Organs Of Mice Research Articles

Preparation and pharmacological evaluation of A3B-type tetraphenyltetrabenzoporphyrins for photodynamic anti-tumor therapy

A series of A3B-type meso-tetraphenyltetrabenzoporphyrin photosensitizers were designed, synthesized and evaluated. All new compounds exhibited an intense Soret band at ∼ 470 nm and three Q bands at ∼ 595, ∼ 648, and ∼ 701 nm. They all had the ability to produce singlet oxygen upon photoexcitation, and photodynamic activities towards human esophageal carcinoma (Eca-109) cancer cells in vitro. Among them, 5 displayed the excellent cytotoxic effect on Eca-109 cells with the cell viability of ∼30 % at the concentration of 0.5 μM under a light dose of 12 J/cm2. 5 also could induce cell apoptosis after laser irradiation, and display superior photodynamic activity towards Eca-109 cells in vivo. Notably, inflammation and nuclear shrinkage of tumors were observed in the 5-PDT group, while no obvious changes were presented in major organs (heart, kidney liver, lung, and spleen) of mice, and the weight of mice were increased steadily during the treatment period. Hence, compound 5 possesses high photodynamic efficacy and good biosafety, and deserves to be considered as a potential photodynamic drug candidate for cancers.

Bioreversible Anionic Cloaking Enables Intracellular Protein Delivery with Ionizable Lipid Nanoparticles.

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic "cloak" to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic β-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.

A xanthan gum and carbomer-codispersed divalent manganese ion-loaded tannic acid nanoparticle adjuvanted inactivated pseudorabies virus vaccine induces balanced humoral and cellular immune responses

Adjuvants including aluminum adjuvant (Alum) and oil-water emulsion have been widely used in inactivated pseudorabies virus (PRV) vaccines to improve their performance, however, they are not sufficient to protect from PRV infection because of the weak immune response and poor Th1-type immune response. Divalent manganese ion (Mn2+) has been reported to increase the cellular immune response significantly. In this work, a xanthan gum and carbomer-dispersed Mn2+-loaded tannic acid-polyethylene glycol (TPMnXC) nanoparticle colloid is developed and used as an adjuvant to improve the performance of the inactivated PRV vaccine. The good in vitro and in vivo biocompatibility of the developed TPMnXC colloid has been confirmed by the cell viability assay, erythrocyte hemolysis, blood routine analysis, and histological analysis of mouse organs and injection site. The TPMnXC-adjuvanted inactivated PRV vaccine (TPMnXC@PRV) significantly promotes higher and more balanced immune responses indicating with an increased specific total IgG antibody and IgG2a/IgG1 ratio, efficient splenocytes proliferation, and elevated Th1- and Th2-type cytokine secretion than those of control groups. Wild PRV challenge experiment is performed using mice as a model animal, achieving a protection rate of up to 86.67 %, which is much higher than those observed from the commercial Alum. This work not only demonstrates the high potentiality of TPMnXC in practical applications but also provides a new way to develop the Mn2+-loaded nanoadjuvant for veterinary vaccines.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

BackgroundProgrammed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.MethodsWe designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.ResultsBinding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.ConclusionsUsing directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.Graphical

An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Investigating the Impact of Circulating miRNAs on Cardiovascular System Remodeling During Pregnancy

During pregnancy, women undergo significant remodeling of their cardiovascular system, a complex process involving various physiological adaptations to ensure an adequate blood supply to the developing fetus. An intriguing area of research focuses on the role of circulating microRNAs, particularly the primate-specific chromosome 19 miRNA cluster (C19MC) and the rodent-specific chromosome 2 miRNA cluster (C2MC), both of which exhibit high expression levels in the placenta. Extracellular vesicles, such as exosomes, play a crucial role in intercellular communication and may carry microRNAs that contribute to the physiological changes observed during pregnancy. However, the influence of circulating C19MC and C2MC microRNAs on maternal cardiovascular system remodeling remains unexplored. We hypothesized that the increased levels of circulating C2MC or C19MC microRNAs during pregnancy might play a role in the maternal cardiovascular system adaptations. To investigate this hypothesis, we conducted a study to determine whether circulating exosomes during pregnancy lead to elevated expression levels of C19MC or C2MC microRNAs in various organs, including the heart, kidney, liver, spleen, lungs, and brain. In our investigation, we utilized three distinct mouse models: wild-type (WT) mice expressing only C2MC, C2MC knock-out (KO) mice, and a novel humanized mouse model, which combined C2MC KO with either a single or double copy of the human C19MC. We extracted total RNA from the organs of these mice and performed cDNA synthesis. Subsequently, we measured the expression levels of two representative microRNAs, miR-517a and miR-466a, corresponding to C19MC and C2MC, respectively, in the organs of pregnant and non-pregnant mice using TaqMan RT-qPCR. Our results indicated no significant differences in the expression levels of miR-517a and miR-466a when normalized to the housekeeping gene snoRNA-202 across different organs in pregnant and non-pregnant mice. To comprehensively assess the expression patterns of all microRNAs within the C19MC and C2MC clusters in various organs of pregnant and non-pregnant mice, further investigation with a larger sample size and small RNA-Seq analysis is warranted. This approach will enable us to explore a broader spectrum of microRNAs and potentially identify specific candidates that may play a crucial role in the essential physiological process of maternal cardiovascular system remodeling. This study is supported by USF Cardiovascular Summer Undergraduate Research Fellowship (SURF) sponsored by the American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Long-term toxicity evaluation of aristolochic acid-IIIa in mice

Aristolochic acid (AA)-IIIa is an AA analog present in Aristolochiaceae plants. To evaluate the chronic toxicity of AA-IIIa, mice were intragastrically administered with media control, 1 mg/kg AA-IIIa, and 10 mg/kg AA-IIIa, and designated as the control (CTL), AA-IIIa low dose (AA-IIIa-L), and AA-IIIa high dose (AA-IIIa-H) groups, respectively. AA-IIIa was administered three times a week, every other day, for 24 weeks (24-week time point). Thereafter, some mice were sacrificed immediately, while others were sacrificed 29 or 50 weeks after AA-IIIa withdrawal (53- or 74-week time point). Serum and organs were collected for biochemical and pathological analyses, respectively. Whole-genome sequencing was performed on the kidney, liver, and stomach tissues of AA-IIIa-treated mice for single-nucleotide polymorphism (SNP) detection. AA-IIIa-H mice died at 66 weeks, and the remaining mice showed moribund conditions at the 69 weeks. AA-IIIa induced minor kidney tubule injury, fibroblast hyperplasia, and forestomach carcinoma in mice. Bladder, intestine, liver, heart, spleen, lung, and testis tissues were not pathologically altered by AA-IIIa. In addition, AA-IIIa increased the C:G > A:T mutation in the kidney; however, no SNP mutation changes were observed in the liver and forestomach tissues of AA-IIIa-H mice at the 24-week time point compared with control mice. Therefore, we suspect that AA-IIIa is potentially mutagenic for mice after overdose and long-term administration. On the other hand, the forestomach is a unique organ in mice, but it does not exist in humans; thus, we hypothesize that the stomach toxicity induced by AA-IIIa is not a suitable reference for toxicological evaluation in humans. We recommend that Aristolochiaceae plants containing AA-IIIa should be properly supervised, and overdosing and long-term administration of drugs containing AA-IIIa should be avoided.

Deciphering the potential of a novel cytostatic autophagy modulator for the treatment of brain cancer along with reduction in brain inflammation

Background and Purpose: Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, with significant failure of current treatment options, with the five-year survival rate of GBM being less than 7%. Temozolomide (TMZ), the standard chemotherapy, leads to severe toxicity, whereas radiation therapy leads to necrosis. Moreover, GBM leads to severe neuroinflammation, leading to severe fatalities and, thus, poor quality of life in patients. Therefore, owing to the pressing need for safer treatment alternatives, we repurposed a novel FDA-approved acetamide, BRZ, which not only exhibited a significant curbing of GBM progression but also reduced neuroinflammation. Thus, our study provides a treatment option that can also lead to better quality of life in GBM patients. Methods: Sulfarhodamine B cytotoxicity assay was performed on human (SF268, SF295, SF188) and murine (CT2A-LUC) GBM cell lines for both BRZ and TMZ. In the quest for figuring out the mode of cell-growth suppression, we performed annexin and acridine orange flow cytometry assays along with b-galactosidase cellular senescence staining assay. Cellular staining was also confirmed using confocal microscopy. Oncogenic modulation due to BRZ was studied by performing western blotting. In order to study the effect of BRZ on neuroinflammation, we examined the modulation of different neuroinflammatory genes on LPS-stimulated human and murine microglia using RT-qPCR and western blotting. We corroborated our in vitro findings by investigating the tumor progression in intracranially-injected GBM tumors in C57/BL6 mice. Mice weight and organ weights were recorded for both vehicle and BRZ-treated mice to assess toxicity profiles. Results: BRZ exhibited ~300 times more cytotoxicity in GBM cells as compared to TMZ, making it a more potent treatment option. BRZ lead to an increase in acridine orange in a concentration-dependent manner, hinting towards autophagic cell death. b-galactosidase staining assay and confocal microscopy exhibited a dose-dependent increase in b-galactosidase, showcasing a probable cytostatic effect. Expression of autophagic markers such as Atg5, Atg101, LC3A/B, and senescence markers b-galactosidase, p16, and p21 were increased due to BRZ confirming a cytostatic autophagic modulation in GBM cells. Immunoblotting revealed oncogenic modulation due to BRZ wherein Notch-1, Gli-1, Akt, and p-STAT3 were significantly downregulated, as observed in western blotting. Neuroinflammatory genes such as CXCL10, IL6, and ISG15 were downregulated in LPS-stimulated microglia using BRZ in a concentration-dependent manner, hinting towards the potential of BRZ in brain inflammation. BRZ significantly reduced the progression of orthotopic GBM tumors in C57/BL6 mice. There was no significant difference observed in body and organ weights in the vehicle and BRZ-treated mice, hinting toward the safety profile of BRZ. Conclusions and future direction: Our study provides a novel and safer treatment option for GBM in the form of a cytostatic autophagy modulator, BRZ, which also reduces neuroinflammation with the potential to increase patient’s quality of life. In the future, we aim to perform ex vivo analyses of mice brain tumors and perform the neuroinflammation study in GBM mice. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Toxicity of Sida rhombifolia L. 96% ethanol extract based on LD50 and macropathological examination of mice's organs

The Indonesian people empirically use Sidaguri (Sida rhombifolia L.) in medicine, such as anti-hyperuricemia. Herbal-based treatment is currently much preferred. Drug metabolism, in general, mainly occurs in the liver, so the possibility of damage to this organ is high. This study aims to determine the acute toxicity category of 96% ethanol extract of sidaguri based on the LD50 value, changes in body weight, changes in organ weight, and macro pathology in the organs of male DDY mice in vivo. The method used in this research was experimental with a fixed dose design following the Indonesian Food and Drug Authority regulations regarding guidelines for in vivo non-clinical toxicity tests. The test group was divided into six dose groups (standard, 50, 300, 2000, 5000, and 15000 mg/kg Body of weight). Observations were made for 24 hours. Observations were continued for 14 days on death parameters, toxicity symptoms, body weight, and relative organ weights using five main organs (heart, lungs, liver, spleen, and kidneys, and pathological examination). The acute toxicity test results showed no death in all dose treatment groups. Macropathological analysis did not show abnormalities in organs in all groups of mice. LD50 value is more than 15000 mg/kg. Sidaguri 96% ethanol extract is safe and Practically non-toxic.

Characterization of Escherichia coli pathogenicity and drug resistance in yolk peritonitis

Yolk Peritonitis can lead to a rapid decline in egg production, which seriously affects the health of laying hens and the profitability of chicken farms. Escherichia coli (E. coli) is the most common cause of yolk peritonitis in laying hens. In this study, bacterial samples were collected from the ovaries and fallopian tubes of laying hens with suspected yolk peritonitis from a laying farm in Jiangsu Province, and their pathogenicity and drug resistance were investigated. Initially, morphological and biochemical detection methods were employed to isolate and identify the pathogenic bacteria. The results showed that a total of 16 strains of E. coli were isolated from laying hens with yolk peritonitis. Subsequently, the drug resistance and pathogenicity of a randomly selected E. coli strain were analyzed and predicted by genome sequencing technology, and the drug resistance of E. coli was verified by drug sensitivity test and PCR. Finally, the virulence was verified by infection experiment in mice. The study revealed that the egg-yolk peritonitis in laying hens was caused by E. coli infection, and the genome sequencing analysis revealed that the bacteria had multidrug resistance and high virulence. The drug susceptibility testing indicates that E. coli exhibited resistance to aminoglycosides, β-lactam, macrolides, fluoroquinolones, and sulfonamides. In this study, resistance genes including KdpE, aadA5, APH(3 ")-ID, APH(6)-ID, and TEM-1 were identified, and their expression levels varied across different stages of bacterial growth. The results of virulence analysis indicated a mortality rate of 50% in mice infected with E. coli at a concentration of 2.985 × 107 CFU/mL. E. coli infection resulted in damage to various tissues and organs in mice, with the intestinal tissue structure being the most severely affected. This study provides a reference for the study of drug resistance mechanisms in E. coli and provides valuable insights into the selection of drugs for the treatment of vitelline peritonitis.

MALDI Imaging Mass Spectrometry Visualizes the Distribution of Antidepressant Duloxetine and Its Major Metabolites in Mouse Brain, Liver, Kidney, and Spleen Tissues.

Imaging mass spectrometry (IMS) is a powerful tool for mapping the spatial distribution of unlabeled drugs and metabolites that may find application in assessing drug delivery, explaining drug efficacy, and identifying potential toxicity. This study focuses on determining the spatial distribution of the antidepressant duloxetine, which is widely prescribed despite common adverse effects (liver injury, constant headaches) whose mechanisms are not fully understood. We used high-resolution IMS with matrix-assisted laser desorption/ionization to examine the distribution of duloxetine and its major metabolites in four mouse organs where it may contribute to efficacy or toxicity: brain, liver, kidney, and spleen. In none of these tissues is duloxetine or its metabolites homogeneously distributed, which has implications for both efficacy and toxicity. We found duloxetine to be similarly distributed in spleen red pulp and white pulp but differentially distributed in different anatomic regions of the liver, kidney, and brain, with dose-dependent patterns. Comparison with hematoxylin and eosin staining of tissue sections reveals that the ion images of endogenous lipids help delineate anatomic regions in the brain and kidney, while heme ion images assist in differentiating regions within the spleen. These endogenous metabolites may serve as a valuable resource for examining the spatial distribution of other drugs in tissues when staining images are not available. These findings may facilitate future mechanistic studies of the therapeutic and adverse effects of duloxetine. In the current work, we did not perform absolute quantification of duloxetine, which will be reported in due course. SIGNIFICANCE STATEMENT: The study utilized imaging mass spectrometry to examine the spatial distribution of duloxetine and its primary metabolites in mouse brain, liver, kidney, and spleen. These results may pave the way for future investigations into the mechanisms behind duloxetine's therapeutic and adverse effects. Furthermore, the mass spectrometry images of specific endogenous metabolites such as heme could be valuable in analyzing the spatial distribution of other drugs within tissues in scenarios where histological staining images are unavailable.

Tissue clearing method in visualization of cancer progression and metastasis.

Since various imaging modalities have been developed, cancer metastasis can be detected from an early stage. However, limitations still exist, especially in terms of spatial resolution. Tissue-clearing technology has emerged as a new imaging modality in cancer research, which has been developed and utilized for a long time mainly in neuroscience field. This method enables us to detect cancer metastatic foci with single-cell resolution at whole mouse body/organ level. On top of that, 3D images of cancer metastasis of whole mouse organs make it easy to understand their characteristics. Recently, further applications of tissue clearing methods were reported in combination with reporter systems, labeling, and machine learning. In this review, we would like to provide an overview of this technique and current applications in cancer research and discuss their potentials and limitations.

Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 μg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 μg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

Peripheral T-cell responses of EphB2- and EphB3-deficient mice in a model of collagen-induced arthritis

Both EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3−/− mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2−/− T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3−/− lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions.

Effects of endogenous DHA milk and exogenous DHA milk on oxidative stress and cognition in SAMP8 mice

In this study, Senescence Accelerated Mice (SAMP8) were supplemented with exogenous DHA milk, endogenous DHA milk, normal milk, or 0.9 % saline solution. Enzyme-linked immunosorbent assay (ELISA), gas chromatography (GC), ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI MS/MS), and Morris water maze were used to characterize the effects of diet on oxidative stress and cognition in SAMP8 mice. Supplementation endogenous DHA milk or exogenous DHA milk can enhance the antioxidant capacity of mice organs. Endogenous DHA milk increased the superoxide dismutase (SOD) activity of mice brain and serum than normal milk and 0.9 % saline solution (P ≤ 0.05), as well as increased SOD activity of mice liver and glutathione peroxidase (GSH-Px) activity of mice brain than normal milk (P ≤ 0.05). Exogenous DHA milk increased SOD activity of mice brain than normal milk and 0.9 % saline solution, as well as increased SOD activity of mice serum than 0.9 % saline solution (P ≤ 0.05). Several polar lipid relative content, such as 18:0/18:2 PS, 17:0 Ceramide, and 20:4 LPC in mice brain was affected by dietary supplementation with DHA-containing milk. Lipid oxidation metabolites in mice brain were not affected by DHA-containing milk. Endogenous DHA milk increased the number of platform location crossing times of mice in the Morris water maze test, compared with Exogenous DHA milk, normal milk, and 0.9 % saline solution (P ≤ 0.05).

Abstract 125: Development of humanized VEGFA models to assess preclinical efficacy of novel therapeutics in vivo

Abstract VEGFA is an essential growth factor required for angiogenesis in the context of normal wound healing as well as malignant tissue growth, ultimately facilitating the delivery of oxygen and nutrients to fuel cellular survival, expansion, and repair. VEGFA signaling has also been reported to exert effects on cancer cells and immune cells directly, with evidence suggesting that the pathway can both support tumor growth and inhibit immune surveillance. Thus, targeting VEGFA has been intensely explored as a therapeutic strategy for several cancer indications. Here, we generated a novel humanized VEGFA model (B-hVEGFA mice) to explore the ability of novel VEGFA-targeting therapeutics to curb cancer cell growth and angiogenesis in vivo. This was achieved by replacing the murine Vegfa gene with the corresponding human VEGFA coding region. We confirmed exclusive expression of human VEGF in lung homogenates of the humanized model at levels comparable to murine VEGF quantified in wild-type C57BL/6 mice. Immunohistochemical analysis at 8 weeks of age demonstrates no significant difference in the CD31+ area in several organs of the humanized mice when compared to wild-type C57BL/6 mice, including kidney, heart, liver, and eye. To assess the functional utility of this model in evaluating VEGFA modulators, tumor studies were conducted: murine colon cancer MC38 cells that were also engineered to express VEGFA in a similar manner (B-hVEGFA MC38 cells) were subcutaneously implanted into homozygous B-hVEGFA mice. Treatment with bevacizumab analog essentially abolished tumor growth in the humanized mice, while control tumors continued to expand over the 3 week study. To further evaluate the role of novel combination therapies or bispecific antibodies targeting VEGFA and PD-1, we also generated a triple humanized model expressing human VEGF, PD-1, and PD-L1 (B-hPD-1/hPD-L1/hVEGFA mice) by crossing B-hVEGFA mice with our B-hPD-1/hPD-L1 mice model, which expresses the extracellular domains of human PD-1 and PD-L1. Flow cytometric analysis of this model is currently underway to validate the model for further efficacy testing. Together, these humanized VEGF models provide robust in vivo platforms to evaluate novel VEGFA therapeutic candidates. Citation Format: Shujin Zhang, Chonghui Liu, Jin Guo, Zhiyuan Shen, James Jin, Bahetiyaer Huwatibieke, Yang Shen. Development of humanized VEGFA models to assess preclinical efficacy of novel therapeutics in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 125.

Abstract 2336: A prototype for a machine-learning assisted image analysis tool for preclinical in vivo studies

Abstract The purpose of this study was to build a software tool to test the feasibility of machine-learning (ML) assisted analysis of in vivo 3D images in oncology research studies. Conventionally, in vivo image segmentations are performed by hand, and are a bottleneck for data analysis, taking time and introducing both inter and intra-user variability. Recently, advances in ML have shown promise in accelerating similar clinical image analysis tasks. Our objective was to work toward making this technology accessible to preclinical cancer researchers. 3D image data across multiple mouse models, organs, modalities, and imaging systems were used. ML segmentation models were trained with user-defined masks on images of mouse bladders (N=134), kidneys (N=131), livers (N=65), and lungs (N=26) from either ultrasound or micro-computed tomography (uCT) scanners. Models were developed using PyTorch/MONAI, hosted in an AWS cloud environment, and served to users via a custom extension for 3D Slicer (www.slicer.org). Performance was assessed as the difference between the human vs. ML organ volumes on a test set unseen by the models during training. Lastly, the functionality of on-the-fly segmentation improvement tools through iterative user feedback was assessed. Results demonstrated that generating segmentations with the prototype ML software took 2-6 seconds per image running on a modern GPU (NVIDIA RTX 3090), compared to 3-7 minutes if performed manually by an expert user (>30x improvement). The Dice scores between the ML generated and human segmentations ranged from 0.81 (liver ultrasound) to 0.84 (bladder ultrasound) but could be improved using ML-based on-the-fly mask improvement tools. Using data from this pilot study, we estimate that the threshold to achieve 90% accuracy on first-pass segmentations is between 200-300 training images, depending on the variance observed in the image data. In summary, the prototype software was able to decrease the manual analysis time across a variety of preclinical in vivo image modalities, which is broadly relevant to preclinical oncology research. The next steps for this project will be to extend the platform to additional orthotopic tumor models (e.g. pancreatic, liver, prostate, etc.) and deploy to the field to enable access to this toolset by preclinical imaging scientists across the world. Citation Format: Adam M. Aji, Thomas M. Kierski, Juan D. Rojas, Hannah Morilak, Olivia J. Kelada, Kathryn H. Gessner, Andrew S. Gdowski, Lucia Kim, William Y. Kim, Ryan C. Gessner, Tomek J. Czernuszewicz. A prototype for a machine-learning assisted image analysis tool for preclinical in vivo studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2336.

A study of the relationship of the dynamics of development and characteristics of chimerism with manifestations of graft-vs.-host disease in the organs of mice after allogeneic transplantation of whole bone marrow

In the сlinical practice, allogeneiс bone marrow transplantation (BMT) is often cause of the graft-versus-host disease (GvHD). GvHD is explained by the fact that T-lymphocytes, which are administered simultaneously with hematopoietic cells during transplantation and after then formed and matured in the timus of the recipient from donor progenitor cells, recognize and attack the cells of the host. However, a complete explanation of the phenomenon of the GvHD does not exists, and the chimerization of the recipient’s organism as a possible cause of damage of its organs is not taken into account. Therefore, the aim of this work was the modeling of allogeneic transplantation of the whole bone marrow (BM, experiment) and comparing its results with syngeneic transplantation (control) basing on the investigation of engraftment of cells of donor origin in the main GvHD target organs. Bone marrow (BM) donors were Tg(ACTB-EGFP)1Osb/J mice carrying a green fluorescent protein gene (EGFP), recipients were the animal of CBA and C57BL/6 inbred strains with age 2–10 months. 1 day before BMT (1.5×107 cells per mouse) all recipients were irradiated at a dose of 6.5 Gy (LD 50/30). After 1, 3, 5, 7, 11, 14, 21, 28, 35 and 55 days the development of chimerism in the liver, skin and colon of animals was examined using a fluorescent microscope. Already in 1 day, single fibroblast-like donor cells were found in the colon, in 7 days – in the skin and liver. 14–28 days after BMT, with donor cells mainly stroma in the liver, in the skin fibroblasts and keratinocytes were formed, in the colon villous cells and also stromal and parenchymal cells of Peyer’s patches which were died off after irradiation were substituted. Unlike control, in the experimental groups GFP+ giant fibroblasts about 30 mkm in length were found in the stroma of the liver, in the skin and in the colon; in the liver there was a lot of GFP+-bulkheads and fibroblast-like Ito’s cells of a very intricate configuration. To 35–55 days after allogeneic BMT cells of the donor origin in the liver and in the villi of the colon began to destroy, the villi became overgrown with GFP+-connective tissue cells and warped, wall of the colon became thin and the skin was fully substituted with a new one (all these things were never observed in the conrol groups). We propose a hypothesis that beside with GvHD traits like thinning of the colon wall and plenty of roundish GFP+-cells on inner surface of the skin, other signs of the studied after allogeneic BMT organs suggest that the cells of the organs which are formed from mesenchymal stem cells of the whole bone marrow become target for the recipient’s T-cells, i.e. suggest existence of host-versus-graft (HVG) reaction. Obvious manifestation of immune reactions after BMT directly coincides with the term of massive engraftment of the studied organs with cells of donor origin and restoration of the host’s own immune system, i.e. the development of chimerism determines the development of organ damage. This explains the events of GvHD from medical practice – atrophy of the mucous membranes, excess production of collagen, sclerosis of the bile ducts, skin damage, colitis – and the timing of its manifestation.

HISTOPATHOLOGICAL FEATURES OF MICE LIVER AND KIDNEY ON ORAL ACUTE TESTING OF WHITE RADISH TUBER LYOPHILISATE

Background and aim: Radish tuber (Raphanus sativus L.) is one of the plant species often consumed by society as a vegetable. Currently, its use is beginning to expand as a medicinal plant, including its use for diabetes, pharyngitis, sinusitis, cardiac fibrosis, diuretics, and bacterial infections. This study aims to determine the histopathology of the liver and kidney organs in male albino mice (Mus musculus) by testing the oral pungency of white radish (Raphanus sativus L.). Methods: This experimental study used 12 mice weighing 25–30 grams, divided into 4 treatment groups: Group 1 was administered aquadest, Group 2 received a dose of 400 mg, Group 3 received 800 mg, and Group 4 received 1600 mg of the medication. The treatment was carried out for 14 days, and then the mice were euthanized to see the histopathology of the hepatic and kidney tissues. Results: In the histopathology of the liver and kidney in treatment groups 1, 2, 3, and 4, the results were normal, mild, moderate, and severe damage, respectively. In kidney histopathology, there are signs of damaged tissue, such as bleeding, edema, and inflamation cells. Conclusion: It can be concluded that the higher the dose of white radish lyophilizate, the greater the potential to cause toxicity to the liver and kidney organs. Peer Review History: Received 3 December 2023; Revised 29 January 2024; Accepted 27 February; Available online 15 March 2024 Academic Editor: Prof. Dr. Gorkem Dulger, Duzce University, Turkey, gorkemdulger@yandex.com Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 8.0/10 Reviewers: Prof. Hassan A.H. Al-Shamahy, Sana'a University, Yemen, shmahe@yemen.net.ye Dr. George Zhu, Tehran University of Medical Sciences, Tehran, Iran, sansan4240732@163.com

A novel antimicrobial peptide with broad-spectrum and exceptional stability derived from the natural peptide Brevicidine

The global issue of antibiotic resistance is increasingly severe, highlighting the urgent necessity for the development of new antibiotics. Brevicidine, a natural cyclic lipopeptide, exhibits remarkable antimicrobial activity against Gram-negative bacteria. In this study, a comprehensive structure-activity relationship of Brevicidine was investigated through 20 newly synthesized cyclic lipopeptide analogs, resulting in the identification of an optimal linear analog 22. The sequence of analog 22 consisted of five d-amino acids and four non-natural amino acid 2,5-diaminovaleric acid (Orn) and conjugated with decanoic acid at N-terminal. Compared to Brevicidine, analog 22 was easier to synthesize, and exerted broad spectrum antimicrobial activity and excellent stability (t1/2 = 40.98 h). Additionally, analog 22 demonstrated a rapid bactericidal effect by permeating non-specifically through the bacterial membranes, thereby minimizing the likelihood of inducing resistance. Moreover, it exhibited remarkable efficacy in combating bacterial biofilms and reversing bacterial resistance to conventional antibiotics. Furthermore, it effectively suppressed the growth of bacteria in vital organs of mice infected with S. aureus ATCC 25923. In conclusion, analog 22 may represent a potential antimicrobial peptide for further optimization.