Fabrication of Transparent Pt-TiO2 Sol and Its Photocatalytic Activity for Hydrogen Evolution.

Titanium dioxide doped with Pt(IV) ions was synthesized via a sol-gel method, incorporating a sol purification process through dialysis. The doped Pt(IV) ions were reduced by UV light irradiation to obtain a transparent Pt-TiO2 sol, with Pt(0) acting as a cocatalyst for hydrogen evolution. The hydrogen evolution activity of Pt-TiO2 sol was evaluated under UV light irradiation using methanol as a sacrificial reagent. 0.05 atom % Pt-TiO2 sol remained in the sol state without precipitation after 10 h of UV light irradiation. The amount of hydrogen produced after 10 h of light irradiation was 1195 µmol, with a turnover number (TON) of 664.1, a turnover frequency (TOF) of 66.4 h-1, and an apparent quantum yield (AQY) of 0.86%. The sol-gel synthesis from the molecular level is thought to result in Pt ions being doped inside the TiO2 particles, maintaining high dispersibility by suppressing particle growth due to the aggregation of Pt(0) during the UV reduction of Pt(IV) ions. These results show that the Pt-TiO2 photocatalyst has a high renewable energy factor because it can be synthesized at room temperature, and the aggregation of Pt-TiO2 particles is suppressed by making full use of a sol-gel method.

Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours.

Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.

PP2A and its adapter protein IER5 induce the DNA-binding ability and target gene expression of E2F1 via dephosphorylation at serine 375.

The transcription factor E2F1 participates in cell cycle control through transcriptional activation of genes that promote S-phase entry. E2F1 is also linked to the expression of proapoptotic genes, and the loss of E2F1 activity facilitates tumor progression by reducing cellular apoptosis. Phosphorylation controlled by protein kinases and phosphatases is the major posttranslational modification and regulates the cellular levels and transactivator function of E2F1. Here, we characterize the regulatory roles of serine-375 (S375), one of the major phosphorylation sites of E2F1. Cyclin-dependent kinases such as CDK8 phosphorylate at S375 of E2F1, which is dephosphorylated by protein phosphatase 2A (PP2A) containing the B55 regulatory subunit. The PP2A adapter protein IER5 binds to both PP2A/B55 and E2F1 and assists dephosphorylation at S375 by PP2A. S375-dephosphorylated E2F1 exhibits higher DNA-binding affinity than the phosphorylated form. Although the promoter regions of proapoptotic genes are less occupied by E2F1 in cells, an increase in S375-dephosphorylated E2F1 induces preferential binding of E2F1 to the proapoptotic gene promoters and their expression. Our data identify PP2A/B55-IER5 as a critical regulator of E2F1 and suggest that the phosphorylation state of E2F1 is an important determinant for the expression of proapoptotic genes.

Enhanced Systemic Antitumour Immunity by Hypofractionated Radiotherapy and Anti-PD-L1 Therapy in Dogs with Pulmonary Metastatic Oral Malignant Melanoma.

Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days (p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.

Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies.

Coagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.

PP2A-B55 and its adapter proteins IER2 and IER5 regulate the activity of RB family proteins and the expression of cell cycle-related genes.

The retinoblastoma (RB) tumour suppressor protein regulates cell proliferation, motility, differentiation and apoptosis. The phosphorylation state of RB is modulated by kinases and phosphatases, and RB exhibits phosphorylation-sensitive interactions with E2F family transcription factors. Here, we characterize RB dephosphorylation by protein phosphatase 2A (PP2A). The growth factor-inducible immediate early response (IER) proteins IER2 and IER5 possess an adapter-like function in which IER proteins bind to both PP2A and its target proteins and enhance PP2A activity towards the proteins. IER2 interacts with RB and facilitates dephosphorylation of RB at T821/T826 by PP2A. In IER2 knockdown cells, elevated phosphorylation of RB resulted in reduced binding of RB to the promoters and derepression of cyclin D1 and p21. IER5 binds to both RB and RB-like 1 (p107/RBL1), enhances dephosphorylation of these proteins by PP2A and represses the expression of various cell cycle-related genes. However, IER2-regulated dephosphorylation at T821/T826 is not necessary for the repression function of RB in cell mobility-related gene expression. Our data identify PP2A adapter proteins as critical regulators of RB family proteins and suggest that the phosphorylation status of RB differentially affects gene expression.

Exploration of serum biomarkers in dogs with malignant melanoma receiving anti-PD-L1 therapy and potential of COX-2 inhibition for combination therapy.

Immune checkpoint inhibitors (ICIs) such as anti-PD-L1 antibodies are widely used to treat human cancers, and growing evidence suggests that ICIs are promising treatments for canine malignancies. However, only some canine oral malignant melanoma (OMM) cases respond to ICIs. To explore biomarkers predictive of survival in dogs with pulmonary metastatic OMM receiving the anti-PD-L1 antibody c4G12 (n = 27), serum concentrations of prostaglandin E2 (PGE2), cytokines, chemokines, and growth factors were measured prior to treatment initiation. Among 12 factors tested, PGE2, interleukin (IL)-12p40, IL-8, monocyte chemotactic protein-1 (MCP-1), and stem cell factor (SCF) were higher in OMM dogs compared to healthy dogs (n = 8). Further, lower baseline serum PGE2, MCP-1, and vascular endothelial growth factor (VEGF)-A concentrations as well as higher IL-2, IL-12, and SCF concentrations predicted prolonged overall survival. These observations suggest that PGE2 confers resistance against anti-PD-L1 therapy through immunosuppression and thus is a candidate target for combination therapy. Indeed, PGE2 suppressed IL-2 and interferon (IFN)-γ production by stimulated canine peripheral blood mononuclear cells (PBMCs), while inhibition of PGE2 biosynthesis using the COX-2 inhibitor meloxicam in combination with c4G12 enhanced Th1 cytokine production by PBMCs. Thus, serum PGE2 may be predictive of c4G12 treatment response, and concomitant use of COX-2 inhibitors may enhance ICI antitumor efficacy.

Eicosapentaenoic acid suppresses cisplatin-induced muscle atrophy by attenuating the up-regulated gene expression of ubiquitin.

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub-proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients.

Canine Transforming Growth Factor-ß Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-ß1 Immunosuppression.

Cancer cells can evade host immune systems via multiple mechanisms. Transforming growth factor beta 1 (TGF-ß1) is an immunosuppressive cytokine that induces regulatory T cell (Tregs) differentiation and is involved in immune evasion mechanisms in cancer. The inhibition of the TGF-ß1 signaling pathway can suppress cancer progression and metastasis through the modulation of anticancer immune responses. However, to best of our knowledge, no implementation of treatments targeting TGF-ß1 has been reported in dog cancers. This study aimed to examine whether TGF-ß1 is upregulated in canine cancers. We measured TGF-ß1 concentrations in culture supernatants of canine melanoma cell lines and in serum samples from dogs with oral malignant melanoma. TGF-ß1 production was observed in several cell lines, and serum TGF-ß1 levels were elevated in dogs with oral malignant melanoma. Interestingly, the addition of recombinant TGF-ß1 to canine peripheral blood mononuclear cell cultures decreased Th1 cytokine production and increased differentiation of CD4+CD25+Foxp3+ lymphocytes, suggesting that TGF-ß1 is immunosuppressive in canine immune systems. We developed a decoy receptor for TGF-ß, namely TGF-ßRII-Ig, by identifying an open reading frame of the canine TGFBR2 gene. TGF-ßRII-Ig was prepared as a recombinant fusion protein of the extracellular region of canine TGF-ßRII and the Fc region of canine IgG-B. As expected, TGF-ßRII-Ig bound to TGF-ß1. In the presence of TGF-ß1, the treatment with TGF-ßRII-Ig increased Th1 cytokine production and decreased the differentiation of CD4+CD25+Foxp3+ lymphocytes. Our results suggest that TGF-ßRII-Ig competitively inhibits the immunosuppressive effects of TGF-ß1 and thereby activates immune responses. This study demonstrated the potential of TGF-ßRII-Ig as a novel biologic for canine melanoma.