Balb Research Articles

Cellular immune response of Staphylococcus aureus enterotoxin B in Balb/c mice through intranasal infection.

Background and Aim:Staphylococcus aureus produces various superantigen exotoxins, including staphylococcal enterotoxin B (SEB). It causes fatal anaphylactic reactions and toxic shock. This study aimed to evaluate the reaction of leukocytes and histopathological changes in the respiratory organs of Balb/c mice after intranasal infection with enterotoxigenic S. aureus (SEB).Materials and Methods:The presence of the seb gene in S. aureus was established in this study using polymerase chain reaction-specific primer. Two groups of 8-week-old male Balb-c mice consist of six mice in each group. The treated group was infected with 50 μL and 100 μL of SEB intranasal on days 1 and 14, respectively. NaCl was administered in the second group and was considered as a control group. Blood samples were collected through the retro-orbital plexus on days 1, 4, 7, 14, and 22 after infections. Total cell counts were analyzed with an independent sample t-test and compared using the statistical package for the social sciences (SPSS) version 16.0 (IBM Corp., NY, USA). The infected tissues of the respiratory organ were observed descriptively and compared to the control group.Results:The seb gene with a molecular size of 478 bp, indicating the SEB strain, is present in S. aureus used in this study. Intranasal administration of SEB showed increased leukocytes, lymphocytes, monocytes, and eosinophils on day 22 post-infection. Significant leukocytosis was seen on days 6 and 14; lymphocytosis on days 1, 4, 6, and 16; and eosinophilia on days 6, 14, and 22 compared with the control group (p > 0.05). In contrast, the neutrophil decreased after an increase of immature band cells compared to the control group, indicating a severe acute infection with SEB. The lungs and trachea of the test group had an inflammatory cell accumulation in the respiratory organ.Conclusion:Intranasal route infection of S. aureus containing seb gene significantly induced the cellular immune response and caused pathological changes in the respiratory tissues of the Balb/c mice model. The hematological changes were aligned with marked pathological changes in the respiratory tract. Balb/c mice could be an excellent experimental model to study toxic and anaphylactic shock against SEB to define the future therapeutic agents.

Effect of the combined application of Lampung Robusta Coffee Extract and Lactobacillus acidophilus on the Ileum and Caecum Histopathology in Salmonella enterica infected Balb/C Mice

Salmonella enterica is a gram-negative bacterium that can cause Salmonellosis and gastroenteritis in humans and animals. Further, this bacterial infection is also associated with the reactive oxygen species (ROS) by lipid peroxidase that can destroy the intestinal cell's membrane. This study aimed to evaluate the preventive effect of the combined application of Lampung Robusta coffee extract and Lactobacillus acidophilus on the Ileum and Caecum Histopathology in Salmonella enterica infected Mice. In this study, male Balb-c mice aged between 8-10 weeks and weight 20-25 grams were used, these experimental animals were divided into six experimental groups namely K- (Negative control without any infection), K+ (Positive control with S. enterica), KL (Only L. acidophilus treated mice), P1, P2, and P3 were given a preventive extract of coffee with a concentration of 250 mg/kg BW, 500 mg/kg BW, and 750 mg/kg BW respectively and L. acidophilus to S.enterica infected mice and arrange in completely Randomized Design. Descriptive histopathological analyses were carried out after HE staining and villi's length and width for ileum's histopathology and counting goblet cells for caecum's histopathology was scored. The results of the study revealed that administration of Robusta Coffee extract @ 250 mg/ kg BW and L. acidophilus has a preventive effect on the ileum and caecum damage caused by salmonellosis.

Abstract 1324: IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist

Abstract Background & Aims: The IL-1 subfamily, IL-36, is increasingly being implicated as a potent cytokine family involved in chronic inflammatory conditions such as psoriasis and IBD. Recent work has also suggested IL-36 cytokines may act as a potential adjuvant in immunotherapy regimens to augment the anti-cancer immune response. Given the pluripotent nature of IL-1 cytokines and the relationship between inflammation and tumorigenesis, we investigated the effects of IL-36 signalling in colorectal cancer (CRC). Methods: IL-36 family expression in CRC progression was investigated by the NCBI GEO transcriptomic database, and then by immunohistochemistry (IHC) and qRT-PCR. Pro-tumorigenic properties such as cancer cell migration, invasion and proliferation were investigated in 2D and 3D models. In vivo models were performed using Balb-c mice which were subcutaneously inoculated with CT26 cells and treated with IP injections of recombinant IL-36R agonists (IL-36α, β, γ) or antagonist (IL-36RN). Tumour tissues were excised and studied by flow cytometry and IHC analysis. Additionally, CRISPR-Cas9 mediated IL-36R KO CT26 cells were generated and subcutaneously injected into Balb-c mice, with changes in tumour growth and immune cell infiltrate investigated. Results: IL-36R expression was shown to significantly increase with stage of disease in the adenoma-carcinoma sequence, including distant metastases. Additionally, expression of all family members was increased in tumour tissue relative to adjacent normal tissue in a separate colon cancer cohort. In vitro IL-36R signalling on cancer cells augmented a pro-tumorigenic phenotype by induction of pro-tumorigenic cytokines/chemokine production, increased cellular migration, invasion and proliferation of tumour cells. In vivo inhibition of IL-36R signalling by both administration of recombinant IL-36RN and through deletion of the IL-36R gene in CT26 cells, resulted in a significant reduction in tumour growth. This reduction was associated with a significant decrease in tumour cell proliferation, as well as an increase in CD8+ T cell infiltrate in IL-36R KO groups. Of note, administration of IL-36R agonists also resulted in reduced tumour growth, albeit to a lesser extent, associated with an increase in CD4+ and CD8+ T cell infiltration. Conclusions: This data indicates that IL-36 family members, similar to other IL-1 family members, have dual functions in colon cancer. In addition, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for CRC patients with IL-36R+ tumour cells. Citation Format: Kevin J. Baker, Charlotte O'Donnell, Micheal O'Riordain, Elizabeth Brint, Maura Bendix, Aileen Houston. IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist [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 1324.

Abstract 1750: Design and development of a novel highly sequence-selective guanine mono-alkylating DNA-interactive ADC payload suitable for solid tumor treatments

Abstract Antibody-Drug Conjugates (ADCs) are a fast-growing class of targeted cancer therapeutic agents with over ten approved and greater than 100 in various stages of clinical development. The pyridinobenzodiazepines (PDDs) are a new class of DNA-interactive sequence-selective Guanine mono-alkylating ADC payloads which contain a sequence recognition component with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). A high potency PDD, FGX15-147, has been developed that contains a bi-aryl moiety capable of forming an intramolecular ‘sigma-hole’ interaction with DNA bases, thus providing the molecule with a unique sequence-selectivity profile when compared to other DNA-interactive agents. DNA footprinting data will be presented indicating that the molecule, which spans 8-9 DNA base pairs in the minor groove, has an increased sequence selectivity compared with related analogues, preferring DNA regions containing two guanines 2-4 base pairs apart. FGX15-147 is highly cytotoxic in vitro (e.g., IC50 = 0.30 nM in SW60; 1.6 nM in LIM1215 and 0.142 nM in SW48, after 96 hours incubation), and is thought to exert its activity through a combination of DNA alkylation and inhibiting the binding of important oncogenic transcription factors such as Hypoxia Inducible Factor (HIF) to DNA (demonstrated through an in vitro Transcription Factor Array assay). HIF is known to be involved in the development of a large number of solid malignancies, including pancreatic cancer. To explore the utility of FGX15-147 as an ADC payload, it was conjugated to trastuzumab in a stochastic manner (DAR 1.6) using a Maleimide-Valine-Alanine linker construct. The resulting ADC exhibited significant in vivo efficacy in a low copy number HER2+ pancreatic cancer Human Tumor Xenograft model using BALB-c mice transplanted with the CAPAN-1 cell line. Complete tumor regression was observed out to 60 days after a single dose of 2 mg/kg comparing favorably to a 10 mg/kg dose of trastuzumab deruxtecan (Enhertu®) in the same study in which substantial tumor regrowth had occurred by 57 days. In a standard mouse model, the FGX15-147-based ADC had a good tolerability profile with a Maximum Tolerated Dose (MTD) of at least 16 mg/kg. Overall, the favorable tolerability and efficacy profile of FGX15-147 in an ADC format suggests that it represents a potentially valuable approach for the treatment of solid tumors. Citation Format: Paolo Andriollo, George Procopiou, Daniella M. di Mascio, Paul J. Jackson, Khondaker M. Rahman, Keith R. Fox, David E. Thurston. Design and development of a novel highly sequence-selective guanine mono-alkylating DNA-interactive ADC payload suitable for solid tumor treatments [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 1750.

SUPLEMENTASI YOGURT FORTIFIKASI KONSENTRASI BERTINGKAT EKSTRAK ROSELLA UNGU TERHADAP KADAR MALONDIALDEHIDA (MDA) DAN GAMBARAN HISTOLOGI GINJAL MENCIT

Purple roselle has ingredients such as organic acids, anthocyanins, polysaccharides, and flavonoids. The antioxidant content in purple roselle has benefits in preventing cell damage due to free radicals. This study was conducted to discoverthe effect of supplementation yogurt fortified with a graded concentration of purple roselle extract on MDA levels and histology of the kidney organs of mice. Laboratory experimental study using 16 male Balb-C mice divided into four groups. The control (C) group was not given any treatment. The treatment groups 1,2, and 3 (T1, T2, T3) were given fortified purple roselle yogurt with concentrations of 5%, 10%, and 15%, respectively. The treatment was carried out for 14 days with a volume of 0.4 ml/day. Kidney organs were collected for histological analysis. Kidney malondialdehydelevels were assayed using the TBARS test and statistically analyzed with a 95% confidence level by one-way ANOVA analysis. Histology score analyzed using the Kruskal-Wallis test followed by the Mann- Whitney test. This study showedthat at concentrations of 10% and 15%, MDA levels of the mice kidneys reduce significantly compared to the control group. The kidney histology score results show some changes in treatment groups compares to control group. The average score decreasing from different treatment group, and the lowest score was group given yogurt fortified roselle extract 15%. The administration of fortified yogurt with purple roselle extract at a concentration of 15% can reduce MDA levels in the kidneys of mice, and the higher the concentration can reduce kidney damage in mice.

The Prembion® pre-biotic improves the impact of anti-CTLA4 immune checkpoint inhibitor in a murine model of malignant pleural mesothelioma

Abstract Objective Immune checkpoint inhibition (ICI) therapy has revolutionized the outcome of certain cancers such as malignant pleural mesothelioma (MPM). However, patient responsiveness to this treatment remains unpredictable. Recently, a role for the gut microbiota composition has emerged for patients to generate a robust immune response against their tumors, following immunotherapy. Here, we studied the impact of Prembion®, a pre-biotic and modulator of the gut microbiota, on tumor control and lymphocyte infiltration in a murine MPM model treated by ICI. Methods Prembion® (diluted into drinking water) was administrated to BALBc mice for 14 days. These animals were then inoculated orthotopically with a syngeneic MPM cell line (AB12-luc cells injected in the pleura) and followed by bioluminescence imaging. We determined the tumor growth and mouse survival in different groups: untreated control, Prembion®, IgG control, anti-PDL-1, anti-CTLA4, Prembion®+anti-PDL-1 and Prembion®+anti-CTLA4. A correlation between tumor response/animal survival and MPM infiltration with CD8+ lymphocytes was also performed by immunohistochemistry. Results Prembion® was well tolerated and did not affect animal weight or activity. Interestingly, Prembion® was as effective as anti-PDL1 and anti-CTLA4 monotherapy on tumor control, prolonging survival by 4.0 ± 1.1 days compared to controls (p<0.05). Moreover Prembion® potentiated anti-CTLA4 efficacy with a significant improvement in mouse survival of the Prembion®+anti-CTLA4 compared to controls (3.6 ± 1.1 days, p<0.05). Additionally, this finding correlated with enhanced MPM infiltration by CD8+ lymphocytes compared to controls (p<0.05). Conclusion Prembion® positively regulated the adaptive immune response against MPM and helped to improve the impact of anti-CTLA4 ICI on MPM. Further work focusing on the gut microbiome changes induced by Prembion® are ongoing to better understand the mechanisms involved.

Anti‐Epileptic Drug and Teratogen Valproic Acid Induces Microglial Cell Death in a Valproic Acid Murine Model of Autism

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects approximately 1 in 44 children in North America, and its association with neuronal connectivity is an area of intense research. Valproic acid (VPA) is a multi‐target drug widely used to treat epilepsy. It is classified as a teratogen and as a histone deacetylase inhibitor (HDACi) and fetal exposure to VPA increases the risk of ASD. The VPA model has been well characterized for behavioral and neuronal deficits, including hyperconnectivity. The cause of hyperconnectivity is poorly understood however, it is speculated that lack of pruning may be partly responsible for this anomaly. Microglia, the principal immune cells of CNS regulate dendrite and synapse formation during early brain development. The effect of VPA on microglia during early development has not been well characterized and may provide potential hints regarding the etiology of this disorder. Preliminary studies from our lab indicate that VPA reduces microglial number during peak periods of postnatal synaptogenesis in the mouse brain primary cortex. Nevertheless, the cause of this reduction is not known. Therefore, in this study, we determined the mechanism for reduced microglial numbers in the VPA model of autism.Pregnant BALBc mice were administered 600 mg/kg of VPA by subcutaneous injection on day 13.5 of gestation. We harvested the mice brains at embryonic (E) day 14, E17, and postnatal day 1 (PD1). Brain tissue was double immunolabeled for microglia (IBA1) and cell death marker (TUNEL). We found a gradual increase in TUNEL staining beginning E14 to PD1 compared to age‐matched controls. The mechanism for reduced microglial number may be due to cell death. Thus, our findings possibly have pathophysiological/pathogenic implications in autism and may, ultimately, lead to the discovery of new therapeutic target(s) for autism.

Role of apoptosis and autophagy in masseter muscle atrophy evoked by Botulinum Toxin injection

IntroductionMusculoskeletal disorders of the masticatory system impact the quality of life and have a high cost of diagnosis and treatment. Masseter muscle hyperactivity is a cause of pain in the chewing apparatus. Botulinum toxin type A (BoNTA) injection is widely used to induce paralysis of the masseter muscle, thereby decreasing impaired muscle activity. However, our laboratory has described in a preclinical model that the injection of BoNTA not only induces paralysis, but also muscle atrophy, which subsequently decreases bone quality. However, it is unknown whether apoptosis or autophagy mechanisms could contribute to muscle atrophy.AimTo evaluate the induction of apoptosis and autophagy in BoNTA‐injected masseter muscle of adult mice.MethodologyUnilateral injection of BoNTA (0.2U/10µl) in the masseter muscle was performed in adult BalbC mice (approved by IACUC‐Universidad de Chile, #21446‐ODO‐UCH). Apoptosis and autophagy markers were evaluated in masseter muscles by immunoblot at 2‐7d post‐injection and immunofluorescence at 7d post‐injection. Autophagy activity was blocked by i.p injection of chloroquine. The data were evaluated with t‐test, one‐way ANOVA test, Mann‐Whitney test, or Kruskal‐Wallis test, as appropriate. The results were expressed as mean ±SEM (n=4‐8; p <0.05).ResultsUnilateral injection of BoNTA did not change the relative levels of apoptosis‐related proteins like cleaved Caspase 3, PARP, and AIF. There was a significant increase in protein levels related to autophagy, such as lipidated LC3 (1.86‐fold), non‐lipidated LC3 (1.66‐fold), and P62 (1.24‐fold) at 7 d post‐injection. Also, there was a strong punctuated stain for LC3 in histological sections of masseter muscle 7d post injection, suggesting autophagic vesicles. However, the injection of chloroquine for blocking the autophagy flux did not improve the accumulation of autophagy proteins in masseter muscle evoked by BoNTA in the induction.ConclusionsThe atrophy of masseter muscle evoked by BoNTA injection is not related to autophagy‐ or apoptosis‐induction. The increase in autophagy markers after BoNTA injection, with no further increase after autophagic flux blockade, suggests that BoNTA may be blocking autophagy in the masseter muscle, perhaps favoring a proteasomal pathway of muscle atrophy.

Botulinum toxin injection in masseter muscle evokes musculoskeletal impairment of the masticatory system

The masticatory system is a finely coordinated machine, where minimal deregulation induces alterations of the whole system. Botulinum toxin type A (BoNTA) injection in masseter muscle is widely used as a clinical/aesthetic procedure. However, the non‐desirable effects of masseter muscle paralysis in neighbor masticatory muscles or mandibular bone are unknown. In the current work, we aimed to evaluate musculoskeletal loss at the masticatory system after BoNTA‐injection in masseter muscle of adult mice.Unilateral paralysis of masseter muscles was induced in male BalbC mice (8 weeks‐old) by single‐injection of BoNTA (0.2U/10 µL) in the right muscle and saline solution in the left muscle. Masticatory muscles (masseter, temporalis, lateral‐ and medial‐pterygoid) and mandibles were dissected after 2‐7‐14d for molecular measurements (qPCR), histology, or morphology (microcomputed tomography). All procedures were approved by the IACUC‐Universidad de Chile (# 20381‐ODO‐UCH). T‐test or Wilcoxon‐test were used for statistical comparisons between BoNTA‐ and Control‐side (n=3‐10, p<0,05).The expression of molecular markers of atrophy (Atrogin1, Murf1) and regeneration (myogenin) significantly increased in the BoNTA‐side, as early as 2‐7d, in the intervened masseter but also in the ipsilateral temporalis and medial pterygoid muscles. There was a time‐dependent reduction in masseter fibers diameter from 2d to 14d. Masseter, temporalis, and medial pterygoid’s volumes significantly reduced in the BoNTA‐injected side at 14d. Interestingly, the volume of lateral pterygoid increased in the BoNTA side, with no change in atrophy/regeneration markers. Microstructural analysis of mandibular condyles demonstrated a progressive bone loss in the BoNTA‐injected side, starting as early as 7d and worsening 14d after the intervention.In conclusion, BoNTA injection in the masseter muscle impairs the whole masticatory system, inducing atrophy of masseter but also the ipsilateral temporalis and medial pterygoid, and even early‐bone loss of the mandibular condyle at the injected side. Then, clinical uses of BoNTA in masticatory muscles should be avoided or carried out under strict morpho‐functional monitoring.

UJI AKTIVITAS EKSTRAK METANOL BIJI JUWET (SYZYGIUM CUMINI (L.) SKEELS) SEBAGAI ANTIHIPERURISEMIA PADA MENCIT HIPERURISEMIA

The purpose of this research was to study the activity of Syzygium cumini seed extract as antihyperuricemics agents on hyperuricemic male Balb-C mice. Twenty four male mice were divided into 6 groups, i.e the first group as a normal control K(N); the second group as a negative control K(-) was treated with CMC Na 1% 0,2 ml/20g/BW orally; the third group as a positive control K(+) was treated with allopurinol 10 mg/kg BW orally; the fourth to sixth groups (P1, P2, and P3) were treated with 200, 400, and 800 mg/kg BW methanol extract of Syzygium cumini seed orally for 8 days. Inducing performed using chicken liver juice 0,2 % b/v for 4 days, melinjo 10 % b/b of standart feed mice for 12 days, and potassium oxonate 250 mg/kg BW i.p was induced in 2 hours before the blood samples collection. The blood samples was taken from vena ophtalmicus. The concentration of uric acid was determined with FS DHBSA. Data was analyzed using One Way Anova dan LSD test. The result showed that activity of methanol extract of Syzygium cumini seed has significant effect (p<0,05) in reducing the concentration of uric acid in mice. The substances estimated has antihyperuricemic activity were flavonoid, alkaloid, terpenoid, saponin, and tanin.

Breast cancer metastasis to brain results in recruitment and activation of microglia through annexin-A1/formyl peptide receptor signaling

BackgroundDespite advancements in therapies, brain metastasis in patients with triple negative subtype of breast cancer remains a therapeutic challenge. Activated microglia are often observed in close proximity to, or within, malignant tumor masses, suggesting a critical role that microglia play in brain tumor progression. Annexin-A1 (ANXA1), a glucocorticoid-regulated protein with immune-regulatory properties, has been implicated in the growth and metastasis of many cancers. Its role in breast cancer-microglia signaling crosstalk is not known.MethodsThe importance of microglia proliferation and activation in breast cancer to brain metastasis was evaluated in MMTV-Wnt1 spontaneous mammary tumor mice and BALBc mice injected with 4T1 murine breast cancer cells into the carotid artery using flow cytometry. 4T1 induced-proliferation and migration of primary microglia and BV2 microglia cells were evaluated using 2D and coculture transwell assays. The requirement of ANXA1 in these functions was examined using a Crispr/Cas9 deletion mutant of ANXA1 in 4T1 breast cancer cells as well as BV2 microglia. Small molecule inhibition of the ANXA1 receptor FPR1 and FPR2 were also examined. The signaling pathways involved in these interactions were assessed using western blotting. The association between lymph node positive recurrence-free patient survival and distant metastasis-free patient survival and ANXA1 and FPR1 and FPR2 expression was examined using TCGA datasets.ResultsMicroglia activation is observed prior to brain metastasis in MMTV-Wnt1 mice with primary and secondary metastasis in the periphery. Metastatic 4T1 mammary cancer cells secrete ANXA1 to promote microglial migration, which in turn, enhances tumor cell migration. Silencing of ANXA1 in 4T1 cells by Crispr/Cas9 deletion, or using inhibitors of FPR1 or FPR2 inhibits microglia migration and leads to reduced activation of STAT3. Finally, elevated ANXA1, FPR1 and FPR2 is significantly associated with poor outcome in lymph node positive patients, particularly, for distant metastasis free patient survival.ConclusionsThe present study uncovered a network encompassing autocrine/paracrine ANXA1 signaling between metastatic mammary cancer cells and microglia that drives microglial recruitment and activation. Inhibition of ANXA1 and/or its receptor may be therapeutically rewarding in the treatment of breast cancer and secondary metastasis to the brain.

Kontrollü Ovaryen Hiperstimulasyon Uterusta Apelin ve Apelin Reseptör Ekspresyonunu Arttırır

Objective; In this study, we investigated whether superovulation, which is frequently used in the clinic as controlled ovarian hyperstimulation, affect the expression of Apelin (APLN) and Apelin Receptor (APJ) in the uterus.
 
 Methods: Twelve female Balb-C mice 6-8 weeks old were used in this study. The groups were established as follows; virgin control female group (Knt) which did not receive any treatment, the pregnant control group (GK) which was on their pregnancy day 1, and the PMSG+hCG group (P+H) that received 5 IU/mouse/intraperitoneal (ip) PMSG and 5 IU/mouse/ip hCG . Vaginal plaque control was performed on the day after mating in the P+H and GK groups, and vaginal plaque-positive mice were accepted at day 0.5 of pregnancy. Mice in the P+H group were sacrificed 42 hours after hCG injection. Uterine tissues were obtained from all groups for immunohistochemical analysis, and paraffin sections were obtained. Adipose tissue was used as a positive control.
 
 Results: In the Knt group, APLN protein expression was present especially in the uterine glands and stromal cells that are located close to the glands. The localization of APJ protein expression was also similar to APLN. In the GK group, APLN and APJ expression levels were similar to the Knt group. However, when the localization of APJ was evaluated, it has been observed that APJ expression was intensely expressed especially in the perimetrial glands of the uterus, which was not present in the Knt group. In the P+H group, APLN expression significantly increased in the endometrial glands and stromal cells compared to the Knt and GK groups (p

Prenatal Stress and Adaptive Behavior of Offspring: The Role of Placental Serotonin

The effect of mild prenatal stress in mice, leading to an increase in the placental serotonin level, on the formation of adaptive behavior in male offspring at the age of 35 days was studied. It was shown that, in BalbC mice, daily immobilization for 1 h during the period from 11 to 14 days of pregnancy led to an increase in placental and fetal serotonin levels on the 15th day of prenatal development. According to “resident–intruder” behavioral test, the prenatally stressed mice showed more reactive behavior in adulthood and low tendency to defend their territory. Thus, placental serotonin, formed under the stress condition, may act as a mediator between the environment and the fetuses and determine the adaptive behavior of offspring.

Examination the effects of chestnut and Manuka Honey for wound healing on mice experimental model

Objective: In this study, the aim is to examine the effects of Chestnut and Manuka honey on wound healing in order to investigate the effectiveness of cost-effective alternative to current approaches in terms of wound care and treatment. Material and Methods: In this study, we used 30 healthy male Balbc mice weighing 18-24 g. We randomly divided the rats into three groups. A control group, a group treated with only Chestnut honey, a group treated with Manuka honey cream. After the wounds were formed in groups, the tissue samples were gathered on the seventh and fourteenth days. Then these samples were examined histologically and immunohistochemically. Results: When the study results were evaluated, statistically significant differences were seen between histological and immune-histochemical findings in wound tissue preparations. On the seventh day, tissue samples showed re-epithelialization (P=0,002), granulation cell density (P=0,003) and angiogenesis (P= 0,003). In the fourteenth day tissue samples, we found epithelialization (P=0,001), granulation cell density (P=0,002) and angiogenesis (P=0,001). In the tissue samples in the seventh and fourteenth days between the groups, we found immino-histochemically, Ki-67 and EGF dyeing percentages as P= 0,004 and P=0,003 respectively. Conclusion: We think chestnut honey may contribute to a shorter wound healing process.

Camouflaged liposomes by 11A4-nanobody for co-delivery of cisplatin and nitroxoline in breast cancer tumors: An in vitro/in vivo study

Nitroxoline (NIT) is a quinoline antibiotic with anti-cancer effects which prevents cell migration by inhibition of cathepsin B enzyme. Cisplatin (CIS) is normally used for various kinds of malignancies such as metastatic breast cancer. The goal of the present study was increasing cellular toxicity and cell migration inhibition by co-delivery of NIT-CIS using targeted liposomes by anti HER2 nanobody. Liposomes were prepared by solvent injection method using cholesterol, soy lecithin and maleimide-polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine (Mal-PEG-DSPE) and targeted by anti HER2 nanobody. The liposomes were optimized for their particle size, zeta potential, drugs loading and release efficiency. Then their cytotoxicity, migration, cellular uptake and apoptosis induction were tested on TUBO (HER2+) and MDA-MB-213 (HER2-) cell lines. The in vivo antitumor activity of CIS-NIT loaded in targeted liposomes was compared with free drugs solution and non-targeted liposomes in TUBO cells induced breast cancer in Balb-c mice. Results indicated that the mixture of CIS and NIT significantly increased cytotoxicity. Moreover, targeted liposomes increased cellular toxicity and cellular uptake in TUBO cells, unlike MDA-MB-213 cells. Cell migration studies implied the effect of NIT combinations on lowering the invasiveness of the cells. Targeted liposomes loaded with CIS-NIT had outstanding tumor suppression efficiency compared to control group, non-targeted liposomes and free drugs.

Abstract PD3-03: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

Abstract Introduction: Triple negative breast cancers (TNBC) are frequently poorly differentiated with higher propensity for metastasis than all other subtypes. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase that mediates transcriptional repression of pro-differentiation genes in normal and neoplastic cells. The oncogenic role of EZH2 through H3K27me3 is well established. However, non-canonical H3K27me3-independent functions are unclear. We have reported that p38 phosphorylates T367 of EZH2, increasing TNBC metastasis. Recent reports show that EZH2 can regulate signaling pathways through direct methylation of proteins suggesting the hypothesis that EZH2 may methylate p38 in TNBC. Methods: Human tissue samples from 16 primary invasive breast carcinomas and matched distant metastasis arrayed in tissue microarrays were interrogated for pEZH2 T367 and p-p38 by immunohistochemistry. To study p38 methylation, we performed LC-MS/MS analyses GST-p38α, and GST-EZH2 were each incubated with or without recombinant PRC2 complex (EZH2/E ED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor. MDA-MB-231, mouse and human primary TNBC cells and murine 4T1 cells were used in funcional assays in vitro and in vivo. EZH2 knockdown and rescue was carried out using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type) or pBabe-myc-EZH2 (T367A). We developed p38 methylation mutants: HA-p38a K139A, HA-p38a K165A, and HA-p38a K130A/K165K. To investigate the importance of EZH2 and p38 enzymatic activities we used the EZH2 methyltransferase inhibitors GSK-343 and EPZ-6438 and the p38 inhibitor SB202190 in vitro and in vivo. For animal studies, MDA-MB-231 and 4T1 cells were orthotopically injected into the right inguinal mammary fat pad of eight-week old NOD/SCID or BALBc mice, respectively. When tumors reached 100 mm3 mice were treated 5 days/week by intraperitoneal injection with SB202190 (1mg/kg/day), EPZ-6438 (10mg/kg/day), combination of SB202190 and EPZ-6438, or control. We monitored tumor growth for 50 days, and harvested primary tumors, lungs, and sites of metastasis for histopathology, Western blot, and immunohistochemistry. Primary xenografts from the control and treatment groups were subjected to RNA sequencing. Results: EZH2 methylates p38 at lysines 139 and 165 leading to enhanced p38 stability and increased invasion, and p38 activation requires T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2 T367, H3K27me3, and p-p38 in vivo, reduces TNBC growth and metastasis, and results in gene expression changes towards a better differentiated phenotype. pEZH2 T367 and p-p38 proteins are significantly coexpressed in human primary and metastatic breast cancer. Conclusions: We provide direct evidence that EZH2 methylates p38 with resultant p38 activation, and that EZH2 phosphorylation at T367 is important for this function. Dual targeting of EZH2 methyltransferase and p38 kinase activities with specific inhibitors reduces breast cancer growth and metastasis indicating a cooperation between EZH2 canonical and non-canonical mechanisms and suggesting therapeutic strategies. Citation Format: Maria E Gonzalez, Shilpa R. Tekula, Talha Anwar, Shoshana A. Leflein, Celina G. Kleer. EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-03.

The Effect of Inhaled Anesthetic Halothane on Liver Cells (Experimental Study of Halothane Toxic Effect in BalbC Mice)

Adverse effects of halothane on the liver range from liver dysfunction to hepatitis, and are more frequent following repeated use. Medical personals are in risk of chronic exposure of wasted halothane. However, there are limited literatures about this effect based on time exposure. To identify and analyze the liver cells changes of halothane exposure to liver cells of Balb/C mice. This experimental study used randomized post-test only control group design in 30 male BalbC mice given 0.011 mg/weight halothane 3 hours daily for two weeks (group 1), four weeks (group 2), six weeks (group 3), six weeks followed by 2 weeks of halothane free (group 4), and without exposure (control group). The liver tissues were HE stained and observed for enlarge, karyorrhexis, and karyolitic of the nucleus. Immunohistochemistry was undertaken to count the percentage of cytocrome-P450 stained brown colour and scored based on its intensity. The difference among groups and between each group were statistically significant (p< 0.05), except between group 2 and 4 (p = 0.078), and between group 3 and 4 (p = 0.522). The difference of cytocrome-P450 expression among groups was not statistically significant (p = 0.266). There was statistically significant difference of abnormal liver cell nucleus changes in groups with different time of halothane exposure (p = 0.000) while no statistically significant difference of cytocrome-P450 expressions (p = 0.266) was found. The nucleus changes were gradually increased with the time of exposure.

Olvanil activates sensory nerve fibers, increases T cell response and decreases metastasis of breast carcinoma

BackgroundInactivation of sensory neurons expressing transient receptor potential vanilloid 1 (TRPV1) enhances breast cancer metastasis. Sensory neurons have profound effects on immune response to a wide range of diseases including cancer. Hence, activation of sensory nerves using feasible approaches such as specific TRPV1 agonists may inhibit breast cancer metastasis through neuroimmune pathways. TRPV1 agonists are considered for the treatment of pain and inflammatory diseases. Methods: We here first determined the effects of four different TRPV1 agonists on proliferation of three different metastatic breast carcinoma cells since TRPV1 is also expressed in cancer cells. Based on the results obtained under in-vitro conditions, brain metastatic breast carcinoma cells (4TBM) implanted orthotopically into the mammary-pad of Balb-c mice followed by olvanil treatment (i.p.). Changes in tumor growth, metastasis and immune response to cancer cells were determined. Results: Olvanil dose-dependently activated sensory nerve fibers and markedly suppressed lung and liver metastasis without altering the growth of primary tumors. Olvanil (5 mg/kg) systemically increased T cell count, enhanced intra-tumoral recruitment of CD8+ T cells and increased IFN-γ response to irradiated cancer cells and Con-A. Anti-inflammatory changes such as increased IL-10 and decrease IL-6 as well as S100A8+ cells were observed following olvanil treatment. Conclusions: Our results show that anti-metastatic effects of olvanil is mainly due to activation of neuro-immune pathways since olvanil dose used here is not high enough to directly activate immune cells. Furthermore, olvanil effectively depletes sensory neuropeptides; hence, olvanil is a good non-pungent alternative to capsaicin.

Abstract P003: Efficacy results of a novel vaccine composed of stimulated and haptenized tumors cells in Balbc mice grafted with murine colon adenocarcinoma CT26 cells

Abstract BACKGROUND: Metastatic colorectal cancer (mCRC) is a major cause of death. Unmet medical need in immunotherapy is high for MSS patients and still present for MSI-H/dMMR patients. STC vaccine (Brenus Pharma) is composed of selected tumor cell lines, stimulated to overexpress TAA/TSA and neoantigens including resistant factors that are further haptenized to form immunogenic hapten-protein complex to educate the immune system to recognize and target the patient's tumor cells expressing the same resistance factors. We report results of two studies aiming to A) evaluate efficacy of a one cell line-based product (CT26) physical stimulated (S=irradiation and heat shock) and/or haptenized (H) w/o immunostimulant (IS=cyclophosphamide + mGM-CSF w/o BCG) and to B) investigate a potential increase of antitumoral effect of 3 cell lines vaccine (3CL-SH made of CT26, CMT-93, LTPA). STUDY DESIGN and METHODS: Female BALB/c mice were subcutaneously grafted with 5.104 CT26-WT cells. In study A, 9 groups (10 mice per group) are allocated to: G1) Control group, G2) IS, G3) CT26-S, G4) CT26-H, G5) CT26-SH, G6) CT26-S+IS, G7) CT26-H+IS, G8) CT26-SH+IS, G9) CT26-SH+IS+BCG. In study B, 5 groups (20 mice per group) are allocated to: G1) Control group, G2) CT26-SH + IS, G3) 3CL-SH, G4) 3CL-SH + IS once a week for 3 weeks and G5) 3CL-SH + IS twice a week for 4 weeks. Treatments were administered subcutaneously. Overall survival (OS) and tumor growth (TG) were recorded until 1000 mm3, safety endpoint or on D41 (study A) or D50 (study B). RESULTS: Stimulated cell-based treatments with IS (CT26-SH + IS) significantly increases OS compared to control group (Study A: G1/G8 p=0.0046 & study B: G1/G2 p=0.0023). Best mOS among groups is observed with 3CL-SH+IS (Study B: G1/G4 p<0.0001 38d vs 27d, Log-rank test). A direct comparison of 3CL-SH+IS and CT26-SH+IS confirmed a highly significant added benefit in favour of the 3 cell lines vaccine (Study B: G2/G4 p=0.0475) compared to the one cell line treatment. In addition, IS reinforced the effect of cell-based treatment, with CT26-SH (Study A: G1/G8, p<0.0001 at D20, Study B: G1/G2 p<0.0001 at D24) and with 3CL-SH (Study B: G4/G3 p=0.0004). Addition of BCG to CT26-SH+IS does not improve efficacy (Study A: G8/G9 p=NS). Results showed a significant impact on TG when cells were both physically stimulated then haptenized, such as CT26-SH (study A: G1/G5, p=0.003 at D20) or 3CL-SH (study B G1/G3 P<0.0001 at D24) compared to the control group. 3CL-SH+IS exhibits a significant efficacy on TG (Study B: G1/G4 p=0.0053 or G1/G5 p=0.0018) and OS whatever the administration schedule No side effect or inflammatory reaction towards the vaccines have been evidenced. CONCLUSION: Brenus STC vaccine based on physical stimulation and haptenization demonstrated a significant anticancer effect in mice with immunostimulant and confirmed a better efficacy of the 3 cell lines vaccine versus a single cell line vaccine. Further studies are ongoing to test the efficacy of STC vaccine in PD1 resistant preclinical model and in combination with SOC. Citation Format: Céline Gongora, Jacqueline Taleb, Benoît Pinteur, Lionel Chalus, Fanny De Luca, Paul Bravetti, François Ghiringhelli. Efficacy results of a novel vaccine composed of stimulated and haptenized tumors cells in Balbc mice grafted with murine colon adenocarcinoma CT26 cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P003.

Effect of a Standardized Hydroalcoholic Extract of Echeveria subrigida on Mice Glycemia and In Vitro Kinetics of α-Glucosidase Inhibition

Leaf extracts of Echeveria subrigida show antioxidant, antibacterial, anti-mutagenic, and α-glucosidase inhibitory activities. The inhibitory α-glucosidase is associated with the content of quercetin-3-O-glucoside and isorhametin-3-O-glucoside. The aims of this study were to analyze the effect on mice glycemia and in vitro kinetics of α-glucosidase inhibition of the hydroalcoholic extract of Echeveria subrigida standardized in the content of isorhametin-3-O-glucoside. The high-performance liquid chromatography method was validated to quantitate isorhametin-3-O-glucoside. Hydroalcoholic extract of Echeveria subrigida identity was characterized by its organoleptic, physicochemical, pharmacological and toxicological parameters. The effects on glycemia were carried out in 6 w old male Balb-C mice and using the following treatments; hydroalcoholic extract of Echeveria subrigida at 50, 100 and 200 mg/kg b.w.; positive control glibenclamide or acarbose at 10 mg/kg b.w. The inhibition kinetics of hydroalcoholic extract of Echeveria subrigida was determined by the Lineweaver-Burk plots. Hydroalcoholic extract of Echeveria subrigida had 4.87±0.14 mg isorhametin-3-O-glucoside/g, complied with the World Health Organization parameters for standardized extracts and induced a mixed inhibition on α-glucosidase. Hydroalcoholic extract of Echeveria subrigida (200 mg/kg b.w) and glibenclamide treatments showed similar percent hypoglycemia, 49.1 % and 52 %, respectively. The anti-hyperglycemic assay showed that similar percent reductions in glucose-levels were found in the treatments with hydroalcoholic extract of Echeveria subrigida (100 mg/kg, 29.32 %; 200 mg/kg, 28.99 %) and acarbose (10 mg/kg, 19.87 %). The standardized hydroalcoholic extract of Echeveria subrigida showed hypoglycemic and anti-hyperglycemic activities, stability, and innocuity, suggesting its potential to prevent/treat diabetes mellitus.