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1.
Heliyon ; 8(9): e10674, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-36164515

RESUMEN

Insect cell lines represent a promising and expanding field as they have several research applications including biotechnology, virology, immunity, toxicology, cell signalling mechanisms and evolution. They constitute a powerful tool having a direct impact on human and veterinary medicine and agriculture. Although more than 1000 cell lines have currently been established from various insect species, Calliphora vicina-derived fly cell lines are lacking. This study was aimed at establishing a new C. vicina embryonic tissue-derived cell line. Adult flies were collected and embryonated eggs were mechanically homogenised and seeded in four types of culture media (L15, Grace's insect medium, Grace's/L15 and DMEM). Cell growth and morphological characteristics were recorded and cytogenetic and molecular patterns were determined. The CV-062020-PPB cell line was established and was shown to have optimal growth in Grace's/L15 medium. CV-062020-PPB cell monolayers that had been sub-cultured over 16 times consisted of firmly adhering cells having different morphologies; a fibroblast-like shape dominated and the karyotype had a 12-chromosome diploid number. RAPD-PCR analysis of the CV-062020-PPB cell line revealed a high similarity index and strong intraspecific relationship with C. vicina adult flies and a weaker relationship with the Lutzomyia longipalpis-derived cell line (Lulo). The CV-062020-PPB cell line constitutes the first cell line obtained from C. vicina embryonic tissues and represents an important basic and applied research tool.

2.
Biomedica ; 42(1): 54-66, 2022 03 01.
Artículo en Inglés, Español | MEDLINE | ID: mdl-35471170

RESUMEN

Introduction: The growing resistance to antibiotics worldwide represents a global threat to public health. The larval excretions and secretions derived from necrophagous flies from the Calliphoridae family could represent a promising source for counteracting their effects. Objective: To compare the antimicrobial activity of Calliphora vicina and Sarconesiopsis magellanica (Diptera: Calliphoridae) native excretions and secretions and those weighing more than 10 kDa and less. Materials and methods: We used the turbidimetry technique for the bioassay; we determined the minimum inhibitory concentration (MIC) for excretions and secretions weighing less than 10 kDa. Results: Calliphora vicina and S. magellanica native excretions and secretions and those weighing less than 10 kDa exhibited potent antibacterial activity against three Staphylococcus aureus strains and four Gram-negative bacteria; those weighing less than 10 kDa were more effective than the native ones in the two species of flies evaluated here. Furthermore, excretions and secretions weighing less than 10 kDa had the same effectiveness, except in the MIC trials where S. magellanica excretions and secretions weighing less than 10 kDa were more potent against all the bacteria evaluated, except for S. aureus ATCC 25923. Excretions and secretions weighing more than 10 kDa did not inhibit bacterial growth. Conclusions: These results potentially validate these substances as an important source for isolating and characterizing antimicrobial agents.


Introducción. La creciente resistencia bacteriana a los antibióticos representa una amenaza mundial de salud pública. Las excreciones y secreciones larvarias derivadas de moscas necrófagas de la familia Calliphoridae podrían configurar una fuente promisoria para contrarrestar sus efectos. Objetivo. Comparar la actividad antimicrobiana de las excreciones y secreciones larvarias nativas, y de las mayores y menores de 10 kDa de Calliphora vicina y Sarconesiopsis magellanica (Diptera: Calliphoridae). Materiales y métodos. El bioensayo se hizo a partir de la técnica de turbidimetría y en el caso de las excreciones y secreciones menores de 10 kDa se determinó la concentración inhibitoria mínima (CIM). Resultados. Las excreciones y secreciones nativas y las menores de 10 kDa de C. vicina y S. magellanica, evidenciaron una potente actividad antibacteriana contra tres cepas de Staphylococcus aureus y cuatro bacterias Gram negativas, siendo las menores de 10 kDa más efectivas que las nativas en las dos especies de moscas evaluadas. Además, las menores de 10 kDa presentaron la misma efectividad, aunque en las pruebas de CIM se observó que las de S. magellanica fueron más potentes en todas las bacterias evaluadas, excepto contra la cepa de S. aureus ATCC 25923. Las mayores de 10 kDa no inhibieron el crecimiento bacteriano. Conclusión. Los resultados validaron, en general, que estas sustancias son fuente importante para el aislamiento y la caracterización de agentes antimicrobianos.


Asunto(s)
Calliphoridae , Dípteros , Animales , Staphylococcus aureus
3.
Biomédica (Bogotá) ; Biomédica (Bogotá);42(1): 54-66, ene.-mar. 2022. tab, graf
Artículo en Español | LILACS | ID: biblio-1374507

RESUMEN

Introducción. La creciente resistencia bacteriana a los antibióticos representa una amenaza mundial de salud pública. Las excreciones y secreciones larvarias derivadas de moscas necrófagas de la familia Calliphoridae podrían configurar una fuente promisoria para contrarrestar sus efectos. Objetivo. Comparar la actividad antimicrobiana de las excreciones y secreciones larvarias nativas, y de las mayores y menores de 10 kDa de Calliphora vicina y Sarconesiopsis magellanica (Diptera: Calliphoridae). Materiales y métodos. El bioensayo se hizo a partir de la técnica de turbidimetría y en el caso de las excreciones y secreciones menores de 10 kDa se determinó la concentración inhibitoria mínima (CIM). Resultados. Las excreciones y secreciones nativas y las menores de 10 kDa de C. vicina y S. magellanica, evidenciaron una potente actividad antibacteriana contra tres cepas de Staphylococcus aureus y cuatro bacterias Gram negativas, siendo las menores de 10 kDa más efectivas que las nativas en las dos especies de moscas evaluadas. Además, las menores de 10 kDa presentaron la misma efectividad, aunque en las pruebas de CIM se observó que las de S. magellanica fueron más potentes en todas las bacterias evaluadas, excepto contra la cepa de S. aureus ATCC 25923. Las mayores de 10 kDa no inhibieron el crecimiento bacteriano. Conclusión. Los resultados validaron, en general, que estas sustancias son fuente importante para el aislamiento y la caracterización de agentes antimicrobianos.


Introduction: The growing resistance to antibiotics worldwide represents a global threat to public health. The larval excretions and secretions derived from necrophagous flies from the Calliphoridae family could represent a promising source for counteracting their effects. Objective: To compare the antimicrobial activity of Calliphora vicina and Sarconesiopsis magellanica (Diptera: Calliphoridae) native excretions and secretions and those weighing more than 10 kDa and less. Materials and methods: We used the turbidimetry technique for the bioassay; we determined the minimum inhibitory concentration (MIC) for excretions and secretions weighing less than 10 kDa. Results: Calliphora vicina and S. magellanica native excretions and secretions and those weighing less than 10 kDa exhibited potent antibacterial activity against three Staphylococcus aureus strains and four Gram-negative bacteria; those weighing less than 10 kDa were more effective than the native ones in the two species of flies evaluated here. Furthermore, excretions and secretions weighing less than 10 kDa had the same effectiveness, except in the MIC trials where S. magellanica excretions and secretions weighing less than 10 kDa were more potent against all the bacteria evaluated, except for S. aureus ATCC 25923. Excretions and secretions weighing more than 10 kDa did not inhibit bacterial growth. Conclusions: These results potentially validate these substances as an important source for isolating and characterizing antimicrobial agents.


Asunto(s)
Modalidades de Secreciones y Excreciones , Dípteros , Bacterias Gramnegativas , Bacterias Grampositivas , Larva , Antibacterianos
4.
Mem Inst Oswaldo Cruz ; 116: e200587, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34287503

RESUMEN

BACKGROUND: The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE: Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS: Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs' physico-chemical properties. Synthetic AMPs' antibacterial activity was evaluated. FINDINGS: Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules' sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS: S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.


Asunto(s)
Dípteros , Animales , Antibacterianos/farmacología , Calliphoridae , Cuerpo Adiposo , Larva , Pruebas de Sensibilidad Microbiana , Proteínas Citotóxicas Formadoras de Poros
5.
Mem Inst Oswaldo Cruz, v. 116, e200587, jun. 2021
Artículo en Inglés | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3904

RESUMEN

BACKGROUND The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs’ physico-chemical properties. Synthetic AMPs’ antibacterial activity was evaluated. FINDINGS Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules’ sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance

6.
Mem. Inst. Oswaldo Cruz ; 116: e200587, 2021. tab, graf
Artículo en Inglés | LILACS | ID: biblio-1287343

RESUMEN

BACKGROUND The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs' physico-chemical properties. Synthetic AMPs' antibacterial activity was evaluated. FINDINGS Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules' sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.


Asunto(s)
Animales , Dípteros , Cuerpo Adiposo , Pruebas de Sensibilidad Microbiana , Proteínas Citotóxicas Formadoras de Poros , Calliphoridae , Larva , Antibacterianos/farmacología
7.
Molecules ; 24(11)2019 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-31159162

RESUMEN

Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 µM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/farmacología , Dípteros/metabolismo , Secuencia de Aminoácidos , Animales , Antibacterianos/biosíntesis , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Bacterias/efectos de los fármacos , Fenómenos Químicos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad
8.
Molecules, v. 24, 2077, maio 2019
Artículo en Inglés | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2770

RESUMEN

Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 µM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.

9.
Front Microbiol ; 9: 2249, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30323791

RESUMEN

Larval therapy (LT) is an alternative treatment for healing chronic wounds; its action is based on debridement, the removal of bacteria, and stimulating granulation tissue. The most important mechanism when using LT for combating infection depends on larval excretions and secretions (ES). Larvae are protected against infection by a spectrum of antimicrobial peptides (AMPs); special interest in AMPs has also risen regarding understanding their role in wound healing since they degrade necrotic tissue and kill different bacteria during LT. Sarconesiopsis magellanica (Diptera: Calliphoridae) is a promising medically-important necrophagous fly. This article reports a small AMP being isolated from S. magellanica ES products for the first time; these products were obtained from third-instar larvae taken from a previously-established colony. ES were fractionated by RP-HPLC using C18 columns for the first analysis; the products were then lyophilised and their antimicrobial activity was characterized by incubation with different bacterial strains. These fractions' primary sequences were determined by mass spectrometry and de novo sequencing; five AMPs were obtained, the Sarconesin fraction was characterized and antibacterial activity was tested in different concentrations with minimum inhibitory concentrations starting at 1.2 µM. Potent inhibitory activity was shown against Gram-negative (Escherichia coli D31, E. coli DH5α, Salmonella enterica ATCC 13314, Pseudomonas aeruginosa 27853) and Gram-positive (Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 12228, Micrococcus luteus A270) bacteria. Sarconesin has a significant similarity with Rho-family GTPases which are important in organelle development, cytoskeletal dynamics, cell movement, and wound repair. The data reported here indicated that Sarconesin could be an alternative candidate for use in therapeutics against Gram-negative and Gram-positive bacterial infections. Our study describes one peptide responsible for antibacterial activity when LT is being used. The results shown here support carrying out further experiments aimed at validating S. magellanica AMPs as novel resources for combating antibacterial resistance.

10.
Front Microbiol, v. 9, 2249, 2018
Artículo en Inglés | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2574

RESUMEN

Larval therapy (LT) is an alternative treatment for healing chronic wounds; its action is based on debridement, the removal of bacteria, and stimulating granulation tissue. The most important mechanism when using LT for combating infection depends on larval excretions and secretions (ES). Larvae are protected against infection by a spectrum of antimicrobial peptides (AMPs); special interest in AMPs has also risen regarding understanding their role in wound healing since they degrade necrotic tissue and kill different bacteria during LT. Sarconesiopsis magellanica (Diptera: Calliphoridae) is a promising medically-important necrophagous fly. This article reports a small AMP being isolated from S. magellanica ES products for the first time; these products were obtained from third-instar larvae taken from a previously-established colony. ES were fractionated by RP-HPLC using C18 columns for the first analysis; the products were then lyophilised and their antimicrobial activity was characterized by incubation with different bacterial strains. These fractions’ primary sequences were determined by mass spectrometry and de novo sequencing; five AMPs were obtained, the Sarconesin fraction was characterized and antibacterial activity was tested in different concentrations with minimum inhibitory concentrations starting at 1.2 µM. Potent inhibitory activity was shown against Gram-negative (Escherichia coli D31, E. coli DH5a, Salmonella enterica ATCC 13314, Pseudomonas aeruginosa 27853) and Gram-positive (Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 12228, Micrococcus luteus A270) bacteria. Sarconesin has a significant similarity with Rho-family GTPases which are important in organelle development, cytoskeletal dynamics, cell movement, and wound repair. The data reported here indicated that Sarconesin could be an alternative candidate for use in therapeutics against Gram-negative and Gram-positive bacterial infections. Our study describes one peptide responsible for antibacterial activity when LT is being used. The results shown here support carrying out further experiments aimed at validating S. magellanica AMPs as novel resources for combating antibacterial resistance.

11.
Acta Trop ; 164: 280-289, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27686957

RESUMEN

This study's main objective was to evaluate the action of larval therapy derived from Lucilia sericata and Sarconesiopsis magellanica (blowflies) regarding Leishmania panamensis using an in vivo model. Eighteen golden hamsters (Mesocricetus auratus) were used; they were divided into 6 groups. The first three groups consisted of 4 animals each; these, in turn, were internally distributed into subgroups consisting of 2 hamsters to be used separately in treatments derived from each blowfly species. Group 1 was used in treating leishmanial lesions with larval therapy (LT), whilst the other two groups were used for evaluating the used of larval excretions and secretions (ES) after the ulcers had formed (group 2) and before they appeared (group 3). The three remaining groups (4, 5 and 6), consisting of two animals, were used as controls in the experiments. Biopsies were taken for histopathological and molecular analysis before, during and after the treatments; biopsies and smears were taken for assessing parasite presence and bacterial co-infection. LT and larval ES proved effective in treating the ulcers caused by the parasite. There were no statistically significant differences between the blowfly species regarding the ulcer cicatrisation parameters. There were granulomas in samples taken from lesions at the end of the treatments. The antibacterial action of larval treatment regarding co-infection in lesions caused by the parasite was also verified. These results potentially validate effective LT treatment against cutaneous leishmaniasis aimed at using it with humans in the future.


Asunto(s)
Terapia Biológica/métodos , Desbridamiento/métodos , Larva , Leishmaniasis Cutánea/terapia , Úlcera/terapia , Animales , Antibacterianos/uso terapéutico , Coinfección , Dípteros , Humanos , Proteínas de Insectos/metabolismo , Leishmania guyanensis , Leishmaniasis Cutánea/parasitología , Mesocricetus , Resultado del Tratamiento , Úlcera/parasitología
12.
Forensic Sci Int ; 233(1-3): 380-6, 2013 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-24314544

RESUMEN

Sarconesiopsis magellanica is a forensically relevant necrophagous blowfly that can aid in determining the post-mortem interval (PMI) as it is the first to colonise decomposing corpses. The blowfly has been reported in several South-American countries including Colombia, in high-altitude regions ranging from 1200 to 3100 m above sea level. The present study reports this blowfly's life cycle and an analysis of its reproductive and population parameters under laboratory conditions for the first time. Six successive generations of flies were produced with an average of 65.38% adults emerging with respect to the total number of puparia. The shortest life cycle from egg to adult emergence was found in individuals fed on a lyophilised liver (LL) diet, while the longest one was found in individuals fed with an egg-powdered milk (E-PM) diet; intermediate values were found when the pig liver (PL) diet was tested. The greatest adult longevity was achieved when the PL diet was used, the LL diet giving the shortest. The population parameters based on the horizontal life table were: net reproductive rate (Ro)=447.752±9.9, mean generational time (Tc)=18.18±0.38, natural population increase rate (r(m))=0.145 and finite population increase rate (λ)=1.398. This blowfly colony represents a valuable asset for both basic and applied studies. Members of the S. magellanica colony so established were used for analysing the life-cycle, reproductive and population parameters, and further medical and forensic application studies are currently underway.


Asunto(s)
Alimentación Animal , Dípteros/crecimiento & desarrollo , Animales , Dípteros/fisiología , Conducta Alimentaria , Larva/crecimiento & desarrollo , Longevidad , Oviposición , Pupa/crecimiento & desarrollo
13.
J Insect Sci ; 13: 130, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24766352

RESUMEN

Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies.


Asunto(s)
Línea Celular , Dípteros/citología , Animales , Técnicas de Cultivo de Célula , Proliferación Celular , Dípteros/genética , Embrión no Mamífero , Técnica del ADN Polimorfo Amplificado Aleatorio
14.
Parasit Vectors ; 5: 142, 2012 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-22805335

RESUMEN

BACKGROUND: Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event. METHODS: Flagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis. RESULTS: The success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0 kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20 µg/ml) and 16% (for HS 10 µg/ml); HBP Mf (35.2% for 10 µg/ml and 25.4% for 20 µg/ml) and HBP Ff (10.0% for 10 µg/ml and 31.4% for 20 µg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces. CONCLUSIONS: The data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Adhesión Celular/fisiología , Leishmania braziliensis/metabolismo , Psychodidae/citología , Animales , Moléculas de Adhesión Celular/genética , Membrana Celular/fisiología , Células Cultivadas , Regulación de la Expresión Génica/fisiología
15.
Parasit Vectors ; 4: 216, 2011 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-22082050

RESUMEN

BACKGROUND: Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. METHODS: Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. FINDINGS: The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. CONCLUSIONS: We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.


Asunto(s)
Adhesión Celular , Interacciones Huésped-Patógeno , Leishmania/patogenicidad , Psychodidae/citología , Animales , Línea Celular , Modelos Animales de Enfermedad , Leishmania/crecimiento & desarrollo , Microscopía
16.
Rev. cienc. salud (Bogotá) ; 6(2): 64-73, ago. 2008. tab, graf
Artículo en Español | LILACS, COLNAL | ID: lil-635932

RESUMEN

En el presente trabajo se evaluó la actividad tóxica de extractos de Eupatorium microphyllum L.F. sobre larvas de IV estadio del mosquito Aedes aegypti (Linneaus), bajo condiciones de laboratorio. Se utilizaron extractos acuosos en concentraciones del 500 mg L-1, 1.500 mg L - 1 y 2.500 mg L-1 y acetónicos en concentraciones de 10 mg L-1, 20 mg L-1, 30 mg L-1, 40 mg L-1 y 50 mg L-1. Los bioensayos se realizaron por triplicado, cada uno con 20 larvas, expuestas durante 24 horas a 150 mL de solución. En todos los ensayos biológicos se emplearon grupos control. En la evaluación de los extractos acetónicos, se empleó un control negativo para evitar que la mortalidad de las larvas ocurriera a causa del solvente. Los extractos acuosos mostraron acción moderadamente baja en la mortalidad de larvas, menor del 20%. Por el contrario, la acción de los extractos acetónicos se observó a 10 y 20 mg L-1, con 15% de mortalidad, mientras que a 30 y 40 mg L-1 se registraron 22 al 38% de mortalidad, en tanto que a 50 mg L-1 la mortalidad fue del 95,4% con resultados estadísticos altamente significativos. Las concentraciones de los extractos acetónicos mostraron ser las más eficientes para el control de los mosquitos seleccionados. Ambos tipos de extractos mostraron efecto tóxico en larvas de A. aegypti ; sin embargo, se observó mayor efecto en los extractos acetónicos en relación con los extractos acuosos de E. microphyllum, lo cual constituye una alternativa viable en la búsqueda de nuevos larvicidas a partir de compuestos naturales.


In the present work the toxic activity of extracts of Eupatorium microphyllum L.F. was evaluated on 4 th instar larvae of the mosquito Aedes aegypti (Linneaus), under laboratory conditions. Aqueous extracts were utilized in concentrations of 500 mg L-1, 1,500 mg L-1 and 2,500 mg L-1 and acetone in concentrations of 10 mg L-1, 20 mg L-1, 30 mg L-1, 40 mg L-1 and 50 mg L-1. The bioassays were carried out for triplicate each one with 20 larvae, exposed for 24 hours to 150 mL of solution. In all the bioassays were employed control groups. In the evaluation of the acetone extracts, a negative control was employed to avoid that the mortality of the larvae to occur on account of the solvent. The Aqueous extracts showed low moderate action in the mortality of larvae, less than 20%. On the contrary, the action of the acetone extracts was observed to 10 and 20 mg L-1 with 15% of mortality, while to 30 and 40 mg L-1 were registered 22 to 38% of mortality. However, to 50 mg L-1 the mortality was of 95.4% with highly significant statistical results. The concentrations of the acetone extracts showed to be the most efficient for the control of the mosquitoes selected. Both types of extracts showed toxic effect in larvae of A. aegypti, nevertheless, greater effect in the acetone extracts was observed relating to the aqueous extracts of E. microphyllum, which constitutes a viable alternative in the search of new larvicides from composed natural.


Asunto(s)
Animales , Eupatorium , Soluciones , Solventes , Bioensayo , Aedes , Toxicidad
17.
Rev. cienc. salud (Bogotá) ; 6(2): 9-24, ago. 2008. ilus, graf, tab
Artículo en Español | LILACS, COLNAL | ID: lil-635928

RESUMEN

Introducción. Durante las últimas dos décadas, la terapia larval ha resurgido como una alternativa confiable y segura para la cura de úlceras cutáneas que no responden a los tratamientos convencionales. Objetivo. Evaluar el uso de las larvas de Lucilia sericata en el tratamiento de heridas infectadas con Pseudomonas aeruginosa en un modelo animal. Materiales y métodos. Se tomaron 12 conejos, los cuales fueron divididos al azar en 3 grupos homogéneos: al primero se le aplicó terapia larval, el segundo se trató con terapia antibiótica y el tercero fue establecido como control. A cada uno de los animales se les realizó una herida, luego se inoculó en ésta una suspensión de P. aeruginosa y, finalmente, al registrarse el desarrollo de la infección, se procedió, en los dos primeros grupos, a los tratamientos correspondientes. Para la evaluación macroscópica de las heridas, se tuvo en cuenta la presencia de edema y exudado, mal olor, inflamación alrededor de la herida y apariencia del tejido de granulación. Al proceso de cicatrización se le hizo seguimiento a través de una técnica dermohistológica. Resultados. Se registraron claras diferencias entre el grupo de animales tratados con terapia larval vs. el grupo tratado con terapia convencional de antibióticos, estableciéndose un periodo de 10 días para alcanzar la cicatrización en el grupo de terapia larval mientras que en el segundo grupo el proceso se cumplió en 20 días. Conclusiones. S e demostró la eficacia de las larvas de L. sericata en el tratamiento de heridas infectadas con P. aeruginosa.


Introduction. During the last two decades the larval therapy has reemerged as a safe and reliable alternative for the healing of cutaneous ulcers that do not respond to the conventional treatments. Objective. To evaluate the use of the larvae of Lucilia sericata as a treatment for infected wounds with Pseudomonas aeruginosa in an animal model. Materials and methods. Twelve rabbits were randomly distributed in 3 groups: the first group was treated with larval therapy; the second was treated with antibiotics therapy and to the third no treatment was applied, therefore was established as a control group. To each animal a wound was artificially induced, and then a suspension of P. aeruginosa was inoculated into the lesion. Finally, every rabbit was evaluated until the infection development was recognized and treatment was set up for the first two groups according with the protocols mentioned above. Macroscopic evaluation of the wounds was based on the presence of edema, exudates, bad odor, inflammation around the wound and the presence of granulation tissue. The healing process was evaluated by monitoring histological changes in the dermal tissue. Results. Differences in the time required for wound healing were observed between the first group treated with larval therapy (10 days) and the second group treated with conventional antibiotics therapy (20 days). Conclusion. The L. sericata larva is and efficient tool as a therapy for infected wounds with P. aeruginosa.


Asunto(s)
Animales , Modelos Animales , Pseudomonas aeruginosa , Terapéutica , Cicatrización de Heridas , Heridas y Lesiones , Larva
18.
Mem Inst Oswaldo Cruz ; 100(6): 519-25, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16302061

RESUMEN

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.


Asunto(s)
Leishmania infantum/crecimiento & desarrollo , Psychodidae/parasitología , Animales , Línea Celular/parasitología , Humanos , Leishmania infantum/ultraestructura , Microscopía Electrónica de Transmisión , Psychodidae/citología
19.
Mem. Inst. Oswaldo Cruz ; 100(6): 519-525, Oct. 2005. tab, graf
Artículo en Inglés | LILACS | ID: lil-417069

RESUMEN

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6 percent) and on day 4 in the J774 cells (51 percent). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.


Asunto(s)
Humanos , Animales , Leishmania infantum/crecimiento & desarrollo , Psychodidae/citología , Línea Celular/parasitología , Leishmania infantum/ultraestructura , Microscopía Electrónica , Psychodidae/parasitología
20.
J Am Mosq Control Assoc ; 21(1): 28-32, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15825758

RESUMEN

The main objective of the present work was to establish and maintain a colony of Ochlerotatus taeniorhynchus, Riohacha strain, under laboratory conditions and to study its life cycle. The colony's insectary was established from adult male and female mosquitoes collected from peripheral neighborhoods of Riohacha, Colombia. Environmental conditions established in the insectary were 26.5 degrees C average temperature, 80% average relative humidity, and 12 h photoperiodicity. Eight continuous generations were taken into account for maintaining the mosquitoes and analyzing their life cycle. The male mosquito's average life cycle was 26.8 days. The female's cycle was 30.8 days. Analysis of each of the biological stages of development (mean days) produced the following results: egg incubation 4.55 +/- 0.291, larvae 8.28 +/- 0.499, pupae 1.32 +/- 0.215, adult male 12.65 +/- 5.920. and adult female 16.73 +/- 6.034. The Riohacha colony has been maintained for 32 generations in 31 months. Comparison of the Riohacha colony with the previously established Cartagena and Barranquilla colonies showed few differences in the duration of stages of the life cycle between strains.


Asunto(s)
Ochlerotatus , Animales , Animales de Laboratorio , Colombia , Femenino , Larva , Masculino , Ochlerotatus/fisiología , Pupa
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