Treatment rationale and protocol design: an investigator-initiated phase II study of combination treatment of nivolumab and TM5614, a PAI-1 inhibitor for previously treated patients with non-small cell lung cancer.

Background: There is no established standard 3rd line treatment for patients with advanced non-small cell lung cancer (NSCLC). Although cytotoxic chemotherapeutic agents that are not used as 1st or 2nd line treatment are administrated as 3rd line treatment, their anti-tumor efficacy is insufficient. Anti-programmed death ligand-1 (PD-L1)/programmed death-1 (PD1) treatment is more effective and less toxic than chemotherapy in anti-PD-L1/PD-1 treatment-naïve patients with NSCLC. Therefore, anti-PD-L1/PD-1 therapy is considered an appropriate 3rd line treatment. However, the anti-tumor efficacy is limited in patients previously treated with anti-PD-L1/PD-1 antibody. Today, new drugs are needed to increase the efficacy of anti-PD-L1/PD-1 antibodies. Methods: This open-label, single-arm, investigator-initiated phase II study is designed to evaluate combination treatment of nivolumab and TM5614, a plasminogen activator inhibitor (PAI-1) inhibitor as 3rd or more line treatment in NSCLC patients who underwent standard treatment. The primary endpoint is the objective response rate and the secondary endpoints are progression-free survival (PFS), overall survival (OS), duration of response (DOR) and safety. Recruitment began in September 2023 and is expected to continue for approximately three years. Discussion: Currently, there is no standard 3rd line treatment for advanced NSCLC, and we hope that the findings of this study will facilitate more effective treatments in this setting. Ethics and dissemination: the study protocol conformed to the ethical principles outlined in the Declaration of Helsinki. All patients will provide written informed consent prior to enrollment. Results will be published in a peer-reviewed publication. Trial Registration: This study is registered to Japan Registry of Clinical Trials with number: jRCT2061230039 (19/July/2023).

Effective-mononuclear cell (E-MNC) therapy alleviates salivary gland damage by suppressing lymphocyte infiltration in Sjögren-like disease.

Introduction: Sjögren syndrome (SS) is an autoimmune disease characterized by salivary gland (SG) destruction leading to loss of secretory function. A hallmark of the disease is the presence of focal lymphocyte infiltration in SGs, which is predominantly composed of T cells. Currently, there are no effective therapies for SS. Recently, we demonstrated that a newly developed therapy using effective-mononuclear cells (E-MNCs) improved the function of radiation-injured SGs due to anti-inflammatory and regenerative effects. In this study, we investigated whether E-MNCs could ameliorate disease development in non-obese diabetic (NOD) mice as a model for primary SS. Methods: E-MNCs were obtained from peripheral blood mononuclear cells (PBMNCs) cultured for 7 days in serum-free medium supplemented with five specific recombinant proteins (5G culture). The anti-inflammatory characteristics of E-MNCs were then analyzed using a co-culture system with CD3/CD28-stimulated PBMNCs. To evaluate the therapeutic efficacy of E-MNCs against SS onset, E-MNCs were transplanted into SGs of NOD mice. Subsequently, saliva secretion, histological, and gene expression analyses of harvested SG were performed to investigate if E-MNCs therapy delays disease development. Results: First, we characterized that both human and mouse E-MNCs exhibited induction of CD11b/CD206-positive cells (M2 macrophages) and that human E-MNCs could inhibit inflammatory gene expressions in CD3/CD28- stimulated PBMNCs. Further analyses revealed that Msr1-and galectin3-positive macrophages (immunomodulatory M2c phenotype) were specifically induced in E-MNCs of both NOD and MHC class I-matched mice. Transplanted E-MNCs induced M2 macrophages and reduced the expression of T cell-derived chemokine-related and inflammatory genes in SG tissue of NOD mice at SS-onset. Then, E-MNCs suppressed the infiltration of CD4-positive T cells and facilitated the maintenance of saliva secretion for up to 12 weeks after E-MNC administration. Discussion: Thus, the immunomodulatory actions of E-MNCs could be part of a therapeutic strategy targeting the early stage of primary SS.

Clinicopathological and Prognostic Analysis of PD-L1 and PD-L2 Expression in Surgically Resected Primary Tongue Squamous Cell Carcinoma.

BACKGROUND/AIM: The expression of tumor-associated programmed death-ligand 1 (PD-L1) predicts clinical responses to PD-1-directed immunotherapy. The expression levels of PD-L2, another PD-1 ligand, and its relationship with responses to PD-1-targeting therapy in oral squamous cell carcinoma (OSCC) remain unclear. Furthermore, the clinicopathological characteristics and prognostic effects of the expression of PD-L1 and PD-L2 in OSCC have not yet been elucidated. MATERIALS AND METHODS: The expression of PD-L1 and PD-L2 was immunohistochemically examined in 98 tongue carcinomas. Furthermore, the expression levels of PD-L1 and PD-L2 in OSCC cell lines and their relationships with those of MMP2 and MMP9 were assessed. RESULTS: The expression levels of PD-L1 and PD-L2 correlated with those of MMP2 and MMP9. The expression of PD-L1 and/or PD-L2 was detected in OSCC cells, and their levels correlated with those of MMP9. The prognosis of patients with PD-L1- and PD-L2-positive tumors was significantly worse. CONCLUSION: PD-L1 and PD-L2 status is potentially a novel predictor of the prognosis of OSCC and provides a rationale for the development of novel immunotherapies.

Phase 1 clinical study of cell therapy with effective-mononuclear cells (E-MNC) for radiogenic xerostomia (first-in-human study) (FIH study on E-MNC therapy for radiogenic xerostomia).

BACKGROUND: Treatment for most patients with head and neck cancers includes ionizing radiation with or without chemotherapy. This treatment causes irreversible damage to salivary glands in the irradiation field accompanied by a loss of fluid-secreting acinar cells and a considerable decrease of saliva secretion. There is currently no adequate conventional treatment for this condition. In recent years, we developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs), and such effectively conditioned PBMNC (E-MNC) therapy has shown promising improvements to the function of radiation-injured salivary glands in preclinical studies. However, the safety and effect of E-NMC therapy have yet assessed in human. The objective of this ongoing first-in-man study is to assess the safety, tolerability, and in part the efficacy of E-MNC therapy for treating radiation-induced xerostomia. METHODS/DESIGN: This phase 1 first-in-man study is an open-label, single-center, two-step dose escalation study. A total of 6 patients, who had no recurrence of head and neck cancer over 5 years following radiation therapy and suffered from radiation-induced xerostomia, will receive a transplantation of E-NMCs derived from autologous PBMNCs to a submandibular gland. The duration of the intervention will be 1 year. To analyze the recovery of salivary secretion, a gum test will be performed. To analyze the recovery of atrophic salivary glands, computed tomography (CT), and magnetic resonance imaging (MRI) of salivary glands will be conducted. The primary endpoint is the safety of the protocol. The secondary endpoints are the changes from baseline in whole saliva secretion and salivary gland atrophy. DISCUSSION: This will be the first clinical study of regenerative therapy using E-MNCs for patients with severe radiation-induced xerostomia. The results of this study are expected to contribute to developing the low-invasive cell-based therapy for radiation-induced xerostomia. TRIAL REGISTRATION: This study was registered with the Japan Registry of Clinical Trials (http://jrct.niph.go.jp) as jRCTb070190057.

Anti-inflammatory and vasculogenic conditioning of peripheral blood mononuclear cells reinforces their therapeutic potential for radiation-injured salivary glands.

BACKGROUND: There are currently no effective treatments available for patients with irreversible loss of salivary gland (SG) function caused by radiation therapy for head and neck cancer. In this study, we have developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs) and investigated whether such effectively conditioned PBMNCs (E-MNCs) could regenerate radiation-injured SGs and ameliorate salivary secretory function in mice. METHODS: Mouse PBMNCs were expanded in primary serum-free culture with five vasculogenic proteins for 5 days, and then the resulting cells (E-MNCs) were analyzed for their characteristics. Subsequently, 5 × 104 E-MNCs (labeled with EGFP in some experiments) were injected intra-glandularly into a mouse model of radiation-injured atrophic submandibular glands. After 2-3 weeks, the submandibular glands were harvested, and then the injected E-MNCs were tracked. Four, 8, and 12 weeks after irradiation (IR), salivary outputs were measured to evaluate the recovery of secretory function, and the gland tissues were harvested for histological and gene expression analyses to clarify the effects of cell transplantation. RESULTS: The resulting E-MNCs contained an enriched population of definitive CD11b/CD206-positive (M2 macrophage-like) cells and showed anti-inflammatory and vasculogenic characteristics. Salivary secretory function in E-MNC-transplanted mice gradually recovered after 4 weeks post-irradiation (post-IR) and reached 3.8-fold higher than that of non-transplanted mice at 12 weeks. EGFP-expressing E-MNCs were detected in a portion of the vascular endothelium and perivascular gland tissues at 2 weeks post-IR, but mainly in some microvessels at 3 weeks. Between 4 and 12 weeks post-IR, mRNA expression and histological analyses revealed that E-MNC transplantation reduced the expression of inflammatory genes and increased the level of tissue-regenerative activities such as stem cell markers, cell proliferation, and blood vessel formation. At 12 weeks post-IR, the areas of acinar and ductal cells regenerated, and the glands had less fibrosis. CONCLUSIONS: This effective conditioning of PBMNCs is a simple, rapid, and efficient method that provides a non-invasive source of therapeutic cells for regenerating radiation-injured atrophic SGs.

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation.

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability.

Nuclear translocation of glutathione S-transferase π is mediated by a non-classical localization signal.

Glutathione S-transferase π (GSTπ), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GSTπ is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GSTπ appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GSTπ was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GSTπ195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GSTπ depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GSTπ, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.

Taste development from health education among schoolchildren: a two-year intervention study.

It is necessary to develop a system of nutritional education which can be understood among schoolchildren who have not yet received a basic education. In the present study, we conducted an educational program for lower-grade schoolchildren, which contained dish selection, an agricultural experience, a cooking experience, and a lecture on digestive absorption. We evaluated the effect of this program on development by measuring taste sensitivity regarding sweet, sour, salty and bitter tastes. For the baseline period, there was no significant difference between the intervention school and the control school in each variable. At follow-up periods, both the intervention and the control schools showed an increasing sense of taste. In the intervention school, development of sensitivity to the sweet, the sour, and the bitter taste was significant. In the control school, development of sensitivity to the sweet and the bitter taste was significant. The increases in the sense of the sour and the bitter tastes and the sum of the four tastes for the intervention subjects were significantly larger than comparable values for the control subjects. These results suggest that the development of taste sensitivity is affected by nutritional education for lower-grade elementary schoolchildren.

[A case of angiosarcoma of pelvis with pulmonary metastases which responded to paclitaxel].

A 60-year-old man was admitted to our hospital complaining of back pain and bloody sputum. Chest CT scan showed characteristic multiple small nodules with central dense opacity and surrounding faint opacity, suggesting lesions with hemorrhage. Bone scintigram and MRI revealed multiple osteolytic lesions in pelvis and lumbar spine. Biopsy of the bone lesion established a diagnosis of angiosarcoma. Chemotherapy with paclitaxel and palliative radiotherapy for the bone were initiated. Pulmonary metastases dramatically diminished after 4 courses of paclitaxel treatment. After eight weeks, the tumor recurred. Salvage chemotherapy of weekly administration of docetaxel yielded limited effects. The patient died of cancer one year after treatment initiation.

Crystal structure of the ferredoxin component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non-heme iron oxygenase system.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.

Dioxin catabolic genes are dispersed on the Terrabacter sp. DBF63 genome.

Reverse transcription-PCR of the dbfA1A2, dbfBC, and pht genes, encoding oxygenase component of multicomponent dioxygenase, meta cleavage enzyme and hydrolase, and phthalate-degrading enzymes, respectively, revealed their role in the aromatic compound degradation by Terrabacter sp. strain DBF63. The specific expression in strain DBF63 cells grown on dibenzofuran (the model compound of dioxin; DF) and/or fluorene (FN) indicated that the DbfA1A2 and DbfBC catalyze the conversion of DF to salicylate, and that the DbfA1A2 and Pht enzymes are involved in FN degradation. Pulsed-field gel electrophoresis analyses revealed that the dbfA1A2 cistron and pht operon were located on the two linear plasmids, pDBF1 (160 kb) and pDBF2 (190 kb), while dbfBC genes were located on the chromosome. Because the pht operon is located immediately upstream of the dbfA1A2 cistron, the dioxin-catabolic genes were dispersed on the genome of strain DBF63, while FN-catabolic genes were gathered on the plasmids.