Leishmania (Leishmania) amazonensis: Experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice

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Experimental Parasitology 120 (2008) 123–125

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Research brief

Leishmania (Leishmania) amazonensis: Experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice Luiz Otávio Pereira Carvalho a, Kátia da Silva Calabrese a, Sylvio Celso Gonçalves da Costa a, Verônica Gonçalves Mendes a, Ana Patrícia de Carvalho da Silva b, Ana Caroline Ericeira Barros b, Solange de Araújo Melo b, Ana Lucia Abreu-Silva b,* a Laboratório de Imunomodulação e Protozoologia do Instituto Oswaldo Cruz/FIOCRUZ, Pavilhão Carlos Chagas, 3° andar, Av. Brasil, 4365, Manguinhos, CEP 20045-900, Rio de Janeiro, Brazil b Departamento de Patologia, Universidade Estadual do Maranhão, Cidade Universitária Paulo VI, S/N, Tirirical, CEP 65054-970, São Luís, Maranhão, Brazil

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Article history: Received 23 January 2008 Received in revised form 4 June 2008 Accepted 5 June 2008 Available online 14 June 2008 Keywords: Mice Cutaneous leishmaniasis Leishmania (L.) Amazonensis Systemic amyloidosis

a b s t r a c t We infected Swiss and C57BL/6 female mice in the left hind footpad with 104 Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Leishmania (L.) amazonensis is one of causative agents of human tegumentary leishmaniasis in New World. It is mainly causes with human cutaneous diffuse leishmaniasis (Lainson and Shaw, 1987), but it can also cause the mucosal form and it is capable of dissemination to internal organs, causing the visceral form. Depending on the mice genetic background of the mice, L. (L.) amazonensis can cause all three forms of leishmaniasis: cutaneous localized, mucocutaneous or visceral leishmaniasis (Barral et al., 1991). Amyloidosis is a disease characterized by extracellular deposition of a b folded protein called amyloid in several different organs and tissues. It has been associated with inflammatory diseases, several chronic infections (Röcken and Shakespeare, 2002; Obici et al., 2005) and parasitic diseases such as leishmaniasis (George et al., 1976; Poli et al., 1991). Secondary amyloidosis (AA) results from the accumulation of amyloid A fibrils formed from an acute phase reactant, serum amyloid AA (Keyur et al., 2006). Amyloidosis is chronic and usually progresses slowly over a number of years. The severity of the disease depends on which organs are affected by the amyloid deposits and can be potentially

* Corresponding author. Fax: +55 98 3257 3676. E-mail address: anabreu@ioc.fiocruz.br (A.L. Abreu-Silva). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.06.004

life-threatening when the kidneys and heart are affected. As the kidney is affected by leishmaniasis, an association of parasitism with amyloidosis can lead to organ dysfunction which results from disruption of tissue architecture by amyloid deposits. However, increasing evidence indicating that amyloidogenic precursor proteins or precursor aggregates have direct cytotoxic effects that also contribute to disease manifestations is emerging (Brenner et al., 2004). The aim of this paper is to describe generalized AA amyloidosis associated with L. (L.) amazonensis infection in both outbred and inbred mice, giving emphasis to lesions observed in ovary and inflammatory reactions in the pancreas and adrenal glands.

2. Materials and methods Twenty female Swiss Webster and twenty female C57BL/6 mice were infected in left hind footpad with 104 L. (L.) amazonensis promastigotes in stationary phase. Ten animals of each strain were clinically followed clinically up monthly, until 240 days post infection when macroscopic lesions were observed. At this time all animals were killed and necropsy was performed. Fragments of skin, draining lymph node, spleen, pancreas, adrenal gland, kidney, liver, and ovary were fixed in 10% formalin, routinely processed for embedding in paraffin wax and stained with Hematoxylin and

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Eosin (H&E) and Gomori’s Trichrome stains. This experiment was conducted in accordance to guidelines for experimental procedures of Fundação Oswaldo Cruz (Process no. P0062-00). 3. Results and discussion On macroscopic examination all ten Swiss mice presented with a nodular non-ulcerated lesion at the site of inoculation and enlargement of liver and spleen, which were pale in color. The kidneys and liver showed miliary whitish yellow foci within parenchyma. Histological study showed that primary lesion consisted of an extensive liquefactive necrosis area and great number of vacuolated and parasitized macrophages, eosinophils, and few lymphocytes (data not shown). The liver consisted of an inflammatory reaction with a deposition of a hyaline and amorphous substance deposition among the hepatic cells, which was confirmed by Congo red staining as amyloid. The same substance was observed in hepatic glomerular tufts and in the splenic marginal zone. All C57BL/6 mice showed lesions at the inoculation site, although only one-third of mice had systemic amyloidosis, including amyloid deposition in both ovarian cortical and medullary regions (Fig. 1). Unexpectedly, an inflammatory infiltrate composed of mononuclear cells in the pancreatic islet and adrenal gland was observed (Fig. 2) but no parasites were seen in these organs. However, as the mice presented with a generalized infection, the parasites must pass through in these tissues, inducing an inflammatory reaction. In secondary inflammation or inflammation-associated amyloid AA amyloidosis, inflammation plays a central role, as patients suffering from various chronic

inflammatory diseases. A major factor responsible for the development of AA amyloidosis is the increased synthesis and subsequent degradation of the precursor protein SAA1 under chronic inflammatory conditions (Ray et al., 2006). Amyloid is a proteinaceous accumulation commonly observed in chronic inflammatory diseases such as tuberculosis, leprosy, rheumatoid arthritis, and other degenerative diseases. Studies have shown that 45% of all generalized amyloidosis are secondary type or AA amyloidosis (Röcken and Shakespeare, 2002). It has been demonstrated that treatment with potassium permanganate is a good method to identify amyloid type. This method is based on amyloid sensitivity or resistance to KMnO4 oxidation. AA amyloid is ‘‘sensitive”, whereas the other types do not lose typical birefringence, even when exposed for a long time to treatment (Bely and Apathy, 2000). Amyloidosis has been described in spleen and liver of dogs naturally infected with L. (L.) infantum (Bely and Apathy, 2000; Poli et al., 1991). In hamsters, this pathology has been reported in adrenal gland (Novoa et al., 1990). Here, inflammatory infiltrates were observed in both medullary and cortical regions, however amyloidosis depositions were not demonstrated, suggesting passage of parasitic antigen though these organs but without evidence of parasites. Previous studies showed that Swiss mice infected with L. (L.) amazonensis amastigotes had developed amyloidosis in spleen, liver, and kidney 10 months post-infection (Barbosa-Santos et al., 1984). In conclusion, our results have shown that leishmaniasis directly or indirectly could lead to dysfunction in reproductive organs, based on the presence of amyloid deposition in the ovary during the chronic phase of this disease.

Fig. 1. Histopathology of liver, kidney, spleen, and ovary from females C57BL/6 mice infected in left hind footpad with 104 L. (L.) amazonensis promastigotes. (A) Liver: Amyloid deposition among hepatic cells (*) associated to inflammatory reaction composed of mononuclear cells (arrow)—Hematoxylin–Eosin—Scale bars 100 lm; (B) Kidney: Amyloid deposits in glomerular tufts (*) and inflammatory reaction (arrow). Hematoxylin–Eosin—Scale bars 100 lm; (C) Spleen: amyloid deposits in the perifollicular area (*)—Gomori’s Trichrome—Scale bars 100 lm; (D) Ovary: amyloid deposition in both cortical and medullar regions (*). Gomori’s Trichrome-Scale bars 100 lm.

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Fig. 2. Histopathology of pancreas and adrenal from females C57BL/6 mice infected in left hind footpad with 104 L. (L.) amazonensis promastigotes—Hematoxylin–Eosin. (A) Pancreas: Inflammatory infiltrates in a pancreatic islet (arrow)—Scale bars 100 lm; (B) In high magnification it was observed mononuclear cells were observed —Scale bars 100 lm; (C) Discrete inflammatory reaction (arrow) in the medulla of the adrenal—Scale bars 100 lm; (D) Inflammatory reaction in the cortical region (arrow) of the adrenal gland—Scale bars 100 lm. Window shows inflammatory cells in high magnification.

Acknowledgments We would like to thank Arlindo Caldeira da Rocha (in memoriam) and Lia Abreu de Souza for histological preparations. Equally we thank the instrumental support given by Call Zeiss of Brazil. This work was supported by grants from Instituto Oswaldo Cruz/ Ministry of Health, Brazil.

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