TGF-β Research Articles

Abstract LB194: Methylation subtypes of skull base chordoma

Abstract Background: Chordoma is a rare bone tumor with a high recurrence partially due to incomplete tumor resection. Recent studies suggest that chordoma is a heterogeneous disease that may comprise of molecular subtypes manifesting distinct clinical outcomes. Molecular classification may improve our knowledge of chordoma biology and patient stratification. However, due to its rareness, tumor profiling data is limited. The aim of this study was to identify molecular subtypes of chordoma that may improve clinical management using methylation profiling data. Methods: Genomic DNA was extracted from fresh frozen tissues in 67 patients with skull-base chordoma from Beijing Tiantian Hospital, China. DNA was then profiled using an Infinium Methylation 850K BeadChip array. CpG sites involving low-quality data (CpG detection with p > 0.01), those on the X and Y chromosomes, and the sites overlapping with single-nucleotide polymorphisms were removed. RNA sequencing (RNA-Seq) data was available for 51 out of 67 tumor samples. Results: We conducted unsupervised clustering analysis using the top 8000 most variable methylation probes and identified two distinct clusters (Cluster 1 n = 40, Cluster 2 n = 27). Thirty-three significant differentially methylated regions (DMRs, p-adj < 0.05) were identified between the two clusters using Bumphunter. Gene set enrichment analysis (GSEA) Hallmark analysis identified four enriched pathways of interest: IL6 JAK STAT3 Signaling, Mitotic Spindle, TGF Beta Signaling, and Apical Junction among genes included in these DMRs (p-adj < 0.05). We used RNA-Seq data to identify significant correlations between methylation probes and gene expression associated with DMR genes (n=30 genes; n=150 probes). We found probes in four genes of interest (HOXA3, MBP, NEDD4L, and TEX14) showed consistent negative correlations with gene expression levels. HOXA3 encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation in pathways that are highly relevant to chordoma development. Previous research has cited HOX complex inhibition in highly therapy resistant chordoma relapses. NEDD4L is associated with regulation of the epithelial NA+ channels and plays an important role in regulating tumor cell function through ubiquitinating substrates. MBP is involved in nervous system development, while the role of TEX14 in cancer is less understood. Conclusion: Our results confirmed findings from previous studies that chordoma is molecularly heterogeneous. In addition to immune pathways, we showed that the two subtypes also demonstrated different epigenetic regulation in mitosis and cell adhesion related genes/pathways. Next, we will expand the analysis by including more samples to increase statistical power and to associate our epigenetic findings with patient outcomes. Our goal is to build a reliable classifier to facilitate clinical management. We will integrate whole genome/exome sequencing data to identify potential epigenetic drivers. Citation Format: Gulzar N. Daya, Yujia Xiong, Mingxuan Li, Hela Koka, Xiaoyu Wang, DiFei Wang, Shilpa Katta, Jiwei Bai, Xiaohong R. Yang. Methylation subtypes of skull base chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB194.

Pre-Transplant Cytokine Levels as Signatures of Microvascular Inflammation in Kidney Allograft Biopsies.

The presence of microvascular inflammation (MVI) characterized by leukocyte margination in the glomeruli (glomerulitis, Banff score 'g') and peritubular capillaries (peritubular capillaritis, Banff score 'ptc') is a hallmark histological feature of antibody-mediated rejection (AMR), even in the absence of circumferential C4d positivity. In this study, we assessed the efficacy of pre-transplant plasma cytokines as an ancillary screening tool to identify MVI in kidney allograft indication biopsies to facilitate better graft survival. This single-center prospective analytical study comprises 38 kidney transplant recipients whose peripheral blood was collected before transplant and assessed for the plasma cytokine concentrations of FOXP3, IL-6, TGF beta, and IL-17 using enzyme-linked immunosorbent assays (ELISA). Histopathological assessment was done in post-transplant indication biopsies, and Banff scores of 'g+ ptc' were calculated to categorize recipients into three MVI groups. The correlational, regression, and ROC curve analyses were used to assess the association and predictive ability of the cytokines with respect to MVI. In our study cohort, 27 recipients had MVI=0, five had MVI=1, and six had MVI≥2. A significant difference in plasma cytokines was observed between these groups, and we found a strong negative correlation of FOXP3 with MVI, whereas a strong positive correlation of IL-6, TGF beta, and IL-17 was recorded with MVI. We have also assessed the predictive ability of these cytokines, FOXP3, IL-6, TGF-beta, and IL-17, through the ROC curve, which showed an AUC of 0.70, 0.76, 0.84, and 0.72, respectively. Our findings suggest that the pre-transplant levels of cytokines FOXP3, IL-6, TGF-beta, and IL-17 could be measured to identify recipients at risk of post-transplant MVI, which could further serve as an additional tool for effective management of the kidney allograft.

TGF-β1 Drives Integrin-Dependent Pericyte Migration and Microvascular Destabilization in Fibrotic Disease

Interactions between endothelial cells (EC) and mural pericytes (PC) are critical to maintaining the stability and function of the microvascular wall. Abnormal interactions between these two cell types are a hallmark of progressive fibrotic diseases, such as systemic sclerosis (SSc, scleroderma). However, the role that PCs play in signaling microvascular dysfunction remains underexplored. It is hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Using pro-inflammatory TNF alpha (TNFα) or fibrotic growth factor TGF beta-1 (TGFβ1), human PC inflammatory and fibrotic phenotypes were evaluated by assessing their migration, matrix deposition, integrin expression, and subsequent effects on endothelial dysfunction. Both TNFα and TGFβ1 treatment altered integrin expression and matrix protein deposition, but only fibrotic TGFβ1 drove PC migration in an integrin-dependent manner. In addition, integrin-dependent PC migration was correlated to changes in EC angiopoietin-2 levels, a marker of vascular instability. Finally, there was evidence of changes in vascular stability corresponding to disease state in human SSc skin. The work presented demonstrates that TNFα and TGFβ1 induce changes in PC integrin expression and matrix deposition that facilitate migration and reduce vascular stability, providing evidence that microvascular destabilization can be an early indicator of tissue fibrosis.

Abstract 2685: Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers

Abstract Innate lymphoid cells (ILCs) and natural killer (NK) cells are a heterogeneous family of innate immune cells that regulate inflammation, immune tolerance and tissue homeostasis in infection, chronic inflammation, metabolic diseases and cancer. The ILC family lacks the expression of antigen-specific receptors and demonstrate effector function by generating cytokines and secreted proteins. In tumor microenvironment, ILCs play a dual role including both tumor regression and tumor promotion, suggesting their diverse phenotypes and functional states. Among the ILC family, ILC1s and NK cells share some features, including the expression of the transcription factor T-bet and the ability to produce IFN-γ. In the present study, we analyzed single-cell RNA sequencing (scRNAseq) data to investigate the landscape and functional status of ILC families in patients with head and neck squamous cell carcinoma (HNSCC). The GSE164690 dataset, including pre-processed scRNAseq data and clinical data, was obtained from Gene Expression Omnibus. A total of 17 pairs of tumor-derived CD45-positive cells and peripheral blood lymphocytes, consisting of 6 HPV-positive HNSCCs and 11 HPV-negative HNSCCs, were analyzed using the Seurat v4 R package. After quality control, a total of 95,809 cells were obtained and clustered into 16 immune cell clusters. Next, a total of 7,278 NK cells were extracted and sub-clustered into 11 clusters. To characterize the 11 clusters, the abundances of NK subsets were estimated by CIBERSORTx. This approach identified eight NK cell clusters, two intraepithelial ILC1 (ieILC1) clusters, and one ieILC1-NK-intermediate (ieILC1-NK-int) cluster. Single-sample GSEA (ssGSEA) analysis was performed to further characterize each subset. ieILC1 clusters expressed tissue-resident markers ITGA1, CD69, and CXCR6, whereas ieILC1-NK-int cluster expressed ILC1 markers CCR7, IL7R, and SELL. Principle component analysis (PCA) based on ssGSEA indicated that ieILC1 clusters and ieILC1-NK-int showed the different PCA distribution from NK clusters. Inflammation and effector function-related pathways, including inflammatory response, interferon-gamma response, and interferon-alpha response, were enriched in ieILC1 clusters compared with the others. Among ieILC1 clusters, several pathways, including hypoxia, TNFA signaling via NFKB, allograft rejection, TGF beta signaling, were more enriched in ieILC1-1 than in ieILC1-2, suggesting that ieILC1-1 is the most immunologically active phenotype among NK subclusters. Of note, ieILC1-2 mainly comprised cells isolated from HPV-negative tumors, whereas ieILC1-1 mainly comprised cells isolated from HPV-positive tumors. In conclusion, we have demonstrated the diverse landscape of NK and ieILC1 cells in patients with HNSCC. Further characterization of the ILC family would lead to a novel therapeutic approach against HNSCC. Citation Format: Hideyuki Takahashi, Shota Ida, Hiroe Tada, Kazuaki Chikamatsu. Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers [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 2685.

Abstract 2914: AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation

Abstract Background: While checkpoint inhibitors have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal objective responses observed. Phenomic has developed a proprietary machine learning/artificial intelligence platform to identify novel stromal targets with superior expression profiles that enable selective targeting of immune activating agents that will relieve these immunosuppressive barriers in difficult to treat indications. Methods: Using our single cell RNA Atlas, we assessed cancer-associated fibroblasts (CAFs) in several solid tumor indications for identification of novel targets, including proteoglycans. Our Atlas was also used to identify immune activating payloads whose cognate receptors were present in indications of interest. Antibodies were generated, and lead clones who demonstrated potent ligand binding and cell staining were used to generate fusion proteins to immune activating ligands. Efficacy and PD were assessed in multiple syngeneic tumor models. Results: Bioinformatic analysis identified a unique subset of pathogenic CAFs, which are TGF beta responsive, secrete several ECM proteins, and their presence tracks with poor outcome and resistance to immunotherapy in several solid tumor types. CTHRC1 was identified as a novel matrix protein highly expressed in this CAF subtype as well as tumor epithelium and is highly selective in a range of tumor types such as ovarian cancer, triple negative breast cancer, and pancreatic ductal adenocarcinoma. While secreted, CTHRC1 is complexed on the cell surface and affords the opportunity to drug this target in a variety of ways. We identified an absence of 4-1BBL expression across indications of interest, and fusion proteins were generated to deliver 4-1BBL via CTHRC1 targeting. In checkpoint-resistant tumor models, we observed significant increases in CD8 T cells and robust anti-tumor activity with anti-CTHRC1-targeted 4-1BBL. Biodistribution studies were conducted using our targeting mAb and demonstrate uptake only in sites of primary and metastatic tumors, even at doses 20-fold higher than those that achieve maximal therapeutic activity, suggesting the potential to minimize the toxicity that has been observed with other 4-1BB agonists. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both pathogenic CAFs and tumor cells that is highly selective for tumors, enabling the therapeutic targeting of immune activating payloads with the potential for safely delivering payloads while limiting toxicity. Given the specificity and selectivity afforded by CTHRC1 expression, ADC and CD3 engager approaches are also being pursued. These data represent novel approaches aimed at breaking down stromal barriers in tumors previously unresponsive to immunotherapies. Citation Format: Christopher Harvey, Elizabeth Koch, Amanda Hanson, Lindsey Rice, Amy Berkley, Kerry White, Reza Saberianfar, Nikolai Suslov, Sam Cooper, Michael Briskin. AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation [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 2914.

Abstract 1704: Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma

Abstract Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, 50% of patients develop metastatic disease responding poorly to chemotherapy, targeted therapy, and immunotherapy. The molecular basis of events leading to initiation of UM is related to mutations in G alpha proteins GNAQ or GNA11, rendering them constitutively active and activating downstream PKC, ERK and AKT pathways sustaining cell proliferation. In our previous work, we showed that NCOA3 regulates proliferation of UM cells in vitro and the growth of UM xenografts in vivo downstream of GNAQ or GNA11. The critical role of NCOA3 in UM is manifested by transcriptional regulation of proliferation-related genes that are massively suppressed by NCOA3 knockout or knockdown. Here, we interrogated transcription factors that NCOA3 interacts with in UM to execute oncogenic programs and identified Melanocyte inducing transcription factor (MITF) as its critical partner. Chromatin immunoprecipitation (ChIP) using an NCOA3 recognizing antibody followed by sequencing was performed to determine NCOA3 binding sites in the human genome. The known transcription binding motifs within NCOA3 peaks were discovered using HOMER algorithm. Co-immunoprecipitation was performed to examine direct NCOA3 and MITF interaction. ChIP sequencing for MITF was performed to determine co-localization of NCOA3 and MITF at chromatin. Proliferation of UM cells after inhibition of MITF by either siRNA mediated knockdown or MITF inhibitor ML329 was evaluated using MTT assay. Functional enrichment analysis of transcriptome was used to identify classes of genes regulated by MITF, with specific attention to the subset regulated by MITF partnered with NCOA3. NCOA3 chromatin peaks are highly enriched in MITF binding motifs, and 77% of the NCOA3 peaks co-localizes with the MITF peaks. NCOA3 co-immunoprecipitates with MITF. Among transcriptional programs regulated by MITF in UM are those related to melanocyte biology, differentiation, and melanin production, but also those related to melanoma growth, especially cell division, DNA replication, myc response, purine and pyrimidine metabolism, and mTOR signaling. Strong suppression of these transcriptional signatures by inhibition of MITF using siRNA-mediated knockdown or pharmacological inhibition correlates with dramatic loss of cell viability. Clinical data indicate that higher expression of MITF-positively regulated genes correlates with lower survival rates of UM patients. Importantly, the transcriptional programs regulated by MITF cooperating with NCOA3 were related to TGF beta signaling, cell adhesion, ECM interaction and actin cytoskeleton. Importance of MITF as an oncogene is manifested in uveal melanoma cell lines and clinical data. Oncogenic function of MITF is partially dependent on its interaction with a transcription co-activator NCOA3. Citation Format: Aleksandra Rusin, Maria E. Echartea, Sai P. Manikonda, Darlene G. Skapura, Christel M. Davis, Erik A. Ehli, Sandra L. Grimm, Cristian Coarfa, Salma Kaochar. Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma [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 1704.

Abstract 294: The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte

Abstract Homeobox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF beta signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7-overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7-HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration. Citation Format: Ahone Gina Akume, Kideok Jin. The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte [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 294.

Abstract 5445: Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach

Abstract Introduction: The cancer stem cell hypothesis explains the heterogeneity, drug resistance, and metastatic potential of Small Cell Lung Cancer (SCLC). To address limited surgical tissue availability, we developed an induced pluripotent stem cell (iPSC) model for investigating SCLC stemness mechanisms and potential novel therapies. Cigarette smoke condensates (CSC) have previously been linked to stem cell activation in lung epithelial cells. We aimed to transform normal lung iPSCs (Lu-iPSC) through prolonged CSC exposure to identify candidate genes in the transition to cancer stem cells. Methods: Four Lu-iPSC clones derived from normal human small airway epithelial cells (SAEC) of two healthy donors were exposed to CSC at different concentrations for short- (7 days) and long-term (6 months) periods. RNA-seq was performed to identify differentially expressed genes (DEGs) relative to respective SAEC. Gene expression data of 23 SCLC cell lines and 38 NSCLC from the public databases was extracted and analyzed relative to SAEC. In vitro tri-lineage differentiation studies, drug sensitivity assays, and phenotypic characterization by flow cytometry was performed in this study. Results: Gene expression profiles of Lu-iPSCs closely resembled those of SCLC rather than NSCLC. Short-term CSC exposure reduced Lu-iPSC proliferation while increasing the expression of key stemness markers such as SALL1 in a dose-dependent manner. Concurrently, CSC exposure downregulated the expression of nodal and its inhibitors, LEFTY1 and LEFTY2, which can be considered as the earliest signs of differentiation events in Lu-iPSC. Long term CSC exposure induced expression of epithelial mesenchymal transition markers including ZEB1 and ZEB2. In-vitro trilineage differentiation studies revealed that CSC particularly decreased expression of endodermal lineage specific markers (GATA4, SOX17, and FOXA2) without affecting mesodermal and ectodermal markers. Importantly, CSC induced the phenotypic expression of several lung cancer stem cell markers (CD44, CD133, and CD338) along with resistance to cisplatin and topotecan. RNA seq analysis of long-term CSC exposed Lu-iPSCs identified 92 common DEGs (53 up and 39 down) relative to their controls. Gene set enrichment analysis on DEGs indicated upregulation of Notch signaling, IGFBPs, TGF beta receptor signaling, and NCAM1 interactions while GABA receptor activation and GPCR signaling were downregulated. Comprehensive RNA seq analysis of CSC exposed and unexposed Lu-iPSCs relative to SCLC and NSCLC, identified potential candidate genes associated with SCLC stemness. Conclusion: Our findings suggest that this iPSC model shows promise for investigating mechanisms mediating stemness in SCLC and identifying candidate genes and their associated pathways as potential targets for preclinical development of novel treatment regimens for these highly lethal neoplasms. Citation Format: Sudheer Kumar Gara, Haitao Wang, Anand Singh, Tuana Tolunay, Vivek Shukla, Ruihong Wang, Mary Zhang, Stephanie Shiffka, David S. Schrump. Unravelling the molecular mechanisms of stemness in small cell lung cancer using an iPSC-based approach [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 5445.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

THE EFFECT OF ALOE VERA TOPICAL GEL EXTRACTION ON TGF-β1 EXPRESSION IN THERMAL BURN PALPEBRA GRADE II RATS MODEL

Abstract
 Introduction & Objectives : Grade II palpebral burns are burns that reach the epidermis and part of the dermis of palpebrae and can have impaired wound healing. TGF β1 has the broadest spectrum of action, affecting all cell types involved in all stages of wound healing. Decreased expression of TGF β1 is associated with impaired wound healing. Aloe Vera has long been known in Indonesia as a healing plant. Aloe Vera extract contains a variety of bioactive compounds that play a role in stimulating wound healing. The administration of Aloe Vera extract on thermal burns aims to reduce inflammation, increase collagen content, and increase the rate of re-epithelization. The administration of Aloe Vera gel extract allows faster healing of thermal burns and rebuilds the vascularity of the wound tissue.
 Methods : Objective: To determine the expression of Transforming Growth Factor Beta 1 (TGF β1) in a rat model of thermal burn injury grade II of the eyelids treated with topical Aloe Vera gel extract with the concentrations of 20%, 40%, Balanced Salt Sodium (BSS) and a control group that was not given burn intervention.
 Results : Methodology: an experimental study with the post-test-only approach with a control group design. 24 Rats have induced grade II eyelid thermal burns, divided into four groups: Normal (without treatment), negative control (BSS), given Aloe vera 20%, 40%. Then TGF β1expression was quantified
 Conclusion : The highest rise in TGF β1expression was found in the group with 40% Aloe vera gel, followed by 20% Aloe vera gel compared to the negative control (BSS Balanced Salt)

Urinary biomarkers in children with neurogenic and non-neurogenic lower urinary tract dysfunction: A systematic review and meta-analysis.

The aim of this systematic review is to assess urinary biomarkers studied in children with neurogenic and non-neurogenic lower urinary tract dysfunction (LUTD). The systematic review was conducted in accordance with the PRISMA guidelines. The screening was performed on PUBMED without any publication date limitation. Only original articles were included. Parameters related to the following topics were obtained: study design, characteristics of participants, number of participants, age, control group, types of biomarkers, measurement technique in urine, subgroup analysis, urodynamic findings, and outcome. Dutch Cochrane Checklist (DCC) and level of evidence by EBRO platform were used for quality assessment. Meta-analysis was performed with the Comprehensive Meta-Analysis Version 4 program. A total of 494 studies were screened and 16 studies were included. 11 (68.75%) were conducted in children with non-neurogenic LUTD and 5 (31.25%) neurogenic LUTD. Nerve growth factor (NGF) was evaluated in 12 studies, brain-derived neurotrophic factor (BDNF) in 5, Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) in 2, transforming growth factor beta-1 (TGF Beta-1) in 2, neutrophil gelatinase-associated lipocalin (NGAL) in 1, and Aquaporin-2 in 1. According to DCC, 10 (62.5%) articles were evaluated on 4 (37.5%) items and 4 articles on 5 items. The average score was 3.91+/-0.56. The level of evidence was found as B for 13 (81.25%) articles and C for 3 (18.75%). In meta-analysis, urinary NGF levels in children with non-neurogenic LUTS were significantly higher than in the healthy control group (Hedges's g = 1.867, standard error = 0.344, variance = 0.119, p = 0.0001). Urinary biomarkers are promising for the future with their noninvasive features. However, prospective studies with larger sample sizes are needed to better understand the potential of urinary biomarkers to reflect urodynamic and clinical findings in children with LUTD.

Abstract B072: SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms

Abstract As increasing numbers of MRIs and other scans are ordered for unrelated diseases, more and more people are being diagnosed with cystic neoplasms of the pancreas. While many or most of these will likely remain benign, intraductal papillary mucinous neoplasms (IPMNs) have a risk of progression to cancer and IPMN is now recognized as another precursor to pancreatic ductal adenocarcinoma. We are investigating the mechanisms by which IPMNs progress from benign to malignant disease in order to identify markers of progression and targets to block that progression. We found that loss of SMAD4 in conjunction with Krasmutation leads to IPMN that quickly becomes invasive in a mouse model. Examining human IPMN tissues, we found that only one in 46 benign IPMN tumors lacked detectable SMAD4 protein. Among 19 cases of invasive IPMNs, the sites of active invasion were SMAD4 negative in 7 cases, nuclear negative/cytoplasm positive in 3 cases (suggesting no active canonical signaling), and a mix of positive and negative cells in 3 cases, implicating complete or partial loss of SMAD-mediated signaling in 68% of actively invading cells. In order to understand how SMAD signaling blocks progression to invasion, we are examining cell lines from a benign mouse model of IPMN. While these lines do not have endogenous TGF beta signaling, they are capable of responding to exogenous TGF beta, suggesting that TGF beta-related ligands may be supplied by the tumor microenvironment rather than the tumor cells themselves. We are currently determining how different TGF beta family members contribute to regulation of growth and invasion in these cells. In summary, loss of the tumor suppressor SMAD4 allows an otherwise benign disease to progress to invasive cancer. Citation Format: Anna L. Means, Hannah Yoo, Quoc-Huy Trinh, Roshini Thiagarajan, Jinghuan Zi, Frank L. Revetta, Marcus C. Tan, Kathleen E. DelGiorno, Mary K. Washington. SMAD4 loss allows progression from benign to malignant disease in intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B072.

Systemic Interleukin-17 A and Transforming Growth Factor β1 in Acute Tonsillitis Patients

The study aim to this research was to figure out what role regulatory cytokines that are a play in patients. suffer from acute tonsillitis disease.to analyses the level of production of cytokine Interleukiny17j andjtransformingjgrowthjfactorjbetaj1 (IL17A and TGF β1 )and c-reactive protein .This study included collected about 92 blood sample from the patients who were the suffering from acute tonsillitis disease and ten (10)control for both genera male and female with age the ranging from the 1-60 years admitted in Al_ Hillah teaching the hospital and the hospital Babylon for birth and children during the a period from November 2017 Marchyy2018 This study included studying of some immunological parameter such as Interleukiny17j(IL-17)andjtransformingjgrowthjfactorjbetaj1(TGF-β)andj CRP The CRP –latex particles are coated with antibodies to human CRP. The CRP latex reagent has been standardized to detect serum CRP level at or above 6 mg/L which is considered the lowest concentration of clinical significance When the latex suspension was mixed with serum containing elevated. The results of CRP in this study show that 37 sample was positive for CRP test and the titer of CRP was higher than normal range CRP titer >5CRP increases when there is inflammation throughout the body . results of CRP test in this study show that’s 38 sample were show positive to these test titer of this test >5 The concentration level of the IL-17jand TGF_βk (systemic )were determined by Enzyme Linked Immune sorbent Assay (Elisa)method . The concentration level of IL-17systemic molecule with mean value (746.40)pg./ml respectively while the control group with mean value (659.76)pg./ml. The concentration level of TGF_β systemic with the mean value is (63.2942)pg./ml respectively while the control group with the mean value (54.6214)pg./ml .

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1β, Transforming Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Keratinocyte-to-macrophage communication exacerbate psoriasiform dermatitis via LRG1-enriched extracellular vesicles.

Rationale: Macrophage-associated inflammation and keratinocytes excessive proliferation and inflammatory cytokines secretion induced by stimulation play an important role in the progression of psoriasiform dermatitis. However, how these two types of cells communicate remains obscure. Methods: We induced a mouse model with experimental psoriasiform dermatitis by Imiquimod (IMQ). To investigate whether damaged keratinocytes promote macrophage polarization and accelerate skin lesions by releasing extracellular vesicle (EV), purified EV were isolated from the primary epidermis of 5-day IMQ-induced psoriasiform dermatitis model mice, and then fluorescence-labeled the EV with PKH67. The EV was injected into the skin of mice treated with IMQ or vehicle 2 days in situ. In addition, we established a co-culture system of the human monocytic cell line (THP-1) and HaCaT, and THP-1/HaCaT conditioned media culture model in vitro respectively. Subsequently, we evaluated the effect of Leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EV on macrophage activation. Results: We demonstrated macrophages can significantly promote keratinocyte inflammation and macrophage polarization may be mediated by intercellular communication with keratinocytes. Interestingly, IMQ-induced 5-day, keratinocyte-derived EV recruited macrophage and enhanced the progression of skin lesions. Similar to results in vivo, EV released from M5-treated HaCaT significantly promotes Interleukin 1β (IL-1β) and Tumor necrosis factor α (TNF-α) expression of THP-1 cells. Importantly, we found that LRG1-enriched EV regulates macrophages via TGF beta Receptor 1 (TGFβR1) dependent process. Conclusion: Our findings indicated a novel mechanism for promoting psoriasiform dermatitis, which could be a potential therapeutic target.

Transcriptome Analysis Revealed the Early Heat Stress Response in the Brain of Chinese Tongue Sole (Cynoglossus semilaevis).

As a common influencing factor in the environment, temperature greatly influences the fish that live in the water all their life. The essential economic fish Chinese tongue sole (Cynoglossus semilaevis), a benthic fish, will experience both physiological and behavioral changes due to increases in temperature. The brain, as the central hub of fish and a crucial regulatory organ, is particularly sensitive to temperature changes and will be affected. However, previous research has mainly concentrated on the impact of temperature on the gonads of C. semilaevis. Instead, our study examines the brain using transcriptomics to investigate specific genes and pathways that can quickly respond to temperature changes. The fish were subjected to various periods of heat stress (1 h, 2 h, 3 h, and 5 h) before extracting the brain for transcriptome analysis. After conducting transcriptomic analyses, we identified distinct genes and pathways in males and females. The pathways were mainly related to cortisol synthesis and secretion, neuroactive ligand-receptor interactions, TGF beta signaling pathway, and JAK/STAT signaling pathway, while the genes included the HSP family, tshr, c-fos, c-jun, cxcr4, camk2b, and igf2. Our study offers valuable insights into the regulation mechanisms of the brain's response to temperature stress.

Иммуновоспалительные механизмы старения мочевого пузыря.

Introduction. Despite the absence of a generally accepted theory explaining the mechanisms of aging, existing scientific data indicate a close relationship between aging and chronic low-grade inflammation (low-grade inflammation). This theory of inflammatory aging (inflame-aging) suggests that aging is accompanied by the development of a chronic inflammatory phenotype, which ultimately leads to fibroplastic transformation of tissues, which manifests itself in a functional deficiency of organs and, in particular, the bladder. Aims. To study the role of free radical oxidation, endothelial dysfunction and low-level age-associated inflammation as universal mechanisms of cellular senescence in the process of age-related detrusor rearrangement in experimental animals. Materials and methods. Two groups of laboratory animals were used in the experiment: group 1 (30-month-old white mongrel rats, n=10) and group 2 (10- month-old white mongrel rats, n=10). Markers of low-level age-associated inflammation (TNF-α, IL-6, IL1RA), endothelial dysfunction (ET-1, IL-1β, ESM1), profibrotic status (TGF beta 1 and CTGF), and free radical oxidation (8-isoprostane) were determined in blood and bladder homogenate, malonic dialdehyde, diene conjugates).The Student's criterion was used to compare two groups with a normal distribution. Results and discussion. It was shown that with age in experimental animals, the levels of inflammation markers (TNF-α, IL-6, IL1RA), endothelial dysfunction (ET-1, IL-1β, ESM1), lipid peroxidation (diene conjugates, silicone dialdehyde) and profibrotic cytokines (TGF beta 1 and CTGF) increased, while the levels of antioxidant activity (catalase) decreased statistically significantly.This pattern was revealed both at the systemic (blood plasma) and organ levels (bladder wall). Conclusion. The results of our experimental study confirm the role of chronic low-level inflammation in fibrosis-mediated inflammatory aging of organs and, in particular, in age-associated bladder reconstruction.

Differential transcriptome response of blood brain barrier spheroids to neuroinvasive Neisseria and Borrelia.

The blood-brain barrier (BBB), a highly regulated interface between the blood and the brain, prevents blood-borne substances and pathogens from entering the CNS. Nevertheless, pathogens like Neisseria meningitidis and Borrelia bavariensis can breach the BBB and infect the brain parenchyma. The self-assembling BBB-spheroids can simulate the cross talk occurring between the cells of the barrier and neuroinvasive pathogens. BBB spheroids were generated by co-culturing human brain microvascular endothelial cells (hBMECs), pericytes and astrocytes. The BBB attributes of spheroids were confirmed by mapping the localization of cells, observing permeability of angiopep2 and non-permeability of dextran. Fluorescent Neisseria, Borrelia or E. coli (non-neuroinvasive) were incubated with spheroids to observe the adherence, invasion and spheroid integrity. Transcriptome analysis with NGS was employed to investigate the response of BBB cells to infections. hBMECs were localized throughout the spheroids, whereas pericytes and astrocytes were concentrated around the core. Within 1 hr of exposure, Neisseria and Borrelia adhered to spheroids, and their microcolonization increased from 5 to 24 hrs. Integrity of spheroids was compromised by both Neisseria and Borrelia, but not by E. coli infection. Transcriptome analysis revealed a significant change in the expression of 781 genes (467 up and 314 down regulated) in spheroids infected with Neisseria, while Borrelia altered the expression of 621 genes (225 up and 396 down regulated). The differentially expressed genes could be clustered into various biological pathways like cell adhesion, extracellular matrix related, metallothionines, members of TGF beta, WNT signaling, and immune response. Among the differentially expressed genes, 455 (48%) genes were inversely expressed during Neisseria and Borrelia infection. The self-assembling spheroids were used to perceive the BBB response to neuroinvasive pathogens - Neisseria and Borrelia. Compromised integrity of spheroids during Neisseria and Borrelia infection as opposed to its intactness and non-adherence of E. coli (non-neuroinvasive) denotes the pathogen dependent fate of BBB. Genes categorized into various biological functions indicated weakened barrier properties of BBB and heightened innate immune response. Inverse expression of 48% genes commonly identified during Neisseria and Borrelia infection exemplifies unique response of BBB to varying neuropathogens.

Maternal diet during gestation affect prostatic tissue component in SHR/Izm offspring.

Numerous studies have investigated the associations between maternal nutritional status and various diseases, with the underlying mechanism often attributed to epigenetic changes. However, limited research has been conducted on the relationship between maternal nutrition and benign prostatic hyperplasia (BPH). In this study, we aimed to explore the potential association between maternal nutrition and BPH using an animal experiment and evaluating the findings through fluorescent immunostaining and genetic analysis. Female spontaneously hypertensive rats (SHR/Izm) were randomly assigned to three groups at the start of pregnancy: a standard diet group (SD; 17% protein, 7% fat), a low-protein diet group (LPD; 6% protein, 7% fat), and a high-fat diet group (HFD; 22% protein, 35% fat). The diets were maintained throughout gestation. After giving birth, both the mothers and their pups were exclusively fed a standard diet. Male pups were euthanized at 48 weeks, and their prostates were removed. The composition of the ventral prostate (VP) was evaluated using fluorescent immunostaining with antibodies for cytokeratin, vimentin, and Ki-67. Microarray analysis, real-time RT-PCR, and DNA methylation analysis using pyrosequencing were performed. Statistical analysis was conducted using one-way ANOVA and Tukey's multiple comparison test, with a significance level set at p < 0.05. Pups in the LPD group exhibited significant underweight from birth (1 day; SD vs.LPD vs.HFD: 4.46 vs. 4.08 vs. 4.35, p = 0.04) until weaning (21 days; SD vs.LPD vs.HFD: 30.8 vs. 27.4 vs. 29.2, p = 0.03). However, they exhibited catch-up growth, and there was no significant difference at 48 weeks (p = 0.84). The epithelial area in the ventral prostate was significantly increased in the LPD group (SD vs.LPD vs.HFD: 39% vs. 48% vs. 37%, p = 0.01), while the stromal area was significantly increased in the HFD group (SD vs.LPD vs.HFD: 11% vs. 11% vs. 15%, p < 0.01). Gene ontology analysis of the gene expression microarray showed increased activity in developmental processes (SD vs.LPD: p = 6.3E-03, SD vs.HFD: p = 7.2E-03), anatomical structure development (SD vs.LPD: p = 6.3E-03, SD vs.HFD: p = 5.3E-03), and cell differentiation (SD vs.LPD: p = 0.018, SD vs.HFD: p = 0.041) in both the LPD and HFD groups. Real-time RT-PCR revealed high expression levels of the transcription factors NFκB (p < 0.01) and Smad3 (p < 0.01) in both the LPD and HFD groups. XIAP, an apoptosis inhibitor, was increased in the LPD group (p = 0.02). The TGF beta pathway, associated with epithelial mesenchymal transition (EMT), and vimentin (p < 0.01) were upregulated in the HFD group. Pyrosequencing DNA methylation analysis of the TGF beta pathway indicated hypomethylation of TGFb1, TGFbR1, and Smad3 in all groups, although there were no significant differences. Our findings suggest that both maternal undernutrition and obesity influence the prostatic development of offspring. Maternal consumption of a low protein diet promotes epithelial hyperplasia through the upregulation of apoptosis inhibitors, while a high fat diet leads to increased stromal growth through the induction of EMT.

Efficacy and Tolerability of Luspatercept in Patients with Lower Risk Myelodysplastic Syndrome and Anemia: A Systematic Review and Meta-Analysis of Phase III Randomized Controlled Trials

Introduction: Erythropoietin Stimulating Agents (ESAs) are the standard-of-care treatment for most patients with lower risk myelodysplastic syndrome (MDS) and anaemia. However, there has been unmet need for those who failed ESAs other than regular blood transfusion. Luspatercept is a recombinant fusion protein which binds TGF beta ligands resulting in reduction of erythroid maturation through reduction of SMAD2 and SMAD3 signaling. In recent years, studies have shown that luspatercept has significantly improved efficacy in patients with lower risk MDS who failed ESAs with some notable adverse events. The purpose of our meta-analysis is to determine efficacy by observing the rate of transfusion independence for 12 weeks or longer and haematological improvement in erythroid (HIE), and tolerability by rate of treatment emergent serious adverse events (SAE), treatment discontinuation (TD) and treatment interruption (TI) or dose reduction and treatment related mortality in patients with lower risk MDS treated with luspatercept. Methods: We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 30 th, 2023. Phase III RCTs utilizing luspatercept in patients with lower risk MDS that mention efficacy i.e. transfusion independence 12 weeks or longer and HIE and tolerability i.e. rate of treatment discontinuation and interruption, dose reduction, treatment emergent serious adverse events, treatment related mortality were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Fixed effects model was applied. Results: 2 phase III RCTs (MEDALIST and COMMANDS) including total 583 patients were eligible in our meta-analysis. MEDALIST trial compared luspatercept vs placebo and COMMANDS trial compared luspatercept vs erythropoietin stimulating agent. The randomization ratios were 2:1 in MEDALIST and 1:1 in COMMANDS trials. Transfusion independence for 12 weeks or longer was noted in 141 (47%) in study arm vs 77 (33.5%) in control arm at RR of 1.67 (95% CI: 1.35 -2.06; P &amp;lt;0.00001). 190 (63.3%) patients in study arm achieved HIE response compared to 88 (38.3%) in control arm at RR of 1.85 (95% CI: 1.53 -2.24; P&amp;lt;0.00001). TD due to TEAE was reported in 21 (6.3%) of luspatercept group vs 10 (4%) in control group with RR of 1.38 (95% CI: 0.67 -2.84; P = 0.39). The incidence for TI or dose reduction due to TEAE was similar between the two groups at 18% with RR of 1.23 (95% CI: 0.88 -1.72; P = 0.22). Treatment emergent SAEs were observed in 116 (35%) in the luspatercept group vs 83 (32.9%) in control group (RR, 1.09; 95% CI: 0.87-1.38; P=0.45). Treatment related mortality was noted in 13 (3.9%) of patients in the study group compared to 16 (6.3%) in the control group with RR of 0.65 (95% CI: 0.31 -1.33; P = 0.24). Conclusions: According to our meta-analysis, luspatercept significantly improved transfusion independent rate and HIE response in patients with lower risk MDS and anaemia compared to control arm. Furthermore, there was no statistically significant difference in the treatment emergent SAEs, treatment discontinuation, treatment interruption or dose reduction and treatment related mortality between the two groups.