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Papers by Carlos Roberto Alves

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome vir... more The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins’ possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemo...

Antimicrobial Agents and Chemotherapy, 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more ... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor.

Journal of Agricultural Science and Technology B, 2014

The coconut fiber is produced in large quantities in Brazil, even though very small quantities ar... more The coconut fiber is produced in large quantities in Brazil, even though very small quantities are being used by some industries, mainly cordage mats and handicrafts. An alternative usage would be the use of these fibers in biosorption of heavy metals from aqueous solutions. This present study aimed to evaluate the potentiality of cadmium biosorption by coconut fiber. The coconut fibers were used at kinetic analysis, influence of pH and adsorption isotherms were also carried out. It can be concluded that there is great potential for the use of coconut fiber, and the optimum pH for adsorption was around 5.0. The adsorption kinetics is fast and equilibrium occurs within 120 min. The Langmuir isotherm was considered the most suitable to describe the experimental data.

Current Drug Targets, 2014

This review presents and discusses the current status and perspectives of leishmaniasis treatment... more This review presents and discusses the current status and perspectives of leishmaniasis treatment, with a special focus on the use of proteinase inhibitors. The history of treatment development, the first-and second-choice modern drugs and the advantages and disadvantages of using proteinases inhibitors as leishmanicidal treatments are presented and discussed. The reports gathered herein confirm the potential usefulness of proteinases inhibitors as an alternative or complement to the current leishmaniasis treatments. They also support the hypothesis that a combined treatment with multiple proteinase inhibitors may be efficient against Leishmania infections in vertebrate hosts.

Antimicrobial agents and chemotherapy, Jan 12, 2015

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum o... more Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapy treatments present many issues, as several adverse side effects and development of resistant strains. Natural compounds have been investigated as potential anti-leishmanial agents and, in the present study, the effects of epoxy-α-lapachone on L. (L.) amazonensis are analyzed. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 hours of exposure. This compound also had an effect in experimentally infected BALB/C, causing a reduction in paw lesions six weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area: 24.9±2.0 mm(2)), when compared to untreated animals 30.8±2.6 mm(2) or animals treated with glucantime 28.3±1.5 mm(2). In addition, the effects of this compound on the serine proteinase act...

Transactions of the Royal Society of Tropical Medicine and Hygiene, 2005

To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multipl... more To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.

Parasitology, 2011

SUMMARYLeishmaniasis is a vector-borne disease and an important public health issue. Glycosaminog... more SUMMARYLeishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) bios...

Memórias do Instituto Oswaldo Cruz, 2008

We report for the first time the expression of multiple protease activities in the first instar l... more We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Journal of Biomedicine and Biotechnology, 2010

American Journal of Immunology, 2005

Previous studies have shown that circulating levels of cytokines are increased in patients with c... more Previous studies have shown that circulating levels of cytokines are increased in patients with congestive heart failure (CHF), resulting in myocardial depression. In this work, we have determined serum levels of TNF-α and IFN-γ by ELISA, detected sTNFR-I by dot ELISA, as well as TNFα, IL-6, IL-10 and granulysin mRNA in unstimulated peripheral blood mononuclear cells (PBMC) by RT-PCR in CHF patients. Such patients were classified using New York Heart Association criteria and compared to a control group of volunteers without CHF. The performed echocardiographic evaluations showed a significant difference between the control group and the patients. Additionally, the ejection fraction and the left ventricular fractional shortening showed a direct relation with functional classes, varying in inverse proportion. Generally, the studied cytokines in serum or PBMC did not correlate either to functional classes or to the presence/absence of CHF. However, the granulysin mRNA was detected in most of the patients tested as compared to controls. Moreover, the qualitative detection of the sTNFR-I also made it possible to discriminate between patients and the control group. Functional classes could be separated because of a direct association between CHF severity and elevated levels of sTNFR-I, defined by intensity of signal in dot-ELISA. Our results suggest that detection of granulysin mRNA as well as the detection of sTNFR-I appear to have a clinical relevance as markers of immune activation in CHF.

Acta Tropica, 2008

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmania... more Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L. braziliensis strains, isolated from patients with distinct clinical manifestations, display different pattern of metalloprotease activities. Following these results, we investigated the cellular localization of these molecules and their relation to the major surface protease (gp63) of Leishmania. Comparative analyses of metalloprotease expression among different clinical isolates as well as an evaluation of the effect of long-term in vitro passage on the expression pattern of these metalloproteases were also performed. Western blot analysis, using an anti-gp63 antibody, revealed polypeptide patterns with a similar profile to that observed in zymographic analysis. Flow cytometry and fluorescence microscopy analyses corroborated the presence of metalloproteases with homologous domains to gp63 in the parasites and revealed differences in the expression level of such molecules among the isolates. The cellular distribution of metalloproteases, assessed by confocal analysis, showed the existence of intracellular metalloproteases with homologous domains to gp63, predominantly located near the flagellar pocket. Finally, it was observed that differential zymographic profiles of metalloproteases exhibited by L. (V.) braziliensis isolates remain unaltered during prolonged in vitro culture, suggesting that the proteolytic activity pattern is a stable phenotypic characteristic of these parasites.

Acta Tropica, 2008

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex as... more In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.

BMC Immunology, 2011

Background Leishmania parasites have been reported to interfere and even subvert their host immun... more Background Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant. Results Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell ...

Scientific Reports

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistan... more Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders—R) and relapse or therapeutic failure (Non-responders—NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one...

Molecules

Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have... more Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have been shown to be promising drugs for Leishmaniases treatment. Although, it is known the action spectrum of both compounds affect the Leishmania spp. multiplication, there are gaps in the molecular binding details of target enzymes related to the parasite’s physiology. Molecular docking assays simulations were performed using DockThor server to predict the preferred orientation of both compounds to form stable complexes with key enzymes of metabolic pathway, electron transport chain, and lipids metabolism of Leishmania spp. This study showed the hit rates of both compounds interacting with lanosterol C-14 demethylase (−8.4 kcal/mol to −7.4 kcal/mol), cytochrome c (−10.2 kcal/mol to −8.8 kcal/mol), and glyceraldehyde-3-phosphate dehydrogenase (−8.5 kcal/mol to −7.5 kcal/mol) according to Leishmania spp. and assessed compounds. The set of molecular evidence reinforces the potential of both...

International Journal of Molecular Sciences

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

International Journal of Molecular Sciences, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Parasitology Research

Current treatment guidelines for leishmaniasis is based on chemotherapy with drugs that show a se... more Current treatment guidelines for leishmaniasis is based on chemotherapy with drugs that show a set of limitations such as high cost, toxicity, difficult route of administration, and lack of efficacy in endemic areas. In this context, phytopharmaceutical products and herbal medicines emerge as promising alternatives for developing new treatment against leishmaniasis. This review discusses the perspectives of leishmaniasis treatment based on natural products and phytotherapy highlighting the Piper genus, especially P. aduncun and P. mollicomum Kunth covering the period of 1998 to 2020. Leishmanicidal activity of pure compounds of Piper spp. [3-(3,4,5-trimethoxyphenyl) propanoic acid, 3-chlorosintenpyridone, 2'-hydroxy-3',4',6'-trimethoxy-chalcone, cardamonin, conocarpan, cubebin, eupomatenoid, flavokavain B, ( +)-(7R,8S)-epoxy-5,6-didehydrokavain, N-[7-(3',4'-methylenedioxypheny l-2(E),4(E)-heptadienoyl-pyrrolidine, N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl-pyrrolidine, piperovatine, pellitorine, and piplartine (piperlongumine)] were proved against the promastigote and amastigote forms of parasite related with cutaneous (L. (L.) amazonensis, L. (V.) braziliensis, and L. (V.) guyanensis) and visceral (L. (L.) donovani, L. (L.) chagasi, and L. (L.) infantum). We also discussed the perspective of leishmaniasis treatment, considering the potential synergism between different promising species of Piper, presenting some interesting interaction possibilities for future studies between plants. Finally, the necessary steps for technological development of phytomedicines and herbal medicines with the desirable quality requirements for medicines are highlighted. The data presented here highlight the use of Piper spp. as source of pharmacological compounds that can lead to effective, safe, and inexpensive treatments for leishmaniasis.

Memórias do Instituto Oswaldo Cruz

BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studi... more BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

BioMed Research International

Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive succes... more Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive success these parasites to the mammalian hosts. Since these enzymes are poorly studied in naturally infected dogs, this work aims to show the differences in metalloprotease and cysteine proteases gene expression in ear edge skin of dogs naturally infected by Leishmania (Leishmania) infantum. A cohort of dogs (n=20) naturally infected by L. (L.) infantum was clinically classified as asymptomatic, oligosymptomatic, and polysymptomatic and the parasite load range estimated. The analysis of proteases expression by RT-PCR in the ear edge skin was also assessed, suggesting more transcripts of proteases in cDNA samples from polysymptomatic dogs than oligosymptomatic and asymptomatic ones. Metalloprotease RT-PCR assays yielded products (202 bp) in all assessed cDNA dog samples. In contrast, cysteine proteases transcripts (227 bp) had shown to be better detected in cDNA samples of polysymptomatic dogs,...

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome vir... more The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins’ possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemo...

Antimicrobial Agents and Chemotherapy, 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more ... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor.

Journal of Agricultural Science and Technology B, 2014

The coconut fiber is produced in large quantities in Brazil, even though very small quantities ar... more The coconut fiber is produced in large quantities in Brazil, even though very small quantities are being used by some industries, mainly cordage mats and handicrafts. An alternative usage would be the use of these fibers in biosorption of heavy metals from aqueous solutions. This present study aimed to evaluate the potentiality of cadmium biosorption by coconut fiber. The coconut fibers were used at kinetic analysis, influence of pH and adsorption isotherms were also carried out. It can be concluded that there is great potential for the use of coconut fiber, and the optimum pH for adsorption was around 5.0. The adsorption kinetics is fast and equilibrium occurs within 120 min. The Langmuir isotherm was considered the most suitable to describe the experimental data.

Current Drug Targets, 2014

This review presents and discusses the current status and perspectives of leishmaniasis treatment... more This review presents and discusses the current status and perspectives of leishmaniasis treatment, with a special focus on the use of proteinase inhibitors. The history of treatment development, the first-and second-choice modern drugs and the advantages and disadvantages of using proteinases inhibitors as leishmanicidal treatments are presented and discussed. The reports gathered herein confirm the potential usefulness of proteinases inhibitors as an alternative or complement to the current leishmaniasis treatments. They also support the hypothesis that a combined treatment with multiple proteinase inhibitors may be efficient against Leishmania infections in vertebrate hosts.

Antimicrobial agents and chemotherapy, Jan 12, 2015

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum o... more Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapy treatments present many issues, as several adverse side effects and development of resistant strains. Natural compounds have been investigated as potential anti-leishmanial agents and, in the present study, the effects of epoxy-α-lapachone on L. (L.) amazonensis are analyzed. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 hours of exposure. This compound also had an effect in experimentally infected BALB/C, causing a reduction in paw lesions six weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area: 24.9±2.0 mm(2)), when compared to untreated animals 30.8±2.6 mm(2) or animals treated with glucantime 28.3±1.5 mm(2). In addition, the effects of this compound on the serine proteinase act...

Transactions of the Royal Society of Tropical Medicine and Hygiene, 2005

To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multipl... more To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.

Parasitology, 2011

SUMMARYLeishmaniasis is a vector-borne disease and an important public health issue. Glycosaminog... more SUMMARYLeishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) bios...

Memórias do Instituto Oswaldo Cruz, 2008

We report for the first time the expression of multiple protease activities in the first instar l... more We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Journal of Biomedicine and Biotechnology, 2010

American Journal of Immunology, 2005

Previous studies have shown that circulating levels of cytokines are increased in patients with c... more Previous studies have shown that circulating levels of cytokines are increased in patients with congestive heart failure (CHF), resulting in myocardial depression. In this work, we have determined serum levels of TNF-α and IFN-γ by ELISA, detected sTNFR-I by dot ELISA, as well as TNFα, IL-6, IL-10 and granulysin mRNA in unstimulated peripheral blood mononuclear cells (PBMC) by RT-PCR in CHF patients. Such patients were classified using New York Heart Association criteria and compared to a control group of volunteers without CHF. The performed echocardiographic evaluations showed a significant difference between the control group and the patients. Additionally, the ejection fraction and the left ventricular fractional shortening showed a direct relation with functional classes, varying in inverse proportion. Generally, the studied cytokines in serum or PBMC did not correlate either to functional classes or to the presence/absence of CHF. However, the granulysin mRNA was detected in most of the patients tested as compared to controls. Moreover, the qualitative detection of the sTNFR-I also made it possible to discriminate between patients and the control group. Functional classes could be separated because of a direct association between CHF severity and elevated levels of sTNFR-I, defined by intensity of signal in dot-ELISA. Our results suggest that detection of granulysin mRNA as well as the detection of sTNFR-I appear to have a clinical relevance as markers of immune activation in CHF.

Acta Tropica, 2008

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmania... more Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L. braziliensis strains, isolated from patients with distinct clinical manifestations, display different pattern of metalloprotease activities. Following these results, we investigated the cellular localization of these molecules and their relation to the major surface protease (gp63) of Leishmania. Comparative analyses of metalloprotease expression among different clinical isolates as well as an evaluation of the effect of long-term in vitro passage on the expression pattern of these metalloproteases were also performed. Western blot analysis, using an anti-gp63 antibody, revealed polypeptide patterns with a similar profile to that observed in zymographic analysis. Flow cytometry and fluorescence microscopy analyses corroborated the presence of metalloproteases with homologous domains to gp63 in the parasites and revealed differences in the expression level of such molecules among the isolates. The cellular distribution of metalloproteases, assessed by confocal analysis, showed the existence of intracellular metalloproteases with homologous domains to gp63, predominantly located near the flagellar pocket. Finally, it was observed that differential zymographic profiles of metalloproteases exhibited by L. (V.) braziliensis isolates remain unaltered during prolonged in vitro culture, suggesting that the proteolytic activity pattern is a stable phenotypic characteristic of these parasites.

Acta Tropica, 2008

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex as... more In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.

BMC Immunology, 2011

Background Leishmania parasites have been reported to interfere and even subvert their host immun... more Background Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant. Results Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell ...

Scientific Reports

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistan... more Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders—R) and relapse or therapeutic failure (Non-responders—NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one...

Molecules

Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have... more Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have been shown to be promising drugs for Leishmaniases treatment. Although, it is known the action spectrum of both compounds affect the Leishmania spp. multiplication, there are gaps in the molecular binding details of target enzymes related to the parasite’s physiology. Molecular docking assays simulations were performed using DockThor server to predict the preferred orientation of both compounds to form stable complexes with key enzymes of metabolic pathway, electron transport chain, and lipids metabolism of Leishmania spp. This study showed the hit rates of both compounds interacting with lanosterol C-14 demethylase (−8.4 kcal/mol to −7.4 kcal/mol), cytochrome c (−10.2 kcal/mol to −8.8 kcal/mol), and glyceraldehyde-3-phosphate dehydrogenase (−8.5 kcal/mol to −7.5 kcal/mol) according to Leishmania spp. and assessed compounds. The set of molecular evidence reinforces the potential of both...

International Journal of Molecular Sciences

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

International Journal of Molecular Sciences, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Parasitology Research

Current treatment guidelines for leishmaniasis is based on chemotherapy with drugs that show a se... more Current treatment guidelines for leishmaniasis is based on chemotherapy with drugs that show a set of limitations such as high cost, toxicity, difficult route of administration, and lack of efficacy in endemic areas. In this context, phytopharmaceutical products and herbal medicines emerge as promising alternatives for developing new treatment against leishmaniasis. This review discusses the perspectives of leishmaniasis treatment based on natural products and phytotherapy highlighting the Piper genus, especially P. aduncun and P. mollicomum Kunth covering the period of 1998 to 2020. Leishmanicidal activity of pure compounds of Piper spp. [3-(3,4,5-trimethoxyphenyl) propanoic acid, 3-chlorosintenpyridone, 2'-hydroxy-3',4',6'-trimethoxy-chalcone, cardamonin, conocarpan, cubebin, eupomatenoid, flavokavain B, ( +)-(7R,8S)-epoxy-5,6-didehydrokavain, N-[7-(3',4'-methylenedioxypheny l-2(E),4(E)-heptadienoyl-pyrrolidine, N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl-pyrrolidine, piperovatine, pellitorine, and piplartine (piperlongumine)] were proved against the promastigote and amastigote forms of parasite related with cutaneous (L. (L.) amazonensis, L. (V.) braziliensis, and L. (V.) guyanensis) and visceral (L. (L.) donovani, L. (L.) chagasi, and L. (L.) infantum). We also discussed the perspective of leishmaniasis treatment, considering the potential synergism between different promising species of Piper, presenting some interesting interaction possibilities for future studies between plants. Finally, the necessary steps for technological development of phytomedicines and herbal medicines with the desirable quality requirements for medicines are highlighted. The data presented here highlight the use of Piper spp. as source of pharmacological compounds that can lead to effective, safe, and inexpensive treatments for leishmaniasis.

Memórias do Instituto Oswaldo Cruz

BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studi... more BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

BioMed Research International

Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive succes... more Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive success these parasites to the mammalian hosts. Since these enzymes are poorly studied in naturally infected dogs, this work aims to show the differences in metalloprotease and cysteine proteases gene expression in ear edge skin of dogs naturally infected by Leishmania (Leishmania) infantum. A cohort of dogs (n=20) naturally infected by L. (L.) infantum was clinically classified as asymptomatic, oligosymptomatic, and polysymptomatic and the parasite load range estimated. The analysis of proteases expression by RT-PCR in the ear edge skin was also assessed, suggesting more transcripts of proteases in cDNA samples from polysymptomatic dogs than oligosymptomatic and asymptomatic ones. Metalloprotease RT-PCR assays yielded products (202 bp) in all assessed cDNA dog samples. In contrast, cysteine proteases transcripts (227 bp) had shown to be better detected in cDNA samples of polysymptomatic dogs,...