Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis

Abstract INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features

ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Leishmanioses do continente americano / Leishmaniasis in the Americas

No Brasil, ela já foi responsável por epidemias em diferentes cidades. Nos últimos dez anos, sua letalidade aumentou em diversas regiões do país. Em várias partes do mundo ela vem se expandindo, inclusive em lugares onde, anteriormente, não havia registro de transmissão. A infecção por parasitos do gênero Leishmania causa uma das doenças tropicais mais negligenciadas da atualidade. Estima-se que existam 350 milhões de pessoas em risco de contrair a infecção, sobretudo nas áreas mais pobres do planeta, e que dois milhões de novos casos de leishmanioses ocorram a cada ano. Esse grave cenário justifica o esforço empreendido pelos organizadores e demais pesquisadores do Instituto Oswaldo Cruz (IOC/Fiocruz) especialistas no assunto, assim como por profissionais de outras unidades da Fiocruz e instituições brasileiras: eles prepararam uma coletânea que compila o conhecimento já existente sobre o assunto, identifica os principais desafios e discute estratégias para enfrentá-los...

Immunohistochemical profile of HIF-1α, VEGF-A, VEGFR2 and MMP9 proteins in tegumentary leishmaniasis / Estudo da expressão imunohistoquímica das proteínas HIF-1α, VEGF-A, VEGFR2 e MMP9 em leishmaniose tegumentar americana

BACKGROUND: Leishmaniasis is one of the most important infectious diseases worldwide. Our study can provide more knowledge about angiogenic and hypoxic events in leishmaniasis. We attempted to verify whether the HIF-1 α protein expression may be associated to VEGF-A, VEGFR2 and MMP9 in leishmanial lesions. OBJECTIVES: Besides understanding the pathway, we performed the correlation of VEGF-A, VEGFR2 and MMP9 proteins. METHODS: In this study, we gathered 54 paraffin blocks taken from skin lesions in patients from northern Minas Gerais, Brazil, with confirmed diagnosis of tegumentary leishmaniasis. Immunohistochemistry was used to evaluate the expression of the proteins. The expression of HIF-1α was categorized into two groups according to the median: HIF-1 α lower and HIF-1 α higher. RESULTS: We observed increase of VEGFR2 and MMP9 protein expressions in HIF-1 α higher group of epithelial cells. Spearman analyses in epithelial cells showed correlation between VEGF-A and MMP9, VEGFR2 and MMP9 protein expression. CONCLUSIONS: HIF-1 α higher group showed increase of VEGFR2 and MMP9 proteins. In epithelial cells, VEGF-A was correlated to MMP9 protein. Furthermore, considering leukocyte cells, VEGFR2 was negatively correlated to MMP9 protein levels. This pathway possibly prepares the cells for a higher activity in a hypoxic or an angiogenic microenvironment. Other in vitro and in vivo studies may clarify the activation mechanism and the response from the proteins HIF-1 α, VEGFR2 and MMP-9 in tegumentary leishmaniasis.

FUNDAMENTOS: A leishmaniose é uma das mais importantes doenças infecciosas em todo o mundo. Em leishmaniose, tem sido sugerido que muitas características da lesão está associado a eventos de hipóxia, podendo este ter um papel fundamental na evolução da doença. OBJETIVO: O presente estudo pode fornecer dados acerca do fenômeno hipóxia e da angiogênese em leishmaniose tegumentar americana. Buscou-se verificar se a expressão da proteína HIF-1 α associa-se à expressão das proteínas VEGF-A, VEGFR2 e MMP9 em lesões de Leishmania sp. MÉTODOS: Neste estudo retrospectivo, foram utilizados 54 blocos de parafina de lesões de leishmaniose tegumentar americana de pacientes do norte de Minas Gerais, Brasil, com diagnóstico confirmado de leishmaniose tegumentar americana. A técnica de imunohistoquimica foi utilizada para avaliação da expressão das proteínas. A expressão da HIF-1α foi categorizada em dois grupos de acordo com a mediana: HIF-1 α abaixo e HIF-1 α acima da mediana. RESULTADOS: Observamos aumento das expressões das proteínas VEGFR2 e MMP9 no grupo HIF-1 α acima da mediana. A análise de Spearman demonstrou correlação entre as proteínas VEGF-A e MMP9, VEGFR2 e MMP9. CONCLUSÃO: Os dados aqui apresentados indicam uma alta expressão da proteína HIF-1 α em LTA. O grupo HIF-1α acima da mediana apresentou maior expressão das proteínas VEGFR2 e MMP9. Foi demonstrada correlação entre as proteínas VEGF-A e MMP9, VEGFR2 e MMP9. Outros estudos in vitro e in vivo devem ser realizados a fim de esclarecer o mecanismo de ativação e resposta das proteínas HIF-1 α, VEGFR2 e MMP-9 em leishmaniose tegumentar americana.

Evaluation of myofibroblasts and its association with TGF-β and IFN-γ in lesions of patients with american tegumentary leishmaniasis / Avaliação de miofibroblastos e sua associação com TGF-β e IFN-γ em lesões de pacientes com leishmaniose tegumentar americana

BACKGROUND: Leishmaniasis is caused by protozoa of Leishmania spp. genus. It is transmitted by the bite of the sand fly insect. It is believed that 12 million people are infected with this disease and that its annual incidence is 2 million; this number is increasing. OBJECTIVES: The present study aimed to evaluate the expression of myofibroblasts through alpha smooth muscle actin labeling, and to analyze their relationship with the expression of the cytokines Interferon gama (IFN-γ) and Transforming growth factor beta (TGF-β1) in lesions of American tegumentary leishmaniasis (ATL). METHODS: For this retrospective study, we gathered 28 patients diagnosed with ATL between 2002 and 2006. We verified α-SMA positivity and performed IFN-γ and TGF-β1 immunolabeling to identify the profile of these cytokines in both positive and negative cases for myofibroblasts, via immunohistochemistry, in order to assess the presence of myofibroblasts,. RESULTS: We observed that out of the 28 cases, 17 (60.71%) were positive for alpha smooth muscle actin, while 11 (39.29%) were negative, and IFN-γ was more expressed than TGF-β1 (p=0.007). The mean percentages of expression of IFN-γ and TGF-β1 in the group negative for alpha smooth muscle actin were different, with an increased expression of IFN-γ (p=0.047). However, in the group positive for alpha smooth muscle actin, there was no difference in cytokine labeling (p>0.05). CONCLUSION: We verified the presence of positive α-SMA stromal cells in the majority of the cases of ATL, indicating the presence of myofibroblasts. In cases negative for alpha smooth muscle actin, an increased expression of IFN-γ compared to TGF-β1 was observed, revealing an inflammatory phase progressing to a healing process.

FUNDAMENTOS: A leishmaniose é causada pelo protozoário do gênero Leishmania spp., sendo transmitida via picada do inseto flebotomíneo. Estima-se que 12 milhões de indivíduos estejam infectados com a doença, sendo a incidência anual de 2 milhões, número este que tende a aumentar. OBJETIVOS: Avaliar a expressão de miofibroblastos através da imunomarcação de actina de músculo liso alfa, e analisar sua relação com a expressão de citocinas IFN-γ e TGF-β1 nas lesões de pacientes com leishmaniose tegumentar americana. MÉTODOS: Trata-se de um estudo retrospectivo, em que foram avaliados 28 pacientes diagnosticados com leishmaniose tegumentar americana durante o período de 2002 a 2006. Na técnica de imuno-histoquímica avaliou-se a presença de miofibroblastos, através do marcador actina de músculo liso alfa, além da imunomarcação do IFN-γ e TGF-β1 para identificar o perfil dessas citocinas nos casos positivos e negativos para miofibroblastos. RESULTADOS: Observou-se que dos 28 casos, 17 (60,71%) foram positivos para actina de músculo liso alfa, enquanto 11 (39,29%) foram negativos. IFN-γ teve uma maior expressão do que TGF-β1 (p=0,007). A porcentagem média de expressão de IFN-γ e TGF-β1 no grupo negativo para actina de músculo liso alfa foi diferente, apresentando uma maior expressão de IFN-γ (p=0.047). Entretanto, o grupo positivo para actina de músculo liso alfa não apresentou uma diferença estatisticamente significativa (p>0,05). CONCLUSÃO: Verificou-se uma expressão de actina de músculo liso alfa nos casos de leishmaniose tegumentar americana, indicando a presença de miofibroblastos. Nos casos negativos para actina de músculo liso alfa, observou-se uma maior expressão de IFN-γ comparando com TGF-β1, revelando que a fase inflamatória está envolvida no processo de cicatrização da lesão.

Expression of TLR2 and TLR4 in lesions of patients with tegumentary american leishmaniasis / Expressão de TLR2 e TLR4 em lesões de pacientes com leishmaniose tegumentar americana

OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-α, anti-IFN-Γ, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 ± 82.46 cell/mm² and 3.26 ± 4.11 cell/mm² respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.

OBJETIVOS: O objetivo deste estudo foi descrever o padrão de expressão dos receptores toll-like 2 e 4 (TLR2 e TLR4) em biópsias de pele de pacientes com leishmaniose tegumentar americana (LTA). MÉTODOS: Este estudo prospectivo avaliou 12 pacientes com LTA causada por Leishmania braziliensis confirmada por reação em cadeia da polimerase. Imunohistoquímica foi realizada para determinar a expressão de TLR2 e TLR4. O número de células NK, células dendríticas e macrófagos foi calculado no tecido. A expressão de citocinas foi determinada usando anti-TNF-α, anti-IFN-Γ, anti-IL-1 e anti-IL-6. Dupla marcação foi usada para determinar a célula responsável pela expressão de TLR2 e TLR4. RESULTADOS: O número de células expressando TLR2 e TLR4 foi 145.48±82.46 cell/mm² e 3.26 ± 4.11 cell/mm² respectivamente (p < 0.05). Não houve correlação entre a quantidade de expressão de TLR2 e TLR4 com a quantidade de citocinas e o número de células NK, macrófagos e células dendríticas. A dupla marcação revelou que o TLR2 é expresso por macrófagos. CONCLUSÃO: Na LTA causada por Leishmania braziliensis, TLR2 é o TLR mais comum na doença ativa, principalmente por macrófagos sem correlação com a quantidade de citocinas e outras células.

Aspectos comparativos da resposta inflamatória em lesões de leishmaniose cutânea localizada e disseminada / Comparative aspects of the inflammatory response in lesions of cutaneous leishmaniasis and disseminated

A forma clínica mais frequente das leishmanioses é a leishmaniose cutânea, que acomete somente a pele e constitui um importante problema de saúde no Brasil. A leishmaniose cutânea caracteriza-se por lesão cutânea ulcerada única: leishmaniose cutânea localizada, que pode regredir espontaneamente ou se disseminar com múltiplas úlceras e pápulas e que aparecem em diferentes locais. A presença de disseminação caracteriza a leishmaniose cutânea disseminada. Investigar, no tecido, células que compõem o infiltrado inflamatório em lesões de pele na leishmaniose e caracterizar a resposta imune e sua correlação com a extensão total da inflamação in situ pode contribuir para aprofundar o entendimento da leishmaniose cutânea. Neste estudo, através da imunomarcação e quantificação de células por imunoistoquímica e HE, e análise da extensão da inflamação, foi comparado a histopatologia e presença de células inflamatórias CD4+, CD8+, CD68+, CD20+, plasmócitos e neutrófilos, e células granzima B+ em biópsias de pacientes com leishmaniose cutânea tardia (úlcera tardia e úlcera recente) e leishmaniose disseminada (úlcera e pápula). A análise dos padrões histomorfológicos mostrou semelhança entre os quatro grupos de biópsias analisados. Avaliando o número e tipo de células presentes, o predomínio foi de macrófagos e infócitos. As úlceras da leishmaniose cutânea clássica e disseminada apresentaram média maior de inflamação, maior frequência de linfócitos T CD4+, T CD8+, macrófagos, linfócitos B CD20+ e plasmócitos que úlceras recentes e pápulas. Estas, por sua vez, ao contrário das úlceras tardias de ambas formas clínicas, tiveram correlação positiva entre o aumento do infiltrado inflamatório e T CD4+ e T CD8+, e não correlação com granzima B e neutrófilos. Os plasmócitos se mostraram elementos quase constantes no infiltrado das lesões em todos os grupos, e sua frequência foi maior que de linfócitos B. Seguindo o mesmo padrão descrito nos linfócitos B, os plasmócitos não apresentam correlação com o influxo de infiltrado inflamatório entre os grupos. A frequência de macrófagos é vista nos dois estágios avaliados da doença e nas duas formas clínicas de forma semelhante, porém com uma tendência para maior presença em lesões de LD. A análise de neutrófilos revelou semelhança da frequência dessas células em todos os tipos de lesões em ambas formas clínicas, com as pápulas tendendo para uma menor quantidade. Portanto, o desenvolvimento das lesões ocorrem com o influxo de células inflamatórias, como linfócitos T CD4+ e T CD8+ e a resposta imune celular é mais intensa em lesões crônicas da leishmaniose cutânea localizada e disseminada do que em lesões localizadas recentes e pápulas da leishmaniose disseminada. Diferenças in situ na resposta inflamatória destas duas formas clínicas e quatro espectros de lesão da leishmaniose humana podem elucidar o papel de células no local da lesão e contribuir para o entendimento da patogênese da leishmaniose.

Cutaneous leishmaniasis is the most frequent clinical form of leishmaniasis, which affects only the skin and is an important health problem in Brazil. Cutaneous leishmaniasis is characterized by single ulcerated skin lesion: localized cutaneous leishmaniasis, which may regress spontaneously or spread with multiple ulcers and papules that appear in different parts of the body. The presence of dissemination features disseminated leishmaniasis. Investigate tissue cells from inflammatory infiltrate in skin lesions in leishmaniasis and characterize the immune response and its correlation with the total extent of inflammation in situ may contribute to deepen the understanding of cutaneous leishmaniasis. In this study, immunostaining and cells quantification by immunohistochemistry and HE and analysis of inflammation extention was compared with histopathology and CD4+, CD8+, CD68+, CD20+, plasma cells, neutrophils and granzyme B+ cells in biopsies of patients with localized cutaneous leishmaniasis (late ulcer and recent ulcer) and disseminated leishmaniasis (ulcers and papules). The histopathological patterns analysis was similar among the four biopsies groups analyzed. Macrophages and lymphocytes were the predominant cells. The ulcers of localized cutaneous leishmaniasis and disseminated leishmaniasis presented higher mean inflammation, increased frequency of CD4+ and CD8+ T cells, macrophages, B lymphocytes CD20+ and plasma cells than recent ulcers and papules. These, in turn, unlike late ulcers of both clinical forms, had positive correlation between the increase in inflammatory infiltrate and CD4+ and CD8+ T cells, differing in Granzyme B and neutrophils. Plasma cells were almost constant in lesion infiltrate in all groups and was higher than the frequency of B lymphocytes. Following the same pattern described in B lymphocytes, plasma cells did not show an association with the influx of inflammatory infiltrate between groups. The frequency of macrophages is seen in both stages of the disease and in the two clinical forms similarly, but with a tendency to be increased in disseminated lesihmaniasis lesions. The analysis revealed similarity of neutrophils frequency in all types of lesions, with papules tending to a lesser extent. Therefore, the development of lesions occur with the influx of inflammatory cells such as CD4+ and CD8+ T cells and cellular immune response is more intense in chronic lesions of localized cutaneous leishmaniasis and disseminated leishmaniasis than recent localized lesions and papules from disseminated leishmaniasis. Differences of inflammatory response in situ in these two clinical forms and four spectra of human leishmaniasis lesions may elucidate the role of cells at the lesion site and contribute to the understanding of leishmaniasis pathogenesis.

Influence of the formulation type (o/w, w/o/w emulsions and ointment) on the topical delivery of paromomycin

Ointments containing paromomycin (PA) have been used for topical treatment of cutaneous leishmaniasis. Although influence of the vehicle on skin permeation is crucial for optimization of formulations containing PA, this aspect has not been investigated experimentally. In this study, we have evaluated the influence of the formulation type [oil-in-water (o/w), water-in-oil-in-water (w/o/w) emulsions and ointment] on in vitro release and skin permeation of PA and experiments were carried out on cellulose acetate membrane and skin biopsies from hairless mice, respectively. PA release from o/w emulsion (51.7 por cento ± 0.9 of dose applied) was faster than that observed for w/o/w emulsion (3.0 por cento ± 0.5)...

Transforming growth factor-ß in tegumentary leishmaniasis

1. The course of infection with the protozoan parasite Leishmania is determined in part by its early replication in macrophages, the exclusive host cells for these organisms. Resistance to and recovery from leishmanial infection is related to cell-mediated immune responses in all forms of human and murine leishmaniasis. 2. Factors contributing to the early inhibition or proliferation of Leishmania are poorly understood, but cytokines such as IFNÔ, IL-10 or transforming growth factor ß (TGF-ß) are known to influence the replication of Leishmania in macrophages. 3. TGF-ß is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Infection of murine or human macrophages by Leishmania induces the production of active TGF-ß. Recombinant TGF-ß added to murine or human macrophage cultures leads to increased intracellular replication of Leishmania. Exogenous TGF-ß administered in vivo promotes enhancement of infection, whereas its neutralization by monoclonal antibodies decreases the level of in vitro infection, and protects susceptible mice. 4. Susceptible animals treated with anti-TGF-ß monoclonal antibodies change their immune response, not increasing the expression of IL-4 while increasing the expression of IFNÔ mRNA in their draining lymph nodes. Resistant animals treated with TGF-ß also change their pattern of immune response as indicated by an increase of the important Th2 cytokine IL-10 mRNA in the draining lymph node. 5. TGF-ß has profound effects on the host response to Leishmania in both mouse and man, and probably is an important parasite escape mechanism