Tesis sobre el tema "Leishmaniasis"

La leishmaniasis es una infección parasitaria que afecta a humanos, animales domésticos y silvestres. Es causada por los miembros del género Leishmania que realizan parte de su ciclo biológico en un hospedador vertebrado, en forma aflagelar o amastigote. Y completan su desarrollo en el tubo digestivo del hospedador invertebrado, en su forma flagelar o promastigote, estos son flebótomos del género Phlebotomus en el Viejo Mundo y Lutzomyia en el Nuevo Mundo (Miró, 2007b; Baneth, 2006, 2007). En varios países de América el principal vector de leishmaniasis visceral es Lutzomyia longipalpis, su distribución abarca desde el sur de México hasta el norte de Argentina y es capturada dentro y fuera de las viviendas humanas (Milleron et al., 2004; Lainson y Rangel, 2005; González et al., 2006; Ramírez et al., 2006; Dantas, 2008; Diniz et al., 2008). La leishmaniasis está distribuida en el sur de Europa, África, Asia, América del sur, centro y recientemente en Estados Unidos y Canadá (Baneth, 2007). En el mundo hay 14 millones de personas infectadas y cada año se registran 2 millones de casos nuevos. De ellos 500,000 viscerales, que provoca más de 50,000 defunciones; y 1´500,000 casos cutáneos. La población en riesgo se eleva a 350 millones de personas y sólo en 33 de los 88 países endémicos la leishmaniasis es una enfermedad de notificación obligatoria (OMS, 2007). Los cambios ecológicos, demográficos y medioambientales relacionados con nuevos proyectos de desarrollo, urbanización y grandes movimientos de población están conduciendo a un aumento a escala mundial de consecuencias sanitarias adversas donde el desarrollo del flebótomo vector se ve favorecido, de tal manera que la aparición de casos parece estar relacionada con la continua deforestación y la expansión urbana, que se ha intensificado en los últimos años. Posicionándose como un problema de salud pública cada vez más acusado en muchas regiones de Latinoamérica, especialmente en Brasil, Colombia y Venezuela, donde anteriormente no se encontraba (Desjeux, 2002; Rondón, 2006; ENY-740S, 2007; Sousa y Pearson, 2009). El rol del perro (Canis familiaris), como reservorio en la transmisión doméstica y peri doméstica de la leishmaniasis humana ha sido reconocida desde que Charles Nicoles, el ganador al premio Nobel, descubrió la enfermedad en perros en Tunisia en 1908. El número de perros infectados en Sud América es estimado en millones con una alta tasa de infección en algunas áreas de Brasil y Venezuela (Killick-Kendrick, 1999; Baneth, 2006, 2007; Sousa y Pearson, 2009). La leishmaniasis canina es una enfermedad zoonótica que existe en cerca de 50 de los 88 países donde la leishmaniasis humana está presente y resulta frecuentemente mortal en humanos y perros no tratados (Baneth, 2006; Coura et al., 2006). En la población canina afectada los signos clínicos son muy variables, podemos observar desde animales aparentemente sanos, hasta otros que manifiestan varios signos clínicos. Esto se debe a la complejidad de los mecanismos patogénicos dependientes del parásito y a la marcada individualidad de la respuesta inmunitaria del hospedador (Miró, 2007b). Los términos que utilizaremos como abreviación para leishmaniasis canina será Lcan, en esta especie la piel se ve afectada en el transcurso de la diseminación de la enfermedad a los órganos internos, manifestándose ambas formas de la enfermedad a la vez (Killick-Kendrick, 1999; Baneth, 2006; Miró, 2007b; OIE, 2008). Para designar la enfermedad según sus manifestaciones clínicas en humanos, usaremos las siguientes abreviaciones: leishmaniasis cutánea (LC), leishmaniasis mucocutánea (LMC) y leishmaniasis visceral (LV) (Killick-Kendrick, 1999; Baneth, 2006). La presente monografía pretende ser un documento actualizado donde se revisa la enfermedad en todos sus aspectos, para aumentar los conocimientos existentes sobre esta zoonosis parasitaria de creciente interés en los últimos veinte años.
Tesis

The proliferation dynamics in vitro of the obligate intracellular protozoan parasite Leishmania donovani was studied for 14 days in resting monolayers of peritoneal macrophages of C57BL/6 $(Lsh sp{ rm s})$ mice inoculated with 5, 50, or 500 promastigotes/cell. Irrespective of the inoculum, only 50 to 65% of the cells became infected initially; only 3 to 6 amastigotes were present in each macrophage initially, suggesting a limited number of parasite ligands on the host cell. The amastigotes did not divide in the first 3 or 4 days after infection and then they actively proliferated from day 5 to day 8; the number of parasites was then reduced. Infection with L. donovani down-regulates immunity and parasite clearance by macrophages, but IL-2-stimulated splenocytes activate leishmanicidal action in vitro in infected peritoneal macrophages and in vivo in C57BL/6 (Lsh$ sp{ rm s})$ mice. IL-2-stimulated splenocytes were most effective when used in the non-proliferating phase of the infection, whereas the anti-leishmanial drug Pentostam was most effective when used during the proliferative phase. Immunochemotherapy was more effective than either treatment alone. Infection with L. donovani abolishes the ability of macrophages to produce the superoxide and INO microbicidal responses; curative treatment with IL-2-stimulated splenocytes restores the ability of infected macrophages to secrete inorganic nitrogen oxides, but not to produce a superoxide response. Pentostam had no effect to stimulate either microbicidal mechanism in infected cells; the drug is, therefore, probably directly toxic to the parasite. These studies have indicated, among other things, that IL-2-stimulated splenocytes rescue infected cells and infected animal from the immunological deficit which L. donovani induces, allowing the re-establishment of those mechanisms that make the macrophage an essential component of the host's protective immune system.

Zoonotic visceral leishmaniasis (ZVL) is a fatal disease caused by the sandfly-borne intracellular protozoan parasite Leishmania infantum, and vaccine development in the reservoir host (the domestic dog) is a current research priority. The aims of this study were (1) to conduct safety and immunogenicity trials of two candidate vaccines in dogs, and (2) to compare and demonstrate the utility of immunological and molecular tools for measurement of vaccine efficacy in naturally exposed dogs. DNA/ modified vaccinia virus Ankara (MVA) prime/boost canine vaccines expressing the Leishmania proteins TRYP and LACK were safe, and elicited a type-1 cytokine response, in vivo delayed-type hypersensitivity and IgG2 class responses, consistent with superior protective immunogenicity of TRYP over LACK. However, inconsistent associations were found between progressive disease in infected dogs and IgG class levels, prompting caution in use of the latter as a proxy for protective immunogenicity. Specific serological responses in vaccinated dogs did not cross-react with an unrelated diagnostic antigen rK39, and responses to crude parasite antigen (CLA) were minimal, enabling serological detection of infection incidence in vaccinated dogs. Particularly in early stage infection, CLA ELISA was more sensitive than rK39 ELISA and an rK39-based rapid diagnostic test, though rK39 serology was sensitive for diagnosis of symptomatic clinical cases. A commercially available PCR kit incorporating a rapid oligochromatographic detection step was tested for the first time in dogs, and proved highly sensitive for detection of ZVL infection in bone marrow, comparable to existing nested PCR methods. Molecular methods were investigated as proxy measures to replace labour-intensive xenodiagnosis for detection of the infectiousness of dogs to biting sand flies. Conventional and real-time PCR of tissues from naturally infected dogs were sensitive tests to identify infectiousness, but showed low to moderate specificity. Recommendations are made to improve the application of molecular methods as proxy measures of infectiousness and hence vaccine efficacy.

In Bihar state, India, the cure rate of antimonial compounds in the treatment of visceral leishmaniasis (VL) has declined from over 85% to less than 50%. This has been attributed to prolonged, widespread misuse of antimonials within the Indian private healthcare system. An alternative resistance hypothesis is that exposure to arsenic in drinking water in this region has resulted in antimony-resistant Leishmania parasites. Leishmania donovani were serially passaged in mice exposed to environmentally-relevant levels of arsenic in drinking water. Arsenic accumulation in organs of these mice was proportional to exposure. After five passages, isolated parasites were refractory to SbV in drug sensitivity assays. Treatment of infected mice with SbV confirmed that these parasites retained resistance in vivo, supporting this hypothesis. A retrospective field study on a cohort of antimony treated VL patients was performed in an arsenic contaminated area of Bihar to evaluate the presence of an increased risk of treatment failure and death in those exposed to arsenic. It demonstrated a significant increased risk of death from VL in arsenic exposed patients but did not indicate a significant relationship between arsenic exposure and antimonial treatment failure. Collectively these data suggest that it is biochemically possible that arsenic contamination may have contributed to the development of antimonial resistance in Bihar although issues of underpower and the retrospective nature of our epidemiological study made it difficult to conclusively demonstrate this. Further research in to the relationships between arsenic exposure and antimonial treatment failure and death in the leishmaniases is warranted.

Les leishmaniosis són un grup de malalties causades per protozous del gènere Leishmania que es transmeten per vectors. La leishmaniosi visceral (LV) humana pot ser mortal si no es tracta, resultant en 26 000-65 000 morts a l’any. Els cànids són el principal reservori i hostes de Leishmania infantum, l’agent causant de la LV zoonòtica a la conca mediterrània. Es desconeixen els mecanismes que regulen el resultat final de la infecció, però és sabut que el sistema immunitari juga un paper clau en el control de la malaltia. Diversos estudis han demostrat que la vitamina D té un rol important en la resposta immune, activant el sistema immunitari innat i modulant la resposta adaptativa. A més, s’ha descrit la relació entre la deficiència de vitamina D i el risc de patir diverses malalties. L’objectiu de la tesis va ser estudiar si la vitamina D té una contribució rellevant en la leishmaniosi canina (LCan). Per això, es va mesurar la concentració de vitamina D en mostres de sèrum d’una població de gossos sans i malalts residents en zona altament endèmica i se’n va estudiar la relació amb paràmetres parasitològics i immunològics. Els gossos malalts presentaven nivells de vitamina D significativament més baixos que els no infectats i que els infectats asimptomàtics. A més, la deficiència de vitamina D es correlacionava amb paràmetres relacionats amb la progressió de la LCan. També vam investigar si variacions genètiques en el locus del gen del receptor de la vitamina D s’associa amb la progressió de la LCan, però les freqüències al·lèliques dels polimorfismes (SNPs) trobats no van resultar ser estadísticament diferents entre grups. Llavors, vam analitzar la concentració de vitamina D en mostres de sèrum preses en diferents períodes de l’any en una cohort de gossos sans. Els resultats van mostrar que no hi ha variació estacional dels nivells de vitamina D en gossos. També es va analitzar retrospectivament la concentració de vitamina D en gossos amb leishmaniosi clínica i gossos no-infectats a l’inici de l’estudi, quan tots els animals eren negatius a Leishmania, i un any després. Mentre que els gossos sans no van mostrar canvis en els nivells de vitamina D durant l’estudi, els que van desenvolupar leishmaniosi van mostrar una reducció significativa al final de l’estudi. Per tant, la concentració de vitamina D no és un factor de risc per desenvolupar LCan, sinó que disminueix amb el curs de la malaltia. Un model in vitro va demostrar que afegir vitamina D activa (1,25(OH)2D3) comporta una reducció significativa de la càrrega de L. infantum en macròfags canins. Analitzant l’expressió de gens relacionats amb la via de la vitamina D en monòcits canins primaris vam demostrar que l’expressió de la β-defensina CBD103 augmenta significativament amb l’addició de 1,25(OH)2D3. Els resultats van corroborar que la vitamina D juga un paper en el control del paràsit. Per últim, es va estudiar la viabilitat de la vitamina D com a adjuvant per potenciar l’efecte d’una vacuna enfront la leishmaniosi. Es va administrar vitamina D conjuntament amb una vacuna d’ADN encapsulada en liposomes a ratolins BALB/c. Dues setmanes després de la vacunació els animals van ser infectats amb L. infantum. Es va mesurar la càrrega parasitària en òrgans diana i es va avaluar la resposta immune abans de la infecció i sis setmanes després. La vacuna no va reduir significativament la càrrega parasitària, però amb la co-administració de vitamina D es va apreciar una tendència a disminuir-la. L’estudi de la resposta immunològica va suggerir que l’augment de limfòcits T CD4+ i CD8+ podrien haver contribuït en la protecció parcial aconseguida per la vacuna amb la vitamina D com a potenciador.
Las leishmaniosis son un grupo de enfermedades causadas por protozoos del género Leishmania que se transmiten por vectores. La leishmaniosi visceral (LV) humana puede ser mortal si no se trata, resultando en 26 000-65 000 muertes por año. Los cánidos son el principal reservorio y huéspedes de Leishmania infantum, el agente causante de la LV zoonótica en la cuenca mediterránea. Se desconoce el mecanismo que regula el resultado final de la infección, pero se sabe que el sistema inmunitario juega un papel clave en el control de la enfermedad. Varios estudios han demostrado que la vitamina D tiene un rol importante en la respuesta inmune, activando el sistema inmunitario innato y modulando la respuesta adaptativa. Además, se ha descrito la relación entre la deficiencia de vitamina D y el riesgo de sufrir algunas enfermedades. El objetivo de la tesis fue estudiar si la vitamina D tiene una contribución relevante en la leishmaniosis canina (LCan). Para ello se determinó la concentración de vitamina D en muestras de suero de una población de perros sanos y enfermos de leishmaniosis residentes en una zona altamente endémica y se estudió la relación de ésta con parámetros parasitológicos e inmunológicos. Los perros enfermos mostraron niveles de vitamina D significativamente más bajos que los no infectados y que los infectados asintomáticos. Además, la deficiencia de vitamina D se correlacionó con parámetros relacionados con la progresión de la enfermedad. También investigamos si las variaciones genéticas en el locus del gen del receptor de la vitamina D se asocia con la progresión de LCan, pero las frecuencias alélicas de los polimorfismos (SNPs) encontrados no resultaron ser estadísticamente diferentes entre grupos. Posteriormente se analizó la concentración de vitamina D en muestras de suero tomadas en diferentes periodos del año en una cohorte de perros sanos. Los resultados mostraron que no hay una variación estacional de los niveles de vitamina D en perros. También se analizó retrospectivamente la concentración de vitamina D en perros con leishmaniosis clínica y perros no infectados al inicio del estudio, cuando todos los animales eran negativos a Leishmania, y un año después. Mientras que los perros sanos no mostraron cambios en los niveles de vitamina D durante el estudio, los que desarrollaron leishmaniosis mostraron una reducción significativa al final del estudio. Por lo tanto, la concentración de vitamina D no es un factor de riesgo para desarrollar LCan, sino que disminuye con el curso de la enfermedad. Un modelo in vitro demostró que añadir vitamina D activa (1,25(OH)2D3) conlleva una reducción significativa de la carga de L. infantum en macrófagos caninos. Analizando la expresión de genes relacionados con la vía de la vitamina D en monocitos caninos primarios demostramos que la expresión de la β-defensina CBD103 aumenta significativamente con la adición de 1,25(OH)2D3. Los resultados corroboraron que la vitamina D juega un papel en el control del parásito. Por últimos, se estudió la viabilidad de la vitamina D como adyuvante para potenciar el efecto de una vacuna frente la leishmaniosis. Se administró vitamina D junto a una vacuna de ADN encapsulada en liposomas a ratones BALB/c. Dos semanas después de la vacunación los animales se infectaron con L. infantum. Se determinó la carga parasitaria en órganos diana y se evaluó la respuesta inmune antes de la infección y seis semanas después. La vacuna no redujo significativamente la carga parasitaria, pero con la coadministración de vitamina D se apreció una tendencia a reducirla. El estudio de la respuesta inmunológica sugirió que el aumento de linfocitos T CD4+ y CD8+ podrían haber contribuido a la protección parcial conseguida cuando se administró vitamina D como potenciador junto a la vacuna.
Leishmaniasis are a group of neglected vector-borne diseases caused by obligate intracellular protozoan parasites of the genus Leishmania. Human visceral leishmaniasis (VL) can be fatal if left untreated, resulting in 26 000-65 000 deaths per year. Canids are the main reservoir and hosts of L. infantum, the causative agent of zoonotic VL in the Mediterranean Basin. The mechanisms that regulate the outcome of the infection are undisclosed, although it is well known that immune system plays a key role in leishmaniasis disease control. Several studies have shown that vitamin D plays an important immunomodulatory role by activating innate immune system and modulating the adaptive immune response. Furthermore, the relationship between vitamin D deficiency and the risk of suffering from a plethora of health disorders has been described. The aim of the thesis was to study if vitamin D have a relevant contribution in canine leishmaniasis (CanL). Because of that, we measured vitamin D concentration in serum samples from a cohort of healthy and ill dogs from a highly endemic area and we have also studied the relationship of vitamin D concentration with parasitological and immunological parameters. The sick dogs presented significantly lower vitamin D levels than their non-infected and the asymptomatic counterparts. In addition, vitamin D deficiency correlated with several parameters linked to leishmaniasis progression. We also aimed to investigate whether genetic variation within the vitamin D receptor gene locus is associated with the progression of CanL, but the allelic frequencies of the four single nucleotide polymorphisms (SNPs) found were not statistically different between groups. Afterwards, we analysed retrospectively vitamin D concentration in serum samples from a cohort of healthy dogs collected in different periods of the year. The results showed that there is not a seasonal variation of vitamin D concentration in dogs. We also analysed retrospectively vitamin D concentration in serum samples from dogs with clinical leishmaniasis and non-infected healthy dogs, in which we measured vitamin D levels at the beginning of the study, when all dogs were negative for Leishmania, and 1 year later. Whereas non-infected dogs showed no changes in vitamin D levels along the study, those developing clinical leishmaniasis showed a significant vitamin D reduction at the end of the study. Therefore, vitamin D concentration is not a risk factor for developing canine leishmaniasis, but it diminishes with the onset of clinical disease. An in vitro model using a canine macrophage cell line proved that adding active vitamin D (1,25(OH)2D3) leads to a significant reduction in L. infantum load. Analyzing expression of genes related to vitamin D pathway on primary canine monocytes, we showed that defensin CBD103 expression was significantly enhanced after active vitamin D addition. The in vitro results corroborated that vitamin D plays a role in parasitic control. Finally, we studied the suitability of vitamin D as an adjuvant to enhance the effect of a DNA vaccine against VL. BALB/c mice were treated with vitamin D concomitantly with a DNA vaccine encapsulated in liposomes. Two weeks after vaccination, the animals were infected with L. infantum parasites. Parasite load was measured in target tissues and immune response was evaluated before challenge and six weeks post-infection. Our DNA vaccine did not significantly reduce parasite load in liver nor spleen, but vitamin D coadministration showed a tendency to diminish parasite load in target organs. The study of cell response in splenocytes suggested that higher levels of CD4+ and CD8+ T cells may be responsible for the partial protection mediated by the DNA vaccine with vitamin D as enhancer.
Universitat Autònoma de Barcelona. Programa de Doctorat en Farmacologia

Leishmaniasis is a group of diseases caused by an obligate protozoan Leishmania and transmitted by sand flies. As a neglected tropical disease (NTD), leishmaniasis disproportionately affects the poorest populations and those living in rural, remote areas or conflict zones with limited or no access to health care. Manifesting in cutaneous, mucocutaneous or visceral symptoms, the diseases’ complexity and diversity across regions contribute to the challenges in the control efforts. Visceral leishmaniasis (VL) is fatal without treatment, and the indelible scars left by cutaneous leishmaniasis (CL) may have important psycho-social impact. Eastern Africa region currently bears most of the world’s VL burden. However, underestimation of true disease burden is likely, as the paucity of data from unstable contexts may contribute to inaccurate disease estimates. Both VL and CL are known to have limited geographic distribution but may show high variability inter- and intra-countries. Population movement due to conflict or drought, combined with weak or poorly functioning health system have led to epidemics and spread in new areas. Without vaccine or effective vector control, the pillar of control strategy in Africa remains diagnosis and treatment. Access to adequate, quality diagnostic and treatment services in Africa is challenging. The rk39 rapid test is less accurate and treatment options are limited. A 17-day combination of antimonial and paromomycin is the first line treatment for VL in the region, requiring prolonged hospitalisation and increased economic burden for the patients and their households. Despite the progress in tackling NTDs, access to care for leishmaniasis is often taken for granted. Especially in Africa, access remains problematic and the current body of literature shows critical evidence gaps. Low coverage of the health services, accessibility and availability of quality care, limited diagnostic and therapeutic options along with inefficient procurement and supply remain significant challenges in the region. Delay in seeking treatment not only increase morbidity and mortality but also sustain transmission. The hypothesis informing the project is that access to care for leishmaniasis in Africa is still inadequate. The general objectives of this thesis are to improve our understanding on access to care in Africa, by documenting availability, affordability and accessibility of care, explore novel ways of enhancing such care, and provide insights into specific elements of access to formulate coherent policy recommendations for leishmaniasis in eastern Africa. Three specific objectives were formulated: the first is to update the disease burden, second to examine access issues ‘upstream' i.e. the R&D process and third, assess access issues ‘downstream’.
La leishmaniasis es un grupo de enfermedades causadas por un protozoo (Leishmania) y transmitidas por flebótomos. Como enfermedad tropical desatendida (NTD, por sus siglas en inglés), la leishmaniasis afecta de manera desproporcionada a las poblaciones más pobres y a las personas que viven en zonas rurales, remotas o en zonas de conflicto con acceso limitado o nulo a la atención médica. Las distintas formas clínicas (cutánea, visceral), la complejidad y la distribución de la leishmaniasis en distintas regiones son algunos de los desafíos para controlar la enfermedad. La leishmaniasis visceral (LV) es mortal si el paciente no recibe tratamiento a tiempo, y las cicatrices dejadas por la leishmaniasis cutánea (LC) pueden tener un importante impacto psicosocial. Actualmente la mayor carga de LV se concentra en la región de África oriental aunque las cifras disponibles son probablemente una subestimación del número real de casos debido a la falta de datos fiables. Se sabe que tanto la LV como la LC tienen una distribución geográfica limitada, pero pueden mostrar una alta variabilidad tanto entre países como entre zonas en un mismo país. Los movimientos poblacionales debidos a conflictos o sequías, combinado con un sistema de salud débil o con un funcionamiento deficiente, provocan la expansión de la enfermedad a nuevas áreas y la aparición de epidemias. Al no existir una vacuna ni un control efectivo de vectores, el control de la leishmaniasis en África se sigue basando en el diagnóstico y el tratamiento de los casos. La hipótesis inicial de este proyecto es que es que el acceso al cuidado de la leishmaniasis en África sigue siendo inadecuado. Los objetivos generales de esta tesis son mejorar el conocimiento sobre el acceso a la atención de los casos de leihmaniasis en África, documentando la disponibilidad, la asequibilidad y la accesibilidad de los servicios sanitarios, explorar nuevas formas de mejorar dicha atención y formular recomendaciones de políticas de acceso al cuidado de la leishmaniasis en África oriental. Los tres objetivos específicos son: actualizar los datos sobre carga de enfermedad así como estudiar los problemas de acceso tanto a nivel de I+D como sobre el terreno.

Visceral Leishmaniasis (VL) is a chronic infectious disease caused by the protozoan parasites Leishmania donovani and L. infantum (chagasi) responsible for significant morbidity and mortality in the developing world. Transmitted by sand fly vectors, Leishmania parasites preferentially infect host macrophages throughout the viscera. Experimental L. donovani infection in genetically susceptible mice elicits a highly organ-specific outcome that mirrors much of the immunopathology observed in human VL patients. In this context, the liver is a site of an acute, resolving infection whereas parasite persistence is established in the chronically infected spleen. Generation of an effective immune response against infection requires IFNγ- and TNF-producing CD4+ T(h1) cells. Functional impairment of this pro-inflammatory subset is noted during chronic infection. Here, we describe 3 distinct pathways by which L. donovani promotes persistence within the host via the impairment of protective Th1 responses. IL-10 is a potent immunoregulatory cytokine well known for its T cell-modulatory activity. IL-10-producing CD4+ Th1 (Tr1) cells were found to contribute to much of the dysregulation observed during chronic L. donovani infection in C57BL/6 mice. While not influencing parasite control, Foxp3+ regulatory T(reg) cells were determined to be required for the induction of Tr1 cell populations during established infection. During chronic infection, however, Treg cells appeared to restrict the expansion of pathogenic Tr1 cells.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa and transmitted by a sand fly vector. There are three main disease manifestations: self-healing but scarring cutaneous leishmaniasis, mucocutaneous leishmaniasis with destruction of the mucosal tissues in the nose, mouth and throat, and visceral leishmaniasis in which parasites disseminate to the bone marrow, liver and spleen, leading to high fever, hepatosplenomegaly, wasting, and death in the absence of treatment. Visceral leishmaniasis is the second most lethal tropical disease after malaria. A key question of leishmaniasis research is why some Leishmania species such as Leishmania major remain at the site of the sand fly bite in cutaneous leishmaniasis while other species such as Leishmania donovani metastasize to visceral organs. The overall goal of this thesis was to identify factors involved in visceral disease pathogenesis. We hypothesized that parasite factors play a determining role in visceral disease and that host characteristics are also involved. First, we examined the function of A2, a protein family already implicated in L. donovani visceralization. A2 is shown to protect against heat shock and oxidative stress, key host defences against visceral leishmaniasis. Second, we studied an atypical L. donovani clinical isolate from Sri Lanka that causes cutaneous rather than visceral leishmaniasis, comparing it to a clinical isolate from a Sri Lankan visceral leishmaniasis patient. Although both strains were equally infective to macrophages in vitro, they caused significantly different disease phenotypes in vivo in mice: only the cutaneous isolate caused footpad swelling while only the visceral isolate led to significant liver and spleen parasitemia. A2 expression was lower in the cutaneous isolate and ectopically expressing an additional A2 gene in the cutaneous isolate partially restored virulence in the visceral organs. Therefore, parasite factors are a key determinant of visceral disease. However, host characteristics and history can also influence the development of visceral leishmaniasis. In Sri Lanka, cutaneous leishmaniasis caused by L. donovani is frequent while visceral disease is rare. We show here that immunization with a cutaneous clinical isolate is associated with a mixed Th1/Th2 response and protects BALB/c mice from visceral leishmaniasis, providing a possible rationale for the low incidence of visceral leishmaniasis in Sri Lanka. Overall, these results represent significant progress into the determinants of visceral leishmaniasis and in particular on the role of the virulence factor A2. A novel candidate for a live-attenuated vaccine against visceral leishmaniasis is also presented here. Given the lack of a human vaccine for leishmaniasis and the limitations of the current treatment options, this work could have a significant impact on disease management in Sri Lanka and on vaccine development.
Les leishmanioses sont un groupe de maladies tropicales causées par le protozoaire Leishmania et transmises par la piqûre de phlébotomes. Les leishmanioses peuvent être divisées en trois formes cliniques: leishmaniose cutanée qui guérit sans traitement mais laisse des cicatrices, leishmaniose mucocutanée avec destruction des muqueuses du nez, de la bouche et de la gorge, et leishmaniose viscérale où les parasites quittent le site de la piqûre et se propagent jusqu'à la moelle osseuse, le foie et la rate. Les symptômes de la leishmaniose viscérale sont une forte fièvre et une hepatosplénomégalie, et ceci peut être fatal en l'absence de traitement. La leishmaniose viscérale a le deuxième plus haut taux de mortalité parmi les maladies tropicales, après la malaria. Un des enjeux majeurs de la recherche sur les leishmanioses est de comprendre pourquoi certaines espèces de Leishmania comme Leishmania major restent au site de la piqûre des phlébotomes dans le cas des leishmanioses cutanées, tandis que Leishmania donovani se propage jusqu'aux organes viscéraux. Le but de cette thèse est d'identifier les facteurs impliqués dans la pathogénèse de la leishmaniose viscérale. L'hypothèse de recherche est que les caractéristiques du parasite jouent un rôle principal dans la leishmaniose viscérale et que les caractéristiques de l'hôte sont également importantes. Nous examinons la fonction de A2, une famille de protéines qui sont impliquées dans la viscéralisation de L. donovani. Nous montrons que A2 protège contre le choc thermique et contre les oxydants, deux défenses clé du système immunitaire contre la leishmaniose viscérale. Ensuite, nous étudions un isolat clinique atypique de L. donovani venu du Sri Lanka qui cause des leishmanioses cutanées au lieu de provoquer des leishmanioses viscérales. Nous comparons cet isolat à un isolat clinique provenant d'un patient sri lankais souffrant de leishmaniose viscérale. Quoique ces deux isolats soient tout aussi infectieux l'un que l'autre in vitro, ils causent des symptômes différents in vivo: seul l'isolat cutané cause l'enflure du coussinet plantaire après injection sous-cutanée chez les souris et seul l'isolat viscéral peut se multiplier dans le foie et la rate. Les niveaux de A2 sont plus bas dans l'isolat cutané et nous démontrons que cela constitue un facteur déterminant de son atténuation. Ces résultats prouvent donc que les caractéristiques du parasite ont une influence majeure dans la pathogénèse des leishmanioses viscérales. Cependant, les caractéristiques de l'hôte et son historique médical peuvent également influencer le développement de cette maladie. Les leishmanioses cutanées sont beaucoup plus fréquentes au Sri Lanka que les leishmanioses viscérales. Nous prouvons ici que la vaccination avec l'isolat cutané mène à une réponse du système immunitaire de type Th1/Th2 mixte et protège contre la leishmaniose viscérale dans un modèle in vivo d'infection de souris BALB/c. Ces résultats proposent un modèle pour expliquer la rareté de la leishmaniose viscérale au Sri Lanka. En conclusion, ces résultats éclaircissent plusieurs facteurs impliqués dans le développement de la leishmaniose viscérale et en particulier le rôle du facteur de virulence A2. Nous présentons également un nouveau candidat de vaccin atténué contre la leishmaniose viscérale. Etant donné l'absence de vaccin humain pour cette maladie et les limites des traitements actuels, cette étude pourrait avoir un impact majeur sur la santé publique au Sri Lanka et sur le développement de vaccins contre la leishmaniose viscérale.

Leishmaniasis and African Trypanosomiasis are diseases caused by the Kinetoplastida parasites of Leishmania sp. and Trypanosoma sp. respectively. Control and management of these diseases, which affect a significant number of people in the tropics and subtropical areas of the world, is beset with numerous problems such as drug toxicity, affordability and the emergence and spread of parasites resistance to most of the routinely used drugs. This situation calls for an urgent search for new drugs that would address these concerns. Based on report of excellent antimicrobial activities against other parasites and the possession of other known good values, analogues of choline and curcumin were thoroughly assessed in this study for their potential as antitrypanosomal and antileishmanial drugs. Standard methods such as the Alamar Blue, propidium iodide and direct microscopy methods were used to determine the susceptibility of the parasites to the different analogues. Toxicity tests were performed to determine the effect of these compounds on Human Embryonic Kidney (HEK) cells. The presence of mediated transport of these compounds across the parasite plasma membrane was investigated using the classical uptake technique. In order to investigate the possible mechanism of antiparasitic action of the compounds, this study employed flow cytometry to assess the mitochondrial membrane potential Ym, as well as parameters such as production of reactive oxygen species (ROS), the permeability of the plasma membrane and any effects of the test copounds on the parasite’s cell cycle. Five out of 7 choline compounds tested in this study had EC50 values of 0.13-1.8 µM against T. brucei, 0.14-6.9 µM against L. major, L. mexicana promastigotes and 1.69-12.9 µM against L. mexicana amastigotes. With regard to the curcuminoid compounds, 35 out of 98 tested were observed to exhibit trypanocidal activity better than the original curcumin with EC50 values between 0.05 and 1 µM. Against Leishmania, most of the compounds displayed higher antiparasitic activity than curcumin but lower than observed against trypanosomes. The activity of choline analogues was very similar against L. mexicana and L. major promastigotes (P>0.05), and much higher than against L. mexicana amastigotes. Interestingly, some of the compounds displayed EC50 values below that of pentamidine, the routinely used drug. Assessment of parasite growth pattern in the presence of choline analogues showed that two of the compounds, T1 and MS1, are fast acting, killing the population of BSF T. b. brucei within 8 h with the onset of cell death at 2-4 hours of treatment. In contrast, the other three choline compounds observed to have antiparasitic activities acted more slowly, completely killing the trypanosome population after more than 30 hours of incubation. However, all the choline compounds appeared to rapidly inhibit trypanosome proliferation. The choline compounds exhibited low toxic effects against HEK cell line T29, with the selectivity index (S.I.) being high for some of the compounds. The curcumin compounds, too, were observed to have generally similar or lower toxicity against the human cells than the parent curcumin compound (AS-HK001), which in itself is not considered toxic and routinely used in food. Investigations on the toxicological and pharmacological effects of the curcumin compounds on the survival and the glutathione and protein content of primary murine hepatocytes showed no significant difference between hepatocyte cells treated with curcuminoid compounds AS-HK001, AS-HK009, and AS-HK014 compared with controls. We also investigated how choline and its analogues enter the trypanmosome. Evidence gathered in this study strongly suggests that unlike in Leishmania species and Plasmodium, choline transporters are not expressed in the bloodstream form of T. b. brucei. It was also conclusively shown that the P2, high affinity pentamidine transporter (HAPT) and low affinity pentamidine transporter (LAPT) do not play any significant role in the uptake of this compound. Lacking radiolabeled forms of the choline analogues, this study could not identify a definitive route of uptake of this class of compounds into the parasite. Analysis of cell cycle progression, by flow cytometry, showed trypanosomes in the G1, S, and G2/M stages. Curcuminoids do not appear to cause any important changes in the proportion of cells in G1, S or G2/M phase of the cell cycle. Cells exposed to various concentrations of some curcumin compounds, such as AS-HK014 and AS-HK096, showed a rapid increase in cell permeability, reaching between 80% and 90% in 4 hours. The permeability was observed to increase with increasing drug concentration and/or the incubation time. Investigations of cell membrane permeability also showed that choline analogues caused plasma membrane defects which could probably lead to cell death. With regard to the effect of the compound on mitochondrial membrane potential Ym the dicationic choline compounds, including M38, G25, T4 and MS1, were observed to have pronounced effects on Ym with an onset as early as 8 h of contact and we believe the mitochondria could be the main target of these compounds rather than indicating the induction of apoptosis, as the action of the test compounds was not associated with the production of reactive oxygen species. Indeed, both choline and curcumin analogues reduced the production of reactive oxygen species in T. b. brucei cultures. Furthermore, there were no major defects in choline phospholipid metabolism upon treatment with the choline compounds, suggesting that phospholipid metabolism is not the target of the anti-trypanocidal activity of these compounds. Preliminary results with infected ICR mice infected with T. b. brucei did not reveal significant in vivo activity of the three curcumin compounds on blood parasitemia when they were injected intra-peritoneally with two doses of 50 mg/kg body weight. With reference to evidence obtained in this study, it can firmly be concluded that analogues of choline and curcumin display highly promising antiparasitic activities and are generally non-toxic to human cells. Information provided in this thesis could therefore assist in the further development of these classes of compounds as lead compounds against kinetoplastid diseases. We strongly recommend that further investigation be carried out to understand the full mechanism of action of these compounds in order to facilitate this strategy.

A general review of cutaneous leishmaniasis (CL) and its management is followed by a retrospective review of 306 cases of CL in British soldiers from Belize. The diagnosis was confirmed by demonstration of the parasite, by histology and/or culture, in 61% of cases. Leishmania braziliensis braziliensis (Lbb) was identified in 78 cases and Leishmania mexicana (Lmm) in a further 29 cases. Only cases in which the parasite was identified were analysed further. A single lesion was present in 71%, usually on the exposed extremities. The mean diameter of the ulcers was 14.4mm and lesions had been present for a mean of 9.9 weeks before treatment started. Those due to Lbb were larger, yet they had been present for a shorter time than those due to Lmm. There were no other distinguishing clinical features between them. Treatment with sodium stibogluconate was effective. A 10 day course of 600-800mg od healed 48.5%, whereas a 14 day course of 600 mg bd healed 63.9%. A total of 24g of sodium stibogluconate healed the ulcers of 90% soldiers irrespective of the regime used. Reports of myalgia, anorexia and malaise were more frequent in those who received the higher daily dose. A transient leucopenia and a rise in serum aminotransferases were noted during treatment. These studies led me to propose some management guidelines. The diagnosis should be confirmed histologically and by culture. Sodium stibogluconate 20mg/kg/day should be administered for 20 days. Patients should be assessed 6 weeks after the completion of treatment both clinically - complete epithelialisation with a flat, non-indurated, scar, and by culture of a biopsy.

Leishmania species are vector-borne protozoan parasites that cause a spectrum of human diseases, with an estimated 12 million people infected in 88 countries. Inflammation plays distinct roles in the different clinical syndromes. Visceral leishmaniasis, in which parasites migrate from the site of infection and proliferate in liver and spleen, is accompanied by systemic immune suppression. Cutaneous leishmaniasis, where parasites remain at the site of inoculation and create a long-term ulcer, is associated with vigorous systemic immunity to the parasite. The innate immune sensing pathways responding to Leishmania spp. parasites are not fully described. NLR proteins are a class of structurally related cytosolic proteins. The most well described NLRs form inflammasome complexes that generate strong inflammatory responses to “danger” signals. Other NLRs do not form inflammasomes and have anti-inflammatory functions. While NLR proteins are known to be important in the immune response to many pathogens, the roles NLR proteins in leishmaniasis have only begun to be investigated. We hypothesized that NLR proteins affect the pathogenesis of leishmaniasis through their ability to modulate inflammatory responses. We hypothesized that inflammasome activation in cutaneous leishmaniasis would be detrimental, leading to greater disease pathology, and that the potential anti-inflammatory functions of the non-inflammasome NLRs, NLRP6, NLRP10, and NLRP12, would be protective, reducing tissue damage. In contrast, we hypothesized that in visceral leishmaniasis greater inflammation due to activation of the inflammasome would be protective and control parasite replication, while the anti-inflammatory NLRs would be permissive to parasite replication in the liver and spleen by contributing to the immunosuppressive strategy of the parasite. We used knockout mouse strains lacking the inflammasome adaptor protein ASC, and several non-inflammasome forming NLRs, to investigate NLR proteins in murine models of visceral or cutaneous leishmaniasis. Our data showed that NLR proteins have important functions in visceral leishmaniasis, where they are essential for appropriate parasite homing and replication in the liver and spleen. In cutaneous leishmaniasis, we found that NLRP10 is essential for controlling inflammation in the skin, limiting lesion development and tissue damage at the site of infection. Taken together our findings show important functions for NLR proteins in leishmaniasis, influencing localized tissue specific inflammation, the adaptive immune responses, and clearance or long term residence of the parasite in the infected organs. This research underscores the importance of localized inflammation at the infection site to the pathogenesis and the course of leishmaniasis.

Al revisar la literatura nacional sobre Leishmaniasis Tegumentaria, no encontramos estudios publicados en población militar, lo cual nos motivó a realizar este trabajo, con el objetivo de determinar características clínicas y epidemiológicas, particulares o generales de la leishmaniasis en población militar del Ejército Peruano. Recientemente en el último Congreso Nacional de Dermatología, los Doctores. Paredes G. y Saavedra M., presentaron en una comunicación libre, un estudio de leismaniasis en efectivos policiales del periodo 1995 al 2001, el cual nos permite comparar resultados en poblaciones similares y plantear conclusiones concretas. Se estudiaron 284 pacientes con diagnóstico de Leishmaniasis Tegumentaria en el Hospital Militar Central entre el periodo de 1997 al 2000. Se observó una frecuencia mayor de la leishmaniasis en 1999 (40.5%) y 2000 (34.5%), años de mayor movilización del personal militar a zonas endémicas. El promedio de edad fue 20.8, todos los pacientes fueron del sexo masculino, la tropa fue mayoritariamente afectada (81.3%). Los lugares de contagio más frecuentes fueron: Junín (43.7%), Loreto (36.3%), Amazonas (4.9%) y Cuzco (4.6%). El tiempo de permanencia en el lugar de contagio de 1 a 2 meses (29.2%), fue el mas frecuente, el factor desencadenante que se reportó principalmente es la picadura de insecto (16.9%) aunque no se conoció el dato en 75.4%, el tiempo de enfermedad al momento del diagnóstico en su mayoría fue de 1 a 2 meses (38%). Una minoría tuvieron antecedente de Leismaniasis (19.4%). Predominó la forma de Leishmaniasis Cutánea (LC) pura (88.4%), la localización preferente de la lesión fue pierna - pie (37.2%), predominando la lesión única (65,1%) y de tipo ulcerativo (76.6%). La localización de la lesión mucosa fue preferentemente el septo (42.4%). La leishmania se encontró en la biopsia en el 46.5% de los casos y el test de leishmanina fue positivo en el 9.5% y negativo en el 11.3%, a 79.2% no se les hizo la prueba. El tratamiento mayormente empleado fue Glucantime intramuscular (58.5%) y la permanencia hospitalaria en la mayoría de casos fue de 1 a 2 meses (50%). Al cruzar variables se evaluó la influencia de unas variables sobre otras, cuando el resultado fue estadísticamente significativo. Así, las leishmaniasis mucosas puras fueron mas frecuentes en pacientes con antecedente de leishmaniasis (17 casos) (p=0.000). Cuando el tiempo de permanencia fue de 1 a 2 meses la posibilidad de encontrar el agente etiológico (48 casos) en el estudio de biopsia fue mayor (p=0.003). Asi mismo cuando el tiempo de enfermedad al momento del diagnóstico fue 1 a 2 meses se encontró mayormente la leishmania en la biopsia (60 casos) (p=0.001). La forma de Leishmaniasis Mucosa (LM) pura y Leishmaniasis Mucocutánea (LMC) en su mayoría correspondieron al departamento de Loreto (14 y 5 casos respectivamente) que correspondieron a la mayoría de las Leishmaniasis Mucosa pura y Leishmaniasis Mucocutánea (p=0.002). Cuando la forma clínica fue Leishmania Cutánea pura se prefirió tratamiento con glucantime intramuscular (153 casos), cuando fue mucosa se prefirió anfotericina (11 casos). El empleo de anfotericina en el tratamiento prolongó la estancia hospitalaria (20 casos) Los resultados obtenidos en el presente estudio se correlacionaron con otros estudios clínico epidemiológicos realizados en el Perú y otros trabajos internacionales, demostrándose que las características son las mismas tanta en la población general como en poblaciones militares, salvo una que otra variable por tratarse de una población sesgada (todos varones y mayormente jóvenes).

Al revisar la literatura nacional sobre Leishmaniasis Tegumentaria, no encontramos estudios publicados en población militar, lo cual nos motivó a realizar este trabajo, con el objetivo de determinar características clínicas y epidemiológicas, particulares o generales de la leishmaniasis en población militar del Ejército Peruano. Recientemente en el último Congreso Nacional de Dermatología, los Doctores. Paredes G. y Saavedra M., presentaron en una comunicación libre, un estudio de leismaniasis en efectivos policiales del periodo 1995 al 2001, el cual nos permite comparar resultados en poblaciones similares y plantear conclusiones concretas. Se estudiaron 284 pacientes con diagnóstico de Leishmaniasis Tegumentaria en el Hospital Militar Central entre el periodo de 1997 al 2000. Se observó una frecuencia mayor de la leishmaniasis en 1999 (40.5%) y 2000 (34.5%), años de mayor movilización del personal militar a zonas endémicas. El promedio de edad fue 20.8, todos los pacientes fueron del sexo masculino, la tropa fue mayoritariamente afectada (81.3%). Los lugares de contagio más frecuentes fueron: Junín (43.7%), Loreto (36.3%), Amazonas (4.9%) y Cuzco (4.6%). El tiempo de permanencia en el lugar de contagio de 1 a 2 meses (29.2%), fue el mas frecuente, el factor desencadenante que se reportó principalmente es la picadura de insecto (16.9%) aunque no se conoció el dato en 75.4%, el tiempo de enfermedad al momento del diagnóstico en su mayoría fue de 1 a 2 meses (38%). Una minoría tuvieron antecedente de Leismaniasis (19.4%). Predominó la forma de Leishmaniasis Cutánea (LC) pura (88.4%), la localización preferente de la lesión fue pierna - pie (37.2%), predominando la lesión única (65,1%) y de tipo ulcerativo (76.6%). La localización de la lesión mucosa fue preferentemente el septo (42.4%). La leishmania se encontró en la biopsia en el 46.5% de los casos y el test de leishmanina fue positivo en el 9.5% y negativo en el 11.3%, a 79.2% no se les hizo la prueba. El tratamiento mayormente empleado fue Glucantime intramuscular (58.5%) y la permanencia hospitalaria en la mayoría de casos fue de 1 a 2 meses (50%). Al cruzar variables se evaluó la influencia de unas variables sobre otras, cuando el resultado fue estadísticamente significativo. Así, las leishmaniasis mucosas puras fueron mas frecuentes en pacientes con antecedente de leishmaniasis (17 casos) (p=0.000). Cuando el tiempo de permanencia fue de 1 a 2 meses la posibilidad de encontrar el agente etiológico (48 casos) en el estudio de biopsia fue mayor (p=0.003). Asi mismo cuando el tiempo de enfermedad al momento del diagnóstico fue 1 a 2 meses se encontró mayormente la leishmania en la biopsia (60 casos) (p=0.001). La forma de Leishmaniasis Mucosa (LM) pura y Leishmaniasis Mucocutánea (LMC) en su mayoría correspondieron al departamento de Loreto (14 y 5 casos respectivamente) que correspondieron a la mayoría de las Leishmaniasis Mucosa pura y Leishmaniasis Mucocutánea (p=0.002). Cuando la forma clínica fue Leishmania Cutánea pura se prefirió tratamiento con glucantime intramuscular (153 casos), cuando fue mucosa se prefirió anfotericina (11 casos). El empleo de anfotericina en el tratamiento prolongó la estancia hospitalaria (20 casos) Los resultados obtenidos en el presente estudio se correlacionaron con otros estudios clínico epidemiológicos realizados en el Perú y otros trabajos internacionales, demostrándose que las características son las mismas tanta en la población general como en poblaciones militares, salvo una que otra variable por tratarse de una población sesgada (todos varones y mayormente jóvenes).
Tesis de segunda especialidad

Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

The present study concerns human genetic susceptibility to two obligate intracellular infections caused by Leishmania braziliensis and Mycobacterium tuberculosis in Peru and Bolivia, and the genetiic epidemiology of the pathogen Mycobacterium tuberculosis in Peru.

There are currently no ideal treatments or acceptable vaccines for cutaneous leishmaniasis, a worldwide health problem caused by infection with a number of species of the dimorphic protozoa Leishmania. Therefore, there is an urgent need to search for simple, safe, effective, and affordable treatments. Imiquimod is an immune-response modifying agent. Recently, 5% imiquimod cream (Aldara(TM)) received approval by the Food and Drug Administration in the United States and is currently available for the treatment of external genital and perianal warts caused by human papillomavirus infection. The antiviral activity of this drug is mediated through stimulation of cytokine release from many cell types including macrophages resulting in a local immune response at the site of application. Moreover, imiquimod has been shown to enhance cell-mediated immune responses (CMIR). Since imiquimod activates macrophages, the exclusive host cells of Leishmania, and stimulates CMIR which are required for host defence against Leishmania, we have investigated the potential of using imiquimod and its related compound, S-28463, as agents for treating leishmaniasis. It is demonstrated within that imiquimod and S-28463 effectively stimulated leishmanicidal activity both in vitro in macrophages and in vivo in a mouse model. These compounds also stimulated signal transduction associated with the induction of nitric oxide synthesis in macrophages. Imiquimod and S-28463 induced leishmanicidal activity in macrophages in the absence of any other cell types. We have demonstrated that S-28463 generated macrophage leishmanicidal activity by inducing genes involved in macrophage activation and inflammatory responses. Finally, we have also performed an analysis on the influence of L. donovani on macrophage gene expression using a cDNA array analysis, a similar methodology to study the effect of S-28463 on macrophage gene expression. Intramacrophage infection with L. donovani was shown to cause general

Leishmaniasis is a serious public health problem throughout the developing world; 350 million people are at risk of infection in 88 countries. It is caused by a protozoa parasite called leishmania. The current therapy for leishmaniasis is limited. Pentavalent antimonials (SbV) have been the recommended drugs for the treatment of leishmaniasis for over 50 years, but long courses, and toxicity limits their use. New approaches for new therapies, including combination therapies, are urgently needed. Imiquimod is a novel compound that activates macrophages to kill leishmania amastigotes in vitro and could reduce disease severity in a mouse model of cutaneous leishmaniasis (CL). It is an agonist for the Toll like receptor 7 (TLR7) on macrophages directing the development of Th1-type immune response required for resolution of leishmaniasis.
A new combined model therapy for CL, was investigated in order to confirm that it was superior to SbV alone in the treatment of CL. A randomized, double-blind, placebo-controlled trial was carried out using Sb V in combination with topical imiquimod (5%) or a placebo cream for the treatment of CL in Peru. The study shows that, when used in combination with SbV, imiquimod significantly shortens the time required for complete cure of CL and may reduce the severity of scarring.
High costs and drug resistance to SbV are a big problem in endemic areas; therefore alternative drugs for SbV are necessary to possibly be used in combination with imiquimod. The findings of these studies represent a significant advance for leishmaniasis, demonstrating that it is possible to translate the rationale for combined therapy based in basic immunological concepts into action in the developing world.

Cutaneous leishmaniasis (CL) is a parasitic disease caused by several species of the protozoan parasite Leishmania and affects approximately 10 million people worldwide. The drugs currently available such as miltefosine, amphotericin B and pentavalent antimonials, are limited by high cost, considerable side effects and restricted efficacy. CL could potentially be treated by a topical formulation. In its simplest form CL consists of a single nodule or papule, typically on exposed body parts, with the parasites residing in the lower epidermis and dermis. A topical treatment would minimise possible adverse effects by reducing the amount of drug taken up in the systemic circulation and would be easy to apply which is important for patient compliance. The overall aim of the work described in this thesis was to explore the use of different drugs in a topical formulation to cure CL. To be efficacious, a topical treatment requires the permeation of the active ingredient through the stratum corneum and into the deeper layers of the skin where the Leishmania parasites resides. Intact skin is a highly effective barrier against xenobiotics. However many skin diseases are known to affect the skin barrier making it more or less permeable to drugs. An understanding of the permeability of the skin hosting the Leishmania parasites is a prerequisite when trying to optimize drug delivery to the skin. Therefore the barrier function of Leishmania infected skin in early stages of CL was characterised with respect to histology, transepidermal water loss and drug permeation. Suitable anti-leishmanial drugs with the potential to permeate into the skin were identified through literature review, taking into account the information obtained from the skin barrier characterisation. The leishmanicidal activity of the drugs was evaluated in an intracellular amastigote-macrophage model and potent compounds were selected for further work. First, the re-formulation of miltefosine, currently used to treat leishmaniasis via oral administration, into a topical formulation was explored. The effects of different solvents on its permeation through and disposition in the skin was evaluated using in vitro Franz diffusion cell permeation studies. Secondly, a drug discovery approach was used to identify lead benzoxaboroles, a set of interesting anti-leishmanial compounds. In the early stages, in vitro ADME studies were conducted to establish basic pharmacokinetic parameters of a range of benzoxaboroles. This information together with previously unpublished data was used to select compounds for further testing such as stability and binding in skin homogenate and permeation evaluation. The three most promising compounds were tested in vivo in a CL model.

Cutaneous leishmaniasis (CL) is a neglected tropical disease. It presents as disfiguring and deforming skin lesions. There is an urgent need for a simple, low-cost and non-toxic treatment for use in resource poor countries. As amphotericin B (AmB) has transformed visceral leishmaniasis treatment, I have developed and optimised a poly (methacrylic acid)-AmB drug (AmB-PMA). This involved the use of a commercially available, non-toxic, well-defined narrow molecular weight distribution polymer, poly (methacrylic acid sodium salt) (PMA) associated with AmB to create AmB-PMA. After chemical synthesis optimisation, AmB-PMA was shown to be less toxic than clinical grade AmB, and it was effective against Leishmania spp. promastigotes and amastigotes in vitro. To determine in vivo efficacy, studies were performed using BALB/c mice. They develop progressive non-healing skin lesions and are unable to control parasite growth. Mice were infected subcutaneously with L. major (LV39) promastigotes and treated locally with AmB-PMA. Studies were then conducted to evaluate the optimum route of administration, treatment frequency and dosing schedules. The results showed a reduction in both parasite burden and lesion size of the infected treated footpad with no adverse toxicity. Cytokine production was determined by quantitative real-time PCR for mRNA at the lesion site and the draining lymph node. I found that the AmB-PMA could effectively heal an established lesion and cure a CL infection. This was associated with: - (i) increased IFN-Y and TNF-α production, (ii) reduced IL-10 and MIP-1β production, when compared to infected, untreated lesions. The success of this treatment approach was also evaluated by histological studies. In addition, treated mice with resolved primary cutaneous lesions mounted a delayed type hypersensitivity response 24 h after challenge of the contra-lateral footpad with L. major. My thesis demonstrates the new opportunity for a cost-effective AmB based polymer drug that can be used locally to cure CL.

Leishmaniasis is a parasitic disease caused by Leishmania spp. Once transmitted to the host, the parasite, being an obligate intracellular pathogen, localizes primarily inside macrophages, where it starts replication. Interferon γ (IFN-γ) is one of the most important mediators produced by Th1 subpopulation and it is responsible for macrophages activation that could lead to parasite control. Neopterin belongs to the chemical group known as pteridines. It is synthesised also by macrophages upon stimulation with interferon-γ and is indicative of immune system activation. Measurement of N concentrations in body fluids like blood, cerebrospinal fluid or urine provides information about the cell-mediated (T helper cells type 1 Th1) immune sistem activation in humans; assuming that, the present study aimed at investigating if N could also be considered as an indicator of a protective reaction (Th1-mediated) of the host against Leishmania, and then if it could be used clinically as prognostic factor in leishmaniotic patients or as helpful parameter in monitoring treated patients, as investigated in men. For these purposes, the study was divided into two phases independent of each other. The first phase of the study aimed to at investigating the possible correlations between sera N values and antibody titers (dosed by immunofluorescence antibody test IFAT) in 74 dogs, both seronegative and seropositive. In the second one, 5 patients affected by leishmaniasis and in treatment for it, were followed for a period of 24 months comparing N values to other parameters (e.g. IFAT, PCRs, citology on fine needle aspiration (FNA) of lymphnodes). Even in the absence of a statistically significant correlation between N values and IFAT titers, serum N appeared to be an interesting marker possibly able to provide an indirect value of Th1/Th2 ratio in leishmaniotic patients. It should also be further investigated its possible role in the early detection of ill patients possibly predisposed to develop renal failure and/or to relapse.

Die vorliegende Studie wurde in einer Gruppe von Dörfern im Ostsudan, Gedaref State, durchgeführt. Bei 100 Patienten mit der Verdachtsdiagnose Kala Azar- oder Post-Kala Azar- Leishmaniose war der Erregernachweis mit der PCR direkt in klinischen Proben, die auf Filterpapier aufgebracht worden waren, ohne vorherige Kultivierung erfolgreich. In dieser PCR wurden die ribosomalen, internal transcribed spacer (ITS1 & ITS2) amplifiziert, weil sie sehr variabel sind, eine klare Speziesidentifizierung gestatten und bei weiterführenden Analysen der PCR-Produkte auch der Nachweis stammspezifischer Unterschiede erwartet werden konnte. Für die Analyse der Diversität von Leishmania donovani-Isolaten aus dem Sudan wurden 4 verschiedene PCR-basierte Methoden eingesetzt: das PCR-Fingerprinting mit unspezifischen Einzelprimern, die RFLP- Analyse des amplifizierten ITS-Locus, ,,single strand conformation polymorphism (SSCP)- Analysen der amplifizierten ITS-Region, des Gens, welches für die Hauptoberflächenprotease (gp63) kodiert, und anonymer DNA- Fragmente sowie Sequenzanalysen der entsprechenden Zielregionen. Das PCR-Fingerprinting und die Restriktionsanalyse der ITS-Region lieferten weitgehend übereinstimmende Fragmentmuster für alle untersuchten L.donovani-Stämme. 12 unterschiedliche Profile wurden bei der SSCP-Analyse des ITS1-Locus für 86 Isolate aus dem Sudan erhalten, während der ITS2-Locus bei diesen Stämmen hochkonserviert war und nur ein Stamm ein unterschiedliches SSCP-Muster aufwies. L. Donovani -Stämme anderer geographischer Herkunft hatten unterschiedliche ITS2-Profile in der SSCP. Für den gp63 - Locus waren 3 polymorphe SSCP-Muster bei 31 untersuchten sudanesischen Isolaten nachweisbar. Für die meisten der anonymen DNA-Fragmente, L510, L413, LK413, L0308 UND L0114, konnten leider nur von 8 kultivierten Stämmen gute PCR-Produkte erhalten werden. Lediglich das Fragment L0110 konnte erfolgreich von 31 auf Filterpapier aufgebrachten Proben direkt amplifiziert werden. Die Suche nach Polymorphismen mit der SSCP ergab keine Unterschiede in diesen anonymen DNA-Regionen, mit Ausnahme des Fragments L0114, das zwei verschiedene Muster aufwies. Die Ergebnisse der SSCP-Analysen und der DNA- Sequenzierung stimmten gut überein, wodurch bestätigt wurde, dass die SSCP genetische Unterschiede auf dem Niveau einzelner Basenaustausche nachweisen kann. Die SSCP- Technik hat Vorteile gegenüber den anderen Methoden, die für die Untersuchung von Sequenzvariationen innerhalb der Spezies L. donovani angewandt wurden. Es konnten keine Korrelationen zwischen der Form der klinischen Manifestation und den Ergebnissen des PCR- Fingerprinting, der ITS-RFLP- und ITS-SSCP- Analysen festgestellt warden. Diese Studie ist von besonderem Nutzen in epidemiologischen Feldstudien, bei denen die Kultivierung der Erreger besonders in Entwicklungsländern extrem schwierig sein kann.
This study was carried out in clusters of villages that represent an endemic focus of visceral leishmaniasis (VL). These villages were located in Gedaref state, eastern Sudan. For diagnostic purposes polymerase chain reaction (PCR) was performed successfully, directly from clinical samples spotted on filter papers with no prior cultivation from 100 patients suspected of having kala-azar or post kala-azar dermal leishmaniasis. Mainly the ribosomal internal transcribed spacer (ITS1 & ITS2) were targeted in PCR because this region is more variable and allows clear species identification and also strain differences could be expected by further analysis of these PCR products. PCR was found to be more sensitive compared to the gold standard microscopic method. Four PCR based approaches were used to analyse diversity within Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single non-specific primers, restriction analysis of the amplified ITS locus (RFLP), single-stranded conformation polymorphism (SSCP) of the ITS region, major surface protease (gp63) gene, anonymous DNA fragments and sequencing of these targeted regions. When PCR fingerprinting and restriction analysis of ITS region were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave 12 different SSCP profiles among the 86 Sudanese isolates, where as the ITS2 locus was highly conserved among the 86 samples with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. The gp63 locus gave 3 polymorphic patterns among 31 Sudanese isolates. Concerning most of the anonymous DNA fragments namely, L510, L413, LK413, L0308 and L0114 unfortunately, we succeeded to get good PCR products only from DNA extracted 8 successful cultures. Only for the fragment L0110 we were able to get good PCR products from 31 samples spotted on filter papers. When these PCR products were investigated for polymorphisms using SSCP no differences were observed with exception of L0114 region, which showed 2 patterns. SSCP analysis correlates well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigating of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics. This study is beneficial particularly in epidemiological studies based on field-work where obtaining cultures can be extremely difficult especially in developing countries.

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Evalúa la eficacia de la mefloquina en una región endémica de leismaniasis cutánea por Leishmania (Viannia) braziliensis, considerando que esta droga, de administración oral, eficaz en el tratamiento de la malaria, con vida media prolongada y efectos colaterales poco frecuentes podría ser menos tóxica y de administración más fácil al ser comparada con los antimoniales pentavalentes. En Corte de Pedra, poblado ubicado en el litoral sur del Estado de Bahia en Brasil, se administró tratamiento, aleatoriamente a diez pacientes portadores de lesiones leishmaniásicas. Ellos fueron subdivididos en dos grupos de cinco pacientes. El primer grupo recibió mefloquina por vía oral a la dosis de 250 mg/día, durante seis días. Luego de un intervalo de tres semanas se repitió el mismo esquema. El segundo grupo recibió antimoniato de meglumina (Glucantime®) diariamente, por vía endovenosa, en la dosis de 20 mg/kg por 20 días. En el grupo tratado con mefloquina solo un paciente cicatrizó la lesión después de, inclusive el segundo ciclo. En este grupo, un paciente con cuatro lesiones presentó una nueva lesión durante el primer ciclo de tratamiento. La evolución de los otros tres fue lenta y luego de nueve semanas ninguno de ellos había presentado cicatrización de la lesión entretanto que permanecían con gran infiltración y signos evidentes de actividad. El otro grupo, tratado con Glucantime® presentó evidente mejoría en el mismo período de tiempo. No hubo evidencia clínica de mejoría en los pacientes con leishmaniasis cutánea tratados con mefloquina.
Trabajo académico

The immunobiology and the control of experimental visceral leishmaniasis were studied in susceptible (C57BL/6J) and resistant (C57L/J) strains of mice. It was found that the resistant/susceptibility phenotype for infection with L. donovani is expressed only by liver macrophage in vitro; the resistant/susceptible phenotypes were transfered reciprocally by bone marrow radiation chimeras. It was also found that the phagocytic activity of macrophages is reduced by the infection and that liver and peritoneal macrophages reflect their specific resistance/susceptibility phenotype following protective or curative treatment with lymphokines: macrophages from resistant C57L/J mice responded better to lymphokine activation. The production of IL-1 by spleen and peritoneal macrophages is inhibited by the infection. Immunosuppression with cyclosporin A (CsA) exacerbated the infection, without affecting phenotype; both CsA-treated strains of mice heal the infection in the absence of IL-1 and IL-2. There were more infected liver macrophages in normal or CsA-treated susceptible C57BL/6J mice and a greater number of amastigotes per cell than in normal or CsA-treated resistant C57L/J mice. CsA treatment did not affect the responsiveness of macrophages to lymphokine activation. IL-2-treated spleen and blood leucocytes from infected animals reduced infection in macrophages infected in vitro. Adoptive immunotherapy in vivo with IL-2-treated spleen cells from infected animals showed significant specific reduction of the parasite load in the liver; daily injections of IL-2 enhanced cure. T lymphocytes are the cells involved in cure; cure is mediated by soluble factors produced by the treated cells, and is specific to Leishmania infection.