Revista Eletrônica de Biologia

REB Volume 2 (1): 82-98, 2009. ISSN 1983-7682 Revista Eletrônica de Biologia Leshmaniose Cutânea e Visceral: Revisão Cutaneous and Visceral Leishmaniasis: a Review Guilherme Malafaia Mestre em Ciências Biológicas Doutorando do Programa de Pós Graduação em Ciências Biológicas. Laboratório de Doença de Chagas, Universidade Federal de Ouro Preto. Campus Morro do Cruzeiro. CEP 35.400-000 Ouro Preto, MG. Rua Vereador Paulo Elias, 8A. Vila Itacolomy, Ouro Preto-MG, CEP: 35.400-000. E-mail: guilherme@nupeb.ufop.br. RESUMO Protozoários do gênero Leishmania são parasitos transmitidos por insetos flebotomíneos e causam um espectro de doenças coletivamente conhecidas como leishmanioses. Estes doenças são consideras um sério problema de saúde pública em muitas partes do mundo, resultando em uma estimativa de 12 milhões de casos a cada ano. Apesar dos avanços no diagnóstico, tratamento e nas pesquisa científica de base, as leishmanioses são negligenciadas e correlacionadas com a pobreza. Assim, neste artigo, baseado em informações oriundas de uma pesquisa bibliográfica, são descritos a epidemiologia e imunologia da doença, ciclo de vida do parasito Leishmania, tratamento e vacinas contra as leishmanioses. Conclui-se que os obstáculos atuais estão ligados a um inadequado controle do vetor, a inexistência de uma 82

vacina eficaz e o insucesso no desenvolvimento de novas drogas contra a doença. Palavras-chaves: Leishmania; leishmaniose cutânea; leishmaniose visceral; imunologia; flebotomíneo; vacina ABSTRACT Protozoan parasites from the genus Leishmania are spread by a sandfly insect vector and cause a variety of diseases collectively known as leishmaniasis. The disease is a significant health problem in many parts of the world resulting in an estimated 12 million new cases each year. Despite tangible advances in diagnosis, treatment, and basic scientific research, leishmaniasis is neglected and embedded in poverty. Thus, in this article, the epidemiology, immunology, life cycle of Leishmania, treatment and vaccine against leishmaniasis are described according to the information found during bibliographic research. The study concludes that current obstacles to prevention and proper management of the disease include inadequate vector (sandfly) control, no vaccine available, and insufficient access to or failure in developing affordable new drugs. Key-words: Leishmania; cutaneous leishmaniasis; visceral leishmaniasis; immunology; sandfly; vaccine 1) INTRODUCTION Leishmaniasis has several diverse clinical manifestations: ulcerative skin lesions, destructive mucosal inflammation, and disseminated visceral infection (kalazar) (Brasil, 2007). Epidemiology, immunopathology, and outcome are similarly diverse, since infection occurs in multiple endemic regions, in both children and adults, and is caused by nearly two-dozen distinct Leishmania species (Desjeux, 2001; Desjeux, 2004). According to Alvar et al. (2004) and McMahon-Pratt & Alexander (2004), variable disease expression has also been shown in naturally infected animals and, especially, experimentally infected animals. Nevertheless, all forms of this protozoal infection share three pathogenetic features: resident tissue macrophages are targeted and support intracellular parasite replication; the host immunoinflammatory response 83

regulates expression and outcome of disease; and persistent tissue infection is characteristic. The World Health Organization (WHO) estimates approximately 12 million affected individuals with an estimated annual incidence of 1,5-2 million, among a susceptible population of approximately 350 million in 88 different countries (WHO, 2008). According to Murray (2002), leishmaniasis is one such infection which rarely shares this limelight and thus remains largely a neglected disease. Despite this, several issues regarding leishmaniasis merit discussion: resistance to conventional drug treatment has developed in certain areas of the world, necessitating a change of first-line agents; rapid, less invasive diagnostic procedures have been developed which are most useful in poorly resourced parts of the world; despite advances in the understanding of the immunology of the disease and the unraveling of the Leishmania genome, a vaccine has not yet been developed; and the extent of disease in different individuals stresses the complex immunology of leishmaniasis, brought to the fore more recently with the advent of HIV Leishmania coinfection and Leishmania to infection associated to malnutrition protein-caloric (Malafaia et al., 2008a,b), and its difficult eradication in this scenario. Thus, this review is based on information from bibliographic research, Entrez-Pubmed searches on leishmaniasis, review articles and papers in their reference lists, and from the authors personal archives. 2. EPIDEMIOLOGY AND LIFE CYCLE OF LEISHMANIA According to WHO (2008), leishmaniasis is endemic in more than 60 countries worldwide including Southern Europe, North Africa, the Middle East, Central and South America and the Indian subcontinent. The burden of disease 84

(90% of cases) is borne by Afghanistan, Pakistan, Syria, Saudi Arabia, Algeria, Iran,, Peru and Brazil in the case of cutaneous leishmaniasis (CL), and by India, Bangladesh, Nepal, Sudan, and Brazil in the case of visceral leishmaniasis (VL) (Desjeux, 2004). In view of this geography, leishmaniasis remains embedded in poverty as a neglected disease (Yamey, 2002). Recently the number of reported cases and geographical areas has increased (Arias et al., 1996) and this has sparked concern regarding the contribution that global warming might have on this observation (Desjeux, 2001; Kuhn, 1999). One of the causative organisms of leishmaniasis, Leishmania donovani, was first described in 1903 by Leishman and Donovan almost simultaneously (Desjeux, 2004). Leishmania is a protozoon, able to infect animals, humans and sandflies. Nine major species of Leishmania involved in human disease are grouped into old world and new world species (Table 1). Each may cause a disease specific to the species and the host response. Every year, an estimated 1,5 to 2 million children and adults develop symptomatic disease (CL 1 to 1,5 million and VL 0,5 million), and the incidence of infection is substantial when subclinical infections are included (Desjeux, 2004). Leishmaniasis is associated with about 2,4 million disability-adjusted life years and around 70 000 deaths per year (Desjeux, 2004). Table 1. Major species of Leishmania causing human disease and their geographic distribution. Species Region Distribution Manifestation L. major Old World Middle East, Africa and Asia Cutaneous L. tropica Mediterranean basin Cutaneous L. aethiopica Ethiopia and Kenya Cutaneous/Mucocut aneous L. infantum Mediterranean basin Visceral L. donovani Africa, India, Arabian Peninsula Visceral 85

Table 1. (continuation) Species Region Distribution Manifestation L. amazonensis New North, Central and Cutaneous World South America L. mexicana South America Cutaneous L. braziliensis South America Cutaneous/Mucocutaneous L. chagasi Central and South America Visceral About 70 different species of sandfly can transmit leishmaniasis (Murray, 2005). The species are mainly Lutzomyia in the Americas and Phlebotomous elsewhere (Mandell et al., 2005). The sandfly characteristically feeds at dusk, and being a weak flier, tends to remain close to its breeding area, not too high from the ground. Different species have different feeding and resting patterns. Leishmania to Infection is more common in men than in women, but this may reflect increased exposure to sandflies. Although disease occurs irrespective of age, children aged 1 to 4 years are particularly at risk of infection and childhood infection may account for more than half of all cases in some of these countries (Grech et al., 2000). Untreated VL carries a mortality of 75 95%, while CL can disseminate to involve the mucosa, resulting in death from secondary infection (Laison & Shaw, 1987). As reviewed by Killinck-Kendrick (1990) and Gossage et al. (2003), the life cycle of Leishmania involves alternation between a mammalian host and a phlebotomine sand fly host. In the mammalian host the developmental biology of the parasite is relatively simple and consistent between species: metacyclic promastigotes (infective forms) are introduced into the skin by the bite of the sand fly. These areresistant to complement attack and they enter local phagocytesrapidly. Transformation into aflagellate amastigotes then occurs and 86

the life history of the infection in humans is perpetuated by this life cycle stage (Handman & Bullen, 2002; Engweda et al., 2004). In contrast the developmental biology of the parasite in the sand fly host is more complex and less well understood. According to Kamhawi (2006), outside the mammalian host, the Leishmania life cycle is confined to the digestive tract of sand flies. Most Leishmania species (subgenus Leishmania) are suprapylarian parasites; that is, their development is restricted to the midgut. Members of the New World Viannia subgenus, such as L. braziliensis, are peripylarian parasites: they enter the hindgut before migrating forward into the midgut. 3. IMMUNOLOGY Most of the experimental immunological data come from mouse models and less is known about the immunology of human leishmaniasis. Although mouse models have been used for the study of both CL and VL, they more closely reflect the situation in human cutaneous leishmaniasis than visceral disease.one knows that the immune response to Leishmania infection is cell mediated (Table 2). The organism lies exclusively intracellular, mainly inside macrophages as replicating amastigotes. The outcome of infection will depend on whether the host mounts primarily a T-helper (Th1 or Th2 response) (Heinzel et al., 1989). In the case of CL, studies in animal models have indicated that effective protection against infection is attributed to the development of a potent CD4 + Th1 type immune response, characterized by the production of IL-12 and IFNc, which subsequently mediates macrophage activation, nitric oxide production and parasite killing (Rogers et al. 2002; Alexander & Bryson, 2005). In contrast, as demonstrated for von Stebut & Udey (2004), clear-cut polarization of T helper cell responses is not evident in human leishmaniasis which shows a mixed Th1 and Th2 immune response. Studies in animals models suggest that 87

the same parasite epitope can induce a Th1 response in animals with resolving infection or a Th2 response in others with disease progression (Reiner et al., 1993). Other animal studies have shown that Th1 and natural-killer (NK) cells produce interferon gamma (IFN-γ), which mediates resistance, whilst interleukin (IL) 4-producing Th2 cells confer susceptibility to infection (Reed & Scoot, 1993). Human studies have also shown that IL-4, a component of the Th2 response, may also be associated with disease progression (Sundar et al., 1997). Table 2. Types of immunity mediated by cells in leishmaniasis. Th1 immune response Th2 immune response T-helper CD4 cells producing Th2 cells produce IL-4, IL-5, IL- IL-2, IL-3 and IFN-γ; 6, IL-10 which favour induction Will promote immune reponses of antibody responses by B that are primarily cell cells; mediated/inflammatory by In leishmaniasis, associated activating cytotoxic T cells, NK with disease progression. cells and macrophages; In leishmaniasis, associated with disease resolution. In the Th1 response, promastigotes attach to reticuloendothelial cells and T helper CD4 cells produce IL-2, IL-3 and IFN-γ which activate macrophages. IL-12 and tumour necrosis factor (TNF) are also important in this type of response. The promastigotes are then phagocytosed by the activated macrophages into vacuoles which then fuse with lysosomes. Host genetics inevitably influence the type of immune response. Studies in mice and humans have shown that genes such as those coding for natural resistance associated macrophage protein 1 (NRAMP1), TNF or the major histocompatibility complex are thought to play a major role in the outcome of infection (Roberts et al., 2000). 88

The parasite itself can affect the macrophage and dendritic cell responses. Specific gene loci such as the A2 gene can code for products which promote L. donovani infectivity (Zijlstra & EI-Hassan, 2001). Thus the interplay between the host-determined delayed-type hypersensitivity, antigen-specific T- cell reactivity, and cytokine secretion, and the type and virulence of the particular infecting Leishmania species determine what type of disease expression develops in the host. However, most studies on experimental VL question about the role of IL- 4 in susceptibility to visceral infection by Leishmania due to the contradictory results reported (Kaye et al., 1991; Saha et al., 1993; Miralles et al., 1994). Our results point to a less significant role for IL-4 in protection against VL, because IL-4 production was not correlated with reduction of the parasite load in mice vaccinated with L. chagasi antigen plus saponin, as reported by Lehmann et al. (2000). The authors observed that in VL a Thl dominated immune response is protective against the L. donovani parasites and, furthermore, that the capacity to produce IFN-γ rather than the presence of IL-4 determines the efficacy of the immune response in susceptible mice. Others studies have shown that protection in the visceral model is associated with a mixed Th1/Th2 pattern. Ghosh et al. (2002) showed that immunization with A2 protein protects mice against L. donovani infection, and this protein induces a mixed Th1/Th2 and a humoral response. Ramiro et al. (2003) showed that LACK immunization in a heterologous prime-boost regime (plasmid DNA and recombinant vector) was able to protect dogs against L. infantum infection, and this protection was associated with an increase in the mrna level of IL-4 and IFN-γ in peripheral blood mononuclear cells. These studies suggest that, although protection in experimental visceral infection demands IFN-γ production response, IL-4 is also necessary in this model. Furthermore, Stager et al. (2003) showed that IL-4 may be beneficial against L. donovani infection, and that mice knockout for the α chain of IL-4 receptor had a 89

retardation of granuloma maturation and an increase in parasite load both in the liver and spleen. 4. TREATMENT OF LEISHMANIASIS The first line of treatment is predominantly based on pentavalent antimonials, a classic treatment in most of the endemic areas. However, increasing resistance to antimonials is a major problem, and this is most evident in North Bihar, India, where the failure rate for this treatment is more than 50% (Murray, 2005; Sundar et al., 2003). The second line treatment includes drugs such as amphotericin B and pentamidine, which are characterized by high efficacy, but are relatively expensive and have severe side effects (Berman, 2003; Davis & Kedzierski, 2005). Amphotericin B is an effective treatment used in Sb(V)- resistant cases. It is toxic and needs to be given for a prolonged period on an inpatient basis. In deciding the best mode of treatment of CL, some facts need to be considered. Old World CL is not a life threatening disease and 90% of patients heal spontaneously within 3 18 months. The outcome of CL in the New World depends on the infecting species and may vary from benign to more severe manifestations. It is thus important to try to identify the infecting species, either by knowing the endemic species of the specific geographical area, or by means of diagnostic procedures. This can throw light on the prognosis and management options. Treatment of CL will accelerate cure and reduce scarring. This is especially important at cosmetically important sites. Options in the treatment of CL include local or systemic treatment. Criteria in favour of local treatment (Blum et al., 2004) include: Old world CL: small, single lesions; lack of risk of development of mucocutaneous leishmaniasis, lack of lymph node metastases; and L. mexicana lesions. New world lesions except L. mexicana, mucosal or 90

lymph node involvement and lesions refractory to local treatment would be indications for systemic treatment. 5. VACCINE AGAINST LEISHMANIASIS According to Kedzierski et al. (2006), leishmaniasis in general, but particularly CL, is probably one of a few parasitic diseases that is most likely to be controlled by vaccines. The relatively uncomplicated leishmanial life cycle and the fact that recovery from a primary infection renders the host resistant to subsequent infections indicate that a successful vaccine is feasible. Extensive evidence from studies in animal models, mainly mice, indicates that solid protection can be achieved upon immunization with defined protein or DNA vaccines. During the past several decades, extensive efforts have been made to search for an effective Leishmania vaccine. Vaccine formulations including killed, live attenuated parasites, recombinant Leishmania proteins or DNA encoding leishmanial proteins, as well as immunomodulators from sandfly saliva have been examined. Although to date, there is no vaccine against Leishmania, several of the vaccine preparations are at advanced stages of clinical testing. In this in case, as discussed for Kedzierski et al., (2006), an ideal anti- Leishmania vaccine would need to possess several attributes, but not all of them may be easily achievable. These include: i) safety; ii) affordability to the populations in need; iii) induction of CD4+ and CD8+ T cell responses and longterm immunological memory that can be boosted by natural infections, thus minimizing the number of immunizations; iv) effectiveness against species causing CL and VL; v) stability at room temperature eliminating the need for a cold chain to preserve potency and; vi) effectiveness as a prophylactic as well as a therapeutic vaccine. 91

There is no effective vaccine for prevention of leishmaniasis. The closest effective alternative to vaccination follows from the traditional leishmanization technique adopted in the Middle East and Eastern Europe. This involved the encouragement of sandfly bites on traditionally unexposed areas of skin, such as the buttocks. The resulting lesion would heal spontaneously, in the process providing immunity against CL in less acceptable areas, such as the face. Early studies recommended using L. tropica inoculations to induce immunity (Berberian, 1939). This technique has been further developed in Iran, where scientists have produced standardised and stable L. major populations which can produce more consistent and acceptable iatrogenically-induced lesions (Davies et al., 2003). The safety and efficacy of live-attenuated and killed vaccines has been debated and shifts in favour of development some of these has been recorded in recent years. Killed vaccines were favored in the 1990s because of safety problems with liveattenuated vaccines; however, recent advances in manipulation of the Leishmania genome may make development of a live attenuated vaccine more feasible (Handman, 2001). Work on recombinant DNA derived antigen vaccines and protein or peptide-based vaccines is a more recent approach, made possible by the Leishmania Genome Project (www.sanger.ac.uk/projects/l_major/). The realization that CD8 cells are as important as CD4 cells in inducing resistance (Belkaid et al., 2002) and maintaining immunity has led to a shift in vaccine research (Rhee et al., 2002). Most vaccine research is targeted against CL; any effectiveness against VL is uncertain. Work on a vaccine against human VL has been less successful, (Khalil et al., 2000) but should be boosted following success with a CL vaccine. While the cost-effectiveness and safety issues can be relatively straightforward to resolve, the induction and maintenance of the required immune responses are much more difficult to solve and cross-species protection may not be achieved by the same vaccine. 92

6. CONCLUSION Based in what it was displayed, leishmaniasis remains a problematic infection requiring either potentially toxic treatments or less toxic, but expensive drugs. However, the availability of newer oral agents may change the way this disease is managed. Relapse may occur, especially in situations where immunosuppression is present; secondary prophylaxis needs to be given in this setting. The combination of Leishmania, HIV and anthroponotic transmission between injecting drug users heralds a potential for higher incidence rates in endemic countries with severe drug abuse problems. In the absence of an effective vaccine, and with extension of endemicity, possibly due to climate change, these problems may become worse. Current obstacles to realistic prevention and proper management include inadequate vector (sandfly) control, no vaccine, and insufficient access to or impetus for developing affordable new drugs. 7. ACKNOWLEDGEMENTS I thank Msc. Aline S. L. Rodrigues for a careful revision of the article and for fruitful advice. 8. REFERENCES 1. ALEXANDER, J.; BRYSON, K. T helper (h)1/th2 and Leishmania: paradox rather than paradigm. Immunology Letters, v. 99, p. 17-23, 2005. 2. ALVAR, J.; CANAVATE, C.; MOLINA, R.; MORENO, J.; NIETO, J. Canine leishmaniasis. Adv Parasitol, v. 57, p. 1-88, 2004. 3. ARIAS, J.R.; MONTEIRO, P.S.; ZICKER, F. The re-emergence of visceral leishmaniasis in Brazil. Emerg Infect Dis, v. 2, p. 145-6, 1996. 93

4. BELKAID, Y.; VON STEBUT, E.; MENDEZ, S., et al. CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J Immunol, v. 168, p. 3992-4000, 2002. 5. BERMAN, J. Current treatment approaches to leishmaniasis. Current Opinion In Infectious Diseases, v. 16, p. 397-401, 2003. 6. BERBERIAN, D.A. Vaccination and immunity against oriental sore. Trans R Soc Trop Med Hyg, v. 33, p. 87-8. 1939. 7. BLUM, J.; DESJEUX, P.; SCHWARTZ, E., et al. Treatment of cutaneous leishmaniasis among travellers. J Antimicrob Chemother, v. 53, p. 158-66, 2004. 8. BRASIL. Ministério da Saúde. Manual de Vigilância da Leishmaniose Tegumentar Americana. Brasília: Editora do Ministério da Saúde, 2007. 9. DAVIES, C.R.; KAYE, P.; CROFT, S.L., et al. Leishmaniasis: new approaches to disease control. BMJ, v. 326, p. 377-82, 2003. 10. DAVIS, A.J.; KEDZIERSKI, L. Recent advances in antileishmanial drug development. Current Opinion in Investigational Drugs, v. 6, p. 163-169, 2005. 11. DESJEUX, P. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis, v. 27, p. 305-18, 2004. 12. DESJEUX, P. The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg, v. 95, p. 239-43, 2001. 13. ENGWEDA, C.R.; ATO, M.; KAYE, P.M. Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends in Parasitology, v. 20, n.11, p. 524-530, Nov. 2004. 14. GHOSH, A.; ZHANG, W.W.; MATLASHEWSKI, G. Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice 94

against Leishmania donovani infections. Vaccine, v. 20, n. 1-2, p. 59-66, Oct. 2001. 15. GOSSAGE, S.M.; ROGERS, M.E.; BATES, P.A. Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle. In J Parasitol, v. 33, p. 1027-1034, 2003. 16. GRECH, V.; MIZZI, J.; MANGION, M., et al. Visceral leishmaniasis in Malta-an 18 year paediatric, population based study. Arch Dis Child, v. 82, p. 381-5, 2000. 17. HANDMAN, E. Leishmaniasis: current status of vaccine development. Clin Microbiol Rev, v. 14, p. 229-43, 2001. 18. HANDMAN, E.; BULLEN, D. Interaction of Leishmania with the host macrophage. Trends in Parasitology, v 18, p. 332-335, 2002. 19. HEINZEL, F.P.; SADICK, M.D.; HOLADAY, B.J., et al. Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets. J Exp Med, v. 169, p. 59-72, 1989. 20. KAMHAWI, S. Phlebotomine sand flies and Leishmania parasite: friends or foes? Trend in Parasitology, v. 22, n. 9, p. 439-445, Jul. 2006. 21. KAYE, C.L.; CURRY, A.J.; BLACKWELL, J.M. Differential production of Th1- and Th2-derived cytokines does not determine the genetically controlled or vaccine-induced rate of cure in murine visceral leishmaniasis. J Immunol, v. 146, p. 2763-2770, 1991. 22. KEDZIERSKI, L.; HANDMAN, E. Leishmania vaccines: progress and problems. Parasitology, v. 133, p. S87-S112, 2006. 23. KHALIL, E.A.G.; El HASSAN, A.M.; ZIJLSTRA, E.E., et al. Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a 95

randomised, double-blind, BCG-controlled trial in Sudan. Lancet, v. 356, p. 1565-9, 2000. 24. KILLICK-KENDRICK, R. The life-cycle of Leishmania in the sandfly with special references to the form infective to the vertebrate host. Annales de Parasitologie Humaine et Compareé, v. 65, n. suppl. 1, p. 37-42, 1990. 25. KUHN, K.G. Global warming and leishmaniasis in Italy. Bull Trop Med Int Health, v. 7, p. 1-2, 1999. 26. LAINSON, R.; SHAW, J.J. Evolution, classification and geographical distribution. In: PETTER, W., KILLICK-KENDRICK, R. (Eds.) The leishmaniasis in biology and medicine. Vol 1 Biology and Epidemiology. New York: Academic Press, 1987. 27. LEHMANN, J.; ENSSLE, K.H.; LEHMANN, I.; EMMENDORFER, A.; LOHMANN-MATTHES, M. L. The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice. J Interferon Cytokine Res, v. 20, p. 63-77, 2000. 28. Malafaia G. O sinergismo entre a desnutrição protéico-calórica e a leishmaniose visceral. Revista Saúde.Com, 2008a (in press). 29. Malafaia G. Protein-energy malnutrition decreases immune response to Leishmania chagasi vaccine in BALB/c mice. Parasite Immunology. 2008b (in press). 30. MANDELL, G.L.; et al. Principles and practice of infectious diseases, 6th edn. Philadelphia, PA: Elsevier Churchill Livingstone, 2005. 31. MCMAHON-PRATT, D.; ALEXANDER, J. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev, v. 201, p. 206-24, 2004. 96

32. MIRALLES, G.D.; STOECKLE, M.Y.; MCDERMOTT, D.F.; FINKELMAN, F.D.; MURRAY, H.W. Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis. Infect Immun, v. 62, p. 1058-1063, 1994. 33. MURRAY, H.W. Kala-azar-progress against a neglected disease. N Engl J Med, v. 22, p. 1793-4, 2002. 34. MURRAY, H.W.; BERMAN, J.D.; DAVIES, C.R.; et al. Advances in leishmaniasis. Lancet, v. 366, p. 1561-77, 2005. 35. RAMIRO, M.J.; ZARATE, J.J.; HANKE, T.; et al. Protection in dogs against visceral leishmaniasis caused by L. infantum is achieved by immunization with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK. Vaccine, v. 21, p. 2474-2484, 2003. 36. REED, S.G.; SCOTT, P. T-cell and cytokine responses in leishmaniasis. Curr Opin Immunol, v. 5, p. 524-31, 1993. 37. RHEE, E.G.; MENDEZ, S.; SHAH, J.A.; et al. Vaccination with heat-killed Leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J Exp Med, v. 195, p. 1565-73, 2002. 38. REINER, S.L.; WANG, Z.E.; HATAM, F.; et al. Th1 and Th2 cell antigen receptors in experimental leishmaniasis. Science, v. 259, p. 1457-60, 1993. 39. ROBERTS, L.J.; HANDMAN, E.; FOOTE, S.J. Leishmaniasis. BMJ, v. 321, p. 801-4, 2000. 40. ROGERS, K.A.; DEKREY, G.K.; MBOW, M.L.; GILLESPIE, R.D.; BRODSKYN, C.I.; TITUS, R.G. Type 1 and type 2 responses to Leishmania major. FEMS Microbiology Letters, v. 209, p. 1-7, 2002. 97

41. SAHA, B.; BASAK, S.K.; ROY, S. Immunobiological studies on experimental visceral leishmaniasis. III. Cytokine-mediated regulation of parasite replication. Scandinavian J Immunol, v. 37, p. 155-158, 1993. 42. STAGER, S.; ALEXANDER, J.; CARTER, K.C.; BROMBACHER, F.; KAYE, P.M. Both interleukin-4 (IL-4) and IL-4 receptor alpha signaling contribute to the development of hepatic granulomas with optimal antileishmanial activity. Infect Immun, v. 71, n. 8, p. 4804-4808, 2003. 43. SUNDAR, S.; REED, S.G.; SHARMA, S.; et al. Circulating T helper 1 (Th1) celland Th2 cell-associated cytokines in Indian patients with visceral leishmaniasis. Am J Trop Med Hyg, v. 56, p. 522-5, 1997. 44. SUNDAR, S.; JHA, T.K.; THAKUR, C.P.; et al. Single-dose liposomal amphotericin B in the treatment of visceral leishmaniasis in India: a multicenter study. Clin Infect Dis, v. 37, p. 800-4, 2003. 45. VON STEBUT, E.; UDEY MC. Requirements fot Th1-dependent immunity against infection with Leishmania major. Microbes and Infection, v. 6, n. 12, p. 1102-1109, Oct. 2004. 46. ZIJLSTRA, E.E.; EL-HASSAN, A.M. Visceral leishmaniasis. Trans R Soc Trop Med Hyg, v. 95, n. Suppl. 1, p. S27-S58, 2001. 47. WORLD HEALTH ORGANIZATION (WHO). Program for the surveillance and control of leishmaniasis. World Health Organization, Geneva, 2002. Disponível em http://www.who.int/tdr/diseases/leish/diseaseinfo.htm. Acesso em: 20 fev. 2008. 48. YAMEY, G. Public sector must develop drugs for neglected diseases. BMJ, v. 324, 2002. 98