Blockade of SIRPα-CD47 axis by anti-SIRPα antibody enhances anti-tumor activity of DXd antibody-drug conjugates.

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs.

Combining SiRPα decoy-coengineered T cells and antibodies augments macrophage-mediated phagocytosis of tumor cells.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

Nono induces Gadd45b to mediate DNA repair.

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.

The Antiviral Activity of Interferon-Induced Transmembrane Proteins and Virus Evasion Strategies.

Interferons (IFNs) are antiviral cytokines that defend against viral infections by inducing the expression of interferon-stimulated genes (ISGs). Interferon-inducible transmembrane proteins (IFITMs) 1, 2, and 3 are crucial ISG products and members of the CD225 protein family. Compelling evidence shows that IFITMs restrict the infection of many unrelated viruses by inhibiting the virus-cell membrane fusion at the virus entry step via the modulation of lipid composition and membrane properties. Meanwhile, viruses can evade IFITMs' restrictions by either directly interacting with IFITMs via viral glycoproteins or by altering the native entry pathway. At the same time, cumulative evidence suggests context-dependent and multifaceted roles of IFITMs in modulating virus infections and cell signaling. Here, we review the diverse antiviral mechanisms of IFITMs, the viral antagonizing strategies, and the regulation of IFITM activity in host cells. The mechanisms behind the antiviral activity of IFITMs could aid the development of broad-spectrum antivirals and enhance preparedness for future pandemics.

Engineered CD47 protects T cells for enhanced antitumour immunity.

Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

Glioblastoma Phagocytic Cell Death: Balancing the Opportunities for Therapeutic Manipulation.

Macrophages and microglia are professional phagocytes that sense and migrate toward "eat-me" signals. The role of phagocytic cells is to maintain homeostasis by engulfing senescent or apoptotic cells, debris, and abnormally aggregated macromolecules. Usually, dying cells send out "find-me" signals, facilitating the recruitment of phagocytes. Healthy cells can also promote or inhibit the phagocytosis phenomenon of macrophages and microglia by tuning the balance between "eat-me" and "don't-eat-me" signals at different stages in their lifespan, while the "don't-eat-me" signals are often hijacked by tumor cells as a mechanism of immune evasion. Using a combination of bioinformatic analysis and spatial profiling, we delineate the balance of the "don't-eat-me" CD47/SIRPα and "eat-me" CALR/STC1 ligand-receptor interactions to guide therapeutic strategies that are being developed for glioblastoma sequestered in the central nervous system (CNS).

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Transglutaminase 3 regulates cutaneous squamous carcinoma differentiation and inhibits progression via PI3K-AKT signaling pathway-mediated Keratin 14 degradation.

Cutaneous squamous carcinoma is the second most common epithelial malignancy, associated with significant morbidity, mortality, and economic burden. However, the mechanisms underlying cSCC remain poorly understood. In this study, we identified TGM3 as a novel cSCC tumor suppressor that acts via the PI3K-AKT axis. RT-qPCR, IHC and western blotting were employed to assess TGM3 levels. TGM3-overexpression/knockdown cSCC cell lines were utilized to detect TGM3's impact on epithelial differentiation as well as tumor cell proliferation, migration, and invasion in vitro. Additionally, subcutaneous xenograft tumor models were employed to examine the effect of TGM3 knockdown on tumor growth in vivo. Finally, molecular and biochemical approaches were employed to gain insight into the tumor-suppressing mechanisms of TGM3. TGM3 expression was increased in well-differentiated cSCC tumors, whereas it was decreased in poor-differentiated cSCC tumors. Loss of TGM3 is associated with poor differentiation and a high recurrence rate in patients with cSCC. TGM3 exhibited tumor-suppressing activity by regulating cell proliferation, migration, and invasion both in vitro and in vivo. As a novel cSCC tumor differentiation marker, TGM3 expression was positively correlated with cell differentiation. In addition, our results demonstrated an interaction between TGM3 and KRT14 that aids in the degradation of KRT14. TGM3 deficiency disrupts keratinocytes differentiation, and ultimately leads to tumorigenesis. Furthermore, RNA-sequence analysis revealed that loss of TGM3 enhanced EMT via the PI3K-AKT signaling pathway. Deguelin, a PI3K-AKT inhibitor, blocked cSCC tumor growth induced by TGM3 knockdown in vivo. Taken together, TGM3 inhibits cSCC tumor growth via PI3K-AKT signaling, which could also serve as a tumor differentiation marker and a potential therapeutic target for cSCC. Proposed model depicted the mechanism by which TGM3 suppress cSCC development. TGM3 reduces the phosphorylation level of AKT and degrades KRT14. In the epithelial cell layer, TGM3 exhibits a characteristic pattern of increasing expression from bottom to top, while KRT14 and pAKT are the opposite. Loss of TGM3 leads to reduced degradation of KRT14 and activation of pAKT, disrupting keratinocyte differentiation, and eventually resulting in the occurrence of low-differentiated cSCC.

GADD45B in the ventral hippocampal CA1 modulates aversive memory acquisition and spatial cognition.

AIMS: This study was designed to investigate the role of growth arrest and DNA damage-inducible ß (GADD45B) in modulating fear memory acquisition and elucidate its underlying mechanisms. MAIN METHODS: Adeno-associated virus (AAV) that knockdown or overexpression GADD45B were injected into ventral hippocampal CA1 (vCA1) by stereotactic, and verified by fluorescence and Western blot. The contextual fear conditioning paradigm was employed to examine the involvement of GADD45B in modulating aversive memory acquisition. The Y-maze and novel location recognition (NLR) tests were used to examine non-aversive cognition. The synaptic plasticity and electrophysiological properties of neurons were measured by slice patch clamp. KEY FINDINGS: Knockdown of GADD45B in the vCA1 significantly enhanced fear memory acquisition, accompanied by an upregulation of long-term potentiation (LTP) expression and intrinsic excitability of vCA1 pyramidal neurons (PNs). Conversely, overexpression of GADD45B produced the opposite effects. Notably, silencing the activity of vCA1 neurons abolished the impact of GADD45B knockdown on fear memory development. Moreover, mice with vCA1 GADD45B overexpression exhibited impaired spatial cognition, whereas mice with GADD45B knockdown did not display such impairment. SIGNIFICANCE: These results provided compelling evidence for the crucial involvement of GADD45B in the formation of aversive memory and spatial cognition.

Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

Gentulizumab, a novel anti-CD47 antibody with potent antitumor activity and demonstrates a favorable safety profile.

BACKGROUND: Targeting CD47/SIRPα axis has emerged as a promising strategy in cancer immunotherapy. Despite the encouraging clinical efficacy observed in hematologic malignancies through CD47-SIRPα blockade, there are safety concerns related to the binding of anti-CD47 antibodies to CD47 on the membrane of peripheral blood cells. METHODS: In order to enhance the selectivity and therapeutic efficacy of the antibody, we developed a humanized anti-CD47 monoclonal antibody called Gentulizumab (GenSci059). The binding capacity of GenSci059 to CD47 was evaluated using flow cytometry and surface plasmon resonance (SPR) methods, the inhibitory effect of GenSci059 on the CD47-SIRPα interaction was evaluated through competitive ELISA assays. The anti-tumor activity of GenSci059 was assessed using in vitro macrophage models and in vivo patient-derived xenograft (PDX) models. To evaluate the safety profile of GenSci059, binding assays were conducted using blood cells. Additionally, we investigated the underlying mechanisms contributing to the weaker binding of GenSci059 to erythrocytes. Finally, toxicity studies were performed in non-human primates to assess the potential risks associated with GenSci059. RESULTS: GenSci059 displayed strong binding to CD47 in both human and monkey, and effectively inhibited the CD47-SIRPα interaction. With doses ranging from 5 to 20 mg/kg, GenSci059 demonstrated potent inhibition of the growth of subcutaneous tumor with the inhibition rates ranged from 30.3% to complete regression. Combination of GenSci059 with 2.5 mg/kg Rituximab at a dose of 2.5 mg/kg showed enhanced tumor inhibition compared to monotherapy, exhibiting synergistic effects. GenSci059 exhibited minimal binding to hRBCs compared to Hu5F9-G4. The binding of GenSci059 to CD47 depended on the cyclization of N-terminal pyroglutamic acid and the spatial conformation of CD47, but was not affected by its glycosylation modifications. A maximum tolerated dose (MTD) of 450 mg/kg was observed for GenSci059, and no significant adverse effects were observed in repeated dosages up to 10 + 300 mg/kg, indicating a favorable safety profile. CONCLUSION: GenSci059 selectively binds to CD47, effectively blocks the CD47/SIRPα axis signaling pathway and enhances the phagocytosis effects of macrophages toward tumor cells. This monoclonal antibody demonstrates potent antitumor activity and exhibits a favorable safety profile, positioning it as a promising and effective therapeutic option for cancer.

IFITM1 and IFITM2 inhibit the replication of senecavirus A by positive feedback with RIG-I signaling pathway.

The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-ß), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus.

Cancer stem cells: The important role of CD markers, Signaling pathways, and MicroRNAs.

For the first time, a subset of small cancer cells identified in acute myeloid leukemia has been termed Cancer Stem Cells (CSCs). These cells are notorious for their robust proliferation, self-renewal abilities, significant tumor-forming potential, spread, and resistance to treatments. CSCs are a global concern, as it found in numerous types of cancer, posing a real-world challenge today. Our review encompasses research on key CSC markers, signaling pathways, and MicroRNA in three types of cancer: breast, colon, and liver. These factors play a critical role in either promoting or inhibiting cancer cell growth. The reviewed studies have shown that as cells undergo malignant transformation, there can be an increase or decrease in the expression of different Cluster of Differentiation (CD) markers on their surface. Furthermore, alterations in essential signaling pathways, such as Wnt and Notch1, may impact CSC proliferation, survival, and movement, while also providing potential targets for cancer therapies. Additionally, some research has focused on MicroRNAs due to their dual role as potential therapeutic biomarkers and their ability to enhance CSCs' response to anti-cancer drugs. MicroRNAs also regulate a wide array of cellular processes, including the self-renewal and pluripotency of CSCs, and influence gene transcription. Thus, these studies indicate that MicroRNAs play a significant role in the malignancy of various tumors. Although the gathered information suggests that specific CSC markers, signaling pathways, and MicroRNAs are influential in determining the destiny of cancer cells and could be advantageous for therapeutic strategies, their precise roles and impacts remain incompletely defined, necessitating further investigation.

The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47.

OBJECTIVE: Innate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). METHODS: We first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. RESULTS: In initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. CONCLUSION: These results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

Structural-functional diversity of CD47 proteoforms.

The ubiquitously expressed transmembrane glycoprotein CD47 participates in various important physiological cell functions, including phagocytosis, apoptosis, proliferation, adhesion, and migration, through interactions with its ligands, including the inhibitory receptor signal regulatory protein α (SIRPα), secreted glycoprotein thrombospondin-1 (TSP-1), and integrins. Elevated expression of CD47 is observed in a wide range of cancer cells as a mechanism for evading the immune system, blocking the interaction between the CD47 and SIRPα is the most advanced and promising therapeutic approach currently investigated in multiple clinical trials. The widely held view that a single type of CD47 protein acts through membrane interactions has been challenged by the discovery of a large cohort of CD47 proteins with cell-, tissue-, and temporal-specific expression and functional profiles. These profiles have been derived from a single gene through alternative splicing and post-translational modifications, such as glycosylation, pyroglutamate modification, glycosaminoglycan modification, and proteolytic cleavage and, to some extent, via specific CD47 clustering in aging and tumor cells and the regulation of its subcellular localization by a pre-translational modification, alternative cleavage and polyadenylation (APA). This review explores the origins and molecular properties of CD47 proteoforms and their roles under physiological and pathological conditions, mentioning the new methods to improve the response to the therapeutic inhibition of CD47-SIRPα immune checkpoints, contributing to the understanding of CD47 proteoform diversity and identification of novel clinical targets and immune-related therapeutic candidates.

Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury by regulating the Cx43.

PURPOSE: To explore the mechanism by which Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury (TBI). METHODS: The oxygen -glucose deprivation/recovery (OGD/R) cell model was constructed to simulate the pathological state of astrocytes in a TBI environment. The viability of astrocytes was measured by CCK-8, and the cytoskeleton changes were observed by Phalloidin- TRITC staining. The expressions of differentiation markers, Cx43 and phosphorylated Cx43 (P-Cx43) of A1/A2 astrocytes were detected by Western blot, and the complement C3 and S100A10 of A1/A2 astrocytes were detected by ELISA. The TBI rat model was established. The water content of brain tissue was measured by dry-wet specific gravity method, the pathological morphology of brain tissue in cortical injury area was observed by HE staining method, ROS was detected by fluorescence quantitative method, Cx43 expression was detected by immunohistochemistry method, and the differentiation markers of A1/A2 astrocytes were detected by immunofluorescence. RESULTS: In the TBI environment, astrocytes showed decreased cell viability, blurred skeleton, and increased expression of Cx43. In TBI rats, the water content of brain tissue increased, the brain tissue in the cortex injury area was seriously damaged, ROS and Cx43 expression were significantly increased, and mainly distributed in A2 astrocytes. Remazolam can reverse the above results after the intervention. CONCLUSION: Remazolam affects the phenotype and function of astrocytes to improve TBI via regulating Cx43, and plays a role in protecting the neurological function of TBI rats.

Titanium Surfaces with a Laser-Produced Microchannel Structure Enhance Pre-Osteoblast Proliferation, Maturation, and Extracellular Mineralization In Vitro.

The clinical success of dental titanium implants is profoundly linked to implant stability and osseointegration, which comprises pre-osteoblast proliferation, osteogenic differentiation, and extracellular mineralization. Because of the bio-inert nature of titanium, surface processing using subtractive or additive methods enhances osseointegration ability but limits the benefit due to accompanying surface contamination. By contrast, laser processing methods increase the roughness of the implant surface without contamination. However, the effects of laser-mediated distinct surface structures on the osteointegration level of osteoblasts are controversial. The role of a titanium surface with a laser-mediated microchannel structure in pre-osteoblast maturation remains unclear. This study aimed to elucidate the effect of laser-produced microchannels on pre-osteoblast maturation. Pre-osteoblast human embryonic palatal mesenchymal cells were seeded on a titanium plate treated with grinding (G), sandblasting with large grit and acid etching (SLA), or laser irradiation (L) for 3-18 days. The proliferation and morphology of pre-osteoblasts were evaluated using a Trypan Blue dye exclusion test and fluorescence microscopy. The mRNA expression, protein expression, and protein secretion of osteogenic differentiation markers in pre-osteoblasts were evaluated using reverse transcriptase quantitative polymerase chain reaction, a Western blot assay, and a multiplex assay, respectively. The extracellular calcium precipitation of pre-osteoblast was measured using Alizarin red S staining. Compared to G- and SLA-treated titanium surfaces, the laser-produced microchannel surfaces enhanced pre-osteoblast proliferation, the expression/secretion of osteogenic differentiation markers, and extracellular calcium precipitation. Laser-treated titanium implants may enhance the pre-osteoblast maturation process and provide extra benefits in clinical application.

Extra Virgin Olive Oil Phenolic Compounds Modulate the Gene Expression of Biomarkers Involved in Fibroblast Proliferation and Differentiation.

Extra virgin olive oil phenolic compounds have been identified as possible biostimulant agents against different pathological processes, including alterations in healing processes. However, there is little evidence on the molecular mechanisms involved in this process. The aim was to analyse the effect of hydroxytyrosol, tyrosol, and oleocanthal on fibroblast gene expression. PCR was used to determine the expression of different differentiation markers, extracellular matrix elements, and growth factors in cultured human fibroblasts CCD-1064Sk treated with different doses of hydroxytyrosol (10-5 M and 10-6 M), tyrosol (10-5 M and 10-6 M), and oleocanthal (10-6 M and 10-7 M). After 24 h of hydroxytyrosol treatment, increased expression of connective tissue growth factor, fibroblast growth factor (FGF), platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor ß1 (TGF-ß1), and their receptors was observed. Tyrosol and olecanthal modulated the expression of FGF and TGFßR1. All phytochemicals tested modified the expression of differentiation markers and extracellular matrix elements, increasing gene expression of actin, fibronectin, decorin, collagen I, and III. Phenolic compounds present in extra virgin olive could have a beneficial effect on tissue regeneration by modulating fibroblast physiology.

Effects of Siegesbeckia herba extract against particulate matter 10 (PM10 ) in skin barrier-disrupted mouse models.

OBJECTIVES: Skin barrier disruption is a significant problem of the older population in an aging society. It is characterized by increased transepidermal water loss and decreased skin water content, and particulate matter (PM) is a social issue that can contribute to the exacerbation of skin inflammation. Thus, addressing this problem is urgent. METHODS: Skin barrier-disrupted mouse models were induced by two methods using acetone application or tape-stripping. This study investigated the antioxidative and anti-inflammatory properties of the Siegesbeckia herba extract (SHE) on PM-induced changes in skin barrier-disrupted mouse models. To examine changes in skin water content, inflammatory cytokines, and keratinocyte differentiation markers, mouse models were treated with vehicle 100 µL, PM10 100 µL (100 µg/mL), SHE 100 µL, or PM10 100 µL (100 µg/mL) plus SHE 100 µL. RESULTS: SHE preserved skin hydration in the skin barrier-disrupted mouse models regardless of the presence of PM10 . SHE also inhibited the upregulation of inflammatory cytokines such as interleukin (IL)-1ß, IL-4, IL-6, IL-8, and tumor necrosis factor-α and normalized the downregulation of keratinocyte differentiation markers against PM10 in skin barrier-disrupted mouse models. CONCLUSIONS: This study elucidated the therapeutic effects of SHE against PM10 in skin barrier-disrupted mouse models.

OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors.

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.