Antiproliferative activity of essential oils from three plants of the Brazilian Cerrado: Campomanesia adamantium (Myrtaceae), Protium ovatum (Burseraceae) and Cardiopetalum calophyllum (Annonaceae) / Atividade antiproliferativa dos óleos essenciais de três plantas do Cerrado brasileiro: Campomanesia adamantium (Myrtaceae), Protium ovatum (Burseraceae) e Cardiopetalum calophyllum (Annonaceae)

Abstract Essential oils, which may be extracted from several parts of plants, have different biological activities. The Brazilian Cerrado has a large variety of plants that yield essential oils, even though many have not been studied yet. Taking into account the biodiversity of this biome, this study aimed at evaluating the antiproliferative activity of essential oils extracted from three species of plants of the Cerrado in Goiás state: Campomanesia adamantium (Cambess.) O. Berg, Protium ovatum (Engl. in Mart.) and Cardiopetalum calophyllum (Schltdl.). Essential oils were extracted from both C. adamantium and C. calophyllum leaves and from P. ovatum leaves and green fruits by hydrodistillation carried out by a Clevenger-type apparatus. The chemical composition of the essential oils was determined by Gas Chromatography coupled to Mass Spectrometry (GC-MS). The following major chemical constituents were identified in the essential oils under investigation: β-myrcene (62.00%), spathulenol (28.78%), germacrene-B (18.27%), β-caryophyllene oxide (16.40%), β-caryophyllene (14.00%), α-pinene (11.30%), viridiflorol (9.99%), limonene (7.30%) and (Z,E)-pharnesol (6.51%). The antiproliferative activity was evaluated in different human tumor cell lines: breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) and glioblastoma (M059J). A normal human cell line was included (GM07492A, lung fibroblasts). Results showed that essential oils from C. adamantium leaves got the lowest values of IC50 in all strains of tumor cells under evaluation. They were significantly lower than the ones of the normal cell line, an evidence of selectivity. It is worth mentioning that this is the first report of the antiproliferative activity of essential oils from C. adamantium , P. ovatum and C. calophyllum against human tumor cells.

Resumo Os óleos essenciais podem ser extraídos de várias partes das plantas e apresentam diversas atividades biológicas. O Cerrado brasileiro possui uma grande variedade de plantas produtoras de óleos essenciais muitas delas ainda não estudadas. Levando-se em consideração a biodiversidade desse bioma, o objetivo deste trabalho foi avaliar a atividade antiproliferativa dos óleos essenciais extraídos de três espécies de plantas ocorrentes no Cerrado do estado de Goiás: Campomanesia adamantium (Cambess.) O. Berg, Protium ovatum (Engl. in Mart.) e Cardiopetalum calophyllum (Schltdl.). Os óleos essenciais foram obtidos das folhas de C. adamantium e C. calophyllum e das folhas e frutos verdes de P. ovatum por hidrodestilação, usando o aparelho do tipo Clevenger. A composição química dos óleos essenciais foi determinada pelo método de Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-EM). Os constituintes químicos majoritários identificados nos óleos essenciais estudados foram: β-mirceno (62,00%), espatulenol (28,78%), germacreno-B (18,27%), óxido de β-cariofileno (16,40%), β-cariofileno (14,00%), α-pineno (11,30%), viridiflorol (9,99%), limoneno (7,30%) e (Z,E)-farnesol (6,51%). A atividade antiproliferativa foi avaliada em diferentes linhagens de células tumorais humanas: adenocarcinoma de mama (MCF-7), adenocarcinoma cervical (HeLa) e gliobastoma (M059J), além de, uma linhagem celular humana normal (GM07492A, fibroblastos pulmonares). O óleo essencial das folhas de C. adamantium exibiu menores valores de CI50 em todas as linhagens celulares tumorais avaliadas, sendo menores que aquele obtido na linhagem celular normal, indicando seletividade. Este é o primeiro relato da atividade antiproliferativa dos óleos essenciais de C. adamantium , P. ovatum e C. calophyllum contra células tumorais humanas.

Antiproliferative activity of essential oils from three plants of the Brazilian Cerrado: Campomanesia adamantium (Myrtaceae), Protium ovatum (Burseraceae) and Cardiopetalum calophyllum (Annonaceae).

Essential oils, which may be extracted from several parts of plants, have different biological activities. The Brazilian Cerrado has a large variety of plants that yield essential oils, even though many have not been studied yet. Taking into account the biodiversity of this biome, this study aimed at evaluating the antiproliferative activity of essential oils extracted from three species of plants of the Cerrado in Goiás state: Campomanesia adamantium (Cambess.) O. Berg, Protium ovatum (Engl. in Mart.) and Cardiopetalum calophyllum (Schltdl.). Essential oils were extracted from both C. adamantium and C. calophyllum leaves and from P. ovatum leaves and green fruits by hydrodistillation carried out by a Clevenger-type apparatus. The chemical composition of the essential oils was determined by Gas Chromatography coupled to Mass Spectrometry (GC-MS). The following major chemical constituents were identified in the essential oils under investigation: ß-myrcene (62.00%), spathulenol (28.78%), germacrene-B (18.27%), ß-caryophyllene oxide (16.40%), ß-caryophyllene (14.00%), α-pinene (11.30%), viridiflorol (9.99%), limonene (7.30%) and (Z,E)-pharnesol (6.51%). The antiproliferative activity was evaluated in different human tumor cell lines: breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) and glioblastoma (M059J). A normal human cell line was included (GM07492A, lung fibroblasts). Results showed that essential oils from C. adamantium leaves got the lowest values of IC50 in all strains of tumor cells under evaluation. They were significantly lower than the ones of the normal cell line, an evidence of selectivity. It is worth mentioning that this is the first report of the antiproliferative activity of essential oils from C. adamantium , P. ovatum and C. calophyllum against human tumor cells.

Antiproliferative activity of essential oils from three plants of the Brazilian Cerrado: Campomanesia adamantium (Myrtaceae), Protium ovatum (Burseraceae) and Cardiopetalum calophyllum (Annonaceae)

Abstract Essential oils, which may be extracted from several parts of plants, have different biological activities. The Brazilian Cerrado has a large variety of plants that yield essential oils, even though many have not been studied yet. Taking into account the biodiversity of this biome, this study aimed at evaluating the antiproliferative activity of essential oils extracted from three species of plants of the Cerrado in Goiás state: Campomanesia adamantium (Cambess.) O. Berg, Protium ovatum (Engl. in Mart.) and Cardiopetalum calophyllum (Schltdl.). Essential oils were extracted from both C. adamantium and C. calophyllum leaves and from P. ovatum leaves and green fruits by hydrodistillation carried out by a Clevenger-type apparatus. The chemical composition of the essential oils was determined by Gas Chromatography coupled to Mass Spectrometry (GC-MS). The following major chemical constituents were identified in the essential oils under investigation: -myrcene (62.00%), spathulenol (28.78%), germacrene-B (18.27%), -caryophyllene oxide (16.40%), -caryophyllene (14.00%), -pinene (11.30%), viridiflorol (9.99%), limonene (7.30%) and (Z,E)-pharnesol (6.51%). The antiproliferative activity was evaluated in different human tumor cell lines: breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) and glioblastoma (M059J). A normal human cell line was included (GM07492A, lung fibroblasts). Results showed that essential oils from C. adamantium leaves got the lowest values of IC50 in all strains of tumor cells under evaluation. They were significantly lower than the ones of the normal cell line, an evidence of selectivity. It is worth mentioning that this is the first report of the antiproliferative activity of essential oils from C. adamantium , P. ovatum and C. calophyllum against human tumor cells.

Resumo Os óleos essenciais podem ser extraídos de várias partes das plantas e apresentam diversas atividades biológicas. O Cerrado brasileiro possui uma grande variedade de plantas produtoras de óleos essenciais muitas delas ainda não estudadas. Levando-se em consideração a biodiversidade desse bioma, o objetivo deste trabalho foi avaliar a atividade antiproliferativa dos óleos essenciais extraídos de três espécies de plantas ocorrentes no Cerrado do estado de Goiás: Campomanesia adamantium (Cambess.) O. Berg, Protium ovatum (Engl. in Mart.) e Cardiopetalum calophyllum (Schltdl.). Os óleos essenciais foram obtidos das folhas de C. adamantium e C. calophyllum e das folhas e frutos verdes de P. ovatum por hidrodestilação, usando o aparelho do tipo Clevenger. A composição química dos óleos essenciais foi determinada pelo método de Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-EM). Os constituintes químicos majoritários identificados nos óleos essenciais estudados foram: -mirceno (62,00%), espatulenol (28,78%), germacreno-B (18,27%), óxido de -cariofileno (16,40%), -cariofileno (14,00%), -pineno (11,30%), viridiflorol (9,99%), limoneno (7,30%) e (Z,E)-farnesol (6,51%). A atividade antiproliferativa foi avaliada em diferentes linhagens de células tumorais humanas: adenocarcinoma de mama (MCF-7), adenocarcinoma cervical (HeLa) e gliobastoma (M059J), além de, uma linhagem celular humana normal (GM07492A, fibroblastos pulmonares). O óleo essencial das folhas de C. adamantium exibiu menores valores de CI50 em todas as linhagens celulares tumorais avaliadas, sendo menores que aquele obtido na linhagem celular normal, indicando seletividade. Este é o primeiro relato da atividade antiproliferativa dos óleos essenciais de C. adamantium , P. ovatum e C. calophyllum contra células tumorais humanas.

Indirect ELISA for diagnosis of Brucella ovis infection in rams / ELISA indireto para diagnóstico da infecção por Brucella ovis em carneiros

Brucella ovis is a major cause of epididymitis in sexually mature rams, resulting in subfertility, infertility, and economic losses for the sheep industry worldwide. The aim of this study was to develop an indirect ELISA (iELISA) using recombinant proteins, namely rBoP59 and rBP26, as antigens for serological diagnosis of B. ovis infection. The BoP59 and BP26 recombinant proteins were expressed in E. coli and purified by affinity chromatography. Antigenicity was tested by Western blot and iELISA. Standardization of iELISA was performed with 500ng and 1µg BoP59 and rBP26 per well, testing serum from uninfected and experimentally infected rams. rBP26 was effective in distinguishing positive from negative rams. The rBP26 iELISA developed in this study is the first to use a completely purified rBP26 as antigen resulting in high sensitivity (100%) and specificity (90.2%), and an overall accuracy equal to 1.0...

Brucella ovis é uma das principais causas de epididimite em carneiros sexualmente maduros, resultando em subfertilidade e infertilidade e consequentes perdas econômicas para a ovinocultura em todo o mundo. O objetivo deste estudo foi desenvolver ELISA indireto (ELISAi), utilizando como antígeno proteínas recombinantes BoP59r e BP26r para diagnóstico da infecção por B. ovis. BoP59r e BP26r foram expressas em E. coli e purificadas por cromatografia de afinidade e a antigenicidade testada por Western blot e ELISAi. A padronização do ELISAi foi realizada testando 500 ng e 1 µg de BoP59r e BP26r por poço e soros de carneiros infectados e não infectados. A BP26r foi eficiente em diferenciar ovinos negativos de positivos. O ELISAi com BP26 desenvolvido neste estudo foi o primeiro a utilizar BP26 completamente purificada como antígeno, resultando em elevada sensibilidade (100%) e sensibilidade (90,2%), com acurácia igual a 1,0...

The mucosal inflammatory response to non-typhoidal Salmonella in the intestine is blunted by IL-10 during concurrent malaria parasite infection.

Coinfection can markedly alter the response to a pathogen, thereby changing its clinical presentation. For example, non-typhoidal Salmonella (NTS) serotypes are associated with gastroenteritis in immunocompetent individuals. In contrast, individuals with severe pediatric malaria can develop bacteremic infections with NTS, during which symptoms of gastroenteritis are commonly absent. Here we report that, in both a ligated ileal loop model and a mouse colitis model, malaria parasites caused a global suppression of gut inflammatory responses and blunted the neutrophil influx that is characteristic of NTS infection. Further, malaria parasite infection led to increased recovery of Salmonella enterica serotype Typhimurium from the draining mesenteric lymph node (MLN) of mice. In the mouse colitis model, blunted intestinal inflammation during NTS infection was independent of anemia but instead required parasite-induced synthesis of interleukin (IL)-10. Blocking of IL-10 in coinfected mice reduced dissemination of S. Typhimurium to the MLN, suggesting that induction of IL-10 contributes to development of disseminated infection. Thus IL-10 produced during the immune response to malaria in this model contributes to suppression of mucosal inflammatory responses to invasive NTS, which may contribute to differences in the clinical presentation of NTS infection in the setting of malaria.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams / Nested PCR espécie-específica para o diagnóstico da infecção por Brucella ovis em carneiros

The aim of the present study was to evaluate a species-specific nested PCR based on a previously described species-specific PCR for detection of B. ovis in semen and urine samples of experimentally infected rams. The performance of the species-specific nested PCR was compared with the results of a genus-specific PCR. Fourteen rams were experimentally infected with the Brucella ovis REO 198 strain and samples of semen and urine were collected every week up to 180 days post infection. Out of 83 semen samples collected, 42 (50.6%) were positive for the species-specific nested PCR, and 23 (27.7%) were positive for the genus-specific PCR. Out of 75 urine samples, 49 (65.3%) were positive for the species-specific nested PCR, whereas 11 (14.6%) were genus-specific PCR positive. Species-specific nested PCR was significantly more sensitive (P<0.001) than the genus-specific PCR in semen and urine from experimentally infected rams. In conclusion, the species-specific nested PCR developed in this study may be used as a diagnostic tool for the detection of B. ovis in semen and urine samples from suspected rams.

O presente estudo objetivou avaliar uma técnica de nested PCR espécie-específica delineada a partir de PCR espécie-específica descrita anteriormente para detecção de B. ovis em sêmen e urina de carneiros infectados experimentalmente. O desempenho da nested PCR espécie-específica foi comparado com os resultados de uma PCR gênero-específica. Quatorze carneiros foram infectados experimentalmente com Brucella ovis REO 198 e amostras de sêmen e de urina foram colhidas semanalmente até 180 dias após a infecção. De 83 amostras de sêmen, 42 (50,6%) foram positivas pela nested PCR espécie-específica, e 23 (27,7%) foram positivas pela PCR gênero-específica. De 75 amostras de urina, 49 (65,3%) foram positivas pela nested PCR espécie-específica, enquanto 11 (14,6%) foram positivas em PCR gênero-específica. A técnica de nested PCR espécie-específica foi significativamente mais sensível (P<0,001) do que a PCR gênero-específica no sêmen e na urina de carneiros infectados experimentalmente. Em conclusão, a nested PCR espécie-específica desenvolvida neste estudo pode ser utilizada como ferramenta de diagnóstico para detecção de B. ovis em sêmen e urina de carneiros suspeitos.

A comparison of two agar gel immunodiffusion methods and a complement fixation test for serologic diagnosis of Brucella ovis infection in experimentally infected rams / Comparação entre dois métodos de imunodifusão em gel de Agar e um método de fixação de complemento para o diagnóstico sorológico da infecção por Brucella ovis em carneiros experimentalmente infectados

A infecção por Brucella ovis é considerada uma das principais causas de epididimite e infertilidade em carneiros, resultando em falhas reprodutivas e perdas econômicas significativas em rebanhos ovinos ao redor do mundo. O estudo teve o objetivo de avaliar três testes sorológicos disponíveis para o diagnóstico da brucelose ovina por B. ovis, utilizando 181 soros ovinos. Amostras de soro provenientes de carneiros experimentalmente infectados foram coletadas ao longo de 192 dias pós-infecção (n=117) e durante o período pré-infecção (n=9). Adicionalmente, amostras de soro foram obtidas de ovinos provenientes de um rebanho livre para B. ovis (n=55). As técnicas de imunodifusão em gel de agar (IDGA), utilizando dois antígenos disponíveis comercialmente, e de fixação de complemento foram comparadas (FC). Foram obtidos resultados de sensibilidade especificidade semelhantes para ambos os métodos de IDGA e ainda, a técnica de IDGA foi mais eficiente do que a da FC para o diagnóstico sorológico da infecção por B. ovis.

Enteric pathology and Salmonella-induced cell death in healthy and SIV-infected rhesus macaques.

The goal of this study was to morphologically characterize a ligated ileal loop model of Salmonella enterica serotype Typhimurium infection in rhesus macaques (Macaca mulatta) and to verify the occurrence of Salmonella-induced cell death in vivo. Eight adult healthy male rhesus macaques were used for ligated ileal loop surgery. Four macaques had been intravenously inoculated with simian immunodeficiency virus (SIV) mac251. Ileal ligated loops were inoculated with wild-type (WT) S. Typhimurium strain IR715 (ATCC14028 nal (r)), an isogenic noninvasive mutant strain (ATCC14028 nal (r) ΔsipAΔsopABDE2), or sterile Luria Bertani broth. Loops were surgically removed at 2, 5, and 8 hours post-inoculation (hpi). Intestinal samples were processed for histopathology, immunohistochemistry for detecting Salmonella, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and transmission electron microscopy. Combined histopathology scores were similar between SIV-infected and control macaques. As expected, the invasion-deficient mutant was less pathogenic than WT S. Typhimurium. Neutrophil infiltrate in the intestinal mucosa correlated with bacterial loads (r = 0.7148; P < .0001) and fluid accumulation (r = 0.6019; P < .0001) in the lumen of the intestinal loops. Immunolabeled WT S. Typhimurium was observed in the epithelium and lamina propria at the tip of the villi at 2 hpi, progressing toward deeper lamina propria at 5-8 hpi. Most TUNEL-positive cells localized to the lamina propria, and some had morphological features of macrophages. Ultrastructurally, bacteria were observed intracellularly in the lamina propria as well as within apoptotic bodies. This study provides morphological evidence of Salmonella-induced cell death in vivo in a relevant nonhuman primate model.

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen.

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10(6) spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.

Pathological, immunohistochemical and bacteriological study of tissues and milk of cows and fetuses experimentally infected with Brucella abortus.

This report describes a pathological, immunohistochemical and bacteriological study of 42 cows and their progeny (aborted fetuses, weak premature calves, and healthy full-term calves) infected at 6-7 months of gestation by conjunctival inoculation with Brucella abortus. Samples were collected at necropsy within 48 h of abortion or parturition. The most significant lesions were necrotizing and suppurative placentitis and lymphohistiocytic mastitis in cows, and fibrinous pleuritis, fibrinous pericarditis and bronchopneumonia in aborted fetuses. B. abortus was isolated more frequently from milk samples than from mammary tissues, and milk samples from cows with mastitis were often infected. Organisms were often demonstrated immunohistochemically and by culture in tissues showing moderate to severe histological changes.