Oxaliplatin-based adjuvant chemotherapy duration (3 versus 6 months) for high-risk stage II colon cancer: the randomized phase III ACHIEVE-2 trial.

BACKGROUND: Oxaliplatin-based adjuvant chemotherapy may be associated with debilitating peripheral sensory neuropathy (PSN) in patients with high-risk stage II colon cancer. This open-label, multicenter, randomized phase III trial was conducted as a prospective pooled analysis to investigate the non-inferiority of 3 versus 6 months of adjuvant oxaliplatin-based chemotherapy. PATIENTS AND METHODS: From 12 February 2014 to 31 January 2017, 525 Asian patients with high-risk stage II colon cancer were randomly assigned to 3- and 6-month treatment arms. The treatment consisted of either modified fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) or capecitabine combined with oxaliplatin (CAPOX). The primary end point was disease-free survival (DFS). The secondary end points were treatment compliance and safety. RESULTS: Of the 525 randomized patients, 11 were not treated. Among the 514 participating patients (255 in the 3-month arm; 259 in the 6-month arm), 432 (84%) received CAPOX, and 184 (36%) presented with T4 as a high-risk factor for recurrence. The 3-year DFS rate was 88.2% in the 3-month arm and 87.9% in the 6-month arm [hazard ratio (HR), 1.12; 95% confidence interval (CI), 0.67-1.87]. With CAPOX, the 3-year DFS rate was 88.2% in the 3-month arm and 88.4% in the 6-month arm (HR, 1.13; 95% CI, 0.65-1.96). The discontinuation rate in the 3- and 6-month arms was 10% and 31% for mFOLFOX6 (P = 0.0193), and 15% and 35% for CAPOX (P < 0.0001), respectively. The incidence of grade ≥2 PSN was significantly lower in the 3-month arm than in the 6-month arm (16% and 43%, respectively, P < 0.0001). CONCLUSIONS: Three months of combination therapy presented significantly less grade ≥2 PSN than the respective 6-month regimen. The shortened therapy duration did not affect the 3-year DFS rate, suggesting that a 3-month course of CAPOX can be an effective treatment option. CLINICAL TRIAL INFORMATION: UMIN Clinical Trials Registry, UMIN000013036 and Japan Registry of Clinical Trials, jRCTs031180128.

Relationship between vancomycin-associated nephrotoxicity and the number of combined nephrotoxic agents.

Vancomycin is associated with nephrotoxicity; however, the influence of the number of combined nephrotoxic agents on the incidence of vancomycin nephrotoxicity has not been clarified. We investigated patient backgrounds in 148 inpatients who received vancomycin treatment. The patients were divided into nephrotoxicity (n=35) and non-nephrotoxicity (n=113) groups. A comparison of the patient backgrounds in the two groups revealed significant differences in weight, changes in serum creatinine before vancomycin administration, blood urea nitrogen to serum creatinine ratio, length of vancomycin therapy, vancomycin trough concentration, and number of combined nephrotoxic agents. Multiple logistic regression analysis using these six factors as autonomous variables showed that the highest vancomycin trough concentration (odds ratio, 1.080; 95% confidence interval, 1.030-1.140; p = 0.003) and the number of combined nephrotoxic agents (odds ratio, 1.590; 95% confidence interval, 1.120-2.260; p = 0.010) were significantly related to nephrotoxicity.

Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1.

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP-CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

Laparoscopic en bloc excision of gastrointestinal stromal tumors of the rectum after neoadjuvant imatinib therapy: anteriorly extended intersphincteric resection combined with partial resection of the prostate.

We herein present a novel technique for laparoscopic en bloc excision involving anteriorly extended intersphincteric resection with partial resection of the posterior lobe of the prostate for large rectal gastrointestinal stromal tumors (GISTs). The sequence of neoadjuvant imatinib therapy and this less invasive surgery for marginally resectable rectal GISTs has the potential to obviate the need for urinary reconstruction and permanent stomas without jeopardizing the tumor margin status.

Hepatocyte growth factor gene therapy of liver cirrhosis in rats.

Liver cirrhosis is the irreversible end result of fibrous scarring and hepatocellular regeneration, characterized by diffuse disorganization of the normal hepatic structure of regenerative nodules and fibrotic tissue. It is associated with prominent morbidity and mortality, and is induced by many factors, including chronic hepatitis virus infections, alcohol drinking and drug abuse. Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, shows mitogenic, motogenic and morphogenic activities for a wide variety of cells. Moreover, HGF plays an essential part in the development and regeneration of the liver, and shows anti-apoptotic activity in hepatocytes. In a rat model of lethal liver cirrhosis produced by dimethylnitrosamine administrations, repeated transfections of the human HGF gene into skeletal muscles induced a high plasma level of human as well as enodogenous rat HGF, and tyrosine phosphorylation of the c-Met/HGF receptor. Transduction with the HGF gene also suppressed the increase of transforming growth factor-beta1 (TGF-beta1), which plays an essential part in the progression of liver cirrhosis, inhibited fibrogenesis and hepatocyte apoptosis, and produced the complete resolution of fibrosis in the cirrhotic liver, thereby improving the survival rate of rats with this severe illness. Thus, HGF gene therapy may be potentially useful for the treatment of patients with liver cirrhosis, which is otherwise fatal and untreatable by conventional therapy.

Integrin alphavbeta3 regulates microfibril assembly in human periodontal ligament cells.

Fibrillin-1 is the major structural component of extracellular microfibrils. However, the mechanism by which extracellular fibrillin-1 assembles into microfibrils is not fully understood. Fibrillin-1 contains the Arg-Gly-Asp (RGD) motif, which may allow binding to RGD-recognizing integrins. We hypothesized that integrin alphavbeta3 on the cell surface of human periodontal ligament (PDL) fibroblasts may influence fibrillin-1 assembly into cell/matrix layers. We treated PDL fibroblasts with an integrin alphavbeta3-specific antagonist to examine fibrillin-1 assembly. Western blotting and immunofluorescence analysis showed that treatment with the integrin alphavbeta3 antagonist at 5 muM clearly abolished fibrillin-1 deposition. These results provide for the first time evidence that integrin alphavbeta3 regulates extracellular assembly of fibrillin-1, thereby modulating cell-mediated homeostasis of microfibrils.

Erythrocyte migration and gap formation in rabbit blood clots in vitro.

Thrombolytic agents must be carried by the blood circulation to thrombi to exert their functions. Structural gaps exist between blood vessels and thrombi or in the area surrounding thrombi. Therefore, information about fundamental gap formation at thrombotic areas is critically important for thrombolytic therapy. We previously reported that t-PA accelerates the activities of bovine erythrocytes and hemoglobin (Hb) towards bovine plasminogen activation. Here, we examined gap generation by observing morphological changes during thrombolytic processes in rabbit blood clots deformation of erythrocytes from blood clots and Hb transfer from erythrocytes to serum in vitro. Rabbit venous blood samples (1 ml) were stored under sterile conditions in glass tubes at 37 degrees C for 2, 24, 48 h, 1, and 2 weeks. We examined clot diameter, erythrocyte diameter and number as well as Hb volume in the serum, as well as histological changes in the clots. The diameter of blood clots did not change until 2 weeks after sampling. Erythrocyte diameter decreased within 48 h and at 2 weeks after sampling at the clot surface (p < 0.001) and interior (p < 0.001). The number of erythrocytes in the serum started to increase starting from 24 h after sampling (p < 0.01). Serum Hb volume also gradually increased from 24 h until 2 weeks after sampling (p < 0.01). The erythrocyte envelope became disrupted and cytoplasm started to flow through pores into the serum at 24 h. The results indicated that blood clots are reduced due to clot retraction, erythrocyte dissociation and cytoplasm leakage without a distinct fibrinolytic reaction. These results indicated that gaps start to form between 2 and 24 h after blood clotting.

Hepatocyte growth factor ameliorates acute graft-versus-host disease and promotes hematopoietic function.

Acute graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation (BMT) and is characterized by hematopoietic dysfunction, immunosuppression, and tissue injury in the skin, liver, and intestinal mucosa. Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, induces mitogenic and antiapoptotic activity in various epithelial cells and promotes hematopoiesis. Working in a murine model of acute GVHD, we performed repeated transfection of the human HGF cDNA into skeletal muscle and showed that this treatment inhibited apoptosis of intestinal epithelial cells and donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by acute GVHD. HGF also markedly suppressed IFN-gamma and TNF-alpha expression in the intestine and liver and decreased the serum IL-12. Furthermore, extramedullary hematopoiesis by donor cells was increased, and the survival rate was improved. These results suggest that HGF may be useful for controlling acute GVHD after allogeneic BMT.

Clinical characteristics and outcome of human herpesvirus-6 encephalitis after allogeneic hematopoietic stem cell transplantation.

In this retrospective analysis using the Transplant Registry Unified Management Program, we identified 145 patients with human herpesvirus (HHV)-6 encephalitis among 6593 recipients. The cumulative incidences of HHV-6 encephalitis at 100 days after transplantation in all patients, recipients of bone marrow or PBSCs and recipients of cord blood were 2.3%, 1.6% and 5.0%, respectively. Risk factors identified in multivariate analysis were male sex, type of transplanted cells (relative risk in cord blood transplantation, 11.09, P<0.001; relative risk in transplantation from HLA-mismatched unrelated donor, 9.48, P<0.001; vs transplantation from HLA-matched related donor) and GvHD prophylaxis by calcineurin inhibitor alone. At 100 days after transplantation, the overall survival rate was 58.3% and 80.5% among patients with and without HHV-6 encephalitis, respectively (P<0.001). Neuropsychological sequelae remained in 57% of 121 evaluated patients. With both foscarnet and ganciclovir, full-dose therapy (foscarnet ⩾180 mg/kg, ganciclovir ⩾10 mg/kg) was associated with better response rate (foscarnet, 93% vs 74%, P=0.044; ganciclovir, 84% vs 58%, P=0.047). HHV-6 encephalitis is not rare not only in cord blood transplant recipients but also in recipients of HLA-mismatched unrelated donors. In this study, development of HHV-6 encephalitis was associated with a poor survival rate, and neurological sequelae remained in many patients.

Expression of Golf in the rat placenta: Possible implication in olfactory receptor transduction.

Olfactory receptors are G-protein coupled receptors and are encoded by an extremely large and diverse family of genes in mammals. There is increasing evidence that olfactory receptors are widely distributed in many organs, suggesting that olfactory receptors do not only recognize airborne odorants but also play important roles in chemotaxis or organ construction in embryo. In this study, we investigated whether olfactory receptors and their transduction molecule, Golf are expressed in the rat placenta. By RT-PCR, we identified 11 different olfactory receptor genes, which are all members of class II, in the rat placenta cDNAs, and our results suggested that particular members of the olfactory receptor gene family might be preferentially expressed in the placenta. By western blot analysis, we demonstrated that Golf protein is expressed in the placenta and its expression levels are developmentally regulated. We found that Golf immunoreactivity is exclusively localized to giant cell trophoblasts and spongiotrophoblast cells. These findings raised a possibility that a particular subset of olfactory receptors might be coupled with Golf and function in giant cell trophoblasts and spongiotrophoblast cells.

Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Presence of human papillomavirus type 16 genome in bladder carcinoma in situ of a patient with mild immunodeficiency.

Using blot hybridization, we analyzed 10 bladder tumors (1 transitional cell carcinoma in situ, 1 adenocarcinoma, and 8 papillary tumors) for the presence of human papillomavirus (HPV) DNA. We detected HPV 16 DNA in a transitional cell carcinoma in situ, whereas no HPV DNA was found in the other bladder tumors. The patient, a 40-year-old female, who harbored HPV 16 DNA in the bladder tumor, had mild immunodeficiency and recently suffered from the bladder tumor, common warts on the right hand, Bowen's disease of the vulva, and severe dysplasia of the vaginal wall. From each of these lesions, we detected the DNA of HPV 16 or an unclassified HPV. HPV DNAs existed in nonintegrated form in all lesions examined. To our knowledge, this is the first case in which a bladder tumor was shown to harbor HPV DNA. However, HPV does not seem to be regularly present in bladder tumor, because we could not detect HPV DNA from the most common bladder tumor, i.e., papillary tumor. Our demonstration of HPV 16 DNA in a transitional cell carcinoma in situ of the bladder suggests that HPV may be associated with some of the bladder tumors of this type.

Hypermethylation of multiple genes in pancreatic adenocarcinoma.

Hypermethylation of CpG islands is a common mechanism by which tumor suppressor genes are inactivated. We studied 45 pancreatic carcinomas and 14 normal pancreata for aberrant DNA methylation of CpG islands of multiple genes and clones using methylation-specific PCR (MSP) and bisulfite-modified sequencing. Using MSP, we detected aberrant methylation of at least one locus in 60% of carcinomas. The genes analyzed included RARbeta (methylated in 20%), p16 (18%), CACNA1G (16%), TIMP-3 (11%), E-cad (7%), THBS1 (7%), hMLH1 (4%), DAP kinase (2%), and MGMT (0%). In addition, aberrant methylation was found in three CpG islands (MINT31, -1, and -2) in 38, 38, and 14% of carcinomas, respectively. Hypermethylation was largely confined to the carcinomas with only three loci (E-cad, DAP kinase, and MINT2) harboring methylation in some normal pancreata (36, 21, and 14%, respectively). Simultaneous methylation of at least four loci was observed in 5 of 36 (14%) pancreatic adenocarcinomas. We defined this subgroup of pancreatic adenocarcinomas as "CpG island-methylator-phenotype positive (CIMP+)." Two of four carcinomas with microsatellite instability harbored promoter hypermethylation of hMLH1, and both cases were CIMP+. Thus, we conclude that many pancreatic carcinomas hypermethylate a small percentage of genes, whereas a subset displays a CIMP+ phenotype.

Identification and characterization of differentially methylated CpG islands in pancreatic carcinoma.

To identify CpG islands differentially methylated in pancreatic adenocarcinoma, we used methylated CpG island amplification (MCA) coupled with representational difference analysis. Of 42 CpG islands identified by MCA/representational difference analysis, 7 CpG islands [methylated in carcinoma of the pancreas (MICP)] were differentially methylated in a panel of eight pancreatic cancer cell lines compared with normal pancreas. In a larger panel of 75 pancreatic adenocarcinomas, these 7 MICPs (ppENK, Cyclin G, ZBP, MICP25, 27, 36, and 38) were methylated in 93, 3, 9, 15, 48, 19, and 41% of cancers, respectively, by methylation-specific PCR but not in any of 15 normal pancreata. In pancreatic cancer cell lines, methylation of ppENK, a gene with known growth suppressive properties, was associated with transcriptional silencing that was reversible with 5-aza-2'-deoxycytidine treatment. Relationships between the methylation patterns of pancreatic adenocarcinomas and their clinicopathological features were also determined. Larger pancreatic cancers and those from older patients (P = 0.017) harbored more methylated loci than smaller tumors and those from younger patients (P = 0.017). ppENK, MICP25, and 27 were variably methylated in normal gastric, duodenal, and colonic mucosae. These data indicate that aberrant methylation of ppENK and its transcriptional repression is a common event in pancreatic carcinogenesis.

Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

Dispersion state of phospholipids and fluorescence production with peroxidation in organic solvents: investigated by time-resolved fluorescence technique.

Fluorescent substances were found to be produced efficiently when phospholipids containing phosphatidylethanolamine (PE) and linoleic chains were autoxidized in non-polar solvents. By using these fluorescent substances as intrinsic probes, the dispersion state of phospholipids was investigated in various organic solvents. Fluorescence anisotropy decay measurements indicated that the aggregation size of phospholipids was much larger in hexane than in chloroform, methanol and tert-butyl alcohol. The average diameter of phospholipid aggregates in hexane was calculated to be 4-6 nm, which was dependent on the lipid composition. A consistent result was obtained when N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-gly cer o-3- phosphoethanolamine (NBD-PE) was used as an extrinsic probe. Comparison of the fluorescence data with small-angle X-ray scattering (SAXS) data suggested that a reverse micellar structure of phospholipids formed in hexane. It was shown that phospholipid aggregation enhanced the extent of peroxidation as well as the production yield of fluorescent substances of phospholipid.

Exclusive expression of transketolase in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea.

Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes.

Relationship between growth temperature of Anacystis nidulans and phase transition temperature of its thylakoid membranes.

The temperatures of the lipid phase transition at which the solid phase disappears were determined by using the X-ray diffraction method in thylakoid membranes of the blue-green alga, Anacystis nidulans. The temperatures were determined as 26 and 16 degrees C for cells grown at 38 and 28 degrees C, respectively.

Structural characterization of the molten globule and native states of apomyoglobin by solution X-ray scattering.

Compactness and shape are two of the critical properties that describe the degree of protein folding. Solution X-ray scattering is an effective technique for measuring these properties quantitatively. Structural characteristics of various conformational states of horse myoglobin were studied in terms of size and shape by solution X-ray scattering. The radius of gyration for native holomyoglobin was 17.5 A, while that of the apomyoglobin native state was 19.7 A. Corresponding to the increase in the radius of gyration, the largest dimension of the molecule also increased from 47.5 A to 62.5 A. Both states are globular in shape. The scattering profiles in the high angle region suggest that the apomyoglobin native state has a distinct tertiary structure, and that packing of alpha-helices in the apomyoglobin native state would be looser than that of holomyoglobin. These observations indicate that the native state of apomyoglobin is expanded from that of holomyoglobin, and that the conformations of the two are not identical. The radii of gyration for the acid-unfolded state and the denaturant-unfolded state were 30 A and 35 A, respectively. Both unfolded states have chain-like conformations without any tertiary structures. The radius of gyration and the largest dimension of the molten globule stabilized by trichloroacetate were 23.1 A and 72.5 A, respectively. The molten globule is expanded from the native state although it is globular, and is much more compact than the unfolded state. The bimodal distance distribution function and scattering profile at high-angle region suggest that the structure of the apomyoglobin molten globule contains a core comprising a cluster of multiple alpha-helices and flaring tail(s), which would be a common structural property of the compact denatured state appearing during the folding process. The compactness of each conformational state is highly correlated with the extent of formation of the alpha-helix.