Rapid and high throughput assessment of cellular immunity against SARS-CoV-2 based on the ex vivo activation of genes in leukocyte assay with whole blood.

During the novel coronavirus outbreak and vaccine development, antibody production garnered major focus as the primary immunogenic response. However, cellular immunity's recent demonstration of comparable or greater significance in controlling infection demands the re-evaluation of the importance of T-cell immunity in SARS-CoV-2 infection. Here, we developed a novel assay, the ex vivo activation of genes in leukocytes (EAGL), which employs short-term whole blood stimulation with the LeukoComplete™ system, to measure ex vivo SARS-CoV-2-specific T cell responses (cellular immunity). This assay measures upregulated mRNA expression related to leukocyte activation 4 h after antigen stimulation. LeukoComplete™ system uses whole blood samples, eliminating the need for pretreatment before analysis. Furthermore, this system's high reproducibility is ensured through a series of operations from mRNA extraction to cDNA synthesis on a 96-well plate. In the performance evaluation using fresh blood from previously SARS-CoV-2-infected and COVID-19-vaccinated individuals, the EAGL assay had a comparable sensitivity and specificity to the ELISpot assay (EAGL: 1.000/1.000; ELISpot: 0.900/0.973). As a simple, high-throughput assay, the EAGL assay is also a quantitative test that is useful in studies with large sample numbers, such as monitoring new vaccine efficacies against novel coronaviruses or epidemiologic studies that require cellular immune testing during viral infection.

Cycle-generative adversarial network-based bone suppression imaging for highly accurate markerless motion tracking of lung tumors for cyberknife irradiation therapy.

PURPOSE: Lung tumor tracking during stereotactic radiotherapy with the CyberKnife can misrecognize tumor location under conditions where similar patterns exist in the search area. This study aimed to develop a technique for bone signal suppression during kV-x-ray imaging. METHODS: Paired CT images were created with or without bony structures using a 4D extended cardiac-torso phantom (XCAT phantom) in 56 cases. Subsequently, 3020 2D x-ray images were generated. Images with bone were input into cycle-consistent adversarial network (CycleGAN) and the bone suppressed images on the XCAT phantom (BSIphantom ) were created. They were then compared to images without bone using the structural similarity index measure (SSIM) and peak signal-to-noise ratio (PSNR). Next, 1000 non-simulated treatment images from real cases were input into the training model, and bone-suppressed images of the patient (BSIpatient ) were created. Zero means normalized cross correlation (ZNCC) by template matching between each of the actual treatment images and BSIpatient were calculated. RESULTS: BSIphantom values were compared to their paired images without bone of the XCAT phantom test data; SSIM and PSNR were 0.90 ± 0.06 and 24.54 ± 4.48, respectively. It was visually confirmed that only bone was selectively suppressed without significantly affecting tumor visualization. The ZNCC values of the actual treatment images and BSIpatient were 0.763 ± 0.136 and 0.773 ± 0.143, respectively. The BSIpatient showed improved recognition accuracy over the actual treatment images. CONCLUSIONS: The proposed bone suppression imaging technique based on CycleGAN improves image recognition, making it possible to achieve highly accurate motion tracking irradiation.

Development of a phantom for assessing the precision of setup in skin mark-less surface-guided radiotherapy.

BACKGROUND: Surface-guided radiotherapy (SGRT) is adopted by several institutions; however, reports on the phantoms used to assess the precision of the SGRT setup are limited. PURPOSE: The purpose of this study was to develop a phantom to verify the accuracy of the irradiation position during skin mark-less SGRT. METHODS: An acrylonitrile butadiene styrene (ABS) plastic cube phantom with a diameter of 150 mm on each side containing a dummy target of 15 mm and two types of body surface-shaped phantoms (breast/face shape) that could be attached to the cube phantom were fabricated. Films can be inserted on four sides of the cubic phantom (left, right, anterior and posterior), and the center of radiation can be calculated by irradiating the dummy target with orthogonal MV beams. Three types of SGRT using a VOXELAN-HEV600M (Electronics Research&Development Corporation, Okayama, Japan) were evaluated using this phantom: (i) SGRTCT-a SGRT set-up based solely on a computed tomography (CT)-reference image. (ii) SGRTCT + CBCT-a method where cone beam computed tomography (CBCT) matching was performed after SGRTCT. (iii) SGRTScan-a resetup technique using a scan reference image obtained after completing the (ii) step. RESULTS: Both the breast and face phantoms were recognized in the SGRT system without problems. SGRTScan ensure precision within 1 mm/1° for breast and face verification, respectively. All SGRT methods showed comparable rotational accuracies with no significant disparities. CONCLUSIONS: The developed phantom was useful for verifying the accuracy of skin mark-less SGRT position matching. The SGRTScan demonstrated the feasibility of achieving skin-mark less SGRT with high accuracy, with deviations of less than 1 mm. Additional research is necessary to evaluate the suitability of the developed phantoms for use in various facilities and systems. This phantom could be used for postal surveys in the future.

Common clinicopathological and immunological features of sarcomatoid carcinoma across organs: A histomorphology-based cross-organ study.

Sarcomatoid carcinoma (SC), which can occur in any organ, is a rare disease. To elucidate common characteristics of SC beyond organs, we evaluated clinicopathological and immunological features of SC defined by the single histological criterion beyond organs compared to randomly matched conventional carcinoma (non-SC) adjusted for the disease stage. Immunological features were assessed by multiplex immunohistochemistry, comparing immune cell density in tumor tissues and tumor programmed death-ligand 1 (PD-L1) expression. A total of 101 patients with SC or non-SC (31 lung, 19 esophagus, 22 pancreas, 15 liver, 4 bile duct, 6 kidney, 2 uterus and 2 ovary) were identified among 7197 patients who underwent surgery at our institute (1997-2020). SC was significantly associated with worse survival (HR: 1.571; 95% CI: 1.084-2.277; P = .017). The frequency of postoperative progression within 6 months was significantly higher for SC patients (54% vs 28%; P = .002). The immune profiling revealed the densities of CD8+ T cells (130 vs 72 cells/mm2 ; P = .004) and tumor-associated macrophages (566 vs 413 cells/mm2 ; P < .0001) and the tumor PD-L1 expression score (40% vs 5%; P < .0001) were significantly higher in SCs than in non-SCs. Among 73 SC patients with postoperative progression, multivariate Cox regression analysis showed that immunotherapy tended to be associated with favorable survival (HR: 0.256; 95% CI: 0.062-1.057; P = .060). Collectively, SCs shared clinicopathological and immunological features across organs. Our study can initiate to standardize the pathological definition of SC and provide a rationale for the investigation and development for this rare disease in a cross-organ manner.

Relationships between physical and immunological tumor microenvironment in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is physically palpated as a hard tumor with an unfavorable prognosis. Assessing physical features and their association with pathological features could help to elucidate the mechanism of physical abnormalities in cancer tissues. A total of 93 patients who underwent radical surgery for pancreatic and bile duct cancers at a single center hospital during a 28-month period were recruited for this study that aimed to estimate the stiffness of PDAC tissues compared to the other neoplasms and assess relationships between tumor stiffness and pathological features. Physical alterations and pathological features of PDAC, with or without preoperative therapy, were analyzed. The immunological tumor microenvironment was evaluated using multiplexed fluorescent immunohistochemistry. The stiffness of PDAC correlated with the ratio of Azan-Mallory staining, α-smooth muscle actin, and collagen I-positive areas of the tumors. Densities of CD8+ T cells and CD204+ macrophages were associated with tumor stiffness in cases without preoperative therapy. Pancreatic ductal adenocarcinoma treated with preoperative therapy was softer than that without, and the association between tumor stiffness and immune cell infiltration was not shown after preoperative therapy. We observed the relationship between tumor stiffness and immunological features in human PDAC for the first time. Immune cell densities in the tumor center were smaller in hard tumors than in soft tumors without preoperative therapies. Preoperative therapy could alter physical and immunological aspects, warranting further study. Understanding of the correlations between physical and immunological aspects could lead to the development of new therapies.

Relationship between microorganisms and volatile components in each fermentation process in the kusaya gravy that plays an important role in the manufacturing of kusaya, a traditional Japanese fermented fish product.

The relationship between the microbiota and volatile components of kusaya gravy involved in the manufacturing of kusaya, a traditional Japanese fermented fish product, in the Izu Islands (Niijima and Hachijojima) and the fermentation processes are not clear. In this study, we aimed to investigate the relationship between the microbiota and volatile compounds involved in the manufacturing and management of kusaya gravy. 16S ribosomal RNA (rRNA) gene-based amplicon sequencing revealed that the microbiota in kusaya gravy was significantly different between the two islands, and the microbiota hardly changed during each fermentation process. Gas chromatography-mass spectrometry analysis also revealed that the volatile components were strongly related to the microbiota in kusaya gravy, with Hachijojima samples containing sulfur-containing compounds and Niijima samples containing short-chain fatty acids. Therefore, our findings suggest that kusaya gravy is a characteristic fermented gravy with a stable microbiota, and the fermented pickling gravy is fermented by microorganisms.

Characterization of the surugamide biosynthetic gene cluster of TUA-NKU25, a Streptomyces diastaticus strain isolated from Kusaya, and its effects on salt-dependent growth.

Kusaya, a traditional Japanese fermented fish product, is known for its high preservability, as it contains natural antibiotics derived from microorganisms, and therefore molds and yeasts do not colonize it easily. In this study, the Streptomyces diastaticus strain TUA-NKU25 was isolated from Kusaya, and its growth as well as the production of antibiotics were investigated. Strain TUA-NKU25 showed advantageous growth characteristics in the presence, but not in the absence, of sodium chloride (NaCl). Antimicrobial assay, high-performance liquid chromatography, and electrospray ionization-mass spectrometry analysis showed that this strain produced surugamide A and uncharacterized antimicrobial compound(s) during growth in the presence of NaCl, suggesting that the biosynthesis of these compounds was upregulated by NaCl. Draft genomic analysis revealed that strain TUA-NKU25 possesses a surugamide biosynthetic gene cluster (sur BGC), although it is incomplete, lacking surB/surC. Phylogenetic analysis of strain TUA-NKU25 and surugamide-producing Streptomyces showed that sur BGC formed a clade distinct from other known groups.

Development of a dynamic motion platform with two independent drive systems for radiotherapy.

BACKGROUND: There are some motion platforms for radiotherapy quality assurance. However, no platform with two drive systems that can move along three axes is available. PURPOSE: The purpose of this study is to develop a dynamic motion platform with two drive systems capable of three-axis motion and to evaluate its motion performance. METHODS: The developed moving platform had two drive systems that use the same equipment. Each axis of the platform used can support a maximum load of 10 kg. The motors for moving the platform in each direction are capable of a drive stroke up to 40 mm. The drive speed is 30 mm/s at maximum load fluctuation. To evaluate the static positional accuracy of this system with an arbitrary input movement, the XYZ position of each axis was measured using a coordinate measuring machine operating from 0 to 40 mm at 10 mm intervals. In addition, the accuracy of dynamic motion was verified with Sine waveform inputs of different patterns to the three axes for approximately 60 s, and they were compared with the resulting detected signals by SyncTrax. RESULTS: The two drive systems were successfully operated on three axes by using independent control systems. For static position, the accuracies were within 0.2 mm, 0.05 mm, and 0.14 mm for lateral, longitudinal, and vertical directions, respectively. For dynamic motion, the mean absolute errors in the X, Y, and Z axes between the platform inputs and SyncTrax detected signals were 0.14 ± 0.10 mm, 0.16 ± 0.12 mm, and 0.16 ± 0.11 mm, respectively. CONCLUSIONS: A new dynamic platform for radiation therapy with two drive systems capable of three-axis motion was developed, and the positional accuracy of the drive axes was confirmed to be less than 0.2 mm.

Evaluation of computed tomography metal artifact and CyberKnife fiducial recognition for novel size fiducial markers.

PURPOSE: This study aimed to compare fiducial markers used in CyberKnife treatment in terms of metal artifact intensity observed in CT images and fiducial recognition in the CyberKnife system affected by patient body thickness and type of marker. METHODS: Five markers, ACCULOC 0.9 mm × 3 mm, Ball type Gold Anchor (GA) 0.28 mm × 10 mm, 0.28 mm × 20 mm, and novel size GA 0.4 mm × 10 mm, 0.4 mm × 20 mm were evaluated. To evaluate metal artifacts of CT images, two types of CT images of water-equivalent gels with each marker were acquired using Aquilion LB CT scanner, one applied SEMAR (SEMAR-on) and the other did not apply this technique (SEMAR-off). The evaluation metric of artifact intensity (MSD ) which represents a variation of CT values were compared for each marker. Next, 5, 15, and 20 cm thickness of Tough Water (TW) was placed on the gel under the condition of overlapping the vertebral phantom in the Target Locating System, and the live image of each marker was acquired to compare fiducial recognition. RESULTS: The mean MSD of SEMAR-off was 78.80, 74.50, 97.25, 83.29, and 149.64 HU for ACCULOC, GA0.28 mm × 10 mm, 20 mm, and 0.40 mm × 10 mm, 20 mm, respectively. In the same manner, that of SEMAR-on was 23.52, 20.26, 26.76, 24.89, and 33.96 HU, respectively. Fiducial recognition decreased in the order of 5, 15, and 20 cm thickness, and GA 0.4 × 20 mm showed the best recognition at thickness of 20 cm TW. CONCLUSIONS: We demonstrated the potential to reduce metal artifacts in the CT image to the same level for all the markers we evaluated by applying SEMAR. Additionally, the fiducial recognition of each marker may vary depending on the thickness of the patient's body. Particularly, we showed that GA 0.40 × 20 mm may have more optimal recognition for CyberKnife treatment in cases of high bodily thickness in comparison to the other markers.

Development of antigen-prediction algorithm for personalized neoantigen vaccine using human leukocyte antigen transgenic mouse.

Immunotherapy is currently recognized as the fourth modality in cancer therapy. CTL can detect cancer cells via complexes involving human leukocyte antigen (HLA) class I molecules and peptides derived from tumor antigens, resulting in antigen-specific cancer rejection. The peptides may be predicted in silico using machine learning-based algorithms. Neopeptides, derived from neoantigens encoded by somatic mutations in cancer cells, are putative immunotherapy targets, as they have high tumor specificity and immunogenicity. Here, we used our pipeline to select 278 neoepitopes with high predictive "SCORE" from the tumor tissues of 46 patients with hepatocellular carcinoma or metastasis of colorectal carcinoma. We validated peptide immunogenicity and specificity by in vivo vaccination with HLA-A2, A24, B35, and B07 transgenic mice using ELISpot assay, in vitro and in vivo killing assays. We statistically evaluated the power of our prediction algorithm and demonstrated the capacity of our pipeline to predict neopeptides (area under the curve = 0.687, P < 0.0001). We also analyzed the potential of long peptides containing the predicted neoepitopes to induce CTLs. Our study indicated that the short peptides predicted using our algorithm may be intrinsically present in tumor cells as cleavage products of long peptides. Thus, we empirically demonstrated that the accuracy and specificity of our prediction tools may be potentially improved in vivo using the HLA transgenic mouse model. Our data will help to design feedback algorithms to improve in silico prediction, potentially allowing researchers to predict peptides for personalized immunotherapy.

Component with abundant immune-related cells in combined hepatocellular cholangiocarcinoma identified by cluster analysis.

Combined hepatocellular cholangiocarcinoma (cHCC-CCA) is a heterogeneous tumor sharing histological features with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). The tumor immune microenvironment (TIME) of cHCC-CCA is unclear. We compared the TIME of cHCC-CCA with that of HCC and iCCA. Twenty-three patients with cHCC-CCA after hepatectomy were evaluated in this study. Twenty-three patients with iCCA and HCC were also included. iCCA was matched for size, and HCC was matched for size and hepatitis virus infection with cHCC-CCA. Immune-related cells among the iCCA-component of cHCC-CCA (C-com), HCC-component of cHCC-CCA (H-com), iCCA, and HCC were assessed using multiplex fluorescence immunohistochemistry. Among C-com, H-com, iCCA, and HCC, multiple comparisons and cluster analysis with k-nearest neighbor algorithms were performed using immunological variables. Although HCC had more T lymphocytes and lower PD-L1 expression than iCCA (P < 0.05), there were no significant differences in immunological variables between C-com and H-com. C-com tended to have more T lymphocytes than iCCA (P = 0.09), and C-com and H-com had fewer macrophages than HCC (P < 0.05). In cluster analysis, all samples were classified into two clusters: one cluster had more immune-related cells than the other, and 12 of 23 H-com and eight of 23 C-com were identified in this cluster. The TIME of C-com and H-com may be similar, and some immunological features in these components were different from those in HCC and some iCCA. Cluster analysis identified components with abundant immune-related cells in cHCC-iCCA.

Sarcomatoid hepatocellular carcinoma is distinct from ordinary hepatocellular carcinoma: Clinicopathologic, transcriptomic and immunologic analyses.

Sarcomatoid hepatocellular carcinoma (SHCC), which was a rare histological subtype of hepatocellular carcinoma (HCC), is currently subclassified as poorly differentiated HCC because of insufficient evidence to define SHCC as a subtype of HCC. We aimed to assess the feasibility of classifying SHCC as a histological subtype of HCC by comprehensively identifying novel and distinct characteristics of SHCC compared to ordinary HCC (OHCC). Fifteen SHCCs (1.4%) defined as HCC with at least a 10% sarcomatous component, 15 randomly disease-stage-matched OHCCs and 163 consecutive OHCCs were extracted from 1106 HCCs in the Pathology Database (1997-2019) of our hospital. SHCC patients showed poor prognosis, and the tumors could be histologically subclassified into the pleomorphic, spindle and giant cell types according to the subtype of carcinomas with sarcomatoid or undifferentiated morphology in other organs. The transcriptomic analysis revealed distinct characteristics of SHCC featuring the upregulation of genes associated with epithelial-to-mesenchymal transition and inflammatory responses. The fluorescent multiplex immunohistochemistry results revealed prominent programmed death-ligand 1 (PD-L1) expression on sarcomatoid tumor cells and higher infiltration of CD4+ and CD8+ T cells in SHCCs compared to OHCCs. The density of CD8+ T cells in the nonsarcomatous component of SHCCs was also higher than that in OHCCs. In conclusion, the comprehensive analyses in our study demonstrated that SHCC is distinct from OHCC in terms of clinicopathologic, transcriptomic and immunologic characteristics. Therefore, it is reasonable to consider SHCC as a histological subtype of HCC.

The immunological impact of preoperative chemoradiotherapy on the tumor microenvironment of pancreatic cancer.

Several therapeutic regimens, including neoadjuvant chemoradiation therapy (NACRT), have been reported to serve as anticancer immune effectors. However, there remain insufficient data regarding the immune response after NACRT in pancreatic ductal adenocarcinoma (PDAC) patients. Data from 40 PDAC patients that underwent surgical resection after NACRT (NACRT group) and 30 PDAC patients that underwent upfront surgery (US group) were analyzed to examine alterations in immune cell counts/distribution using a multiplexed fluorescent immunohistochemistry system. All immune cells were more abundant in the cancer stroma than in the cancer cell nest regardless of preoperative therapy. Although the stromal counts of CD4+ T cells, CD20+ B cells, and Foxp3+ T cells in the NACRT group were drastically decreased in comparison with those of the US group, counts of these cell types in the cancer cell nest were not significantly different between the two groups. In contrast, CD204+ macrophage counts in the cancer stroma were similar between the NACRT and US groups, while those in the cancer cell nests were significantly reduced in the NACRT group. Following multivariate analysis, only a high CD204+ macrophage count in the cancer cell nest remained an independent predictor of shorter relapse-free survival (odds ratio = 2.37; P = .033). NACRT for PDAC decreased overall immune cell counts, but these changes were heterogeneous within the cancer cell nests and cancer stroma. The CD204+ macrophage count in the cancer cell nest is an independent predictor of early disease recurrence in PDAC patients after NACRT.

Carbonylation of skin collagen induced by reaction with methylglyoxal.

Our previous studies have shown that glycerin, which is present at high concentrations in moisturizers and skin lotions, gradually oxidizes to produce methylglyoxal (MGO). In this study, we observed that MGO-treated porcine dermis type-I collagen was carbonylated in an MGO concentration- and time-dependent manner. Furthermore, we examined the structure of advanced glycation end products (AGEs) induced by MGO reacting with type-I collagen. Our findings demonstrate that the α chains of collagen reacted with MGO and easily transformed into a modified protein containing a methylglyoxal-derived hydroimidazolone (MG-H1) moiety in a concentration- and time-dependent manner. Moreover, porcine skin proteins underwent carbonylation when the skin section was treated with MGO for four weeks. Analysis of the structure of AGEs on the carbonylated proteins extracted from MGO-treated skin sections revealed that skin collagen had been converted to MG-H1-modified protein. These novel findings suggest that continuous application of MGO to the skin leads to carbonylation of proteins, which may cause prompt accumulation of MG-H1-modified dermis collagen, thereby resulting in morphological and functional changes of collagen in the skin.

External validation of the albumin, C-reactive protein and lactate dehydrogenase model in patients with metastatic renal cell carcinoma receiving second-line axitinib therapy in a Japanese multi-center cohort.

PURPOSE: To externally validate the utility of the albumin, C-reactive protein and lactate dehydrogenase model to predict the overall survival of previously treated metastatic renal cell carcinoma patients. PATIENTS AND METHODS: The ability of the albumin, C-reactive protein and lactate dehydrogenase model to predict overall survival was validated and compared with those of other prognostication models using data from 421 metastatic renal cell carcinoma patients receiving second-line axitinib therapy at 36 hospitals belonging to the Japan Urologic Oncology Group. RESULTS: The following factors in this cohort were independently associated with poor overall survival in a multivariate analysis: a low Karnofsky performance status, <1 year from diagnosis to targeted therapy, a high neutrophil count, and low albumin, elevated C-reactive protein, and elevated lactate dehydrogenase, and the Japan Urologic Oncology Group model was newly developed based on the presence/absence of these independent factors. In this cohort, 151 (35.9%), 125 (27.7%) and 145 (34.4%) patients were classified into the favorable, intermediate and poor risk groups, respectively, according to the albumin, C-reactive protein and lactate dehydrogenase model; however, the proportions of patients in the intermediate risk group stratified by the Japan Urologic Oncology Group, Memorial Sloan Kettering Cancer Center and International Metastatic Renal Cell Carcinoma Database Consortium models were >50%. The superiority of the albumin, C-reactive protein and lactate dehydrogenase model to the Memorial Sloan Kettering Cancer Center and International Metastatic Renal Cell Carcinoma Database Consortium models, but not the Japan Urologic Oncology Group model, was demonstrated by multiple statistical analyses. CONCLUSIONS: The utility of the albumin, C-reactive protein and lactate dehydrogenase model as a simple and objective prognostication tool was successfully validated using data from 421 metastatic renal cell carcinoma patients receiving second-line axitinib.

Ki67 expression and localization of T cells after neoadjuvant therapies as reliable predictive markers in rectal cancer.

Chemoradiotherapy (CRT) is the standard neoadjuvant therapy for locally advanced rectal cancer (RC). However, neoadjuvant chemotherapy (NAC) also shows favorable outcomes. Although the immunological environment of RC has been thoroughly discussed, the effect of NAC on it is less clear. Here, we investigated the immunological microenvironment, including T cell infiltration, activation, and topological distribution, of resected RC tissue after neoadjuvant therapies and evaluated the correlation between T cell subsets and patient prognosis. Rectal cancer patients (n = 188) were enrolled and categorized into 3 groups, namely CRT (n = 41), NAC (n = 46), and control (surgery alone; n = 101) groups. Characterization of residual carcinoma cells and T cell subsets in resected tissues was performed using multiplex fluorescence immunohistochemistry. The densities of total and activated (Ki67high ) T cells in tissues after NAC, but not CRT, were higher than in control. In both CRT and NAC groups, patients presenting with higher treatment effects showed aggressive infiltration of T cell subsets into carcinomas. Multivariate analyses of pathological and immunological features and prognosis revealed that carcinoma Ki67high CD4+ T cells after CRT and stromal Ki67high CD8+ T cells after NAC are important prognostic factors, respectively. Our results suggest that evaluation of T cell activation with Ki67 expression and its tumor localization can be used to determine the prognosis of advanced RC after neoadjuvant therapies.

Plasma and tumoral glypican-3 levels are correlated in patients with hepatitis C virus-related hepatocellular carcinoma.

Glypican-3 (GPC3) is a cancer antigen expressed in approximately 80% of hepatocellular carcinomas (HCC) and is secreted into the blood. To confirm the effectiveness of GPC3 as a biomarker in HCC, we analyzed the relationship between GPC3 expression levels in cancer cells and in blood in 56 patients with HCC. Preoperative plasma GPC3 levels were determined with an immunoassay, and expression of GPC3 in resected tumors was analyzed by immunohistochemical staining. Median plasma GPC3 level in all HCC cases was 4.6 pg/mL, and tended to be higher in patients with hepatitis C virus (HCV)-related HCC (HCV group) (9.9 pg/mL) than in patients with hepatitis B virus (HBV)-related HCC (HBV group) (2.6 pg/mL) or in those without virus infection (None group) (3.0 pg/mL), suggesting that the virus type most likely influences GPC3 secretion. Median percentage of GPC3+ cells in tumors was also higher in the HCV (26.2%) and HBV (11.1%) groups than in the None group (4.2%). In the HCV group, there was a positive correlation between the two parameters (r = 0.66, P < .01). Moreover, receiver operating characteristic analysis predicted >10% GPC3+ cells in a tumor if the cut-off value was 6.8 pg/mL (sensitivity 80%, specificity 100%; area under the curve 0.875, 95% confidence interval 0.726-1) in the HCV group. Plasma concentration of GPC3 could be a predictive marker of tumoral GPC3 expression in patients with HCV-related HCC, suggesting a useful biomarker for immunotherapies targeting GPC3, although larger-scale validations are needed.

Direct Observation of Cell Surface Sialylation by Atomic Force Microscopy Employing Boronic Acid-Sialic Acid Reversible Interaction.

Tracing cell surface sialylation dynamics at a scale of the glycolipoprotein microdomain (lipid rafts) formations remains an intriguing challenge of cellular biology. Here, we demonstrate that this goal is accessible, taking advantage of a boronic acid (BA)-based reversible molecular recognition chemistry. A BA-end-functionalized poly(ethylene glycol) was decorated onto an atomic force microscopy (AFM) cantilever, which provided a dynamic and sialic acid (SA)-specific imaging mode. Using this technique, we were able to heat map the SA expression levels not only on protein-decorated substrates but also directly on the cell surfaces, with a submicrometer scale resolution that may be relevant to that of the lipid rafts formation. The SA specificity and the binding reversibility of the probe were confirmed from its pH-dependent characteristics and an inhibition assay using free state SA. This finding may provide a noninvasive means for assessing a variety of SA-involved glycosylation dynamics spanning from physiology to pathology.

Oxidative Formation of Methylglyoxal in Glycerol Preparations during Storage.

Methylglyoxal (MGO) is a reactive α-dicarbonyl compound that causes carbonylation of protein and DNA through the pathways of the Maillard reaction. It is known that MGO is physiologically involved in renal dysfunction, vascular disorders, and the acceleration of aging. In this study, we showed for the first time, that a trace amount of MGO was present as an impurity in glycerol preparations used as external medicines and intravenous infusions, when kept unused. The concentration of MGO in the glycerol solutions, diluted to a concentration of 20%, significantly increased after storage for one month when compared to the MGO concentration immediately after opening. Following storage for 6 months at 25°C, MGO concentration increased by about 300 times (approx. 170 µM), and at 40°C, it increased by about 600 times (approx. 350 µM). In the case of intravenous infusion preparations containing 10% glycerol, the MGO concentration increased by 4-15 times (approx. 70 µM) after 2 months of storage at 40°C, and reached over 200 µM after 6 months. Results from the present study showed that glycerol in pharmaceutical preparations is gradually oxidized to form MGO via autoxidation, depending on the temperature and dissolved oxygen content. Thus, we suggest that precautions should be taken when storing glycerol preparations in bottles or plastic containers, with respect to the storage temperature and sealability to prevent MGO formation due to oxidation of glycerol.

Effect of a hydrogel spacer on the intrafractional prostate motion during CyberKnife treatment for prostate cancer.

The purpose of this study was to evaluate the effect of a hydrogel spacer on intrafractional prostate motion during CyberKnife treatment. The retrospective study enrolled 24 patients (with the hydrogel spacer = 12, without the hydrogel spacer = 12) with two fiducial markers. Regarding intrafractional prostate motion, the offset values (mm) of three axes (X-axis; superior [+] to inferior [-], Y-axis; right [+] to left [-], Z-axis; posterior [+] to anterior [-]) obtained from fiducial markers position between a digitally reconstructed radiographs images and live images in the Target Locating System were used, and extracted from generated log files. The mean values of the offset and each standard deviation were calculated for each patient, and both the groups were compared. For all the patients, a total of 2204 offset values and timestamps (without the hydrogel spacer group: 1065, with the hydrogel spacer group: 1139) were recorded for the X-, Y-, and Z-axes, respectively. The offset values (mean ± standard deviation) for the X-, Y-, and Z-axes were -0.04 ± 0.92 mm, -0.03 ± 0.97 mm (P = 0.66), 0.02 ± 0.51, -0.02 ± 0.49 mm (P = 0.50), and 0.56 ± 0.97 mm, 0.34 ± 1.07 mm (P = 0.14), in patients inserted without or with the hydrogel spacer, respectively. There was no effect of a hydrogel spacer on the intrafractional prostate motion in the three axes during CyberKnife treatment for prostate cancer.