Diagnostic potential of SDHB mRNA contained in serum extracellular vesicles among patients with prostate cancer.

BACKGROUND: Androgen receptor signaling inhibitors(ARSIs) have been used to treat patients with metastatic prostate cancer (PC) and castration-resistant prostate cancer (CRPC). In this study, we aimed to identify novel serum extracellular vesicle (EV)-based biomarkers to diagnose ARSI-resistance and therapeutic targets for ARSI-resistant CRPC. METHODS: Total RNA contained in serum EVs from 5 cases of CRPC before ARSI treatment and after acquiring ARSI-resistance was subjected to RNA-sequencing. The expression changes of selected RNAs contained in EVs were confirmed in 48 cases of benign prostatic hyperplasia (BPH) and 107 PC using reverse transcription-quantitative PCR (RT-qPCR) and compared with tissue RNA expression using public datasets. RESULTS: RNA-sequencing revealed that mitochondrial oxidative phosphorylation (OXPHOS)-related genes were increased in EVs after acquiring ARSI-resistance. Among them, RT-qPCR and datasets analysis demonstrated that SDHB mRNA was upregulated after acquiring ARSI-resistance in EVs and ARSI-exposed PC tissue compared to ARSI-naïve EVs and tissue, respectively. SDHB mRNA levels both in EVs and tissue were increased in localized PC compared with BPH and decreased in advanced PC. Tissue expression of SDHB mRNA was significantly correlated with those of other OXPHOS-related genes. SDHB mRNA in EVs (EV-SDHB) was elevated among 3 out of 7 ARSI-treating patients with stable PSA levels who later progressed to ARSI-resistant CRPC. CONCLUSIONS: The levels of OXPHOS-related mRNAs in EVs correlated with those in PC tissue, among which SDHB mRNA was found to be a novel biomarker to diagnose ARSI-resistance. EV-SDHB may be useful for early diagnosis of ARSI-resistance.

Difference in activated partial thromboplastin time values with two different reagents according to C-reactive protein values.

BACKGROUND: Activated partial thromboplastin time (APTT) is susceptible to reagent composition. This study aimed to investigate a large number of specimens and determine the cause of discrepancies. METHOD: This study included 18,994 subjects who underwent coagulation tests at our hospital from May 2020 to December 2020. Measuring reagents included HemosIL SynthASil APTT (APTT-SS, Instrumentation Laboratory) and Coagpia APTT-N (APTT-N, Sekisui Medical). RESULTS: A total of 451 patients demonstrated APTT-N of >39 seconds and an APTT-N/SS ratio of >1.3. A C-reactive protein (CRP) level of ≥1.4 mg/L demonstrated a significant positive correlation, with a higher APTT-N/SS indicating higher CRP levels. All 28 subjects receiving no anticoagulants and who had remaining specimens underwent a cross-mixing test (CMT). Of them, 17 were suspected for lupus anticoagulant (LA) by both the waveform shape and the index of circulating anticoagulant (ICA) value, 6 by the ICA value, and 5 were difficult to determine. CONCLUSION: This study revealed that the APTT-N prolongation correlated with CRP degree and the transient involvement of LA in CMT results due to CRP. This study indicated various reactivities depending on the assay reagents used. Further testing is warranted if LA is suspected, considering the patient's background.

Humoral and Cellular Response Induced by Primary Series and Booster Doses of mRNA Coronavirus Disease 2019 Vaccine in Patients with Cardiovascular Disease: A Longitudinal Study.

Preexisting cardiovascular disease (CVD) is a pivotal risk factor for severe coronavirus disease 2019 (COVID-19). We investigated the longitudinal (over 1 year and 9 months) humoral and cellular responses to primary series and booster doses of mRNA COVID-19 vaccines in patients with CVD. Twenty-six patients with CVD who received monovalent mRNA COVID-19 vaccines were enrolled in this study. Peripheral blood samples were serially drawn nine times from each patient. IgG against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) was measured using an enzyme-linked immunosorbent assay. The numbers of interferon-γ-releasing cells in response to SARS-CoV-2 peptides were measured using an enzyme-linked immunospot assay. The RBD-IgG titers increased 2 weeks after the primary series and booster vaccination and waned 6 months after vaccination. The S1-specific T cell responses in patients aged < 75 years were favorable before and after booster doses; however, the Omicron BA.1-specific T cell responses were poor. These results suggest that regular vaccination is useful to maintain long-term antibody levels and has implications for booster dose strategies in patients with CVD. Additional booster doses, including Omicron variant-adapted mRNA vaccines, may be recommended for patients with CVD, regardless of age.

PROX1 is a regulator of neuroendocrine-related gene expression in lung carcinoid.

Lung neuroendocrine neoplasms (NENs) are a diverse group of tumors characterized by neuroendocrine (NE) differentiation. Among lung NENs, lung carcinoid (LC) is a rare tumor with unique characteristics. Recent research has highlighted the importance of transcription factors (TFs) in establishing gene expression programs in lung NENs such as small cell lung carcinoma. However, the TFs that control the gene expression of LC are largely unknown. In this study, we report the expression and potential function of a TF called Prospero homeobox protein1 (PROX1) in LC. Publicly available transcriptome data suggested that PROX1 was highly expressed in LC tissues, which was confirmed by immunohistochemical analysis on a tissue microarray. Knockdown of PROX1 did not impact the cellular viability of an LC-derived cell line, NCI-H727. Meanwhile, transcriptome analysis revealed that PROX1 knockdown altered the expression of genes involved in NE differentiation. ASCL1, CHGA, CALCA, and LINC00261 were suggested as downstream genes of PROX1. These findings indicate that PROX1 may play an important role in the NE identity of LC by regulating the expression of key target genes.

Consistency Analysis of the UF-1500 and UF-5000 Automated Urine Particle Analyzers.

BACKGROUND: The aim was to evaluate the consistency of the results between the UF-1500 and UF-5000, fully automated urine particle analyzers. METHODS: A total of 554 randomly selected inpatient and outpatient urine samples were collected for analysis using the UF-1500, the UF-5000, and by manual microscopic examination. The coincidence rate, intraday repeatability, and interday reproducibility were evaluated on the UF-1500 and UF-5000. To analyze the review flags from the UF-1500, the UF-1500 results were compared to manual microscopy as the gold standard. RESULTS: The repeatability of red blood cells (RBCs), white blood cells (WBCs), epithelial cells (ECs), casts, and bacteria using the UF-1500 and UF-5000 is expressed as the relative standard deviations of the intraday and inter-day measurements. For the UF-1500, the relative standard deviation values ranged from 5.9% to 12.6% and 4.9% to 17.2% for the low and 1.6% to 9.3% and 2.3% to 16.9% for the high samples, respectively. The correlation co-efficient for RBCs, WBCs, ECs, SECs, casts, crystals, and bacteria for the UF-1500 were 0.981, 0.993, 0.968, 0.963, 0.821, 0.783, and 0.992, respectively. Review samples from the UF-1500 were confirmed by microscopic examination. Review flags for all 554 samples included 3 samples with "DEBRIS High" and 23 samples with "RBCs/YLC Abnormal classification". CONCLUSIONS: The identification of various urine components by both instruments meets laboratory requirements. These two instruments with different performances have specific characteristics and should be used based upon the needs of each laboratory.

Multifaced roles of the long non-coding RNA DRAIC in cancer progression.

Long non-coding RNAs (lncRNA) are functional RNAs, with over 200 nucleotides in length and lacking protein-coding potential. Studies have indicated that lncRNAs are important gene regulators under physiological conditions. Aberrant lncRNA expression is associated with the initiation and progression of various diseases, including cancers. High-throughput transcriptome analyses have revealed thousands of lncRNAs as putative tumor suppressors or promoters in various cancers, but the detailed molecular mechanisms of each lncRNA remain unclear. Downregulated RNA In Cancer, inhibitor of cell invasion and migration (DRAIC) (also known as LOC145837 and RP11-279F6.1) is a lncRNA that inhibits or promotes cancer progression with several modes of action. DRAIC was originally identified as a tumor-suppressive lncRNA in prostate adenocarcinoma. Subsequent studies also revealed that it has an anti-tumor role in glioblastoma, triple-negative breast cancer, and stomach adenocarcinoma. However, DRAIC exhibits oncogenic functions in other malignancies, such as lung adenocarcinoma and esophageal carcinoma, indicating its highly context-dependent effects on cancer progression and clinical outcomes. DRAIC and its associated pathways regulate various biological processes, including proliferation, invasion, metastasis, autophagy, and neuroendocrine function. This review introduces the multifaceted roles of DRAIC, particularly in cancer progression, and discusses its biological significance and clinical implications.

Chromogenic in situ hybridization reveals specific expression pattern of long non-coding RNA DRAIC in formalin-fixed paraffin-embedded specimen.

Long non-coding RNA (lncRNA) plays an important role in the regulation of gene expression in normal and cancer cells. We previously discovered a novel tumor-suppressive lncRNA, DRAIC, in prostate cancer cells. Subsequent studies have demonstrated that DRAIC is dysregulated in various malignancies and exhibits a tumor-suppressive or pro-oncogenic function. However, details regarding its expression pattern in normal and cancerous tissues remain largely unknown. In this study, we performed chromogenic in situ hybridization (CISH) using RNAscope technology to assess DRAIC expression in formalin-fixed paraffin-embedded (FFPE) specimens. In the neuroendocrine-differentiated cancer cell line VMRC-LCD, CISH revealed a diffuse localization of DRAIC in the cytoplasm as well as specific accumulation in the nuclear compartment. DRAIC expression was comprehensively analyzed using tissue microarrays containing 89 normal and 155 tumor tissue samples. DRAIC was weakly expressed in normal epithelial cells of the colon, bronchiole, kidney, prostate, and testis. Conversely, DRAIC was moderately to highly expressed in some cancer tissues, including prostate adenocarcinoma, invasive ductal carcinoma of the breast, neuroendocrine carcinoma of the esophagus, lung adenocarcinoma, and small cell lung carcinoma. While DRAIC knockdown did not affect VMRC-LCD cellular viability and invasive ability, gene expression related to the neuroendocrine and cancer-related pathways was altered. Our expression analysis revealed the specific expression pattern of DRAIC in normal and cancerous FFPE tissues. The results presented here may lead to the elucidation of additional novel functions of DRAIC.

Comparison of the Serial Humoral Immune Response according to the Immunosuppressive Treatment after SARS-CoV-2 mRNA Vaccination.

Objective The objective of this study was to estimate the humoral immune response evaluated by immunoglobulin G (IgG) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD-IgG) following the third mRNA coronavirus disease 2019 (COVID-19) vaccination in patients with kidney disease who received immunosuppressive treatment. Methods The primary outcome was RBD-IgG levels after the third SARS-CoV-2 vaccination. The primary comparison was the RBD-IgG levels between patients with kidney disease who received immunosuppressive treatment (n=124) and those who did not (n=33). Results The RBD-IgG levels were significantly lower in the patients with kidney disease who received immunosuppressive treatment than in those who did not receive immunosuppressive treatment. The RBD-IgG levels were lower in patients treated with glucocorticoid monotherapy than in those who did not receive immunosuppressive treatment. Even in patients who received ≤5 mg prednisolone, the RBD-IgG levels were significantly lower. Nine of the 10 patients who received rituximab within one year before the first vaccination did not experience seroconversion after the third vaccination. Meanwhile, all nine patients who received rituximab only after the second vaccination experienced seroconversion, even if B cell recovery was insufficient. Patients treated with mycophenolate mofetil plus glucocorticoid plus belimumab had significantly lower RBD-IgG levels than those treated with mycophenolate mofetil plus glucocorticoid. Conclusion The RBD-IgG levels were lower in patients with kidney disease who received immunosuppressive treatment than in those who did not receive immunosuppressive treatment. Low-dose glucocorticoid monotherapy affected the humoral immune response following the third mRNA COVID-19 vaccination.

Resting echocardiographic parameters can exclude significant coronary artery disease: A comparison with coronary computed tomography angiography.

INTRODUCTION: Coronary computed tomography angiography (CCTA) is known to have a high negative predictive value (NPV) in identifying coronary artery disease (CAD). This study aimed to examine whether resting echocardiographic parameters could exclude significant CAD on CCTA. METHODS: We recruited 142 patients who had undergone both CCTA and echocardiography within a 3-month window. Based on the CCTA findings, patients were divided into two groups: Group A (non-significant CAD, defined as all coronary segments having <50% stenosis) and Group B (significant CAD). Resting echocardiographic parameters were compared between the two groups to identify predictors of non-significant CAD on CCTA. RESULTS: A total 92 patients (mean age, 68 ± 13 years; males, 62%) were eligible for this study; 50 in Group A and 42 in Group B. Among the various echo parameters, left atrial volume index (LAVI) and left ventricular (LV) global longitudinal strain (GLS) were significantly lower in Group A (23.5 ± 7.6 vs. 33.6 ± 7.4 mL/m2 , p < .001; -20.2 ± 1.8% vs. -16.8 ± 2.0%, p < .001, respectively). Analysis of the receiver operating characteristic curve revealed that the cutoff value to exclude significant CAD on CCTA was 29.0 mL/m2 for LAVI (NPV 80.8%) and -18.1% for GLS (NPV 80.7%). The NPV increased to 95.0% when these parameters were combined (LAVI < 29.0 mL/m2 and GLS < -18.1%). CONCLUSION: The combination of resting LAVI and GLS was clinically useful in excluding significant CAD via CCTA.

H19 in Serum Extracellular Vesicles Reflects Resistance to AR Axis-targeted Therapy Among CRPC Patients.

BACKGROUND/AIM: We aimed to evaluate the changes of androgen receptor (AR) signaling-related long non-coding RNAs (lncRNAs) in serum extracellular vesicles (EVs) from prostate cancer (PC) patients, in order to identify novel biomarkers for AR axis-targeted therapy (ARAT)-resistance among castration-resistant PC (CRPC) patients. PATIENTS AND METHODS: EVs were isolated from 2 patients before and after acquiring ARAT-resistance. RNA profiling of EVs was performed by RNA-sequencing. The expression levels of selected lncRNAs in EVs were analyzed by digital droplet PCR (ddPCR) in 58 localized and 14 metastatic PC patients at diagnosis, 7 ARAT-naïve and 6 ARAT-resistant CRPC patients. LncRNA H19 expression in PC tissue was examined using published data. In order to analyze the role of H19, the prognosis was analyzed in PC patients and proteomic analysis was performed in 22Rv1 PC cells. RESULTS: RNA-sequencing revealed that AR-regulated RNAs were most enriched in EVs after acquiring ARAT-resistance. Among them, up-regulation of AR signaling-related lncRNAs (PCAT1, H19, HOXA-11AS, ZEB1-AS1, ARLNC1, PART1, CTBP1-AS and PCA3) was confirmed by ddPCR. H19 contained in EVs (EV-H19) was significantly increased among ARAT-resistant patients compared to ARAT-naïve CRPC or metastatic PC patients. In PC tissue, H19 was negatively correlated with AR protein and AR-activity score and up-regulated in neuroendocrine CRPC tissue with low AR expression. Furthermore, EV-H19 expression was significantly associated with worse outcome to androgen-deprivation therapy. Proteomic analysis demonstrated that H19 knockdown enhanced PC-related protein expression. CONCLUSION: EV-H19 may negatively correlate with AR-signaling activity and could be a marker to diagnose ARAT-resistance among CRPC patients.

Transcriptome of Oral Cancer Cells Adapted to Suspension Culture Is Potentially Related to Cancer Progressive Phenotypes.

BACKGROUND/AIM: Cervical lymph node metastasis worsens oral cancer prognosis. Cancer cells with high metastatic ability can delay or resist apoptosis and survive in the floating condition during circulation. The involved genes and pathways in this process remain largely unknown. This study aimed to establish an oral cancer cell line adapted to suspension culture by in vitro selection and perform gene expression analysis. MATERIALS AND METHODS: The oral cancer cell subline adapted to suspension culture was isolated by in vitro selection from the oral cancer cell line, HSC-3. The transcriptome profiles of HSC-3 and its subline were compared using gene expression microarrays. Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis (GSEA), and Ingenuity Pathway Analysis (IPA) were performed to predict the involved pathways and molecules in cancer progression. RESULTS: The subline was designated as HSC-3S5 The cellular viability of HSC-3S5 cells at the suspension culture was higher than that of HSC-3 cells. A total of 961 genes were differentially expressed between HSC-3 and HSC-3S5 cells under the threshold cut-off (FDR-adjusted p-value of <0.05 and absolute fold change of >1.5). GO terms, such as growth regulation, were enriched in the DEGs. GSEA revealed the association between the DEGs and significant gene sets, including metastasis and stemness. IPA predicted that the proliferation-related pathways were enhanced while the apoptotic pathway was inhibited in HSC-3S5 cells compared to HSC-3 cells. CONCLUSION: Our transcriptome analysis revealed several potentially activated pathways and molecules in the floating-adapted oral cancer cells and indicated molecular implications for cancer progression.

CD8+ Regulatory T Cells Induced by Lipopolysaccharide Improve Mouse Endotoxin Shock.

Sepsis is a systemic inflammatory disease caused by a bacterial infection that leads to severe mortality, especially in elderly patients, because of an excessive immune response and impaired regulatory functions. Antibiotic treatment is widely accepted as the first-line therapy for sepsis; however, its excessive use has led to the emergence of multidrug-resistant bacteria in patients with sepsis. Therefore, immunotherapy may be effective in treating sepsis. Although CD8+ regulatory T cells (Tregs) are known to have immunomodulatory effects in various inflammatory diseases, their role during sepsis remains unclear. In this study, we investigated the role of CD8+ Tregs in an LPS-induced endotoxic shock model in young (8-12 wk old) and aged (18-20 mo old) mice. The adoptive transfer of CD8+ Tregs into LPS-treated young mice improved the survival rate of LPS-induced endotoxic shock. Moreover, the number of CD8+ Tregs in LPS-treated young mice increased through the induction of IL-15 produced by CD11c+ cells. In contrast, LPS-treated aged mice showed a reduced induction of CD8+ Tregs owing to the limited production of IL-15. Furthermore, CD8+ Tregs induced by treatment with the rIL-15/IL-15Rα complex prevented LPS-induced body wight loss and tissue injury in aged mice. In this study, to our knowledge, the induction of CD8+ Tregs as novel immunotherapy or adjuvant therapy for endotoxic shock might reduce the uncontrolled immune response and ultimately improve the outcomes of endotoxic shock.

Dataset dependency of low-density lipoprotein-cholesterol estimation by machine learning.

OBJECTIVES: We evaluated the applicability of a machine learning-based low-density lipoprotein-cholesterol (LDL-C) estimation method and the influence of the characteristics of the training datasets. METHODS: Three training datasets were chosen from training datasets: health check-up participants at the Resource Center for Health Science (N = 2664), clinical patients at Gifu University Hospital (N = 7409), and clinical patients at Fujita Health University Hospital (N = 14,842). Nine different machine learning models were constructed through hyperparameter tuning and 10-fold cross-validation. Another test dataset of another 3711 clinical patients at Fujita Health University Hospital was selected as the test set used for comparing and validating the model against the Friedewald formula and the Martin method. RESULTS: The coefficients of determination of the models trained on the health check-up dataset produced coefficients of determination that were equal to or inferior to those of the Martin method. In contrast, the coefficients of determination of several models trained on clinical patients exceeded those of the Martin method. The means of the differences and the convergences to the direct method were higher for the models trained on the clinical patients' dataset than for those trained on the health check-up participants' dataset. The models trained on the latter dataset tended to overestimate the 2019 ESC/EAS Guideline for LDL-cholesterol classification. CONCLUSION: Although machine learning models provide valuable method for LDL-C estimates, they should be trained on datasets with matched characteristics. The versatility of machine learning methods is another important consideration.

Serum angiotensin-converting enzyme levels indicating early sarcoidosis diagnosis and immunosuppressive therapy efficacy.

AIMS: This study aimed to determine the new cut-off value of serum angiotensin-converting enzyme (ACE) levels for detecting patients with sarcoidosis and to examine the change in ACE levels after the initiation of immunosuppressive therapy. METHODS AND RESULTS: We retrospectively examined patients in whom serum ACE levels were measured for suspected sarcoidosis between 2009 and 2020 in our institution. For patients diagnosed with sarcoidosis, changes in ACE levels were also observed. Of the 3781 patients (51.1% men, 60.1 ± 17.0 years old), 477 were excluded for taking ACE inhibitors and/or immunosuppression agents or those with any diseases affecting serum ACE levels. In 3304 patients including 215 with sarcoidosis, serum ACE levels were 19.6 IU/L [interquartile range, 15.1-31.5] in patients with sarcoidosis and 10.7 [8.4-16.5] in those without sarcoidosis (P < 0.01), and the best cut-off value was 14.7 IU/L with 0.865 of the area under the curves. Compared with the current ACE cut-off of 21.4, the sensitivity improved from 42.3 to 78.1 at the new cut-off, although specificity slightly decreased from 98.6 to 81.7. The ACE level significantly decreased more in those with immunosuppression therapy than in those without it (P for interaction <0.01), although it decreased in both groups (P < 0.01). CONCLUSIONS: Because the sensitivity for detecting sarcoidosis is comparatively low at the current standard value, further examinations are needed for patients suspected of sarcoidosis with relatively high ACE levels in the normal range. In patients with sarcoidosis, ACE levels decreased after the initiation of immunosuppression therapy.

Spermidine enhances the efficacy of adjuvant in HBV vaccination in mice.

Background: Various vaccine adjuvants have been developed to eliminate HBV from patients with chronic HBV infection. In addition, spermidine (SPD), a type of polyamine, has been reported to enhance the activity of immune cells. In the present study, we investigated whether the combination of SPD and vaccine adjuvant enhances the HBV antigen-specific immune response to HBV vaccination. Methods: Wild-type and HBV-transgenic (HBV-Tg) mice were vaccinated 2 or 3 times. SPD was orally administered in drinking water. Cyclic guanosine monophosphate-AMP (cGAMP) and nanoparticulate CpG-ODN (K3-SPG) were used as the HBV vaccine adjuvants. The HBV antigen-specific immune response was evaluated by measuring the HBsAb titer in blood collected over time and the number of interferon-γ producing cells by enzyme-linked immunospot assay. Results: The administration of HBsAg + cGAMP + SPD or HBsAg + K3-SPG + SPD significantly enhanced HBsAg-specific interferon-γ production by CD8 T cells from wild-type and HBV-Tg mice. The administration of HBsAg, cGAMP, and SPD increased serum HBsAb levels in wild-type and HBV-Tg mice. In HBV-Tg mice, the administration of SPD + cGAMP or SPD + K3-SPG with HBV vaccination significantly reduced HBsAg levels in the liver and serum. CONCLUSIONS: These results indicate that the combination of HBV vaccine adjuvant and SPD induces a stronger humoral and cellular immune response through T-cell activation. These treatments may support the development of a strategy to completely eliminate HBV.

Administration of spermidine attenuates concanavalin A-induced liver injury.

A previous study revealed that treatment with the anticoagulant heparin attenuated concanavalin A (ConA)-induced liver injury. The administration of spermidine (SPD) increased urokinase-type plasminogen activator (uPA) levels in the serum. uPA is clinically used for the treatment of some thrombotic diseases such as cerebral infarction. Therefore, SPD may attenuate ConA-induced liver injury that is exacerbated by blood coagulation. The present study investigated the effect of SPD on liver injury in mice with autoimmune hepatopathy induced by ConA. A model of liver injury was created by intravenous injection of ConA into mice. SPD was administered in free drinking water and was biochemically and pathologically examined over time. The administration of SPD to ConA-treated mice significantly reduced liver injury. However, SPD treatment upregulated the mRNA expression of TNF-α and IFN-ϒ in the livers of ConA-treated mice. In contrast, the mRNA expression of tissue factor in the livers of SPD-treated mice was decreased after ConA injection. The frequency of lymphocytes and lymphocyte activation were not affected by SPD administration in ConA-treated mice. SPD treatment increased uPA levels in the serum and decreased the level of D-dimer in ConA-treated mice. Moreover, SPD decreased fibrin in the livers of ConA-treated mice. These results indicated that SPD treatment increased anticoagulant ability by increasing of uPA and attenuated ConA-induced liver injury.

Absence of indoleamine 2,3­dioxygenase 2 promotes liver regeneration after partial hepatectomy in mice.

The partial loss of liver due to liver transplantation or acute liver failure induces rapid liver regeneration. Recently, we reported that the selective inhibition of indoleamine 2,3­dioxygenase (Ido) 1 promotes early liver regeneration. However, the role of Ido2 in liver regeneration remains unclear. Wild­type (WT) and Ido2­deficient (Ido2­KO) mice were subjected to 70% partial hepatectomy (PHx). Hepatocyte growth was measured using immunostaining. The mRNA expression of inflammatory cytokines and production of kynurenine in intrahepatic mononuclear cells (MNCs) were analyzed using reverse transcription­quantitative PCR and high­performance liquid chromatography. The activation of NF­κB was determined by both immunocytochemistry and western blotting analysis. The ratio of liver to body weight and the frequency of proliferation cells after PHx were significantly higher in Ido2­KO mice compared with in WT mice. The expression of IL­6 and TNF­α in MNCs were transiently increased in Ido2­KO mice. The nuclear transport of NF­κB was significantly higher in peritoneal macrophages of Ido2­KO mice compared with WT mice. These results suggested that Ido2 deficiency resulted in transiently increased production of inflammatory cytokines through the activation of NF­kB, thereby promoting liver regeneration. Therefore, the regulation of Ido2 expression in MNCs may play a therapeutic role in liver regeneration under injury and disease conditions.

Testicular teratomagenesis from primordial germ cells with overexpression of germinal center-associated nuclear protein.

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganpTg mice). In the GANP-teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganpTg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells.

Cytomorphology and Gene Expression Signatures of Anchorage-independent Aggregations of Oral Cancer Cells.

BACKGROUND/AIM: Cancer cells with high anchorage independence can survive and proliferate in the absence of adhesion to the extracellular matrix. Under anchorage-independent conditions, cancer cells adhere to each other and form aggregates to overcome various stresses. In this study, we investigated the cytomorphology and gene expression signatures of oral cancer cell aggregates. MATERIALS AND METHODS: Two oral cancer-derived cell lines, SAS and HSC-3 cells, were cultured in a low-attachment plate and their cytomorphologies were observed. The transcriptome between attached and detached SAS cells was examined using gene expression microarrays. Subsequently, gene enrichment analysis and Ingenuity Pathway Analysis were performed. Gene expression changes under attached, detached, and re-attached conditions were measured via RT-qPCR. RESULTS: While SAS cells formed multiple round-shaped aggregates, HSC-3 cells, which had lower anchorage independence, did not form aggregates efficiently. Each SAS cell in the aggregate was linked by desmosomes and tight junctions. Comparative transcriptomic analysis revealed 1,698 differentially expressed genes (DEGs) between attached and detached SAS cells. The DEGs were associated with various functions and processes, including cell adhesion. Moreover, under the detached condition, the expression of some epithelial genes (DSC3, DSP, CLDN1 and OCLN) were up-regulated. The changes in both cytomorphology and epithelial gene expression under the detached condition overall returned to their original ones when cells re-attached. CONCLUSION: The results suggest specific cytomorphological and gene expression changes in oral cancer cell aggregates. Our findings provide insights into the mechanisms underlying anchorage-independent oral cancer cell aggregation and reveal previously unknown potential diagnostic and therapeutic molecules.

Treatment with hepatocyte transplantation in a novel mouse model of persistent liver failure.

Background and Aim: Cell-based transplantation therapy using hepatocytes, hepatic stem cells, hepatocyte-like cells induced from stem cells, etc. is thought to be an attractive alternative to liver transplantation, and have been studied to date. For its clinical application, however, it is extremely important to develop a model that reproduces the pathological conditions with indication for treatment and enables the study for the ideal treatment strategy. Methods: The transgenic mice which express the thymidine kinase (TK) gene of human herpes simplex virus (HSV) in their hepatocytes with normal immunity has been developed (designated as HSVtk). After ganciclovir (GCV) administration which injure TK-expressing hepatocytes, the primary hepatocytes (PHs) isolated from green fluorescent protein (GFP) transgenic mouse (GFP-tg) were transplanted to HSVtk intrasplenically, and replacement index (RI) with transplanted PHs in the liver, liver histology, and mRNA expressions in the liver were analyzed up to 8 weeks after transplantation. Results: HSVtk without PH transplantation after GCV administration developed persistent liver failure with degenerated hepatocytes, persistent elevation of ALT and hepatic p16 mRNA levels, suggesting the existence of cellular senescence in the base of the disease. When autologous GFP-PHs were transplanted to HSVtk, the transplanted cells were successfully engrafted in the liver. Eight weeks after transplantation, serum ALT levels and liver histology were almost normalized, while RIs varied from 19.8 to 73.8%. Since the hepatic p16 mRNA levels were decreased significantly in these mice, the senescence of hepatocytes associated with liver injury was thought to be resolved. On the other hand, allogenic GFP-PHs transplanted to HSVtk were eliminated as early as 1 week after transplantation. In these mice, hepatic p16 mRNA levels were significantly increased at 8 weeks after transplantation, suggesting the aggravation of hepatocyte senescence. FK506 administration to HSVtk protected the transplanted hepatocytes with allogenic background from rejection at 2 weeks after transplantation, but the condition of mice and the senescent status in the liver seemed worsened. Conclusions: The mouse model with HSVtk/GCV system was useful for studying the mechanism of liver regeneration and the immune rejection responses in the hepatocyte transplantation treatment. It may also be utilized to develop the effective remedies to avoid immune rejection.