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Leishmaniasis Silvia Reni B. Uliana. Leishmania Family Trypanosomatidae Genus Leishmania Sub-genera Leishmania Viannia More than 30 species already described.

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Presentation on theme: "Leishmaniasis Silvia Reni B. Uliana. Leishmania Family Trypanosomatidae Genus Leishmania Sub-genera Leishmania Viannia More than 30 species already described."— Presentation transcript:

1 Leishmaniasis Silvia Reni B. Uliana

2 Leishmania Family Trypanosomatidae Genus Leishmania Sub-genera Leishmania Viannia More than 30 species already described cutaneous leishmaniasis visceral leishmaniasis

3 Geographical Distribution 14 million cases each year, distributed in 88 countries (350 million people at risk). cutaneous leishmaniasis visceral leishmaniasis

4 Visceral leishmaniasis / AIDS

5 Life cycle Mammal Insect bite Reservoir Amastigote Promastigote Assexual (MAN)

6 Promastigotes Extracellular forms living in the intestine of the mosquito insect In the mosquito’s gut they go through a differentiation process called metacyclogenesis.

7 Amastigote Intracellular forms “Ideal” host cell: macrophage Amastigotes are found inside macrophages at various sites: skin, lymph nodes, spleen, liver, bone marrow.

8 Mecanismo de infecção O vetor ingere macrófagos infectados com o sangue ingerido Os amastigotas são liberados no intestino do vetor e transformam- se em promastigotas procíclicos. Estes se reproduzem inicialmente por divisão binária e posteriormente se diferenciam em promastigotas metacíclicos. O macrófago parasitado eventualmente se rompe, liberando amastigotas que passam a infectar outros macrófagos, circulantes ou teciduais. O hospedeiro vertebrado se infecta pela picada do vetor. Os promastigotas invadem macrófagos circulantes ou teciduais e se reproduzem como amastigotas.

9 Clinical syndromes Cutaneous leishmaniasis – L. braziliensis, L. amazonensis, L. guyanensis L. major Diffuse leishmaniasis – L. amazonensis Mucocutaneous leishmaniasis – L. braziliensis Visceral leishmaniasis – L. chagasi, L. donovani New World species Old World species

10 Cutaneous leishmaniasis L. amazonensis L. mexicana L. guyanensis L. braziliensis Reservoir: wild rodents, marsupials (opossum), sloth, anteater

11 Diffuse leishmaniasis Reservoir: wild rodents L. amazonensis

12 Mucocutaneous leishmaniasis Reservoir: ? Wild rodents L. braziliensis

13 Visceral leishmaniasis L. chagasi L. donovani L. infantum Reservoir: fox, dog, humans

14 Some tricks performed by Leishmania S.C. Ilgoutz, M.J. McConville / International Journal for Parasitology 31 (2001) 899±

15 Some tricks performed by Leishmania -LPG multiplication inside the gut – adherence - procyclic being “available” to take the next ride - metacyclic form McConville MJ in Molecular Biology of Parasitic Protozoa, ed. DF Smith, M Parsons

16 O’Garra and Arai (2000) Trends Cell Biol. Immune system Complement Lysis complex C3bi

17 LPG and complement the innate defense mechanisms… lysis complex

18 “getting home…” - the macrophage! inhibition of the respiratory burst Interaction with C3bi receptor via complement and gp63 burst

19 Fusion with lisossomes: gp63 is a protease (other cysteino-proteases) LPG and other GL induce the production of TGF-  and IL-10 AND… inhibit the production of NO “shall we make it cosy?…” – the parasitophorous vacuole “the macrophage army is ready…” DISEASE

20 Diagnosis Biopsy – low sensitivity which species ? Culture – slow isoenzymes, monoclonal antibodies PCR ELISA – cross-reaction, sensitivity

21 Treatment pentavalent antimonials anphotericin B TOXICITY TIME RESISTANCE

22 RESERVOIR - wild PATIENT - treatment VACCINE CONTROL VECTOR – partially effective in urban areas

23 Clinical syndromes Cutaneous leishmaniasis – L. braziliensis, L. amazonensis, L. guyanensis L. major Diffuse leishmaniasis – L. amazonensis Mucocutaneous leishmaniasis – L. braziliensis Visceral leishmaniasis – L. chagasi, L. donovani New World species Old World species What is the difference ? Why do they sometimes behave “badly” ?

24 L. donovani L. tarentolae C. fasciculata T. cruzi T. brucei E. gracilis L. (L.) amazonensis L. (L.) chagasi L. (L.) donovani L. tarentolae Crithidia fasciculata Orlando et al 2002 MIOC Briones et al 1992, MBP

25 Immune-deficiency associated Primary infection (with or without symptoms) AIDS Transplantation “reactivation” (atypical clinical picture) Persistence ? Where ? How ?

26 VIRULENCE Ilgoutz and McConville (2001) Int J Parasitol How do they do all this ?

27 L.maj MEMKNLLGKH KIVSVNGKPA PAGVTVEFKA SENSGSVYMH L.don....H......V.L...R G......H.. L.ama....S.I..R RVL....R TIQ L.maj AKVANIMNGP LKLANRKLSG ALVSTMMLGS DDLMNIENAL L.don.R Q.R.E L.ama.N...F...Q...G T L.maj SQGFMEGMTY TVKDGGKLTL QSKTHIIMLV PA* L.don K....* L.ama I....D......H K.N..T.K....* 2.1 kb L S rRNA Characterisation of the meta 1 gene in L. major Single copy gene RNA expression upregulated in metacyclic promastigotes Gene conserved in both Old and New World Leishmania species

28 Deduced meta 1 aminoacid sequence No significant aminoacid or nucleotide identity with any known protein. No glycosilation sites. Hydrophilicity plot with no distinguishing features. Predicted molecular mass kDa.

29 What is the function of the meta 1 protein in Leishmania ? A- Lines with double replacement of the meta 1 gene were not viable ESSENTIAL GENE

30 B- Overexpressing the meta 1 protein High level expression of the L. major meta 1 protein was obtained in L. amazonensis promastigotes, by stable maintenance of an episome. The levels of expression were high in both log phase and stationary phase promastigotes. L.maj LaHE _ _ _ kDa L S A L S A AI M

31 C- Testing the recombinant lines for a phenotype No change in growth curves No differences in in vitro infection of macrophages

32 Lesion Index Weeks BALB/c 10 7 promastigotes WT ARHyg Hyg3’ HE A BALC/c 10 6 amastigotes Lesion Index Weeks WT HE B C57Bl/ promastigotes Lesion index Weeks WT HE C Meta 1 overexpressor in vivo

33

34 L. major Chromosome 16


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