Proliferation Marker Ki-67 Research Articles

Evaluation of epithelial-to-mesenchymal transition and Ki-67 index in aggressive papillary thyroid cancer

ObjectivesThe present study aims to characterize immunohistochemical features of markers associated with Epithelial–Mesenchymal Transition (EMT) and proliferative activity that could lead to death in Papillary Thyroid Cancer (PTC). MethodsClinical data and tumor material were retrospectively collected. The patients were separated into death from PTC (Group 1), metastatic cases with indolent behavior (Group 2) and non-metastatic indolent PTC (Group 3). Immunohistochemical assessment of E-cadherin, β-catenin, Vimentin, ZEB-1 and Ki-67 was performed in each tumor and a semiquantitative estimation of the percentage of expression was fulfilled on the best marking area at high of the tumor invasion front. Results31 patients were included, 15 that died from PTC (Group 1), 6 in Group 2 and 10 in Group 3. The proliferative marker Ki-67 showed a significant difference in its expression in the tumor invasion front between the groups, specifically between groups 1 and 3 (p = 0.006). On the other hand, EMT-related immunohistochemical markers did not show significant difference in their percentage of expression, since loss of E-cadherin, β-catenin and Vimentin was observed in most cases at the invasion front. ConclusionPatients that died from PTC had a significantly higher Ki-67 labelling index compared to patients with indolent disease (cutoff of 11%). Ki-67 may have a role as a prognostic marker and could be considered for routine use in PTC. Level of evidence4.

Synergistic anti-tumorigenic effect of diosmetin in combination with 5-fluorouracil on human colon cancer xenografts in nude mice

5-Fluorouracil (5-FU) is frequently used to treat colorectal cancer (CRC), but its clinical application is limited by its toxicity. Natural compounds have been combined with chemotherapeutic drugs to reduce chemotherapy-related toxicity. Diosmetin, a natural flavonoid, has demonstrated anticancer effects against CRC. This study investigated diosmetin's potential in combination with 5-FU using a murine model of HCT-116 colon cancer xenografts in nu/nu nude mice. HCT-116 cells were injected into the right flanks of mice, and once tumors reached a size of 50 mm3, the mice were treated with diosmetin (100 mg/kg), 5-FU (30 mg/kg), or a combination of both at two dose levels (100 + 30 mg/kg and 50 + 15 mg/kg) for 4 weeks. Blood and tumors were collected on the final day for further analysis. Mice treated with the higher combination dose exhibited the smallest tumor volume (330.91 ± 88.49 mm3). Biochemistry and histology analysis showed no toxicity or abnormalities in the liver, kidney, and heart with the combination therapy. Immunohistochemistry results revealed a notable reduction in the proliferation marker (Ki67) and inflammation marker (TLR4) in tumors from high-dose combination-treated mice. Moreover, immunofluorescence data indicated increased levels of apoptotic markers (Bax, Caspase-3, p53, p21) and downregulation of anti-apoptotic protein (Bcl-2) in the high-dose combination group. The findings suggest that 100 mg/kg of diosmetin combined with 30 mg/kg 5-FU significantly reduced tumor volume and had a less toxic effect on the heart compared to 5-FU monotherapy.

The oxytocin-oxytocin receptors system — a new pathogenetic mechanism in the development of diabetic phenotype of heart failure with preserved ejection fraction in women

Aim. To determine the pathogenetic role of the oxytocinergic system in the development of myocardium structural and functional changes in women with heart failure with preserved ejection fraction (HFpEF) associated with type 2 diabetes mellitus (DM2T) (diabetic phenotype of HFpEF).Material and methods. The study included 60 women aged 67.0±4.9 years with HFpEF stage I-IIA, FC I-III, 30 of them had DM2T who were admitted for elective coronary artery bypass grafting. The development of HFpEF is caused by coronary artery disease (CAD) and arterial hypertension (AH). Prior to surgery, all patients underwent a standard examination, blood levels of NT-proBNP, oxytocin (Ox), echocardiography were determined to find the types of left ventricular (LV) myocardial remodeling and diastolic dysfunction (DD). Myocardium biopsies of the right atrium auricle obtained during coronary bypass surgery were studied by microscopy, morphometry and immunohistochemistry (the expression of oxytocin receptors (OxR), a marker of proliferation ki-67).Results. According to echocardiography, eccentric LV hypertrophy (46.7/36.7%) and DD type 2 (47/17%, p=0.003) prevailed in the group of women with the diabetic phenotype of HFpEF. A higher content of NT-proBNP (480.72±241.87/434.46±282.78 ng/ml, p=0.06) and a lower concentration of Ox (102.11±35.89/320.37±294.71 pg/ml, p=0.0016) in blood serum were established, as well as an increase in the number of cardiomyocytes (CMC) with a high expression level OxR (63.69±19.47/12.16±23.09%, p=0.000) in patients with the diabetic phenotype of HFpEF. Negative associations were determined between the blood level of Ox and the CMC diameter (r=-0.10, p=0.020), the area of their cytoplasm (r=-0.16, p=0.000) and the area of the nuclei (r=-0.11, p=0.015) in patients of both groups. A decrease in Ox concentration in the blood of patients with diabetic phenotype of HFpEF was accompanied by an increase in the number of CMCs with a high level of OxR expression (r=-0.63, p=0.000).Conclusion. The study has shown the important involvement of oxytocinergic signaling pathways in the HFpEF pathogenesis. HFpEF associated with DM2T in women was characterized by more unfavorable structural and functional changes in the myocardium, a significant increase in the number of hypertrophied CMCs with a high level of OxR expression and Ox decrease in blood serum. The mechanisms of the first-established significant increase in the content of Ox in the blood of patients with HFpEF without diabetes and its significant decrease in patients with diabetic phenotype of HFpEF leading to more pronounced structural and functional changes in the myocardium, require further study.

Bio-Pathological Functions of Posttranslational Modifications of Histological Biomarkers in Breast Cancer.

Proteins are the most common types of biomarkers used in breast cancer (BC) theranostics and management. By definition, a biomarker must be a relevant, objective, stable, and quantifiable biomolecule or other parameter, but proteins are known to exhibit the most variate and profound structural and functional variation. Thus, the proteome is highly dynamic and permanently reshaped and readapted, according to changing microenvironments, to maintain the local cell and tissue homeostasis. It is known that protein posttranslational modifications (PTMs) can affect all aspects of protein function. In this review, we focused our analysis on the different types of PTMs of histological biomarkers in BC. Thus, we analyzed the most common PTMs, including phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, palmitoylation, myristoylation, and glycosylation/sialylation/fucosylation of transcription factors, proliferation marker Ki-67, plasma membrane proteins, and histone modifications. Most of these PTMs occur in the presence of cellular stress. We emphasized that these PTMs interfere with these biomarkers maintenance, turnover and lifespan, nuclear or subcellular localization, structure and function, stabilization or inactivation, initiation or silencing of genomic and non-genomic pathways, including transcriptional activities or signaling pathways, mitosis, proteostasis, cell-cell and cell-extracellular matrix (ECM) interactions, membrane trafficking, and PPIs. Moreover, PTMs of these biomarkers orchestrate all hallmark pathways that are dysregulated in BC, playing both pro- and/or antitumoral and context-specific roles in DNA damage, repair and genomic stability, inactivation/activation of tumor-suppressor genes and oncogenes, phenotypic plasticity, epigenetic regulation of gene expression and non-mutational reprogramming, proliferative signaling, endocytosis, cell death, dysregulated TME, invasion and metastasis, including epithelial-mesenchymal/mesenchymal-epithelial transition (EMT/MET), and resistance to therapy or reversal of multidrug therapy resistance. PTMs occur in the nucleus but also at the plasma membrane and cytoplasmic level and induce biomarker translocation with opposite effects. Analysis of protein PTMs allows for the discovery and validation of new biomarkers in BC, mainly for early diagnosis, like extracellular vesicle glycosylation, which may be considered as a potential source of circulating cancer biomarkers.

Chemical Composition, Antioxidant Capacity, and Anticancerous Effects against Human Lung Cancer Cells of a Terpenoid-Rich Fraction of Inula viscosa.

Inula viscosa is a widely used plant in traditional Mediterranean and Middle Eastern medicine for various illnesses. I. viscosa has been shown to have anticancer effects against various cancers, but its effects against lung cancer have been under limited investigation. At the same time, I. viscosa is rich in terpenoids whose anti-lung cancer effects have been poorly investigated. This study aimed to examine the potential anticancer properties of methanolic and aqueous extracts of stems and leaves of I. viscosa and its terpenoid-rich fraction against human lung cancer A549 cells. Results showed that the methanolic extracts of I. viscosa had significantly higher polyphenol and flavonoid content and radical scavenging capacity than the aqueous extracts. In addition, leaves methanolic extracts (IVLM) caused the highest reduction in viability of A549 cells among all the extracts. IVLM also reduced the viability of human ovarian SK-OV-3, breast MCF-7, liver HepG2, and colorectal HCT116 cancer cells. A terpenoid-rich I. viscosa fraction (IVL DCM), prepared by liquid-liquid separation of IVLM in dichloromethane (DCM), displayed a substantial reduction in the viability of A549 cells (IC50 = 27.8 ± 1.5 µg/mL at 48 h) and the panel of tested cancerous cell lines but was not cytotoxic to normal human embryonic fibroblasts (HDFn). The assessment of IVL DCM phytochemical constituents using GC-MS analysis revealed 21 metabolites, highlighting an enrichment in terpenoids, such as lupeol and its derivatives, caryophyllene oxide, betulin, and isopulegol, known to exhibit proapoptotic and antimetastatic functions. IVL DCM also showed robust antioxidant capacity and decent polyphenol and flavonoid contents. Furthermore, Western blotting analysis indicated that IVL DCM reduced proliferation (reduction of proliferation marker Ki67 and induction of proliferation inhibitor proteins P21 and P27), contaminant with P38 MAP kinase activation, and induced the intrinsic apoptotic pathway (P53/BCL2/BAX/Caspase3/PARP) in A549 cells. IVL DCM also reduced the migration of A549 cells, potentially by reducing FAK activation. Future identification of anticancer metabolites of IVL DCM, especially terpenoids, is recommended. These data place I. viscosa as a new resource of herbal anticancer agents.

EEF1B2 regulates the proliferation and apoptosis of human spermatogonial stem cell lines through TAF4B

BackgroundSpermatogonial stem cells (SSCs) are essential for male fertility, maintaining sperm production throughout life. While mouse SSCs have been studied extensively, the mechanisms regulating human SSCs are less understood. ObjectivesTo investigate the role of EEF1B2 in regulating human SSC proliferation and apoptosis. Material and methodsSingle cell RNA sequencing (scRNA-seq) analysis was utilized to investigate the differentially expressed genes of SSC. The distribution of EEF1B2 in the human testis was examined using immunofluorescence and immunohistochemistry techniques. Cell proliferation, DNA replication, and self-renewal were analyzed using CCK8, EdU, Western blot, and flow cytometry. RNA sequencing was employed to analyze the downstream target molecules and signaling pathways of EEF1B2. ResultsIn this study, we analyzed single-cell sequencing data from human testicular samples and identified EEF1B2 as a protein highly expressed in SSCs, with expression decreasing during development. Immunohistochemistry and immunofluorescence confirmed this pattern and showed co-localization with the proliferation marker KI67. Knockdown of EEF1B2 in human SSC lines impaired proliferation and viability, reducing self-renewal proteins like PLZF and CCNE1. RNA sequencing revealed decreased TAF4B following EEF1B2 knockdown, which could be rescued by replenishing TAF4B. Testicular SSCs from non-obstructive azoospermia (NOA) patients also showed reduced EEF1B2. Discussion and conclusionOur findings reveal a novel regulatory mechanism involving EEF1B2 and TAF4B in human SSCs, suggesting EEF1B2 deficiency may contribute to male infertility.

Altered memory CCR6+ Th17-polarised T-cell function and biology in people with HIV under successful antiretroviral therapy and HIV elite controllers

Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6+ Th17-polarised CD4+ T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied. mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6+CD4+ T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and invitro functional assays. Decreased Th17cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production invitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6+ T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6+ T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6+ T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67. These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17cells. This study was funded by the Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Pogostemon cablin Extract Promotes Wound Healing through OR2AT4 Activation and Exhibits Anti-Inflammatory Activity.

Skin healing occurs through an intricate process called wound healing which comprises four phases: coagulation and hemostasis, inflammation, cellular proliferation, and remodeling. Chronic wounds often arise because of prolonged or excessive inflammation, which hinders the healing process and wound closure. Despite the recognized efficacy of Pogostemon cablin (patchouli) in wound healing, the precise mechanism of action of Pogostemon cablin extract (PCE) on inflammation and wound healing remains poorly understood. In this study, we investigated the effects of PCE on cell proliferation and wound healing, as well as its anti-inflammatory activity, using in vitro experiments. We found that PCE increased cell proliferation and expression of the cell proliferation marker Ki67 and accelerated wound healing in human keratinocytes through the activation of OR2AT4. Furthermore, PCE exhibited anti-inflammatory effects by decreasing the levels of pro-inflammatory cytokines interleukin-6 and -8 in lipopolysaccharide-treated and TNF-α-exposed THP-1 and HaCaT cells, respectively. Overall, these findings suggest that PCE holds therapeutic potential by promoting cell proliferation, facilitating wound healing, and exerting anti-inflammatory effects.

Revealing Cellular Heterogeneity and Key Regulatory Factors of Triple-Negative Breast Cancer through Single-Cell RNA Sequencing.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). TNBC has a poor prognosis due to high intratumoral heterogeneity and metastasis, pointing to the need to explore distinct molecular subtypes and gene regulatory networks. The scRNA-seq data of five primary BC samples were downloaded from the Gene Expression Omnibus (GEO) database. Clustering was performed based on filtered and normalized data using the Seurat R package to identify marker genes, which were subsequently annotated to each subset using the CellMarker database. AUCell R package was applied to calculate the hallmark score for each epithelial cell. Marker genes of each subset were screened with FindAllMarkers and their biological functions were analyzed using the Database for Annotation Visualization and Integrated Discovery (DAVID) database. Next, cell-cell communication was performed with the CellChat R package. To identify the key regulatory genes, single-cell regulatory network inference and clustering (SCENIC) analysis was conducted. Finally, the expression and potential biological functions of the key regulatory factors were verified through cellular experiments. A total of 29,101 cells were classified into nine cell subsets, namely, Fibroblasts, Fibroepithelial cells, Epithelial cells 1, Epithelial cells 2, Epithelial cells 3, Endothelial cells, T cells, Plasma B cells and Macrophages. Particularly, the epithelial cells had a higher proportion and higher transforming growth factor-β (TGF-β) activity in the TNBC pathotype as compared to the non-TNBC pathotype. Furthermore, four epithelial cell subsets (marked as Stearoyl-CoA Desaturase (SCD1), marker of proliferation Ki67 (MKI67), Annexin A3 (ANXA3) and aquaporin 5 (AQP5)) were identified as having the greatest impact on the TNBC pathotype. Cell-cell interaction analysis revealed that ANXA3-epithelial cell subset suppressed the T cell function through different mechanisms. C-fos gene (FOS) and X-box binding protein 1 (XBP1) were considered critical regulons involved in TNBC progression. Notably, cellular experiments demonstrated that silencing XBP1 and overexpressing FOS inhibited cancer cell invasion. The four epithelial cell subsets and two critical regulons identified based on the scRNA-seq data could help explore the underlying intratumoral heterogeneity molecular mechanism and develop effective therapies for TNBC.

PTBP1 is a potential indicator of disease progression in acute myeloid leukemia.

This study aimed to verify a novel potential indicator of disease progression in acute myeloid leukemia (AML) patients. Bone marrow samples were collected from 27 AML patients and 27 controls without hematological malignancies. Polypyrimidine tract-binding protein 1 (PTBP1) expression in bone marrow samples was measured, and the association of PTBP1 with the French-American-British (FAB) classification, cytogenetics, risk stratification, and complete remission (CR) rate was analyzed. The correlation between PTBP1 and Ki-67/p53 expression in AML patients was ultimately evaluated. The results showed that PTBP1 mRNA and protein levels were greater in AML patients than in controls. PTBP1 expression was able to distinguish between AML patients and controls (area under the curve, 0.8601; 95% confidence interval, 0.7632-0.9570). Furthermore, PTBP1 expression was associated with an increased frequency of internal tandem duplication mutations within FMS-like tyrosine kinase-3 (FLT3) and a complex karyotype, while PTBP1 expression was not correlated with FAB classification, monosomal karyotype, isolated biallelic CCAAT/enhancer-binding protein α (CEBPA) mutation, or nucleophosmin 1 (NPM1) mutation in patients with AML. Moreover, PTBP1 expression was associated with a poorer prognosis according to risk stratification and a lower CR rate in AML patients. In addition, PTBP1 expression was positively correlated with the expression of the proliferation marker Ki-67 and negatively correlated with the expression of the apoptosis marker p53 in AML patients. Overall, PTBP1 is a viable biomarker that contributes to the risk prediction and the determination of potential drug targets for AML.

Neoadjuvant cabozantinib restores CD8+ T cells in patients with locally advanced non-metastatic clear cell renal cell carcinoma: a phase 2 trial.

Cabozantinib is an oral multikinase inhibitor approved for treatment in metastatic renal cell carcinoma (RCC). We hypothesized that neoadjuvant cabozantinib could downstage localized tumors, facilitating partial nephrectomy, and facilitating surgery in patients with locally advanced tumors that would require significant adjacent organ resection. We, therefore, conducted a phase 2, single-arm trial of cabozantinib treatment for 12 weeks in 17 patients with locally advanced biopsy-proven non-metastatic clear cell RCC before surgical resection. Six patients (35%) experienced a partial response, and 11 patients (65%) had stable disease. We identified that plasma cell-free DNA (cfDNA), VEGF, c-MET, Gas6, and AXL were significantly increased while VEGFR2 decreased during cabozantinib treatments. There was a trend towards CD8+ T cells becoming activated in the blood, expressing the proliferation marker Ki67 and activation markers HLA-DR and CD38. Cabozantinib treatment depleted myeloid populations acutely. Importantly, immune niches made up of the stem-like CD8+ T cells and antigen presenting cells were increased in every patient. These data suggest that cabozantinib treatment was clinically active and safe in the neoadjuvant setting in patients with locally advanced non-metastatic clear cell RCC and activated the anti-tumor CD8+ T cell response. The trial is registered at ClinicalTrials.gov under registration no. NCT04022343.

Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

Investigation of the relationship between COVID-19 and pancreatic cancer using bioinformatics and systems biology approaches.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, poses a huge threat to human health. Pancreatic cancer (PC) is a malignant tumor with high mortality. Research suggests that infection with SARS-CoV-2 may increase disease severity and risk of death in patients with pancreatic cancer, while pancreatic cancer may also increase the likelihood of contracting SARS-CoV-2, but the link is unclear. This study investigated the transcriptional profiles of COVID-19 and PC patients, along with their respective healthy controls, using bioinformatics and systems biology approaches to uncover the molecular mechanisms linking the 2 diseases. Specifically, gene expression data for COVID-19 and PC patients were obtained from the Gene Expression Omnibus datasets, and common differentially expressed genes (DEGs) were identified. Gene ontology and pathway enrichment analyses were performed on the common DEGs to elucidate the regulatory relationships between the diseases. Additionally, hub genes were identified by constructing a protein-protein interaction network from the shared DEGs. Using these hub genes, we conducted regulatory network analyses of microRNA/transcription factors-genes relationships, and predicted potential drugs for treating COVID-19 and PC. A total of 1722 and 2979 DEGs were identified from the transcriptome data of PC (GSE119794) and COVID-19 (GSE196822), respectively. Among these, 236 common DEGs were found between COVID-19 and PC based on protein-protein interaction analysis. Functional enrichment analysis indicated that these shared DEGs were involved in pathways related to viral genome replication and tumorigenesis. Additionally, 10 hub genes, including extra spindle pole bodies like 1, holliday junction recognition protein, marker of proliferation Ki-67, kinesin family member 4A, cyclin-dependent kinase 1, topoisomerase II alpha, cyclin B2, ubiquitin-conjugating enzyme E2 C, aurora kinase B, and targeting protein for Xklp2, were identified. Regulatory network analysis revealed 42 transcription factors and 23 microRNAs as transcriptional regulatory signals. Importantly, lucanthone, etoposide, troglitazone, resveratrol, calcitriol, ciclopirox, dasatinib, enterolactone, methotrexate, and irinotecan emerged as potential therapeutic agents against both COVID-19 and PC. This study unveils potential shared pathogenic mechanisms between PC and COVID-19, offering novel insights for future research and therapeutic strategies for the treatment of PC and SARS-CoV-2 infection.

Human mesenchymal stem cells-derived microvesicles increase oligodendrogenesis and neurogenesis of cultured adult neural stem cells

Mesenchymal stem cells (MSCs) are involved in tissue repair and anti-inflammatory activities and have shown promising therapeutic efficiency in different animal models of neurodegenerative disorders. Microvesicles (MVs), implicated in cellular communication, are secreted from MSCs and play a key role in determining the fate of cell differentiation. Our study examines the effect of human umbilical cord MSC-derived MVs (hUC-MSC MVs) on the proliferation and differentiation potential of adult neural stem cells (NSCs). Results showed that 0.2 μg MSC derived MVs significantly increased the viability of NSCs and their proliferation, as demonstrated by an increase in the number of neurospheres and their derived cells, compared to controls. In addition, all hUC-MSC MVs concentrations (0.1, 0.2 and 0.4 µg) induced the differentiation of NSCs toward precursors (Olig2 + ) and mature oligodendrocytes (MBP+). This increase in mature oligodendrocytes was inversely proportional to the dose of MVs. Moreover, hUC-MSC MVs induced the differentiation of NSCs into neurons (β-tubulin + ), in a dose-dependent manner, but had no effect on astrocytes (GFAP+). Furthermore, treatment of NSCs with hUC-MSC MVs (0.1 and 0.2 μg) significantly increased the expression levels of the proliferation marker Ki67 gene, compared to controls. Finally, hUC-MSC MVs (0.1 μg) significantly increased the expression level of Sox10 transcripts; but not Pax6 gene, demonstrating an increased NSC ability to differentiate into oligodendrocytes. In conclusion, our study showed that hUC-MSC MVs increased NSC proliferation in vitro and induced NSC differentiation into oligodendrocytes and neurons, but not astrocytes.

IL-2 Complexed With Anti–IL-2 Antibody Expands the Maternal T-Regulatory Cell Pool and Alleviates Fetal Loss in Abortion-Prone Mice

Regulatory T (Treg) cells are essential for immune tolerance of embryo implantation and insufficient Treg cells are implicated in early pregnancy loss. An abortion-prone mouse model was utilized to evaluate the utility of IL-2 complexed with JES6-1 anti-IL-2 antibody (IL-2/JES6-1) to boost uterine Treg cells and improve reproductive success. IL-2/JES6-1 but not IL-2/IgG control administered in the peri-conception phase to CBA/J females mated with DBA/2 males elicited a >2-fold increase in the proportion of CD4+ T cells expressing FOXP3, and an increase in the ratio of FOXP3+ Treg cells to FOXP3- T conventional cells, in the uterus and its draining lymph nodes at embryo implantation that was sustained into mid-gestation. An attenuated phenotype was evident in both thymic-derived and peripheral Treg cells with elevated CTLA4, CD25, and FOXP3 indicating improved suppressive function, as well as increased proliferative marker Ki67. IL-2/JES6-1 treatment reduced fetal loss from 31% to 10%, but this was accompanied by a 6% reduction in late gestation fetal weight, despite comparable placental size and architecture. Similar effects of IL-2/JES6-1 on Treg cells and fetal growth were seen in CBA/J females with healthy pregnancies sired by BALB/c males. These findings show that expanding the uterine Treg cell pool through targeting IL-2 signaling is a strategy worthy of further investigation for mitigating immune-mediated fetal loss.

Synergistic Enhancement of Chemotherapy-Induced Cell Death and Antitumor Efficacy against Tumoral T-Cell Lymphoblasts by IMMUNEPOTENT CRP.

T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), present significant challenges to treatment due to their aggressive nature and chemoresistance. Chemotherapies remain a mainstay for their management, but the aggressiveness of these cancers and their associated toxicities pose limitations. Immunepotent CRP (ICRP), a bovine dialyzable leukocyte extract, has shown promise in inducing cytotoxicity against various cancer types, including hematological cancers. In this study, we investigated the combined effect of ICRP with a panel of chemotherapies on cell line models of T-ALL and T-LBL (CEM and L5178Y-R cells, respectively) and its impact on immune system cells (peripheral blood mononuclear cells, splenic and bone marrow cells). Our findings demonstrate that combining ICRP with chemotherapies enhances cytotoxicity against tumoral T-cell lymphoblasts. ICRP + Cyclophosphamide (CTX) cytotoxicity is induced through a caspase-, reactive oxygen species (ROS)-, and calcium-dependent mechanism involving the loss of mitochondrial membrane potential, an increase in ROS production, and caspase activation. Low doses of ICRP in combination with CTX spare non-tumoral immune cells, overcome the bone marrow-induced resistance to CTX cell death, and improves the CTX antitumor effect in vivo in syngeneic Balb/c mice challenged with L5178Y-R. This led to a reduction in tumor volume and a decrease in Ki-67 proliferation marker expression and the granulocyte/lymphocyte ratio. These results set the basis for further research into the clinical application of ICRP in combination with chemotherapeutic regimens for improving outcomes in T-cell malignancies.

Huangqin tang alleviates colitis-associated colorectal cancer via amino acids homeostasisand PI3K/AKT/mtor pathway modulation

Ethnopharmacological relevanceHuangqin Tang (HQT), a traditional Chinese medicine formula, is commonly used in clinical practice for the treatment of inflammatory bowel diseases. It has been reported that HQT exerts antitumor effects on colitis-associated colorectal cancer (CAC). However, the mechanism by which HQT interferes with the inflammation-to-cancer transformation remains unclear. Aims of the studyThe purpose of this study was to dynamically evaluate the efficacy of HQT in alleviating or delaying CAC and to reveal the underlying mechanism. MethodsWe established a mouse model of CAC using azoxymethane combined with 1.5% dextran sodium sulphate. The efficacy of HQT was evaluated based on pathological sections and serum biochemical indices. Subsequently, amino acids (AAs) metabolism analyses were performed using ultra-performance liquid chromatography-tandem mass spectrometry, and the phosphatidylinositol 3 kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway was detected by western blotting. ResultsThe data demonstrated that HQT could alleviate the development of CAC in the animal model. HQT effectively reduced the inflammatory response, particularly interleukin-6 (IL-6), in the inflammation induction stage, as well as in the stages of proliferation initiation and tumorigenesis. During the proliferation initiation and tumorigenesis stages, immunohistochemistry staining showed that the expression of the proliferation marker Ki67 was reduced, while apoptosis was increased in the HQT group. Accordingly, HQT substantially decreased the levels of specific AAs in the colon with CAC, including glutamic acid, glutamine, arginine, and isoleucine. Furthermore, HQT significantly inhibited the activated PI3K/AKT/mTOR pathway, which may contribute to suppression of cell proliferation and enhancement of apoptosis. ConclusionHQT is effective in alleviating and delaying the colon “inflammation-to-cancer”. The mechanism of action may involve HQT maintained AAs metabolism homeostasis and regulated PI3K/AKT/mTOR pathway, so as to maintain the balance between proliferation and apoptosis, and then interfere in the occurrence and development of CAC.

B Cell Lymphocytes as a Potential Source of Breast Carcinoma Marker Candidates.

Despite advances in the genomic classification of breast cancer, current clinical tests and treatment decisions are commonly based on protein-level information. Nowadays breast cancer clinical treatment selection is based on the immunohistochemical (IHC) determination of four protein biomarkers: Estrogen Receptor 1 (ESR1), Progesterone Receptor (PGR), Human Epidermal Growth Factor Receptor 2 (HER2), and proliferation marker Ki-67. The prognostic correlation of tumor-infiltrating T cells has been widely studied in breast cancer, but tumor-infiltrating B cells have not received so much attention. We aimed to find a correlation between immunohistochemical results and a proteomic approach in measuring the expression of proteins isolated from B-cell lymphocytes in peripheral blood samples. Shotgun proteomic analysis was chosen for its key advantage over other proteomic methods, which is its comprehensive and untargeted approach to analyzing proteins. This approach facilitates better characterization of disease-associated changes at the protein level. We identified 18 proteins in B cell lymphocytes with a significant fold change of more than 2, which have promising potential to serve as breast cancer biomarkers in the future.

P-629 In vivo follicular activation (IVFA) through repeated ovarian punction: results of an experimental study in a premature ovarian insufficiency rodent model

Abstract Study question Can the new IVFA therapy effectively promote ovarian follicle development and increase oocyte number in a premature ovarian insufficiency (POI) animal model. Summary answer The IVFA technique reveals favourable reproductive outcomes in the animal model, demonstrating increased oocyte retrieval without compromising quality, making a promising advancement in fertility restoration. What is known already POI is characterized by the loss of the primordial follicular pool before the age of 40. Ovarian fragmentation for in vitro follicular activation (IVA) is a technique based on the disruption of the ovarian extracellular matrix, subsequently promoting actin polymerization and interfering with the evolutionarily conserved tumour suppressor Salvador-Hippo-Warts (SHW) signalling pathway. This has been proven to increase expression of downstream growth factors, thus promoting the primordial follicle growth and the generation of mature oocytes. Yet, this procedure is invasive and expensive. Consequently, a demand exists for less invasive in vivo therapies. Study design, size, duration Experimental animal study. Sixty 6-week-old C57BL/6 female mice were divided into control (CT) and POI groups. Each group, was further divided into three subsets (n = 10/group): without ovarian punctures (CT-0 and POI-0), 15 punctures (CT-15 and POI-15), and 22 punctures (CT-22 and POI-22). Twenty-one days after POI induction, the new IVFA technique was performed on the anaesthetized animals. Subsequently, controlled ovarian stimulation (COS) was initiated, followed by euthanasia for the oocyte retrieval and histological ovarian evaluation. Participants/materials, setting, methods POI condition was induced with intraperitoneally injections of 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan. The IVFA technique involved ovarian punctures using a 30-gauge needle. COS was induced with 10 IU of PMSG and 10 IU of hCG 48 hours later, leading to euthanasia for oocytes and ovaries collection. Ovaries (n = 6/group) were fixed for follicular counts and immunohistochemistry. Oocyte quality assessment included reactive oxygen species (ROS) levels, mitochondrial fluorescence (Mitotracker), and spindle analysis. Main results and the role of chance The control groups subjected to the IVFA technique (CT-15 and CT-22) demonstrated significantly higher mean total oocyte counts compared to the non-intervention group (CT-0) (30.0±9.8 and 27.7±7.9 vs 18.1±11.7, respectively; p-value<0.05 in both comparisons). Similarly, within the POI model, interventions (POI-15 and POI-22) showed significantly elevated mean total oocyte counts than the non-intervention group (POI-0) (8.1±4.4 and 7.2±2.8 vs 3.8±3.6, respectively; p-value<0.05). The intensity of ROS fluorescence, Mitotracker fluorescence, and spindle analysis in retrieved oocytes were not affected by the IVFA therapy, thereby preserving oocyte quality. Notably, the percentage of growing follicles was significantly increased in the intervention groups compared to the non-intervention groups in the control (60±5.7% and 58.1±4.7% vs 41.5±4.5%; p-value<0.001). In the POI model, statistically significant differences were observed for POI-15 vs POI-0 (57.8±9% vs 41.6±13.3%; p-value<0.05), but not for POI-22 vs POI-0 (51.5±14.2% vs 41.6±13.3%, p=n.s.) Moreover, the global Ki67 proliferation marker demonstrated a significant increase in the percentage of proliferative cells in the CT-15 intervention group compared to the CT-0 group (53±11.2% vs 46.1±15.1; p < 0.01). Finally, in the POI model, the percentage of Ki-67 positive cells was also significantly higher in both IVFA groups (39.4±18.5% and 38.1±17.3% vs 32±17.5%, respectively; p < 0.05). Limitations, reasons for caution Since this is an animal model of POI, further validation of the new therapy with human ovarian samples would be required. The magnitude of the effect could vary due to inter-species variability. Wider implications of the findings The positive results of the new therapy in promoting follicle development and increasing oocyte retrieval, without compromising quality, have significant implications for developing new fertility treatments. If validated in humans, this approach could offer a promising solution for women with POI. Trial registration number not applicable

Retracing RAS signaling by correlating protein expression in different subtypes of neurofibromatosis 1-associated nerve sheath tumors.

Expression patterns of key proteins involved in RAS signaling and connected pathways were determined and correlated to possibly provide information for therapeutic application of RAS inhibitors in neurofibromatosis type 1 (NF1)-associated peripheral nerve sheath tumors (PNST). Clinical variables (age, sex), histological parameters (cell density, mitoses), and expression of immunohistochemically evaluated ligand and receptor proteins (neuregulin 1 (NRG1), ErbB2, ErbB3), RAS pathway proteins (mTor, Rho, phosphorylated MEK), transcription factors (Pax7, Sox9), and proliferation marker Ki-67, were correlated in cutaneous (CNF, n = 136), diffuse (DNF, n = 123)/diffuse plexiform (DPNF, n = 113), and plexiform neurofibroma (PNF, n = 126), and in malignant PNST (MPNST, n = 22). In CNF, NRG1 correlated with Ki-67 and Pax7. Further, mTOR correlated with ErbB3, Sox9, Pax7, and Ki-67. In DNF/DPNF, expression of NRG1 correlated with pMEK and Pax7. mTOR correlated with pMEK, Sox9, and Pax7. Noteworthy, pMEK was weakly expressed in some DNF but not in DPNF. ErbB3 correlated with mTor and Ki-67. Furthermore, Rho correlated with Pax7 and Ki-67. In PNF, ErbB3 expression was associated with Sox9, mTOR, pMEK, and Pax7 as well as mTOR with Sox9 and Pax7, Rho with pMEK and Pax7, and pMEK with Pax7 and Sox9. In MPNST, only few correlations were observed, ErbB2 correlated with Ki-67, and Rho with pMEK. Signaling networks of the RAS pathway could be retraced by correlation analysis of protein expression in subgroups of NF1 associated benign PNST. In regard to treatment of PNST, MEK inhibitors, which are presently evaluated for PNF, may possibly also be effective to some extent in DNF.