Search results for: reporter mice

Authors: Martyna Chotomska, Aleksandra Studzinska, Marta Lisowska, Justyna Szubert, Aleksandra Tabis, Jacek Bania, Arkadiusz Miazek

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Objectives: Assessing antigen-specific T cell responses is of utmost importance for the pre-clinical testing of prototype vaccines against intracellular pathogens and tumor antigens. Mainly two types of in vitro assays are used for this purpose 1) enzyme-linked immunospot (ELISpot) and 2) intracellular cytokine staining (ICS). Both are time-consuming, relatively expensive, and require manual dexterity. Here, we assess if a straightforward detection of luciferase activity in blood samples of transgenic reporter mice expressing a secreted Lucia luciferase under the transcriptional control of IFN-γ promoter parallels the sensitivity of IFNγ ELISpot assay. Methods: IFN-γ-LUCIA mouse strain carrying multiple copies of Lucia luciferase transgene under the transcriptional control of IFNγ minimal promoter were generated by pronuclear injection of linear DNA. The specificity of transgene expression and mobilization was assessed in vitro using transgenic splenocytes exposed to various mitogens. The IFN-γ-LUCIA mice were immunized with 50mg of ovalbumin (OVA) emulsified in incomplete Freund’s adjuvant three times every two weeks by subcutaneous injections. Blood samples were collected before and five days after each immunization. Luciferase activity was assessed in blood serum. Peripheral blood mononuclear cells were separated and assessed for frequencies of OVA-specific IFNγ-secreting T cells. Results: We show that in vitro cultured splenocytes of IFN-γ-LUCIA mice respond by 2 and 3 fold increase in secreted luciferase activity to T cell mitogens concanavalin A and phorbol myristate acetate, respectively but fail to respond to B cell-stimulating E.coli lipopolysaccharide. Immunization of IFN-γ-LUCIA mice with OVA leads to over 4 fold increase in luciferase activity in blood serum five days post-immunization with a barely detectable increase in OVA-specific, IFNγ-secreting T cells by ELISpot. Second and third immunizations, further increase the luciferase activity and coincidently also increase the frequencies of OVA-specific T cells by ELISpot. Conclusions: We conclude that minimally invasive monitoring of luciferase secretions in blood serum of IFN-γ-LUCIA mice constitutes a sensitive method for evaluating primary and memory Th1 responses to protein antigens. As such, this method may complement existing methods for rapid immunogenicity assessment of prototype vaccines.

Keywords: ELISpot, immunogenicity, interferon-gamma, reporter mice, vaccines

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Authors: Kathy Stein, Mariola Lysson, Anja Schmidt, Beatrix Schumak, Sabine Specht, Hicham Bouabe, Jürgen Heesemann, Axel Roers, Joerg C. Kalff, Sven Wehner

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Objective: Postoperative ileus (POI) is a common complication of abdominal surgery. Monocyte infiltration is a hallmark of POI. The polarization of macrophages/monocytes in this process is not well understood. We aimed to investigate if and how M2 macrophage/monocyte differentiation is involved in POI pathogenesis. Design: POI was induced by intestinal manipulation (IM). C57Bl/6, CCR2-/-, IL-10 reporter (ITIB), IL-10-/- and LysMcre/IL-10fl/fl mice underwent IM. At various points in time leukocyte influx, gene and protein expression of cytokines, chemokines and M2 differentiation markers and intestinal motility were analyzed. Results: IM induced the postoperative expression of the M2 markers Arginase-1 and YM-1, predominantly in F4/80+Ly6C+ monocytes. Gene expression analyses indicated an IL-10-dependent, IL-4-independent M2 polarization of these monocytes. IL-10 dependency of M2 differentiation was confirmed in IL-10 deficient mice. Leukocytes, in the order of infiltrating monocytes, neutrophils, and resident macrophages were the main IL-10 producers during POI. IL-10 producing monocytes as well as M2 marker expression were almost absent in CCR2-deficient mice. However, postoperative IL-10 expression was not altered in CCR2-/- mice. The loss of M2 polarized monocytes neither protected CCR2-/- mice from nor affected resolution of POI. In contrast, IL-10 deficiency reduced postoperative neutrophil numbers and ameliorated POI. IL-10Ra expression was strongly induced in neutrophils but not in monocytes. Conclusion: We conclude that IL-10 counteracts POI resolution by activating IL-10Ra-expressing neutrophils in the late phase of disease while IL-10-dependent M2 differentiation is not pivotal to POI manifestation and resolution.

Keywords: interleukin-10, macrophages, neutrophils, postoperative ileus

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Authors: Ngoc Tu Truong, Nuong T. Bui, Ben Rao, Ya L. Shen

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Quorum sensing is a phenomenon present in many gram-negative bacteria that allows bacterial communication and controlled expression of a large suite of genes through quorum sensing signals - N-acyl homoserine lactones (AHLs). In order to investigate the ability of the rhlR/rhlI quorum sensing system in Pseudomonas aeruginosa to express the ß-Galactosidase reporter gene, an engineered E. coli strain EpHL02, was genetically engineered. This engineered E. coli strain EpHL02 responded to the presence of the N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone to express the ß-Galactosidase reporter gene at a concentration limit of 5x10⁻⁸ M. This was also found to be comparable to AHLs extraction from Serratia marcescens H31. Moreover, we examined this ability of this engineered E. coli strain for respond of AHLs from extractions of Pseudomonas aeruginosa ATCC9027. The results demonstrated that the rhlR/rhlI quorum sensing system can express the ß-Galactosidase reporter gene by using the N-butanoyl homoserine lactone, N-hexanoyl homoserine lactone and AHLs from extractions of Serratia marcescens H31 and Pseudomonas aeruginosa ATCC9027 in the engineered E. coli strain EpHL02.

Keywords: N-butanoyl homoserine lactone, C4-HSL, N-hexanoyl homoserine lactone, C6-HSL, Pseudomonas aeruginosa, quorum sensing, Serratia marcescens, ß-galactosidase reporter gene

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Authors: Eun Gyeong Lee, Ji Young Park, Hyun Ae Woo

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Reactive oxygen species (ROS) production is involved in ischemia/reperfusion (I/R) injury in kidney of mice. Oxidative stress develops from an imbalance between ROS production and reduced antioxidant defenses. Many enzymatic and nonenzymatic antioxidant systems including peroxiredoxins (Prxs) are present in kidney to maintain an appropriate level of ROS and prevent oxidative damage. Prxs are a family of peroxidases that reduce peroxides, with a conserved cysteine residue serving as the site of oxidation by peroxides. In this study, we examined the protective role of Prx V against I/R-induced acute kidney injury (AKI) using Prx V wild type (WT) and knockout (KO) mice. We compared the response of Prx V WT and KO mice in mice model of I/R injury. Renal structure, functions, oxidative stress markers, protein levels of oxidative damage marker were worse in Prx V KO mice. Ablation of Prx V enhanced susceptibility to I/R-induced oxidative stress. Prx V KO mice were seen to have more severe renal damage than Prx V WT mice in mice model of I/R injury. Our results demonstrate that Prx V is protective against I/R-induced AKI.

Keywords: peroxiredoxin, ischemia/reperfusion, kidney, oxidative stress

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Authors: Teng Zhang, Shenshi Huang, Gang Xu, Xuelin Zhang, Shouxiang Lu

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With the acceleration of urbanization and the increasing of the population in the city, the evacuation of pedestrians suffering from disaster environments such as fire in a room or other limited space becomes a vital issue in modern society. Mice have been used in experimental crowd evacuation in recent years for its good similarities to human in physical structure and stress reaction. In this study, the effect of experience or memory on the collective behavior of mice was explored. To help mice familiarize themselves with the design of the space and the stimulus caused by smoke, we trained them repeatedly for 2 days so that they can escape from the emergency conditions as soon as possible. The escape pattern, trajectories, walking speed, turning angle and mean individual escape time of mice in each training trail were analyzed. We found that mice can build memory quickly after the first trial on the first day. On the second day, the evacuation of mice was maintained in a stable and efficient state. Meanwhile, the group with size of 30 (G30) had a shorter mean individual escape time compared with G12. Furthermore, we tested the experience of evacuation skill of mice after several days. The results showed that the mice can hold the experience or memory over 3 weeks. We proposed the importance of experience of evacuation skill and the research of training methods in experimental evacuation of mice. The results can deepen our understanding of collective behavior of mice and conduce to the establishment of animal models in the study of pedestrian crowd dynamics in emergency conditions.

Keywords: experience, evacuation, mice, group size, behavior

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Authors: Sheila M. Wicks, Gail Mahady, Udesh Patel, Joanna Michel, Armando Caceres

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Introduction: The genus piperaceae contains approximately 1000 species of herbs scrubs small trees and hanging vines distributed in both hemispheres. During our ethno medical work in Guatemala of the 27 plant families documented for us e by the Qeqchi Maya for reproductive disorders the most prominent were the Piperaceae (15%) and Menispermiaceae. Our Previous work showed that extracts from form Piper and Cissampelos species bound to both and progesterone and the estrogen receptors. In this work active extracts from Piper aeruginosibaccum Trelease, P auritum, P tuerckheimii and Cissampels tropaeolifolia were tested in functionalized cell based assays including a SEAP reporter gene and by qPCR of ER-responsive gene expression in MCF-7cells. In the reporter gene assay P aeruginosibaccum was estrogenic and enhanced E2 EFFECTS IN MCF-7 CELLS. P. tuerckheimi was not estrogenic alone but significantly enhanced the effects of E2 on SEAP reporter gene expression. Both altered mRNA expression of E2 responsive genes in MCF-7. Methods: this is collaborative project between University of Illinois at Chicago and University of San Carlos Guatemala City. 144 spices of plants were collected in Guatemala of which 57 used to treat a variety of women's reproductive health. The Genus Piperaraceae contains approximately 1000 species of herbs scrubs and small trees. Active extracts of the plants were tested in functionalized in cell-based bioassays including SEAP reporter genes. Results demonstrated altered mRNA expression of E2 responsive genes in MC-7 cells plants were collected in Guatemala of which 57 used. Conclusion of the 5 plants tested all were shown to contain components of binding to estrogenic receptor to a greater or lesser degree. These effects support the use of QEqchi Maya women in Guatemala for reproductive.

Keywords: reporter genes, MC7, guatemala piperaceae, reproductive health

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Authors: Ji Hyung Park, Dong-Hyun Seo, Young-Jin Jung, Han Sung Kim

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The aim of this study is to evaluate the effects of the laser and partial vibration stimulation on the mice tibia with morphological characteristics. Twenty female C57BL/6 mice (12 weeks old) were used for the experiment. The study was carried out on four groups of animals each consisting of five mice. Four groups of mice were ovariectomized. Animals were scanned at 0 and 2 weeks after ovariectomy by using micro-computed tomography to estimate morphological characteristics of tibial trabecular bone. Morphological analysis showed that structural parameters of multi-stimuli group appear significantly better phase in BV/TV, BS/BV, Tb.Th, Tb.N, Tb.Sp, and Tb.pf than single stimulation groups. However, single stimulation groups didn’t show significant effect on tibia with Sham group. This study suggests that multi-stimuli may restrain the change as the degenerate phase on osteoporosis in the mice tibia.

Keywords: laser, partial vibration, osteoporosis, in-vivo micro-CT, mice

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Authors: Anne R. Fernandez, Mohammad Akmal Adnan, Tanes Prasat, Indu Bala Jaganath, Brian Kirby, Kamalan Jeevaratnam

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Introduction: Plants from the Phyllantus genus have been used indigenously for the treatment of a variety of ailments for generations. A cocktail of phytonutrients prepared from a plant of the genus Phyllanthus has demonstrated the potential to alleviate ailments which include cardiovascular disorders. In this study, we investigated the cholesterol modulating properties of a highly purified proprietary extract of a Phyllanthus species in hypercholesteraemic mice. Methods: Hypercholesteraemia was induced in ICR mice by ad-libitum feeding of high fat diet daily for six weeks. The mice were then divided into 3 groups and force fed with 10mg/kg of atorvastatin, 200mg/kg of the proprietary Phyllanthus extract and water respectively. Blood samples were taken at the end of fourth week of treatment by a tail prick. At the end of the eighth week of treatment, mice were sacrificed and serum levels of total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides were measured. Results: The mean cholesterol levels in the mice fed with high fat diet were 44% (p < 0.05) higher than the mice on normal diet thus validating the model developed. The plasma HDL was significantly elevated in mice treated with the formulation (p ˂ 0.05) in comparison to the statin-treated and control mice. The total cholesterol levels in the mice treated with the proprietary extract were reduced significantly (p < 0.05) at the end of 4 weeks of treatment in comparison to the mice treated with atorvastatin. By the end of 8 weeks of treatment, there was no significant difference in the cholesterol levels of the mice in all groups. Conclusion: These results demonstrate that this proprietary extract from Phyllanthus species has the beneficial effect of reducing total cholesterol level more rapidly than atorvastatin and increasing HDL levels. Since an increase in the HDL cholesterol can reduce the risk of heart disease, this proprietary extract is a useful and safe therapeutic option compared to atorvastatin.

Keywords: high-density lipoprotein, hypercholesteraemic mice model, ICR mice, Phyllanthus spp.

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Authors: Yao Huang, Hongjie Yu, Fangrong Xu, Longbiao Cai, Qiqiang He

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The Klotho gene has been found to be involved in cardiovascular health, and epigenetic mechanism has risen as good candidates to understand the role of lifestyle factors in obesity. The aim of this study was to investigate the effect of exercise intervention on the expression and DNA methylation of Klotho gene in high-fat diet induced obese mice. C57BL/6 male mice were fed a normal diet (ND) or a high-fat diet (HFD) for 12 weeks. HFD induced obese mice were divided into secondary group (SED) and exercise group (EX) randomly. The treadmill exercise was performed in EX group for 8 weeks. The expression and DNA methylation of Klotho were evaluated by Western blot, RT-PCR, and Methylation-specific PCR. Results indicated that Klotho protein and mRNA expression were significantly lower in the SED group than those in the ND and EX groups (P<0.01), whereas no significant difference, was found between ND group and EX group (P>0.05). Furthermore, mice in the ND group and SED group showed significantly lower levels of completely methylated Klotho DNA in ND group (0%) and SED group (50%) compared with the EX group (90%), and unmethylated Klotho DNA level in ND group (80%) was significantly higher than those in the SED (0%) and EX (0%) groups. These results suggested that exercise leads to increased Klotho expression and reduced Klotho DNA methylation level in HFD induced obese mice.

Keywords: DNA methylation, exercise intervention, klotho, obese mice

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Authors: Daniel P. Kisangau, Ken M. Hosea, Herbert V. M. Lyaruu, Cosam C. Josep, Zakaria H. Mbwambo, Pax J. Masimba

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Crude extracts of Dracaena steudneri bark (DSB), Sapium ellipticum bark (SEB) and Capparis erythrocarpos root (CER) were investigated for their antifungal activity in immunocompromised mice infected with Candida albicans in an in vivo mice infection model. The results revealed a substantial dose dependency in all treatments given, with mice survival to the end of the experiment correlating well to the dose levels. At a dose of 400 mg/kg, C. erythrocarpos was the most effective with mice survival of 60% and organ burden clearance ranging from 64.0%-99.9% (P<0.0001) in all treatments. At the same dose, the least effective plant was S. ellipticum which had a mice survival of 20% and organ burden clearance ranging from 78.0%-96.6 (P>0.05). Mice survival for D. steudneri was 30% with organ burden clearance ranging from 89.0%-99.9% (P<0.05). All mice receiving no active treatment died before ten days post infection. In all treatment groups, there was a steady decline in mean weights of mice immediately after immunosuppression followed by gradual recovery in some cases which appeared to be dose dependent a few days post infection. Thus, extracts of D. steudneri and C. erythrocarpos portrayed the most significant potential as sources of antifungal drugs.

Keywords: antifungal activity, medicinal plants, candida albicans, East Africa

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Authors: Josephine Hai, Jie Jiang, Karen M. Lyons

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Of the musculoskeletal tissues, tendon is least understood in terms of biological development. The current study examines Cysteine-rich angiogenic inducer 61, or CCN1, a member of the CCN family of secreted matricellular proteins that regulate cell behavior via intercellular signaling. Though CCN1 is notable in limiting fibrosis by inducing senescence in fibroblasts, little is known about its role in normal fibrous tissue, where it may be essential to the development of ECM-rich structures like tendons. We found that CCN1 knockout mice (using limb-specific Prx1-Cre) exhibited clubfoot and waddling gaits, a unique phenotype not described in any other mutant to date. Histological analysis showed that the Achilles and patellar tendons, where we previously found high CCN1 expression in adult reporter mice, were thicker and denser in the Prx1-Cre knockouts than in their wildtype littermates. We then hypothesized that CCN1 is required directly in tendon progenitor cells for normal tendon development and generated tendon-specific CCN1 knockout mice using Scx-Cre. We observed similar Achilles/patellar tendon morphology among the Scx-Cre and Prx1-Cre mutants, indicating that the phenotype is a direct result of CCN1’s loss in tendon. To further study phenotype onset and progression, we will histologically characterize these tendons across different developmental time-points. We will also perform RNA-seq and qPCR to analyze tenocyte gene expression and expect fibrotic marker upregulation in the Scx-Cre mutants if CCN1 is required to maintain a normal tendon phenotype. Thus, our study aims to elucidate the molecular mechanisms underlying tendon formation and maintenance. Understanding tendons at the most basic level invites novel approaches to tendon repair.

Keywords: development, matricellular, musculoskeletal, tendon

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Authors: Shiva Emami, Jenny Persson, Bengt Johansson Lindbom

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Group A streptococcus (GAS) is a prevalent human pathogen, causing a wide range of infections and diseases. One of the most well-known virulence factors in GAS is M protein, a surface protein that facilitates bacterial invasion. In this study, we used a recombinant GAS strain (GAS-2W) expressing M protein containing a hyper immunogenic peptide (2W). Mice were immunized three times with heat-killed-GAS subcutaneously at three weeks intervals. Three weeks post last immunization, mice were challenged intraperitoneally with a lethal dose of live GAS. In order to investigate the impact of IFN-ƴ and antibodies in protection against GAS infection, we used a mouse model knock-out for IFN-ƴ (IFN-ƴ KO). We observed immunization with GAS-2W strain can increase protection against GAS infection in mice compared with the original GAS strain. Higher levels of antibodies against M1 protein were measured in GAS-2W-immunized mice. There was also a significant increase in IgG2c response in mice immunized with GAS2W. By using IFN-ƴ KO mice, we showed that not a high level of total IgG, but IgG2c was correlated with protection through the i.p challenge. It also emphasizes the importance of IFN-ƴ cytokine to combat GAS by isotype switching to IgG2c (which is opsonic for phagocytosis). Our data indicate the crucial role of IFN-ƴ in the protective immune response that, together with IgG2c, can induce protection against GAS.

Keywords: Group A streptococcus, IgG2c, IFN-γ, protection

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Authors: P. O. Ughachukwu, P. O. Okonkwo, P. C. Unekwe, J. O. Ogamba

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Background: The prevalence of hepatitis B viral infection is high worldwide with liver cirrhosis and hepatocellular carcinoma as important complications. Cases of poor antibody response to hepatitis B vaccination abound. Immunosuppression, especially from glucocorticoids, is often cited as a cause of poor antibody response and there are documented evidences of irrational administration of glucocorticoids to children and adults. The study was, therefore, designed to find out if administration of glucocorticoids affects immune response to vaccination against hepatitis B in mice. Methods: Mice of both sexes were randomly divided into 2 groups. Daily intramuscular methylprednisolone injections, (15 mg kg-1), were given to the test group while sterile deionized water (0.1ml) was given to control mice for 30 days. On day 6 all mice were given 2 μg (0.1ml) hepatitis B vaccine and a booster dose on day 27. On day 34, blood samples were collected and analyzed for anti-HBs titres using enzyme-linked immunosorbent assay (ELISA). Statistical analysis was done using Graph Pad Prism 5.0 and the results taken as statistically significant at p value < 0.05. Results: There were positive serum anti-HBs responses in all mice groups but the differences in titres were not statistically significant. Conclusions: At the dosages and length of exposure used in this study, methylprednisolone injection did not significantly inhibit anti-HBs response in mice following immunization against hepatitis B virus. By extrapolation, methylprednisolone, when used in the usual clinical doses and duration of therapy, is not likely to inhibit immune response to hepatitis B vaccinations in man.

Keywords: anti-HBs, hepatitis B vaccine, immune response, methylprednisolone, mice

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Authors: Rubaiyea Uddin

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The hippocampus, located in the limbic system, is most commonly known for its role in memory and spatial navigation (as cited in Brain Reward and Pathways). It maintains an especially important role in specifically episodic and declarative memory. The hippocampus has also recently been linked to dopamine, the reward pathway’s primary neurotransmitter. Since research has found that dopamine also contributes to memory consolidation and hippocampal plasticity, this neurotransmitter is potentially responsible for contributing to the hippocampus’s role in memory formation. In this experiment we tested to see the effect of tactile cues on spatial navigation for eight different mice. We used a radial arm that had one designated 'reward' arm containing sucrose. The presence or absence of bedding was our tactile cue. We attempted to see if the memory of that cue would enhance the mice’s memory of having received the reward in that arm. The results from our study showed there was no significant response from the use of tactile cues on spatial navigation on our 129 mice. Tactile cues therefore do not influence spatial navigation.

Keywords: mice, radial arm maze, memory, spatial navigation, tactile cues, hippocampus, reward, sensory skills, Alzheimer’s, neurodegnerative disease

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Authors: Hanjun Zhao

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Limited efficacy of current antivirals and antiviral-resistant mutations impair anti-influenza treatment. Here, we evaluated the in vitro and in vivo antiviral effect of three defective interfering genes (DIG-3) of influenza virus. Virus replication was significantly reduced in 293T and A549 cells transfected with DIG-3. Mice transfected with DIG-3 encoded by jetPEI-vector, as prophylaxis and therapeutics against A(H7N7) virus respectively, had significantly better survivals (80% and 50%) than control mice (0%). We further developed a dual-functional peptide TAT-P1, which delivers DIG-3 with high transfection efficiency and concomitantly exerts antiviral activity by preventing endosomal acidification. TAT-P1/DIG-3 was more effective than jetPEI/DIG-3 in treating A(H7N7) or A(H1N1)pdm09-infected mice and showed potent prophylactic protection on A(H7N7) or A(H1N1)pdm09-infected mice. The addition of P1 peptide, preventing endosomal acidification, could enhance the protection of TAT-P1/DIG-3 on A(H1N1)pdm09-infected mice. Dual-functional TAT-P1 with DIG-3 can effectively protect or treat mice infected by avian and seasonal influenza virus infection.

Keywords: antiviral peptide, dual-functional peptide, defective interfering genes, influenza virus

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Authors: F. Motamedi Sedeh, Sh. Chahardoli, H. Mahravani, N. Harzandi, M. Sotoodeh, S. K. Shafaei

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Foot-and-mouth disease virus (FMDV) is the most economically important disease of livestock. The aim of the study is inactivation of FMD virus type O/IRN/2010 by electron beam without antigenic changes as electron radio vaccine. The BALB/C mice were divided into three groups, each group containing five mice. Three groups of mice were inoculated with conventional vaccine and electron beam irradiated vaccine FMDV type O/IRN/2010 subcutaneously three weeks interval, the final group as negative control. The sera were separated from the blood samples of mice 14 days after last vaccination and tested for the presence of antibodies against FMDV type O/IRN/2010 by serum neutralization test. The Serum Neutralization Test (SNT) was carried out and antibody titration was calculated according to the Kraber protocol. The results of the SNT in three groups of mice showed the titration of neutralizing antibody in the vaccinated mice groups; electron radio vaccine and conventional vaccine were significantly higher than negative control group (P<0.05). Therefore, the radio vaccine is a good candidate to immunize animals against FMDV type O/IRN/2010.

Keywords: FMDV type O/IRN/2010, neutralizing antibody response, electron beam, radio vaccine

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Authors: Lívia Pimentel Sant'ana Dourado, Raquel Das Neves Almeida, Luís Henrique Costa Corrêa Neto, Nayara Soares, Kelly Grace Magalhães

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Introduction: Obesity and high-fat diet intake have a crucial impact on immune cells and inflammatory profile, highlighting an emerging realization that obesity is an inflammatory disease. In the present work, we aimed to characterize the role of caspase-1/11 and NLRP3 inflammasome in the establishment of mice obesity and modulation of inflammatory lipid metabolism induced by high fat diet intake. Methods and results: Wild type, caspase-1/11 and NLRP3 knockout mice were fed with standard fat diet (SFD) or high fat diet (HFD) for 90 days. The weight of animals was measured weekly to monitor the weight gain. After 90 days, the blood, peritoneal lavage cells, heart and liver were collected from mice studied here. Cytokines were measured in serum by ELISA and analyzed in spectrophotometry. Lipid antigen presentation molecule CD1d expression, reactive oxygen species (ROS) generation and lipid droplets biogenesis were analyzed in cells from mice peritoneal cavity by flow cytometry. Liver histopathology was performed for morphological evaluation of the organ. The absence of caspase-1/11, but not NLRP3, in mice fed with HFD favored the mice weight gain, increased liver size, induced development of hepatic steatosis and IL-12 secretion in mice compared to mice fed with SFD. In addition, caspase-1/11 knockout mice fed with HFD presented an increased CD1d molecule expression, as well as higher levels of lipid droplets biogenesis and ROS generation compared to wild type mice also fed with HFD. Conclusion: Our data suggest that caspase-1/11 knockout mice have greater susceptibility to obesity as well as increased activation of lipid metabolism and inflammatory markers.

Keywords: caspase 1, caspase 11, inflamassome, obesity, lipids

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Authors: Mhuji Kilonzo

Abstract:

Acute oral toxicity of Mystroxylon aethiopicum root bark aqueous was evaluated in albino mice of either sex. In this study, five groups of mice were orally treated with doses of 1000, 2000, 3000, 4000 and 5000 mg/kg body weight of the crude extract. The mortality, signs of toxicity and body weights were observed individually for two weeks. At the end of the two weeks study, all animals were sacrificed, and the hematological and biochemical parameters, as well as organ weights relative to body weight of each animal, were determined. No mortality, signs of toxicity and abnormalities in vital organs were observed in the entire period of study for both treated and control groups of mice. Additionally, there were no significant changes (p > 0.05) in the blood hematology and biochemical analysis. However, the body weights of all mice increased significantly. The Mystroxylon aethiopicum root bark aqueous extract were found to have a high safe margin when administered orally. Hence, the extract can be utilized for pharmaceutical formulations.

Keywords: acute oral toxicity, albino mice, Mystroxylon aethiopicum, safety

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Authors: Rubaiyea Uddin, Rebecca Taylor, Emily Levesque

Abstract:

The hippocampus, located in the limbic system, is most commonly known for its role in memory and spatial navigation (as cited in Brain Reward and Pathways). It maintains an especially important role in specifically episodic and declarative memory. The hippocampus has also recently been linked to dopamine, the reward pathway’s primary neurotransmitter. Since research has found that dopamine also contributes to memory consolidation and hippocampal plasticity, this neurotransmitter is potentially responsible for contributing to the hippocampus’s role in memory formation. In this experiment we tested to see the effect of tactile cues on spatial navigation for eight different mice. We used a radial arm that had one designated “reward” arm containing sucrose. The presence or absence of bedding was our tactile cue. We attempted to see if the memory of that cue would enhance the mice’s memory of having received the reward in that arm. The results from our study showed there was no significant response from the use of tactile cues on spatial navigation on our 129 mice. Tactile cues therefore do not influence spatial navigation.

Keywords: mice, radial arm maze, memory, spatial navigation, tactile cues, hippocampus, reward, sensory skills, Alzheimer's, neuro-degenerative diseases

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Authors: Meiling Yu, Nadia Rouatbi, Khuloud T. Al-Jamal

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Background: Treatment of neurological disorders with modern medical and surgical approaches remains difficult. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. The treatment of brain diseases with gene therapy requires the gene-editing tool to be delivered efficiently to the central nervous system. In this study, we explored the efficiency of different delivery routes, namely intravenous (i.v.), intra-cranial (i.c.), and intra-nasal (i.n.), to deliver stable nucleic acid-lipid particles (SNALPs) containing gene-editing tools namely Cas9 mRNA and sgRNA encoding for GFP as a reporter protein. We hypothesise that SNALPs can reach the brain and perform gene-editing to different extents depending on the administration route. Intranasal administration (i.n.) offers an attractive and non-invasive way to access the brain circumventing the blood–brain barrier. Successful delivery of gene-editing tools to the brain offers a great opportunity for therapeutic target validation and nucleic acids therapeutics delivery to improve treatment options for a range of neurodegenerative diseases. In this study, we utilised Rosa26-Cas9 knock-in mice, expressing GFP, to study brain distribution and gene-editing efficiency of SNALPs after i.v.; i.c. and i.n. routes of administration. Methods: Single guide RNA (sgRNA) against GFP has been designed and validated by in vitro nuclease assay. SNALPs were formulated and characterised using dynamic light scattering. The encapsulation efficiency of nucleic acids (NA) was measured by RiboGreen™ assay. SNALPs were incubated in serum to assess their ability to protect NA from degradation. Rosa26-Cas9 knock-in mice were i.v., i.n., or i.c. administered with SNALPs to test in vivo gene-editing (GFP knockout) efficiency. SNALPs were given as three doses of 0.64 mg/kg sgGFP following i.v. and i.n. or a single dose of 0.25 mg/kg sgGFP following i.c.. knockout efficiency was assessed after seven days using Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis. In vivo, the biodistribution of DiR labelled SNALPs (SNALPs-DiR) was assessed at 24h post-administration using IVIS Lumina Series III. Results: Serum-stable SNALPs produced were 130-140 nm in diameter with ~90% nucleic acid loading efficiency. SNALPs could reach and stay in the brain for up to 24h following i.v.; i.n. and i.c. administration. Decreasing GFP expression (around 50% after i.v. and i.c. and 20% following i.n.) was confirmed by optical imaging. Despite the small number of mice used, ICE analysis confirmed GFP knockout in mice brains. Additional studies are currently taking place to increase mice numbers. Conclusion: Results confirmed efficient gene knockout achieved by SNALPs in Rosa26-Cas9 knock-in mice expressing GFP following different routes of administrations in the following order i.v.= i.c.> i.n. Each of the administration routes has its pros and cons. The next stages of the project involve assessing gene-editing efficiency in wild-type mice and replacing GFP as a model target with therapeutic target genes implicated in Motor Neuron Disease pathology.

Keywords: CRISPR, nanoparticles, brain diseases, administration routes

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Authors: Pooja Khanal, Huaming Zhang

Abstract:

Labeling of issues/bugs, also known as bug classification, plays a vital role in software engineering. Some known labels/classes of bugs are 'User Interface', 'Security', and 'API'. Most of the time, when a reporter reports a bug, they try to assign some predefined label to it. Those issues are reported for a project, and each project is a repository in GitHub/GitLab, which contains multiple issues. There are many software project repositories -ranging from individual projects to commercial projects. The labels assigned for different repositories may be dependent on various factors like human instinct, generalization of labels, label assignment policy followed by the reporter, etc. While the reporter of the issue may instinctively give that issue a label, another person reporting the same issue may label it differently. This way, it is not known mathematically if a label in one repository is similar or different to the label in another repository. Hence, the primary goal of this research is to find the semantic differences between bug labeling of different repositories via machine learning. Independent optimal classifiers for individual repositories are built first using the text features from the reported issues. The optimal classifiers may include a combination of multiple classifiers stacked together. Then, those classifiers are used to cross-test other repositories which leads the result to be deduced mathematically. The produce of this ongoing research includes a formalized open-source GitHub issues database that is used to deduce the similarity of the labels pertaining to the different repositories.

Keywords: bug classification, bug labels, GitHub issues, semantic differences

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Authors: Bouaziz H., Soudania N., Essafia M., Ben Amara I., Hakim A., Jamoussi K., Zeghal Km, Zeghal N.

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In this investigation, we have evaluated the effects of arsenic trioxide on hepatic function in pregnant and lactating Swiss albino mice and their suckling pups. Experiments were carried out on female mice given 175 ppm As2O3 in their drinking water from the 14th day of pregnancy until day 14 after delivery. Our results showed a significant decrease in plasma levels of total protein and albumin, cholesterol and triglyceride in As2O3 treated mice and their pups. The hyperbilirubinemia and the increased plasma total alkaline phosphatase activity suggested the presence of cholestasis. Transaminase activities as well as lactate deshydrogenase activity in plasma, known as biomarkers of hepatocellular injury, were elevated indicating hepatic cells’damage after treatment with As2O3. Exposure to arsenic led to an increase of liver thiobarbituric acid reactive substances level along with a concomitant decrease in the activities of superoxide dismutase, catalase and glutathione peroxidase and in glutathione.

Keywords: antioxidant status, arsenic trioxide, hepatotoxicity, mice, oxidative stress

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Authors: Sonam Agarwal, Renu Bist

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Current study emphasizes the toxic effects of dichlorvos on serum in terms of oxidative stress. Meanwhile, a protective action of bacoside A and bromelain was investigated against the biochemical alterations in serum. The experimental design included six groups of mice: saline was given as a vehicle to the control mice (group I). Mice belonging to groups II, III and IV, were administered with dichlorvos (40 mg/kg b.w.), bromelain and bacoside A, respectively. The fifth group received a combination of bromelain and bacoside A. In group VI, Bacoside A, and bromelain were administered 20 minutes prior to exposure of dichlorvos. Thiobarbituric acid reactive substances (TBARS), protein carbonyl content (PCC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) level were used as biochemical test of toxic action for dichlorvos intoxication. Significantly increased TBARS and PCC level in second group suggests that dichlorvos enhances the production of free radicals in serum of mice (p< 0.05). Antioxidants treatment significantly decreased the levels of TBARS and PCC (p< 0.05). Dichlorvos administration causes a significant reduction in the level of CAT, SOD, GPx and GSH (p< 0.05) which was restored significantly by co-administration of bromelain and Bacoside A in dichlorvos exposed mice (p< 0.05). Treatment of bromelain and Bacoside A in combination served as better scavengers of toxicity induced by dichlorvos.

Keywords: bacoside A, bromelain, dichlorvos, serum

Procedia PDF Downloads 349

Authors: Milda Nainyte, Viktoras Masevicius

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Biological methylation is a methyl group transfer from S-adenosyl-L-methionine (AdoMet) onto N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules. The reaction is catalyzed by enzymes called AdoMet-dependent methyltransferases (MTases), which represent more than 3 % of the proteins in the cell. As a general mechanism, the methyl group from AdoMet replaces a hydrogen atom of nucleophilic center producing methylated DNA and S-adenosyl-L-homocysteine (AdoHcy). Recently, DNA methyltransferases have been used for the sequence-specific, covalent labeling of biopolymers. Two types of MTase catalyzed labeling of biopolymers are known, referred as two-step and one-step. During two-step labeling, an alkylating fragment is transferred onto DNA in a sequence-specific manner and then the reporter group, such as biotin, is attached for selective visualization using suitable chemistries of coupling. This approach of labeling is quite difficult and the chemical hitching does not always proceed at 100 %, but in the second step the variety of reporter groups can be selected and that gives the flexibility for this labeling method. In the one-step labeling, AdoMet analog is designed with the reporter group already attached to the functional group. Thus, the one-step labeling method would be more comfortable tool for labeling of biopolymers in order to prevent additional chemical reactions and selection of reaction conditions. Also, time costs would be reduced. However, effective AdoMet analog appropriate for one-step labeling of biopolymers and containing cleavable bond, required for reduction of PCR interferation, is still not known. To expand the practical utility of this important enzymatic reaction, cofactors with activated sulfonium-bound side-chains have been produced and can serve as surrogate cofactors for a variety of wild-type and mutant DNA and RNA MTases enabling covalent attachment of these chains to their target sites in DNA, RNA or proteins (the approach named methyltransferase-directed Transfer of Activated Groups, mTAG). Compounds containing hex-2-yn-1-yl moiety has proved to be efficient alkylating agents for labeling of DNA. Herein we describe synthetic procedures for the preparation of N-biotinoyl-N’-(pent-4-ynoyl)cystamine starting from the coupling of cystamine with pentynoic acid and finally attaching the biotin as a reporter group. The synthesis of the first AdoMet based cofactor containing a cleavable reporter group and appropriate for one-step labeling was developed.

Keywords: adoMet analogs, DNA alkylation, cofactor, methyltransferases

Procedia PDF Downloads 195

Authors: Erica Fellin, Albrecht Schulte-Hostedde

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As the climate warms, the black-legged tick’s (Ixodes scapularis) range expands further north in Ontario, Canada, reaching new host populations that have not previously interacted with this blood-feeding parasite. Peromyscus mice in these northern areas are unfamiliar and inexperienced to the effects of these ticks compared to their southern counterparts that have adapted to living with these organisms. The purpose of this study was to see if there is a difference in physiology between these two groups – deer mice living in areas where tick populations have established and deer mice living in black-legged tick-free environments – looking specifically to see if there is significant variation in hemoglobin levels, which can negatively impact how these mice function in their environment. Along with this, a comparison of the parasite community structure on these mice hosts was analyzed to see if ticks change the composition of these micro-environments. Blood samples were collected from individual mice from populations where black-legged ticks were either present or absent to assess haemoglobin levels. At the same time, ectoparasites were collected from these same mice to determine parasite loads and species diversity. Haemoglobin levels were found to be lower when tick loads were high, and parasite diversity appeared to be higher when ticks were absent. Since black-legged ticks are carriers of many pathogens that can be passed on to humans, including Lyme’s disease, it is important to understand their movement and distribution across Ontario as well as their interactions with their hosts (and co-occurring parasites) in their environments.

Keywords: community ecology, hematology, hosts, parasites

Procedia PDF Downloads 141

Authors: Agatha Swasti Ayuning Tyas

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Hyperglycemia in Diabetes Mellitus (DM) is an important factor in cellular and vascular damage, which is caused by activation of C Protein Kinase, polyol and hexosamine track, and production of Advanced Glycation End-Products (AGE). Those mentioned before causes the accumulation of Reactive Oxygen Species (ROS). Oxidative stress increases the expression of proinflammatory factors IL-6 as one of many signs of endothelial disfunction. Genistein in soy milk has a high immunomodulator potential. Goat milk contains amino acids which have antioxidative potential. Fermented kefir has an anti-inflammatory activity which believed will also contribute in potentiating goat milk and soy milk. This study is a quasi-experimental posttest-only research to 30 Wistar mice. This study compared the levels of IL-6 between healthy Wistar mice group (G1) and 4 DM Wistar mice with intervention and grouped as follows: mice without treatment (G2), mice treated with 100% goat milk kefir (G3), mice treated with combination of 50% goat milk kefir and 50% soy milk kefir (G4), and mice treated with 100% soy milk kefir (G5). DM animal models were induced with Streptozotocin & Nicotinamide to achieve hyperglycemic condition. Goat milk kefir and soy milk kefir are given at a dose of 2 mL/kg body weight/day for four weeks to intervention groups. Blood glucose was analyzed by the GOD-POD principle. IL-6 was analyzed by enzyme-linked sandwich ELISA. The level of IL-6 in DM untreated control group (G2) showed a significant difference from the group treated with the combination of 50% goat milk kefir and 50% soy milk kefir (G3) (p=0,006) and the group treated with 100% soy milk kefir (G5) (p=0,009). Whereas the difference of IL-6 in group treated with 100% goat milk kefir (G3) was not significant (p=0,131). There is also synergism between glucose level and IL-6 in intervention groups treated with combination of 50% goat milk kefir and 50% soy milk kefir (G3) and the group treated with 100% soy milk kefir (G5). Combination of 50 % goat milk kefir and 50% soy milk kefir and administration of 100% soy milk kefir alone can control the level of IL-6 remained low in DM Wistar mice induced with streptozocin and nicotinamide.

Keywords: diabetes mellitus, goat milk kefir, soy milk kefir, interleukin 6

Procedia PDF Downloads 285

Authors: Idowu R. Akomolafe, Naven Chetty

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Background: The widespread medical application of ionizing radiation has raised public concern about radiation exposure and, thus, associated cancer risk. The production of reactive oxygen species and free radicals as a result of radiation exposure can cause severe damage to deoxyribonucleic acid (DNA) of cells, thus leading to biological effect. Radiotherapy is an excellent modality in the treatment of cancerous cells, comes with a few challenges. A significant challenge is the exposure of healthy cells surrounding the tumour to radiation. The last few decades have witnessed lots of attention shifted to plants, herbs, and natural product as an alternative to synthetic compound for radioprotection. Thus, the study investigated the radioprotective efficacy of Costus afer against whole-body radiation-induced haematological, histopathological disorder in mice. Materials and Method: Fifty-four mice were randomly divided into nine groups. Animals were pretreated with the extract of Costus afer by oral gavage for six days before irradiation. Control: 6 mice received feed and water only; 6 mice received feed, water, and 3Gy; 6 mice received feed, water, and 6Gy; experimental: 6 mice received 250 mg/kg extract; 6 mice received 500 mg/kg extract; 6 mice received 250 mg/kg extract and 3Gy; 6 mice received 500 mg/kg extract and 3Gy; 6 mice received 250 mg/kg extract and 6Gy; 6 mice received 500 mg/kg extract and 6Gy in addition to feeding and water. The irradiation was done at the Radiotherapy and Oncology Department of Grey's Hospital using linear accelerator (LINAC). Thirty-six mice were sacrificed by cervical dislocation 48 hours after irradiation, and blood was collected for haematology tests. Also, the liver and kidney of the sacrificed mice were surgically removed for histopathology tests. The remaining eighteen (18) mice were used for mortality and survival studies. Data were analysed by one-way ANOVA, followed by Tukey's multiple comparison test. Results: Prior administration of Costus afer extract decreased the symptoms of radiation sickness and caused a significant delay in the mortality as demonstrated in the experimental mice. The first mortality was recorded on day-5 post irradiation, and this happened to the group E- that is, mice that received 6Gy but no extract. There was significant protection in the experimental mice, as demonstrated in the blood counts against hematopoietic and gastrointestinal damage when compared with the control. The protection was seen in the increase in blood counts of experimental animals and the number of survivor. The protection offered by Costus afer may be due to its ability to scavenge free radicals and restore gastrointestinal and bone marrow damage produced by radiation. Conclusions: The study has demonstrated that exposure of mice to radiation could cause modifications in the haematological and histopathological parameters of irradiated mice. However, the changes were relieved by the methanol extract of Costus afer, probably through its free radical scavenging and antioxidant properties.

Keywords: costus afer, hematological, mortality, radioprotection, radiotherapy

Procedia PDF Downloads 140

Authors: Yousry El-Sayed Elbolkiny, Mohamed Labib Salem

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Background: Rosemary (ROLE) and costus (SLRE) have been established to show antioxidant effects. Aim: This study aimed to evaluate the ameliorating effects of ROLE and SLRE on the side effects induced by cisplatin (CIS) in tumor-bearing mice. Materials and Methods: Extracts of ROLE and SLRE were examined for their phytochemical activities. To evaluate their anti-tumor effects, mice were inoculated intraperitoneally (i.p.) with 2.5x105 Ehrlich ascites carcinoma (EAC) and then treated i.p. with CIS at days 3-7 and with ROLE (dose) or SLRE (dose) at days 3-14. Mice were sacrificed on day 14 for CBC and T-cell analyses. Results: Phytochemical analysis revealed that both ROLE and SLRE showed similar antioxidant activities. Treatment of EAC-bearing mice with CIS-induced antitumor efficacy of about 90%. Treatment with CIS in combination with ROLE or SLRE did not further enhance the antitumor activity of CIS. However, co-administration of ROLE or SLRE with CIS significantly increased the antitumor efficacy of CIS. Flow cytometric analysis showed that the numbers of CD4+ and CD8+ T cells were decreased in EAC-bearing mice after treatment with CIS. Treatment with both ROLE and SLRE improved the number of these cells. Conclusion: Combinatorial treatment with rosemary and costus can enhance the antitumor activity of CIS

Keywords: CBC, cisplantin, costus, rosemary

Procedia PDF Downloads 48

Authors: Firas Rashad Al-Samarai

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Introduction: Benign prostatic hyperplasia (BPH) is a common condition as men get older. An enlarged prostate gland can cause uncomfortable urinary symptoms, such as blocking the flow of urine out of the bladder. It can also cause bladder, urinary tract, or kidney problems. Objective: to evaluate the efficacy and interaction of tamsulosin with finasteride treatment on induced benign prostate hyperplasia (BPH) in mice. Methods: BPH was induced by subcutaneous injection of testosterone propionate (20 mg/kg) for 30 days. Eighty-five mice were divided into five groups. The first group (G1): twenty-five mice induced BPH treated with tamsulosin orally and divided into five equal subgroups with doses (0.017, 0.052, 0.087, 0. 123, and 0.158) mg/kg, the second group (G2): twenty-five mice induced BPH treated with finasteride orally and divided into five equal subgroups with doses (0.175, 0.527, 0.878, 1.23, and 1.580) mg/kg. the third group (G3): twenty-five mice induced BPH treated with a combination of tamsulosin with finasteride orally, and divided into five equal subgroups with doses (0.0085, 0.0875), (0.026, 0.2635), (0.0435, 0.439) , (0.0615, 0.615) and ( 0.079 , 0.790 ) mg/kg respectively. Fourth group (G4): five mice induced BPH and treated distilled water. Fifth group (G5): five mice were not inducing BPH and without any treatment. Results: The results showed a gradual significant increase in prostate weight % and prostate index % Inhibitions until reached saturation in the last two doses of tamsulosin, finasteride, and combination groups, the maximum effective dose of tamsulosin and finasteride were (0.156) and (1.495) mg/kg respectively. Moreover, the effective dose of the combination (tamsulosin and finasteride) was estimated (0.06876, 0.6876) mg/kg, respectively, as well as the type of interaction was synergism and the value of the combination index was 0.046. Conclusions: We concluded that the combination of tamsulosin with finasteride showed a synergistic effect in BPH treatment by minimizing the side effect of each drug as s result of decreasing the dose of each one.

Keywords: Tamsulosin, Finasteride, combination, BPH

Procedia PDF Downloads 76

Authors: Singh Baljinder, Sharma Navneet

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Foeniculum vulgare Miller is a biennial medicinal and aromatic plant belonging to the family Apiaceae (Umbelliferaceae). It is a hardy, perennial–umbelliferous herb with yellow flowers and feathery leaves. The aim is to study the control of blood glucose in alloxan induced diabetic mice.Method used for extraction was continuous hot percolation method in which Soxhlet apparatus was used.95%ethanol was used as solvent. Male albino mice weighing about 20-25 g obtained from Guru Angad Dev University of Veterinary Science, Ludhiana were used for the study. Diabetes was induced by a single i.p. injection of 125 mg/kg of alloxan monohydrate in sterile saline (11). After 48 h, animals with serum glucose level above 200 mg/dl (diabetic) were selected for the study. Blood samples from mice were collected by retro-orbital puncture (ROP) technique. Serum glucose levels were determined by glucose oxidase and peroxidase method. Single administration (single dose) of aqueous extract of fennel (25, 50, and 100 mg/kg, p.o.) in diabetic Swiss albino mice, showed reduction in serum glucose level after 45 min. Maximum reduction in serum glucose level was seen at doses of 100 mg/kg. Aqueous extract of fennel in all doses except 25 mg/kg did not cause any significant decrease in blood glucose. It may be said that the aqueous extract of fennel decreased the serum glucose level and improved glucose tolerance owing to the presence of aldehyde moiety. The aqueous extract of fennel has antihyperglycemic activity as it lowers serum glucose level in diabetic mice.

Keywords: Foeniculum vulgare Miller, antihyperglycemic, diabetic mice, Umbelliferaceae

Procedia PDF Downloads 286