Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-06-25Bitstream added on 2014-06-13T19:50:59Z : No. of bitstreams: 1 soares_mjv_me_jabo.pdf: 1304188 bytes, checksum: 544b5360dfa9669aa67bc62df0ffaa9a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O cão é um importante reservatório do agente etiológico da leishmaniose visceral (LV). Este estudo compreendeu a avaliação de aspectos clínico-laboratoriais, histopatologia renal (cap. 2) e uso da PCR na identificação do agente infectante em baço, fígado, linfonodo e rins em cães com LV (cap. 3). No primeiro estudo, foram utilizados 34 cães soropositivos sintomáticos (caso), e 17 assintomáticos (controle), machos ou fêmeas, com raças e idades variadas, provenientes de Teresina-PI. Os cães foram submetidos a eutanásia. Após, foram coletados fragmentos de baço, fígado, linfonodo e rins. Fragmentos renais foram preparados por técnicas histológicas e corados com HE e PAS. Ao exame físico, foi freqüente hipertrofia de linfonodos (85,29%) e úlceras cutâneas (35,29%) no grupo caso. Verificou-se anemia em 55,88% dos cães no grupo caso e 11,76% no controle. A avaliação histopatológica renal no grupo caso, revelou que 61,76% dos cães apresentaram glomerulonefrite (GN) membranoproliferativa e, no controle, 17,65%. GN proliferativa mesangial ocorreu em 32,35% dos cães do grupo caso e em 64,70% do controle. Foram encontradas formas amastigotas de Leishmania em meio ao infiltrado inflamatório em um dos cães do grupo caso. No segundo estudo, utilizaram-se 25 cães soropositivos para LV sintomáticos (caso) e 15 assintomáticos (controle). As PCRs foram realizadas em termociclador automático, utilizando-se primers específicos para o gênero Leishmania. Os 25 cães do grupo caso, foram positivos nas PCRs de baço, ou fígado ou linfonodo, contra dois cães do grupo controle. Por meio das PCRs em rins, detectou-se DNA de Leishmania em oito animais (32%) do grupo caso. O estudo permitiu concluir que na LV canina, independentemente de haver sinais detectados ao exame físico, os rins freqüentemente estão acometidos e que a PCR é um exame mais preciso que a histopatologia convencional, na detecção da presença do parasita. / The dog is an important reservoir of visceral leishmaniasis (VL) etiological agent. This study included clinical and laboratorial evaluation, renal histopathology (chapter 2) and the use of PCR in the identification of the infective agent on the spleen, liver, lynphonodes and kidney in dogs with VL (chapter 3). In the first study, it were used 34 symptomatic soropositives dogs (case), and 17 asymptomatic (control), male or female, with different breeds and ages, coming from Teresina-PI. The dogs were submitted to euthanasia. Right after, samples from spleen, liver, lymphonodes and kidneys were collected. Renal fragments were prepared using histological techniques and stained with HE and PAS. At physical exam, it was frequent lymphonodes hypertrophy (85.29%) and cutaneous ulcers (35,29%) in the case group. Anaemia was presented in 55.88% of the dogs from case group and 11.76% from control. The renal histopathology evaluation of the case group showed that 61.76% of the dogs presented membranoproliferative glomerulonephritis (GN) and, at the control group, 17.65%. Mesangial proliferative glomerulonephritis occurred in 32.35% of the dogs from case group and in 64.70% from control. In one of the dogs from case group, it was found amastigotes forms of Leishmania in the middle of the inflamatory infiltrated. In the second study, it were used 25 symptomatics soropositives dogs for VL (case) and 15 asymptomatic (control). PCRs were accomplished at an automatic termocycler using specific primers for Leishmania. All 25 dogs from case group were positives in PCRs of spleen, or liver or lymphonode, and only two from the control group. Through the PCRs of the kidneys, it was detected DNA of Leishmania in eight animals (32%) from case group... (complete abstract, access undermentioned eletronic address)

Made available in DSpace on 2016-09-27T13:39:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-04. Added 1 bitstream(s) on 2016-09-27T13:45:08Z : No. of bitstreams: 1 000870645_20170104.pdf: 242400 bytes, checksum: 9d69e8bb5f77471c03c024ed5ca56917 (MD5) Bitstreams deleted on 2017-01-06T13:21:19Z: 000870645_20170104.pdf,. Added 1 bitstream(s) on 2017-01-06T13:22:02Z : No. of bitstreams: 1 000870645.pdf: 525092 bytes, checksum: 20064957cf347be120a7f3cbbb1f7137 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dogs infected with Leishmania infantum showed a reduction in the number of lymphocytes T. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family, it is expressed by cells of the immune system and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces deactivation of T cells or apoptosis. This study aimed to evaluate the PD-1 expression and its ligands as well as their role in T lymphocyte induction of apoptosis, TNF-α and IL-4 secretion, nitric oxide production and parasite load in dogs with visceral leishmaniasis. We observed that in canine visceral leishmaniasis PD-1 and its ligands involved in induction of apoptosis of T lymphocytes, are regulating the nitric oxide production, TNF-α, IL-4, as well as the parasitic load. This study helps to clarify the mechanism of immune response and immunotherapeutic drugs such as blocking monoclonal antibodies that can influence the LVC / FAPESP: 2013/066849

Orientador: Valéria Marçal Felix de Lima / Resumo: PD-1 ligado a PD-L1 ou PD-L2, desencadeia sinais incapacitantes às células T, podendo estar envolvidos na supressão das respostas imunes na LVC. Investigamos a expressão de PD-1 e seus ligantes no baço na LVC, e sua correlação com a carga parasitária. Para confirmar o seu papel regulador na leishmaniose, estes receptores foram avaliados após infecção in vitro por L. infantum. Em cães infectados, foi avaliado o bloqueio destes receptores na produção de citocinas e seus efeitos no sobrenadante da cultura, de linfoproliferação com antígeno solúvel (AtgS) de L. infantum. Foram selecionados 23 cães saudáveis e 54 com LV. PD-1 e seus ligantes foram determinados por imunohistoquímica em tecidos esplênicos. A carga parasitária de cães infectados foi avaliada por PCR em tempo real. As citocinas nos sobrenadantes de cultura foram determinados por ELISA de captura e o NO por Griess. A Linfoproliferação das células do linfonodo e expressão de PD-1 e seus ligantes após infecção in vitro por L. infantum foram avaliadas por citometria de fluxo. Observou-se que a infecção por L. infantum, aumenta a expressão de PD1 e seus ligantes. Uma correlação positiva foi observada entre a expressão de PD-1 e PD-L1 em linfócitos B e o aumento da carga parasitária. O bloqueio destes receptores restaura a proliferação de linfócitos, aumenta a produção de NO e diminui a IL-4 e IL-10. L. infantum modula a expressão de PD-1 e seus ligantes na LVC, suprimi a linfoproliferação, alterando a produção de citocinas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: PD-1 when bound to PD-L1 or PD-L2, triggers signals disabling T cells, and they may be involved in the suppression of cellular immune responses in CVL. We investigated the expression of PD-1 and its ligands in spleen in CVL, and its correlation with parasite burden in this tissue, to confirm its regulatory role in leishmaniasis. These receptors were evaluated after in vitro infection by L. infantum. In infected dogs, we evaluated the blocking of these receptors in the production of cytokines in the effects of supernatant of cultured cells, and antigen specific lymphoproliferation. We selected 23 healthy dogs and 54 with VL. PD-1 and its ligands were determined by immunohistochemistry in splenic tissues. Parasitic load of infected dogs was evaluated by RT-PCR. Cytokines in culture supernatants were determined by capture ELISA and NO by Griess. Lymphoproliferation lymph nodes and expression of PD-1 and its ligand after in vitro infection by L. infantum was evaluated by flow cytometry. We observed that L. infantum infection increases expression of PD-1 and its ligands. A positive correlation was observed between the expression of PD-1 and PD-L1 B lymphocytes and an increase in parasite load. Blockade these receptors restores lymphocyte proliferation, increases the production of NO and decreases IL-4 and IL-10. In conclusion, L. infantum modulates expression of PD-1 and its ligands in CVL, suppressing lymphoproliferative response and altering cytokine production, which in turn ca... (Complete abstract click electronic access below) / Doutor

Resumo:Cães infectados com Leishmania infantum, apresentam uma redução do número de linfócitos T. PD-1(programmed cell death 1) um novo membro da família B7-CD28, é expresso por células do sistema imune e sua ligação a PD-L1 (CD274) ou PD-L2 (CD273) induz à desativação dos linfócitos T ou à apoptose. O presente estudo teve como objetivo avaliar a expressão de PD-1 e seus ligantes bem como sua função na indução de apoptose de linfócitos T, secreção de TNF-α e IL-4, óxido nítrico e na carga parasitária em cães com leishmaniose visceral. Observamos que na leishmaniose visceral canina PD-1 e seus ligantes participam na indução da apoptose celular de linfócitos T, estão regulando a produção de óxido nítrico, TNF-α, IL-4, além da carga parasitária. Esse estudo ajuda a esclarecer o mecanismo da resposta imunológica e drogas imunoterapeuticas como anticorpos monoclonais bloqueadores que podem influenciar na LVC / Abstract:Dogs infected with Leishmania infantum showed a reduction in the number of lymphocytes T. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family, it is expressed by cells of the immune system and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces deactivation of T cells or apoptosis. This study aimed to evaluate the PD-1 expression and its ligands as well as their role in T lymphocyte induction of apoptosis, TNF-α and IL-4 secretion, nitric oxide production and parasite load in dogs with visceral leishmaniasis. We observed that in canine visceral leishmaniasis PD-1 and its ligands involved in induction of apoptosis of T lymphocytes, are regulating the nitric oxide production, TNF-α, IL-4, as well as the parasitic load. This study helps to clarify the mechanism of immune response and immunotherapeutic drugs such as blocking monoclonal antibodies that can influence the LVC / Orientador:Valéria Marçal Felix de Lima / Banca:Alexandra Ivo de Medeiros / Banca:Caris Maroni Nunes / Mestre

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2015. / Submitted by Ana Cristina Barbosa da Silva (annabds@hotmail.com) on 2015-04-06T15:45:47Z No. of bitstreams: 1 2015_LucianaRibeiroCamargos.pdf: 1062457 bytes, checksum: 99c86f7162abaa43b4510ea18e316de1 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2015-04-14T20:34:30Z (GMT) No. of bitstreams: 1 2015_LucianaRibeiroCamargos.pdf: 1062457 bytes, checksum: 99c86f7162abaa43b4510ea18e316de1 (MD5) / Made available in DSpace on 2015-04-14T20:34:30Z (GMT). No. of bitstreams: 1 2015_LucianaRibeiroCamargos.pdf: 1062457 bytes, checksum: 99c86f7162abaa43b4510ea18e316de1 (MD5) / A Leishmaniose Visceral Canina é uma doença que acomete, dentre outros órgãos, a pele. Esse estudo teve como objetivo quantificar as alterações morfológicas e histológicas presentes nas várias apresentações cutâneas de cães naturalmente infectados. Para isso foram avaliados 31 fragmentos de pele de 20 animais com dermatite esfoliativa seca, alopecia, hipotricose, crosta, nódulo, úlcera, eritema, despigmentação, hiperpigmentação e/ou hiperqueratose. Foram determinados o padrão microscópico inflamatório e não inflamatório de cada lesão, e realizada a mensuração da intensidade inflamatória, parasitológica e de colagenólise, e a histomorfometria por meio da detecção e diferenciação dos colágenos do tipo I e III. As regiões acometidas foram cabeça (40,35%), membros (21,05%), tórax (15,8%), difusa (8,77%), abdômen (5,26%), pelve (5,26%) e cervical (3,51%), sendo a dermatite crostosa e a hipotricose as macroleões mais encontradas. Houve o predomínio de infiltrado histiocítico e a maioria dos fragmentos apresentaram intensidade inflamatória acentuada, sendo a inflamação visualizada principalmente em região peri/intra-anexo. A forma amastigota foi visualizada em 48,38% dos casos. Colagenólise, crostas e hiperqueratose ortoqueratótica foram as lesões não inflamatórias mais encontradas. Houve predominância do colágeno maduro (60,31% + 2,906) em relação ao imaturo (8,720% + 1,187) em região de derme e esses dados foram estatisticamente significativos. Houve um discreto aumento de colágeno do tipo III e diminuição do colágeno do tipo I, ao se comparar amostras com e sem presença de amastigotas. Concluiu-se que a presença do infiltrado inflamatório em cães com LVC no DF, contribuiu para as mudanças ocorridas na matriz extracelular. / The Canine Visceral Leishmaniasis is a disease that affects, among other organs, the skin. This study aimed to quantify the morphological and histological changes present in various skin samples from naturally infected dogs. To achieve the proposed objective were evaluated 31 skin flakes of 20 animals with dry exfoliative dermatitis, alopecia, hypotrichosis, crust, nodules, ulcers, erythema, depigmentation, hyperpigmentation and/or hyperkeratosis. Inflammatory and non-inflammatory microscopic pattern of each lesion, the measurement of inflammatory intensity, parasitological and collagenolysis, and histomorphometry through the detection and differentiation of type I and III collagens were performed. The regions affected were head (40.35%), members (21.05%), chest (15.8%), diffuse (8.77%), abdomen (5.26%), pelvis (5.26%) and cervical (3.51%), the crusted dermatitis and hypotrichosis large lesions were the most frequent injuries. There was a predominance of histiocytic infiltrate and most of the fragments showed severe inflammatory intensity, with inflammation viewed mainly in peri/intra-annexed region. The amastigotes were seen in 48.38% of cases. Collagenolysis, crusts and orthokeratotic hyperkeratosis were the most frequent non-inflammatory lesions. There was a predominance of mature collagen (60.31% + 2.906) compared to immature (8,720 + 1,187%) in the dermis region and these data were statistically significant. There was a slight increase in collagen type III and decreased collagen type I, when comparing samples with and without the presence of amastigotes. It was concluded that the presence of inflammatory infiltrate in dogs with CVL in DF contributed to the changes in the extracellular matrix.

Orientadora: Iêda Maria Begnini. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Exatas e Naturais, Programa de Pós-Graduação em Química.

Orientador: Ieda Maria Begnini. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Exatas e Naturais, Programa de Pós-Graduação em Química.

Orientador: Ricardo Andrade Rebelo. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Exatas e Naturais, Programa de Pós-Graduação em Química.

Despite huge suffering caused by cutaneous leishmaniasis (CL), there is no effective and affordable treatment strategy against CL and no licensed vaccines. The current treatments show limited efficacy and high toxicity. Improved therapies through discovery of novel drugs and/or an alternative treatment approaches are/is urgently needed. We aimed at identifying a novel antileishmanial agent and developing an innovative nanoparticle (NP) based platform for safe and effective treatments against CL. We discovered that polyhexanide (PHMB), a widely used antimicrobial polymer and wound antisepsis, shows an inherent antileishmanial activity at submicromolar concentrations. PHMB appears to kill L. major parasites via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation. However, host chromosomes binding appear to be limited by exclusion from mammalian cell nuclei. Moreover, we attempted to establish effective drug delivery systems that overcome the various shortcomings in the present treatment of CL. In this scenario, we initially studied the cellular interactions of NPs and their uptake mechanisms into mammalian cells before applying them in drug delivery system. We obtained clear evidence for the involvement of multiple endocytic routes to internalize NPs. Physicochemical properties of NPs, cell type, temperature and pathogenesis of the target diseases were shown to be determinant factors. Thereafter, a mechanism based host- and pathogen-directed combination therapy comprising PHMB and CpG ODN immunomodulator was established for overall synergistic effect against CL. It simultaneously targets the pathogen and the host immunity with effective delivery system. The results show that PHMB binds to CpG ODN and form stable nanopolyplexes for efficient cell entry and therapy. The nanopolyplexes displayed enhanced cellular uptake and antileishmanial potency while drastically reducing the toxicity against mammalian cells. In conclusion, our findings clearly indicate that PHMB can be used as effective candidate drug against CL and as non-viral delivery of immunomodulatorynucleic acids. Moreover, our proof-of concept study showed nanomedicine approaches are effective strategy to challenge CL and other human diseases. / Obgleich enorme Leiden mit der kutanen Leishmaniose einhergehen stehen bis dato keine wirkungsvollen und erschwinglichen Therapien oder zugelassene Impfstoffe zur Verfügung. Die derzeitigen Behandlungsmethoden sind kaum effektiv und zeichnen sich vor allem durch ihre enormen Nebenwirkungen aus. Aus diesem Grund ist die Erforschung neuartiger Wirkstoffe und Therapieansätze gegen kutane Leishmaniose zwingend notwendig. Die vorliegende Arbeit beschreibt die Entdeckung eines neuen antileishmanialen Wirkstoffes und die Etablierung eines innovativen und auf Nanopartikeln basierenden Verfahrens zur sicheren und effizienten Behandlung der kutanen Leishmaniose. Das Polyhexanid, welches bereits Verwendung als antimikrobielles Polymer und als Wundantiseptikum findet, weist bereits in submikromolaren Konzentrationen eine immanente antileishmaniale Wirkung auf. Den Beobachtungen zu Folge beeinflusst das Polyhexanid die Integrität der parasitären Zellmembran und führt zur selektiven Chromosomenkondensation des Parasiten Leishmania major. Eine potentielle Chromosomenmodifikation in der Säugetierzelle wird durch den Ausschluss des Polyhexanides aus dem Zellkern verhindert. Um die zahlreichen Mängel der aktuellen Behandlungsmethoden gegen kutane Leishmaniose zu überwinden, wurde zudem ein effizientes System der Wirkstoffabgabe etabliert. Diesbezüglich wurden zunächst die zellulären Wechselwirkungen der Nanopartikel und deren Aufnahme in die Säugtierzelle untersucht ehe diese als Vehikel für den Wirkstoff verwendet wurden. Es konnte gezeigt werden, dass die Nanopartikel über mehrere endozytische Wege internalisiert werden. Physikochemische Eigenschaften der Nanopartikel, der Zelltyp, die Temperatur und erregerspezifische Pathogenese gehören zu den beeinflussenden Faktoren. Daraufhin wurde eine Kombinationstherapie bestehend aus Polyhexaniden und dem unmethylierten Immunmodulator Zystein-Phosphat-Guanin Oligodeoxynukleotid mit synergistischen antileishmanialen Auswirkungen, etabliert. Dies gestattet eine gegen den Erreger zielgerichtete Behandlung und die zeitgleiche Stimulierung der Wirtsimmunität. Die Bildung eines stabilen Nanopolyplexes bestehend aus dem Polyhexanid und dem oben genannten Immunmodulator befähigen die effiziente Aufnahme in die Zelle und somit die Behandlung. Der Nanopolyplex ermöglicht eine verbesserte Aufnahme in die Zelle und antileishmaniale Wirksamkeit wohingegen die Toxizität gegenüber Säugetierzellen drastisch reduziert ist. Zusammenfassend lässt sich feststellen, dass Polyhexanide als effizienter Wirkstoffkandidat gegen kutane Leishmaniose und als nicht-viraler Träger von immunmodulatorischen Nukleinsäuren zu betrachten sind. Zugleich wurde gezeigt, dass die Nanomedizin einen wertvollen Beitrag zur Bekämpfung der kutanen Leishmaniose und sicherlich auch anderer Krankheitserregern leisten kann.

Universiẗat, Diss., 2005--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2004.