Mitochondrial energy metabolism in baby hamster kidney (BHK-21/C13) cells treated with karnozin extra® / Metabolismo energético mitocondrial en células de riñón de hámster bebé (BHK-21 / C13) tratadas con karnozin extra®

SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.

RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.

A comprehensive morphometric study of visceral and subcutaneous adipose tissue depots in mice, hamsters and rats / Un estudio morfométrico completo de los depósitos de tejido adiposo visceral y subcutáneo en ratones, hámsters y ratas

SUMMARY: Adipose tissue morphology of different fat tissue depots can be described using the number of adipocytes and cell surface of adipocytes. This study deals with characteristics and morphometric analysis of white and brown adipose tissue depots in healthy adult laboratory mice, hamsters and rats of both sexes. The number of unilocular adipocytes in white adipose tissue differs from one adipose tissue depot to another, with the largest number of adipocytes in mice and a similar number in hamsters and rats. The smallest surface area and the largest percentage of small unilocular adipocytes were found in mice. White adipose tissue in hamsters and rats was predominantly made out of a larger percentage of medium-sized adipocytes and a smaller percentage of small and medium-sized adipocytes. Uncoupling protein 1 positive multilocular adipocytes were found in classic brown adipose tissue depots with larger percentages in mice (93.20 %) and hamsters (91.30 %), while rats had a smaller percentage (78.10 %). In white and brown adipose tissue, significant differences between species and both sexes within the same species were found, indicating the influence of sexual dimorphism. The presented morphometric results could serve as a basis for further studies concerning experimental animal models of metabolic disorders and obesity.

RESUMEN: La morfología del tejido adiposo de diferentes depósitos de tejido graso se puede describir utilizando el número de adipocitos y la superficie celular de los adipocitos. Este estudio analiza las características y el análisis morfométrico de los depósitos de tejido adiposo blanco y marrón en ratones, hamsters y ratas de laboratorio, adultos sanos de ambos sexos. El número de adipocitos uniloculares en el tejido adiposo blanco difiere de un depósito de tejido adiposo a otro, con el mayor número de adipocitos en ratones y un número similar en hámsteres y ratas. La superficie más pequeña y el mayor porcentaje de adipocitos uniloculares pequeños se encontraron en ratones. El tejido adiposo blanco en hámsteres y ratas estaba compuesto predominantemente por un mayor porcentaje de adipocitos de tamaño mediano y un porcentaje menor de adipocitos de tamaño pequeño y mediano. Los adipocitos multiloculares positivos para la proteína desacopladora 1 se encontraron en depósitos de tejido adiposo marrón clásico con mayores porcentajes en ratones (93,20 %) y hámsters (91,30 %), mientras que las ratas tenían un porcentaje menor (78,10 %). En el tejido adiposo blanco y pardo se encontraron diferencias significativas entre especies y entre ambos sexos dentro de una misma especie, lo que indica la influencia del dimorfismo sexual. Los resultados morfométricos presentados podrían servir como base para futuros estudios sobre modelos animales experimentales de trastornos metabólicos y obesidad.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Identification and sequence analysis of prolactin receptor and its differential expression profile at various developmental stages in striped hamsters

Prolactin (PRL) plays critical roles in regulation of biological functions with the binding of specific prolactin receptor (PRLR). Revealing the expression patterns of PRLR at different developmental stages is beneficial to better understand the role of PRL and its mechanism of action in striped hamsters. In this study, the cDNA sequence of PRLR (2866-base-pairs) was harvested from the pituitary of mature female striped hamsters (Cricetulus barabensis) that contains an 834-base-pair 5′-untranslated region (1-834 bp), a 1848-base-pair open reading frame (835-2682 bp), and a 184-base-pair 3′-untranslated region (2683-2866). The 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids. In the mature PRLR, two prolactin-binding motifs, 12 cysteines, and five potential Asn-linked glycosylation sites were detected. Our results showed that the PRLR mRNA quantity in the hypothalamus, pituitary, ovaries, or testis was developmental-stage-dependent, with the highest level at sub-adult stage and the lowest level at old stage. We also found that PRLR mRNAs were highest in pituitary, medium level in hypothalamus, and lowest in ovaries or testis. PRLR mRNAs were significantly higher in males than in females, except in the hypothalamus and pituitary from 7-week-old striped hamsters. Moreover, the PRLR mRNAs in the hypothalamus, pituitary, and ovaries or testis were positively correlated with the expression levels of GnRH in the hypothalamus. These results indicated that the PRLR has conserved domain in striped hamster, but also possesses specific character. PRLR has multiple biological functions including positively regulating reproduction in the striped hamster.

Effect of topical 2% eucalyptus extract on 5-fu-induced oral mucositis in male golden hamsters

Abstract Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p<0.05). Also, a lower malondialdehyde level and higher myeloperoxidase and glutathione activities were detected in the eucalyptus group in comparison to the calendula and control groups (p<0.001). The thickness of the mucosa and submucosa increased in the eucalyptus group. The numerical density of the fibroblast and the volume density of the collagen significantly increased in the eucalyptus group. In conclusion, the use of eucalyptus hydroalcoholic extract may be associated with reduced intensity of oral mucositis, diminished concentration of malondialdehyde, increased activity of myeloperoxidase and glutathione, increased volume of mucosa and submucosa, increased fibroblast and collagen in the induced oral mucositis in golden hamsters undergoing 5-FU consumption.

Resumo A mucosite oral é uma complicação inflamatória comum em pacientes com câncer. Este estudo avaliou os marcadores histopatológicos, estereológicos e antioxidantes de Eucalyptus 2% na mucosite oral induzida em hamsters dourados machos. Neste estudo em animais, a mucosite oral foi induzida em 30 hamsters golden masculinos por 5-FU (60 mg / kg) nos dias 0, 5 e 10 do estudo. A bolsa da bochecha foi arranhada com uma agulha estéril uma vez ao dia nos dias 3 e 4. Nos dias 14 a 17, os grupos de gel de eucalipto a 2% e curativos à base de gel foram tratados e comparados com um grupo controle. Foram avaliados os escores histopatológicos e estereológicos e o conteúdo de malondialdeído na bolsa, bem como as atividades de glutationa e mieloperoxidase no tecido da bolsa. Os escores histopatológicos de mucosite foram menores no grupo de gel de eucalipto a 2% do que os do gel e do grupo controle (p <0,05). Além disso, um nível mais baixo de malondialdeído e maiores atividades de mieloperoxidase e glutationa foram detectadas no grupo tratado com eucalipto em comparação aos grupos à base de gel e controle (p <0,001). A espessura da mucosa e submucosa aumentou no grupo Eucalyptus. A densidade numérica do fibroblasto e a densidade do volume do colágeno aumentaram significativamente nos grupos tratados com eucalipto. Em conclusão, o uso do extrato hidroalcoólico de Eucalyptus pode estar associado a menor intensidade de mucosite oral, diminuição da concentração de malondialdeído, aumento da atividade de mieloperoxidase e glutationa, aumento do volume de mucosa e submucosa, aumento de fibroblastos e colágeno na mucosite oral induzida em hamsters dourados em consumo de 5 UF.

DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Oxyurid nematodes of pet rodents in Slovakia - a neglected zoonotic threat / Nematódeos oxiurídeos de roedores de estimação na Eslováquia - uma ameaça zoonótica negligenciada

Abstract The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.

Resumo O papel dos roedores como reservatórios de helmintos de importância para a saúde pública não é bem conhecido. O potencial zoonótico de Syphacia spp. foi confirmado; portanto, o estudo teve como objetivo estimar a ocorrência de nematóides oxiurídeos em pequenos roedores de pet shops e clubes de reprodução na Eslováquia. Amostras fecais de 586 roedores mantidos em 133 gaiolas foram coletadas entre 2016 e 2018 e examinadas pelo método de flotação de Faust. Foram detectadas quatro espécies de nematódeos oxiurídeos, Syphacia muris, S. obvelata, Aspiculuris tetraptera e Paraspidodera uncinata, A. tetraptera foi encontrado nas amostras fecais de todas as espécies de roedores incluídas nesta pesquisa. O número de gaiolas positivas variou de 5,4% em hamsters a 70,0% em camundongos. A prevalência de Syphacia muris foi maior nos gerbilos da Mongólia, onde até 75,0% das gaiolas foram positivas; S. obvelata foi encontrada em 26,7% das gaiolas com camundongos, 25,0% das gaiolas com gerbilos da Mongólia e 3,2% das gaiolas com ratos. A alta prevalência de Syphacia spp. em todas as espécies animais aponta o risco de infecção para os seres humanos. Animais oferecidos para venda estão frequentemente em contato próximo com seres humanos; portanto, eles devem ser regularmente testados quanto a parasitas e, então, efetivamente desparasitados.

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.

Investigating associations between intestinal alterations and parasite load according to Bifidobacterium spp. and Lactobacillus spp. abundance in the gut microbiota of hamsters infected by Leishmania infantum

BACKGROUND Visceral leishmaniasis (VL) is a tropical neglected disease with high associated rates of mortality. Several studies have highlighted the importance of the intestinal tract (IT) and gut microbiota (GM) in the host immunological defense. Data in the literature on parasite life cycle and host immune defense against VL are scarce regarding the effects of infection on the IT and GM. OBJECTIVES This study aimed to investigate changes observed in the colon of Leishmania infantum-infected hamsters, including alterations in the enteric nervous system (ENS) and GM (specifically, levels of bifidobacteria and lactobacilli). METHODS Male hamsters were inoculated with L. infantum and euthanised at four or eight months post-infection. Intestines were processed for histological analysis and GM analysis. Quantitative polymerase chain reaction (qPCR) was performed to quantify each group of bacteria: Bifidobacterium spp. (Bf) and Lactobacillus spp (LacB). FINDINGS Infected hamsters showed histoarchitectural loss in the colon wall, with increased thickness in the submucosa and the mucosa layer, as well as greater numbers of intraepithelial lymphocytes. Forms suggestive of amastigotes were seen inside mononuclear cells. L. infantum infection induced changes in ENS, as evidenced by increases in the area of colonic enteric ganglia. Despite the absence of changes in the levels of Bf and LacB during the course of infection, the relative abundance of these bacteria was associated with parasite load and histological alterations. MAIN CONCLUSIONS Our results indicate that L. infantum infection leads to important changes in the colon and suggest that bacteria in the GM play a protective role.

Comparative assessment of the replication efficiency of dengue, yellow fever, and chikungunya arboviruses in some insect and mammalian cell lines

Abstract INTRODUCTION: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. METHODS: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. RESULTS: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. CONCLUSIONS: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication.

Amifostine reduces inflammation and protects against 5-fluorouracil-induced oral mucositis and hyposalivation

Oral mucositis (OM) is a common and dose-limiting side effect of cancer treatment, including 5-fluorouracil (5-FU) and radiotherapy. The efficacy of the therapeutic measures to prevent OM is limited and disease prevention is not fully observable. Amifostine is a cytoprotective agent with a described anti-inflammatory potential. It is clinically used to reduce radiotherapy and chemotherapy-associated xerostomia. This study investigated the protective effect of amifostine on an experimental model of OM. Hamsters were divided into six groups: saline control group (5 mL/kg), mechanical trauma (scratches) of the right cheek pouch; 5-FU (60 and 40 mg/kg, ip, respectively, administered on days 1 and 2); amifostine (12.5, 25, or 50 mg/kg) + 5-FU + scratches. Salivation rate was assessed and the animals were euthanized on day 10 for the analysis of macroscopic and microscopic injury by scores. Tissue samples were harvested for the measurement of neutrophil infiltration and detection of inflammatory markers by ELISA and immunohistochemistry. 5-FU induced pronounced hyposalivation, which was prevented by amifostine (P<0.05). In addition, 5-FU injection caused pronounced tissue injury accompanied by increased neutrophil accumulation, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) tissue levels, and positive immunostaining for TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS). Interestingly, amifostine prevented the inflammatory reaction and consequently improved macroscopic and microscopic damage (P<0.05 vs 5-FU group). Amifostine reduced inflammation and protected against 5-FU-associated oral mucositis and hyposalivation.

Red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells / Própolis vermelha e L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256

ABSTRACT Objective: To evaluate the effect of red propolis and L-lysine on angiogenesis and tumor growth in a new model of hamster cheek pouch inoculated with Walker 256 tumor cells. Methods: The study consisted of two experiments with four groups each (total: 57 hamsters). In the experiment 1, the animals were inoculated with Walker tumor cells, followed by administration of test substances (red propolis 200mg/5mL/kg or L-lysine 150mg/kg) or control substances (gum arabic 5mL/kg or water 5mL/kg) for 10 days. The animals in the experiment 2 received red propolis, L-lysine, gum arabic or water at the same doses, for 33 days prior to inoculation of Walker tumor cells, followed by 10 days of treatment with the same substances. Based on single-plane images, angiogenesis was quantified (mean vascular area), in percentage, and tumor area (mm2) and perimeter (mm). Results: In the experiment 1, compared to animals receiving water, the mean vascular area expressed in percentage was significantly smaller in animal treated with propolis (p<0.05) and L-lysine (p<0.001). Conclusion: Both red propolis and L-lysine inhibited tumor angiogenesis in the new hamster cheek pouch model when administered after tumor inoculation.

RESUMO Objetivo: Avaliar o efeito da própolis vermelha e da L-lisina na angiogênese e no crescimento tumoral em novo modelo de bolsa jugal de hamster inoculada com células de tumor de Walker 256. Métodos: O estudo consistiu em dois experimentos com quatro grupos cada (total: 57 hamsters). No experimento 1, os animais foram inoculados com células de tumor de Walker, tendo em seguida administradas as substâncias teste (própolis vermelha 200mg/5mL/kg ou L-lisina 150mg/kg) ou controle (goma arábica 5mL/kg ou água 5mL/kg) por 10 dias. Os animais do experimento 2 receberam própolis vermelha, L-lisina, goma arábica ou água nas mesmas doses, por 33 dias antes do inóculo das células de tumor de Walker, seguido por 10 dias de tratamento com as mesmas substâncias. Baseado em imagens em plano único, foram quantificados a angiogênese (área vascular média), em termos percentuais, e a área (mm2) e o perímetro (mm) do tumor. Resultados: Comparada aos animais que receberam água, a área vascular média, expressa em percentagem, foi significativamente menor nos animais tratados com própolis (p<0,05) e com L-lisina (p<0,001). Conclusão: Tanto a própolis vermelha quanto a L-lisina inibiram a angiogênese no novo modelo de bolsa jugal de hamsters, quando administradas após a inoculação do tumor.

Effect of red propolis on hamster cheek pouch angiogenesis in a new sponge implant model

Abstract Purpose: To evaluate the effects of red propolis on cheek pouch angiogenesis in a hamster new model sponge implant. Methods: Forty eight animals divided into eight groups. (Groups I-IV), the animals were treated for 15 days before and 10 days after sponge implantation. (Groups V-VIII), the animals were treated for 10 days after sponge implantation (GI and GV: red propolis 100 mg/kg, GII and GVI: celecoxib 20 mg/kg, GIII and GVII: 1% gum arabic 5 mL/kg, GIV and GVIII: distilled water 5 mL/kg). On the 11th day of implantation, the animals were anesthetized for stereoscopic microscopic imaging and morphometric quantification of angiogenesis (SQAN), followed by histopathological evaluation (H&E). Results: In the SQAN analysis, no significant difference was found between the groups. However, on histology, propolis was found reduce the population of mastocytes in the qualitative analyses (p = 0,013) in the quantitative analyses to reduce the number of blood vessels (p = 0,007), and increase the macrophage count (p = 0,001). Conclusion: Red propolis inhibited inflammatory angiogenesis when administered before andcontinuously after sponge implant, and was shown to have immunomodulating effects on inflammatory cells (mastocytes and macrophages) in a new sponge implant hamster model.

Maternal infection by Leishmania braziliensis in hamster does not influence the course of disease in progeny / A infecção materna por Leishmania braziliensis em hamster não influencia o curso da doença na prole

Introduction: Immunoglobulins, soluble antigens, cells, cytokines and other immune system products can be transferred from infected mother to her offspring, leading to suppression or stimulation of immune response. Objective: To evaluate the influence of gender and maternal infection with Leishmania braziliensis in the course of the disease in the offspring of hamsters. Methods: Offspring born from infected mother (IMO) or non-infected mother (NIMO) by Leishmania braziliensis, both sexes, was infected with the same strain of the mother after 30 days of life and followed for 18 weeks. We evaluated the thickness of the lesion, parasite load and histology of the lesions. Results: The number of parasite in both lesions and lymph node of IMO offspring showed a significant reduction in the 5th week post-infection compared to the NIMO offspring; however, this did not correspond to clinical symptoms. Histopathological analysis revealed that in the IMO offspring, the inflammatory process was more prominent. In relation to gender, it was observed that the male offspring showed lesion thickness and higher parasite burden than females. Conclusion: Maternal infection by L. braziliensis in hamsters does not appear to influence the course of the disease in the homologous offspring infection, as well as the male offspring presented augmented susceptibility to L. braziliensis infection regardless of whether they were born from IMO or NIMO. Also, the reduction of the granuloma index in the IMO offspring, together with the higher inflammatory response, suggests a less effective cellular response in the chronic phase of the disease in these animals. (AU)

Introdução: Imunoglobulinas, antígenos solúveis, células, citocinas e outros produtos do sistema imune podem ser transferidos de mãe infectada para a sua prole, levando à supressão ou estimulação da resposta imune. Objetivo: Avaliar a influência do gênero e a infecção materna por Leishmania braziliensis no curso da doença na prole de hamsters. Métodos: Filhotes nascidos de mãe infectada (MI) e mãe não infectada (MNI) por L. braziliensis, ambos os sexos, foram infectados com a mesma cepa da mãe após 30 dias de vida e acompanhados por 18 semanas. Avaliou-se a espessura da lesão, a carga parasitária e os aspectos histopatológicos das lesões. Resultados: A carga parasitária (lesões e linfonodo de drenagem das lesões) da prole nascida de MI mostrou diminuição significativa na 5a semana pós-infecção, comparada àquela nascida de MNI, no entanto, esta diminuição não correspondeu aos sintomas clínicos. A análise histopatológica revelou que na prole nascida de MI, o processo inflamatório mostrou-se mais proeminente. Em relação ao gênero observou-se que os filhotes machos apresentaram espessura das lesões e carga parasitária maiores do que as fêmeas. Conclusão: A infecção materna por L. braziliensis parece não influenciar o curso da doença na infecção homóloga da prole, bem como os filhotes machos apresentaram aumentada susceptibilidade à infecção por L. braziliensis, independente se eles nasceram de MI ou MNI. Além disso, a redução no index de granulomas na prole nascida de MI, em conjunto com a maior resposta inflamatória, sugere uma resposta celular menos efetiva na fase crônica da doença nestes animais. (AU)

Role of the route of leukotrienes in an experimental model of oral mucositis induced by 5-fluorouracil

Abstract Purpose: To investigate the participation of cysteinyl leukotrienes in the pathophysiology of oral mucositis. Methods: Oral mucositis was induced in hamsters using 5-fluorouracil (5-FU; 60 and 40 mg/kg; i.p., on days 1 and 2, respectively, and with excoriations in jugal mucosa on day 4). Montelukast (10, 20, or 40 mg/kg/d; gavage), MK886 (3 mg/kg/d, i.p.), or saline or celecoxib (7.5 mg/kg/d; i.p.) was administered 1 h prior to 5-FU and daily, until the fourth (MK886) or tenth day, when the animals were euthanized and their jugal mucosa was collected for macroscopic, histopathological, and immunohistochemical evaluation. Results: Neither montelukast nor MK-886 prevented the oral mucositis induced by 5-FU, as observed by histopathological evaluation. In addition, we did not find significant differences in the expression of inducible nitric oxide synthase-2, cyclooxygenase-2, or interleukin (IL)-1β between the experimental and control groups. However, we did observe a significant decrease in tumor necrosis factor (TNF)-α expression for all doses of montelukast; we also observed a significant decrease in IL-10 with 40 mg/kg/d and MK 886. Conclusions: Cysteinyl leukotrienes do not play an important role in experimental oral mucositis induced by 5-FU. There is a modulating action specifically on TNF-α.

Ultrastructural alterations in Schistosoma mansoni juvenile and adult male worms after in vitro incubation with primaquine

BACKGROUND Praziquantel has been cited as the only drug for treating schistosomiasis. However, concerns over drug resistance have encouraged the search for novel drug leads. The antimalarial drug primaquine possesses interesting anti-schistosmal properties. OBJECTIVES This study is the first to document the potential role of primaquine as a schistosomicide and the ultrastructural changes induced by primaquine on juvenile or adult male worms of Schistosoma mansoni. METHODS Ultrastructural alterations in the tegumental surface of 21-day-old juvenile and adult male worms of S. mansoni were demonstrated following primaquine treatment at different concentrations (2, 5, 10, 15, and 20 µg/mL) and incubation periods (1, 3, 6, 24, and 48 h) in vitro, using both scanning and transmission electron microscopy. FINDINGS At low concentrations (2, 5, and 10 µg/mL) both juvenile and adult male worms were alive after 24 h of incubation, whereas contraction, paralysis, and death of all worms were observed after 24 h of drug exposure at 20 µg/mL. The tegument of juvenile and adult male worms treated with primaquine exhibited erosion, peeling, and sloughing. Furthermore, extensive damage of both tegumental and subtegumental layers included embedded spines, and shrinkage of muscles with vacuoles. The in vitro results confirmed that primaquine has dose-dependent effects with 20 µg/mL as the most effective concentration in a short incubation period. MAIN CONCLUSIONS The schistosomicidal activity of primaquine indicates that this drug possesses moderate in vitro activity against juvenile and adult male worms, since it caused high mortality and tegumental alterations. This study confirmed that the antimalarial drug primaquine possesses anti-schistosomal activity. Further investigation is needed to elucidate its mechanism of action.

Evaluation of cytogenotoxicity, antioxidant and hypoglycemiant activities of isolate compounds from Mansoa hirsuta D.C. (Bignoniaceae)

ABSTRACT Mansoa hirsuta (Bignoniaceae) is a native plant from caatinga in Brazilian semiarid. This plant has been locally used as antimicrobial and hypoglycemiant agents, but their action mechanisms and toxicity remain largely unknown. Therefore, we evaluated the composition and antioxidant, cytoprotective and hypoglycemiant effects of raw extract, fractions and compounds from leaves of M. hirsuta. The cytogenotoxic effects of ursolic and oleanolic acids, the main phytotherapic components of this plant, were assessed. The raw extract and fractions presented steroids, saponins, flavonols, flavanonols, flavanones, xanthones, phenols, tannins, anthocyanins, anthocyanidins and flavonoids. The ethyl acetate fraction inhibited efficiently the cascade of lipid peroxidation while the hydroalcoholic fraction was richer in total phenols and more efficient in capturing 2,2-diphenyl-1-picrylhydrazyl (·DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS·+) radicals. The isolated fraction of M. hirsuta also inhibited the α-amylase activity. Cytotoxic effects were absent in both raw extract and fractions while ursolic+oleanolic acids were efficient in protecting cells after exposure to hydrogen peroxide. Moreover, this mixture of acid shad no significant interference on the mitotic index and frequency of nuclear and/or chromosomal abnormalities in Allium cepa test. Therefore, M. hirsuta represents a potential source of phytochemicals against inflammatory and oxidative pathologies, including diabetes.

In vitro and in vivo antileishmanial activity of Artemisia annua L. leaf powder and its potential usefulness in the treatment of uncomplicated cutaneous leishmaniasis in humans

ABSTRACT INTRODUCTION: Cutaneous leishmaniasis (CL) is a tropical disease that affects millions of individuals worldwide. The current drugs for CL may be effective but have serious side effects; hence, alternatives are urgently needed. Although plant-derived materials are used for the treatment of various diseases in 80% of the global population, the validation of these products is essential. Gelatin capsules containing dried Artemisia annua leaf powder were recently developed as a new herbal formulation (totum) for the oral treatment of malaria and other parasitic diseases. Here, we aimed to determine the usefulness of A. annua gel capsules in CL. METHODS: The antileishmanial activity and cytotoxicity of A. annua L. capsules was determined via in vitro and in vivo studies. Moreover, a preliminary evaluation of its therapeutic potential as antileishmanial treatment in humans was conducted in 2 patients with uncomplicated CL. RESULTS: Artemisia annua capsules showed moderate in vitro activity in amastigotes of Leishmania (Viannia) panamensis; no cytotoxicity in U-937 macrophages or genotoxicity in human lymphocytes was observed. Five of 6 (83.3%) hamsters treated with A. annua capsules (500mg/kg/day) for 30 days were cured, and the 2 examined patients were cured 45 days after initiation of treatment with 30g of A. annua capsules, without any adverse reactions. Both patients remained disease-free 26 and 24 months after treatment completion. CONCLUSION: Capsules of A. annua L. represent an effective treatment for uncomplicated CL, although further randomized controlled trials are needed to validate its efficacy and safety.

Avaliação de biodisponibilidade e mecanismos de ação hipocolesterolemizante de peptídeos do amaranto (Amaranthus cruentus L. BRS-Alegria) / Evaluation of bioavailability and hypocholesterolemic mechanisms of peptides from Amaranth (Amaranthus cruentus L. BRS-Alegria)

Introdução: Doenças cardiovasculares constituem importante causa de morte em todo mundo e a hipercolesterolemia está diretamente relacionada a este problema de saúde pública. A dieta desempenha papel importante neste processo e alguns alimentos, como o amaranto (Amaranthus cruentus L. BRSAlegria), têm mostrado capacidade de redução do colesterol plasmático. Estudos sugerem que este efeito está relacionado a peptídeos liberados durante a digestão das proteínas, os quais atuam na modulação do metabolismo lipídico. Considerando-se que os efeitos da digestão gastrointestinal e da absorção destes peptídeos são claramente complexos torna-se importante a realização de estudos visando avaliar bioacessibilidade e mecanismos de ação destes peptídeos nos locais alvo do organismo. Objetivo: Analisar a biodisponibilidade de peptídeos em modelos animais após ingestão de isolado proteico de amaranto e relacioná-la com parâmetros ligados ao metabolismo do colesterol. Métodos: O amaranto teve sua proteína isolada. Os peptídeos da proteína do amaranto foram analisados após digestão in vitro. Dois experimentos in vivo foram conduzidos: um de fase aguda e outro de média duração. No primeiro, o isolado proteico de amaranto foi administrado a ratos e os peptídeos no sangue foram monitorados por 2 horas para verificação de fragmentos que resistissem à digestão gastrointestinal. O experimento in vivo 2 consistiu na alimentação de 3 grupos de hamster, um com dieta recomendada pela AIN93 (grupo N) e dois com dietas hipercolesterolêmicas por 21 dias, contendo a proteína de amaranto como única proteína da ração (grupo I), comparada ao controle de caseína (grupo H). Neste experimento foram analisados no plasma: peptídeos, colesterol total e frações; nas fezes: colesterol total e ácidos biliares; no fígado: colesterol, lipídeos totais, ácidos graxos, atividade enzimática da Hmgcr, expressão de Hmgcr, Srebf2, Lxr, Abca1, Abcg8 e Ampk. Resultados e discussão: Foram identificados fragmentos peptídicos provenientes da digestão in vitro do isolado proteico de amaranto, e outras dezenas de sequencias peptídicas em ratos após administração aguda de amaranto foram analisadas. Destaca-se a identificação do peptídeo ALGV, presente em proteína do amaranto de acordo com banco de dados, e similar a fragmentos com ação hipocolesterolemizante. No sangue de hamsters foram encontrados seis peptídeos com 100 por cento de cobertura e similaridade a base de dados de proteínas de amaranto, merecendo investigação sobre seus efeitos. Verificou-se que o isolado proteico de amaranto foi capaz de suprimir a hipercolesterolemia quando a dieta hipercolesterolemizante foi introduzida em paralelo a este ingrediente, com valores inferiores em 72 por cento (triglicerídeos), 64 por cento (colesterol total), 80 por cento (LDL-c) do grupo I em relação ao grupo H. Foi observada ainda menor concentração de colesterol e lipídeos totais no fígado dos animais do grupo I em relação ao grupo H (177 x 464 mg de colesterol/100 g de tecido; 2,06 x 2,86 g de lipídeos/100 g de tecido, respectivamente). Parâmetros lipídicos do sangue, das fezes e do fígado foram similares aos do grupo N, cuja dieta seguiu a preconização para roedores. Foi observada maior excreção de colesterol total no grupo I em relação ao grupo H, mas não houve maior excreção de ácidos biliares nas fezes. Não houve mudança na expressão dos genes analisados neste estudo, mas o amaranto reduziu a atividade da enzima Hmgcr. Postulase que parâmetros como expressão de Ldlr e atividade da Acat sejam alterados pela ingestão de amaranto. O perfil de ácidos graxos também foi modificado de forma a se assimilar ao grupo N, porém deve-se verificar parâmetros inflamatórios devido à maior proporção de ácido araquidônico em relação aos demais grupos estudados. Conclusão: Verifica-se biodisponibilidade dos peptídeos do amaranto e ação hipocolesterolemizante e hipolipemiante em diversas vias metabólicas, promovendo proteção cardiovascular

Introduction: Cardiovascular diseases are important causes of death worldwide, and hypercholesterolemia is directly related to this public health problem. Diet plays an important role in this process and some foods such as amaranth (Amaranthus cruentus L. BRS-Alegria) have been shown to reduce plasma cholesterol. Studies suggest that this effect is related to peptides released during the digestion of proteins, which would play an important role in the modulation of lipid metabolism. Considering that the effects of gastrointestinal digestion and the absorption of these peptides are clearly complex, it is important to carry out studies aiming to evaluate their bioaccessibility and evaluation of the mechanisms of action of these peptides in the target sites of the organism. Objective: To analyze the bioavailability of peptides in animal models after ingestion of amaranth protein isolate and to relate it to parameters associated to cholesterol metabolism. Methods: The amaranth was crushed, the flour was defatted and its protein isolated. Amaranth peptides were analysed after in vitro digestion. Two in vivo experiments were conducted: one of acute phase and one of medium duration. In the first, the amaranth protein isolate was administered to rats and the peptides in the blood were monitored for 2 hours to check for fragments that resisted gastrointestinal digestion. The in vivo experiment 2 consisted of feeding three groups of hamsters, one with a diet recommended by AIN93 (group N) and two with hypercholesterolemic diets for 21 days, containing amaranth protein as the only dietary protein (group I), compared to casein control (group H). In this experiment were analyzed in the plasma: peptides, total cholesterol and fractions; In feces: total cholesterol and bile acids; In the liver: cholesterol, total lipids, fatty acids, Hmgcr enzymatic activity, Hmgcr expression, Srebf-2, Lxr, Abca1, Abcg8 and Ampk. Results and discussion: Peptide fragments from the in vitro digestion of amaranth protein isolate were identified and other dozens of peptide sequences were found in rats after acute amaranth administration. A higher number of peptides were found in the serum in relation to the plasma of the animals. Remarkably, ALGV peptide was found in serum of rats. This peptide is present in amaranth protein, according to databases, and is similar to fragments that present hypocholesterolemic action. In the blood of hamsters it could be found six peptides with 100 per cent coverage and similarity to the database of amaranth proteins, deserving investigation about their effects. Amaranth protein was able to suppress hypercholesterolemia when the hypercholesterolemic diet was introduced in parallel with this ingredient, with values lower for group I in 72 per cent (triglycerides), 64 per cent (total cholesterol), 80 per cent (LDL-c) in relation to the H group. A lower concentration of cholesterol and total lipids were observed in the liver of the group I compared to the H group (177 x 464 mg cholesterol / 100 g of tissue, 2.06 x 2,86 g lipids / 100 g of tissue, respectively). Lipid parameters of blood, faeces and liver were similar to those of group N, whose diet followed the recommendation for rodents. There was greater excretion of total cholesterol in group I in relation to group H, but there was no greater excretion of bile acids in feces, indicating that the effect of amaranth protein may be due to increased transintestinal cholesterol excretion, decreased micellar solubilization of cholesterol and / or modification in the expression of cholesterol transport related proteins in the intestine. There was no change in the expression of the genes analyzed in this study, but amaranth reduced the activity of the Hmgcr enzyme. It is postulated that parameters such as Ldlr expression and Acat activity are altered by amaranth intake. The fatty acid profile was also modified in order to assimilate to the N group, but inflammatory parameters related to amaranth intake should be verified due to the higher proportion of arachidonic acid in relation to the higher proportion of arachidonic acid in relation to the other groups studied. Conclusion: The bioavailability of amaranth peptides and hypocholesterolemic and hypolipidemic activity in several metabolic pathways is verified, therefore promoting cardiovascular protection

Endocrine integration of fetal ovaries grafted under kidney capsule of unilaterally or bilaterally orchidectomized male hamster / Integración endocrina de ovarios fetales trasplantados bajo la cápsula renal de hámster machos uni o bilateralmente castrados

Mammalian ovary development undergoes important changes during the perinatal period, moment when follicles are assembled and start to develop in a process not well known, involving endocrine and paracrine factors. In order to investigate the effect of two different hormonal environments on the early development of the ovary, we used an autologous transplant model in which Syrian hamster fetal ovaries were grafted under the kidney capsule of males hosts previously unilaterally or bilaterally orchidectomized. After 35 days of graft, ovaries and kidney parenchyme of the host male did not present signs of rejection. Ovaries contained primordial, primary follicles, secondary follicles and few tertiary follicles with morphological features similar to ovaries of control females of 35 days of age. Healthy primary and secondary follicles of experimental groups had frequency distribution and size similar to control ovaries but tertiary follicles were scarce in control as well as in grafts where they were mainly atretic. PCNA, marker of proliferation, was immuno detected in granulosa cells of growing follicles and the marker of apoptosis, Caspase 3 active, was evident mainly in secondary follicles. Immunoreactivity for steroidogenic proteins, StAR, 3-bHSD and aromatase detected in the follicular wall cells and the decreased serum levels of FSH without important changes in testosterone in bilateral orchidectomized males that received ovarian graft, and testosterone decreased without changes in FSH levels in unilateral orchidectomized males (UO) with ovarian graft, all together suggest the effect of steroid hormones produced by the ovary. In conclusion, the experimental model of autologous transplant presents evidence of early ovary development under the kidney capsule and its functional integration to the endocrine axis of the host male.

El desarrollo del ovario en mamíferos sufre importantes cambios durante el periodo perinatal, momento en el cual los folículos se ensamblan y comienzan a desarrollarse en un proceso no muy dilucidado que involucra señales endocrinas y paracrinas. Con el objetivo de investigar el efecto de dos ambientes hormonales sobre el desarrollo temprano del ovario de hamster, usamos un modelo de trasplante autólogo en el que ovarios fetales fueron trasplantados bajo la cápsula renal de machos receptores previamente castrados y hemicastrados. Después de 35 días de trasplante, los ovarios y el parénquima renal de los machos receptores no presentaron señales de rechazo. El ovario presentó folículos primordiales, primarios, secundarios y algunos folículos terciarios con características morfológicas similares a los ovarios de hembras controles de 35 días de edad. Folículos primarios y secundarios sanos de ambos grupos experimentales se encontraron en frecuencia y tamaño similar al de ovarios controles, los folículos terciarios fueron escasos tanto en controles como en ovarios trasplantados, siendo en éstos principalmente atrésicos. PCNA, un marcador de proliferación celular, fue detectado por inmunohistoquímica en células granulosas de folículos en crecimiento, mientras que caspasa 3 activa, un marcador de apoptosis, fue evidente en folículos secundarios. Por otra parte, inmunoreactividad para proteínas esteroidogénicas, StAR, 3-bHSD y aromatasa, fue detectada en la pared folicular. Esta observación, junto a la disminución de niveles séricos de FSH, sin cambios importantes en los niveles de testosterona en machos castrados que recibieron trasplantes ováricos, y la disminución en los niveles de testosterona sin cambios en los niveles de FSH en machos hemicastrados con trasplantes ováricos, sugiere que el ovario no solo produce hormonas esteroidales sino que además éstas modifican los niveles hormonales del macho receptor del trasplante. En conclusión, este modelo de trasplante autólogo agrega información del desarrollo ovárico temprano cuando éste se desarrolla bajo la cápsula renal de machos entregando evidencia de la integración funcional del ovario trasplantado al eje endocrino de los machos receptores.