Balb-c Mouse Research Articles

Comparative Macroscopic and Morphometric Analysis of Mandible in Different Mouse (Balb-c; Cd-1; C57bl/6) and Rat Strains (Wistar Albino; Sprague Dawley; Wag/Rij)

Since the morphology of the mandible varies among animal species, it is the subject of many studies. The aim of this study was to investigate the macroanatomical and morphometric aspects of the mandible in three different rat (Spague Dawley, Wistar Albino, Wig/Rij) and mouse strains (Balb-c, C57bl/6, CD-1). In this study, which was conducted using a total of 96 mandibles, 8 females and 8 males from each species, measurements were taken from 6 parameters. Similarities and differences between strains and sexes were revealed by statistical analyses. The mandibles were examined under a stereomicroscope. For the Balb-c strain, the termination of the process of the angularis at the level of the process of the condylaris and the shallowness of the masseteric tubercle; for Wig/Rij rats, the proc. angularis, proc. The termination of the condylaris level, the prominent masseteric tubercle and the localization of the molar teeth were macroscopically revealed as distinctive anatomical structures. Specific measurement parameters were proc. coronoideus height for Balb-c mouse; mandible length and lower M3 crown length for CD-1 mouse, while no specific measurement point was available for C57bl/6 mouse. In rats, proc. coronoideus height was the specific measurement point for Wig/Rij rat; lower M3 crown length was decisive for Sprague Dawley. Mandible length parameter was specific for all rat strains. It was concluded that strain determination could be made by looking at these distinctive parameters.

Development and Characterization of a Novel Microwave Plasma Source for Enhanced Healing in Wound Treatment

Our study explores the potential of a novel microwave plasma source for enhancing wound healing in BALB-C mouse models. Chronic wounds, particularly in diabetic individuals, present significant challenges due to impaired regenerative capacity. Cold Atmospheric Plasma (CAP) has emerged as a promising approach, offering diverse therapeutic benefits. However, its specific efficacy in the context of diabetic wounds remains underexplored. We developed and characterized a microwave plasma source optimized for wound treatment, inducing acute wounds and treating them with CAP in a controlled experimental setup. The treated group exhibited accelerated wound closure compared to controls, suggesting CAP’s potential to enhance the healing process. Our findings underscore CAP’s multifaceted impact on the wound healing cascade, highlighting its ability to promote angiogenesis, modulate inflammatory responses, and exhibit antimicrobial properties. These results position CAP as a promising intervention in acute wound management, paving the way for further exploration of its therapeutic potential in clinical settings.

From genes to phenotype: investigating the link between smad2rs29725537:c>a and high take off in mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación, Gobierno de España. Introduction and Purpose High take off (HTO) is a congenital anomaly with a prevalence of 0.36% that can cause severe ischemia and sudden cardiac death. The C57Bl/6 (B6) mouse strain shows around 60% incidence of HTO, whereas it is absent in the Balb/c (BC) strain. The aim of this study was to identify the genes responsible for the development of HTO. Methods Following an inheritance study, a genetic linkage study was performed in B6C mice using 134 microsatellites (MIT). After selection of Smad2 as a candidate gene, a genotype-phenotype study was performed by PCR-RFLP genotyping of 293 B6C mice. The process of coronariogenesis was examined in E13.5 and E14.5 mouse embryos of B6 (n = 27) and BC (n = 23) strains with specific markers for the vascular plexuses involved in coronariogenesis (PROX1 and ERG 1/2/3) and for SMAD2. Finally, the SMAD2 and pSMAD2 protein expression levels were analyzed by western blot in ventricle, liver, and kidney of B6C mice. Results The incidence of HTO in B6, BC and B6C mice were concordant with an inheritance pattern mediated by a dominant causal allele and dominant modifier in the BC strain that reduces the incidence of HTO. The genetic linkage study revealed the association of a region between markers MIT-49 and 106 on chromosome 18 with the presence of HTO. This region contained a total of 10 genes, of which Smad2 was selected as a candidate gene because of its functions in aortic root formation. Comparative sequence analysis of Smad2 between the B6 and BC mouse strains uncovered an intronic SNP (Smad2rs29725537:C>A or Smad2C>A) that predicts and error in the splicing of intron 10-11. A statistically significant association (p<0.05) between the Smad2C>A allele and the presence of HTO was found. pSMAD2 was detected in embryonic coronary primordia, the subepicardial aortic plexus (SAP) and the primary coronary plexus. Around 60% of B6 examined embryos showed an exacerbated SAP compared to the BC embryos. Additionally, we detected a differential expression of total SMAD2, but not of pSMAD2, in different organs according to the Smad2C>A allele genotype (C/C > A/C > A/A). Conclusions We conclude that: 1) Smad2C>A allelic variant is associated with HTO in mice, suggesting it as a major causal factor. 2) Smad2 altered expression may affect the establishment of the embryonic coronary primordia leading to a mispositioned ostium. 3) the Smad2C>A allele has a systemic effect on total SMAD2 expression without affecting its phosphorylation level, which suggest a compensatory mechanism of expression that maintain the normal activity of pSMAD2 in most tissues.

Inhibiting Mitochondrial RNA Degradosome Complex SUV3 and PNPT1 Increases dsRNA in the Cytoplasm, Triggers Viral Mimicry Response and Sensitizes AML Cells to Immune Mediated Killing

Eukaryotic cells have two separate genomes; nuclear DNA organized in chromosomes, and circular mitochondrial DNA located within mitochondria. Mitochondrial DNA is comprised of a double-stranded circular genome that is 16.6 kB in length, lacks introns, and encodes two rRNAs, 22 t-RNAs and 13 of the 90 proteins in the mitochondrial respiratory chain. To maintain homeostasis, mitochondria possess RNA degradation machinery to regulate mitochondrial RNA turnover. The ATP-dependent helicase, SUV3 (encoded by the gene SUPV3L1) and the exonuclease PNPase (encoded by the gene PNPT1) function in a complex to degrade mitochondrial dsRNA. We identified PNPT1 and SUPV3L1 through an unbiased in-silico screen using bioinformatics platform to identify novel targets in AML. Our analysis revealed a strong correlation between these genes and ontologies of exogenous dsRNA, response to viruses, and RNA catabolic processes. PNPase and SUV3 protein were increased in 7/7 AML patient samples and 10/10 of AML cell lines compared to the normal hematopoietic cells by immunoblotting. Analysis of the TARGET AML dataset revealed that AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall survival and event-free survival respectively. We knocked down or knocked out PNPT1 and SUPV3L1 with shRNA or sgRNA in AML cells, to study the importance of these genes in AML. Genetic knockdown or knockout of PNPT1 or SUPV3L1 decreased the growth and viability of OCI-AML2, TEX, K562, U937, NB4 and 8227 leukemia cells. Moreover, SUPV3L1 & PNPT1 ranked top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (https://depmap.org/portal). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of AML cells. Demonstrating the functional importance of PNPT1 & SUPV3L1 on leukemia-initiating cells in vivo, genetic knockdown of PNPT1 and SUPV3L1 significantly reduced engraftment of TEX cells into the marrow of immune-deficient mice. Finally, primary AML cells with SUPV3L1 knockdown had reduced engraftment into the marrow of immune-deficient mice. Whereas, the knockdown of SUPV3L did not alter the primary engraftment of CD34+ enriched cord blood cells. Mechanistically, knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased cytoplasmic dsRNA in 143B cells, but not Rho(0) 143B cells that lack mitochondrial DNA, demonstrating a mitochondrial source for the increased dsRNA. Increased cytoplasmic dsRNA can mimic viral infection and trigger a type 1 Interferon response. Knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) associated with a type 1 interferon response compared to control. We also observed that PNPT1 and SUPV3L1 knockdown in AML cells sensitized the leukemic cells to T-cell mediated killing upon co-culturing with double negative T cells. In addition, using an immunocompetent Balbc mouse model, we demonstrated endogenous T cells were required to observe the greatest reduction in tumor burden after SUPV3L1 knockdown. In summary, the mitochondrial RNA degradosome complex SUPV3L1 and PNPT1 are overexpressed in AML and are essential for AML cells and stem/progenitors. These enzymes regulate the levels of mitochondrial dsRNA and their inhibition leads to a viral mimicry response and heightened sensitivity to immune-mediated killing.

Repeated radon exposure induced epithelial-mesenchymal transition-like transformation via disruption of p53-dependent mitochondrial function.

As a human carcinogen, radon and its progeny are the second most important risk factor for lung cancer after smoking. The tumor suppressor gene, p53, is reported to play an important role in the maintenance of mitochondrial function. In this work, we investigated the association between p53 and p53-responsive signaling pathways and radon-induced carcinogenesis. After repeated radon exposure, the malignant characteristics, cell cycle arrest, cell apoptotic rate, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) level, mitochondrial DNA (mtDNA) copy number as well as indicative biomarkers involved in mitochondrial energy metabolism were evaluated in BEAS-2B cells or BALB-c mouse lung tissue. Radon exposure induced epithelial-mesenchymal transition (EMT)-like transformation in BEAS-2B cells, as indicated by increased cell proliferation and migration. Additional mitochondrial alterations, including decreased ATP content, increased ROS levels, mtDNA copy numbers, cell apoptosis, and G2/M cell cycle arrest were observed. Radon exposure caused an energy generation shift from aerobic respiration to glycolysis as reflected by increased expression of TIGAR and p53R2 proteins and decreased expression of SCO2 protein in BEAS-2B cells, and increased expression of p53, SCO2 and TIGAR proteins in mouse lung tissue, respectively. The effects of p53 deficiency on the prevention of mitochondrial dysfunction suggested a protective role of p53 in radon-induced malignant-like features in BEAS-2B cells. Repeated radon exposure induced EMT-like transformation in BEAS-2B cells via disruption of mitochondrial function. Activation of p53 and p53-responsive signaling pathways in BEAS-2B cells and BALB-c mice may confer a protective mechanism for radon-induced lung injury.

Abstract 5195: Humanized CD3E,D,G mice: An ideal model for efficacy study of CD3-bispecific antibodies

Abstract CD3-targeting bispecific antibodies (CD3 bsAb) are designed to simultaneously bind to T cells and tumor cell antigens, leading to T-cell activation, proliferation, and tumor cell death. To create a better animal model for studies of CD3-TCBs, we developed the BALBc-hCD3EDG model by gene editing technology, Briefly, a large human genomic fragment containing CD3E,CD3D,CD3G as well as regulatory elements was introduced into the BALBc mouse genome and animals carrying human CD3E,D,G integration were selected to establish the new strain. The BALBc-hCD3EDG mice have a normal immune system and successfully express human CD3E,CD3D,CD3G on the T cell surface. As expected, cytotoxicity of activity T cells by redirecting with CD3-CD20 bsAb was observed in in vitro T cell-mediated killing assays using an A20 cell expressing human CD20. Furthermore, the growth of subcutaneously engrafted CT26 and hCD20-A20 tumors in BALBc-hCD3EDG mice, was strongly inhibited by CD3-TCB antibody treatment. In conclusion, the CD3EDG humanized mice are ideal models for preclinical studies of bispecific antibodies and other immunotherapies. Citation Format: xiaoliu Yang, cunxiang Ju, mingkun zhang, jing zhao. Humanized CD3E,D,G mice: An ideal model for efficacy study of CD3-bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5195.

Biochemical and histopathological investigation of the protective effects of melatonin and vitamin E against the damage caused by acetamiprid in Balb-c mouse testicles at light and electron microscopic level.

The protective effects of melatonin (Mel) and vitamin E (Vit E) against the negative effects of acetamiprid (Acmp) on testicles, reproductive hormones, and oxidative stress parameters were investigated in the present study. A total of 50 Balb-c male mice were used in 7 groups; 6 mice in the control groups (distilled water, corn oil, ethanol), and 8 in other groups (Acmp, Acmp + Mel, Acmp + Vit E, Acmp + Vit E + Mel). After the experiment, which lasted 21days, hematoxylin eosin (H&E), periodic acid Schiff (PAS), and caspase-3 immunohistochemical (IHC) staining was performed on the testicular tissues. Also, the tissues were examined ultrastructurally with the transmission electron microscopy (TEM). In the Acmp group, there were decreased seminiferous tubule diameter and epithelial thickness, epithelial degeneration, decreased spermatozoa in the lumen, decreased PAS-positive staining in the seminiferous epithelial basement membrane, edema in the interstitial area, and hydropic degeneration in Leydig cells. Caspase-3 immunoreactivity was higher than in the other groups. TEM examination showed degeneration in tubule cells, lysosomal accumulation in cells of the spermatogenic line, vacuolizations with myelin figures, and necrosis. Hydropic degeneration, electron-dense lipid vacuoles, and chromatolysis were evident in the Leydig cell cytoplasm. In Sertoli cells, electron-dense lysosomal deposits were noted. In biochemical terms, there were decreased tissue glutathione (GSH) and total antioxidant status (TAS), and increased malondialdehyde (MDA) and total oxidant status (TOS). Plasma luteinizing hormone (LH), follicular stimulating hormone (FSH), and testosterone levels were decreased. In the groups with melatonin, vitamin E, and both were applied together, tissue damage, and apoptotic cell death were reduced at both light microscopic and ultrastructural levels. In biochemical terms, there were decreased oxidative parameters and increased hormonal parameters. It was found that vitamin E was more effective in decreasing oxidative parameters and increasing antioxidative parameters when compared to melatonin, and hormonal parameters increased at a higher level in the Acmp + Vit E group than in all groups. As a result, it was found that exposure to Acmp caused damage to testicular tissue, induced oxidative stress in testicles, and decreased plasma LH, FSH, and testosterone levels, and although vitamin E is more effective than melatonin in preventing this damage, both are effective.

Involvement of Smad2 Allelic Variants in Murine Coronary Artery High Take‐off Development

The abnormal origin of a coronary artery from the ascending aorta, above the sinotubular junction or high take-off (HTO) is a rare congenital anomaly, which is associated with sudden cardiac death in approximately 1.5% of cases. HTO has been reported in 62.8% of mice from the C57Bl/6 (B6) strain, while it is absent in the Balb/c (BC) mouse strain. We have observed that the incidences of HTO in hybrid mice are intermediate between those of the parental animals (F1: 26.32%; F1xF1: 20%; F1xB6: 46.34%; F1xBC: 9.80%). In addition, we found that these incidences fit with a mode of inheritance consisting in the influence of a major causal dominant allele, present in the B6 strain, and a modifier dominant allele in the BC strain that reduces by half the incidence of HTO. In order to identify chromosomal regions including genes responsible for HTO development, we have carried out a genetic linkage study through the generation of a congenic strain, using microsatellite genetic markers. A region of 2,699,738 bp in chromosome 18 between the markers MIT-49 and MIT-106 was found statistically associated with HTO occurrence. This chromosomal region included a total of 9 genes (Smad2, Skor2, Ier3pi, Hdhd2, Katnal2, Pias2, St8sia5, Loxhd1, Rnf165). Among these, Smad2 was selected as a candidate gene, due to its multiple biological functions related to cardiovascular system development, specifically to aortic root formation and coronariogenesis. The comparative analysis of the Smad2 sequence between B6 and BC mouse strains revealed the presence of an intronic SNP (Smad2rs29725537:C>A or Smad2C>A) located in the 5’ splicing zone of intron 10-11. Sequence analysis predicted a possible error in Smad2 10-11 intron splicing caused by the B6 missense variant (Smad2C>A). After genotyping a total of 104 B6xBC F2 to F5 hybrid individuals of the developing congenic strain, by means of PCR-RFLP using Smad2 specific primers, we found a statistically significant (p<0.05) association between the allele Smad2C>A and HTO occurrence. We conclude that HTO is an inherited trait in mice, probably caused by a dominant allele and a dominant modifier allele. Our genotype-phenotype association study points to the Smad2rs29725537:C>A allele as the main causal factor for HTO in mice. Further studies are required to 1) confirm the role of the Smad2C>A SNP in murine HTO development; 2) check whether human HTO development is also influenced by the Smad2C>A allele; and 3) investigate the possible involvement of the Smad2C>A allele in the development of additional traits, particularly those differentially expressed between mice of the B6 and BC strains.

Single Low Dose of Cocaine-Structural Brain Injury Without Metabolic and Behavioral Changes.

Chronic cocaine use has been shown to lead to neurotoxicity in rodents and humans, being associated with high morbidity and mortality rates. However, recreational use, which may lead to addictive behavior, is often neglected. This occurs, in part, due to the belief that exposure to low doses of cocaine comes with no brain damage risk. Cocaine addicts have shown glucose metabolism changes related to dopamine brain activity and reduced volume of striatal gray matter. This work aims to evaluate the morphological brain changes underlying metabolic and locomotor behavioral outcome, in response to a single low dose of cocaine in a pre-clinical study. In this context, a Balb-c mouse model has been chosen, and animals were injected with a single dose of cocaine (0.5 mg/kg). Control animals were injected with saline. A behavioral test, positron emission tomography (PET) imaging, and anatomopathological studies were conducted with this low dose of cocaine, to study functional, metabolic, and morphological brain changes, respectively. Animals exposed to this cocaine dose showed similar open field activity and brain metabolic activity as compared with controls. However, histological analysis showed alterations in the prefrontal cortex and hippocampus of mice exposed to cocaine. For the first time, it has been demonstrated that a single low dose of cocaine, which can cause no locomotor behavioral and brain metabolic changes, can induce structural damage. These brain changes must always be considered regardless of the dosage used. It is essential to alert the population even against the consumption of low doses of cocaine.

Microwave plasma torch for wound treatment

Cold atmospheric pressure plasma (CAP) sources have recently been proven to be an effective therapeutic source regarding wound healing. The most preferred and used plasma devices at this moment are the well-known dielectric barrier discharges (DBD) and free jet devices. In this work, we studied a low temperature plasma torch at atmospheric pressure sustained by a travelling electromagnetic wave excited by surfatron type wave launcher coupled to solid state microwave generator. This plasma source allows variation of discharge conditions: geometric parameters (discharge tube inner diameter and thickness), wave power and gas flow velocity which varies the main plasma parameters (length, gas temperature, concentration of charged particles and reactive species, UV and microwave radiation). Appropriate combination of the parameters lead to the low temperature plasma torch obtaining a gas temperature up to 30–37°C, suitable for in vivo treatment of BALB-C mouse models. The purpose of this research is to study the discharge conditions leading to acceleration of wound healing at short treatment times with relatively low gas flow and microwave power

Elimination of ESAT-6 and CFP-10 from a candidate vaccine against bovine tuberculosis impaired its protection efficacy in the BALBc mouse model.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that mainly affects cattle. Although vaccination is the most effective strategy to control bTB, it may interfere with the diagnosis of the infection. Therefore, ancillary tests to differentiate vaccinated from infected animals (DIVA) are essential in a cattle vaccination scenario. ESAT-6 and CFP-10 are the most promissory DIVA antigens. In this study, we deleted esat6 and cfp10 genes from the M. bovis Δ mce2 live-attenuated vaccine candidate and evaluated its protection level against bTB in BALBc mice. We found that the M. bovis strain mutant in mce2, esat-6 and cfp-10 failed to confer protection against virulent M. bovis challenge in a mouse model of tuberculosis. This result highlights the relevant role of ESAT-6 and CFP-10 in the induction of protective immune response against M. bovis infection and reveals the need of evaluating different strategies to compensate for the lack of these DIVA antigens in new vaccine formulations.

The Effect of Hydroalcoholic Extract of Amaranthus Retroflexus L. Seeds on Cox2 and Her2 Genes on the Induced Colorectal Cancer in an Animal Model of Balbc Mouse

Background: for many centuries, herbs and herbal extracts have been used as a treatment for diseases. Today, many medicines are derived from herbal sources. Herbs for treatment purposes are the source of many modern medicine treatments. Materials and Methods: In this experimental study, the hydroalcoholic extract of Amaranthus Retroflexus L. seeds was prepared, then the required concentrations for the experimental stages were prepared and MTT test was performed on the CT26 and MRC5 cells. Simultaneously, tumor induction was performed in mice. The clone tissue was sampled, RNA was extracted, and finally, the cDNA was implemented. The results of gene expression changes were evaluated by RealTime technique with the t-test and ANOVA. Results: Amaranthus Retroflexus L. seed extract reduced the survival and proliferation of cancer cells. The decrease in cell survival depends on concentration, and this decrease is significant (p

Sauromatum guttatum extract promotes wound healing and tissue regeneration in a burn mouse model via up-regulation of growth factors

Contexts: Sauromatum guttatum (Wall.) Schott (Araceae) has been traditionally used for the treatment of wounds.Objectives: This study evaluates the healing and tissue regeneration potential of S. guttatum extract in burn wounds.Materials and methods: S. guttatum extract was analysed using various chemical tests, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Moreover, the extract was tested against burn associated bacteria and minimum inhibitory concentration (MIC) was also calculated. Wound healing and tissue regeneration potential was assessed using a thermally induced burn BALBc mouse model. S. guttatum extract (2% w/w) prepared in petroleum jelly, vehicle and positive control [silver sulfadiazine (SD)] groups was applied three times a day. The treatment was continued for 15 d and wound closure was measured and photographed on day 5, 10 and 15. The burnt tissues excised from wounds were subjected to histological and comparative gene expression analysis.Results: The results of the chemical tests indicated the presence of alkaloids, saponins, phenols, phytosterols, tannins, and flavonoids, while TLC and HPLC analysis indicated the presence of various compounds. The extract showed excellent activity against the tested pathogens. The lowest MIC (125 µg/mL) was observed against Staphylococcus aureus. A considerable decrease in wound area (72%) was observed in extract-treated group. Histological examination of extract-treated group showed good signs of wound healing with complete re-epithelialization and better tissue regeneration. Comparative gene expression analysis revealed the up-regulation of wound healing related PDGF, EGF and FGF genes.Conclusions: S. guttatum extract may be used to isolate bioactive constituents for the treatment of burn wounds.

Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza

BackgroundInfectious coryza (IC) is an acute respiratory disease of growing chickens and layers caused by Avibacterium paragallinarum. The development of tools that allow rapid pathogen detection is necessary in order to avoid disease dissemination and economic losses in poultry. An Av. paragallinarum-specific Ma-4 epitope of the TonB-dependent transporter (TBDT) was selected using bioinformatic tools in order to immunize a BalbC mouse and to produce monoclonal antibodies to be used in a lateral flow test (LFT) developed for Av. paragallinarum detection in chicken nasal mucus samples.ResultsThe 1G7G8 monoclonal antibody was able to detect TBDT in Av. paragallinarum cultures (serogroups: A, B and C) by Western blot and indirect ELISA assay. Consequently, we developed a self-pairing prototype LFT. The limit of detection of the prototype LFT using Av. paragallinarum cultures was 1 × 104 colony-forming units (CFU)/mL. Thirty-five nasal mucus samples from chickens suspected of having infectious coryza were evaluated for the LFT detection capacity and compared with bacterial isolation (B.I) and polymerase chain reaction (PCR). Comparative indicators such as sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive values (NPV) and the kappa index (K) were obtained. The values were 100.0% Se, 50% Sp, 65.4% PPV, 100% NPV, and 0.49 K and 83.9% Se, 100% Sp, 100% PPV, 44.4% NPV, and 0.54 K for the comparison of the LFT with B.I and PCR, respectively. Additionally, the LFT allowed the detection of Av. paragallinarum from coinfection cases of Av. paragallinarum with Gallibacterium anatis.ConclusionsThe results indicate that the self-pairing prototype LFT is suitable for the detection of TBDT in Av. paragallinarum cultures as well as in field samples such as nasal mucus from Av. paragallinarum-infected chickens. Therefore, this prototype LFT could be considered a rapid and promising tool to be used in farm conditions for Av. paragallinarum diagnosis.

P-24 - Benznidazole induce oxidative stress, DNA damage and apoptosis in Balb-c mice-bearing Ehrlich ascites tumor

Benznidazole (BZN), through its nucleophilic metabolites enable ROS generation and oxidative stress induction triggers death of Chagas disease parasites. In this sense, increase of intracellular ROS in cancer cells arise as smart therapeutic target based in deficiency on redox capability of cancer cells. The aim of this study was to evaluate ROS induction by BZN and its effect against Ehrlich ascites carcinoma in Balb-c mouse. Three treated groups: Dimethyl sulfoxide 1% (Negative Control - NC); BZN (10 mg/kg/day) and Methotrexate (2.5 mg/kg/day; Positive Control). After 9 days of treatment, ascitic liquid samples were collected to evaluate intracellular ROS, Calcium Uptake, DNA damage, cell cycle and types of cell death. Results showed increased 1.7-fold ROS level in BZN treatment comparatively to NC, Calcium Uptake increased 1.6-fold comparatively to NC and MTX. Also increased 7.6-fold DNA fragmentation compared to NC and 1.4 to MTX. The BZN showed important arrest cell cycle in G1 1.3-fold when compared NC and 2.7-fold to MTX. Apoptosis increase 2.9-fold in BNZ when compared NC and 1.3-fold to MTX. The data obtained suggest that the induction of oxidative stress by BZN could lead to oxidation and fragmentation DNA with consequent cell cycle arrest and triggers apoptosis.

P-25 - Docking studies predict GPx inhibitory mechanisms behind antitumor activity of supercritical carbon dioxide black pepper bioactive fractions rich in piperine

Supercritical carbon dioxide extracts rich in piperine (SFE) (300 bar; 40 °C) solubilize black pepper (Piper nigrum) bioactive phytochemicals with antitumor effect. The aim of this study was to evaluate pro-oxidant status induced by SFE and its effect against Ehrlich ascites carcinoma in Balb-c mouse. Balb/c mice bearing-Ehrlich ascites carcinoma cells (EAC) (male; 22±2 g) received intraperitoneally SFE (100 mg/kg/d; 9 days) or saline. EAC inhibition/survival, antioxidant enzymes activity and GSH content were evaluated. USCF Chimera 1.11.2 /Autodock Vina 1.1.2 default parameters prepared/docked Glutathione Peroxidase (PDB 2OBI; 1.55 A) and piperine (PubChem CID 638024). SFE inhibit EAC (60%), increasing mice survival (46%) and enhancing activity of superoxide dismutase (4 fold), catalase (56 fold) and glutathione reductase (88%) meanwhile decreased glutatione peroxidase/GPx activity (75%) with GSH depletion (84%). Piperine-GPx poses showed H bonds and hydrophobic interactions near catalytic residues (Cys/Gln/Trp). Therefore, antitumor activity of SFE enhanced EAC pro-oxidative status related with antioxidant enzymes response insufficient to overcome induced oxidative stress. And protein docking suggest the GPx-Piperine interactions could contributed to inhibition of GPx.

Preclinical tools for the evaluation of tuberculosis treatment regimens for children.

Tuberculosis (TB) treatment regimens have been extrapolated from adults to children. However, pediatric disease merits different treatment strategies to avoid under- or over-treatment. While animal models have been pivotal in identifying effective regimens for adult disease, pediatric TB is heterogeneous and cannot be represented by a single preclinical model. Infants and young children most commonly have disseminated disease or tuberculous meningitis (TBM), school-aged children have paucibacillary disease, and adolescents have adult-like cavitary lung disease. Models simulating these forms of pediatric TB have been developed, but their utility in assessing treatment regimens is in the early stages. Disseminated, intracellular disease can be partly reproduced by an in vitro pharmacodynamic system, TBM by a pediatric rabbit model of TBM, paucibacillary TB by the balbC mouse model, and cavitary disease by a rabbit model and a C3HeB/FeJ mouse model of pulmonary TB. Although there is no one-size-fits-all preclinical 'pediatric TB model', these models can be employed to study drug distribution to the sites of disease and, coupled with translational modeling, used to help select and optimize regimens for testing in children. Use of these models may accelerate the development of regimens for rare or hard-to-treat TB, namely drug-resistant TB and TBM.

A novel in vivo model of puncture‐induced iris neovascularization

PurposeTo assess iris neovascularization by uveal puncture of the mouse eye and determine the role of angiogenic factors during iris neovascularization.MethodsUveal punctures were performed on BalbC mouse eyes to induce iris angiogenesis. VEGF‐blockage was used as an anti‐angiogenic treatment, while normoxia‐ and hypoxia‐conditioned media from retinal pigment epithelium (RPE) cells was used as an angiogenic‐inducer in this model. Iris vasculature was determined in vivo by noninvasive methods. Iris blood vessels were stained for platelet endothelial cell adhesion molecule‐1 and vascular sprouts were counted as markers of angiogenesis. Expression of angiogenic and inflammatory factors in the puncture‐induced model were determined by qPCR and western blot.ResultsPunctures led to increased neovascularization and sprouting of the iris. qPCR and protein analysis showed an increase of angiogenic factors, particularly in the plasminogen‐activating receptor and inflammatory systems. VEGF‐blockage partly reduced iris neovascularization, and treatment with hypoxia‐conditioned RPE medium led to a statistically significant increase in iris neovascularization.ConclusionsThis study presents the first evidence of a puncture‐induced iris angiogenesis model in the mouse. In a broader context, this novel in vivo model of neovascularization has the potential for noninvasive evaluation of angiogenesis modulating substances.

A novel in vivo model of puncture-induced iris neovascularization.

PurposeTo assess iris neovascularization by uveal puncture of the mouse eye and determine the role of angiogenic factors during iris neovascularization.MethodsUveal punctures were performed on BalbC mouse eyes to induce iris angiogenesis. VEGF-blockage was used as an anti-angiogenic treatment, while normoxia- and hypoxia-conditioned media from retinal pigment epithelium (RPE) cells was used as an angiogenic-inducer in this model. Iris vasculature was determined in vivo by noninvasive methods. Iris blood vessels were stained for platelet endothelial cell adhesion molecule-1 and vascular sprouts were counted as markers of angiogenesis. Expression of angiogenic and inflammatory factors in the puncture-induced model were determined by qPCR and western blot.ResultsPunctures led to increased neovascularization and sprouting of the iris. qPCR and protein analysis showed an increase of angiogenic factors, particularly in the plasminogen-activating receptor and inflammatory systems. VEGF-blockage partly reduced iris neovascularization, and treatment with hypoxia-conditioned RPE medium led to a statistically significant increase in iris neovascularization.ConclusionsThis study presents the first evidence of a puncture-induced iris angiogenesis model in the mouse. In a broader context, this novel in vivo model of neovascularization has the potential for noninvasive evaluation of angiogenesis modulating substances.

Lipopolysaccharide‐Induced Activation of Bone Marrow‐Derived Macrophages Disrupts Brain Microvascular Endothelial Cell Function in a Mouse Model of Psychological Resilience

Psychological resilience, as measured by the ability of an individual to overcome the development of psychological disorders following exposure to stressful or traumatic events, varies significantly from individual to individual. Inflammation, and the breakdown of the blood‐brain barrier (BBB) are thought to be key factors in the development and progression of psychological disorders such as depression, schizophrenia, and post‐traumatic stress disorder. The activation of macrophages is associated with the disruption of barrier function of the BBB, and thus may play an important role in determining resilience to stress. It is unknown whether the macrophage phenotype and/or BBB phenotype of resilient individuals is significantly different from that of non‐resilient individuals, or whether these phenotypic differences and their interaction contribute to the degree of resilience in individuals. In this study, we tested the hypothesis that resilient individuals possess macrophage and blood brain barrier phenotypes that are more resistant to the activating effects of lipopolysaccharide (LPS), than non‐resilient individuals. We used Electric Cell‐substrate Impedance Signaling (ECIS) technology (Applied BioPhysics, Troy, NY) to measure transendothelial electrical resistance (TEER) of cultured brain microvascular endothelial cells of the resilient C57BL6 mouse (BMECC57BL6), and the non‐resilient BalbC mouse (BMECBalbC). BMECs were plated at a density of 4.8 × 104 cells/cm2, and allowed to establish a monolayer for 48 hours. BMECs were then treated with LPS derived from E. coli (0, 10, or 100 ng/ml). After 24 hours, bone marrow‐derived macrophages of C57BL6 mice (BMM C57BL6) or BalbC mice (BMM BalbC) (4.0 × 104 cells/cm2) were added to the BMEC monolayer. TEER was continuously recorded for an additional 72 hours. Barrier function (Rb), membrane capacitance (Cm), and constraint on current flow beneath the cells (α) were derived via ECIS mathematical modeling. At baseline, BMECC57BL6 had significantly lower Rb (1.90±0.26 versus 2.81±0.26 Ωcm2, p = 0.03, n = 7) and α (7.01±0.14 versus 8.01±0.16 Ω0.5cm, p = 0.0006, n =7), and significantly higher Cm (1.42±0.03 versus 1.22±0.02 μF/cm2, p = 0.0003, n = 7) when compared to BMECBalbC. LPS treatment alone or BMM alone did not have significantly differential effects on any of these parameters between BMECC57BL6 and BMECBalbC; however, in the presence of 100 ng/ml LPS, only α value of BMECBalbC significantly declined over the 6 hours after addition of BMM C57BL6 (from 7.50±0.211 to 6.71±0.221 Ω0.5cm; one‐way ANOVA, p = 0.02, n = 4), or BMM BalbC (from 7.54±0.17 to 6.16±0.17 Ω0.5cm; one‐way ANOVA, p &gt; 0.0001, n = 4). These findings indicate that variability in psychological resilience among individuals may be associated with functional differences in blood brain barrier and macrophages.Support or Funding InformationThe views expressed in this presentation are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of Defense, or the U.S. government.