Expression Of TGF-beta Research Articles

Bicalutamide Reveals Immunomodulatory Effects in Prostate Cancer by Regulating Immunogenic Dendritic Cell Maturation

Prostate cancer poses a significant global health challenge, ranking as the second most prevalent and fifth most lethal malignancy among males. The intricate interplay between androgen signaling and the immune microenvironment underscores the complexity of prostate cancer progression. Notably, androgen receptor (AR) signaling has been shown to affect immune response mediated by tumor antigen-presenting dendritic cells (DCs). Therefore, this study aimed to explore the potential of Bicalutamide, a nonsteroidal anti-androgen, in modulating DCs-mediated immune responses. Peripheral blood mononuclear cells (PBMCs) were isolated, and monocytes were extracted, followed by their differentiation into immature dendritic cells (iDCs) using GM-CSF and IL-4. Harvested tumor cell lysates from human prostate cancer cells were then utilized to induce the transformation of iDCs into mature dendritic cells (mDCs). Then, mDCs were treated with non-toxic concentration of Bicalutamide determined by annexin V/PI assay. The morphological characteristics of mDCs were investigated using an inverted light microscope. Flow cytometry was used to determine the cell surface expression of molecular markers of DC maturation, and qRT-PCR was employed to evaluate expression levels of proinflammatory genes involved in DC maturation. The obtained results indicated that Bicalutamide treatment of monocyte-derived mDCs induces an immunogenic and matured phenotype, marked by increased expression of CD86 and HLA-DR. Besides, qRT-PCR results evidenced that Bicalutamide decreased the expression of anti-inflammatory genes, including Interleukin-10 (IL-10) and TGF-beta, as an indication of immunogenic DCs. These findings suggest that beyond its established anti-androgen role, Bicalutamide may exert anti-tumor effects through the modulation of DCs-mediated immune responses. This novel immunomodulatory feature holds promise for the development of novel therapies, including combination therapies, in prostate cancer treatment.

Intracavernosal mesenchymal stem cell therapy in ischaemic priapism: an experimental study.

The most common form of priapism is ischaemic and its prevalence in men has increased in recent years as a result of intracavernosal drug use. Currently, there is no approved specific treatment for ischaemic priapism other than cavernosal aspiration, which can only provide detumescence. This study aims to evaluate the efficacy of intracavernosal mesenchymal stem cell (MSC) therapy in an ischaemic priapism model. Thirty male Wistar albino rats were divided into three groups: sham (n = 6), priapism (n = 12) and priapism + MSC treatment (n = 12). The experimental groups were also divided into 1 and 12h subgroups of ischaemic priapism. The experimental model was created using a vacuum erection device and constrictive tape technique, and intracavernosal MSC were applied immediately after the tape was removed. After 4weeks, intracavernosal pressures (ICPs) and systemic mean arterial pressure (MAP) were measured. Penectomy was then performed to assess histopathological and molecular changes in the rats' penile tissues. In the ischaemic priapism model, MSC therapy showed significant improvements in peak and mean ICPs and mean ICP/MAP ratio. Histopathological analysis showed significant increases in smooth-muscle/collagen ratio and e-NOS and n-NOS expression. Although there was a decrease in fibrosis, it was not significant. At the molecular level, there were significant decreases in TGF-beta and VEGF mRNA expression, whilst NGF and BDNF mRNA-expression levels showed significant increases with MSC therapy. In terms of ICPs, the therapy showed more significant improvements in short-term priapism. However, when looking at histopathological and molecular parameters, the therapy had positive effects on a wider range of parameters in the long-term priapism. MSC treatment improved cavernosal physiology and had positive effects at the histopathological and molecular level in the ischaemic priapism model.

#2884 Dose-dependent EGR1 expression in the rat renal medulla after chronic NSAID treatment

Abstract Background and Aims Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in clinical medicine. Chronic use of NSAIDs is associated with reduced renal medullary blood flow, potentially causing severe and irreversible damage including papilla necrosis. We have previously demonstrated that chronic hyperosmolar injury in the renal medulla induces the expression of pro-fibrotic TGF-beta1 and EGR1. However, the impact of chronic NSAID treatment on EGR1 and pro-fibrotic factors in renal cortex and medulla remains unexplored. Thus, we aimed to analyze the effects of chronic NSAID treatment in the rat renal medulla. Method Male Wistar rats (250-260 grams) were orally treated twice daily for two weeks with various NSAIDs: indomethacin (IND, 2 mg/kg), naproxen (NAP, 10 or 20 mg/kg), celecoxib (CEL, 10 and 30 mg/kg), or vehicle (1% hydroxyethylcellulose). Left kidney was removed for histology and immunohistochemistry, the right kidney medulla was separated for mRNA analysis. Kruskal-Wallis test was performed for statistical analysis. Results NSAIDs caused significant tubular dilatation in all treated groups except IND. CEL (10, 30 mg/kg) and NAP (10 mg/kg) markedly increased epithelial atrophy. NAP 10 and 30 mg/kg induced a dose-dependent, 2-fold and 3.2-fold TIMP1 mRNA over-expression, respectively. Only NAP 20 mg/kg induced significant, 2.5-fold EGR1 mRNA expression. Both IND and CEL increased a 2-fold EGR1 protein expression in the medulla, but NAP induced a dose-dependent 5- and 10-fold increase of EGR1 protein. Conclusion Our study show that EGR1 and TIMP1 might play an important role in the development of NSAID induced tissue injury in the rat renal medulla. Further studies are needed to shed light on the molecular pathogenesis.

Mesenchymal stem cell-derived exosomes decrease Hyperplasia in Psoriasis by inducing transforming growth factor β2 (TGF-β2).

Psoriasis, a chronic inflammatory skin disease, is increasingly effectively managed with the targeted immunotherapy; however, long-term immunotherapy carries health risks, and loss of response. Therefore, we need to develop the alternative treatment strategies. Mesenchymal stem/stromal cell (M.S.C.) exosomes stand out for their remarkable immunomodulatory properties, gaining widespread recognition. This study investigated whether M.S.C. exosomes can reduce psoriasis-induced hyperplasia by inducing Transforming Growth Factor beta 2 (TGF-beta2) signaling. Exosomes were isolated from M.S.C.s by ultracentrifugation. Then, scanning electron microscopy was used for the morphology of exosomes. To ascertain the exosome concentration, the Bradford test was used. To ascertain the cellular toxicity of exosomes in Human Umbilical Vein Endothelial Cells ( H.U.V.E.C), an MTT experiment was then conducted. Real-time PCR was used to quantify TGF beta2 expression levels, whereas an ELISA immunosorbent assay was used to determine the protein concentration of TGF beta2. In this study, the exosomes of 15-30nm in size that were uniform, and cup-shaped were isolated. Moreover, the IC50 value for this Treatment was calculated to be 181.750µg/ml. The concentration of TGF-β2 gene in the target cells significantly increased following Treatment with the exosomes. Furthermore, the expression level of the studied gene significantly increased due to the Treatment. Upregulating the expression of TGF-β2 in psoriatic cells via TGF-β2 signaling is one way exosomes can help reduce hyperplasia.

Small airways morphological alterations associated with functional impairment in lymphangioleiomyomatosis

BackgroundLymphangioleiomyomatosis (LAM) is a rare neoplastic and cystic pulmonary disease characterized by abnormal proliferation of the so-called LAM cells. Despite the functional obstructive pattern observed in most patients, few studies investigated the morphological changes in the small airways, most of them in patients with severe and advanced LAM undergoing lung transplantation. Understanding the morphological changes in the airways that may occur early in the disease can help us understand the pathophysiology of disease progression and understand the rationale for possible therapeutic approaches, such as the use of bronchodilators. Our study aimed to characterize the morphological alterations of the small airways in patients with LAM with different severities compared to controls, and their association with variables at the pulmonary function test and with LAM Histological Score (LHS).MethodsThirty-nine women with LAM who had undergone open lung biopsy or lung transplantation, and nine controls were evaluated. The histological severity of the disease was assessed as LHS, based on the percentage of tissue involvement by cysts and infiltration by LAM cells. The following morphometric parameters were obtained: airway thickness, airway closure index, collagen and airway smooth muscle content, airway epithelial TGF-β expression, and infiltration of LAM cells and inflammatory cells within the small airway walls.ResultsThe age of patients with LAM was 39 ± 8 years, with FEV1 and DLCO of 62 ± 30% predicted and 62 ± 32% predicted, respectively. Patients with LAM had increased small airway closure index, collagen and smooth muscle content, and epithelial TGF-beta expression compared with controls. Patients with LAM with the more severe LHS and with greater functional severity (FEV1 ≤ 30%) presented higher thicknesses of the airways. Bronchiolar inflammation was mild; infiltration of the small airway walls by LAM cells was rare. LHS was associated with an obstructive pattern, air trapping, and reduced DLCO, whereas small airway wall thickness was associated with FEV1, FVC, and collagen content.ConclusionLAM is associated with small airway remodelling and partial airway closure, with structural alterations observed at different airway compartments. Functional impairment in LAM is associated with airway remodelling and, most importantly, with histological severity (LHS).

Fibrotic effect of monoculture, conditioned media and coculture of fibroblast NIH3T3 cells on collagen secretion and mRNA TGF-beta expressions

Fibrotic effect of monoculture, conditioned media and coculture of fibroblast NIH3T3 cells on collagen secretion and mRNA TGF-beta expressions

#5741 POLYPLOID TUBULAR CELLS INITIATE A TGF-BETA CONTROLLED LOOP THAT SUSTAINS POLYPLOIDIZATION AND TUBULOINTERSTITIAL CROSSTALK DURING AKI-CKD TRANSITION

Abstract Background and Aims Acute Kidney Injury (AKI) is characterized by a sudden kidney failure accompanied by a transient decrease of kidney functionality. It is regarded as an important risk factor for chronic kidney disease (CKD) development, but the link is still elusive. Renal fibrosis, especially tubulointerstitial fibrosis, is the final manifestation of CKD and is characterized by an excessive synthesis and deposition of extracellular matrix associated with inflammatory infiltration, tubular epithelial (TC) cell damage and fibroblast activation. Although no targeted therapy yet exists to slow the progression of tubulointerstitial fibrosis, recent findings contributed to clarify the cellular and molecular mechanisms underlying its development and progression, posing TC at the center of this process. Accordingly, we have recently demonstrated that fibrosis and senescence are trade-offs of TC polyploidy occurring immediately after AKI to support fast kidney function recovery, but promoting consequent CKD. However, the mechanisms turning TC polyploidy to senescence and fibrosis still need to be elucidated. In this study, we propose that TC polyploidy is the primary driver of CKD progression after AKI. Methods Polyploid TC are characterized by an increased DNA content in the absence of cell division. To discriminate polyploid cells from actively proliferating cells, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology. AKI was triggered by unilateral ischemia reperfusion injury (IRI) or glycerol-induced rhabdomyolysis. This technology allows to follow the cell cycle phasing of living cells. Cell sorting and cytofluorimetric techniques were employed to isolate and characterize a subpopulation of polyploid TC that progressively accumulate DNA damage after AKI. These results were further corroborated by single cell RNA-sequencing (scRNA-seq) analyses in vitro and in vivo. Results In this study, we found that immediately after AKI, expression of cell cycle markers mostly identifies a population of DNA damaged polyploid TC. Employing transgenic mouse models and single cell RNA-sequencing we showed that after AKI, polyploid TC accumulate DNA damage and survive eventually resting in the G1 phase of cell cycle, while diploid cells do not survive DNA damage. This suggests that after AKI, polyploidization is a means to survive injury. Sorting of DNA-damaged polyploid TC showed that they express p21 and acquire a pro-fibrotic phenotype culminating in TGF-beta expression. in vitro analysis and single cell RNA-sequencing evidenced that TGF-beta directly promoted polyploidization, demonstrating that TC polyploidization is a self-sustained mechanism. Interactome analysis by single cell RNA-sequencing revealed that TGF-beta signaling fostered a reciprocal activation loop among polyploid TC, macrophages and fibroblasts to sustain kidney fibrosis and promote CKD progression. Conclusion Collectively, this study contributes to the ongoing revision of the paradigm of kidney tubule response to AKI supporting the existence of a tubulointerstitial crosstalk mediated by TGF-beta signaling produced by polyploid TC following DNA damage. Moreover, this study identifies polyploid TC as central actors during CKD progression with important implications for the development of novel targeted therapy to block CKD.

Down-regulation of the Smad signaling by circZBTB46 via the Smad2-PDLIM5 axis to inhibit type I collagen expression.

Abnormal type I collagen (COL1) expression is associated with the development of many cardiovascular diseases. The TGF-beta/Smad signaling pathway and circRNAs have been shown to regulate COL1 gene expression, but the underlying molecular mechanisms are still not fully understood. Gain- and loss-of-function experiments were prformed to study the effect of circZBTB46 on the expression of alpha 2 chain of type I collagen (COL1A2). Co-immunoprecipitation assay was performed to observe the interaction between two proteins. RNA immunoprecipitation assay and biotin pull-down assay were performed to observe the interaction of circZBTB46 with PDLIM5. In this study, we investigated the role of circZBTB46 in regulating COL1A2 expression in human vascular smooth muscle cells (VSMCs). We found that circZBTB46 is expressed in VSMCs and that TGF-beta inhibits circZBTB46 formation by downregulating KLF4 expression through activation of the Smad signaling pathway. CircZBTB46 inhibits the expression of COL1A2 induced by TGF-beta. Mechanistically, circZBTB46 mediates the interaction between Smad2 and PDLIM5, resulting in the inhibition of Smad signaling and the subsequent downregulation of COL1A2 expression. Furthermore, we found that the expression of TGF-beta and COL1A2 is decreased, while circZBTB46 expression is increased in human abdominal aortic aneurysm tissues, indicating that circZBTB46-mediated regulation of TGF-beta/Smad signaling and COL1A2 synthesis in VSMCs plays a crucial role in vascular homeostasis and aneurysm development. CircZBTB46 was identified as a novel inhibitor of COL1 synthesis in VSMCs, highlighting the importance of circZBTB46 and PDLIM5 in regulating TGF-beta/Smad signaling and COL1A2 expression.

MWF RAT AS A NOVEL ANIMAL MODEL OF CARDIORENAL SYNDROME TYPE IV

Objective: Cardiorenal syndrome type 4 (CRS type 4) is characterized by primary chronic kidney disease (CKD) leading to cardiac dysfunction and an increase in cardiovascular events. There is an unmet need for suitable animal models of CRS to further define the pathogenesis and progression of CRS to test new therapeutic strategies. We aim to determine if CKD in aged Munich Wistar Frömter (MWF) rat may translate to cardiovascular alterations of CRS. Design and method: Profibrotic markers and angiotensin receptor (ATR) expression was measured by RT-q-PCR in kidney, aorta and heart from 22 and 45-week-old (aged) male MWF rats (n = 7-10/group). Results: Aged MWF showed higher renal expression of tubular damage markers (Kim-1 and Ngal) and profibrotic factors (Col1A1, Col3A1, TGF-beta) together with lower levels of AT2R. Thoracic aorta also exhibited increased expression of profibrotic CTGF-1 and Col1-A1, as well as a lower AT2R expression. Both age groups were hypertensive with similar systolic and diastolic blood pressure levels. However, aged MWF had an increased PWV and heart weight, together with increased intrinsic arterial stiffness and an outward hypertrophic remodeling in mesenteric arteries. In 45-week-old MWF, levels of cardiac CTGF-1, AT1R and AT2R were higher, whereas TGF-beta expression was lower. No differences were observed for Col1A1 or Col3A1. Conclusions: The presence of renal and vascular fibrosis, hypertension, arterial stiffness and cardiac hypertrophy, together with inflammation, vascular remodeling and early profibrotic cardiac environment, potentially counterregulated by AT2R make aged-MWF rat an useful model for CRS study.

The Effects of Mesenchymal Stem Cells on the Gene Expression of TGF-beta and IFN-gamma in Patients with Rheumatoid Arthritis.

The therapeutic and immunomodulatory potential of mesenchymal stem cells (MSCs) in rheumatoid arthritis (RA) has attracted considerable scientific attention in recent decades. This study aimed to evaluate the expression of genes encoding interleukin (IL)4 and IL10, as well as interferon-gamma (IFNG) and transforming growth factor beta (TGFB1) in refractory RA patients following intravenous injection of autologous bone marrow-derived MSCs (BM-MSCs). This study was registered in Iranian Registry of Clinical Trials (IRCT) (2015102824760N1) and ClinicalTrials.gov (identifier: NCT03333681). Blood samples were taken from 13 patients before and 1 and 6 months after the MSC injection to evaluate the clinical manifestations, paraclinical factors, and expression of IL4, IL10, IFNG, and TGFB1 genes employing the SYBR Green real-time reverse-transcriptase polymerase chain reaction (RT-PCR) technique. There was a significant increase in the expression of TGFB1 at 1 and 6 months after the MSC injection compared to that in the baseline, while the expression of IL4 and IL10 did not change significantly. On the other hand, the expression of IFNG increased significantly after 1 month but decreased significantly at 6 months compared to 1 month after the intervention. Nevertheless, it showed no significant decrease compared to the baseline. A significant decrease was observed for the expression of IFNG 6 months after the injection compared to that after 1 month, which was in concordance with the rise in the expression of the TGFB1 gene. A significant change in the gene expression of TGFB1 and IFNG in our study wasconsistent with the amelioration of clinical manifestations, suggesting a mechanism of action for MSCs in the treatment of RA.

Abstract 5141: Macrophage transcriptomic signature validation in scRNA seq and overall survival differences in urothelial carcinoma

Abstract Introduction: While the prognostic value of immune system biomarkers has been well explored, role of cancer associated macrophages in bladder cancer(BC) is unclear due to lack of consistent results, coupled with tissue nonspecific transcriptomic signature. Methodology: Data acquisition from 3,936 patients of The Cancer Genome Atlas (TCGA) in addition to Gene Expression Omninus (GEO) data sets GSE13507, GSE16945, GSE48277, GSE32894, GSE149582, as well as European Genotype Phenotype EGAS000001004507 was obtained. Tumor associated macrophages M1 and M2 were defined utilizing 188 and 159 gene expression profiles via xCell computational algorithm. To obtain bladder tissue residence macrophage signatures, scRNA seq was performed on tissue collected from surgical resection of 5 HG bladder cancer patients and 2 healthy controls. CellRanger software package with default parameters was utilized giving a total of 8,068 cells. FindAllMarkers was utilized for integration of DEG genes (cancer vs control) among the cluster and identification of BC macrophage specific markers. Endpoint of OS were measured in months from the time of cystectomy to follow up. Results: Surprisingly, presence of M1 infiltration was found to be associated with improved OS in two out of eight cohorts only (GSE32894, GSE70691, p<0.001), while the remainder detected no significant difference. Similarly, no association with OS was detected among all 8 cohorts with M2 infiltration. To improve our understanding of tissue specific markers of macrophage population in bladder, we then analyzed macrophage clusters detected within scRNAseq of BC patients compared to healthy. Gene ontology enrichment analysis of functions within tumor specific macrophages demonstrated an exaggerated expression of mTORC1 signaling, PI3K/AKT/mTOR signaling, TGF beta and EGF receptor pathways compared to non-cancer controls. Reanalysis of 22 tissue specific markers for M1/M2 infiltration showed no difference in any of the 8 cohorts with M1 high vs low infiltration. Similarly, addition of M2 infiltration as predictive marker yielded no further associations with OS in all but one cohort (GSE32894, p=0.00041), where high M2 tumor presence was associated with improved OS. Conclusion: Our study represents the largest TAMs evaluation of BC across 8 cohorts with additional scRNA seq exploration of tissue specific signatures, demonstrating no association with OS in bladder cancer. Citation Format: Laura Bukavina, Spencer Bell, Daniel Geynisman, Ilaha Isali, Daniel Ranti, John Sfakianos, Henkel Valentine, Adam Calaway, Alexander Kutikov, Andres Correa, Robert Uzzo, Lee Ponsky, Philip Abbosh. Macrophage transcriptomic signature validation in scRNA seq and overall survival differences in urothelial carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5141.

Abstract 6415: CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel

Abstract Background: Regulatory T cells (Tregs) have a strong immunosuppressive function, inhibit anti-tumor immunity, and promote the occurrence and development of tumors. In addition, a high infiltration of Treg cells is associated with poor survival in various types of cancer. Since CD25 is highly expressed on Treg cells, it is a potential target protein for Treg depletion. Methods: The binding affinity(Kd) of CD25 aptamer to rhCD25 protein was calculated through bio-layer interferometry(BLI). The cell binding of Cy5-labelled CD25 aptamer was detected by flow cytometry, and the internalization was visualized by confocal microscopy. The cell internalization rate was determined using the MFI value of flow cytometry. Competitive ELISA, western blot, and qPCR were performed to investigate the effect of CD25 aptamer on CD25/IL-2 binding. In vitro cytotoxicity of prototype-ApDC was determined by CCK-8 assay. Moreover, CD25-dependent cytotoxicity of prototype-ApDC was evaluated by Karpas299(Treg-like):HuT78(Teff-like) 1:1 co-culture model using FACS analysis. Results: CD25 aptamer binds human CD25 protein with 4.64nM affinity. CD25 aptamer showed selective binding and internalization to CD25-positive Karpas299 and had no interaction with CD25-negative Daudi. The internalization half-time of CD25 aptamer in karpas299 was determined to 9.9 min (95% CI 8.2~12.6min). Interestingly, CD25 aptamer was identified as IL-2R antagonist by evaluating competitive ELISA and the level of phosphor-STAT5 and downstream mRNA expression of TGF-beta. The IC50 value of prototype-ApDC was 24nM to Karpas299 while >1uM to Daudi. These results show that prototype-ApDC has selective cytotoxicity against CD25-expressing cells. Furthermore, in a study of an in vitro co-culture model mimicking the Treg-rich intratumoral T cell environment, prototype-ApDC treatment induced CD25-specific depletion resulting in escalating the ratio of HuT89/Karpas299 with increasing exposure time. Conclusions: Our CD25-targeted aptamer and its drug-conjugated ApDC have demonstrated the selective blocking of IL-2R-STAT5 signaling and induction of CD25-positive cell-specific depletion, respectively. Moreover, in the co-culture system of the Treg-like and Teff-like cells, CD25-ApDC also proved its cytotoxic selectivity. Therefore, one potential strategy in cancer patients is to use CD25-ApDC with excellent tumor permeability as an anticancer agent with a mechanism of Treg cell depletion through specific targeting of CD25 overexpressed in tumor-infiltrating Treg cells. Citation Format: Daekyun Lee, Ju-hyung Kang, Inu Song. CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6415.

PD25-01 MACROPHAGE TRANSCRIPTOMIC SIGNATURE VALIDATION IN SCRNA SEQ AND OVERALL SURVIVAL DIFFERENCES IN UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023PD25-01 MACROPHAGE TRANSCRIPTOMIC SIGNATURE VALIDATION IN SCRNA SEQ AND OVERALL SURVIVAL DIFFERENCES IN UROTHELIAL CARCINOMA Laura Bukavina, Imran Safder, Spencer Bell, Daniel Geynishman, Ilaha Isali, Daniel Ranti, John Sfakianos, Henkel Valentine, Adam Calaway, Alexander Kutikov, Andres Correa, Robert Uzzo, Lee Ponsky, and Philip Abbosh Laura BukavinaLaura Bukavina More articles by this author , Imran SafderImran Safder More articles by this author , Spencer BellSpencer Bell More articles by this author , Daniel GeynishmanDaniel Geynishman More articles by this author , Ilaha IsaliIlaha Isali More articles by this author , Daniel RantiDaniel Ranti More articles by this author , John SfakianosJohn Sfakianos More articles by this author , Henkel ValentineHenkel Valentine More articles by this author , Adam CalawayAdam Calaway More articles by this author , Alexander KutikovAlexander Kutikov More articles by this author , Andres CorreaAndres Correa More articles by this author , Robert UzzoRobert Uzzo More articles by this author , Lee PonskyLee Ponsky More articles by this author , and Philip AbboshPhilip Abbosh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: While the prognostic value of immune system biomarkers has been well explored, role of cancer associated macrophages in BC is unclear due to lack of consistent results, coupled with tissue nonspecific transcriptomic signature. METHODS: Data acquisition from 3,936 patients of The Cancer Genome Atlas (TCGA) in addition to Gene Expression Omninus (GEO) data sets GSE13507, GSE16945, GSE48277, GSE32894, GSE149582, as well as European Genotype Phenotype EGAS000001004507 was obtained (Figure 1A). To obtain bladder tissue residence macrophage signatures, scRNA seq was performed. Endpoint of OS were measured in months from the time of cystectomy to follow up. Statistical analysis was performed using R software. RESULTS: Based on previous literature highlighting “M1” macrophage role in creating an anti-tumor environment, we expected “M1” high tumors to correlate with improved survival, while “M2” tumors with worse OS. Surprisingly, presence of M1 infiltration was found to be associated with improved OS in two out of eight cohorts only (GSE32894, GSE70691, p<0.001), while the remainder detected no significant difference. To improve our understanding of tissue specific markers of macrophage population in bladder, we then analyzed macrophage clusters detected within scRNAseq of BC patients compared to healthy. A total of 8 populations of macrophages were detected among all cell types, with 22 unique macrophage markers present in BC only patients. (Figure 1 C, D).Reanalysis of 22 tissue specific markers for M1/M2 infiltration showed no difference in any of the 8 cohorts with M1 high vs low infiltration. Gene ontology enrichment analysis of functions within tumor specific macrophages demonstrated an exaggerated expression of mTORC1 signaling, PI3K/AKT/mTOR signaling, TGF beta and EGF receptor pathways compared to non-cancer controls. (Figure 1E). CONCLUSIONS: Our study represents the largest TAMs evaluation of BC across 8 cohorts with additional scRNA seq exploration of tissue specific signatures, demonstrating no association with OS. While these results imply that overall TAMs may not be utilized as a predictive marker for OS in bladder cancer, studies of TAM subtypes/populations are indicated to assess their role as prognostic indicators and therapeutic targets. Source of Funding: AUA Research Scholars © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e729 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Laura Bukavina More articles by this author Imran Safder More articles by this author Spencer Bell More articles by this author Daniel Geynishman More articles by this author Ilaha Isali More articles by this author Daniel Ranti More articles by this author John Sfakianos More articles by this author Henkel Valentine More articles by this author Adam Calaway More articles by this author Alexander Kutikov More articles by this author Andres Correa More articles by this author Robert Uzzo More articles by this author Lee Ponsky More articles by this author Philip Abbosh More articles by this author Expand All Advertisement PDF downloadLoading ...

Matrix Metalloproteinase-2, Matrix Metalloproteinase-9, and Transforming Growth Factor Beta 1 Levels in Escherichia coli-Infected Rats with Acute Pyelonephritis

Objective: To investigate the expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and transforming growth factor beta 1 (TGF beta 1) in the kidney tissue of rats with pyelonephritis and their relationship with pyelonephritis by establishing a rat model of acute pyelonephritis. Methods: 80 male Wistar rats were randomly divided into a control group and an experimental group, with 40 rats each. The rats of the control group were injected with and saline and those of the experimental group were injected with 10 ?g/mL Escherichia coli (E. coli) and saline (1:100); the solutions for both groups were administered every 3 days for 7 days. The expressions of MMP-2, MMP-9 and TGF beta 1 in the kidney tissues of rats in each group were observed. Results: The expression of MMP-9 and TGF beta 1in the kidney tissue of rat acute pyelonephritis model rats was significantly higher than those of the control group (P &lt; 0.01); the MMP-9 mRNA content in the kidney tissue of the experimental group was significantly higher than that of the control group (P &lt; 0.05); the TGF beta 1 mRNA content in the renal tissue of the experimental group increased significantly compared to the (P &lt; 0.05); MMP-2, MMP-9 and TGF beta 1 began to express in the early stage of pyelonephritis until the complete formation of renal pelvic edema. The difference between groups was statistically significant (P &lt; 0.01). Conclusion: MMP-9 and TGF beta 1 are important factors regulating renal tubular epithelial cell injury and inflammatory response.

Role of TGF-beta1 and TNF-alpha1 produced by neoplastic cells in the pathogenesis of fibrosis in patients with hematologic neoplasms.

We conducted this study with the objective of elucidating the mechanism of development of fibrosis in hematologic neoplasms and develop treatments for these patients. Among the suggested mechanisms of development of fibrosis is cases of hematologic neoplasms is the production of TGF-beta1 (transforming growth factor-beta-1) and TNF-alpha1 (tumor necrotizing factor-alpha-1) by the tumor cells, both of which are fibrosis-stimulating cytokines that act on fibroblasts to promote fibrosis. However, there are few reports based on human clinical pathology studies. We conducted an immunohistochemical study on paraffin-embedded formalin-fixed specimens obtained from 104 patients with various pathologic conditions (acute leukemia, malignant lymphoma, inflammation, cancer, etc.). The association of tissue fibrosis with positive immunohistochemistry for TGF- beta1 and/or TNF-alpha1, TGF-beta1 was found to be strongly associated with tissue fibrosis, and in cases with positive immunohistochemistry for TGF-beta1, the odds ratio for fibrosis was 12.8, which was significantly high. Combined positivity for TGF-beta1 and TNF-alpha1 was also associated with a significant odds ratio for fibrosis of 3.4, suggesting that TGF-beta1 expression is an important prerequisite. TGF-beta1 has been suggested as playing a relatively important role in tissue fibrosis. Future clinical application of these cytokines for both diagnosis and treatment is expected.

Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes.

Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)-CB1R agonist, rimonabant-CB1R antagonist, John W. Huffman (JWH133)-CB2R agonist and CB2R antagonist-6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation.

Platelet-rich fibrin (PRF) graft and amniotic membrane graft on transforming growth factor-β (TGF-β) and type 1 collagen post conjunctival excision

Background: Conjunctiva, when injured, can heal spontaneously. However, in extensive wounds, wound healing is often accompanied by scarring and wound contraction. Currently, for cases of disease and trauma to the surface of the eyeball, it can be reconstructed using the amniotic membrane, but there are still weaknesses in the use of amnion. Platelet-rich fibrin (PRF) is currently being developed for the reconstruction of ocular surface wounds. Methods: A total of 20 male New Zealand rabbits (20 eyes) were divided into two groups. In the first group, conjunctival excision and PRF membrane graft suturing were performed on the right eye conjunctival defect, while the second group had an amniotic membrane sutured on the right eye conjunctival defect. On the 14th day, enucleation was carried out and continued with histopathological examination using immunohistochemistry using TGF antibodies and type 1 collagen to evaluate the levels of TGF and type 1 collagen in the conjunctiva. Results: The results after 14 days of treatment showed that there was a very significant difference in TGF Beta expression between the AMT group and the PRF group (p = 0.007*; &lt;0.05). However, in the expression of type 1 collagen, there was no significant difference in the expression of type 1 collagen between the AMT group and the PRF group (p = 0.791; &gt;0.05). Conclusion: There were differences in the expression of TGF-β and type 1 collagen in the administration of the PRF membrane compared to the administration of the amniotic membrane after conjunctival excision of experimental male New Zealand rabbits on day 14.

Evaluation of In-vivo Anticancer Activity Choerospondias axillaris in Swiss Albino Mice Models

Background and Objective: The fruits of the plant, Choerospondias axillaris are one of the richest sources of flavonoids phenol and vitamin C that have been shown to possess a variety of biological activities. The present study was aimed to study the potential anti-cancer activity of the methanolic extract of fruit of Choerospondias axillaris using the in-vivo model to scientifically validate the folkloric use of the Choerospondias axillaris.&#x0D; Study Design: In-vivo model.&#x0D; Place and Duration of the Study: Department of pharmacology, Karnataka College of pharmacy, Bangalore India, between October 2021 to June 2022.&#x0D; Methods: Anti-cancer property of Choerospondias axillaris was evaluated against DMBA/croton oil induced skin tumorigenesis in Swiss albino mice. A single topical application of DMBA (100g/100l of acetone), followed 2 weeks later by repeated application of croton oil (1% in acetone three times a week) for 16 weeks. In contrast, animals treated orally with Choerospondias axillaris 200mg/kg/b.w. (group IV) and orally with Choerospondias axillaris 400mg/kg/b.w. (group V) and 5 Flu 10mg/kg (group III). The following parameters like; body weight, tumor incidence, cumulative number of tumors, tumor yield, average latency period, number of papillomas, haematological parameters, Serum Zinc and C-Reactive Protein, anti-oxidants enzyme, pro-inflammatory cytokines &amp; Histopathological of Tumor skin studies were observed.&#x0D; Results: 100 percent tumor incidence exhibited in group II (DMBA/croton oil) whereas the group IV (Choerospondias axillaris 200mg/kg) and group V (Choerospondias axillaris 400mg/kg) and group III (5 Flu 10mg/kg) exhibited 50, 33.7 and 42.5% tumor incidence, which significantly lesser when compared to than group II (Toxic control). The cumulative number of papillomas during the observation period of 16 weeks was significantly decreased in the Choerospondias axillaris treated groups IV, V and III (9, 4 and 6 no’s tumor respectively) in comparison to 18 cumulative numbers of papillomas in carcinogen control group. The average latent period significantly increased in the Toxic control group to 3.2, 4.3, 6.5 and 5.4 in group II, IV, V and III respectively. Tumor yield were significantly lesser (1.5, 0.66 and 1.0) as compared to DMBA/Croton oil treated control. Furthermore, the level of LPO was significantly increased whereas, the activity of CAT level were significantly decreased in skin tissue of DMBA/Croton oil treated animals compared Choerospondias axillaris treated animals. Similarly, NLR (&lt; 3.0), ESR, Serum Zinc and CRP have got improved in treated with Choerospondias axillaris. Protein expression of TGF-beta, IFN-G, TNF-alpha and IL-6 have shown Improvement in markers indicating a reduction in inflammation and immune imbalance in treated with Choerospondias axillaris compared with DMBA/Croton oil where shown moderate immune suppression.&#x0D; Conclusion: Based on the results it can be concluded that the test drug Choerospondias axillaris could be a potential candidate for the treatment of skin cancer.

The Traditional Chinese Medicine Gedan Jiangya Decoction Alleviates Left Ventricular Hypertrophy via Suppressing the Ras/ERK1/2 Signaling Pathway.

Gedan Jiangya Decoction (GJD), a Chinese herbal medicine composed of six botanical medicines, was designed to treat hypertension (patent published number (CN114246896A)). The overexpression of the ERK (extracellular signal-regulated kinase) signaling pathway is essential in developing left ventricular hypertrophy (LVH). This study aimed to evaluate GJD's effects on LVH in spontaneously hypertensive rats (SHRs) and examine its potential mechanisms on Ras/ERK1/2 pathway regulation. Thirty-five ten-week-old SHRs were randomly assigned to one of five groups: GJD low dosage, medium dose, high dose, model, and captopril. Wistar-Kyoto (WKY) rats served as the control group. All rats received a 6-week treatment. The following parameters were measured: systolic (SBP) and diastolic blood pressure (DBP), left ventricular mass index (LVMI), and serum TGF-beta1. The pathologic structure was determined by H & E staining and Masson. TGF-beta1, Ras, ERK1/2, and C-Fos levels were determined using western blotting and real-time qPCR. SBP, DBP, and LVMI were reduced significantly in the GJD group compared with the model group. GJD inhibited TGF-beta1, Ras, ERK1/2, and C-Fos expression in LVH. In conclusion, GJD reduced the Ras/ERK1/2 pathway expression, which decreased hypertension-induced heart hypertrophy. GJD may protect hypertension-induced myocardial hypertrophy by altering gene expression patterns in the heart.

Bioactivity of Biomass and Crude Exopolysaccharides Obtained by Controlled Submerged Cultivation of Medicinal Mushroom Trametes versicolor.

The aim of this study is to characterize the bioactivity of mycelial biomass and crude exopolysaccharides (EPS) produced by Trametes versicolor NBIMCC 8939 and to reveal its nutraceutical potential. The EPS (1.58 g/L) were isolated from a culture broth. The macrofungal biomass was rich in protein, insoluble dietary fibers and glucans. The amino acid composition of the biomass was analyzed and 18 amino acids were detected. Three mycelial biomass extracts were prepared and the highest total polyphenol content (16.11 ± 0.14 mg GAE/g DW) and the total flavonoid content (5.15 ± 0.03 mg QE/g DW) were found in the water extract. The results indicated that the obtained EPS were heteropolysaccharides with glucose as the main building monosaccharide and minor amounts of mannose, xylose, galactose, fucose and glucuronic acid. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the complex structure of the crude EPS. Five probiotic lactic acid bacteria strains were used for the determination of the prebiotic effect of the crude EPS. The anti-inflammatory potential was tested in vitro using cell line HT-29. The significant decrease of IL-1 and IL-8 and increase of TGF-beta expression revealed anti-inflammatory potential of the crude exopolysaccharides from T. versicolor.