The Comprehensive Characterization of B7-H3 Expression in the Tumor Microenvironment of Lung Squamous Cell Carcinoma: A Retrospective Study.

Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer; therefore, new therapeutic approaches are required to improve the prognosis of LSCC. Although immunotherapies targeting B7 family molecules were explored as treatments for several cancer types, the expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated in detail. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression using 110 surgically resected pathological specimens retrospectively. We examined the correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells. High B7-H3 expression in tumor cells was associated with a better prognosis and a significant increase in the number of CD163+PD-L1+ macrophages. Quantitative analysis revealed that there is a positive correlation between B7-H3 and PD-L1 expression in tumor and stromal cells, as well as in intratumoral tumor-infiltrating lymphocytes and tumor-associated macrophages in the same cells. CD68+, CD163+, and CK+ cells with PD-L1+ phenotypes had higher B7-H3 expression compared to PD-L1- cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies.

Spatial and Quantitative Analysis of Tumor-Associated Macrophages: Intratumoral CD163-/PD-L1+ TAMs as a Marker of Favorable Clinical Outcomes in Triple-Negative Breast Cancer.

Tumor-associated macrophages (TAMs) and abnormalities in cancer cells affect cancer progression and response to therapy. TAMs are a major component of the tumor microenvironment (TME) in breast cancer, with their invasion affecting clinical outcomes. Programmed death-ligand 1 (PD-L1), a target of immune checkpoint inhibitors, acts as a suppressive signal for the surrounding immune system; however, its expression and effect on TAMs and the clinical outcome in breast cancer are unknown. In this study, we used high-throughput multiple immunohistochemistry to spatially and quantitatively analyze TAMs. We subjected 81 breast cancer specimens to immunostaining for CD68, CD163, PD-1, PD-L1, CD20, and pan-CK. In both stromal and intratumoral areas, the triple-negative subtype had significantly more CD68/CD163, CD68/PD-L1, and CD163/PD-L1 double-positive cells than the estrogen receptor (ER)/progesterone receptor (PR) subtype. Interestingly, a higher number of CD68+/PD-L1+/CK-/CD163- TAMs in the intratumoral area was correlated with a favorable recurrence rate (p = 0.048). These findings indicated that the specific subpopulation and localization of TAMs in the TME affect clinical outcomes in breast cancer.

Quantitative high-throughput analysis of tumor infiltrating lymphocytes in breast cancer.

In breast cancer (BC), the development of cancer immunotherapy including immune checkpoint inhibitors has progressed. Tumor infiltrating lymphocytes (TILs) is one of the important factors for an immune response between tumor cells and immune cells in the tumor microenvironment, and the presence of TILs has been identified as predictors of response to chemotherapy. However, because complex mechanisms underlies the crosstalk between immune cells and cancer cells, the relationship between immune profiles in the tumor microenvironment and the efficacy of the immune checkpoint blocked has been unclear. Moreover, in many cases of breast cancer, the quantitative analysis of TILs and immuno-modification markers in a single tissue section are not studied. Therefore, we quantified detailed subsets of tumor infiltrating lymphocytes (TILs) from BC tissues and compared among BC subtypes. The TILs of BC tissues from 86 patients were classified using multiplex immunohistochemistry and an artificial intelligence-based analysis system based on T-cell subset markers, immunomodification markers, and the localization of TILs. The levels of CD4/PD1 and CD8/PD1 double-positive stromal TILs were significantly lower in the HER2- BC subtype (p <0.01 and p <0.05, respectively). In triple-negative breast cancer (TNBC), single marker-positive intratumoral TILs did not affect prognosis, however CD4/PDL1, CD8/PD1, and CD8/PDL1 double-positive TILs were significantly associated with TNBC recurrence (p<0.05, p<0.01, and p<0.001, respectively). TIL profiles differed among different BC subtypes, suggesting that the localization of TILs and their tumor-specific subsets influence the BC microenvironment.

Long-term safety and effectiveness of stiripentol in patients with Dravet syndrome: Interim report of a post-marketing surveillance study in Japan.

BACKGROUND: A post-marketing surveillance study is investigating the safety and effectiveness of stiripentol during real-world clinical use in Japanese patients with Dravet syndrome (DS). METHODS: The safety and effectiveness of stiripentol were prospectively investigated over 104 weeks in all patients with DS who were administered the drug from November 2012 through July 2019 in Japan. Patients administered stiripentol for the first time after its approval were defined as "new patients," and those who continued to take the drug after participating in domestic clinical studies were defined as "continuous-use patients." The responder rate was defined as the proportion of patients with a ≥50 % decrease in seizure episodes at the time of assessment of stiripentol effectiveness compared with the 4 weeks before starting stiripentol. Overall improvement was evaluated by the physician in charge based on the comprehensive assessment of the patient's condition after stiripentol treatment. RESULTS: Of 411 patients whose information was collected, 410 patients (376 new and 34 continuous-use) were included in the safety analysis set, and 409 (376 new and 33 continuous-use) were included in the effectiveness analysis set. The median age of new patients was 7 years (range: 0.5-50 years) at the time of stiripentol initiation; 99 % of patients were taking concomitant sodium valproate and 93 % clobazam. Adverse drug reactions occurred in 70 % of new patients; the most common were somnolence (39 %) and loss of appetite (25 %). No new safety concerns due to stiripentol were observed. The responder rate in new patients was 43 % (110/257 patients) for convulsive seizures (tonic-clonic and/or clonic convulsions), 55 % (58/105 patients) for focal impaired awareness seizures, and 62 % (56/90 patients) for generalized myoclonic seizures and/or generalized atypical absence seizures. Overall improvement (after 104 weeks or at the time of drug discontinuation) was rated as marked or moderate in 160/353 of new patients (45 %). CONCLUSION: Stiripentol is safe and effective during long-term use in patients with DS in routine clinical practice.

Effect of educational environments on nursing faculty members' perceptions regarding oral care.

AIM: To investigate nursing faculty members' perceptions of oral care and to identify the effects of an educational environment on their perceptions. METHODS: A cross-sectional questionnaire regarding oral care was conducted at eight nursing schools in Japan. A total of 156 (71.6%) faculty members participated in this study. Their perceptions of oral care practice were compared by using a statistical analysis according to the length of work experience, teaching field, school, and facility setting (defined as whether the school had an affiliated hospital with a dental department). RESULTS: Almost all of the faculty members were nurses and most were female. Almost all perceived that oral care was effective in the prevention of aspiration pneumonia and frailty. There were significant differences by teaching field in the nurses' perceptions regarding with whom they should collaborate to conduct oral care and there were significant differences by school in the nurses' perceptions regarding who should provide oral care, where it should be provided, and what kinds of knowledge are important for practice. Perceptions of low involvement in oral care were significantly associated with the schools having an affiliated university hospital with a dental department. CONCLUSION: There were different perceptions regarding oral care among nursing faculty members and their perceptions might have been affected by their educational environment. Therefore, it is suggested that oral care education should be standardized and nursing faculty members should standardize the curriculum regarding oral care for nursing students.

Biochemical characterization of thermostable ß-1,4-mannanase belonging to the glycoside hydrolase family 134 from Aspergillus oryzae.

A ß-1,4-mannanase, termed AoMan134A, that belongs to the GH 134 family was identified in the filamentous fungus Aspergillus oryzae. Recombinant AoMan134A was expressed in Pichia pastoris, and the purified enzyme produced mannobiose, mannotriose, mannotetraose, and mannopentaose from galactose-free ß-mannan, with mannotriose being the predominant reaction product. The catalytic efficiency (k cat/K m ) of AoMan134A was 6.8-fold higher toward galactomannan from locust bean gum, than toward galactomannan from guar gum, but similar toward galactomannan from locust bean gum and glucomannan from konjac flour. After incubation at 70°C for 120 min, the activity of AoMan134A toward glucomannan decreased to 50% of the maximal activity at 30°C. AoMan134A retained 50% of its ß-1,4-mannanase activity after heating at 90°C for 30 min, indicating that AoMan134A is thermostable. Furthermore, AoMan134A was stable within a neutral-to-alkaline pH range, as well as exhibiting stability in the presence of a range of organic solvents, detergents, and metal ions. These findings suggest that AoMan134A could be useful in a diverse range of industries where conversion of ß-mannans is of prime importance.

[Strategies to Cope with the Shortage of Technologists: Facing the Mass-Retirement of the 'Baby-Boomer' Generation].

In Japan, the primary 'baby-boomer' generation, born between 1947 and 1949, is now in its retirement. This has caused a marked shortage of human resources nationwide. Clinical laboratory technologists are no exception, and many clinical laboratories in Japanese healthcare facilities are struggling with management because the number of new graduates, i.e., newly licensed technologists, is mostly fixed and, therefore, their recruitment is becoming more and more competitive. Our laboratory is now facing a wave of mass-retirement associated with our history. In addition, in the early 2000s, there was almost no position for new graduates replacing those retiring because of the change in the social healthcare system as well as our hospital's policy. This resulted in uneven numbers of technologists in generations, and it seemed to be getting worse. Fortunately, five years ago, the direction of social health care was changed and lots of positions became available as a result. We have been trying to recruit new graduates and experienced technologists as well, and were able to hire 18 people. Among them, 8 were non-freshmen. The generation gap has been mostly resolved. We will continue to make our laboratory more attractive not just to new graduates but also to experienced technologists, especially those who wish to return to work after a several-year absence to raise their children. We believe that this will energize our laboratory.

Immune adjuvant effect of Juzentaihoto, a Japanese traditional herbal medicine, on tumor vaccine therapy in a mouse model.

Japanese traditional herbal medicine (Kampo) have been used to improve the general physical condition after surgery and to mitigate the side effects of radiation and chemotherapy in tumor patients. Juzentaihoto (JTT) consists of ten medical herbs, and is also called Shi-Quan-Da-Bu-Tang in Chinese herbal medicine. Among Kampo medicines, JTT has especially gained attention as a biological response modifier. Currently, clinical trials of various tumor vaccine therapies are being performed world-wide. However, tumor antigens that are inoculated as vaccines do not have high immunogenicity; thus, it is difficult to obtain an effective therapeutic effect. Thus, it is necessary to develop a tumor vaccine adjuvant that is more potent and very safe. In the present study, we examined the efficacy of JTT as an oral adjuvant when given together with tumor vaccines. As a result, JTT enhanced the phagocytic ability of OVA antigen and the presentation ability of OVA antigen in dendritic cells in vitro. Furthermore, tumor growth was markedly decreased, and the survival period was significantly prolonged in mice inoculated with mouse lymphoma, which is expressed with tumor model antigen. In conclusion, these findings suggest that JTT can be used with tumor vaccines as an immune adjuvant.

Novel ß-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans.

Many filamentous fungi produce ß-mannan-degrading ß-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel ß-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free ß-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with ß-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-ß-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward ß-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when ß-mannans were the sole carbon source, indicating that Man134A is involved in ß-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of ß-mannan degradation that is enhanced by the novel ß-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-ß-1,4-mannanases.

[Achievements in the objectives of a practicum as seen through changes in awareness of nursing and care techniques in a basic nursing practice].

This study aims to elucidate the level of achievement and the challenges in a basic nursing practicum for second-year nursing students through the association method. Analysis revealed various descriptions of learning through the practicum training experiences in terms of understanding the healthcare environment, planning and implementing daily life assistance, and involvement in building a supportive relationship with a care recipient. In this sense, we can assume that the goal of the practicum was achieved. The students' attitude toward learning showed an emphasis on knowledge, basics, reasoning, and acquisition of knowledge through experience. At the same time, however, we did not observe descriptions of developing the healthcare environment, understanding the methods of developing healthcare plans, or respect for privacy, thus not indicating whether the goal was achieved. In the future, it is necessary to additionally examine specific details of students' learning through analyzing their reports.

A phagocytotic inducer from herbal constituent, pentagalloylglucose enhances lipoplex-mediated gene transfection in dendritic cells.

Antigen-presenting cells are key vehicles for delivering antigens in tumor immunotherapy, and the most potent of them are dendritic cells (DCs). Recent studies have demonstrated the usefulness of DCs genetically modified by lipofection in tumor immune therapy, although sufficient gene transduction into DCs is quite difficult. Here, we show that Paeoniae radix, herbal medicine, and the constituent, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), have an attractive function to enhance phagocytosis in murine dendritic cell lines, DC2.4 cells. In particular, PGG in combination with lipofectin (LPF) enhanced phagocytic activity. Furthermore, PGG enhanced lipofection efficacy in DC2.4 cells, but not in colorectal carcinoma cell lines, Colon26. In other words, PGG synergistically enhanced the effect of lipofectin-dependent phagocytosis on phagocytic cells. Hence, according to our data, PGG could be an effective aid in lipofection using dendritic cells. Furthermore, these findings provide an expectation that constituents from herbal plant enhance lipofection efficacy.

Anti-tumor effect of combining CC chemokine 22 and an anti-CD25 antibody on myeloma cells implanted subcutaneously into mice.

Chemokines are known to have anti-tumor effects due to their chemoattractant properties, which stimulate the accumulation of infiltrating immune cells in tumors. CCL22 (macrophage-derived chemokine, MDC) attracts killer T?cells, helper T cells and antigen-presenting cells expressing the CCL22 receptor, CCR4. Thus, CCL22 gene expression results in the accumulation of these cells in tumors, and has been shown to suppress lung and colon cancer growth in mice. In the present study, early-stage subcutaneous tumor growth in a mouse multiple myeloma cell line stably expressing CCL22 (MPC-CCL22) was decreased compared to tumor growth in control cells (MPC-mock). However, the final extent of tumor growth in these cell lines was almost equivalent. Regulatory T cells, which express CD25, CD4 and CCR4, are known to cause immune disruption. We therefore investigated the association of regulatory T cells with the progressive decrease in CCL22 anti-tumor effect observed in late-stage experimental multiple myelomas. Tumor growth in MPC-CCL22 cells was observed to drastically decrease, to the point of complete tumor regression, when CD4 or CD25 T cells were depleted. Here, we document the drastic anti-tumor effect of a combination of CCL22 and anti-CD25 antibody on multiple myeloma cells.

Health support program for family members with hospitalized child.

In 2001, we began conducting Health Support Programs on the pediatric ward of Miyazaki Medical College Hospital. The program is designed to help family members who accompany and care for their children who are ill and hospitalized to practice some physical movements for their own benefit. An interview survey was undertaken to clarify the effects and the purpose of the program by evaluating subjective data from program participants, as well as by evaluating changes in participants' blood pressure rates. Findings indicate that the program's effects include "reduction of stress and/or lack of exercise," "refreshing effect," "comfort and/or exhilaration," "relaxation," and "alleviation of physical symptoms." We also have noted that mothers become more aware of their own health issues and that the program offers an opportunity to reexamine their health conditions. Furtherrnore, we have learned that for parents and children who participate together, the program offers a fun environment, and that, on occasion, mothers are the ones who are cared for in the program.