TGF-β1 Gene Research Articles

Effect of Polymeric Nanoparticles with Entrapped Fish Oil or Mupirocin on Skin Wound Healing Using a Porcine Model.

The utilization of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) with entrapped fish oil (FO) loaded in collagen-based scaffolds for cutaneous wound healing using a porcine model is unique for the present study. Full-depth cutaneous excisions (5 × 5 cm) on the pig dorsa were treated with pure collagen scaffold (control, C), empty PLGA NPs (NP), FO, mupirocin (MUP), PLGA NPs with entrapped FO (NP/FO) and PLGA NPs with entrapped MUP (NP/MUP). The following markers were evaluated on days 0, 3, 7, 14 and 21 post-excision: collagen, hydroxyproline (HP), angiogenesis and expressions of the COX2, EGF, COL1A1, COL1A3, TGFB1, VEGFA, CCL5 and CCR5 genes. The hypothesis that NP/FO treatment is superior to FO alone and that it is comparable to NP/MUP was tested. NP/FO treatment increased HP in comparison with both FO alone and NP/MUP (day 14) but decreased (p < 0.05) angiogenesis in comparison with FO alone (day 3). NP/FO increased (p < 0.05) the expression of the CCR5 gene (day 3) and tended (p > 0.05) to increase the expressions of the EGF (day 7, day 14), TGFB1 (day 21) and CCL5 (day 7, day 21) genes as compared with NP/MUP. NP/FO can be suggested as a suitable alternative to NP/MUP in cutaneous wound treatment.

Non-viral CRISPR activation system targeting VEGF-A and TGF-β1 for enhanced osteogenesis of pre-osteoblasts implanted with dual-crosslinked hydrogel.

Healing of large calvarial bone defects remains challenge but may be improved by stimulating bone regeneration of implanted cells. The aim of this study is to specially co-activate transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF-A) genes expressions in pre-osteoblast MC3T3-E1 cells through the non-viral CRISPR activation (CRISPRa) system to promote osteogenesis. A cationic copolymer carrying nucleus localizing peptides and proton sponge groups dimethyl-histidine was synthesized to deliver CRISPRa system into MC3T3-E1 cells with high cellular uptake, lysosomal escape, and nuclear translocation, which activated VEGF-A and TGF-β1 genes expressions and thereby additively or synergistically induced several osteogenic genes expressions. A tunable dual-crosslinked hydrogel was developed to implant the above engineered cells into mice calvaria bone defect site to promote bone healing in vivo. The combination of multi-genes activation through non-viral CRISPRa system and tunable dual-crosslinked hydrogel provides a versatile strategy for promoting bone healing with synergistic effect.

Diluted Lycopodium Induced Cell Death and Clinical Improvement in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common cancer with high incidence rate, and 5-year survival rate in HCC is less than 20%. Thus, in search of newer anticancer agents effective in HCC, we have explored possible usefulness of an alternative medicine Lycopodium against the human liver cancer cell line, HepG2 along with its clinical efficacy. The HepG2 cell line was challenged with Lycopodium 6C (diluted Lycopodium &lt;1pg /mL available as alternative medicine) along with vehicle alcohol control in 24 hours. The cytopathic effect and viability test with methylene blue stain were observed. The cells were harvested for total RNA extraction, and gene expression levels of targeted cytokines -Interferon gamma (IFN γ); Interleukins - IL-6, IL-8, IL-10, IL-1β, Transforming Growth Factor- TGF-β1, TGF-β3 and Tumor Necrosis Factor alpha (TNF-α) by RT-PCR were studied. DNA fragmentation assay and cell viability assay by MTT method were also tested. After ethical permission we applied this medicine as adjunct therapy to observe any beneficial role of the medicine. Statistically significant changes of IL-10, IL-1β and TGF-β3 were observed after challenge with Lycopodium 6C. The IL-10 gene expression in malignant cells was significantly reduced with Lycopodium 6C; however, the expression is more with vehicle alcohol compared to normal control set. Thus, the medicine could decrease the excessive IL-10 gene expression to a moderate level. IL-1β and TGF-β3 gene up-regulation by the vehicle alcohol were also mitigated by the medicine Lycopodium 6C. Mild DNA fragmentation was also seen in cancer cells after challenge with the medicine. Two cases suffering from hepatocellular carcinoma showed much clinical improvement after therapy with this medicine. Lycopodium 6C may act as a supporting alternative medication for treating HCC.

Assessment of the TGFB1 gene expression and methylation status of the promoter region in patients with colorectal cancer

The aim of this study was to evaluate the expression of the TGFB1 gene encoding the TGF-β1 cytokine in 64 patients, and then to compare it with clinico-pathological features. The study also investigated whether the regulation of the gene expression is caused by methylation of the promoter region between − 235 and + 22 nucleotide from the start of transcription. The dependence of the relative level of the TGFB1 gene expression on the clinical advancement according to the TNM classifications was shown. Additionally, the individual grades of the T and M features of the TNM classification differed in the relative transcript levels of the TGFB1 gene. Moreover, the higher relative expression level of the studied gene was associated with a lack of vascular invasion by cancer cells and presence of lymphocytes in the neoplastic tissue. The obtained results may indicate a possible impact of the gene on the process of carcinogenesis in colorectal cancer and reduction of its expression level may be one of the factors contributing to progression of the disease.

Molecular characterization and frequency of transforming growth factor beta 1 gene polymorphism and its relation to bone complications in Egyptian patients with β-thalassemia

Objective The objective of this study was to determine the frequency of transforming growth factor beta 1 (TGFβ1) C-509T gene polymorphism and its relation to bone complications in patients with β-thalassemia major in Egypt. Background Osteoporosis is the most prevalent bone complication in patients with β-thalassemia major despite regular blood transfusions and iron chelation therapy. It is characterized by low bone mineral density (BMD) resulting in reduced bone strength and increased risk of fractures. Genetic factors play an important role in the determination of BMD. The TGFβ1 gene, which encodes TGFβ1, is a strong candidate for susceptibility to osteoporosis, and several studies have reported associations between BMD and different polymorphisms of TGFβ1, although these studies have yielded conflicting results. Study design and methods Single nucleotide polymorphism in the TGFβ1 gene promoter (C-509T) was investigated in 100 regularly treated Egyptian children with β-thalassemia major by PCR/RFLP genotyping. BMD was measured by dual-energy radiograph absorptiometry and expressed as Z score. Results The frequency of TGFβ1 gene polymorphism C-509T genotypes in all studied patients was 6% for homozygous CC, 85% for heterozygous CT, and 9% for homozygous TT. C allele frequency was 48.5%, whereas T allele frequency was 51.5%. BMD Z score was significantly higher in TT genotype compared with CC genotype, with P value less than 0.05. Patients were grouped on the basis of BMD Z score: 51 (51%) patients with BMD deficit (Z score &lt;−1) and 49 (49%) with normal BMD (Z score ≥−1). TGFβ1 gene polymorphism C-509T genotypes were distributed differently between the two groups; the TT genotype frequency was lower in patients with BMD deficit (P&lt;0.05). Conclusion TGFB1 gene polymorphism C-509T is associated with BMD and genetic susceptibility to osteoporosis and may play a role in the pathogenesis and modification of bone complication in β-thalassemia major. BMD deficit is common in Egyptian children with β-thalassemia major. Analysis of this polymorphism at an early age could help in identification of thalassemic children at risk of osteoporosis and early management. However, large-scale studies are required to confirm these findings.

O-137 Evaluation of the effect of seminal plasma exosomes from unexplained infertile men on the expression of implantation-related genes

Abstract Study question Do exosomes from seminal plasma of men with unexplained infertility affect endometrial receptivity? Summary answer Seminal plasma exosomes from unexplained infertile men alter the expression of genes and signaling pathways related to endometrial receptivity. What is known already Unexplained infertile Men are a challenging group with no obvious cause of infertility. Many Complicated mechanisms play a crucial role in preparing the receptive endometrium for embryo implantation. The exosomes of seminal plasma are a heterogeneous cargo of nucleic acids, proteins, and lipids that participate in sperm maturation and function by binding to sperm. It is now apparent that the seminal plasma and its exosomes play a complex role in controlling the female reproductive system. There is no study investigating the effect of Unexplained seminal plasma exosomes on endometrial receptivity. Study design, size, duration Samples were collected from 10 men with normal semen parameters from couples undergoing infertility treatment and from 10 donor men with proven fertility and analyzed according to WHO -2010 criteria. Endometrial biopsy tissue was collected from 6 women in the proliferative phase of a natural menstrual cycle. Endometrial epithelial cells were cultured immediately after tissue processing and treated with 100 μg/ml seminal exosomes for (6 and 24 hours); then analyzed for gene expression. Participants/materials, setting, methods Exosomes isolated by an ultracentrifuge and characterized size and ultrastructure with dynamic light scattering and transmission electron microscopy. Western blot analysis showed the presence of the exosome markers. After incubation of exosomes with DiI dye, cytoplasmic localization of exosomes detected by fluorescence microscopy. The endometrial receptivity and signaling pathway genes were analyzed by qRT-PCR and immunocytochemistry. Image J and Prism software were used to quantify and analyze the data. P-value &amp;lt;0.05 was considered statistically significant. Main results and the role of chance Compared with the embryo donation group, the mRNA levels of MUC1, LIF, G-CSF, CX3CL1, and VEGF were significantly downregulated in the endometrium treated with unexplained infertile exosomes, especially after 24 hours of treatment. Mean mRNA levels of IL6, IL8, and TGFB genes in the unexplained infertile group changed compared with the embryo donation group but were not significant (p &amp;gt; 0.05). Immunocytochemical analysis showed that MUC1 and LIFR protein levels were increased in endometrial epithelial cells. Limitations, reasons for caution larger sample size and exosomal compositions assessment will be useful to identify the effect of exosomes on the endometrium. Wider implications of the findings Alterations in the expression of genes associated with endometrial receptivity by exosomes of the unexplained infertile men compared with embryo donation could be one of the causes of infertility in these individuals. Presumably, the atypical composition of exosomes has a different effect on sperm functions and endometrial genes expression. Trial registration number Not applicable

Cross talk between lysine methyltransferase Smyd2 and TGF-β-Smad3 signaling promotes renal fibrosis in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited genetic disorder that is caused by mutations in PKD1 or PKD2 genes and is characterized by renal fluid-filled cyst formation and interstitial fibrosis. PKD1 gene mutation results in the upregulation of SET (suppressor of variegation, enhancer of zeste, trithorax) and MYND (myeloid-nervy-DEAF1) domain-containing lysine methyltransferase 2 (SMYD2) in kidneys from Pkd1 mutant mice and patients with ADPKD. However, the role and mechanism of Smyd2 in the regulation of renal fibrosis in ADPKD remains elusive. In the present study, we showed that 1) expression of Smyd2 can be regulated by transforming growth factor (TGF)-β-Smad3 in normal rat kidney 49F (NRK-49F) cells and mouse fibroblast NIH3T3 cells; 2) knockdown of Smyd2 and inhibition of Smyd2 with its specific inhibitor, AZ505, decreases TGF-β-induced expression of α-smooth muscle actin, fibronectin, collagen type 1 and 3, and plasminogen activator inhibitor-1 in NRK-49F cells; 3) Smyd2 regulates the transcription of fibrotic marker genes through binding on the promoters of those genes or through methylating histone H3 to indirectly regulate the expression of those genes; and 4) knockout and inhibition of Smyd2 significantly decreases renal fibrosis in Pkd1 knockout mice, supporting that targeting Smyd2 can not only delay cyst growth but also attenuate renal fibrosis in ADPKD. This study identified a cross talk between TGF-β signaling and Smyd2 in the regulation of fibrotic gene transcription and activation of fibroblasts in cystic kidneys, suggesting that targeting Smyd2 with AZ505 is a potential therapeutic strategy for ADPKD treatment.NEW & NOTEWORTHY Here, we identified a cross talk between SET and MYND domain-containing lysine methyltransferase 2 (Smyd2) and transforming growth factor (TGF)-β-Smad3 signaling and a synergistic feedback loop between them, in which TGF-β stimulates expression of Smyd2 in a Smad3-dependent manner, and upregulation of Smyd2 regulates the transcription of TGF-β and other fibrotic marker genes through direct binding on their promoters or methylating histone H3 indirectly to regulate the transcription of those genes in fibroblasts. Thus, the Smyd2-TGF-β-Smad3-Smyd2 signaling axis plays an important role in promoting renal fibrosis, and targeting Smyd2 with its specific inhibitor should not only delay cyst growth but also ameliorate renal fibrosis in ADPKD.

Increased glucocorticoid metabolism in diabetic kidney disease.

AimsGlomerular damage indicated by proteinuria is a main symptom in diabetic nephropathy. Mineralocorticoid receptor (MR) antagonists (MRAs) are beneficial irrespective of aldosterone availability. Thus, we hypothesized an alternatively activated MR to promote glomerular damage in proteinuric diabetic nephropathy. Specifically, we aimed first to demonstrate the presence of steroid hormones serving as alternative MR targets in type II diabetic patients with proteinuric kidney disease, second whether MR selectivity was modified, third to characterize MR and glucocorticoid receptor (GR) expression and activity in glomerular cell types exposed to eu- and hyperglycemic conditions, fourth to characterize the pro-fibrotic potential of primary human renal mesangial cells (HRMC) upon stimulation with aldosterone and cortisol, and fifth to specify the involvement of the MR and/or GR in pro-fibrotic signaling.Materials and methodsUrinary steroid hormone profiles of patients with diabetic kidney disease were analyzed by gas chromatography–mass spectrometry and compared to an age and gender matched healthy control group taken out of a population study. In both cohorts, the activity of the MR pre-receptor enzyme 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2), which inactivates cortisol to prevent it from binding to the MR, was assessed to define a change in MR selectivity. Expression of HSD11B2, MR and GR was quantified in HRMC and primary human renal glomerular endothelial cells (HRGEC). Activity of MR and GR was explored in HRMC by measuring the MR/GR down-stream signal SGK1 and the pro-fibrotic genes TGFB1, FN1 and COL1A1 in normal and high glucose conditions with the MR/GR agonists aldosterone/cortisol and the MR/GR antagonists spironolactone/RU486.ResultsPatients with diabetic kidney disease excreted more tetrahydroaldosterone than the control group reaching significance in men. The excretion of MR-agonistic steroid hormones was only increased for 18-hydroxytetrahydrocorticosterone in diabetic women. The excretion of most glucocorticoids was higher in the diabetic cohort. Higher apparent systemic HSD11B2 activity suggested less activation of the MR by cortisol in diabetic patients. Both cell types, HRMC and HRGEC, lacked expression of HSD11B2. Hyperglycemic conditions did not change MR and GR expression and activity. Stimulation with both aldosterone and cortisol promoted upregulation of pro-fibrotic genes in HRMC. This effect of MR and/or GR activation was more pronounced in high glucose conditions and partially inhibited by MRAs and GR antagonists.ConclusionsIn patients with diabetic kidney disease alternative MR activation is conceivable as cortisol and cortisone metabolites are increased. Systemic availability of active metabolites is counteracted via an increased HSD11B2 activity. As this cortisol deactivation is absent in HRMC and HRGEC, cortisol binding to the MR is enabled. Both, cortisol and aldosterone stimulation led to an increased expression of pro-fibrotic genes in HRMC. This mechanism was related to the MR as well as the GR and more marked in high glucose conditions linking the benefit of MRAs in diabetic kidney disease to these findings.

Analysis of associations of genetic markers with the development of congenital scoliosis

Objective. To study the associations of single-nucleotide polymorphisms: rs6570507 in GPR126 gene, rs1800795 in IL-6 gene, rs1800469 in TGFB1 gene, rs731236 in VDR gene, rs625039 and rs11598564 polymorphisms in LBX1 gene, and rs12946942 in SOX9 gene with congenital scoliosis.Material and Methods. The study included 90 patients with verified congenital anomalies of the spine (single and multiple malformations of the spine, ICD-10 Code: Q76.3) and 157 clinically healthy volunteers without diagnosed spinal deformity and without family history of spinal malformations or osteoarticular system diseases. Molecular genetic testing was performed by PCR with real-time registration of a signal from the developed oligonucleotides used to determine rs6570507, rs1800795, rs1800469, rs625039, rs11598564, rs12946942, and rs731236 polymorphisms. Reference sequences were selected from the dbSNP database, and sequence design was performed on the BLAST platform. Data analysis was performed using the R free software computing environment. Data were compared using Pearson’s c2 test, and 95 % confidence interval limits were calculated to assess the significance of OR.Results. Statistically significant association of the G allele and GG genotype of the rs1800795 polymorphism in the interleukin-6 gene with congenital scoliosis was found in group of Russian patients (p &lt; 0.001). No significant association of alleles and genotypes of polymorphic variants of rs6570507, rs1800469, rs625039, rs11598564, rs12946942, and rs731236 with congenital scoliosis was found.Conclusion. The rs1800795 polymorphism can be considered as a promising marker for molecular genetic diagnostics of congenital scoliosis.

Is human umbilical cord mesenchymal stem cell-derived conditioned medium effective against oxidative and inflammatory status in CCl4-induced acute liver injury?

IntroductionAcute liver injury (ALI) is diagnosed by detection of elevated liver enzymes within six months after liver damage. Mesenchymal stem cells (MSCs) have recently been considered a beneficial strategy for treating various diseases due to healing secretory factors. Therapeutic effects of human umbilical cord MSCs-derived conditioned medium (hMSC-CM) were evaluated on CCl4-induced ALI. Materials and methodsTwenty-four male Wistar rats were divided into groups including N (received saline), ALI (received CCl4), RPMI (received CCl4 and RPMI medium), and ALI-CM (received CCl4 and hMSC-CM) groups. The expression of TNF-α and TGFβ-1 genes was evaluated with qPCR. Hepatic levels of TNF-α and TGF-β were measured by ELISA. Total antioxidant capacity (TAC), total oxidant status (TOS), malondialdehyde (MDA), glutathione peroxidase (GPx) activity, and catalase (CAT) activity were also assayed. Hematoxylin-eosin (H&E), Masson's trichrome, reticulin, and Periodic Acid–Schiff (PAS) stainings were conducted to evaluate tissue lesions. ResultsCCl4 increased expression of TNF-α and TGF-1β at both mRNA and protein levels, while hMSC-CM decreased these parameters in the ALI-CM group. TAC levels significantly decreased in the ALI group, and CCl4 increased TOS and MDA levels compared with the N group. hMSC-CM treatment led to the return of these parameters to their baseline levels. GPx and CAT activity in the ALI group were significantly lower than in the N group and hMSC-CM reduced these parameters to the baseline in the ALI-CM group. hMSC-CM modulated CCl4-induced tissue lesions. ConclusionThe present study suggests hMSC-CM probably improves CCl4-induced ALI through its antioxidant and anti-inflammatory effects.

Antiulcer Potential of Psidium guajava Seed Extract Supported by Metabolic Profiling and Molecular Docking

One of the most severe human health problems is gastric ulceration. The main aim of our study is to explore the gastroprotective effect of the Psidium guajava seeds extract (PGE). Metabolic profiling based on LC-HRMS for the extract led to the dereplication of 23 compounds (1–23). We carried out a gastric ulcer model induced by indomethacin in male albino rats in vivo and the extract of PGE was investigated at a dose of 300 mg/kg in comparison to cimetidine (100 mg/kg). Furthermore, the assessment of gastric mucosal lesions and histopathology investigation of gastric tissue was done. It has been proved that Psidium guajava seeds significantly decreased the ulcer index and protected the mucosa from lesions. The antiulcer effect of Psidium guajava seed extract, which has the power of reducing the ensuing inflammatory reactions, can counteract the inflammation induced by indomethacin by the downregulation of relative genes expression (IL-1β, IL-6, and TNF-α). Moreover, PGE significantly downregulated the increased COX-2, TGF-β, and IGF-1 relative genes expression, confirming its beneficial effect in ulcer healing. Moreover, the possible PGE antioxidant potential was determined by in vitro assays using hydrogen peroxide and superoxide radical scavenging and revealed high antioxidant potential. Additionally, on the putatively annotated metabolites, an in silico study was conducted, which emphasized the extract’s antiulcer properties might be attributed to several sterols such as stigmasterol and campesterol. The present study provided evidence of Psidium guajava seeds considered as a potential natural gastroprotective agent.

Silver nanoparticles induced hepatoxicity via the apoptotic/antiapoptotic pathway with activation of TGFβ-1 and α-SMA triggered liver fibrosis in Sprague Dawley rats

Despite the extraordinary use of silver nanoparticles (AgNPs) in medicinal purposes and the food industry, there is rising worry about potential hazards to human health and the environment. The existing study aims to assess the hepatotoxic effects of different dosages of AgNPs by evaluating hematobiochemical parameters, oxidative stress, liver morphological alterations, immunohistochemical staining, and gene expression to clarify the mechanism of AgNPs’ hepatic toxic potential. Forty male Sprague Dawley rats were randomly assigned into control and three AgNPs intraperitoneally treated groups 0.25, 0.5, and 1 mg/kg b.w. daily for 15 and 30 days. AgNP exposure reduced body weight, caused haematological abnormalities, and enhanced hepatic oxidative and nitrosative stress with depletion of the hepatic GSH level. Serum hepatic injury biomarkers with pathological hepatic lesions where cholangiopathy emerges as the main hepatic alteration in a dosage- and duration-dependent manner were also elevated. Furthermore, immunohistochemical labelling of apoptotic markers demonstrated that Bcl-2 was significantly downregulated while caspase-3 was significantly upregulated. In conclusion, the hepatotoxic impact of AgNPs may be regulated by two mechanisms, implying the apoptotic/antiapoptotic pathway via raising BAX and inhibiting Bcl-2 expression levels in a dose-dependent manner. The TGF-β1 and α-SMA pathway which triggered fibrosis with incorporation of iNOS which consequently activates the inflammatory process were also elevated. To our knowledge, there has been no prior report on the experimental administration of AgNPs in three different dosages for short and long durations in rats with the assessment of Bcl-2, BAX, iNOS, TGF-β1, and α-SMA gene expressions.Graphical abstract

Polymorphismes 869C> T et 915 G>C du TGF-β1 dans la rétinopathie du diabète de type 1 chez la population algérienne

Diabetic retinopathy (DR) is characterized by chronic low-grade inflammation in which the effects of genetic factors is well established. The objective of our study is to explore an association of the 869C>T and 915G>C polymorphisms of the TGF-β1 gene with type 1 diabetic retinopathy in the Algerian population. A case-control study was carried out in which the SNPs 869C>T and 915G>C of the TGF-β1 gene were analysed by the PCR-SSP technique. We compared the distribution of allelic and genotypic frequencies between patients with and without retinopathy and looked for an association between these polymorphisms and certain clinical characteristics of and risk factors for diabetic retinopathy. A significant increase in the frequencies of the C allele (P=0.03) and GG genotype (P=0.007) of the 915 G>C polymorphism were found, respectively, in patients without and with retinopathy. However, no significant difference was found for allelic and genotypic frequencies of the 869C>T SNP (all P>0.05) or associations between genotypes and clinical characteristics or risk factors for DR. Our preliminary results suggest that the C allele of the 915 G>C polymorphism of TGF-β1 is protective against type 1 diabetic retinopathy in the Algerian population, while the GG genotype could confer susceptibility to it.

Abstract 2515: Estrogen regulates the immune microenvironment of colorectal cancer liver metastases

Abstract Liver metastases (LM) remain a major cause of cancer-related death and many cancers preferably metastasize to the liver due to its unique anatomical location, rich blood supply and immune-tolerant microenvironment. Up to 50% of colorectal carcinoma patients develop LM during their disease and this is associated with a poor prognosis. Our laboratory previously identified a sexual dimorphism in the regulation of the immune microenvironment (IME) of LM and showed that estrogen could promote the accumulation of myeloid-derived suppressor cells (MDSCs) and Tregs in the liver in response to invading cancer cells. The objective of this study was to elucidate the role of estrogen in the recruitment and polarization of monocytes/macrophages and in the accumulation and phenotype of natural killer (NK) cells in the IME of colorectal carcinoma LM (CRCLM). Moreover, we sought to determine the therapeutic potential of Selective Estrogen Receptor Degrader (SERD) therapy in tumor bearing female mice. In estrogen-competent female mice bearing CRCLM, we found increased gene and protein expression of the immunosuppressive cytokine TGF-β, and inversely a decrease in the pro-inflammatory cytokine TNF-alpha. Furthermore, we identified a significant increase of newly recruited, immunosuppressive CD68+F4/80+CD163+ M2 macrophages, as compared to estrogen-depleted (ovariectomized) mice. The recruitment of pro-inflammatory CD68+F4/80+TNF-alpha+ M1 macrophages, and the accumulation of the highly cytotoxic NK1.1+ CD49a+ liver NK cells were increased in estrogen-depleted female mice and could be restored upon estrogen supplementation. Moreover, treatment of female mice with the SERD Fulvestrant, markedly reduced the number of CRCLM compared to vehicle-treated mice, and significantly reduced the cell frequencies and counts of recruited myeloid cells and M2 macrophages in the liver. Taken together, our results identify estrogen as a critical regulator of an immunosuppressive tumor ME in the liver and a potential therapeutic target. Funding by the Canadian Institute of Health Research is gratefully acknowledged. Citation Format: Yasmine Benslimane, Sarah Lapin, Julien Chambon, Matthew Leibovitch, Stéphanie Perrino, Pnina Brodt. Estrogen regulates the immune microenvironment of colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2515.

Abstract 2117: IFN-γ signaling in myeloid cells regulates pancreatic cancer growth and progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by low survival, early metastasis, and rapidly emerging resistance to therapy. Interferon gamma (IFN-γ) signaling pathway is one of the key regulators of the tumor microenvironment in numerous cancer settings and its possible key role in myeloid cells is suggested. Here we investigated the IFN-y signaling in myeloid cells during PDAC development, progression, and metastasis using mice with conditional ablation of IFNgR2 in myeloid cells (IFNgR2-Delta-Mye). We performed an orthotopical injection of cells derived from conditional murine pancreatic model (Pdx1Cre-Kras-P53, or “KPC” cells) into IFNgR2-LysMCre (IFNgR2-Delta-Mye) mice. Ablation of IFN-γ signaling in myeloid cells led to a higher tumor burden compared to controls. Subsequent RNA sequence analysis demonstrated that IFN-γR2 deficiency in myeloid cells resulted in increased TGFβ1 gene expression as well as changes in metabolic and growth factor pathways in macrophages isolated from tumor tissue. Immunofluorescent analysis demonstrated that IFN-γR2 deletion in myeloid cells led to changes in cancer cell differentiation, resulting in a loss of characteristic epithelial histological pattern and shifting it towards more aggressive phenotype, possible via TGFβ1 dependent mechanism. Using murine model of liver metastasis, we found that loss of IFN-γR2 in myeloid cells leads to increased metastatic outgrowth of PDAC KPC cells. This effect was partially dependent on the gut microbiota, as shown by “separated and co-housed mice” experiments. Deficiency of IFN-γR2 in myeloid cells resulted in increased recruitment and infiltration of monocytes in normal and tumor/metastatic liver tissues. Our data identifies myeloid cell type specific IFN-γ signaling pathway as an important regulator of pancreatic cancer progression and metastasis, that can be potentially exploited as a novel immunooncology target. Citation Format: Elena Ivleva, Natalia Andreeva, Sergei Grivennikov. IFN-γ signaling in myeloid cells regulates pancreatic cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2117.

Cytokine TGFβ Gene Polymorphism in Asthma: TGF-Related SNP Analysis Enhances the Prediction of Disease Diagnosis (A Case-Control Study With Multivariable Data-Mining Model Development).

IntroductionTGF-β and its receptors play a crucial role in asthma pathogenesis and bronchial remodeling in the course of the disease. TGF-β1, TGF-β2, and TGF-β3 isoforms are responsible for chronic inflammation, bronchial hyperreactivity, myofibroblast activation, fibrosis, bronchial remodeling, and change the expression of approximately 1000 genes in asthma. TGF-β SNPs are associated with the elevated plasma level of TGF-β1, an increased level of total IgE, and an increased risk of remodeling of bronchi.MethodsThe analysis of selected TGF-β1, TGF-β2, TGF-β3-related single-nucleotide polymorphisms (SNP) was conducted on 652 DNA samples with an application of the MassARRAY® using the mass spectrometry (MALDI-TOF MS). Dataset was randomly split into training (80%) and validation sets (20%). For both asthma diagnosis and severity prediction, the C5.0 modelling with hyperparameter optimization was conducted on: clinical and SNP data (Clinical+TGF), only clinical (OnlyClinical) and minimum redundancy feature selection set (MRMR). Area under ROC (AUCROC) curves were compared using DeLong’s test.ResultsMinor allele carriers (MACs) in SNP rs2009112 [OR=1.85 (95%CI:1.11-3.1), p=0.016], rs2796821 [OR=1.72 (95%CI:1.1-2.69), p=0.017] and rs2796822 [OR=1.71 (95%CI:1.07-2.71), p=0.022] demonstrated an increased odds of severe asthma. Clinical+TGF model presented better diagnostic potential than OnlyClinical model in both training (p=0.0009) and validation (AUCROC=0.87 vs. 0.80,p=0.0052). At the same time, the MRMR model was not worse than the Clinical+TGF model (p=0.3607 on the training set, p=0.1590 on the validation set), while it was better in comparison with the Only Clinical model (p=0.0010 on the training set, p=0.0235 on validation set, AUCROC=0.85 vs. 0.87). On validation set Clinical+TGF model allowed for asthma diagnosis prediction with 88.4% sensitivity and 73.8% specificity.DiscussionDerived predictive models suggest the analysis of selected SNPs in TGF-β genes in combination with clinical factors could predict asthma diagnosis with high sensitivity and specificity, however, the benefit of SNP analysis in severity prediction was not shown.

Neonatal presentation of Loeys-Dietz syndrome: two case reports and review of the literature

BackgroundLoeys-Dietz syndrome (LDS) is a rare connective tissue disorder characterized by cardiovascular manifestations, especially aortic dilatations and arterial tortuosity, craniofacial and skeletal features, joint laxity or contractures, skin abnormalities, hypotonia and motor delay. Its diagnosis is established by the identification of a pathogenic variant in TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 or TGFB3 genes. In newborns and toddlers, vascular complications such as aneurism rupture, aortic dissection, and intracerebral incidents, can occur already in the weeks of life. To avoid these events, it is crucial to precociously identify this condition and to start an apunderwent a surgical procedurepropriate treatment which, depending on the severity of the vascular involvement, might be medical or surgical.Case presentationWe report two cases of Loeys-Dietz syndrome precociously diagnosed. The first describes a male, born at 38 + 1 weeks of gestation, with hypotonia, joint hypermobility, arachnodactyly, and fingers joint contractures, as well as senile appearance and facial dysmorphisms. In the suspect of a connective tissue disorder, an echocardiography was performed and revealed an aortic root dilatation of 13 mm (Z score + 3). A trio based Whole Exome Sequencing found a novel de novo variant in the TGFBR2 gene. Despite the onset of a low-dose angiotensin receptor blocker therapy, the aneurysm progressed. The second case describes a female, born at 41 + 3 weeks of gestation. During the neonatal examination a cleft palate was noticed, as well as minor dysmorphisms. Since the family history was suspicious for connective tissue disorders, a genetic panel was performed and identified a pathogenetic variant in TGFB3 gene. In this case, the echocardiography revealed no abnormalities.ConclusionsIn addition to our cases, we identified 14 subjects with neonatal LDS in the medical literature. All of them had aortic involvement. Skeletal and face abnormalities, including eyes and palate malformations, were also highly frequent. Overall, 10 subjects required medical therapy to avoid aneurysm progression, and 8 patients underwent surgical procedures.Benefits of an early diagnosis of LDS are various and imply a potential modification of the natural history of the disease with early interventions on its complications.

Serum concentration and mRNA expression of Transforming Growth Factor-Beta 1 (TGF-β1) in stunted and non- stunted toddlers

West Nusa Tenggara Province has the fifth-highest prevalence of stunting cases in Indonesia. So far, limited research is available to understand the likelihood of stunting in this region. Transforming Growth Factor-Beta 1 (TGF-β1), an immunoregulatory cytokine, may affect the stunting progression. Knowledge of messenger mRNA expression in the TGF-β1 gene in stunted toddlers could help to determine therapeutic targets to catch up on their growth. This study compared the expression of TGF- β1 mRNA and TGF-β1 concentrations in the stunted and the non-stunted toddlers. The nutritional status of all participants was also gathered and linked to the stunting issue. A cross-sectional study was conducted on 48 toddlers aged 12-36 months. The stunting case was defined as a Z-Score of less than-2 of length/height for age according to WHO. The serum TGF-β1 and TGF-β1 gene mRNA were measured using ELISA and RT-PCR, respectively. The nutritional status data were collected through interviewer-administered structured questionnaire to the toddlers' parents and 48-h food recalls. Descriptive analyses were applied to determine the distribution of participants' macronutrient and micronutrient levels. Results show that there were significant differences in expressions of the TGF- β1 gene mRNA of the stunted and the non-stunted toddlers. The expression of the TGF- β1 mRNA gene in the non-stunted toddlers was also higher with 13.7±0.859 fold change than those of the stunted toddlers with 9.01±1.76 fold change with a p-value <0.001. The serum TGF-β1 concentrations in the stunted toddler (6.20±3.60pg/ml) were significantly lower than the ones in the non-stunted toddlers (14.3±1.05pg/ml) with p-value <0.001. However, there was no clear relationship between the likelihood of stunting and the nutritional status from the obtained data. Overall findings demonstrate the significantly lower both the TGF-β1 gene mRNA expression and serum TGF-β1 for the stunted toddlers than the non-stunted toddlers, impacting bone formation and resorption. The outcomes of this study encourage the development of interventional therapy for stunted toddlers by increasing the serum TGF-β1 concentrations.

Concomitant Expression of IL-6 and TGF-β Cytokines and their Receptors in Peripheral Blood of Patients with Multiple Sclerosis: The Effects of INFβ Drugs.

Concomitant signals from IL-6 and TGF-β have a central role in the Th17 cells development and differentiation, and these cells are the main promoters of demyelinating inflammation in the central nervous system (CNS) resulting in multiple sclerosis (MS). To evaluate the simultaneous IL-6 and TGF-β gene and their receptor protein expression in patients with Relapsing-Remitting (RR)-MS. IL-6 and TGF-β mRNA and their receptor expression on the surface of CD4+T cells were evaluated using real-time PCR (RT-PCR) and flow cytometry, respectively. The IL-6 mRNA expression in patients with RRMS was significantly higher than in the controls (p= 0.019). When patients who did not receive any other treatment were compared with the controls, the significant difference was substantial (p=0.006). The TGF-β mRNA expression in patients was lower than in the controls (p = 0.03). However, in patients receiving IFNβ, it increased compared with the other patients (p= 0.036). There was no difference in cytokine receptor expression between patients and the control group. Our data conclude an increase and decrease in mRNA expression levels of IL-6 and TGF-β in patients with RRMS, respectively. Moreover, there were no significant differences in receptor expression of either cytokines. Based on our data the balance of TGF and IL-6 appears to have a positive impact on the disease control.

Pan-Cancer analysis of the mutation frequency of genes in TGF-beta pathway.

e15113 Background: The TGF-β signaling pathway governs key cellular processes under physiologic conditions and deregulates in cancer, including a diverse and varied set of gene responses that range from cytostatic and apoptotic tumor-suppressive ones in early-stage tumors to proliferative, invasive, angiogenic, and oncogenic ones in advanced cancer. However, the genomics profiles of TGF-β signaling pathway genes are unclear. In this study, we evaluated the genomic features in Chinese pan-cancer patients and investigated the mutation frequency of genes in the TGF-β pathway. Methods: A total of 5420 patients consisting of 22 cancer types were enrolled in this study. Utilizing target next-generation sequencing (NGS) platform, genomic profiling of 599 cancer-related genes, including genes of TGF-β pathway (SMAD2/3/4, PDGFRB, CDH1, MDM2, CDKN1A, and CDKN2B), was performed on tumor tissue or blood samples. Results: We observed 25.57% (1386/5420) of cases harbored at least one alteration in the TGF-β pathway genes. The mutation frequency ranges 9.09% to 39.77% among the 22 cancer types, including 39.77% (247/621) in gastric cancer, 39.31 (320/814) in bowel cancer, 36.84% (7/19) in endometrial cancer, 11.11% (8/72) in esophageal cancer, 10.46% (7/67) in kidney cancer, and 9.09% (2/22) in the gastrointestinal stromal tumor, respectively. The mutation of tumor-promoting effects genes SMAD2/3/4, PDGFRB, CDH1and MDM2 in the TGF-β pathway were found in all cancer types and mainly occurred in CNS tumor, gastrointestinal stromal tumor, pancreatic cancer, sarcoma, gastric cancer, and bowel cancer. The tumor-suppressing effective genes CDKN1A and CDKN2B primarily mutated in melanoma, endometrial cancer, and kidney cancer. Approximately 21.77% of patients had CNV in the TGF-β pathway of all tumor types. The most frequently mutated TGF-β genes were MDM2 and CDKN2B, mainly altered in sarcoma, melanoma, and CNS Tumor. Conclusions: We found a unique genomic feature of the TGF-β pathway of Chinese pan-cancer patients. Targeting mutant of TGF-β pathway genes remains a potentially effective treatment in the future.