Mesenchymal stromal cells plus basiliximab improve the response of steroid-refractory acute graft-versus-host disease as a second-line therapy: a multicentre, randomized, controlled trial.

BACKGROUND: For patients with steroid-refractory acute graft-versus-host disease (SR-aGVHD), effective second-line regimens are urgently needed. Mesenchymal stromal cells (MSCs) have been used as salvage regimens for SR-aGVHD in the past. However, clinical trials and an overall understanding of the molecular mechanisms of MSCs combined with basiliximab for SR-aGVHD are limited, especially in haploidentical haemopoietic stem cell transplantation (HID HSCT). METHODS: The primary endpoint of this multicentre, randomized, controlled trial was the 4-week complete response (CR) rate of SR-aGVHD. A total of 130 patients with SR-aGVHD were assigned in a 1:1 randomization schedule to the MSC group (receiving basiliximab plus MSCs) or control group (receiving basiliximab alone) (NCT04738981). RESULTS: Most enrolled patients (96.2%) received HID HSCT. The 4-week CR rate of SR-aGVHD in the MSC group was obviously better than that in the control group (83.1% vs. 55.4%, P = 0.001). However, for the overall response rates at week 4, the two groups were comparable. More patients in the control group used ≥ 6 doses of basiliximab (4.6% vs. 20%, P = 0.008). We collected blood samples from 19 consecutive patients and evaluated MSC-derived immunosuppressive cytokines, including HO1, GAL1, GAL9, TNFIA6, PGE2, PDL1, TGF-ß and HGF. Compared to the levels before MSC infusion, the HO1 (P = 0.0072) and TGF-ß (P = 0.0243) levels increased significantly 1 day after MSC infusion. At 7 days after MSC infusion, the levels of HO1, GAL1, TNFIA6 and TGF-ß tended to increase; however, the differences were not statistically significant. Although the 52-week cumulative incidence of cGVHD in the MSC group was comparable to that in the control group, fewer patients in the MSC group developed cGVHD involving ≥3 organs (14.3% vs. 43.6%, P = 0.006). MSCs were well tolerated, no infusion-related adverse events (AEs) occurred and other AEs were also comparable between the two groups. However, patients with malignant haematological diseases in the MSC group had a higher 52-week disease-free survival rate than those in the control group (84.8% vs. 65.9%, P = 0.031). CONCLUSIONS: For SR-aGVHD after allo-HSCT, especially HID HSCT, the combination of MSCs and basiliximab as the second-line therapy led to significantly better 4-week CR rates than basiliximab alone. The addition of MSCs not only did not increase toxicity but also provided a survival benefit.

Curcumin Suppresses M2 Macrophage-derived Paclitaxel Chemoresistance through Inhibition of PI3K-AKT/STAT3 Signaling.

BACKGROUND: Breast cancer is the leading cancer in women worldwide. The development of chemoresistance that leads to recurrence and mortality remains a major concern. M2-type tumor-associated macrophages (TAMs), present in the breast tumor microenvironment, secrete various cytokines and growth factors that induce chemoresistance. Curcumin, isolated from Curcuma longa, is known to sensitize cancer cells and increase the efficacy of standard chemotherapeutic agents. However, the effect of curcumin on the chemoresistancegenerating ability of M2 TAMs is not known. OBJECTIVE: The study aimed to determine whether curcumin could modulate M2 macrophages and suppress their ability to induce resistance to paclitaxel in breast cancer cells. METHODS: THP-1 cells were differentiated to M2 macrophages using PMA and IL-4/IL-13 in the presence or absence of curcumin in vitro. The effect of the conditioned medium of M2 macrophages on inducing resistance towards paclitaxel in MCF-7 or MDA-MB-231 cells was analyzed by cell proliferation assay, cell cycle analysis, wound healing and transwell migration assays. RT-PCR analysis was used to determine the mRNA expression of anti-inflammatory cytokines in M2 macrophages. The effect of curcumin on TGF-ß, pAKT, and pSTAT3 in M2 macrophages was analyzed by western blotting. RESULTS: Our data revealed that the M2 macrophages polarized in the presence of curcumin lacked the ability to generate chemoresistance to paclitaxel in breast cancer cell lines. Transcriptomic analysis revealed the expression of TGF-ß to be highest amongst M2 macrophage-secreted cytokines. We observed that purified recombinant TGF-ß generated chemoresistance in breast cancer cells. We found that curcumin treatment abrogated the expression of TGF-ß in M2 macrophages and suppressed their ability to induce chemoresistance in breast cancer cells. STITCH analysis showed strong interaction between curcumin and AKT/STAT3 pathway. Mechanistically, curcumin inhibited PI3K/AKT/STAT3 signaling in M2 macrophages. Western blot analysis revealed that M2 TAM CM, but not curcumin-treated macrophage CM, activated COX2/NF-κB in breast cancer cells. CONCLUSION: Our results showed that curcumin reduced the chemoresistance-generating ability of M2 TAMs. The study has revealed a non-cancer cell-autonomous mechanism by which curcumin partly overcomes the chemoresistance of paclitaxel in breast cancer.

Nintedanib solid lipid nanoparticles improve oral bioavailability and ameliorate pulmonary fibrosis in vitro and in vivo models.

Nintedanib (NIN) and pirfenidone are the only approved drugs for the treatment of Idiopathic Pulmonary Fibrosis (IPF). However, NIN and pirfenidone have low oral bioavailability and limited therapeutic potential, requiring higher dosages to increase their efficacy, which causes significant liver and gastrointestinal toxicities. In this study, we aimed to develop nintedanib-loaded solid lipid nanoparticles (NIN-SLN) to improve the oral bioavailability and therapeutic potential against TGF-ß-induced differentiation in IPF fibroblasts and bleomycin (BLM)-induced lung fibrosis in rat models. NIN-SLN was prepared using a double-emulsification method and characterization studies (Particle size, zeta potential, entrapment efficiency and other parameters) were performed using various techniques. NIN-SLN treatment significantly (p < 0.001) downregulated α-SMA and COL3A1 expression in TGF-ß stimulated DHLF and LL29 cells. NIN-SLN showed a 2.87-fold increase in the bioavailability of NIN and also improved the NIN levels in lung tissues compared to NIN alone. Pharmacodynamic investigation revealed that NIN-SLN (50 mg/Kg) treatment significantly attenuated BLM-induced lung fibrosis by inhibiting epithelial-to-mesenchymal-transition (EMT), extracellular matrix remodelling, and collagen deposition compared to free NIN. Additionally, in the BLM model of fibrosis, NIN-SLN greatly improved the BLM-caused pathological changes, attenuated the NIN-induced gastrointestinal abnormalities, and significantly improved the lung functional indices compared to free NIN. Collectively, NIN-SLN could be a promising nanoformulation for the management of pulmonary fibrosis.

Bone regeneration in critical-size defects of the mandible using biomechanically adapted CAD/CAM hybrid scaffolds: An in vivo study in miniature pigs.

The study aimed to analyze bone regeneration in critical-size defects using hybrid scaffolds biomechanically adapted to the specific defect and adding the growth factor rhBMP-2. For this animal study, ten minipigs underwent bilateral defects in the corpus mandibulae and were subsequently treated with novel cylindrical hybrid scaffolds. These scaffolds were designed digitally to suit the biomechanical requirements of the mandibular defect, utilizing finite element analysis. The scaffolds comprised zirconium dioxide-tricalcium phosphate (ZrO2-TCP) support struts and TCP foam ceramics. One scaffold in each animal was loaded with rhBMP-2 (100 µg/cm³), while the other served as an unloaded negative control. Fluorescent dyes were administered every 2 weeks, and computed tomography (CT) scans were conducted every 4 weeks. Euthanasia was performed after 3 months, and samples were collected for examination using micro-CT and histological evaluation of both hard and soft tissue. Intravital CT examinations revealed minor changes in radiographic density from 4 to 12 weeks postoperatively. In the group treated with rhBMP-2, radiographic density shifted from 2513 ± 128 (mean ± SD) to 2606 ± 115 Hounsfield units (HU), while the group without rhBMP-2 showed a change from 2430 ± 131 to 2601 ± 67 HU. Prior to implantation, the radiological density of samples measured 1508 ± 30 mg HA/cm³, whereas post-mortem densities were 1346 ± 71 mg HA/cm³ in the rhBMP-2 group and 1282 ± 91 mg HA/cm³ in the control group (p = 0.045), as indicated by micro-CT measurements. The histological assessment demonstrated successful ossification in all specimens. The newly formed bone area proportion was significantly greater in the rhBMP-2 group (48 ± 10%) compared with the control group without rhBMP-2 (42 ± 9%, p = 0.03). The mean area proportion of remaining TCP foam was 23 ± 8% with rhBMP-2 and 24 ± 10% without rhBMP-2. Successful bone regeneration was accomplished by implanting hybrid scaffolds into critical-size mandibular defects. Loading these scaffolds with rhBMP-2 led to enhanced bone regeneration and a uniform distribution of new bone formation within the hybrid scaffolds. Further studies are required to determine the adaptability of hybrid scaffolds for larger and potentially segmental defects in the maxillofacial region.

Vactosertib potently improves anti-tumor properties of 5-FU for colon cancer.

BACKGROUND: Several studies have shown that the TGF-ß signaling pathway plays a critical role in colorectal cancer (CRC) pathogenesis. The aim of the current study is to investigate the therapeutic potential of Vactosertib (EW-7197), a selective inhibitor of TGF-ß receptor type I, either alone or in combination with the standard first-line chemotherapeutic treatment, 5-Fluorouracil (5-FU), in CRC progression in both cellular and animal models. METHODS: Real-Time PCR, Zymography, enzyme-linked immunosorbent assay (ELISA), Hematoxylin and Eosin (H&E) tissue staining, and Flow cytometry techniques were applied to determine the anti-tumor properties of this novel TGF-ß inhibitor in in vitro (CT-26 cell line) and in vivo (inbred BALB/C mice) samples. RESULTS: Our findings showed that Vactosertib decreased cell proliferation and induced spheroid shrinkage. Moreover, this inhibitor suppressed the cell cycle and its administration either alone or in combination with 5-FU induced apoptosis by regulating the expression of p53 and BAX proteins. It also improved 5-FU anti-cancer effects by decreasing the tumor volume and weight, increasing tumor necrosis, and regulating tumor fibrosis and inflammation in an animal model. Vactosertib also enhanced the inhibitory effect of 5-FU on invasive behavior of CRC cells by upregulating the expression of E-cadherin and inhibiting MMP-9 enzymatic activity. CONCLUSION: This study demonstrating the potent anti-tumor effects of Vactosertib against CRC progression. Our results clearly suggest that this inhibitor could be a promising agent reducing CRC tumor progression when administered either alone or in combination with standard treatment in CRC patients.

Targeted delivery of CD163+ macrophage-derived small extracellular vesicles via RGD peptides promote vascular regeneration and stabilization after spinal cord injury.

Targeted delivery of small extracellular vesicles (sEVs) with low immunogenicity and fewer undesirable side effects are needed for spinal cord injury (SCI) therapy. Here, we show that RGD (Arg-Gly-Asp) peptide-decorated CD163+ macrophage-derived sEVs can deliver TGF-ß to the neovascular endothelial cells of the injured site and improve neurological function after SCI. CD163+ macrophages are M2 macrophages that express TGF-ß and are reported to promote angiogenesis and vascular stabilization in various diseases. Enriched TGF-ß EVs were crucial in angiogenesis and tissue repair. However, TGF-ß also boosts the formation of fibrous or glial scars, detrimental to neurological recovery. Our results found RGD-modified CD163+ sEVs accumulated in the injured region and were taken up by neovascular endothelial cells. Furthermore, RGD-CD163+ sEVs promoted vascular regeneration and stabilization in vitro and in vivo, resulting in substantial functional recovery post-SCI. These data suggest that RGD-CD163+ sEVs may be a potential strategy for treating SCI.

Tetrahydroberberrubine prevents peritoneal adhesion by suppressing inflammation and extracellular matrix accumulation.

Peritoneal adhesion is a common abdominal surgical complication that induces abdominal haemorrhage, intestinal obstruction, infertility, and so forth. The high morbidity and recurrence rate of this disease indicate the need for novel therapeutic approaches. Here, we revealed the protective roles of tetrahydroberberrubine (THBru), a novel derivative of berberine (BBR), in preventing peritoneal adhesion and identified its underlying mechanism in vivo and in vitro. Abrasive surgery was used to create a peritoneal adhesion rat model. We found that THBru administration markedly ameliorated peritoneal adhesion, as indicated by a lowered adhesion score and ameliorated caecal tissue damage. By comparison, THBru exhibited more potent anti-adhesion effects than BBR at the same dose. Mechanistically, THBru inhibited inflammation and extracellular matrix (ECM) accumulation in the microenvironment of adhesion tissue. THBru suppressed the expression of inflammatory cytokines including interleukin-1ß (IL-1ß), IL-6, transforming growth factor ß (TGF-ß), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1), by regulating the transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) and TAK1/nuclear factor κB (NF-κB) signaling pathways. However, THBru promoted the activation of MMP-3 by directly blocking the TIMP-1 activation core and subsequently decreased collagen deposition. Taken together, this study identifies THBru as an effective anti-adhesion agent that regulates diverse mechanisms, thereby outlining its potential therapeutic implications for the treatment of peritoneal adhesion.

Pulmonary fibrosis and type-17 immunity.

Fibrosis of the lung can occur in idiopathic pulmonary fibrosis, collagen vascular diseases, and hypersensitivity pneumonitis, among other diseases. Transforming growth factor (TGF)-ß, vascular epithelial growth factor, fibroblast growth factor, and platelet-derived growth factor contribute to the pathophysiology of fibrosis. TGF-ß and other cytokines, including interleukin (IL)-1ß, IL-6, and IL-23, activate type-17 immunity, which is involved in pulmonary fibrosis. The components of type-17 immunity include type-17 helper T cells, γδT cells, IL-17A-producing CD8-positive T cells, invariant NKT cells, and group 3 innate lymphoid cells. IL-17A, the main cytokine of type-17 immunity, is able to induce the epithelial-mesenchymal transition in epithelial cells via a production of TGF-ß, directly stimulate fibroblasts and fibrocytes, and inhibit autophagy, which otherwise protects against pulmonary fibrosis. IL-23 induces type-17 immunity and plays an important role in the acute exacerbation of pulmonary fibrosis. Clinical studies have also linked type-17 immunity to the pathogenesis of pulmonary fibrosis. Consequently, targeting type-17 immunity may serve as a new therapeutic strategy to prevent the development or exacerbation of pulmonary fibrosis.

Recruitment of regulatory T cells with rCCL17 promotes M2 microglia/macrophage polarization through TGFß/TGFßR/Smad2/3 pathway in a mouse model of intracerebral hemorrhage.

AIMS: Intracerebral hemorrhage (ICH) is a severe neurological condition with high mortality and morbidity. Microglia activation and peripheral inflammatory cells infiltration play an important role in ICH prognosis. Previous studies demonstrated that regulatory T cells (Tregs) ameliorated neuroinflammation following experimental ICH. However, the molecular mechanism underlying such effects of Tregs remains unclear. The objective was to examine how Tregs recruitment induced by recombinant CC chemokine ligand 17 (rCCL17) influences microglia/macrophage polarization in an intrastriatal autologous blood injection ICH animal model, and to determine if TGFß/TGFß-R/Smad2/3 pathway was involved. METHODS: 380 adult CD1 mice (male, eight weeks old) were subjected to sham surgery or autologous blood injection induced ICH. A CD25-specific mouse antibody or isotype control mAb was injected intraventricular (i.c.v) 48 h prior to ICH induction to deplete Tregs. rCCL17, a CC chemokine receptor 4 (CCR4) ligand, was delivered intranasally at 1 h post-ICH. SB431542, a specific inhibitor of TGF-ß was administered intraperitoneally 1 h before ICH induction. Following the ICH, neurobehavioral testing, brain edema, hematoma volume, hemoglobin content, western blotting, double immunofluorescence labeling, and immunohistochemistry were performed. RESULTS: Endogenous expressions of CCL17, Tregs marker Foxp3, and the number of Tregs in perihematomal region increased following ICH. Tregs depletion with a CD25 antibody aggravated neurological deficits and brain edema, increased inflammatory cytokines, neutrophil infiltration, oxidative stress, and reduced the rate of hematoma resolution in ICH mice. rCCL17 treatment increased the number of Tregs in the brain, ameliorated neurological deficits and brain edema after ICH, and promoted microglia/macrophage polarization toward M2 phenotype which was reversed with CD25 antibody. Moreover, rCCL17 increased the expressions of brain TGF-ß/phosphorylated-Smad2/3 which was abrogated with the selective TGFß inhibitor SB431542. CONCLUSIONS: rCCL17-mediated Tregs recruitment may be a potential therapeutic strategy to promote M2 microglia/macrophages polarization and alleviate early brain injury following ICH.

Bone morphogenetic proteins in spine surgery: a scientometric analysis to date.

OBJECTIVE: Bone morphogenetic proteins (BMPs) are part of the transforming growth factor-beta superfamily and are involved in bone formation and repair. In spine surgery, recombinant human BMP (rhBMP) is used as an alternative to autografts for spinal fusions. This study aimed to evaluate bibliometric parameters and citations of the literature on BMPs to provide an overview of how the field has evolved. METHODS: A comprehensive search of the literature was conducted using Elsevier's Scopus database to capture all the published and indexed studies relevant to BMPs from 1955 to the present. A discrete set of validated bibliometric parameters was extracted and analyzed. All statistical analyses were performed using R 4.1.1. RESULTS: The 100 most cited articles were published between 1994 and 2018 by 472 unique authors in 40 sources (e.g., journals and books). On average, there were 279 citations per publication and 17.69 citations per publication per year. The United States had the publications with the most citations (n = 23,761), followed by Hong Kong (n = 580) and the United Kingdom (n = 490). The three institutions in the United States with the greatest number of publications in the field were Emory University (n = 14), Hughston Clinic (n = 9), Hospital for Special Surgery (n = 6), and University of California (n = 6). CONCLUSIONS: The authors evaluated and characterized the 100 most cited articles about BMP. Most of the publications were clinical in nature and focused on BMP's application in spine surgery. While early scientific efforts focused on basic science research to advance the understanding of BMP's mechanism of action in promoting bone formation, the majority of the more recent publications are clinically focused. It will be beneficial to conduct more controlled clinical trials to compare the outcomes of BMP use with other methods.

Can a low dosage of recombinant human bone morphogenetic protein-2 loaded on collagen sponge induce ectopic bone?

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is one of the growth factors that may induce the formation of new bone. The aim was to determine the efficacy of low doses of rhBMP-2 for bone regeneration using a collagen sponge as a carrier. Three doses of rhBMP-2 (1.167, 0.117, and 0.039 mg/mL) were combined with an absorbable collagen sponge (ACS) as a delivery vehicle. The rhBMP-2/ACS implants were placed in the subcutaneous tissues of rat backs. X-ray microcomputed tomography (micro-CT) and histological analysis were used to evaluate bone formation. The samples treated with 1.167 mg/mL of rhBMP-2 showed greater bone formation than the samples treated with 0.117 mg/mL of rhBMP-2 four weeks after surgery. However, there was no evidence of bone formation in the samples that were treated with 0.039 mg/mL of rhBMP-2. It was found that rhBMP-2 was osteogenic even at one-tenth of its manufacturer's recommended concentration (1.167 mg/mL), indicating its potential for clinical use at lower concentrations.

Creation and preclinical evaluation of a novel mussel-inspired, biomimetic, bioactive bone graft scaffold: direct comparison with Infuse bone graft using a rat model of spinal fusion.

OBJECTIVE: Infuse bone graft is a widely used osteoinductive adjuvant; however, the simple collagen sponge scaffold used in the implant has minimal inherent osteoinductive properties and poorly controls the delivery of the adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). In this study, the authors sought to create a novel bone graft substitute material that overcomes the limitations of Infuse and compare the ability of this material with that of Infuse to facilitate union following spine surgery in a clinically translatable rat model of spinal fusion. METHODS: The authors created a polydopamine (PDA)-infused, porous, homogeneously dispersed solid mixture of extracellular matrix and calcium phosphates (BioMim-PDA) and then compared the efficacy of this material directly with Infuse in the setting of different concentrations of rhBMP-2 using a rat model of spinal fusion. Sixty male Sprague Dawley rats were randomly assigned to each of six equal groups: 1) collagen + 0.2 µg rhBMP-2/side, 2) BioMim-PDA + 0.2 µg rhBMP-2/side, 3) collagen + 2.0 µg rhBMP-2/side, 4) BioMim-PDA + 2.0 µg rhBMP-2/side, 5) collagen + 20 µg rhBMP-2/side, and 6) BioMim-PDA + 20 µg rhBMP-2/side. All animals underwent posterolateral intertransverse process fusion at L4-5 using the assigned bone graft. Animals were euthanized 8 weeks postoperatively, and their lumbar spines were analyzed via microcomputed tomography (µCT) and histology. Spinal fusion was defined as continuous bridging bone bilaterally across the fusion site evaluated via µCT. RESULTS: The fusion rate was 100% in all groups except group 1 (70%) and group 4 (90%). Use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater results for bone volume (BV), percentage BV, and trabecular number, as well as significantly smaller trabecular separation, compared with the use of the collagen sponge with 2.0 µg rhBMP-2. The same results were observed when the use of BioMim-PDA with 2.0 µg rhBMP-2 was compared with the use of the collagen sponge with 20 µg rhBMP-2. CONCLUSIONS: Implantation of rhBMP-2-adsorbed BioMim-PDA scaffolds resulted in BV and bone quality superior to that afforded by treatment with rhBMP-2 concentrations 10-fold higher implanted on a conventional collagen sponge. Using BioMim-PDA (vs a collagen sponge) for rhBMP-2 delivery could significantly lower the amount of rhBMP-2 required for successful bone grafting clinically, improving device safety and decreasing costs.

Stability and Activity of Interferon Beta to Treat Idiopathic Pulmonary Fibrosis with Different Nebulizer Technologies.

Background: Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by lung scarring, which results in breathing difficulty. Currently, patients with IPF exhibit a poor survival rate and have access to very limited therapeutic options. Interferon beta (IFN-ß) has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsing forms of multiple sclerosis, and it has also been shown to exhibit therapeutic potential in IPF. However, clinical use of IFN-ß did not lead to improved overall survival in IPF patients in existing studies. One possibility is the limited efficiency of IFN-ß delivery through intravenous or subcutaneous injection. Materials and Methods: The aerosol particle size distribution was determined with a laser diffraction particle size analyzer to characterize the droplet size and fine particle fraction generated by three types of nebulizers: jet, ultrasonic, and mesh. A breathing simulator was used to assess the delivery efficiency of IFN-ß, and the temperature in the medication reservoirs was monitored with a thermocouple during nebulization. To further evaluate the antifibrotic activity of IFN-ß pre- and postnebulization, bleomycin (BLM)- or transforming growth factor-beta (TGF-ß)-treated human lung fibroblast (HLF) cells were used. Cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Transwell migration assay and Q-PCR analysis were used to evaluate cell migration and the myofibroblast differentiation ability, respectively. IFN-ß protein samples were prepared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, and the expression of IFN-ß was assessed by western blotting. Results: Among the current drug delivery systems, aerosolized medication has shown increased efficacy of drug delivery for treating respiratory diseases when compared with parenteral drugs. It was found that neither the structural integrity nor the biological function of nebulized IFN-ß was compromised by the nebulization process of the mesh nebulizer. In addition, in BLM dose-response or TGF-ß-induced lung fibroblast proliferation assays, these effects could be reversed by both parenteral and inhaled IFN-ß nebulized with the mesh nebulizer. Nebulized IFN-ß with the mesh nebulizer also significantly inhibited the migration and myofibroblast differentiation ability of TGF-ß-treated HLF cells. Conclusions: The investigations revealed the potential efficacy of IFN-ß in the treatment of IPF with the mesh nebulizer, demonstrating the higher efficiency of IFN-ß delivered through the mesh nebulizer.

Myelodysplastic syndromes with ring sideroblasts.

Myelodysplastic syndromes (MDS) are acquired bone marrow malignant disorders characterized by ineffective hematopoiesis, resulting from a complex interaction between genetic and epigenetic mutations, alterations of the marrow microenvironment, and the immune system. In 2001, the World Health Organization (WHO) proposed a classification that integrates morphologic and genetic information, considering the MDS with ring sideroblasts (MDS-RS) as a distinct entity. Considering the strong association between MDS-RS and SF3B1 mutation and its importance in the development of MDS, the last WHO classification replaced the prior entity of MDS-RS with MDS with SF3B1 mutation. Several studies were performed to explore this genotype-phenotype correlation. Mutant SF3B1 protein deregulates the expression of genes implicated in developing hematopoietic stem and progenitor cells. Of paramount importance are PPOX and ABCB7 involved in iron metabolism. Another essential role in hemopoiesis is played by the transforming growth factor-beta (TGF-ß) receptor. This gene exerts its effects on SMAD pathways, regulating hematopoiesis through effects on balancing proliferation and apoptosis cell inactivity, differentiation, and migration. Luspatercept (ACE-536) is a soluble fusion protein that inhibits molecules in the TGF-ß superfamily. Since its structure resembles the TGF-ß family receptor, it catches TGF-ß superfamily ligands before binding to the receptor, resulting in reduced activation of SMAD signaling, thus enabling erythroid maturation. Luspatercept was investigated in the phase III trial MEDALIST, showing promising efficacy in treating anemia compared to placebo. Nowadays, further studies are needed to explore the real potential of luspatercept, investigating the biological features likely associated with treatment response, the potential use in combination treatments, and its role in the treatment of naïve MDS.

Bintrafusp Alfa, a Bifunctional Fusion Protein Targeting TGF-ß and PD-L1, in Patients With Non-Small Cell Lung Cancer Resistant or Refractory to Immune Checkpoint Inhibitors.

BACKGROUND: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor beta receptor II (a TGF-ß "trap") fused to a human immunoglobulin G1 monoclonal antibody blocking programmed cell death 1 ligand 1 (PD-L1). We report the efficacy and safety in patients with non-small cell lung cancer (NSCLC) that progressed following anti-PD-(L)1 therapy. MATERIALS AND METHODS: In this expansion cohort of NCT02517398-a global, open-label, phase I trial-adults with advanced NSCLC that progressed following chemotherapy and was primary refractory or had acquired resistance to anti-PD-(L)1 treatment received intravenous bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. The primary endpoint was best overall response (by Response Evaluation Criteria in Solid Tumors version 1.1 adjudicated by independent review committee); secondary endpoints included safety. RESULTS: Eighty-three eligible patients (62 [74.7%] treated with ≥3 prior therapies) received bintrafusp alfa. Four patients (3 primary refractory, 1 acquired resistant) had confirmed partial responses (objective response rate, 4.8%; 95% CI, 1.3%-11.9%), and 9 had stable disease. Tumor cell PD-L1 expression was not associated with response. Nineteen patients (22.9%) experienced grade ≥3 treatment-related adverse events, most commonly asthenia (3 [3.6%]) and fatigue, eczema, and pruritus (2 each [2.4%]). One patient had grade 4 amylase increased. One patient died during treatment for pneumonia before initiation of bintrafusp alfa. CONCLUSION: Although the primary endpoint was not met, bintrafusp alfa showed some clinical activity and a manageable safety profile in patients with heavily pretreated NSCLC, including prior anti-PD-(L)1 therapy. Tumor responses occurred irrespective of whether disease was primary refractory or had acquired resistance to prior anti-PD-(L)1 therapy.

Preclinical studies on the use of a P-selectin-blocking monoclonal antibody to halt progression of myelofibrosis in the Gata1low mouse model.

The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-ß (TGF-ß) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-ß and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients.

Paeonol impacts ovarian cancer cell proliferation, migration, invasion and apoptosis via modulating the transforming growth factor beta/smad3 signaling pathway.

Paeonol (2-hydroxy-4-methoxyphenylacetophenone) is a natural phenolic component isolated from the root bark of peony with multiple pharmacological activities and has been proven to have anti-cancer effects. The objective of this study is to investigate the influence mechanism of paeonol on the proliferatory and apoptotic activities of ovarian cancer (OC) cells by modulating the transforming growth factor beta (TGF-ß)/Smad3 pathway. The SKOV3 cells were pretreated with various concentrations of paeonol (0, 25, 50, 100, 200, 400 µg/mL) for 48 hours to determine the optimal experimental concentration of paeonol. Following this, the TGF-ß overexpression vector was constructed and transfected into the SKOV3 cells. The assessment of cell proliferation, invasion, and migration was conducted through MTT, colony formation, flow cytometry, transwell, and wound-healing experiments. The detection of TGF-ß/Smad3 pathway-related proteins and apoptosis-related proteins (B-cell lymphoma (Bcl-2) Bcl-2-associated X protein (Bax)) was performed using Western blot analysis. Paeonol exhibited a significant inhibitory effect on SKOV3 cell viability when administered at concentrations ranging from 50-400 µg/mL, with an IC50 value of 200 µg/mL. Within the concentration range of 50 to 200 µg/mL, paeonol exhibited a dose-dependent effect on the progression of SKOV3 cells, including a reduction in the anti-apoptotic protein Bcl-2, an increase in the pro-apoptotic protein Bax (P<0.05), inhibition of cell migration and invasion (P<0.05), and promotion of cell apoptosis (P<0.05), particularly at a concentration of 200 µg/mL. These effects were found to be more pronounced. The aforementioned effects of paeonol can be ascribed to its inhibition of the TGFß/Smad3 pathway, according to a mechanistic viewpoint. It is noteworthy that the inhibitory impact of paeonol on SKOV3 cell progression is counteracted by the elevation of TGF-ß levels following overexpression. We conclude that paeonol exerts regulatory effects on the TGF-ß/Smad3 pathway, leading to the inhibition of proliferation, migration, and invasion of OC cells, thereby attenuating malignant behavior of cancer cells.

Comparative evaluation of interleukin-10, transforming growth factor-ß, and interleukin-17 in gastrointestinal tuberculosis and crohn's disease.

Background: Gastrointestinal tuberculosis (GITB) and Crohn's disease (CD) are close mimickers and difficult to discriminate. Recent work has focused on the immunological differences between GITB and CD based on cytokines related to T-regulatory cells and Th17 cells. In the present cross-sectional study, suspected cases of GITB or CD underwent extensive clinical, radiological, endoscopic, histological, and microbiological assessment. The diagnosis was based on standard criteria and response to antitubercular therapy endoscopically. Methods: Interleukin (IL)-10, transforming growth factor-ß (TGF-ß), and IL-17 were measured and compared between GITB and CD along with other parameters. Fisher's exact test and Mann-Whitney U test were used as per the data type. Results: Of the 27 patients, 11 had CD, 9 had GITB, and 7 had other conditions. Chronic diarrhea, involvement of left and long segments of the colon, and aphthous ulcers were significantly more frequent in CD; however, transverse ulcers were in GITB. IL-10 was reduced in both GITB (median-interquartile range [IQR] 9.54 [3.65-24.04]) and CD (median-IQR 13.28 [6.91-22.50]) compared to control (median-IQR 26.72 [10.34-35.43]). TGF-ß showed little variation, but IL-17 was below the detection limit in most cases. None of these cytokines were significantly different between CD and GITB. The sensitivity and specificity of multiplex Mycobacterium tuberculosis-polymerase chain reaction were 44.44% and 100%, respectively. Conclusion: Serum cytokine profiling (IL-10, IL-17, and TGF-ß) could not significantly differentiate GITB and CD. Moreover, extensive molecular, transcriptomic, chemokines, and cytokine analyses may shed light on these aspects.

[Digoxin alleviates pulmonary fibrosis by regulating phosphatidylinositol-3-kinase/Akt signaling through inhibiting the activation of fibroblast: an in vivo and in vitro experiment].

OBJECTIVE: To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments. METHODS: (1) In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-I and COL-III) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-ß (TGF-ß) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. (2) In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-ß for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting. RESULTS: (1) In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-ß and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-ß and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA (A value): 5.37±1.10 vs. 9.51±1.66, TGF-ß protein (TGF-ß/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. (2) In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-ß was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01]. CONCLUSIONS: Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.

Roles of TGF-ß in cancer hallmarks and emerging onco-therapeutic design.

Transforming growth factor-beta (TGF-ß) is a double-edged sword in cancer treatment because of its pivotal yet complex and roles played during cancer initiation/development. Current anti-cancer strategies involving TGF-ß largely view TGF-ß as an onco-therapeutic target that not only substantially hinders its full utilisation for cancer control, but also considerably restricts innovations in this field. Thereby, how to take advantages of therapeutically favourable properties of TGF-ß for cancer management represents an interesting and less investigated problem. Here, by categorising cancer hallmarks into four critical transition events and one enabling characteristic controlling cancer initiation and progression, and delineating TGF-ß complexities according to these cancer traits, we identify the suppressive role of TGF-ß in tumour initiation and early-stage progression and its promotive functionalities in cancer metastasis as well as other cancer hallmarks. We also propose the feasibility and possible scenarios of combining cold atmospheric plasma (CAP) with onco-therapeutics utilising TGF-ß for cancer control given the intrinsic properties of CAP against cancer hallmarks.