in inferior incisor teeth was monitored using a thermographic camera. Two laser treatment parameters were investigated: continuous emission mode (CW) with P = 2.5 W and pulsed emission mode (PL) with average power?P = 1.25 W. The morphological changes of irradiated samples were analyzed by SEM for both treatment modes and compared with a non-irradiated control group. The apical marginal leakage was measured after the immersion in methylene blue solution for irradiated (CW; P = 2.5 W) and non-irradiated samples sealed by one of the following endodontic sealers: N-Rickert, AH Plus or Apexit. The reduction in microbiological activity after conventional endodontic therapy and after laser irradiation was evaluated in primary endodontic infection using culture and PCR techniques, where the control group used conventional 0.5 % NaOCl solution and calcium hydroxide treatment was compared with laser irradiation (CW; P = 2.5 W). The reduction of viable anaerobic microorganisms, the quantity of E. faecalis and Lactobacillus per samples, and the rate of E. faecalis and Lactobacillus per viable microorganism were analyzed. The temperature rise at external root surface during root canal irradiation in both continuous and pulsed emission modes was demonstrated not exceed the safety limit of 10 ºC. The maximum median temperature variation was 8.6 °C (CW) at the continuous emission mode and 1.6 °C at pulsed mode (PL) (Wilcoxon, p < 0.05). The optimal ?resting time? between each of five irradiation cycles was 12 determined to be 20 seconds to allow for tissue cooling and hence to prevent potential dangerous rises in the tissue temperature. SEM analysis revealed melting of dentine and closure of dentinal tubules especially at apical third for both irradiation conditions. The apical marginal leakage was significant reduced in the irradiated samples sealed with Apexit (Kruskal Wallis, p < 0.05). The prevalence of the viable anaerobic microorganisms exhibited reduced with time in both the control and laser groups with no statistical difference (Mann-Whitney p > 0.05). The Lactobacillus rate per viable microorganisms in the control group was 22 % at the beginning of the treatment and decreasing to zero at the end. This microorganism was not detected in laser group. The rate of Enterococcus per viable microorganism ranged 36 % to 50 % in the control group and 22 % to 0 % in laser group at the begin and the end of treatments, respectively. The conclusion is that diode laser technology is suitable for applications in endodontic therapy under the conditions tested in this study, however, the biological effects are similar to well done conventional endodontic therapy.

L’épidémiologie moléculaire des virus VIH-1 en France est caractérisée par une augmentation constante de la diversité virologique et de la fréquence des virus de sous-types non-B chez les patients en primo-infection. Entre 1997 et 2007, 28.4% des 591 patients suivis étaient infectés par des virus de sous-types non-B. De plus, 49 patients (8.3%) étaient infectés par des souches de sous-types différents sur les gènes pol et env, témoignant d’évènements de recombinaisons entre ces gènes. Ces virus recombinants étaient isolés à la fois chez des patients originaires d’Afrique sub-saharienne (28.3%) et des sujets caucasiens (6.3%). Ces résultats témoignent des échanges de souches virales entre les populations d'origine africaine et caucasienne, contribuant encore à augmenter la diversité virologique dans ces deux populations. Parmi 131 virus de sous-types non-B, 12.2% étaient classés comme ayant un tropisme CXCR4 par méthode génotypique, mais seulement 0.8% par méthode phénotypique, indiquant d’une part la faible proportion de virus non-B de tropisme CXCR4 en primo-infection en France, et d’autre part le manque de spécificité des méthodes génotypiques de détermination du tropisme pour ces sous-types, rendant nécessaire la mise au point d’autres algorithmes spécifiques pour ces virus.L’analyse de 987 virus isolés dans la cohorte entre 1999 et 2010 a mis en évidence que 12.7% d’entre eux étaient regroupés en "clusters" de transmission. Les patients en primo-infection contribuent donc de façon significative à la propagation de l’épidémie de VIH en France, particulièrement les hommes homo/bi-sexuels, avec une fréquence augmentant au cours de la période récente (2006-2010).La comparaison des quasi-espèces virales circulant concomitamment chez 8 patients en primo-infection (« receveurs ») et leurs 8 partenaires sexuels respectifs (« donneurs ») a révélé dans tous les cas la transmission d’un virus unique, présent de façon minoritaire parmi les sous-populations virales du donneur. La transmission virale muqueuse implique donc une sélection génétique drastique. / High genetic diversity is a major characteristics of HIV-1. In France, although subtype B strains are still predominant, the proportion of non-B viruses isolated in patients at the time of primary HIV-1 (PHI) infection increases over time. Between 1997 and 2007, 28.4% of patients were infected with non-B subtypes strains. Forty-nine viruses showed different phylogenies between the pol and env genes, indicating that recombinations have occurred in 8.3% of cases. These recombinants were isolated both in patients from Sub-Saharan Africa (28.3%) and in white subjects (6.3%).The phenotypic analysis of viral tropism of 131 non-B strains showed a very low (0.8%) proportion of CXCR4-tropic strains (X4 strains) at the time of PHI. Compared to phenotypic tests, genotypic predictions can overestimate (12.2% versus 0.8%) the proportion of X4 strains in non-B subtypes.The phylogenetic analysis of 987 strains isolated in 1999-2010 showed that 12.7% of PHI cosegregated into 56 transmission chains. PHIs are a significant source of onward transmission, especially in men having sex with men, with increasing frequency during the recent years (10.2% in 1999-2006 versus 15.2% in 2006-2010, p=0.02).The comparison of the viral quasispecies isolated in plasma and PBMC samples from 8 patients at the time of PHI ("recipients") and their transmitting partners ("donors") suggested that a severe genetic bottleneck occurrs during HIV-1 heterosexual and homosexual transmission. Indeed, we observed in all cases the transmission of a single variant, which was derived from an infrequent variant population within the blood of the donor. The proportion of X4 quasispecies in donors were higher in case of X4 versus CCR5-tropic viral transmission, suggesting that X4 transmission may be associated with a threshold of X4 circulating quasispecies in donors.