Leishmania amazonensis resistance in murine macrophages: Analysis of possible mechanisms.

Leishmaniasis encompass a group of infectious parasitic diseases occurring in 97 endemic countries where over one billion people live in areas at risk of infection. It is in the World Health Organization list of neglected diseases and it is considered a serious public health problem, with more than 20,000 deaths a year and high morbidity. Infection by protozoa from the genus Leishmania can cause several forms of the disease, which may vary from a self-healing ulcer to fatal visceral infection. Leishmania species, as well as host immune response and genetics can modulate the course of the disease. Leishmania sp are obligatory intracellular parasites that have macrophages as their main host cell. Depending on the activation phenotype, these cells may have distinct roles in disease development, acting in parasite control or proliferation. Therefore, the purpose of this work was to analyze Leishmania amazonensis infection in primary macrophage cells obtained from mice with two distinct genetic backgrounds, ie. different susceptibility to the infection; evaluating the cause for that difference. After infection, peritoneal macrophages from the resistant C3H/He strain presented lower parasite load when compared to susceptible BALB/c macrophages. The same was also true when cells received a Th2 stimulus after infection, but the difference was abrogated under Th1 stimulus. Nitric oxide production and arginase activity was different between the strains under Th1 or Th2 stimulus, respectively, but iNOS inhibition was unable to suppress C3H/He resistance. Hydrogen peroxide production was also higher in C3H/He than BALB/c under Th1 stimulus, but it could not account for differences in susceptibility. These results led us to conclude that, although they have an important role in parasite control, neither NO nor H2O2 production can explain C3H/He resistance to infection. Other studies are needed to uncover different mechanisms of resistance/susceptibility to L. amazonensis

Extracellular matrix alterations in experimental Leishmania amazonensis infection in susceptible and resistant mice

Submitted by Sandra Infurna ([email protected]) on 2017-04-09T17:30:35Z No. of bitstreams: 1 celeste_souza_etal_IOC_2012.pdf: 4071688 bytes, checksum: be9a7b5c819ca7994ef54d3f4bad9150 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-04-09T17:53:02Z (GMT) No. of bitstreams: 1 celeste_souza_etal_IOC_2012.pdf: 4071688 bytes, checksum: be9a7b5c819ca7994ef54d3f4bad9150 (MD5)Made available in DSpace on 2017-04-09T17:53:02Z (GMT). No. of bitstreams: 1 celeste_souza_etal_IOC_2012.pdf: 4071688 bytes, checksum: be9a7b5c819ca7994ef54d3f4bad9150 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ, Brasil / Universidade Estadual do Maranhão. Departamento de Patologia. São Luis, MA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ, Brasil.Leishmania is inoculated, by the bite of an infected sandfly, into the skin of the host, where the promastigotes are phagocyted by dermal macrophages. The dermal region comprises cells and abundant extracellular matrix. Studies show that matrix metalloproteinases play an important role in host defense responses against pathogens in mammals and that their activities lead to the production of antimicrobial peptides. The aim of this study is to evaluate the changes in the distribution of fibronectin and laminin as well as in the elastic system fibres during the course of infection caused by Leishmania amazonensis in mice with distinct genetic backgrounds of susceptibility to this parasite. The results showed that BALB/c presented an enhancement of fibronectin during the course of infection when compared to their control group while the infected or non-infected C3H.He showed a decrease of this protein at end of the experiment. Laminin, on the other hand, remained unaltered in both strains. Also in both BALB/c and C3H.He mice the elastic and elaunin fibres remained unchanged while the oxytalan fibres decreased along the experiment. Ninety days after the infection C3H.He mice had recovered their capacity to produce oxytalan fibres

Temporizin and Temporizin-1 Peptides as Novel Candidates for Eliminating Trypanosoma cruzi

Submitted by Sandra Infurna ([email protected]) on 2017-01-03T09:56:42Z No. of bitstreams: 1 katia_calabrese_etal_IOC_2016.pdf: 3404748 bytes, checksum: fb6b61ca57828307152d5ab9081cb4d4 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-01-03T10:15:50Z (GMT) No. of bitstreams: 1 katia_calabrese_etal_IOC_2016.pdf: 3404748 bytes, checksum: fb6b61ca57828307152d5ab9081cb4d4 (MD5)Made available in DSpace on 2017-01-03T10:15:50Z (GMT). No. of bitstreams: 1 katia_calabrese_etal_IOC_2016.pdf: 3404748 bytes, checksum: fb6b61ca57828307152d5ab9081cb4d4 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde (CDTS). Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Ciência e Tecnologia para a Inovação em Doenças Negligenciadas (INCT-IDN). Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica Experimental e Computacional de Produtos Farmacêuticos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz, Instituto Oswaldo Cruz. Laboratório de Toxoplasmose e outras Protozoonoses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Adolfo Lutz. Seção de Microscopia Eletrônica. Araçatuba, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Comunicação Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde (CDTS). Rio de Janeiro, RJ. Brasil / Universidade Federal Fluminense. Instituto de Biologia. Departamento de Biologia Celular e Molecular. Niterói, RJ, Brasil.Tropical diseases caused by parasitic infections continue to cause socioeconomic distress worldwide. Among these, Chagas disease has become a great concern because of globalization. Caused by Trypanosoma cruzi, there is an increasing need to discover new, more effective methods to manage infections that minimize disease onset. Antimicrobial peptides represent a possible solution to this challenge. As effector molecules of the innate immune response against pathogens, they are the first line of defense found in all multi-cellular organisms. In amphibians, temporins are a large family of antimicrobial peptides found in skin secretions. Their functional roles and modes of action present unique properties that indicate possible candidates for therapeutic applications. Here, we investigated the trypanocide activity of temporizin and temporizin-1. Temporizin is an artificial, hybrid peptide containing the N-terminal region of temporin A, the pore-forming region of gramicidin and a Cterminus consisting of alternating leucine and lysine. Temporizin-1 is a modification of temporizin with a reduction in the region responsible for insertion into membranes. Their activities were evaluated in a cell permeabilization assay by flow cytometry, an LDH release assay, electron microscopy, an MTT assay and patch clamp experiments. Both temporizin and temporizin-1 demonstrated toxicity against T. cruzi with temporizin displaying slightly more potency. At concentrations up to 100 μg/ ml, both peptides exhibited low toxicity in J774 cells, a macrophage lineage cell line, and no toxicity was observed in mouse primary peritoneal macrophages. In contrast, the peptides showed some toxicity in rat adenoma GH3 cells and Jurkat human lymphoma cells with temporizin-1 displaying lower toxicity. In summary, a shortened form of the hybrid temporizin peptide, temporizin-1, was efficient at killing T. cruzi and it has low toxicity in wild-type mammalian cells. These data suggest that temporizin-1 might be a candidate for Chagas disease therapy

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor β (TGFβ), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1β (IL-1β) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis

Oral Outbreak of Chagas Disease in Santa Catarina, Brazil: Experimental Evaluation of a Patient’s Strain

Submitted by sandra infurna ([email protected]) on 2015-11-18T16:00:59Z No. of bitstreams: 1 daiana_hardoim_etal_IOC_2015.pdf: 5139072 bytes, checksum: d738a4074ee287dca068c2c74d703aaa (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2015-11-24T23:07:26Z (GMT) No. of bitstreams: 1 daiana_hardoim_etal_IOC_2015.pdf: 5139072 bytes, checksum: d738a4074ee287dca068c2c74d703aaa (MD5)Made available in DSpace on 2015-11-24T23:07:26Z (GMT). No. of bitstreams: 1 daiana_hardoim_etal_IOC_2015.pdf: 5139072 bytes, checksum: d738a4074ee287dca068c2c74d703aaa (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Ciência e Tecnologia em Doenças Negligenciadas. Centro de Desenvolvimento Tecnológico em Saúde (CDTS). Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Universidade Estadual do Maranhão. Departamento de Patologia. São Luiz, MA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunomodulação e Protozoologia, Rio de Janeiro, RJ, Brasil.Chagas disease is a worldwide public health problem. Although the vectorial transmission of Chagas disease has been controlled in Brazil there are other ways of transmission, such as the ingestion of T. cruzi contaminated food, which ensures the continuation of this zoonosis. Here, we demonstrate the influence of the inoculation route on the establishment and development of the SC2005 T. cruzi strain infection in mice. Groups of Swiss mice were infected intragastrically (IG) or intraperitoneally (IP) with the T. cruzi SC2005 strain derived from an outbreak of oral Chagas disease. The results revealed that 100% of IP infected mice showed parasitemia, while just 36% of IG infected showed the presence of the parasite in blood. The parasitemia peaks were later and less intense in the IG infected mice. Mortality of the IP infected animals was more intense and earlier when compared to the IG infected mice. In the IP infected mice leucopenia occurred in the early infection followed by leucocytosis, correlating positively with the increase of the parasites. However, in the IG infected mice only an increase in monocytes was observed, which was positively correlated with the increase of the parasites. Histopathological analyses revealed a myotropic pattern of the SC2005 strain with the presence of inflammatory infiltrates and parasites in different organs of the animals infected by both routes as well as fibrosis foci and collagen redistribution. The flow cytometric analysis demonstrated a fluctuation of the T lymphocyte population in the blood, spleen and mesenteric lymph nodes of the infected animals. T. cruzi DNA associated with the presence of inflammatory infiltrates was detected by PCR in the esophagus, stomach and intestine of all infected mice. These findings are important for the understanding of the pathogenesis of T. cruzi infection by both inoculation routes

Temporizin causes moderate toxicity in mammalian J774 cells.

<p>J774 cell line was incubated at 37°C for 60 minutes in the presence of temporizin (0.1 and 10 μg/ml; Panel A) or gramicidin (0.1 and 10 μg/ml; Panel B), respectively. C: A quantification of the results obtained by PI entry assay after treating J774 cells with temporizin, gramicidin and benznidazole at different concentrations for 60, 90 and 1440 minutes. Gramicidin and temporizin toxicity was quantified by LDH Release Assay. The values represent the mean ± SD of four to six experiments (cell permeabilization assay) and three experiments (LDH release assay) performed on different days. *p<0.05, **p<0.01 compared with the corresponding negative control.</p

Temporizin-1 possesses toxicity against <i>T</i>. <i>cruzi</i>.

<p>A: <i>T</i>. <i>cruzi</i> was incubated at 37°C for 60 minutes for a flow cytometry assay. A single dose of 1 μg/ml was applied to compare the efficiency of the gramicidin (A2), temporizin A (A3) and temporizin-1 (A4) peptides in killing <i>T</i>. <i>cruzi</i>. B: A quantification of the results obtained in A. C: MTT assay using varied concentrations of temporizin and temporizin-1 for 60 minutes. D: PI entry quantification after the treatment with crescent temporizin and temporizin-1 doses for 60 minutes. E: Temporal assay after treatment with 1 μg/ml temporizin and temporizin-1. The values represent the mean ± SD of three to five experiments (flow cytometry and cell permeabilization assay) and three experiments (MTT assay) performed on different days. *p<0.05, **p<0.01 compared with the corresponding negative control.</p

Temporizin-1 induces a macroscopic ionic current in the HEK-293 mammalian cell line.

<p>A: Macroscopic ionic currents of a whole-cell patch from HEK-293 cells induced by different temporizin-1 concentrations. The ionic currents (far right) elicited by temporizin-1 are indicated by vertical arrows. The holding potential ranged from +100 mv to -100 mV. B: The mean I-V relations for the whole-cell configuration. C: Whole-cell recordings obtained from HEK-293 cells as a function of the varied temporizin-1 concentrations. D: Single channel recordings at distinct temporizin-1 concentrations. The values represent the means ± SD of six experiments performed on different days.</p

The amino acid sequences of temporin A, gramicidin, poly-leu, temporizin and temporizin-1 peptides.

<p>The entire sequences of the peptides are shown. The temporizin peptide was formed from the junction of the N-terminal temporizin A with the portion of the gramicidin pore-forming section and the C-terminus of the poly-leu peptide. Temporizin-1 has a reduction in the portion responsible for gramicidin pore formation.</p