Freie Universiẗat, Diss., 2004--Berlin. / Dateiformat: zip, dateien im PDF-Format. _ Erscheinungsjahr an der Haupttitelstelle: 2004.

Treponema pallidum ssp. pallidum is the causative agent of syphilis, a multi-stage bacterial infection, transmitted sexually or from mother-to-child, with an unparalleled range of symptoms arising from the ability of treponemes to penetrate any tissue and cross immune privileged endothelial barriers to access the brain, the eye, and the fetus. Further, without treatment T. pallidum evades immune clearance and persists within the host to establish a chronic infection. These characteristics suggest that T. pallidum may have evolved unique mechanisms for immune escape and to mediate host-cell interactions. The findings presented in this dissertation contribute to our knowledge of T. pallidum pathogenesis by investigating a previously unexplored host-cell interaction, between T. pallidum and human platelets. These results validate the hypothesis that, as a pathogen which successfully utilizes vascular dissemination, T. pallidum would not only encounter, but interact with human platelets, complex cells now viewed as vascular sentinels that participate in many host-pathogen interactions. This is the first study to demonstrate that T. pallidum interacts with human platelets and to characterize and quantify these interactions using high resolution microscope imaging techniques (video and frame analysis). These interactions were shown to be complex, reversible and mediated by motile treponemes localizing to stationary, (slide-adhered) activated platelets, versus to free-floating, inactive platelets. In addition, it was found that T. pallidum discriminates between the level of platelet activation and preferentially localized to the most activated platelet. Treponema pallidum was also able to induce platelet activation following an extended lag period. Modified chemotaxis assays quantified by flow cytometry, were used to investigate the migration of T. pallidum in response to the plasma of platelets differentially activated with infection-relevant host components (thrombin, collagen). The results herein reveal that T. pallidum discriminates between different mechanisms of platelet activation, with a significant preference towards the secretions of collagen-activated platelets (under these experimental conditions), compared with that of inactive or thrombin-activated platelets. Previously, T. pallidum chemotaxis had been investigated through genomic characterization and molecular interaction studies with recombinant proteins. This investigation is the first time live T. pallidum was utilized for in vitro chemotaxis assays and is also the first study of pathogen chemotaxis in response to the secretions of differentially activated platelets. The body of work in this dissertation provides a foundation to further investigate the role of T. pallidum-platelet interactions during infection, adding a new host-cell interaction to our understanding of T. pallidum pathogenesis. The evidence that the molecular gradients of host components can affect T. pallidum migration suggests an important role for chemotaxis during T. pallidum infection. Together, the characterization of platelet-interactions and treponeme chemotaxis in response to host components, adds to our knowledge of T. pallidum-host interactions, and eludes to additional pathogenic strategies that may facilitate T. pallidum dissemination and immune evasion. / Graduate / 2022-01-14

No description available.

A dermatite digital (DD) é uma das principais doenças podais que acomete bovinos leiteiros, destacando-se negativamente pela queda na produção o volume de leite produzido e pelo seu oneroso tratamento. Como alternativa ao uso de antibióticos, a terapia fotodinâmica (PDT) se apresenta como uma opção interessante no tratamento de infecções cutâneas em animais de produção, evitando a seleção de micro-organimos resistentes. O objetivo deste estudo foi investigar o uso da PDT para tratar a DD em bovinos leiteiros. Para compor os grupos experimentais, 20 lesões de DD, localizadas nos membros pélvicos de 16 animais, foram tratadas com PDT (350mW/Led; = 660nm ± 10nm; A = 13,20 cm2; I = 120mW/cm2) associado à aplicação tópica do fotossenbilizador azul de metileno (300µM; apresentação em gel; com 5 min de tempo de pré-irradiação) ou com aplicação tópica de oxitetraciclina (500 mg em solução à 20%). Cada lesão foi tratada duas vezes com intervalo de 14 dias entre elas. A caracterização macroscópica das lesões foi realizada no momento inicial, e fotografias digitais foram realizadas semanalmente para análise da redução da área das lesões. Além disto, foram coletadas biópsias de cada lesão nos dias 1, 7, 21 e 28, sendo posteriormente analisadas para diferentes colorações histológicas, as quais objetivaram a detecção de micro-organismos e a análise comparativa do processo de cicatrização. Ainda, biópsias de pele realizadas na região dos bulbos dos talões de 10 animais que não apresentavam lesões foram usadas como controle histológico. Em ambos os tratamentos houve redução da área da lesão, sendo que não houve diferença significante entre os tratamentos. Apesar da regressão significante ao longo do período, no último dia de avaliação, três animais tratados com oxitetraciclina ainda apresentavam pequenas lesões, algo que não foi observado nos animais tratados com PDT. Adicionalmente, o tratamento com oxitetraciclina resultou em leve aumento de colágeno do tipo III e diminuição de colágeno do tipo I, enquanto a PDT promoveu aumento significante da área total de colágeno, em especial do tipo I, em relação ao momento inicial. Espiroquetas foram observadas em todas as lesões, no momento que antecedeu os tratamentos, entretanto não foram visualizadas na pele dos animais saudáveis. Ao término do período de avaliação, cinco lesões tratadas com oxitetraciclina ainda apresentavam espiroquetas, enquanto no grupo tratado com PDT não foi possível evidenciar estes micro-organismos em nenhuma das lesões. Portanto, a PDT mostrou ser uma opção mais eficaz do que a oxitetraciclina no tratamento da DD. / Bovine digital dermatitis (BDD) stands out as one of the most important diseases that affect dairy cows, being responsible for negative economic implications. Photodynamic therapy (PDT) has emerged as an interesting therapeutic option to treat superficial infections on livestock animals, avoiding the overuse of antibiotics, and consequently, the selection of multidrug-resistant pathogens. The aim of this study was to investigate the use of PDT to treat BDD. Twenty BDD lesions located in the pelvic limbs of 16 animals were treated by PDT (350mW / Led; = 660nm ± 10nm; A = 13, 20 cm2, I = 120mW/cm2) associated with topical application of methylene blue photosensitizer (300µM; gel presentation; 5 min of pre-irradiation time) or topical application of oxytetracycline (500 mg in 20% solution). Each lesion was treated twice with an interval of 14 days. Lesions were individually classified, and digital photographs were weekly performed to evaluate the reduction of lesions. Additionally, biopsies of each lesion were collected on days 1, 7, 21 and 28; and different histological stains were used to detect microorganisms and compare the healing process after treatmens. Yet, skin biopsies from 10 healthy animals, colleted in the area between heel bulbs, were used as controls. In both treatments, we observed the reduction of lesions area, but there were no significant difference between them. At the last day, three animals treated by topical oxytetracycline still presented small lesions, whereas no lesions were evidenced in the group treated by PDT. The treatment with oxytetracycline resulted in a slight increase in type III collagen and decreased type I collagen levels, while PDT treatment led to a significant increase in the total area of both collagen (mostly type I). Spirochetes were observed in all lesions before treatments but were not detected in the skin of healthy animals. At the end of evaluation period, five lesions treated by topical oxytetracycline still presented spirochetes, whereas in PDT group no spirochetes were evidenced. Therefore, PDT was shown to be a more effective option for BDD when it was compared to topical oxytetracycline treatment.

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No. of bitstreams: 2 Dissertação - Elisângela Franciscon Naves - 2018.pdf: 4013641 bytes, checksum: fcb8f472c325bd54638ccd6b1ef20093 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Individuals deprived of their liberty are at high risk for syphilis, due to sexual and non-sexual risk behaviors, such as inconsistent condom use, sex for money and / or drugs, and multiple sexual partnerships. The objective of this study was to investigate the seroprevalence of syphilis infection in deprived men in the southern region of Goiás. Between September 2017 and May 2018, a cross-sectional study was conducted on 194 men deprived of liberty from a regional prison south of the state of Goiás. All participants were interviewed about socio-demographic characteristics, sexual and non-sexual risk behaviors for syphilis. Next, blood samples were collected for the detection of anti-T. pallidum antibodies by immunochromatographic treponemal test (rapid test). Positive samples were retested by the Venereal Disease Research Laboratory / VDRL. Active syphilis was considered positive in the rapid test and titers of VDRL ≥ 1: 8. Of the 194 samples tested by immunochromatographic examination, 11.8% (95% CI: 7.8-17.3%) were positive and 7.7% (95% CI: 4.7-12.5%) presented VDRL ≥ 1: 8. The prevalence of exposure to Treponema pallidum was 23 times higher than that estimated in Brazilian conscripts (95% CI: 0.45-0.61) and 8 times higher than that found in blood donors from São Paulo (95% CI: 1.31 -1.36). In multiple regression analysis, the prevalence of exposure to T. pallidum was 36% (RPaj: 0.39; 95% CI: 0.18-0.86) in individuals who reported sexual relations with sex workers when compared to those who did not have this characteristic. In addition, it was observed that the prevalence of active syphilis increased with increasing prison time (RPaj: 1.02, 95% CI: 1.00-1.08, p-value = 0.030). The results of this investigation show a high prevalence of syphilis in the men deprived of liberty investigated and indicate the need to implement strategies for prevention and control of infection in such individuals, including educational health actions, rapid and efficient diagnosis and appropriate treatment of cases of active syphilis. / Indivíduos privados de liberdade apresentam elevado risco para sífilis, devido aos comportamentos de risco sexuais e não sexuais, como uso inconsistente do preservativo, troca de sexo por dinheiro e/ou drogas e múltiplas parcerias sexuais. O objetivo desse estudo foi investigar a soroprevalência da infecção por sífilis em homens privados de liberdade, região sul de Goiás. Entre setembro de 2017 a maio de 2018, um estudo de corte transversal foi conduzido em 194 homens privados de liberdade de um presidio regional do sul do estado de Goiás. Todos os participantes foram entrevistados sobre características sociodemograficas, comportamentos sexuais e não sexuais de risco para sífilis. A seguir, foram coletadas amostras de sangue para detecção de anticorpos anti- T. pallidum através do teste treponêmico imunocromatográfico (teste rápido). As amostras positivas foram retestadas pelo Venereal Disease Research Laboratory/Laboratório de Pesquisa de Doenças Venéreas (VDRL). Considerou-se sífilis ativa positividade no teste rápido e títulos de VDRL ≥ 1:8. Das 194 amostras testadas pelo exame imunocromatográfico, 11,8% (IC 95%: 7,8-17,3%) foram positivas e 7,7% (IC 95%: 4,7-12,5%) apresentaram títulos de VDRL ≥ 1:8. A prevalência de exposição ao Treponema pallidum foi 23 vezes superior à estimada em conscritos brasileiros (IC 95%: 0,45-0,61) e 8 vezes maior ao encontrado em doadores de sangue de São Paulo (IC 95%: 1,31-1,36). Em análise de regressão múltipla, a prevalência de exposição ao T. pallidum foi 36% (RPaj: 0,39; IC 95%: 0,18-0,86) em indivíduos que relataram relações sexuais com profissionais do sexo quando comparado aqueles que não apresentavam essa característica. Além disso, observou-se que a prevalência da sífilis ativa aumentou com o aumento do tempo de prisão (RPaj:1,02; IC 95%: 1,00-1,08; p-valor= 0,030). Os resultados dessa investigação evidenciam elevada prevalência de sífilis nos homens privados de liberdade investigados e indicam a necessidade de implementação de estratégias de prevenção e controle da infecção em tais indivíduos, incluindo ações educativas em saúde, diagnóstico rápido e eficiente além do tratamento adequado dos casos de sífilis ativa.

O diagnóstico de neurosífilis é freqüentemente dependente dos resultados dos testes serológicos e alterações no líquido cefalorraquidiano, mas a confiabilidade desses resultados em pacientes com infecção pelo HIV-1 tem sido questionada especialmente em pacientes assintomáticos com sífilis latente. O estudo se propõe avaliar a presença de DNA do T. pallidum no LCR de pacientes assintomáticos infectados pelo HIV, com o diagnóstico de sífilis. Amostras de LCR foram coletadas de 12 pacientes infectados pelo HIV atendidos em um terciário localizado no sul do Brasil , durante o período de 2012 a 2013. A presença de DNA do T. pallidum foram analisadas nas amostras de LCR pelo método de PCR “seminested”. Dados demográficos dos pacientes, parâmetros bioquímicos, celularidade e VDRL do LCR e linfócitos T-CD4 também foram analisados. Nas amostras de LCR de cinco dos 12 pacientes (40%) foram detectados o DNA do T. pallidum . Inesperadamente, nestes doentes, os níveis de contagem de células, proteína e glicose no LCR foram normais. Além disso , nenhuma destas cinco amostras de CSF apresentou uma reacção positiva VDRL. Os títulos de VDRL no soro foram semelhantes entre pacientes positivos e negativos para a presença T. pallidum DNA no LCR. A maioria dos pacientes com DNA de T. pallidum detectável apresentaram baixos títulos de VDRL no soro. O VDRL sérico elevado com título de 1:64 foi observada em apenas um paciente. Nossos resultados demostraram que os pacientes assintomáticos infectados pelo HIV com evidência de sífilis latente e LCR normais podem apresentar DNA de T. pallidum detectável no LCR. A detecção do DNA do T. pallidum pelo nosso seminested PCR pode fornecer informações adicionais além da análise convencional do LCR para o diagnóstico de neurossífilis. presença do DNA de T. pallidum no LCR em pacientes infectados pelo HIV com sífilis latente e resultados de LCR normais pode determinar uma mudança terapêutica do uso de penicilana benzatina intramuscular para o de penicilina cristalina intravenosa aquosa para o tratamento da sífilis. / Neurosyphilis diagnosis is frequently dependent upon the results of serological tests and cerebrospinal fluid abnormalities, but the reliability of findings in patients with HIV-1 infection has been questioned, especially asymptomatic patients with latent syphilis, We present the data on the presence of T. pallidum DNA in CSF from asymptomatic HIV-infected patients with the diagnosis of syphilis. CSF and serum samples were collected from 12 HIV-infected patients attending a tertiary care located in southern Brazil, during the period 2012 to 2013. In CSF samples from five of 12 patients (40%), we detected T. pallidum DNA. Unexpectedly, in these patients, CSF cell count, protein and glucose levels were normal. In addition, none of these 5 CSF samples presented a positive VDRL reaction. Serum VDRL titers were similar between patients with positive and negative CSF T. pallidum DNA. Most patients with detectable T. pallidum DNA presented low serum VDRL titers. Serum VDRL titer of 1:64 was observed in one patient. Our results have shown that asymptomatic HIV-infected patients with evidence of latent syphilis and normal CSF might present detectable T. pallidum DNA in the CSF. The detection of T. pallidum DNA by our seminested PCR provide additional information beyond conventional CSF analysis for diagnosis of neurosyphilis. The detection of T. pallidum DNA in the CSF despite normal CSF findings in HIV-infected patients could also provide a different therapeutic approach including the use of intravenous aqueous crystalline penicillin.

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 111-134).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 147-179).

O diagnóstico de neurosífilis é freqüentemente dependente dos resultados dos testes serológicos e alterações no líquido cefalorraquidiano, mas a confiabilidade desses resultados em pacientes com infecção pelo HIV-1 tem sido questionada especialmente em pacientes assintomáticos com sífilis latente. O estudo se propõe avaliar a presença de DNA do T. pallidum no LCR de pacientes assintomáticos infectados pelo HIV, com o diagnóstico de sífilis. Amostras de LCR foram coletadas de 12 pacientes infectados pelo HIV atendidos em um terciário localizado no sul do Brasil , durante o período de 2012 a 2013. A presença de DNA do T. pallidum foram analisadas nas amostras de LCR pelo método de PCR “seminested”. Dados demográficos dos pacientes, parâmetros bioquímicos, celularidade e VDRL do LCR e linfócitos T-CD4 também foram analisados. Nas amostras de LCR de cinco dos 12 pacientes (40%) foram detectados o DNA do T. pallidum . Inesperadamente, nestes doentes, os níveis de contagem de células, proteína e glicose no LCR foram normais. Além disso , nenhuma destas cinco amostras de CSF apresentou uma reacção positiva VDRL. Os títulos de VDRL no soro foram semelhantes entre pacientes positivos e negativos para a presença T. pallidum DNA no LCR. A maioria dos pacientes com DNA de T. pallidum detectável apresentaram baixos títulos de VDRL no soro. O VDRL sérico elevado com título de 1:64 foi observada em apenas um paciente. Nossos resultados demostraram que os pacientes assintomáticos infectados pelo HIV com evidência de sífilis latente e LCR normais podem apresentar DNA de T. pallidum detectável no LCR. A detecção do DNA do T. pallidum pelo nosso seminested PCR pode fornecer informações adicionais além da análise convencional do LCR para o diagnóstico de neurossífilis. presença do DNA de T. pallidum no LCR em pacientes infectados pelo HIV com sífilis latente e resultados de LCR normais pode determinar uma mudança terapêutica do uso de penicilana benzatina intramuscular para o de penicilina cristalina intravenosa aquosa para o tratamento da sífilis. / Neurosyphilis diagnosis is frequently dependent upon the results of serological tests and cerebrospinal fluid abnormalities, but the reliability of findings in patients with HIV-1 infection has been questioned, especially asymptomatic patients with latent syphilis, We present the data on the presence of T. pallidum DNA in CSF from asymptomatic HIV-infected patients with the diagnosis of syphilis. CSF and serum samples were collected from 12 HIV-infected patients attending a tertiary care located in southern Brazil, during the period 2012 to 2013. In CSF samples from five of 12 patients (40%), we detected T. pallidum DNA. Unexpectedly, in these patients, CSF cell count, protein and glucose levels were normal. In addition, none of these 5 CSF samples presented a positive VDRL reaction. Serum VDRL titers were similar between patients with positive and negative CSF T. pallidum DNA. Most patients with detectable T. pallidum DNA presented low serum VDRL titers. Serum VDRL titer of 1:64 was observed in one patient. Our results have shown that asymptomatic HIV-infected patients with evidence of latent syphilis and normal CSF might present detectable T. pallidum DNA in the CSF. The detection of T. pallidum DNA by our seminested PCR provide additional information beyond conventional CSF analysis for diagnosis of neurosyphilis. The detection of T. pallidum DNA in the CSF despite normal CSF findings in HIV-infected patients could also provide a different therapeutic approach including the use of intravenous aqueous crystalline penicillin.