Yoshiki Yasukochi

Background: Anti-programmed death-1 (PD-1)/anti-PD-ligand-1 (PD-L1) pathway inhibition is a standard regimen for advanced urothelial carcinoma (UC); however, its limited efficacy has been reflected in reported medium response rates. This study explored the role of next-generation coinhibitory receptors (IRs; lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain 3 (TIM-3), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT)) and their ligands (LGs) in the response to PD-(L)1 blockade therapy and the oncological outcomes in patients with UC. Methods: We investigated metastatic UC cases who underwent PD-(L)1 therapy (cohort 1: n=348, cohort 2: n=89, and cohort 4: n=29) or advanced UC cases involving surgery (cohort 3: n=293 and cohort 5: n=90). We assessed the mRNA expression profiles and corresponding clinical information regarding IRs and LGs using cohorts 1, 2, and 3. Additionally, we elucidated the spatial features of these targeted markers using multiplex immunohistochemistry (mIHC) on formalin-fixed paraffin-embedded samples from cohorts 4 and 5. Survival, differential expressed gene, and Gene Set Enrichment analyses were performed. For mIHC, quantitative analyses were also performed to correlate immune and tumor cell densities with patient survival. Results: LAG-3 expression was strongly associated with the responsiveness of PD-(L)1 blockade compared with the expression of TIM-3 and TIGIT. In tumors with high LAG-3 levels, the increased expression of fibrinogen-like protein 1 (FGL1) had a significantly negative effect on the response to PD-(L)1 blockade and overall survival. Moreover, high FGL1 levels were associated with elevated CD4⁺ regulatory T-cell gene signatures and the upregulation of CD39 and neuropilin-1, with both indicating CD8⁺ T-cell exhaustion. mIHC analyses revealed that patients with stromal CD8⁺LAG-3⁺cells^high–tumor FGL1⁺cells^high exhibited a significant negative correlation with survival rates compared with those with stromal CD8⁺LAG-3⁺cells^high–tumor FGL1⁺cells^low. Conclusions: LAG-3 expression and high FGL1 coexpression are important predictive factors of adverse oncological outcomes related to the presence of immunosuppressive contextures. These findings are hypothesis-generating, warranting further mechanistic and clinical studies aimed to evaluate LAG-3/FGL1 blockade in UC.

Raman spectroscopy was applied to study the structural differences between herpes simplex virus Type I (HSV-1) and Epstein–Barr virus (EBV). Raman spectra were first collected with statistical validity on clusters of the respective virions and analyzed according to principal component analysis (PCA). Then, average spectra were computed and a machine-learning approach applied to deconvolute them into sub-band components in order to perform comparative analyses. The Raman results revealed marked structural differences between the two viral strains, which could mainly be traced back to the massive presence of carbohydrates in the glycoproteins of EBV virions. Clear differences could also be recorded for selected tyrosine and tryptophan Raman bands sensitive to pH at the virion/environment interface. According to the observed spectral differences, Raman signatures of known biomolecules were interpreted to link structural differences with the viral functions of the two strains. The present study confirms the unique ability of Raman spectroscopy for answering structural questions at the molecular level in virology and, despite the structural complexity of viral structures, its capacity to readily and reliably differentiate between different virus types and strains.

Background: Cold-induced vasodilation (CIVD) occurs after blood vessels in the skin are constricted due to local cold exposure. Although many CIVD studies have been conducted, the underlying molecular mechanisms are yet to be clarified. Therefore, we explored genetic variants associated with CIVD response using the largest-scale dataset reported to date in a CIVD study involving wavelet analysis; thus, the findings improve our understanding of the molecular mechanisms that regulate the CIVD response. Methods: We performed wavelet analysis of three skin blood flow signals [endothelial nitric oxide (eNO)-independent, eNO-dependent, and neurogenic activities] during finger cold-water immersion at 5 °C in 94 Japanese young adults. Additionally, we conducted genome-wide association studies of CIVD using saliva samples collected from the participants. Results: We found that the mean wavelet amplitudes of eNO-independent and neurogenic activities significantly increased and decreased prior to CIVD, respectively. Our results also implied that as many as ~ 10% of the Japanese subjects did not show an apparent CIVD response. Our genome-wide association studies of CIVD using ~ 4,040,000 imputed data found no apparent CIVD-related genetic variants; however, we identified 10 genetic variants, including 2 functional genes (COL4A2 and PRLR) that are associated with notable blunted eNO-independent and neurogenic activity responses in individuals without CIVD response during local cold exposure. Conclusions: Our findings indicate that individuals without CIVD response differentiated by genotypes with COL4A2 and PRLR genetic variants exhibited notable blunted eNO-independent and neurogenic activity responses during local cold exposure.

Loss of the polybromo-1 (PBRM1) protein has been expected as a possible biomarker for clear cell renal cell carcinoma (ccRCC). There is little knowledge about how PBRM1 immunohistochemical expression correlates with the histomorphological features of ccRCC and the endothelial expression of tumor vasculature. The present study evaluates the association of architectural patterns with the PBRM1 expression of cancer cells using a cohort of 425 patients with nonmetastatic ccRCC. Furthermore, we separately assessed the PBRM1 expression of the endothelial cells and evaluated the correlation between the expression of cancer cells and endothelial cells. PBRM1 loss in cancer cells was observed in 148 (34.8%) patients. In the correlation analysis between architectural patterns and PBRM1 expression, macrocyst/microcystic, tubular/acinar, and compact/small nested were positively correlated with PBRM1 expression, whereas alveolar/large nested, thick trabecular/insular, papillary/pseudopapillary, solid sheets, and sarcomatoid/rhabdoid were negatively correlated with PBRM1 expression. PBRM1 expression in vascular endothelial cells correlated with the expression of cancer cells (correlation coefficient = 0.834, p < 0.001). PBRM1 loss in both cancer and endothelial cells was associated with a lower recurrence-free survival rate (p < 0.001). Our PBRM1 expression profile indicated that PBRM1 expression in both cancer and endothelial cells may be regulated in an orchestrated manner.

The three-tier immunophenotype (desert, excluded, and inflamed) and the four-tier immunophenotype (cold, immunosuppressed, excluded, and hot) have been linked to prognosis and immunotherapy response. This study aims to evaluate whether immunophenotypes of clear cell renal cell carcinoma, identified on hematoxylin and eosin-stained slides, correlate with gene expression signatures related to cancer immunity, and clinical outcomes. We evaluated tumor-associated immune cells (TAICs) status using three methodologies: three-tier immunophenotype based on the location of TAICs, four-tier immunophenotype considering both the location and degree of TAICs and inflammation score focusing only on the degree of TAICs, using a localized clear cell renal cell carcinoma cohort (n = 436) and The Cancer Genome Atlas (TCGA)-KIRC cohort (n = 162). We evaluated the association of the TAICs status assessed by three methodologies with CD8 and PD-L1 immunohistochemistry and immune gene expression signatures by TCGA RNA-sequencing data. All three methodologies correlated with immunohistochemical and immune gene expression signatures. The inflammation score and the four-tier immunophenotype showed similarly higher accuracy in predicting recurrence-free survival and overall survival compared to the three-tier immunophenotype. In conclusion, a simple histologic assessment of TIACs may predict clinical outcomes and immunotherapy responses.

Background: Physiological thermoregulatory systems in humans have been a key factor for adaptation to local environments after their exodus from Africa, particularly, to cold environments outside Africa. Recent studies using high-throughput sequencing have identified various genes responsible for cold adaptation. However, the molecular mechanisms underlying initial thermoregulation in response to acute cold exposure remain unclear. Therefore, we investigated transcriptional profiles of six young Japanese male adults exposed to acute cold stress.Methods: In a climatic chamber, the air temperature was maintained at 28°C for 65 min and was then gradually decreased to 19°C for 70 min. Saliva samples were obtained from the subjects at 28°C before and after 19°C cold exposure and were used for RNA sequencing.Results: In the cold exposure experiment, expression levels of 14 genes were significantly changed [false discovery rate (FDR) < 0.05] although the degree of transcriptional changes was not high due to experimental conditions or blunted transcriptional reaction in saliva to cold stress. As a result, differential gene expression analyses detected the cathepsin L (CTSL) gene to be significantly upregulated, with FDR < 0.05 and log₂fold change value > 1; thus, this gene was identified as a differentially expressed gene. Given that the cathepsin L protein is related to invasion of the novel coronavirus (SARS-CoV-2), mild cold stress might alter the susceptibility to coronavirus disease-19 in humans. The gene ontology enrichment analysis for 14 genes with FDR < 0.05 suggested that immune-related molecules could be activated by mild cold stress.Conclusions: The results obtained from this study indicate thatCTSLexpression levels can be altered by acute mild cold stress.

Background: The thermoregulatory responses during simultaneous exposure to hypoxia and cold are not well understood owing to the opposite reactions of vasomotor tone in these two environments. Therefore, the purpose of this study was to investigate the influences of hypobaric hypoxia on various thermoregulatory responses, including skin blood flow (SkBF) during cold exposure. Methods: Ten subjects participated in two experimental conditions: normobaric normoxia with cold (NC, barometric pressure (P_B) = 760 mmHg) and hypobaric hypoxia with cold (HC, P_B = 493 mmHg). The air temperature was maintained at 28 °C for 65 min and gradually decreased to 19 °C for both conditions. The total duration of the experiment was 135 min. Results: The saturation of percutaneous oxygen (SpO₂) was maintained at 98–99% in NC condition, but decreased to around 84% in HC condition. The rectal and mean skin temperatures showed no significant differences between the conditions; however, the forehead temperature was higher in HC condition than in NC condition. The pulse rate increased in HC condition, and there was a strong negative relationship between SpO₂ and pulse rate (r = − 0.860, p = 0.013). SkBF and blood pressure showed no significant differences between the two conditions. Conclusion: These results suggest that hypobaric hypoxia during cold exposure did not alter the overall thermoregulatory responses. However, hypobaric hypoxia did affect pulse rate regardless of cold exposure.

After the genomic era, the development of high-throughput sequencing technologies has allowed us to advance our understanding of genetic variants responsible for adaptation to high altitude in humans. However, transcriptomic characteristics associated with phenotypic plasticity conferring tolerance to acute hypobaric hypoxic stress remain unclear. To elucidate the effects of hypobaric hypoxic stress on transcriptional variability, we aimed to describe transcriptomic profiles in response to acute hypobaric hypoxia in humans. In a hypobaric hypoxic chamber, young Japanese males were exposed to a barometric pressure of 493 mmHg (hypobaric hypoxia) for 75 min after resting for 30 min at the pressure of 760 mmHg (normobaric normoxia) at 28°C. Saliva samples of the subjects were collected before and after hypobaric hypoxia exposure, to be used for RNA sequencing. Differential gene expression analysis identified 30 significantly upregulated genes and some of these genes may be involved in biological processes influencing hematological or immunological responses to hypobaric hypoxic stress. We also confirmed the absence of any significant transcriptional fluctuations in the analysis of basal transcriptomic profiles under no-stimulus conditions, suggesting that the 30 genes were actually upregulated by hypobaric hypoxia exposure. In conclusion, our findings showed that the transcriptional profiles of Japanese individuals can be rapidly changed as a result of acute hypobaric hypoxia, and this change may influence the phenotypic plasticity of lowland individuals for acclimatization to a hypobaric hypoxic environment. Therefore, the results obtained in this study shed light on the transcriptional mechanisms underlying high-altitude acclimatization in humans.

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.