RESUMO
Recent studies indicate that Acanthamoeba spp. may play a significant role in kidney dysfunction. The aim of the study was to examine the levels of kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and monocyte chemotactic protein 1 (MCP-1), as well as an activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively) in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. The levels of KIM-1, NGAL, and MCP-1 were analyzed by enzyme-linked immunosorbent assay (ELISA), and the activity of MMPs was determined by gelatin zymography. The elevated KIM-1 level was found in the kidneys of immunocompetent mice at the beginning of Acanthamoeba spp. infection. In the immunosuppressed mice, the KIM-1 level was statistically different. The statistically decreased NGAL level was found in the kidneys of immunocompetent mice compared to the uninfected mice. In the immunocompromised mice, we found statistically significant differences in MCP-1 levels between the uninfected and infected groups. There was an increase in the expression of both MMP-2 and MMP-9 in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. compared to the uninfected mice. The results indicate that KIM-1, NGAL, MCP-1, MMP-2, MMP-9, and MMP-9/NGAL might be promising biomarkers of renal acanthamoebiasis.
Assuntos
Acanthamoeba , Amebíase/metabolismo , Biomarcadores/metabolismo , Nefropatias/metabolismo , Amebíase/diagnóstico , Amebíase/parasitologia , Animais , Quimiocina CCL2/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Nefropatias/diagnóstico , Nefropatias/parasitologia , Lipocalina-2/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos BALB CRESUMO
The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.
Assuntos
Acanthamoeba/fisiologia , Amebíase/tratamento farmacológico , Artemisia annua/química , Pneumopatias Parasitárias/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Receptores Toll-Like/metabolismo , Amebíase/metabolismo , Animais , DNA Complementar/metabolismo , Imuno-Histoquímica , Pulmão/parasitologia , Pulmão/patologia , Pneumopatias Parasitárias/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/farmacologia , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/genéticaRESUMO
Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.
Assuntos
Entamoeba histolytica/efeitos dos fármacos , Entamebíase/tratamento farmacológico , Escherichia coli/fisiologia , Ácido Oxaloacético/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Amebíase/tratamento farmacológico , Amebíase/metabolismo , Amebíase/parasitologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/parasitologia , Células Cultivadas , Entamebíase/metabolismo , Entamebíase/parasitologia , Células HeLa , Humanos , Intestino Grosso/efeitos dos fármacos , Intestino Grosso/metabolismo , Intestino Grosso/parasitologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBARESUMO
Free-living amebae are ubiquitous in our environment, but rarely cause cutaneous infection. Balamuthia mandrillaris has a predilection for infecting skin of the central face. Infection may be restricted to the skin or associated with life-threatening central nervous system (CNS) involvement. We report a case of a 91-year-old woman, who presented with a non-healing red plaque over her right cheek. Several punch biopsies exhibited non-specific granulomatous inflammation without demonstrable fungi or mycobacteria in histochemical stains. She was treated empirically for granulomatous rosacea, but the lesion continued to progress. A larger incisional biopsy was performed in which amebae were observed in hematoxylin-eosin stained sections. These were retrospectively apparent in the prior punch biopsy specimens. Immunohistochemistry and polymerase chain reaction studies identified the organisms as Balamuthia mandrillaris. Cutaneous infection by B. mandrillaris is a rare condition that is sometimes complicated by life-threatening CNS involvement and which often evades timely diagnosis due to its rarity and nonspecific clinical manifestations. Moreover, these amebae are easily overlooked in histopathologic sections because of their small number and their resemblance to histiocytes. Dermatopathologists should be familiar with the histopathologic appearance of these organisms and include balamuthiasis and other amebic infections in the differential diagnosis of granulomatous dermatitis.
Assuntos
Amebíase , Balamuthia mandrillaris , Bochecha , Dermatopatias Parasitárias , Idoso de 80 Anos ou mais , Amebíase/metabolismo , Amebíase/patologia , Bochecha/parasitologia , Bochecha/patologia , Feminino , Humanos , Imuno-Histoquímica , Dermatopatias Parasitárias/metabolismo , Dermatopatias Parasitárias/patologiaRESUMO
Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Antimaláricos/farmacologia , Artemisininas/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Serina/metabolismo , Ceratite por Acanthamoeba/tratamento farmacológico , Ceratite por Acanthamoeba/metabolismo , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/metabolismo , Amebíase/tratamento farmacológico , Amebíase/metabolismo , Amebíase/parasitologia , Animais , Apoptose/efeitos dos fármacos , Artemeter , Proliferação de Células/efeitos dos fármacos , Encefalite/tratamento farmacológico , Encefalite/metabolismo , Encefalite/parasitologia , Fosfoglicerato Desidrogenase/metabolismo , Proteômica/métodos , Transaminases/metabolismo , Trofozoítos/parasitologiaRESUMO
Tumour necrosis factor-alpha (TNF-alpha) is a key mediator of inflammation during amoebiasis of humans and mice. Atlantic salmon (Salmo salar L.) are also susceptible to infection by amoebae (Neoparamoeba spp.), inflicting a condition known as amoebic gill disease (AGD). Here, the role of TNF-alpha in AGD-pathogenesis was examined. Two Atlantic salmon TNF-alpha transcripts designated TNF-alpha1 and TNF-alpha2 together with their respective genes were cloned and sequenced. TNF-alpha1 is 1379 bp and consists of a 738 bp open reading frame (ORF) translating into a predicted protein of 246 amino acids. TNF-alpha2 is 1412 bp containing an ORF and translated protein the same lengths as TNF-alpha1. An anti-rainbow trout TNF-alpha polyclonal antibody that bound recombinant Atlantic salmon TNF-alpha1 and TNF-alpha2 was used to detect constitutive and inducible expression of TNF-alpha in various tissues. The anti-TNF-alpha antibody bound to a TNF-like protein approximately 60 kDa that was constitutively expressed in a number of tissues in healthy Atlantic salmon. However, this protein was not detected in lysates from mitogen-stimulated head kidney leucocytes, despite up-regulation of TNF-alpha mRNAs under the same conditions. During the early onset of AGD in Atlantic salmon, there were no demonstrable differences in the gill tissue expression of TNF-alpha1, TNF-alpha2 nor the interleukin-1 beta (IL-1beta), inducible nitric oxide synthase (iNOS) and interferon gamma (IFN-gamma) mRNAs compared to tissue from healthy fish. In Atlantic salmon with advanced AGD, IL-1beta but not TNF-alpha1 or TNF-alpha2 mRNAs was up-regulated and was lesion-restricted. Given that Neoparamoeba spp. modulated both TNF-alpha2 and IL-1beta in head kidney leucocytes in vitro, it appears that rather than being recalcitrant to Neoparamoeba spp.-mediated TNF-alpha expression, either the parasite can influence the cytokine response during infection, there is ineffective signalling for TNF-alpha expression, or there are too few cells at the site of infection with the capacity to produce TNF-alpha. These data support our previous observation that IL-1beta mRNA expression is up-regulated in AGD-affected tissue and that TNF-alpha is not intrinsic in AGD-pathogenesis.
Assuntos
Amebíase/veterinária , Doenças dos Peixes/metabolismo , Regulação da Expressão Gênica , Salmo salar/metabolismo , Fator de Necrose Tumoral alfa/genética , Amebíase/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/análise , Anticorpos/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Brânquias/parasitologia , Interleucina-1alfa/genética , Interleucina-1beta/genética , Leucócitos/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Fator de Necrose Tumoral alfa/sangue , Regulação para CimaRESUMO
Neoparamoeba spp. are amphizoic amoebae with the capacity to colonize the gills of some marine fish, causing AGD. Here, the gill tissue transcriptome response of Atlantic salmon (Salmo salar L.) to AGD is described. Tanks housing Atlantic salmon were inoculated with Neoparamoeba spp. and fish sampled at time points up to 8 days postinoculation (pi.). Gill tissues were taken from AGD-affected fish, and a DNA microarray was used to compare global gene expression against tissues from AGD-unaffected fish. A total of 206 genes, representing 190 unique transcripts, were reproducibly identified as up- or downregulated in response to Neoparamoeba spp. infection. Informative transcripts having GO biological process identifiers were grouped according to function. Although a number of genes were placed into each category, no distinct patterns were observed. One Atlantic salmon cDNA that was upregulated in infected gill relative to noninfected gill at 114 and 189 h pi. showed significant identity with the Xenopus, mouse, and human anterior gradient-2 (AG-2) homologs. Two Atlantic salmon AG-2 mRNA transcripts, designated asAG-2/1 and asAG-2/2, were cloned, sequenced, and shown to be predominantly expressed in the gill, intestine, and brain of a healthy fish. In AGD-affected fish, differential asAG-2 expression was confirmed in samples used for microarray analyses as well as in AGD-affected gill tissue taken from fish in an independent experiment. The asAG-2 upregulation was restricted to AGD lesions relative to unaffected tissue from the same gill arch, while p53 tumor suppressor protein mRNA was concurrently downregulated in AGD lesions. Differential expression of p53-regulated transcripts, proliferating cell nuclear antigen and growth arrest and DNA damage-inducible gene-45beta (GADD45beta) in AGD lesions, suggests a role for p53 in AGD pathogenesis. Thus AGD may represent a novel model for comparative analysis of p53 and p53-regulated pathways.
Assuntos
Amebíase/veterinária , Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Brânquias/metabolismo , Salmo salar/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Amebíase/etiologia , Amebíase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Análise por Conglomerados , Bases de Dados Genéticas , Doenças dos Peixes/etiologia , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Brânquias/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Salmo salar/genética , Proteína Supressora de Tumor p53/genética , Proteínas GADD45RESUMO
Granulomatous amebic encephalitis (GAE), or meningoencephalitis due to Acanthamoeba spp. and leptomyxid ameba are uncommon CNS infections that generally occur in immunocompromised hosts. We describe a case of GAE caused by Balamuthia mandrillaris previously designated as a leptomyxid ameba, in an apparently healthy 14-year-old Venezuelan boy. This case was characterized by sudden onset of seizures, focal neurologic signs and by a prolonged clinical course (from November 1992 to March 1993). Neuroimaging studies showed cerebral hypodense lesions in cerebral hemispheres, brain stem and cerebellum. Microscopically, we found a chronic granulomatous inflammatory reaction with necrotizing angiitis, large numbers of amebic trophozoites and few cysts in perivascular spaces and within necrotic CNS tissue. The amebas were identified as B. mandrillaris based on their immunofluorescence reactivity with the anti-B. mandrillaris serum. So far, 30 cases of GAE due to B. mandrillaris have been recognized in humans, two in AIDS patients. No visceral involvement by free-living amebas or any other significant abnormality was observed. This patient developed "spontaneous" GAE, but it remains possible that an undiagnosed abnormality in cell-mediated immunity or a deficient humoral immune response may explain the susceptibility of this patient to this opportunistic infection.
Assuntos
Amebíase , Encefalite/microbiologia , Granuloma/microbiologia , Adolescente , Amebíase/metabolismo , Amebíase/patologia , Cadáver , Encefalite/patologia , Granuloma/patologia , Humanos , MasculinoRESUMO
We have previously shown that Entamoeba histolytica lysates contain the neurohormones serotonin, neurotensin, immunoreactive substance P, and probably acetylcholine, and that amebic lysates inhibit sodium and chloride absorption and stimulate chloride secretion in the rat descending colon as measured by the Ussing chamber-voltage clamp technique. We now demonstrate that these transport effects have both calcium-dependent and calcium-independent components. In addition, arachidonic acid metabolites of the cyclooxygenase pathway are probably involved in the Entamoeba histolytica-induced changes in colonic transport that are not dependent on Ca++ entry. Prostaglandin E2 (10(-5) M), indomethacin (10(-6) M), piroxicam (5 x 10(-5) M), and mepacrine (10(-4) M) partially inhibited the amebic lysate effect on active transport in the rat descending colon. In addition, verapamil (10(-4) M) partially inhibited the effect of amebic lysates. The effect of verapamil was additive with that of indomethacin, totally blocking the effect of amebic lysate on short-circuit current. However, amebic lysates do not contain prostaglandin E2 as measured by sensitive radioimmunoassay. Amebic lysates stimulated prostaglandin E2 release from rat colonic mucosal strips. Amebic lysate significantly increased colonic cyclic adenosine monophosphate content. Piroxicam inhibited the lysate-induced increase in colonic cyclic adenosine monophosphate content. These results indicate that although amebic lysate does not contain prostaglandin E2, it caused arachidonic acid metabolites to be produced by the cyclooxygenase pathway, and these are probably involved in the Entamoeba histolytica-induced changes in colonic transport. Neurohormones in Entamoeba histolytica may act directly on colonic tissue to stimulate intestinal secretion, probably via a Ca+(+)-dependent mechanism that is blockable by verapamil, or indirectly via stimulation of prostaglandin E2 generation and release from the rat colon via a cyclic adenosine monophosphate-dependent mechanism. These effects appear separate. The cyclic adenosine monophosphate-dependent secretion is the predominant mechanism in this model of colonic amebic diarrhea.
Assuntos
Amebíase/metabolismo , Cálcio/fisiologia , Colo/metabolismo , Dinoprostona/fisiologia , Entamebíase/metabolismo , Animais , Transporte Biológico , AMP Cíclico/metabolismo , Dinoprostona/antagonistas & inibidores , Entamoeba histolytica , Mucosa Intestinal/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
Hepatic iron concentrations were measured in 60 Black patients who had died of amoebiasis. There were 18 infants and young children, 30 adult males and 12 adult females. The mean hepatic iron concentration in infants was normal (0,11% dry weight), while those in adult males and females were significantly raised (0,64% and 0,30% dry weight respectively). The figures for adults are somewhat higher than those previously found in Black subjects, suggesting that iron overload is more common in patients with amoebiasis than in the general population. The relevance of these findings to the pathogenesis of amoebiasis is not clear but the relationship may well not be a direct one, since hepatic iron stores were no larger in patients with liver abscesses than in subjects without.