Midazolam exhibits antitumour and anti-inflammatory effects in a mouse model of pancreatic ductal adenocarcinoma.

BACKGROUND: Anaesthesia and perioperative management contribute to long-term outcomes of patients with cancer, including pancreatic ductal adenocarcinoma. We assessed the antitumour, anti-inflammatory, and analgesic effects of midazolam on LSL-KrasG12D/+;Trp53flox/flox;Pdx-1cre/+ transgenic mice with pancreatic ductal adenocarcinoma. METHODS: Six-week-old transgenic mice were administered midazolam 30 mg kg-1 day-1 p.o. (n=13); midazolam 30 mg kg-1 day-1 with 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) 3 mg kg-1 day-1 i.p., a peripheral benzodiazepine receptor antagonist (n=10); or vehicle (water; n=14) until the humane endpoint. Cancer-associated pain was evaluated using hunching score and mouse grimace scale. Tumour stage and immuno-inflammatory status were determined histopathologically. Anti-proliferative and apoptotic potentials of midazolam were investigated using mouse pancreatic ductal adenocarcinoma cell lines. RESULTS: Midazolam significantly inhibited tumour size and proliferative index of Ki-67 and cyclins in pancreatic ductal adenocarcinoma, which was blocked by administration of PK11195. Local myeloperoxidase+ tumour-associated neutrophils, arginase-1+ M2-like tumour-associated macrophages, and CD11b+Ly-6G+ polymorphonuclear myeloid-derived suppressor cells were reduced by midazolam, which was antagonised by administration of PK11195. Hunching and mouse grimace scale were improved by midazolam, whereas the scores increased with midazolam+PK11195 treatment. Plasma pro-inflammatory cytokines, such as interleukin-6 and CC chemokine ligand (CCL)2, CCL3, and CCL5, were reduced by midazolam, whereas these cytokines increased with PK11195. Midazolam inhibited pancreatic ductal adenocarcinoma proliferation through downregulation of cyclins and cyclin-dependent kinases and induced apoptosis in vitro. CONCLUSIONS: These results suggest that midazolam inhibits pancreatic ductal adenocarcinoma proliferation and local infiltration of tumour-associated neutrophils, tumour-associated macrophages, and polymorphonuclear myeloid-derived suppressor cells, thereby inhibiting pancreatic ductal adenocarcinoma progression.

Induction of cell death in pancreatic ductal adenocarcinoma by indirubin 3'-oxime and 5-methoxyindirubin 3'-oxime in vitro and in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis. To identify potential effective therapeutic drugs for PDAC, we established a screening system based on spheroid formation using 170#3 mouse PDAC cells with or without fibroblasts. We found that indirubin 3'-oxime (Indox) and 5-methoxyindirubin 3'-oxime (5MeOIndox) inhibited PDAC cell proliferation. Furthermore, PDAC xenograft growth was also inhibited in BALB/c nu/nu mice after administration of Indox and 5MeOIndox. Both phosphorylated CDK1 and cyclin B1 levels in 170#3 cells were significantly reduced by treatment with Indox and 5MeOIndox in vitro and in vivo. Cell cycle analysis revealed that 5MeOIndox, but not Indox, induced G2/M arrest. Annexin V-propidium iodide double-staining analysis demonstrated that Indox induced abundant non-apoptotic cell death of 170#3 cells, while 5MeOIndox predominantly induced early apoptosis, indicating that the cytotoxicity of 5MeOIndox is lower than that of Indox. These results suggest that one mechanism of 5MeOIndox is to induce G2/M arrest of PDAC cells via inhibition of CDK1/cyclin B1 levels, thereby leading to apoptosis. Our findings suggest 5MeOIndox as a potential useful anticancer agent in PDAC.

Regional variation of white matter development in the cat brain revealed by ex vivo diffusion MR tractography.

Three-dimensional reconstruction of developing fiber pathways is essential to assessing the developmental course of fiber pathways in the whole brain. We applied diffusion spectrum imaging (DSI) tractography to five juvenile ex vivo cat brains at postnatal day (P) 35, when the degree of myelination varies across brain regions. We quantified diffusion properties (fractional anisotropy [FA] and apparent diffusion coefficient [ADC]) and other measurements (number, volume, and voxel count) on reconstructed pathways for projection (cortico-spinal and thalamo-cortical), corpus callosal, limbic (cingulum and fornix), and association (cortico-cortical) pathways, and characterized regional differences in maturation patterns by assessing diffusion properties. FA values were significantly higher in cortico-cortical pathways within the right hemisphere compared to those within the left hemisphere, while the other measurements for the cortico-cortical pathways within the hemisphere did not show asymmetry. ADC values were not asymmetric in both types of pathways. Interestingly, tract count and volume were significantly larger in the left thalamo-cortical pathways compared to the right thalamo-cortical pathways. The bilateral thalamo-cortical pathways showed high FA values compared to the other fiber pathways. On the other hand, ADC values did not show any differences across pathways studied. These results demonstrate that DSI tractography successfully depicted regional variations of white matter tracts during development when myelination is incomplete. Low FA and high ADC values in the cingulum bundle suggest that the cingulum bundle is less mature than the others at this developmental stage.

Effects of ADH1B and ALDH2 Genetic Polymorphisms on Alcohol Elimination Rates and Salivary Acetaldehyde Levels in Intoxicated Japanese Alcoholic Men.

BACKGROUND: The genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are associated with the risk of alcoholism and upper aerodigestive tract cancer in alcoholics. Salivary ethanol (sEtOH) levels are well correlated with blood EtOH levels. METHODS: To study the effects of ADH1B and ALDH2 genotypes on the alcohol elimination rate (AER) and salivary acetaldehyde (sAcH) levels, we measured the sEtOH and sAcH levels twice at a 1-hour intervals in 99 intoxicated Japanese alcoholic men who had stopped drinking for 4 or more hours. RESULTS: The initial sEtOH levels did not differ between the ADH1B*2 group (n = 50) and the ADH1B*1/*1 group (n = 49) (median: 0.617 vs. 0.762 mg/ml). The salivary AER (sAER) increased as the sEtOH levels increased (p < 0.0001). After stratification according to the sEtOH levels (<0.4, 0.4 to 0.99, and ≥1.00 mg/ml), the median sAER of the ADH1B*2 group was 0.075, 0.188, and 0.228 mg/ml/h, respectively, and that of the ADH1B*1/*1 group was 0.037, 0.115, and 0.233 mg/ml/h, respectively. The sAER of the ADH1B*2 group was faster than that of the ADH1B*1/*1 group overall (p = 0.001) and when the sEtOH category was 0.4 to 0.99 mg/ml (p < 0.0001). The ADH1B genotype and the sEtOH levels had an interaction effect on the sAER (p = 0.036). A multiple linear regression analysis with a stepwise procedure selected the ADH1B*2 allele (p = 0.004) and the sEtOH levels (p < 0.0001) as positive predictors of sAER. The sAER did not differ according to the ALDH2 genotype. The sAcH levels were higher than the blood AcH levels reported in alcoholics, probably because of AcH production by oral microorganisms. The sAcH of the ALDH2*1/*2 group (n = 18) was higher than that of the ALDH2*1/*1 group (n = 81) overall (p = 0.0008) and when the corresponding sEtOH category was ≥1.00 mg/ml (median: 3.195 vs. 1.776 µg/ml, p = 0.009). A multiple linear regression analysis selected the ALDH2*1/*2 and the sEtOH levels as positive predictors of the sAcH levels (p < 0.0001). CONCLUSIONS: The enhanced AER in ADH1B*2 carriers and the increased sAcH levels in ALDH2*1/*2 carriers among intoxicated alcoholics provide possible mechanisms explaining how each genetic polymorphism affects the risk of alcoholism and upper aerodigestive tract cancer.

How do resident stem cells repair the damaged myocardium?

It has been a decade since the monumental discovery of resident stem cells in the mammalian heart, and the following studies witnessed the continuous turnover of cardiomyocytes and vascular cells, maintaining the homeostasis of the organ. Recently, the autologous administration of c-kit-positive cardiac stem cells in patients with ischemic heart failure has led to an incredible outcome; the left ventricular ejection fraction of the cell-treated group improved from 30% at the baseline to 38% after one year and to 42% after two years of cell injection. The potential underlying mechanisms, before and after cell infusion, are explored and discussed in this article. Some of them are related to the intrinsic property of the resident stem cells, such as direct differentiation, paracrine action, and immunomodulatory function, whereas others involve environmental factors, leading to cellular reverse remodeling and to the natural selection of "juvenile" cells. It has now been demonstrated that cardiac stem cells for therapeutic purposes can be prepared from tiny biopsied specimens of the failing heart as well as from frozen tissues, which may remarkably expand the repertoire of the strategy against various cardiovascular disorders, including non-ischemic cardiomyopathy and congenital heart diseases. Further translational investigations are needed to explore these possibilities.

[Verification and Validation on Single Nucleotide Polymorphism Analysis of Alcohol Metabolism-Related Genes ADH1B and ALDH2, Using Dried-Saliva Samples].

We have developed a new method for unprocessed biological specimens as templates directly into the TaqMan assay. Saliva was needed to be put on a water-soluble paper and dried, because foreign substances, such as a filter paper, hinder fluorescence detection through the assay. Genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) polymorphisms was, subsequently, performed by TaqMan PCR assay using dried saliva in the present investigation. The optimized technique was tested on 114 samples of alcoholic patients. The PCR-RFLP methods with purified DNA from blood samples were employed for validation of the assay. Upon validation, complete concordance was observed between the two independent results. These results highlight the ability of TaqMan PCR assays using dried saliva on water-soluble paper in genotyping of ADH1B and ALDH2 genes. Our results showed a rapid, simple, reliable, and cost-effective method for SNP genotyping of mutations in ADH1B and ALDH2 genes. This will be very useful for large-scale association studies in various fields. [Original].

Environmentally benign process for the preparation of antimicrobial α-methylene-ß-hydroxy-γ-butyrolactone (tulipalin B) from tulip biomass.

Tulipalin B (α-methylene-ß-hydroxy-γ-butyrolactone, PaB) is an antimicrobial natural product occurring in tulip (Tulipa gesneriana). PaB is directly formed from the precursor glucose ester 6-tuliposide B (PosB) by endogenous Pos-converting enzyme (TCE). Despite the potential usefulness of antibacterial PaB in various industrial applications, lack of facile synthetic schemes hampers its practical use. Herein, we describe an environmentally benign and facile process for the preparation of PaB using tulip biomass materials based on one-step enzyme reaction catalyzed by TCE without the use of petroleum-derived solvents. By screening 115 tulip cultivars, we found three elite cultivars, which accumulated PosB almost exclusively in flower tissues. The flower extracts with aqueous ethanol were partially purified with activated charcoal and subjected to the enzyme reaction with reusable immobilized TCE prepared from bulb crude extracts. The reaction was completed in a few hours at room temperature, and PaB was purified with activated charcoal and ethanol in a batch-wise manner.

Clinicopathological characteristics of anaplastic carcinoma of the pancreas with rhabdoid features.

Undifferentiated (anaplastic) carcinoma with rhabdoid features is a rare and aggressive subtype of pancreatic carcinoma. Here, we report the clinical, histological, and immunohistochemical phenotypes in six autopsy cases of anaplastic carcinoma with rhabdoid features. The patients ranged between 44 and 76 years of age (median, 61 years) and consisted of four males and two females. All patients except one case died within 3 months of diagnosis, as these tumors were found at an advanced stage and were chemoresistant. At autopsy, tumor masses measuring 4-22 cm in maximum diameter were mainly located in the pancreatic body and tail. Microscopically, all cases showed anaplastic carcinoma with rhabdoid features that were discohesive with round to polygonal eosinophilic cytoplasm with occasional inclusions, and that had vesicular nuclei, and prominent nucleoli. Immunohistochemistry showed that the rhabdoid cells, particularly the inclusions, were strongly positive for pan-cytokeratin (AE1/AE3) and vimentin. Meanwhile, downregulation or aberrant cytoplasmic localization with focal aggregation of E-cadherin, ß-catenin, and EMA were frequently observed in the rhabdoid cells. Moreover, the intracytoplasmic inclusions were labeled with selective autophagy-related molecules including p62/SQSTM1, ubiquitin, and kelch-like ECH-associated protein 1 (KEAP1). In addition, nuclear factor erythroid 2-related factor 2 (NRF2) and overexpression of its target molecule multidrug resistance-associated protein 1 (MRP1) were commonly observed in the rhabdoid cells. Therefore, these results suggest that p62-mediated aggregation of ubiquitinated intermediate filaments and membranous proteins is an important phenomenon in the rhabdoid phenotype. Indeed, the ubiquitinated aggregates of p62 and KEAP1 would induce activation of NRF2 and upregulation of MRP1, leading to potential chemoresistance of anaplastic carcinoma with rhabdoid features.

Myocyte renewal and therapeutic myocardial regeneration using various progenitor cells.

Whereas the demand on effective treatment options for chronic heart failure is dramatically increasing, the recent recognition of physiological and pathological myocyte turnover in the adult human heart provided a fundamental basis for the therapeutic regeneration. Divergent modalities were experimentally introduced to this field, and selected ones have been applied clinically; the history began with skeletal myoblasts and bone-marrow-derived cells, and lately mesenchymal stem/stromal cells and resident cardiac cells joined the repertoire. Among them, autologous transplantation of c-kit-positive cardiac stem cells in patients with chronic ventricular dysfunction resulted in an outstanding outcome with long-lasting effects without increasing major adverse events. To further optimize currently available approaches, we have to consider multiple factors, such as the targeting disease, the cell population and number to be administered, and the timing and the route of cell delivery. Exploration of the consequence of the previous clinical trials would allow us to envision an ideal cellular therapy for various cardiovascular disorders.

Development of cerebellar connectivity in human fetal brains revealed by high angular resolution diffusion tractography.

High angular resolution diffusion imaging (HARDI) tractography has provided insights into major white matter pathways and cortical development in the human fetal cerebrum. Our objective in this study was to further apply HARDI tracography to the developing human cerebellum ranging from fetal to adult stages, to outline in broad strokes the 3-dimensional development of white matter and local gray matter organization in the cerebellum. We imaged intact fixed fetal cerebellum specimens at 17 gestational weeks (W), 21W, 31W, 36W, and 38W along with an adult cerebellum for comparison. At the earliest gestational age studied (17W), coherent pathways that formed the superior, middle, and inferior cerebellar peduncles were already detected, but pathways between deep cerebellar nuclei and the cortex were not observed until after 38W. At 36-38W, we identified emerging regional specification of the middle cerebellar peduncle. In the cerebellar cortex, we observed disappearance of radial organization in the sagittal orientation during the studied developmental stages similar to our previous observations in developing cerebral cortex. In contrast, in the axial orientation, cerebellar cortical pathways emerged first sparsely (31W) and then with increased prominence at 36-38W with pathways detected both in the radial and tangential directions to the cortical surface. The cerebellar vermis first contained only pathways tangential to the long axes of folia (17-21W), but pathways parallel to the long axes of folia emerged between 21 and 31W. Our results show the potential for HARDI tractography to image developing human cerebellar connectivity.

Therapeutic application of cardiac stem cells and other cell types.

Various researches on regenerative medicine were carried out experimentally, and selected modalities have been introduced to the clinical arena. Meanwhile, the presence of resident stem cells in the heart and their role in physiological cell turnover were demonstrated. So far skeletal myoblasts, bone marrow-derived cells, mesenchymal stromal cells, and resident cardiac cells have been applied for therapeutic myocardial regeneration. Among them, autologous transplantation of c-kit-positive cardiac stem cells in congestive heart failure patients resulted in an outstanding outcome, with long-lasting beneficial effects without major adverse events. By reviewing these clinical trials, an endeavor was made to seek for an ideal cellular therapy for cardiovascular diseases.

Thioredoxin-interacting protein mediates ER stress-induced ß cell death through initiation of the inflammasome.

Recent clinical and experimental evidence suggests that endoplasmic reticulum (ER) stress contributes to the life-and-death decisions of ß cells during the progression of type 1 and type 2 diabetes. Although crosstalk between inflammation and ER stress has been suggested to play a significant role in ß cell dysfunction and death, a key molecule connecting ER stress to inflammation has not been identified. Here we report that thioredoxin-interacting protein (TXNIP) is a critical signaling node that links ER stress and inflammation. TXNIP is induced by ER stress through the PERK and IRE1 pathways, induces IL-1ß mRNA transcription, activates IL-1ß production by the NLRP3 inflammasome, and mediates ER stress-mediated ß cell death. Collectively, our results suggest that TXNIP is a potential therapeutic target for diabetes and ER stress-related human diseases such as Wolfram syndrome.

Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells.

Wolfram syndrome is an autosomal-recessive disorder characterized by insulin-dependent diabetes mellitus, caused by nonautoimmune loss of beta cells, and neurological dysfunctions. We have previously shown that mutations in the Wolfram syndrome 1 (WFS1) gene cause Wolfram syndrome and that WFS1 has a protective function against ER stress. However, it remained to be determined how WFS1 mitigates ER stress. Here we have shown in rodent and human cell lines that WFS1 negatively regulates a key transcription factor involved in ER stress signaling, activating transcription factor 6alpha (ATF6alpha), through the ubiquitin-proteasome pathway. WFS1 suppressed expression of ATF6alpha target genes and repressed ATF6alpha-mediated activation of the ER stress response element (ERSE) promoter. Moreover, WFS1 stabilized the E3 ubiquitin ligase HRD1, brought ATF6alpha to the proteasome, and enhanced its ubiquitination and proteasome-mediated degradation, leading to suppression of ER stress signaling. Consistent with these data, beta cells from WFS1-deficient mice and lymphocytes from patients with Wolfram syndrome exhibited dysregulated ER stress signaling through upregulation of ATF6alpha and downregulation of HRD1. These results reveal a role for WFS1 in the negative regulation of ER stress signaling and in the pathogenesis of diseases involving chronic, unresolvable ER stress, such as pancreatic beta cell death in diabetes.

Effect of a comprehensive lifestyle modification program on the bone density of male heavy drinkers.

BACKGROUND: Heavy alcohol drinking is implicated in osteoporosis. Although abstinence is rapidly followed by a restoration of osteoblastic activity, little is known about the contributions of alcohol-related factors or the effectiveness of a lifestyle modification program (LMP) on bone density. METHODS: We conducted a study of 138 male alcoholic patients to investigate whether drinking history and concurrent factors were associated with the bone density of the calcaneus. A 2.5-months LMP in an institutionalized setting was completed by 20 of them, and its effect on bone density, serum parathyroid hormone (PTH), and 1.25-(OH)(2) vitamin D levels were assessed. RESULTS: The patients had a high prevalence of daytime drinking (93.5%), continuous drinking (84.1%), and current smoking (82.0%) with mean duration of alcohol abuse of 30.0 +/- 12.8 years. The patients had lower bone density than a reference control group (Z-scores: -0.45 +/- 1.02). Multiple stepwise regression analysis identified age, poor activities of daily living (ADL), continuous drinking, absence of liver cirrhosis, depression, and dementia as determinants of low bone density. The bone density of the 20 participants in the LMP improved 2.3% (p = 0.0003) with a more ameliorating effect on bone density than a conventional abstinence therapy (p = 0.014 for interventional effect). The upper normal range of PTH levels at baseline were significantly decreased, and 1.25-(OH)(2) vitamin D levels also had a trend toward decrease during the abstinence. CONCLUSIONS: Alcoholic patients may have many complications such as poor ADL and dementia, which are independently associated with decreased bone density. The results of this study support the idea that comprehensive approach to lifestyle factors to minimize risk of osteoporosis is the best way to improve bone density.

Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis.

Glypican-3 (GPC3) is one of carcinoembryonic antigens known to be overexpressed in hepatocellular carcinoma (HCC). It has been suggested that GPC3 may be related to the development of HCC in a background of chronic hepatitis (CH) and liver cirrhosis (LC). Therefore, in an attempt to establish an early diagnostic marker of HCC, we quantified the number of GPC3-specific CTLs in the peripheral blood of CH and LC patients. We selected CH and LC patients who were HCV-RNA (+) or HBs antigen (+) within 6 months prior to the study and had no HCC nodules as detected by imaging. A total of 56 patients with CH and LC, and 45 patients with HLA-A24+ or HLA-A2+ were enrolled for this investigation. After isolation of mononuclear cells from each patient's peripheral blood specimens, we performed ELISPOT assay using HLA-A24- and HLA-A2-restricted GPC3 peptides. In the ELISPOT assay, GPC3-specific CTLs were detected in 10 of the 45 CH and LC cases (22%). In addition, the plasma titers of anti-GPC3 IgG were increased in the CH and LC patients as compared with those in healthy donors. GPC3-specific CTLs were found to be present not only in patients with HCC, but also in patients with CH and LC. This suggests the possibility of GPC3-specific CTLs serving as a marker for the early diagnosis of imaging-invisible HCC.

Dendritic cell based personalized immunotherapy based on cancer antigen research.

Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8+ CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intratumoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.

Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis.

PURPOSE: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. EXPERIMENTAL DESIGN: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis-like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. RESULTS: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. CONCLUSION: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.