Co-culture Studies Research Articles

Nanotextured titanium inhibits bacterial activity and supports cell growth on 2D and 3D substrate: A co-culture study

Medical implant-associated infections pose a significant challenge to modern medicine, with aseptic loosening and bacterial infiltration being the primary causes of implant failure. While nanostructured surfaces have demonstrated promising antibacterial properties, the translation of their efficacy from 2D to 3D substrates remains a challenge. Here, we used scalable alkaline etching to fabricate nanospike and nanonetwork topologies on 2D and laser powder-bed fusion printed 3D titanium. The fabricated surfaces were compared with regard to their antibacterial properties against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and mesenchymal stromal cell responses with and without the presence of bacteria. Finite elemental analysis assessed the mechanical properties and permeability of the 3D substrate. Our findings suggest that 3D nanostructured surfaces have potential to both prevent implant infections and allow host cell integration. This work represents a significant step towards developing effective and scalable fabrication methods on 3D substrates with consistent and reproducible antibacterial activity, with important implications for the future of medical implant technology.

Tissue factor overexpression promotes resistance to KRAS-G12C inhibition in non-small cell lung cancer

The recently approved KRASG12C mutation-specific inhibitors sotorasib and adagrasib (KRASG12C-I) represent a promising therapy for KRASG12C-driven non-small cell lung cancer (NSCLC). However, many eligible patients do not benefit due to intrinsic or acquired drug resistance. Tissue factor (TF) is overexpressed in KRAS-mutated (KRASmut) NSCLC and is the target of the FDA-approved ADC Tivdak. Here, we employed HuSC1-39, the parent antibody of a clinical stage TF-ADC (NCT04843709), to investigate the role of TF in KRASmut NSCLC. We found that patients with TF-overexpression had poor survival, elevated P-ERK/P-AKT activity levels and low immune effector cell infiltration in the tumor. In a panel of KRASG12C cell lines, KRASG12C-I response correlated with suppression of TF mRNA, which was not observed in resistant cells. In the drug resistant cells, TF-overexpression relied on an mTORC2-mediated and proteasome-dependent pathway. Combination treatment of HuSC1-39 or mTORC1/2 inhibitor MTI-31 with KRASG12C-I each produced synergistic antitumor efficacy in cell culture and in an orthotopic lung tumor model. TF-depletion in the resistant cells diminished epithelial mesenchymal transition, reduced tumor growth and greatly sensitized KRASG12C-I response. Moreover, employing immunohistochemistry and coculture studies, we demonstrated that HuSC1-39 or MTI-31 reset the tumor microenvironment and restore KRASG12C-I sensitivity by reshaping an M1-like macrophage profile with greatly enhanced phagocytic capacity toward tumor cell killing. Thus, we have identified the TF/mTORC2 axis as a critical new mechanism for triggering immunosuppression and KRASG12C-I resistance. We propose that targeting this axis with HuSC1-39 or MTI-31 will improve KRASG12C-I response in KRAS-driven NSCLC.

Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice

BackgroundRadiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF.MethodsUsing a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy.ResultsProphylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts.ConclusionsOur study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Plasma-derived exosomal miR-326, a prognostic biomarker and novel candidate for treatment of drug resistant pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a cancer with high incidence rate in pediatrics and drug resistance is a major clinical concern for ALL treatment. The current study was designed to evaluate the role of exosomal miR-326 in diagnosis and treatment of children with B-ALL. Exosomes were isolated from plasma samples of 30 patients and B-ALL cell lines followed by characterization, using nanoparticle tracking analysis, immunoblotting assay and electron microscopy. qPCR showed significantly increased levels of miR-326 in patients exosomes compared with non-cancer controls (P < 0.05, AUC = 0.7500). Moreover, a comparison between the sensitive and drug resistant patients revealed a prognostic value for the exosomal miR326 (P < 0.05, AUC = 0.7755). Co-culture studies on drug resistant patient primary cells and B-ALL cell lines suggested that exosomes with high miR-326 level act as vehicles for reducing cells viability. B-ALL cell line transfection with naked miR-326 mimic confirmed the results, and fluorescence microscopy validated uptake and internalization of exosomes by target cells. The novel introduced features of the exosomal miR-326 address a non-invasive way of diagnosing primary drug resistance in pediatric ALL and advocates a novel therapeutic strategy for this cancer.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.

Novel anti-inflammatory effects of the IL-1 receptor in kidney myeloid cells following ischemic AKI.

Introduction: Acute kidney injury (AKI) is one of the most common causes of organ failure in critically ill patients. Following AKI, the canonical pro-inflammatory cytokine interleukin-1β (IL-1β) is released predominantly from activated myeloid cells and binds to the interleukin-1 receptor R1 (IL-1R1) on leukocytes and kidney parenchymal cells. IL-1R1 on kidney tubular cells is known to amplify the immune response and exacerbate AKI. However, the specific role of IL-1R1 on myeloid cells during AKI is poorly understood. The objective of the present study was to elucidate the function of myeloid cell IL-1R1 during AKI. As IL-1R1 is known to signal through the pro-inflammatory Toll-like receptor (TLR)/MyD88 pathway, we hypothesized that myeloid cells expressing IL-1R1 would exacerbate AKI. Methods: IL-1R1 was selectively depleted in CD11c+-expressing myeloid cells with CD11cCre + /IL-1R1 fl/fl (Myel KO) mice. Myel KO and littermate controls (CD11cCre - /IL-1R1 fl/fl-Myel WT) were subjected to kidney ischemia/reperfusion (I/R) injury. Kidney injury was assessed by blood urea nitrogen (BUN), serum creatinine and injury marker neutrophil gelatinase-associated lipocalin (NGAL) protein expression. Renal tubular cells (RTC) were co-cultured with CD11c+ bone marrow-derived dendritic cells (BMDC) from Myel KO and Myel WT mice. Results: Surprisingly, compared to Myel WT mice, Myel KO mice displayed exaggerated I/R-induced kidney injury, as measured by elevated levels of serum creatinine and BUN, and kidney NGAL protein expression. In support of these findings, in vitro co-culture studies showed that RTC co-cultured with Myel KO BMDC (in the presence of IL-1β) exhibited higher mRNA levels of the kidney injury marker NGAL than those co-cultured with Myel WT BMDC. In addition, we observed that IL-1R1 on Myel WT BMDC preferentially augmented the expression of anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra/Il1rn), effects that were largely abrogated in Myel KO BMDC. Furthermore, recombinant IL-1Ra could rescue IL-1β-induced tubular cell injury. Discussion: Our findings suggest a novel function of IL-1R1 is to serve as a critical negative feedback regulator of IL-1 signaling in CD11c+ myeloid cells to dampen inflammation to limit AKI. Our results lend further support for cell-specific, as opposed to global, targeting of immunomodulatory agents.

Age‐Related Enhanced BMP Signaling Elicits APOE‐Isoform Dependent Effects in IPSC Model of Alzheimer’s Disease

AbstractBackgroundAging is the strongest risk factor for developing Alzheimer’s Disease (AD), and APOE is the largest genetic risk factor for developing AD. Bone morphogenic protein 4 (BMP4) expression and subsequent signaling increases 10‐fold with age and is increased in AD patients compared to age‐matched controls. Enhanced BMP signaling in aged and AD brains suggest a potential molecular cascade involved in AD pathogenesis. A potential interaction exists between increased BMP signaling across lifespan APOE expression that contribute to risk of AD.MethodAD patient derived iPSC’s (APOE3/E4 genotype) and isogenic controls (APOE3/E3 genotype) were differentiated into astrocytes and neurons. Cells were grown for 35 days before treatment with BMP4 or vehicle for 3 days prior to fixation, protein/media collection, or preparation for single cell RNA sequencing (scRNAseq). For coculture studies, neurons and astrocytes of the same patient background were differentiated separately prior to combined plating and BMP treatment. Cocultures were in matched (APOE3/E4 neurons with APOE3/E4 astrocytes) or mismatched (APOE3/E3 neurons with APOE3/E4 astrocytes) conditions to understand the non‐cell autonomous effects of APOE genotype.ResultUpon BMP treatment, APOE3/E4 compared to APOE3/E3 astrocytes exhibit accumulation of lipid droplets with changes in lipid composition. Increased free cholesterol levels as well as triglyceride accumulation is observed in APOE3/E4 astrocytes and is further exacerbated with BMP treatment. RNA‐sequencing analysis reveals marked decreased expression in genes related to oxidative phosphorylation and increased cholesterol synthesis and metabolism in APOE3/E4 astrocytes that is modulated by BMP treatment. In a non‐cell autonomous fashion, APOE3/E3 neurons, when paired with APOE3/E4 astrocytes, express exacerbated pTau levels and aberrant metabolic processes in a BMP‐dependent manner.ConclusionOur results indicate an interaction between genetic and aging factors in cellular processes relevant to AD. Enhanced BMP signaling confers aberrant lipid composition and metabolic processing in APOE3/E4 astrocytes when compared to APOE3/E3. Subsequent BMP‐dependent non‐cell autonomous effects in APOE3/E3 neurons are observed when cocultured with APOE3/E4 astrocytes. This data suggests age‐associated increase in BMP signaling may modulate APOE isoform‐dependent metabolic processes and increase risk for APOE‐mediated pathological cascades.

Tetrazole derivatives as potent immunomodulatory agents in tumor microenvironment

Twenty-seven compounds bearing a tetrazole ring as a central unit have been designed, synthetized and biologically evaluated. Studies have been performed in order to compare the effect of tetrazole derivatives bearing amine electron-donor or nitro electron-acceptor groups. The antiproliferative activity has been determined in monoculture studies on tumor cell lines HT-29, A-549, MCF-7 and on non-tumor cell line HEK-293 as well as in co-culture studies (HT-29/THP-1). All the compounds have been studied as PD-L1 (Programmed Death Ligand 1), VEGFR-2 (Vascular Endothelial Growth Factor 2), CD-47 (Cluster of Differentiation 47) and c-Myc inhibitors. The effect on TNF-α secretion has also been determined. Bromoderivatives 23, 24 and chloroderivatives 26, 27 have demonstrated an apoptotic effect on HT-29 cancer cells. Compounds bearing an amine group have shown very promising effects as TME immunomodulatory agents.

Adipocyte retinoic acid receptor α prevents obesity and steatohepatitis by regulating energy expenditure and lipogenesis.

The adipose tissue-liver axis is a major regulator of the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Retinoic acid signaling plays an important role in development and metabolism. However, little is known about the role of adipose retinoic acid signaling in the development of obesity-associated NAFLD. In this work, the aim was to investigate whether and how retinoic acid receptor alpha (RARα) regulated the development of obesity and NAFLD. RARα expression in adipose tissue of db/db or ob/ob mice was determined. Rarαfl/fl mice and adipocyte-specific Rarα-/- (RarαAdi-/- ) mice were fed a chow diet for 1 year or high-fat diet (HFD) for 20 weeks. Primary adipocytes and primary hepatocytes were co-cultured. Metabolic regulation and inflammatory response were characterized. RARα expression was reduced in adipose tissue of db/db or ob/ob mice. RarαAdi-/- mice had increased obesity and steatohepatitis (NASH) when fed a chow diet or HFD. Loss of adipocyte RARα induced lipogenesis and inflammation in adipose tissue and the liver and reduced thermogenesis. In the co-culture studies, loss of RARα in adipocytes induced inflammatory and lipogenic programs in hepatocytes. The data demonstrate that RARα in adipocytes prevents obesity and NASH via inhibiting lipogenesis and inflammation and inducing energy expenditure.

Epicardial adipose tissue, metabolic disorders, and cardiovascular diseases: recent advances classified by research methodologies.

Epicardial adipose tissue (EAT) is located between the myocardium and visceral pericardium. The unique anatomy and physiology of the EAT determines its great potential in locally influencing adjacent tissues such as the myocardium and coronary arteries. Classified by research methodologies, this study reviews the latest research progress on the role of EAT in cardiovascular diseases (CVDs), particularly in patients with metabolic disorders. Studies based on imaging techniques demonstrated that increased EAT amount in patients with metabolic disorders is associated with higher risk of CVDs and increased mortality. Then, in-depth profiling studies indicate that remodeled EAT may serve as a local mediator of the deleterious effects of cardiometabolic conditions and plays a crucial role in CVDs. Further, in vitro coculture studies provided preliminary evidence that the paracrine effect of remodeled EAT on adjacent cardiomyocytes can promote the occurrence and progression of CVDs. Considering the important role of EAT in CVDs, targeting EAT might be a potential strategy to reduce cardiovascular risks. Several interventions have been proved effective in reducing EAT amount. Our review provides valuable insights of the relationship between EAT, metabolic disorders, and CVDs, as well as an overview of the methodological constructs of EAT-related studies.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Development of protein-polymer conjugate nanoparticles for modulation of dendritic cell phenotype and antigen-specific CD4 T cell responses.

Polymeric nanoparticles (NPs) comprised of poly(lactic-co-glycolic acid) (PLGA) have found success in modulating antigen (Ag)-specific T cell responses for the treatment multiple immunological diseases. Common methods by which Ags are associated with NPs are through encapsulation and surface conjugation; however, these methods suffer from several limitations, including uncontrolled Ag loading, burst release, and potential immune recognition. To overcome these limitations and study the relationship between NP design parameters and modulation of innate and Ag-specific adaptive immune cell responses, we developed ovalbumin (OVA) protein-PLGA bioconjugate NPs (acNP-OVA). OVA was first modified by conjugation with multiple PLGA polymers to synthesize OVA-PLGA conjugates, followed by precise combination with unmodified PLGA to form acNP-OVA with well-defined Ag loadings, reduced burst release, and reduced antibody recognition. Expression of MHC II, CD80, and CD86 on bone marrow-derived dendritic cells (BMDCs) increased as a function of acNP-OVA Ag loading. NanoString studies using BMDCs showed that PLGA NPs generally induced anti-inflammatory gene expression profiles independent of the Ag delivery method, where S100a9, Sell, and Ppbp were most significantly reduced. Co-culture studies using acNP-OVA-treated BMDCs and OT-II CD4+ T cells revealed that Ag-specific T cell activation, expansion, and differentiation were dependent on Ag loading and formulation parameters. CD25 expression was induced using acNP-OVA with the lowest Ag loading; however, the induction of robust CD4+ T cell proliferative and cytokine responses required acNP-OVA formulations with higher Ag loading, which was supported using a regulatory T cell (Treg) induction assay. The distinct differences in Ag loading required to achieve various T cell responses supported the concept of an Ag loading threshold for Ag-specific immunotherapy. We anticipate this work will help guide NP designs and aid in the future development of NP-based immunotherapies for Ag-specific immunomodulation.

P06.03.A IMMORTALIZED MACROPHAGES AS A TOOL TO STUDY PHAGOCYTOSIS AND POLARIZATION IN VITRO

Abstract BACKGROUND Macrophages, an important element of the tumour-microenvironment (TME), are classified based on polarization state: tumour inhibiting ‘classically-activated macrophages’ M1 and tumour promoting ‘alternatively-activated macrophages’ M2. It is wellknown that the glioblastoma (GBM) TME is dominated by immunosuppressive M2 macrophages contributing to tumor growth by promoting angiogenesis, invasion and immunosuppression. However, macrophage culture is difficult to maintain as the cells do not proliferate and expand, making co-culture studies challenging. In this work, we successfully immortalized and expanded bone marrow derived mouse macrophages (BMDMs) that can be used as a tool to study phagocytosis and polarization in co-culture with tumor cells. MATERIAL AND METHODS Primary macrophages, obtained from the bone marrow of C57/BL6 mice, were transduced with two lentiviral vectors: hTERT with antibiotic hygromycin (Hy) and Simian virus 40 (SV40) with mCherry selective markers. Viral-titers were prepared by co-transfecting HEK293T with hTERT and SV40 constructs and plasmids MDL-X (Gag/Pol):Vpx. Vpx is a virion-packaged accessory protein that induces degradation of SAMHD1, a GTP- activated dNTP hydrolase that inhibits lentiviral infection of macrophages. BMDMs transduced with hTERT were subjected to gradient Hy selection after 5 days infection and SV40.mCherry positive cells were sorted by flow cytometry. To assess phagocytotic capacity, BMDMs were incubated with apoptotic bodies of a murine GBM cell-line (GL261) which was treated with HSV-TK/ganciclovir (GCV) suicide gene therapy. Phagocytosis was measured by fluorescence microscopy and flow cytometry. Further, BMDMs were externally induced with either E. coli lipopolysaccharide (LPS) and interferon gamma (IFNγ) or interleukin 4 (IL4) to activate M1 and M2 polarization. Flow cytometry was used to detect iNOS, CD40 and CD86 markers for M1 phenotype and Arginase1 and CD206 for M2. RESULTS hTERT.MDL-X.Vpx.Hy BMDM showed no clear signs of immortalization with limited proliferation followed by cell death. In contrast, SV40.MDL-X.Vpx.mCherry BMDM exhibited proliferative expansion from single colony, efficient freeze-thaw cycles and were used for further studies. BMDMs showed successful phagocytosis of apoptotic bodies from HSV-tk/GCV treated GL261 cells with an increasing capacity upto 18 hours of application. The phagocytic capacity was higher compared to commercial cell lines such as BV2 and Raw 264.7. Immortalized BMDMs tend to have a M2 phenotype without external induction and it is possible to activate M1 phenotype by LPS and IFNγ induction. CONCLUSION Here, we successfully immortalized bone-marrow derived macrophages and showed that they can be used as a tool to study macrophage polarization and phagocytic capability in a tumor immunotherapy context.

BAP31 Knockout in Macrophages Affects CD4+T Cell Activation through Upregulation of MHC Class II Molecule.

The differentiation of CD4+T cells is a crucial component of the immune response. The spleen and thymus, as immune organs, are closely associated with the differentiation and development of T cells. Previous studies have suggested that BAP31 may play a role in modulating T cell activation, but the specific impact of BAP31 on T cells through macrophages remains uncertain. In this study, we present evidence that BAP31 macrophage conditional knockout (BAP31-MCKO) mice display an enlarged spleen and thymus, accompanied by activated clustering and disrupted differentiation of CD4+T cells. In vitro co-culture studies were conducted to investigate the impact of BAP31-MCKO on the activation and differentiation of CD4+T cells. The examination of costimulatory molecule expression in BMDMs and RAW 264.7 cells, based on the endoplasmic reticulum function of BAP31, revealed an increase in the expression of antigen presenting molecules, particularly MHC-II molecule, in the absence of BAP31 in BMDMs or RAW264.7 cells. These findings suggest that BAP31 plays a role in the activation and differentiation of CD4+T cells by regulating the MHC class II molecule on macrophages. These results provide further support for the importance of BAP31 in developing interaction between macrophages and CD4+T cells.

Investigation of human hair keratin-based nanofibrous scaffold for skin tissue engineering application.

Keratin-based nanofibers were fabricated using the electrospinning technique, and their potential as scaffolds for tissue engineering was investigated. Keratin, extracted from the human hair, was blended with poly(vinyl alcohol) (PVA) in an aqueous medium. Morphological characterizations of the fabricated PVA-keratin nanofiber (PK-NF) random and aligned scaffolds performed using a scanning electron microscope (SEM) revealed the formation of uniform and randomly oriented nanofibers with an interconnected three-dimensional network structure. The mean diameter of the nanofibers ranged from 100 to 250nm. Functional groups and structural studies were done by infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. FTIR study suggested that PVA interacted with keratin by hydrogen bonding. Moreover, the in vitro cell culture study could suggest that PK-NF scaffolds were non-cytotoxic by supporting the growth of murine embryonic stem cells (ESCs), human keratinocytes (HaCaT), and dermal fibroblast (NHDF) cell lines. Further, the immunocytochemical characterization revealed the successful infiltration, adhesion, and growth of ESCs, HaCaT, and NHDF cells seeded on PK-NF scaffolds. However, there was no noteworthy difference observed concerning cell growth and viability irrespective of the random and aligned internal fibril arrangement of the PK-NF scaffolds. The infiltration and growth pattern of HaCaT and NHDF cells adjacent to each other in a 3D co-culture study mimicked that of epidermal and dermal skin cells and indeed underscored the potential of PK-NFs as a scaffold for skin tissue engineering.

Discovery of deoxyandrographolide and its novel effect on vascular senescence by targeting HDAC1.

Aconitum carmichaelii (Fuzi) is a traditional Chinese medicine that has been widely used in the clinic to save the dying life for over several thousand years. However, the medicinal components of Fuzi in treating vascular senescence (VS) and its potential mechanism remain unclear. In this study, a network pharmacology method was used to explore the possible components and further validated by experiments to get a candidate compound, deoxyandrographolide (DA). DA restrains aging biomarkers, such as p16, p21, γH2A.X, and p53 in vitro and in vivo blood co-culture studies. Histone deacetylase 1 (HDAC1), mouse double minute2 (MDM2), cyclin-dependent kinase 4, and mechanistic target of rapamycin kinase (mTOR) are predicted to be the possible targets of DA based on virtual screening. Subsequent bio-layer interferometry results indicated that DA showed good affinity capability with HDAC1. DA enhances the protein expression of HDAC1 in the angiotensin II-induced senescence process by inhibiting its ubiquitination degradation. Loss of HDAC1 by CRISPR/Cas9 leads to the disappearance of DA's anti-aging property. The enhancement of HDAC1 represses H3K4me3 (a biomarker of chromosomal activity) and improves chromosome stability. RNA sequencing results also confirmed our hypothesis. Our evidence illuminated that DA may achieve as a novel compound in the treatment of VS by improving chromosome stability.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like Tcell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in exvivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Resolving neutrophils due to TRAM deletion renders protection against experimental sepsis.

Proper inflammation resolution is crucial to prevent runaway inflammation during sepsis and reduce sepsis-related mortality/morbidity. Previous studies suggest that deleting TRAM, a key TLR4 signaling adaptor, can reprogram the first inflammatory responder cell-neutrophil from an inflammatory state to a resolving state. In this study, we aim to examine the therapeutic potential of TRAM-deficient neutrophils in vivo with recipient mice undergoing experimental sepsis. Wild-type or Tram-/- mice were intraperitoneally injected with cecal slurry to induce either severe or mild sepsis. Phenotypic examinations of sepsis and neutrophil characteristics were examined in vivo and ex vivo. The propagations of resolution from donor neutrophils to recipient cells such as monocytes, T cells, and endothelial cells were examined through co-culture assays in vitro. The efficacies of Tram-/- neutrophils in reducing inflammation were studied by transfusing either wild-type or Tram-/- neutrophils into septic recipient mice. Tram-/- septic mice had improved survival and attenuated injuries within the lung and kidney tissues as compared to wild-type septic mice. Wild-type septic mice transfused with Tram-/- resolving neutrophils exhibited reduced multi-organ damages and improved cellular homeostasis. In vitro co-culture studies revealed that donor Tram-/- neutrophils can effectively propagate cellular homeostasis to co-cultured neighboring monocytes, neutrophils, T cells as well as endothelial cells. Neutrophils with TRAM deletion render effective reprogramming into a resolving state beneficial for ameliorating experimental sepsis, with therapeutic potential in propagating cellular and tissue homeostasis as well as treating sepsis.

Tumor-Associated Macrophages Affect the Tumor Microenvironment and Radioresistance via the Upregulation of CXCL6/CXCR2 in Hepatocellular Carcinoma.

Hepatocellular carcinoma is the sixth most diagnosed malignancy and the fourth most common cause of cancer-related mortality globally. Despite progress in the treatment of liver cancer, nonsurgical treatments remain unsatisfactory, and only 15% of early-stage cases are surgically operable. Radiotherapy (RT) is a non-surgical treatment option for liver cancer when other traditional treatment methods are ineffective. However, RT has certain limitations, including eliciting poor therapeutic effects in patients with advanced and recurrent tumors. Tumor-associated macrophages (TAMs) are major inflammatory cells in the tumor microenvironment that are key to tumor development, angiogenesis, invasion, and metastasis, and they play an essential role in RT responses. We used big data analysis to determine the potential of targeting CXCL6/CXCR2. We enrolled 50 patients with liver cancer who received RT at our hospital. Tumor tissue samples were examined for any relationship between CXCL6/CXCR2 activity and patient prognosis. Using a cell coculture system (Transwell), we cocultured Huh7 liver cancer cells and THP-1 monocytes with and without CXCL6/CXCR2 small interfering RNA for 72 h. The overexpression of CXCL6/CXCR2 was highly correlated with mortality. Our tissue study indicated a positive correlation between CXCL6/CXCR2 and M2-TAMs subsets. The coculture study demonstrated that THP-1 monocytes can secrete CXCL6, which acts on the CXCR2 receptor on the surface of Huh7 cells and activates IFN-g/p38 MAPK/NF-κB signals to promote the epithelial-mesenchymal transition and radio-resistance. Modulating the TAM/CXCL6/CXCR2 tumor immune signaling axis may be a new treatment strategy for the effective eradication of radiotherapy-resistant hepatocellular carcinoma cells.