Search results for: ICR mice

Authors: Manal M. Kame, Zeinab A. Demerdash, Hanan G. El-Baz, Salwa M. Hassan, Faten M. Salah, Wafaa Mansour, Olfat Hammam

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Cord blood (CB) derived Unrestricted Somatic Stem Cells (USSCs) with their multipotentiality hold great promise in liver regeneration. This work aims at evaluation of the therapeutic potentiality of USSCs in two experimental models of chronic liver injury induced either by S. mansoni infection in balb/c mice or CCL4 injection in hamsters. Isolation, propagation, and characterization of USSCs from CB samples were performed. USSCs were induced to differentiate into osteoblasts, adipocytes and hepatocyte-like cells. Cells of the third passage were transplanted in two models of liver fibrosis: (1) Twenty hamsters were induced to liver fibrosis by repeated i. p. injection of 100 μl CCl4 /hamster for 8 weeks. This model was designed as; 10 hamsters with liver fibrosis and treated with i.h. injection of 3x106 USSCs (USSCs transplanted group), 10 hamsters with liver fibrosis (pathological control group), and 10 hamsters with healthy livers (normal control group). (2) Murine chronics S.mansoni model: twenty mice were induced to liver fibrosis with S. mansoni ceracariae (60 cercariae/ mouse) using the tail immersion method and left for 12 weeks. This model was designed as; 10 mice with liver fibrosis were transplanted with i. v. injection of 1×106 USCCs (USSCs transplanted group). Other 2 groups were designed as in hamsters model. Animals were sacrificed 12 weeks after USSCs transplantation, and their liver sections were examined for detection of human hepatocyte-like cells by immunohistochemistry staining. Moreover, liver sections were examined for fibrosis level, and fibrotic indices were calculated. Sera of sacrificed animals were tested for liver functions. CB USSCs, with fibroblast-like morphology, expressed high levels of CD44, CD90, CD73 and CD105 and were negative for CD34, CD45, and HLA-DR. USSCs showed high expression of transcripts for Oct4 and Sox2 and were in vitro differentiated into osteoblasts, adipocytes. In both animal models, in vitro induced hepatocyte-like cells were confirmed by cytoplasmic expression of glycogen, alpha-fetoprotein, and cytokeratin18. Livers of USSCs transplanted group showed engraftment with human hepatocyte-like cells as proved by cytoplasmic expression of human alpha-fetoprotein, cytokeratin18, and OV6. In addition, livers of this group showed less fibrosis than the pathological control group. Liver functions in the form of serum AST & ALT level and serum total bilirubin level were significantly lowered in USSCs transplanted group than pathological control group (p < 0.001). Moreover, the fibrotic index was significantly lower (p< 0.001) in USSCs transplanted group than pathological control group. In addition liver sections, of i. v. injection of 1×106 USCCs of mice, stained with either H&E or sirius red showed diminished granuloma size and a relative decrease in hepatic fibrosis. Our experimental liver fibrosis models transplanted with CB-USSCs showed liver engraftment with human hepatocyte-like cells as well as signs of liver regeneration in the form of improvement in liver function assays and fibrosis level. These data provide hope that human CB- derived USSCs are introduced as multipotent stem cells with great potentiality in regenerative medicine & strengthens the concept of cellular therapy for the treatment of liver fibrosis.

Keywords: cord blood, liver fibrosis, stem cells, transplantation

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Authors: Tomás Sobrino, Lara García-Varela, Marta Aramburu-Núñez, Mónica Castro, Noemí Gómez-Lado, Mariña Rodríguez-Arrizabalaga, Antía Custodia, Juan Manuel Pías-Peleteiro, José Manuel Aldrey, Daniel Romaus-Sanjurjo, Ángeles Almeida, Pablo Aguiar, Alberto Ouro

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Introduction: Alzheimer’s disease (AD) and other tauopathies are primary causes of dementia, causing progressive cognitive deterioration that entails serious repercussions for the patients' performance of daily tasks. Currently, there is no effective approach for the early diagnosis and treatment of AD and tauopathies. This study suggests a theragnostic approach based on the importance of phosphorylated tau protein (p-Tau) in the early pathophysiological processes of AD. We have developed a novel theragnostic monoclonal antibody (mAb) to provide both diagnostic and therapeutic effects. Methods/Results: We have developed a p-Tau mAb, which was doped with deferoxamine for radiolabeling with Zirconium-89 (89Zr) for PET imaging, as well as fluorescence dies for immunofluorescence assays. The p-Tau mAb was evaluated in vitro for toxicity by MTT assay, LDH activity, propidium iodide/Annexin V assay, caspase-3, and mitochondrial membrane potential (MMP) assay in both mouse endothelial cell line (bEnd.3) and cortical primary neurons cell cultures. Importantly, non-toxic effects (up to concentrations of p-Tau mAb greater than 100 ug/mL) were detected. In vivo experiments in the tauopathy model mice (PS19) show that the 89Zr-pTau-mAb and 89Zr-Fragments-pTau-mAb are stable in circulation for up to 10 days without toxic effects. However, only less than 0.2% reached the brain, so further strategies have to be designed for crossing the Brain-Blood-Barrier (BBB). Moreover, an intraparenchymal treatment strategy was carried out. The PS19 mice were operated to implement osmotic pumps (Alzet 1004) at two different times, at 4 and 7 months, to stimulate the controlled release for one month each of the B6 antibody or the IgG1 control antibody. We demonstrated that B6-treated mice maintained their motor and memory abilities significantly compared with IgG1 treatment. In addition, we observed a significant reduction in p-Tau deposits in the brain. Conclusions /Discussion: A theragnostic pTau-mAb was developed. Moreover, we demonstrated that our p-Tau mAb recognizes very-early pathology forms of p-Tau by non-invasive techniques, such as PET. In addition, p-Tau mAb has non-toxic effects, both in vitro and in vivo. Although the p-Tau mAb is stable in circulation, only 0.2% achieve the brain. However, direct intraventricular treatment significantly reduces cognitive impairment in Alzheimer's animal models, as well as the accumulation of toxic p-Tau species.

Keywords: alzheimer's disease, theragnosis, tau, PET, immunotherapy, tauopathies

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Authors: Chanya Inprasit, Ching-Liang Hsieh, Yi-Wen Lin

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Obesity (OB) is a hazardous global health problem that has been increasing in prevalence, more severely in last decade. It is the mainly resultant from the imbalance between food consumption and energy expenditure, which is concordant with a modern lifestyle, implying an increase in calories with poorer quality of food intake accompanied by a decrease in physical activities. Obesity does not concern the appearance only but is also a major factor contributing to poor physiology, psychology, society and economic issues. Moreover, OB induces low-grade inflammation in the body through the regulatory effect it enacts on the adipocyte function. Various alternative treatments were investigated for body weight control, including Acupoint Catgut Embedding (ACE). ACE is the implantation of absorbable catgut sutures at specific acupoints, displaying durable and potent stimulation and thereby reducing the treatment frequency. Our study utilized a mouse model to exclude any psychological factors of OB and ACE treatment. High-fat diet and body weight were measured once a week before subjects in ACE and Sham group received the ACE treatment or placebo treatment. We hypothesized that ACE can control body weight through the interaction of the TRPV1 pathways, as TRPV1 accordingly responds to inflammatory factors. The results of body weight variation show a significant decrease in body weight in ACE group compared with the baseline of control and Sham group. Meanwhile, converse results were explored in TRPV1 knockout mice, where a significant maintenance of normal body weight throughout the experiment period was observed. There was no significant difference in food consumption of each group. These finding indicated that TRPV1 pathways and its associated pathways may be involved in the maintenance of body weight, which can be controlled by ACE treatment of genetic manipulation.

Keywords: acupoint catgut embedding, obesity, hypothalamus, TRPV1

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Authors: Golnar Hasheminasab, Mehrdad Faizi, Mona Khoramjouy

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Introduction: Benzodiazepines (BZDs) have pharmacological effects, including anxiolytic, sedative-hypnotic, anticonvulsant, and muscle relaxant properties. However, they have adverse effects such as interaction with alcohol, ataxia, impaired learning, and psychological and physical dependence. According to the structure of zolpidem and on the basis of the structure-activity relationship of BZD receptor ligands, six novel derivatives of 2-fluorobenzyloxy 4,6- diphenylpyramidin-2-ol have been synthesized. We studied the hypnotic, sedative, and anticonvulsant effects of the novel compounds. Method: In this study, we used male mice (18 to 25 g). All the substances were injected intraperitoneally. The hypnotic effect of the compounds was examined by pentobarbital induced sleeping test. The locomotor activities and sedative effects of the novel compounds were evaluated by open field and loss of righting reflex test, respectively. The anticonvulsant effects of the novel compounds were assessed by PTZ and MES tests. Results: In the pentobarbital induced sleeping and open field tests, compound 4-(2-((2-fluorobenzyl)oxy)phenyl)-6-(p-tolyl) pyrimidine-2-ol with ED50=14.20 mg/kg and ED50=47.88 mg/kg, respectively, was the most effective compound. None of the novel compounds showed a significant anticonvulsant effect in the PTZ test. In MES test, compound 4-(2-((2-fluorobenzyl)oxy)phenyl)-6-(p-tolyl)pyrimidine-2-ol with ED50=12.92 mg/kg was the most effective compound. Flumazenil blocked the sedation and hypnosis of all the compounds. Conclusion: All of the novel derivatives showed significant sedative-hypnotic activities and caused the reduction of locomotor activities. The results show that the methyl lipophilic substitutes on the phenyl ring of 4,6-diphenylpyramidin-2-ol derivatives can increase the sedative and hypnotic effects of the derivatives. Flumazenil antagonized the sedative, and the hypnotic effects of the compounds indicate that BZD receptors are involved in the effects.

Keywords: BZD, sedative, hyptonic, anticonvulsant, zolpidem, MES, PTZ, benzodiazepine, locomotor activities, pentobarbital induced sleeping tests

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Authors: Kuan-Hao Tsui, Li-Te Lin, Chia-Jung Li

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The combination of Y-27632 and Vitamin C significantly enhances the quality of aging germ cells by reducing reactive oxygen species (ROS) production, restoring mitochondrial membrane potential balance, and promoting mitochondrial fusion. The age-related decline in oocyte quality contributes to reduced fertility, increased aneuploidy, and diminished embryo quality, with mitochondrial dysfunction in both oocytes and granulosa cells being a key factor in this decline. Experiments on aging germ cells investigated the effects of the Y-27632 and Vitamin C combination. In vivo studies involved aged mice to assess oocyte maturation and ROS accumulation during culture. The assessment included mitochondrial activity, ROS levels, mitochondrial membrane potential, and mitochondrial dynamics. Cellular energy metabolism and ATP production were also measured. The combination treatment effectively addressed mitochondrial dysfunction and regulated cellular energy metabolism, promoting oxygen respiration and increasing ATP production. In aged mice, this supplement treatment enhanced in vitro oocyte maturation and prevented ROS accumulation in aging oocytes during culture. While these findings are promising, further research is needed to explore the long-term effects and potential side effects of the Y-27632 and Vitamin C combination. Additionally, translating these findings to human subjects requires careful consideration. Overall, the study suggests that the Y-27632 and Vitamin C combination could be a promising intervention to mitigate aging-related dysfunction in germ cells, potentially enhancing oocyte quality, particularly in the context of in vitro fertilization.

Keywords: ovarian aging, supplements, ROS, mitochondria

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Authors: Ximena Calle, Plinio Cantero-López, Felipe Muñoz-Córdova, Mayarling-Francisca Troncoso, Sergio Lavandero, Valentina Parra

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MUL1, a mitochondrial E3 ubiquitin ligase anchored to the outer mitochondrial membrane, is highly expressed in the heart. MUL1 is involved in multiple biological pathways associated with mitochondrial dynamics. Increased MUL1 affects the balance between fission and fusion, affecting mitochondrial function, which plays a crucial role in myocardial function. Therefore, it is interesting to evaluate the effect of cardiac-specific overexpression of MUL1 on myocardial function. Aim: To determine heart functionality in a mouse model with cardio-specific overexpression MUL1 protein. Methods and Results: Male C57BL/Tg transgenic mice with cardiomyocyte-specific overexpression of MUL1 (n=10) and control (n=4) were evaluated at 12, 27, and 35 weeks of age. Glucose tolerance curve determination was performed after a 6-hours fast to assess metabolic capacity, treadmill test, and systolic, and diastolic pressure was evaluated by the mouse tail-cuff blood pressure system equipment. The result showed no glucose tolerance curve, and the treadmill test demonstrated no significant changes between groups. However, substantial changes in diastolic function were observed by ultrasound and determination of cardiac hypertrophy proteins by western blot. Conclusions: Cardio-specific overexpression of MUL1 in mice without any treatment affects diastolic cardiac function, thus showing the important role contributed by MUL1 in the heart. Future research should evaluate the effect of cardiomyocyte-specific overexpression of MUL1 in pathological conditions such as a high-fat diet is one of the main risk factors for cardiovascular disease.

Keywords: diastolic dysfunction, hypertrophy cardiac, mitochondrial E3 ubiquitin ligase 1, MUL1

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Authors: Masaaki Ii, Takashi Saito, Yasuhiko Tabata, Shintaro Nemoto

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Background: Clinical trials of autologous adipose-derived stem cell (AdSC) therapy for ischemic heart diseases (IHD) are now on-going. We have investigated the hypothesis that combination of AdSCs and statin, an agent with pleiotropic effects, could augment the therapeutic effect on myocardial infarction (MI). Methods and Results: Human AdSC functions with different doses of simvastatin-conjugated nanoparticle (STNP) uptake were evaluated by in vitro assays. STNP promoted the migration activity without changing the proliferation activity, and also up-regulated growth factors. Next, MI was induced by LAD ligation in nude mice, and the mice were assigned in the following groups 3 days after MI: 1) PBS (control), 2) NP-AdSCs (50000 cells), 3) STNP, and 4) STNP-AdSCs (50000 cells). Cardiac functional recovery assessed by echocardiography was improved at 4 weeks after surgery in STNP-AdSC group. Masson’s trichrome-stained sections revealed that LV fibrosis length was reduced, and the number of TUNEL-positive cardiomyocytes was less in STNP-AdSC group. Surprisingly, a number of de novo endogenous Nkx-2.5/GATA4 positive immature cardiomyocytes as well as massive vascular formation were observed in outer layer of infarcted myocardium despite of a few recruited/retained transfused STNP-AdSCs 4 weeks after MI in STNP-AdSC group. Finally, massive myocardial regeneration was observed 8 weeks after MI. Conclusions: Intravenously injected small number of statin nanoparticle-loaded hAdSCs exhibited a potent therapeutic effect inducing endogenous cardiac tissue regeneration.

Keywords: statin, drug delivery system, stem cells, cardiac regeneration

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Authors: Bernice Lottering, Yi-Wen Lin

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Chronic pain and depression have an estimated 80% rate of comorbidity with unsatisfactory treatment interventions signifying the importance of developing effective therapeutic interventions for a serious chronic condition affecting a large majority of the global population. Chronic pain is defined as persistent pain presenting for over 3 months. This disease state increases the risk of developing depression in comparison to healthy individuals. In the current study, complete Freund’s adjuvant (CFA) was used to induce cell-mediated chronic inflammatory pain in a murine model. Significant mechanical and thermal hyperalgesia was induced, alongside observable depression-like behaviors. These conditions were attenuated through the use of electroacupuncture (EA). Similarly, these effects were also investigated with respect to the transient receptor potential vanilloid 1 (TRPV1), by analyzing the effects of TRPV1 gene deletion on the comorbidity of chronic pain and depression. The expression of the TRPV1 inflammatory response, and related downstream molecules, including protein kinases (PKs), mitogen-activated protein kinase (MAPKs), and transcriptional factors, were significantly reduced in the thalamus, prefrontal cortex (PFC), hippocampus, and periaqueductal gray (PAG) of CFA-treated mice. In addition, phosphorylated N-methyl-D-aspartate (NMDA) receptor 1 was also found to be reduced in the aforementioned areas, suggesting potential application and validity in a clinical setting. Our study determined the prospective therapeutic effects of EA in the treatment of chronic inflammatory pain and depression comorbidity and provides a novel and detailed mechanism underlying EA-mediated analgesia. These findings may be relevant in the utilization of clinical intervention approaches related to chronic pain and depression comorbidity.

Keywords: chronic pain, depression, NMDA, prefrontal cortex, TRPV1

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Authors: Naveen Kumar B. T., Anuj Tyagi, Niraj Kumar Singh, Visanu Boonyawiwat, Shanthanagouda A. H., Orawan Boodde, Shankar K. M., Prakash Patil, Shubhkaramjeet Kaur

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Since 2009, Acute Hepatopancreatic Necrosis Disease (AHPND) outbreaks have increased rapidly, and these have led to the major economic losses to the global shrimp industry. In comparison to other treatments, passive immunity and monoclonal antibody (MAb) based farmer level kit have proved their importance in controlling and treating the diseases in the shrimp industry. In the present study, MAbs were produced against the recombinant PirB protein Vibrio parahaemolyticus strain causing AHPND. Briefly, Balb/C mice were immunized with rPirB at 15 days interval, and antibody titer was determined by ELISA. Spleen cells from mice showing high antibody titer were fused with SP2O myeloma cells for hybridoma production. Among 130 hybridomas, four showed high antibody titer and positive reactivity in an immunoblot assay. In Western blot assay, three out of four MAbs (4C4, 2C2 and 4G3) showed reactivity to rPirB protein. However, in the natural host, only Mab clone 4G3 show strong reactivity (with a strain of V. parahemolyticus causing EMS/AHPND). These clones also showed reactivity with less than 20 kDa proteins in AHPND free V. parahaemolyticus (Thailand stain). Further, on from MAb 4G3 clone, four panels of single cell MAbs clones (G3F5, G3B8, G3H2, and G3D6) were produced of which three showed strong positive reactivity to rPirB protein in the Western blot. These MAbs have potential for controlling and prevention of the AHPND through passive immunity and development of filed level rapid diagnostic kits.

Keywords: shrimp, economic loss, AHPND, MAb

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Authors: Jeanne Leblond, Warren Viricel, Amira Mbarek

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Although several products have reached the market, gene therapeutics are still in their first stages and require optimization. It is possible to improve their lacking efficiency by the use of carefully engineered vectors, able to carry the genetic material through each of the biological barriers they need to cross. In particular, getting inside the cell is a major challenge, because these hydrophilic nucleic acids have to cross the lipid-rich plasmatic and/or endosomal membrane, before being degraded into lysosomes. It takes less than one hour for newly endocytosed liposomes to reach highly acidic lysosomes, meaning that the degradation of the carried gene occurs rapidly, thus limiting the transfection efficiency. We propose to use a new pH-sensitive lipid able to change its conformation upon protonation at endosomal pH values, leading to the disruption of the lipidic bilayer and thus to the fast release of the nucleic acids into the cytosol. It is expected that this new pH-sensitive mechanism promote endosomal escape of the gene, thereby its transfection efficiency. The main challenge of this work was to design a preparation presenting fast-responding lipidic bilayer destabilization properties at endosomal pH 5 while remaining stable at blood pH value and during storage. A series of pH-sensitive lipids able to perform a conformational switch upon acidification were designed and synthesized. Liposomes containing these switchable lipids, as well as co-lipids were prepared and characterized. The liposomes were stable at 4°C and pH 7.4 for several months. Incubation with siRNA led to the full entrapment of nucleic acids as soon as the positive/negative charge ratio was superior to 2. The best liposomal formulation demonstrated a silencing efficiency up to 10% on HeLa cells, very similar to a commercial agent, with a lowest toxicity than the commercial agent. Using flow cytometry and microscopy assays, we demonstrated that drop of pH was required for the transfection efficiency, since bafilomycin blocked the transfection efficiency. Additional evidence was brought by the synthesis of a negative control lipid, which was unable to switch its conformation, and consequently exhibited no transfection ability. Mechanistic studies revealed that the uptake was mediated through endocytosis, by clathrin and caveolae pathways, as reported for previous lipid nanoparticle systems. This potent system was used for the treatment of hypercholesterolemia. The switchable lipids were able to knockdown PCSK9 expression on human hepatocytes (Huh-7). Its efficiency is currently evaluated on in vivo mice model of PCSK9 KO mice. In summary, we designed and optimized a new cationic pH-sensitive lipid for gene delivery. Its transfection efficiency is similar to the best available commercial agent, without the usually associated toxicity. The promising results lead to its use for the treatment of hypercholesterolemia on a mice model. Anticancer applications and pulmonary chronic disease are also currently investigated.

Keywords: liposomes, siRNA, pH-sensitive, molecular switch

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Authors: Katie M. Clark, Adriana A. Tienda, Krista C. Paffenroth, Lindsey N. Brigante, Daniel C. Colvin, Jose Maldonado, Erin S. Calipari, Fiona E. Harrison

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Manganese (Mn) is an essential trace element required for human health and is important in antioxidant defenses, as well as in the development and function of dopaminergic neurons. However, chronic low-level Mn exposure, such as through contaminated drinking water, poses risks that may contribute to neurodevelopmental and neurodegenerative conditions, including attention deficit hyperactivity disorder (ADHD). Pharmacological inhibition of the dopamine transporter (DAT) blocks reuptake, elevates synaptic dopamine, and alleviates ADHD symptoms. This study aimed to determine whether Mn exposure in juvenile mice modifies their response to DAT blockers, amphetamine, and methylphenidate and utilize neuroimaging methods to visualize and quantify Mn distribution across dopaminergic brain regions. Male and female heterozygous DATᵀ³⁵⁶ᴹ and wild-type littermates were randomly assigned to receive control (2.5% Stevia) or high Manganese (2.5 mg/ml Mn + 2.5% Stevia) via water ad libitum from weaning (21-28 days) for 4-5 weeks. Mice underwent repeated testing in locomotor activity chambers for three consecutive days (60 mins.) to ensure that they were fully habituated to the environments. On the fourth day, a 3-hour activity session was conducted following treatment with amphetamine (3 mg/kg) or methylphenidate (5 mg/kg). The second drug was administered in a second 3-hour activity session following a 1-week washout period. Following the washout, the mice were given one last injection of amphetamine and euthanized one hour later. Using the ex-vivo brains, magnetic resonance relaxometry (MRR) was performed on a 7Telsa imaging system to map T1- and T2-weighted (T1W, T2W) relaxation times. Mn inherent paramagnetic properties shorten both T1W and T2W times, which enhances the signal intensity and contrast, enabling effective visualization of Mn accumulation in the entire brain. A subset of mice was treated with amphetamine 1 hour before euthanasia. SmartSPIM light sheet microscopy with cleared whole brains and cFos and tyrosine hydroxylase (TH) labeling enabled an unbiased automated counting and densitometric analysis of TH and cFos positive cells. Immunohistochemistry was conducted to measure synaptic protein markers and quantify changes in neurotransmitter regulation. Mn exposure elevated Mn brain levels and potentiated stimulant effects in males. The globus pallidus, substantia nigra, thalamus, and striatum exhibited more pronounced T1W shortening, indicating regional susceptibility to Mn accumulation (p<0.0001, 2-Way ANOVA). In the cleared whole brains, initial analyses suggest that TH and c-Fos co-staining mirrors behavioral data with decreased co-staining in DATT356M+/- mice. Ongoing studies will identify the molecular basis of the effect of Mn, including changes to DAergic metabolism and transport and post-translational modification to the DAT. These findings demonstrate that alterations in T1W relaxation times, as measured by MRR, may serve as an early biomarker for Mn neurotoxicity. This neuroimaging approach exhibits remarkable accuracy in identifying Mn-susceptible brain regions, with a spatial resolution and sensitivity that surpasses current conventional dissection and mass spectrometry approaches. The capability to label and map TH and cFos expression across the entire brain provides insights into whole-brain neuronal activation and its connections to functional neural circuits and behavior following amphetamine and methylphenidate administration.

Keywords: manganese, environmental toxicology, dopamine dysfunction, biomarkers, drinking water, light sheet microscopy, magnetic resonance relaxometry (MRR)

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Authors: Djebbar Atmani, Dina Kilani, Tristan Richard

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Medicinal plants are an important source for the discovery of potential new substances for use in medicine and food. Pistacia lentiscus, Fraxinus angustifolia and Clematis flammula, plants growing in the Mediterranean basin, are widely used in traditional medicine. Therefore, the present study was designed to investigate their antioxidant, anti-inflammatory, antidiabetic, anti-mutagenic/genotoxic and neuroprotective potential and identification of active compounds using appropriate methodology. Plant extracts and fractions exhibited high scavenging capacity against known radicals, enhanced superoxide dismutase and catalase activitiesand restored blood glucose levels, in vivo, to normal values, in agreement with the in vitro enzymatic inhibition data, through inhibition of amylase and glucosidase activities. Administration of Pistacia lentiscus extracts significantly decreased carrageenan-induced mice paw oedema and reduced effectively IL-1β levels in cell culture, whereas Fraxinus angustifolia extracts showed good healing capacity against wounds when applied topically on rabbits. Pistacia lentiscus and Fraxinus angustifolia extracts showed good neuro-protection and restored cognitive functions in mice, while Clematis flammula extracts showed potent anti-ulcerogenic activity associated to a promising anti-mutagenic/genotoxic activity. HPLC-MS and NMR analyses allowed the identification and structural elucidation of several known and new anthocyanins, flavonols and flavanols. Therefore, Pistacia lentiscus, Fraxinus angustifolia and Clematis flammulacould be used in palliative treatments against inflammatory conditions and diabetes complications, as well as against deterioration of cognitive functions.

Keywords: pistacia lentiscus, clematis flammula, fraxinus angustifolia, phenolic compounds, biological activity

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Authors: Chodidjah, Edi Dharmana, Hardhono, Sarjadi

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Breast cancer treatment options including surgery, radiation therapy, chemotherapy, and immunotherapy have not been effective. Besides, they have side effects. Keladi Tikus (Typhonium flagelliforme) has been shown to improve immune system, suppress tumor growth and induce apoptosis. One of the parameters for immune system, tumor growth and apoptosis is IFNγ (Interferon γ), VEGF (Vascular Endothelial Growth Factor) and Caspase 3 respectively. The aim of this study was to examine the effect of the administration of Keladi Tikus tuber extract at the dose of 200 mg/kgBW, 400 mg/KgBW, and 800 mg/kgBW on the level of IFNγ, VEGF and caspase 3 expression. In this experimental study using post test randomized control group design, 24 CH3 mice with tumor were randomly divided into 4 groups including control group and treated groups: Treated with 0.2 cc extract of Keladi Tikus at the dose of 200 mg/kgBW, 400 mg/kgBW, 800 mg/kgBW, respectively for 30 days. On day 31 the lymphatic tissue was taken and evaluated for its level of IFNγ, using ELISA. The tumor tissue was taken and subjected to immunohistochemistry staining for VEGF and caspase 3 expression evaluation. The data on IFNγ, VEGF and Caspase 3 expression were analyzed using One Way Anova with significant level of 0.05. One Way Anova resulted in p<0.05. LSD test showed that the level of IFNγ and Caspase 3 for control group was different from that of treated groups. There was no significant different between the treated group of 400 mg/KgBW and 800mg/KgBW. VEGF expressions for all the treated groups were significant. In conclusion, the oral administration of ethanolic extract of Keladi Tikus (Typhonium flagelliforme) at the dose of 200mg/kgBW, 400 mg/kgBW,800 mg/kgBW increases IFNγ, Caspase 3 and decreases VEGF expression in C3H mice with adenocarsinoma mamma.

Keywords: Typhonium flagelliforme, IFNγ, caspase 3, VEGF

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Authors: Marcela Parra-Vargas, Ana Sandoval-Rodriguez, Roberto Rodriguez-Echevarria, Jose Dominguez-Rosales, Juan Armendariz-Borunda

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Non-alcoholic fatty liver disease (NAFLD) is characterized by an excess of hepatic lipids, and it is to author’s best knowledge, the most prevalent chronic liver disorder. Anthocyanin-rich food consumption is linked to health benefits in metabolic disorders associated with obesity and NAFLD, although the precise functional role of anthocyanidin delphinidin (Dp) has yet to be established. The aim of this study was to investigate the effect of the Dp in NAFLD metabolic alterations by evaluating prevention or amelioration of hepatic lipid accumulation, as well as molecular mechanisms in two experimental obesity-related models of NALFD. In vitro: HepG2 cells were incubated with sodium palmitate (PA, 1 mM) to induce lipotoxic damage, and concomitantly treated with Dp (180 uM) for 24 h. Subsequently, total lipid accumulation was measured by colorimetric staining with Oil Red O, and total intrahepatic triglycerides were determined by an enzymatic assay. To assess molecular mechanisms, cells were pre-treated with PA for 24 h and then exposed to Dp for 1 h. In vivo: four-week-old male C57BL/6Nhsd mice were allocated in two main groups. Mice were fed with standard diet (control) or high-fat and high-carbohydrate diet (45% fat, HFD) for 16 wk to induce NAFLD. Then HFD was divided into subgroups: one treated orally with Dp (15 mg/kg bw, HFD-Dp) every day for 4 wk, while HFD group treated with vehicle (DMSO). Weight and fasting glucose were recorded weekly, while dietary ingestion was measured daily. Insulin tolerance test was performed at the end of treatment. Liver histology was evaluated with H&E and Masson’s trichrome stain. RT-PCR was used to evaluate gene expression and Western Blot to determine levels of protein in both experimental models. Parametric data were analyzed with one-way ANOVA and Tukey’s post-hoc test. Kruskal-Wallis and Mann-Whitney U test for non-parametric data, and P < 0.5 were considered significant. Dp prevented hepatic lipid accumulation by PA in HepG2 hepatocytes. Furthermore, Dp down-regulated gene expression of SREBP1c, FAS, and CPT1a without modifying AMPK phosphorylation levels. In vivo, Dp oral administration did not ameliorate lipid metabolic alterations raised by HFD. Adiposity, dietary ingestion, fasting glucose, and insulin sensitivity after Dp treatment remained similar to HFD group. Histological analysis showed hepatic damage in HFD groups and no differences between HFD and HFD-Dp groups were found. Hepatic gene expression of ACC and FAS were not altered by HFD. SREBP1c was similar in both HFD and HFD-Dp groups. No significant changes were observed in SREBP1c, ACC, and FAS adipose tissue gene expression by HFD or Dp treatment. Additionally, immunoblotting analysis revealed no changes in pathway SIRT1-LKB-AMPK and PPAR alpha by both HFD groups compared to control. In conclusion, the antioxidant Dp may provoke beneficial effects in the prevention of hepatic lipid accumulation. Nevertheless, the oral dose administrated in mice that simulated the total intake of anthocyanins consumed daily by humans has no effect as a treatment on hepatic lipid metabolic alterations and histological abnormalities associated with exposure to chronic HFD. A healthy lifestyle with regular intake of antioxidants such as anthocyanins may prevent metabolic alterations in NAFLD.

Keywords: anthocyanins, antioxidants, delphinidin, non-alcoholic fatty liver disease, obesity

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Authors: Miao Zhang, Limin Liu, Feng Zhi, Panpan Niu, Mengya Yang, Xuemei Zhu, Ying Diao, Jun Wang, Ying Zhao

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Background and Aims: Clinical studies have demonstrated that serum semicarbazide-sensitive amine oxidase (SSAO) activities positively correlate with the progression of atherosclerosis. The aim of the present study is to investigate the effect of SSAO inactivation on the development of atherosclerosis. Methods: Female LDLr knockout (KO) mice were given the Western-type diet for 6 and 9 weeks to induce the formation of early and advanced lesions, and semicarbazide (SCZ, 0.125%) was added into the drinking water to inactivate SSAO in vivo. Results: Despite no impact on plasma total cholesterol levels, abrogation of SSAO by SCZ not only resulted in the enlargement of both early (1.5-fold, p=0.0043) and advanced (1.8-fold, p=0.0013) atherosclerotic lesions, but also led to reduced/increased lesion contents of macrophages/smooth muscle cells (SMCs) (macrophage: ~0.74-fold, p=0.0002(early)/0.0016(advanced); SMC: ~1.55-fold, p=0.0003(early) /0.0001(advanced)), respectively. Moreover, SSAO inactivation inhibited the migration of circulating monocytes into peripheral tissues and reduced the amount of circulating Ly6Chigh monocytes (0.7-fold, p=0.0001), which may account for the reduced macrophage content in lesions. In contrast, the increased number of SMCs in lesions of SCZ-treated mice is attributed to an augmented synthetic vascular SMC phenotype switch as evidenced by the increased proliferation of SMCs and accumulation of collagens in vivo. Conclusion: SSAO inactivation by SCZ promotes the phenotypic switch of SMCs and the development of atherosclerosis. The enzymatic activity of SSAO may thus represent a potential target in the prevention and/or treatment of atherosclerosis.

Keywords: atherosclerosis, phenotype switch of smooth muscle cells, SSAO/VAP-1, semicarbazide

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Authors: A. S. Lunev, A. A. Larenkov, O. E. Klementyeva, G. E. Kodina

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Background and aims: 68Ga-citrate is one of prospective radiopharmaceutical for PET-imaging of inflammation and infection. 68Ga-citrate is 67Ga-citrate analogue using since 1970s for SPECT-imaging. There's known rebinding reaction occurs past Ga-citrate injection and gallium (similar iron Fe3+) binds with blood transferrin. Then radiolabeled protein complex is delivered to pathological foci (inflammation/infection sites). But excessive gallium bindings with transferrin are cause of slow blood clearance, long accumulation time in foci (24-72 h) and exception of application possibility of the short-lived gallium-68 (T½ = 68 min). Injection of additional chemical agents (e.g. Fe3+ compounds) competing with radioactive gallium to the blood transferrin joining (blocking of its metal binding capacity) is one of the ways to solve formulated problem. This phenomenon can be used for correction of 68Ga-citrate pharmacokinetics for increasing of the blood clearance and accumulation in foci. The aim of real studying is research of effect of stable Fe-citrate on 68Ga-citrate tissue distribution. Materials and methods: 68Ga-citrate without/with extra injection of stable Fe-citrate (III) was injected nonlinear mice with inflammation models (aseptic soft tissue inflammation, lung infection, osteomyelitis). PET/X-RAY Genisys4 (Sofie Bioscience, USA) was used for non-invasive PET imaging (for 30, 60, 120 min past injection 68Ga-citrate) with subsequent reconstruction of imaging and their analysis (value of clearance, distribution volume). Scanning time is 10 min. Results and conclusions: I. v. injection of stable Fe-citrate blocks the metal-binding capability of transferrin serum and allows decreasing gallium-68 radioactivity in blood significantly and increasing accumulation in inflammation (3-5 time). It allows receiving more informative PET-images of inflammation early (for 30-60 min after injection). Pharmacokinetic parameters prove it. Noted there is no statistically significant difference between 68Ga-citrate accumulation for different inflammation model because PET imaging is indication of pathological processes and is not their identification.

Keywords: 68Ga-citrate, Fe-citrate, PET imaging, mice, inflammation, infection

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Authors: Vandana Mohan, Ashwani Koul

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Aim: To evaluate the potential of Aqueous Tinospora cordifolia stem extract (Aq.Tc) and Arabinogalactan (AG) on pulmonary carcinogenesis and associated tumor markers. Background: Lung cancer is one of the most frequent malignancy with high mortality rate due to limitation of early detection resulting in low cure rates. Current research effort focuses on identifying some blood-based biomarkers like CEA, ctDNA and LDH which may have potential to detect cancer at an early stage, evaluation of therapeutic response and its recurrence. Medicinal plants and their active components have been widely investigated for their anticancer potentials. Aqueous preparation of T. Cordifolia extract is enriched in the polysaccharide fraction i.e., AG when compared with other types of extract. Moreover, reports are available of polysaccharide fraction of T. Cordifolia in in vitro lung cancer models which showed profound anti-metastatic activity against these cell lines. However, not much has been explored about its effect in in vivo lung cancer models and the underlying mechanism involved. Experimental Design: Mice were randomly segregated into six groups. Group I animals served as control. Group II animals were administered with Aq. Tc extract (200 mg/kg b.w.) p.o.on the alternate days. Group III animals were fed with AG (7.5 mg/kg b.w.) p.o. on the alternate days (thrice a week). Group IV animals were installed with Benzo(a)pyrene (50 mg/kg b.w.), i.p. twice within an interval of two weeks. Group V animals received Aq. Tc extract as in group II along with it B(a)P was installed after two weeks of Aq. Tc administration following the same protocol as for group IV. Group VI animals received AG as in group III along with it B(a)P was installed after two weeks of AG administration. Results: Administration of B(a)P to mice resulted in increased tumor incidence, multiplicity and pulmonary somatic index with concomitant increase in serum/plasma markers like CEA, ctDNA, LDH and TNF-α.Aq.Tc and AG supplementation significantly attenuated these alterations at different stages of tumorigenesis thereby showing potent anti-cancer effect in lung cancer. A pronounced decrease in serum/plasma markers were observed in animals treated with Aq.Tc as compared to those fed with AG. Also, extensive hyperproliferation of alveolar epithelium was prominent in B(a)P induced lung tumors. However, treatment of Aq.Tc and AG to lung tumor bearing mice exhibited reduced alveolar damage evident from decreased number of hyperchromatic irregular nuclei. A direct correlation between the concentration of tumor markers and the intensity of lung cancer was observed in animals bearing cancer co-treated with Aq.Tc and AG. Conclusion: These findings substantiate the chemopreventive potential of Aq.Tc and AG against lung tumorigenesis. Interestingly, Aq.Tc was found to be more effective in modulating the cancer as reflected by various observations which may be attributed to the synergism offered by various components of Aq.Tc. Further studies are in progress to understand the underlined mechanism in inhibiting lung tumorigenesis by Aq.Tc and AG.

Keywords: Arabinogalactan, Benzo(a)pyrene B(a)P, carcinoembryonic antigen (CEA), circulating tumor DNA (ctDNA), lactate dehydrogenase (LDH), Tinospora cordifolia

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Authors: Nana Katsitadze, Tamar Atanelishvili, Mariam Kutateladze, Alexandre Tushishvili

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In the modern world, tourism has emerged as one of the most powerful economic sectors, and due to its high economic performance, it is attractive to the countries with various levels of economic development. The purpose of the present paper, dedicated to discussing the current problems of tourism development, is to find ways which will contribute to bringing more benefits to the country from the sector. Georgia has been successfully developing leisure tourism for the last ten years, and at the next stage of development business, tourism gains particular importance for Georgia as a means of mitigating the negative socio-economic effects caused by the seasonality of tourism and as a high-cost tourism market. Therefore, the object of the paper is to study the factors that contribute to the development of business tourism. The paper uses the research methods such as system analysis, synthesis, analogy, as well as historical, comparative, economic, and statistical methods of analysis. The information base for the research is made up of the statistics on the functioning of the tourism market of Georgia and foreign countries as well as official data provided by international organizations in the field of tourism. Based on the experience of business tourism around the world and identifying the successful start of business tourism development in Georgia and its causing factors, a business tourism development model for Georgia has been developed. The model might be useful as a methodological material for developing a business tourism development concept for the countries with limited financial resources but rich in tourism resources like Georgia. On the initial stage of development (in absence of conventional centers), the suggested concept of business tourism development involves organizing small and medium-sized meetings both in large cities and in regions by using high-class hotel infrastructure and event management services. Relocation of small meetings to the regions encourages inclusive development of the sector based on increasing the awareness of these regions as tourist sites as well as the increase in employment and sales of other tourism or consumer products. Business tourism increases the number of hotel visitors in the non-seasonal period and improves hotel performance indicators, which enhances the attractiveness of investing in the hotel business. According to the present concept of business tourism development, at the initial stage, development of business tourism is based on the existing markets, including internal market, neighboring markets and the markets of geographically relatively near countries and at the next stage, the concept involves generating tourists from other relatively distant target markets. As a result, by gaining experience in business tourism, enhancing professionalism, increasing awareness and stimulating infrastructure development, the country will prepare the basis to move to a higher stage of tourism development. In addition, the experience showed that for attracting large customers, peculiarities of the field require activation of state policy and active use of marketing mechanisms and tools of the state.

Keywords: hotel industry development, MICE model, MICE strategy, MICE tourism in Georgia

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Authors: Vladimir Berezin, Andrey Bogoyavlenskiy, Pavel Alexyuk, Madina Alexyuk, Aizhan Turmagambetova, Irina Zaitseva, Nadezhda Sokolova, Elmira Omirtaeva

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Introduction: Triterpene saponins of plant origin are one of the most promising candidates for elaboration of novel adjuvants. Due to the combination of immunostimulating activity and the capacity interact with amphipathic molecules with formation of highly immunogenic nanocomplexes, triterpene saponins could serve as a good adjuvant/delivery system for vaccine use. In the research presented adjuvants on the base of nanocomplexes contained triterpene saponins isolated from Glycyrrhiza glabra and Saponaria officinalis plants indigenous to Kazakhstan were elaborated for influenza vaccine use. Methods: Purified triterpene saponins 'Glabilox' and 'SO1' with low toxicity and high immunostimulatory activity were isolated from plants Glycyrrhiza glabra L. and Saponaria officinalis L. by high-performance liquid chromatography (HPLC) and identified using electrospray ionization mass spectrometry (ESI-MS). Influenza virus A/St-Petersburg/5/09 (H1N1) propagated in 9-days old chicken embryos was concentrated and purified by centrifugation in sucrose gradient. Nanocomplexes contained lipids, and triterpene saponins Glabilox or SO1 were prepared by dialysis technique. Immunostimulating activity of experimental vaccine preparations was studied in vaccination/challenge experiments in mice. Results: Humoral and cellular immune responses and protection against influenza virus infection were examined after single subcutaneous and intranasal immunization. Mice were immunized subunit influenza vaccine (HA+NA) or whole virus inactivated influenza vaccine in doses 3.0/5.0/10.0 µg antigen/animal mixed with adjuvant in dose 15.0 µg/animal. Sera were taken 14-21 days following single immunization and mice challenged by A/St-Petersburg/5/09 influenza virus in dose 100 EID₅₀. Study of experimental influenza vaccine preparations in animal immunization experiments has shown that subcutaneous and intranasal immunization with subunit influenza vaccine mixed with nanocomplexes contained Glabilox or SO1 saponins stimulated high levels of humoral immune response (IgM, IgA, IgG1, IgG2a, and IgG2b antibody) and cellular immune response (IL-2, IL-4, IL-10, and IFN-γ cytokines) and resulted 80-90% protection against lethal influenza infection. Also, single intranasal and single subcutaneous immunization with whole virus inactivated influenza vaccine mixed with nanoparticulated adjuvants stimulated high levels of humoral and cellular immune responses and provided 100% protection against lethal influenza infection. Conclusion: The results of study have shown that nanocomplexes contained purified triterpene saponins Glabilox and SO1 isolated from plants indigenous to Kazakhstan can stimulate a broad spectrum of humoral and cellular immune responses and induce protection against lethal influenza infection. Both elaborated adjuvants are promising for incorporation to influenza vaccine intended for subcutaneous and intranasal routes of immunization.

Keywords: influenza vaccine, adjuvants, triterpene saponins, immunostimulating activity

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Authors: Konstantin Yu. Moiseev, Antonina F. Budnik, Andrey I. Emanuilov, Petr M. Masliukov

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The vast majority of sympathetic ganglionic neurons are catecholaminergic. Some sympathetic neurons lack catecholamines and mostly use acetylcholine as their main neurotransmitter. Some cholinergic postganglionic neurons also express neuronal nitric oxide synthase (nNOS). Preganglionic sympathetic neurons are cholinergic and most of them are also nNOS-immunoreactive (IR). The purpose of this study was to gain further insight into the neuroplasticity of sympathetic neurons during postnatal ontogenesis by comparing the development of pre- and postganglionic neurons expressing nNOS in different mammals. nNOS was investigated by immunohistochemistry in the sympathetic superior cervical ganglion (SCG), stellate ganglion (SG), celiac ganglion (CG) and spinal cord from rats, mice and cats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 2-month-old and 2-year-old). In rats and mice, nNOS-positive neurons were not found in sympathetic ganglia from birth onwards. In cats, non-catecholaminergic nNOS-IR sympathetic ganglionic neurons are present from the moment of birth. In all studied age groups, substantial populations of nNOS-IR cells (up to 8.3%) was found in the SG, with a much smaller population found in the SCG (<1%) and only few cells observed in the CG. The percentage of nNOS-IR neurons in the CG and SCG did not significantly change during development. The proportion of nNOS-IR neuron profiles in the SG increased in first 20 days of life from 2.3±0.15% to 8.3±0.56%. In the SG, percentages of nNOS-IR sympathetic neurons colocalizing vasoactive intestinal peptide increased in the first 20 days of life. Choline acetyltransferase (ChAT)-IR and calcitonin gene-related peptide-IR neurons were not observed in the sympathetic ganglia of newborn animals and did not appear until 10 days after birth. In the SG of newborn and 10-day-old kittens, the majority of NOS-IR neurons were calbindin (CB)-IR, whereas in the SCG and CG of cats of all age groups and in the SG of 30-day-old and older kittens, the vast majority of NOS-IR neurons lacked CB. In newborn mammals, the most of sympathetic preganglionic neurons in the nucleus intermediolateralis thoracolumbalis pars principalis (nucl.ILp) were nNOS-IR. The percentage of nNOS-IR neurons decreased and the same parameter of ChAT-IR neurons increased during the development. We conclude that the development of nNOS-IR preganglionic and ganglionic sympathetic neurons in different mammals has time and species differences.

Keywords: sympathetic neuron, nitric oxide synthase, immunohistochemistry, development

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Authors: Wan-Ting Kuo, Yi-Wen Liu, Hsiao-Sheng Liu

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Annual incidence of bladder cancer increases in the world and occurs frequently in the male. Most common type is transitional cell carcinoma (TCC) which is treated by transurethral resection followed by intravesical administration of agents. In clinical treatment of bladder cancer, chemotherapeutic drugs-induced apoptosis is always used in patients. However, cancers usually develop resistance to chemotherapeutic drugs and often lead to aggressive tumors with worse clinical outcomes. Approximate 70% TCC recurs and 30% recurrent tumors progress to high-grade invasive tumors, indicating that new therapeutic agents are urgently needed to improve the successful rate of overall treatment. Nonapoptotic program cell death may assist to overcome worse clinical outcomes. Autophagy which is one of the nonapoptotic pathways provides another option for bladder cancer patients. Autophagy is reported as a potent anticancer therapy in some cancers. First of all, we established a mouse orthotopic bladder tumor formation model in order to create a similar tumor microenvironment. IVIS system and micro-ultrasound were utilized to noninvasively monitor tumor formation. In addition, we carried out intravesical treatment in our animal model to be consistent with human clinical treatment. In our study, we carried out intravesical instillation of the autophagy inducer in mouse orthotopic bladder tumor to observe tumor formation by noninvasive IVIS system and micro-ultrasound. Our results showed that bladder tumor formation is suppressed by the autophagy inducer, and there are no significant side effects in the physiology of mice. Furthermore, the autophagy inducer upregulated autophagy in bladder tissues of the treated mice was confirmed by Western blot, immunohistochemistry, and immunofluorescence. In conclusion, we reveal that a novel autophagy inducer with low side effects suppresses bladder tumor formation in our mouse orthotopic bladder tumor model, and it provides another therapeutic approach in bladder cancer patients.

Keywords: bladder cancer, transitional cell carcinoma, orthotopic bladder tumor formation model, autophagy

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Authors: Marwa Elkalla, E. Ali Abdelfadil, Ali. Mohamed. M. Sami, Ali M. Abdel-Monem

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To assess the impact of the blood collection technique on clinical pathology markers and to establish reference intervals, a study was performed using normal, healthy C57BL/6 mice. Both sexes were employed, and they were randomly assigned to different groups depending on the phlebotomy technique used. The blood was drawn in one of four ways: intracardiac (IC), caudal vena cava (VC), caudal vena cava (VC) plus a peritoneal collection of any extravasated blood, or retroorbital phlebotomy (RO). Several serum biochemistries, such as a liver function test, a complete blood count with differentials, and a platelet count, were analysed from the blood and serum samples analysed. Red blood cell count, haemoglobin (p >0.002), hematocrit, alkaline phosphatase, albumin, total protein, and creatinine were all significantly greater in female mice. Platelet counts, specific white blood cell numbers (total, neutrophil, lymphocyte, and eosinophil counts), globulin, amylase, and the BUN/creatinine ratio were all greater in males. The VC approach seemed marginally superior to the IC approach for the characteristics under consideration and was linked to the least variation among both sexes. Transaminase levels showed the greatest variation between study groups. The aspartate aminotransferase (AST) values were linked with decreased fluctuation for the VC approach, but the alanine aminotransferase (ALT) values were similar between the IC and VC groups. There was a lot of diversity and range in transaminase levels between the MC and RO groups. We found that the RO approach, the only one tested that allowed for repeated sample collection, yielded acceptable ALT readings. The findings show that the test results are significantly affected by the phlebotomy technique and that the VC or IC techniques provide the most reliable data. When organising a study and comparing data to reference ranges, the ranges supplied here by collection method and sex can be utilised to determine the best approach to data collection. The authors suggest establishing norms based on the procedures used by each individual researcher in his or her own lab.

Keywords: clinical, pathology, blood, effect

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Authors: Parul Kulshreshtha, Subrata Sinha, Rakesh Bhatnagar

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This study was conducted by taking B. anthracis as a model pathogen. On infecting a host, B. anthracis secretes three proteins, namely, protective antigen (PA, 83kDa), edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). These three proteins are the components of two anthrax toxins. PA binds to the cell surface receptors, namely, tumor endothelial marker (TEM) 8 and capillary morphogenesis protein (CMG) 2. TEM8 and CMG2 interact with LDL-receptor related protein (LRP) 6 for endocytosis of EF and LF. On entering the cell, EF acts as a calmodulin-dependent adenylate cyclase that causes a prolonged increase of cytosolic cyclic adenosine monophosphate (cAMP). LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MAPKK/MEK) close to their N-terminus. By secreting these two toxins, B.anthracis ascertains death of the host. Once the systemic levels of the toxins rise, antibiotics alone cannot save the host. Therefore, toxin-specific inhibitors have to be developed. In this wake, monoclonal antibodies have been developed for the neutralization of toxic effects of anthrax toxins. We created hybridomas by using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor of B. anthracis) to obtain anti-toxin antibodies. Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immunized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies from all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H8 and H10) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). The protective efficacy of H7, H8, H10 and H11 was investigated. H7, H8 and H10 were found to be protective. H11 was found to have disease enhancing characteristics in-vitro and in mouse model of challenge with B. anthracis. In this study the disease enhancing character of H11 monoclonal antibody and anti-rLFn polyclonal sera was investigated. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature both in-vitro and in-vivo. But combination of H11 with LETscFv (an scFv with VH and VL identical to H10 but lacking Fc region) could not abrogate the disease-enhancing character of H11 mAb. Therefore it was concluded that for suppression of disease enhancement, Fc portion was absolutely essential for interaction of H10 with H11. Our study indicates that the protective potential of an antibody depends equally on its idiotype/ antigen specificity and its isotype. A number of monoclonal and engineered antibodies are being explored as immunotherapeutics but it is absolutely essential to characterize each one for their individual and combined protective potential. Although new in the sphere of toxin-based diseases, it is extremely important to characterize the disease-enhancing nature of polyclonal as well as monoclonal antibodies. This is because several anti-viral therapeutics and vaccines have failed in the face of this phenomenon. The passive –immunotherapy thus needs to be well formulated to avoid any contraindications.

Keywords: immunotherapy, polyclonal, monoclonal, antibody-dependent disease enhancement

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Authors: Aastha Arora, Vikas Bhuria, Saurabh Singh, Uma Pathak, Shweta Mathur, Puja P. Hazari, Rajat Sandhir, Ravi Soni, Anant N. Bhatt, Bilikere S. Dwarakanath

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Radiotherapy is an effective curative and palliative option for patients with thoracic malignancies. However, lung injury, comprising of pneumonitis and fibrosis, remains a significant clin¬ical complication of thoracic radiation, thus making it a dose-limiting factor. Also, injury to the lung is often reported as part of multi-organ failure in victims of accidental radiation exposures. Radiation induced inflammatory response in the lung, characterized by leukocyte infiltration and vascular changes, is an important contributing factor for the injury. Therefore, countermeasure agents to attenuate radiation induced inflammatory response are considered as an important approach to prevent chronic lung damage. Although Amifostine, the widely used, FDA approved radio-protector, has been found to reduce the radiation induced pneumonitis during radiation therapy of non-small cell lung carcinoma, its application during mass and field exposure is limited due to associated toxicity and ineffectiveness with the oral administration. The amifostine analogue (DRDE-30) overcomes this limitation as it is orally effective in reducing the mortality of whole body irradiated mice. The current study was undertaken to investigate the potential of DRDE-30 to ameliorate radiation induced lung damage. DRDE-30 was administered intra-peritoneally, 30 minutes prior to 13.5 Gy thoracic (60Co-gamma) radiation in C57BL/6 mice. Broncheo- alveolar lavage fluid (BALF) and lung tissues were harvested at 12 and 24 weeks post irradiation for studying inflammatory and fibrotic markers. Lactate dehydrogenase (LDH) leakage, leukocyte count and protein content in BALF were used as parameters to evaluate lung vascular permeability. Inflammatory cell signaling (p38 phosphorylation) and anti-oxidant status (MnSOD and Catalase level) was assessed by Western blot, while X-ray CT scan, H & E staining and trichrome staining were done to study the lung architecture and collagen deposition. Irradiation of the lung increased the total protein content, LDH leakage and total leukocyte count in the BALF, reflecting endothelial barrier dysfunction. These disruptive effects were significantly abolished by DRDE-30, which appear to be linked to the DRDE-30 mediated abrogation of activation of the redox-sensitive pro- inflammatory signaling cascade, the MAPK pathway. Concurrent administration of DRDE-30 with radiation inhibited radiation-induced oxidative stress by strengthening the anti-oxidant defense system and abrogated p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and macrophage recruitment to the lungs. Histopathological examination (by H & E staining) of the lung showed radiation-induced inflammation of the lungs, characterized by cellular infiltration, interstitial oedema, alveolar wall thickening, perivascular fibrosis and obstruction of alveolar spaces, which were all reduced by pre-administration of DRDE-30. Structural analysis with X-ray CT indicated lung architecture (linked to the degree of opacity) comparable to un-irradiated mice that correlated well with the lung morphology and reduced collagen deposition. Reduction in the radiation-induced inflammation and fibrosis brought about by DRDE-30 resulted in a profound increase in animal survival (72 % in the combination vs 24% with radiation) observed at the end of 24 weeks following irradiation. These findings establish the potential of the Amifostine analogue, DRDE-30, in reducing radiation induced pulmonary injury by attenuating the inflammatory and fibrotic responses.

Keywords: amifostine, fibrosis, inflammation, lung injury radiation

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Authors: Muhammad Awais Afzal, Lorena Tuchscherr De Hauschopp, Christian Hübner

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Introduction: Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway, which can be induced by extrinsic and intrinsic stressors in living systems to adapt to fluctuating environmental conditions. In the context of inflammatory stress, autophagy contributes to the elimination of invading pathogens, the regulation of innate and adaptive immune mechanisms, and regulation of inflammasome activity as well as tissue damage repair. Lysosomes can be recycled from autolysosomes by the process of autophagic lysosome reformation (ALR), which depends on the presence of several proteins including Spatacsin. Thus ALR contributes to the replenishment of lysosomes that are available for fusion with autophagosomes in situations of increased autophagic turnover, e.g., during bacterial infections, inflammatory stress or sepsis. Objectives: We aimed to assess whether ALR plays a role for cell survival in an in-vitro bacterial infection model. Methods: Mouse embryonic fibroblasts (MEFs) were isolated from wild-type mice and Spatacsin (Spg11-/-) knockout mice. Wild-type MEFs and Spg11-/- MEFs were infected with Staphylococcus aureus (multiplication of infection (MOI) used was 10). After 8 and 16 hours of infection, cell viability was assessed on BD flow cytometer through propidium iodide intake. Bacterial intake by cells was also calculated by plating cell lysates on blood agar plates. Results: in-vitro infection of MEFs with Staphylococcus aureus showed a marked decrease of cell viability in ALR deficient Spatacsin knockout (Spg11-/-) MEFs after 16 hours of infection as compared to wild-type MEFs (n=3 independent experiments; p < 0.0001) although no difference was observed for bacterial intake by both genotypes. Conclusion: Suggesting that ALR is important for the defense of invading pathogens e.g. S. aureus, we observed a marked increase of cell death in an in-vitro infection model in cells with compromised ALR.

Keywords: autophagy, autophagic lysosome reformation, bacterial infections, Staphylococcus aureus

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Authors: Bernice Jeanne Lottering, Yi-Wen Lin

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Introduction: Chronic pain has a definitive lack of objective parameters in the measurement and treatment efficacy of diseases such as Fibromyalgia (FM). Persistent widespread pain and generalized tenderness are the characteristic symptoms affecting a large majority of the global population, particularly females. This disease has indicated a refractory tendency to conventional treatment ventures, largely resultant from a lack of etiological and pathogenic understanding of the disease development. Emerging evidence indicates that the central nervous system (CNS) plays a critical role in the amplification of pain signals and the neurotransmitters associated therewith. Various stimuli have been found to activate the channels existent on nociceptor terminals, thereby actuating nociceptive impulses along the pain pathways. The transient receptor potential vanalloid 1 (TRPV1) channel functions as a molecular integrator for numerous sensory inputs, such as nociception, and was explored in the current study. Current intervention approaches face a multitude challenges, ranging from effective therapeutic interventions to the limitation of pathognomonic criteria resultant from incomplete understanding and partial evidence on the mechanisms of action of FM. It remains unclear whether electroacupuncture (EA) plays an integral role in the functioning of the TRPV1 pathway, and whether or not it can reduce the chronic pain induced by FM. Aims: The aim of this study was to explore the mechanisms underlying the activation and modulation of the TRPV1 channel pathway in a cold stress model of FM applied to a murine model. Furthermore, the effect of EA in the treatment of mechanical and thermal pain, as expressed in FM was also to be investigated. Methods: 18 C57BL/6 wild type and 6 TRPV1 knockout (KO) mice, aged 8-12 weeks, were exposed to an intermittent cold stress-induced fibromyalgia-like pain model, with or without EA treatment at ZusanLi ST36 (2Hz/20min) on day 3 to 5. Von Frey and Hargreaves behaviour tests were implemented in order to analyze the mechanical and thermal pain thresholds on day 0, 3 and 5 in control group (C), FM group (FM), FM mice with EA treated group (FM + EA) and FM in KO group. Results: An increase in mechanical and thermal hyperalgesia was observed in the FM, EA and KO groups when compared to the control group. This initial increase was reduced in the EA group, which directs focus at the treatment efficacy of EA in nociceptive sensitization, and the analgesic effect EA has attenuating FM associated pain. Discussion: An increase in the nociceptive sensitization was observed through higher withdrawal thresholds in the von Frey mechanical test and the Hargreaves thermal test. TRPV1 function in mice has been scientifically associated with these nociceptive conduits, and the increased behaviour test results suggest that TRPV1 upregulation is central to the FM induced hyperalgesia. This data was supported by the decrease in sensitivity observed in results of the TRPV1 KO group. Moreover, the treatment of EA showed a decrease in this FM induced nociceptive sensitization, suggesting TRPV1 upregulation and overexpression can be attenuated by EA at bilateral ST36. This evidence compellingly implies that the analgesic effect of EA is associated with TRPV1 downregulation.

Keywords: fibromyalgia, electroacupuncture, TRPV1, nociception

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Authors: Syeda Hira, Muhammad Gulfraz

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Background: Liver diseases cause serious health issues. Plants contain active compounds that significantly help in the treatment of various diseases. Ficus carica is traditionally used for the treatment of liver diseases. The purpose of the present study was the isolation and identification of active components from F.carica leaves which are responsible for hepatoprotective activity. Methods: The study was designed to identify the most active hepatoprotective sub-fraction from ethyl acetate fraction of Ficus carica by in vitro study and evaluation of its in vivo hepatoprotective effect in animal models. Ethyl acetate fraction was subjected to column, and a total of eight sub-fractions were obtained. In vitro, the hepatoprotective effect of all sub-fractions was determined on HepG2 cell lines. Toxicity was induced by CCl₄ (Carbon tetrachloride), and silymarin was used as a positive control. On the basis of the results, the most active sub-fraction was subjected to LC-MS and FT-IR analysis for the identification of bioactive compounds. In vivo, the hepatoprotective effect was determined in mice. Toxicity was induced by CCl₄; at the end of the experiment, biochemical parameters such as ALT, AST, ALP, bilirubin, and total protein were estimated in serum. Histopathology of liver tissues was also done. Results: Sub-fraction FVI exhibited significant (P<0.05) hepatoprotective activity as compared to other sub-fractions, which was almost similar to the standard drug silymarin. Six known bioactive compounds were identified from this sub-fraction after LC-MS analysis. In vivo, the hepatoprotective activity of sub-fraction FVI was evaluated in CCl₄-induced toxicated mice. Administration of CCl₄ significantly increased level of ALT (Alanine transaminase), AST (Aspartate aminotransferase), ALP (Alkaline phosphatase), and bilirubin and decreased the total protein. Treatment with sub-fraction FVI significantly (p<0.05) reversed the level of these biomarkers toward normal at both doses of 25 mg/kg and 50 mg/kg. Conclusion: Our findings confirmed the hepatoprotective effect of ethyl acetate fraction of F.carica. It could be a good candidate for the development of a natural hepatoprotective drug; pre-clinical investigation on ethyl acetate fraction is recommended.

Keywords: Ficus carica, hepatoprotective, CCl₄, bioactive compounds, liver markers

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Authors: Melina, Jumardi Jumardi, Erwin Erwin, Sri Nuraminah, Andi Nasruddin

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The rice field rat (Rattus argentiventer Robinson and Kloss) is a pest causing the greatest yield loss of rice plants, especially in lowland agroecosystems with intensive cropping patterns (2-3 plantings per year). Field mice damage rice plants at all stages of growth, from seedling to harvest, even in storage warehouses. Severe damage with yield loss of up to 100% occurs if rats attack rice at the generative stage because the plants are no longer able to recover by forming new tillers. Farmers mainly use rodenticides in the form of poisoned baits or as fumigants, which are applied to rat burrow holes. This practice is generally less effective because mice are able to avoid the poison or become resistant after several exposures to it. In addition, excessive use of rodenticides can have negative impacts on the environment and non-target organisms. For this reason, this research was conducted to evaluate the effectiveness of fences as an environmentally friendly mechanical control method in reducing rice yield losses due to rat attacks. This study used a factorial randomized block design. The first factor was the fence material, namely galvanized zinc plate and plastic. The second factor was the height of the fence, namely 25, 50, 75, and 100 cm from the ground level. Each treatment combination was repeated five times. Data shows that zinc fences with a height of 75 and 100 cm are able to provide full protection to plants from rat infestations throughout the planting season. However, zinc fences with a height of 25 and 50 cm failed to prevent rat attacks. Plastic fences with a height of 25 and 50 cm failed to prevent rat attacks during the planting season, whereas 75 and 100 cm were able to prevent rat attacks until all the crops outside of the fence had been eaten by rats. The rat managed to get into the fence by biting the plastic fence close to the ground. Thus, the research results show that fences made of zinc plate with a height of at least 75 cm from the ground surface are effective in preventing plant damage caused by rats. To our knowledge, this research is the first to quantify the effectiveness of fences as a control of field rodents.

Keywords: rice field rat, Rattus argentiventer, fence, rice

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Authors: Manjusha Choudhary

Abstract:

Objective: Cassia occidentalis Linn. belongs to Family Caesalpiniaceae is a common weed scattered from the foothills of Himalayas to West Bengal, South India, Burma, and Sri Lanka. It is used widely in folklore medicine in India as laxative, expectorant, analgesic, anti-malarial, hepatoprotective, relaxant, anti-inflammatory and antidiabetic. The present study was carried out to investigate the hypoglycaemic and hypolipidemic activities of ethanolic extract of Cassia occidentalis stem bark. Methods: Stem bark extract of Cassia occidentalis (SBCO) was administered orally at 250 and 500 mg/kg doses to normal and streptozotocin (STZ) induced type-2 diabetic mice. Various parameters like fasting blood glucose (FBG) level, serum cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides (TG), total protein, urea, creatinine, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) levels and physical parameters like change in body weight, food intake, water intake were performed for the evaluation of antidiabetic effects. Results: Both the doses of extract caused a marked decrease in FBG levels in STZ induced type 2 diabetic mice. Administration of SBCO led to the decrease in the blood glucose, food intake, water intake, organ weight, SGOT, SGPT levels with significant value and increased the levels of TG, HDL cholesterol, creatinine, cholesterol, total protein with a significant value (p < 0.05-0.01). The decrease in body weight induced by STZ was restored to normal with a significant value (p < 0.01) at both doses. Conclusion: Present study reveals that SBCO possess potent hypoglycaemic and hypolipidemic activities and supports the folklore use of the stem bark of plant as antidiabetic agent.

Keywords: Cassia occidentalis, diabetes, folklore, herbs, hypoglycemia, streptozotocin

Procedia PDF Downloads 407

Authors: Nandita Ghosh, Shinjini Mitra, Ena Ray Banerjee

Abstract:

Atopic dermatitis (AD) is a chronic disease of the skin, involving itchy, reddish, and scaly lesions. It mainly affects children and has a high prevalence in developing countries. The AD may occur due to environmental or genetic factors. There is no permanent cure for the AD. Currently, all therapeutic strategies involve methods to simply alleviate the symptoms, and include lotions and corticosteroids, which have adverse effects. Use of phytochemicals and natural products has not yet been exploited fully. The particle used in this study is derived from Cyamopsis tetragonoloba, an edible polysaccharide with a galactomannan component. The mannose component mainly increases its specificity towards cellular uptake by mannose receptors, highly expressed by the macrophage. The aim of this study was to determine the therapeutic effect of guar gum nanoparticles (GN) in vitro and in vivo in the AD. To assess the wound healing capacity of the guar gum nanoparticle (GN), we first treated adherent NIH3T3 cells, with a scratch injury, with GN. GN successfully healed the wound caused by the scratch. In the in vivo experiment, Balb/c mice ear were topically treated with oxazolone (oxa) to induce AD and then were topically treated with GN. The ear thickness was increased significantly till day 28 on the treatment of Oxa. The GN application showed a significant decrease in the thickness as assessed on day 28. The total cell count of skin cells showed fold increase when treated with oxa, was again decreased on topical application of GN on the affected skin. The eosinophil count, as assessed by Giemsa staining was also increased when treated with oxa, GN application led to a significant decrease. The IgE level was assessed in the serum samples which showed that GN helped in restoring the alleviated IgE level. The T helper cells and the macrophage population showed increased percentage when treated with oxa, the GN application. This was examined by flow cytometry. The H&E staining of the ear tissue showed epidermal thickness in the oxa treated mice, GN application showed reduced cellular filtration followed by epidermal thickness. Thus our assays showed that GN was successful in alleviating the disease caused by Oxa when administered topically.

Keywords: allergen, inflammation, nanodrug, wound

Procedia PDF Downloads 243