Multicenter prospective trial of total gastrectomy versus proximal gastrectomy for upper third cT1 gastric cancer.

BACKGROUND: The appropriate surgical procedure for patients with upper third early gastric cancer is controversial. We compared total gastrectomy (TG) with proximal gastrectomy (PG) in this patient population. METHODS: A multicenter, non-randomized trial was conducted, with patients treated with PG or TG. We compared short- and long-term outcomes between these procedures. RESULTS: Between 2009 and 2014, we enrolled 254 patients from 22 institutions; data from 252 were included in the analysis. These 252 patients were assigned to either the PG (n = 159) or TG (n = 93) group. Percentage of body weight loss (%BWL) at 1 year after surgery, i.e., the primary endpoint, in the PG group was significantly less than that of the TG group (- 12.8% versus - 16.9%; p = 0.0001). For short-term outcomes, operation time was significantly shorter for PG than TG (252 min versus 303 min; p < 0.0001), but there were no group-dependent differences in blood loss and postoperative complications. For long-term outcomes, incidence of reflux esophagitis in the PG group was significantly higher than that of the TG group (14.5% versus 5.4%; p = 0.02), while there were no differences in the incidence of anastomotic stenosis between the two (5.7% versus 5.4%; p = 0.92). Overall patient survival rates were similar between the two groups (3-year survival rates: 96% versus 92% in the PG and TG groups, respectively; p = 0.49). CONCLUSIONS: Patients who underwent PG were better able to control weight loss without worsening the prognosis, relative to those in the TG group. Optimization of a reconstruction method to reduce reflux in PG patients will be important.

Malignant neoplasm in kidney transplantation.

BACKGROUND: The kidney recipient is at a higher risk for cancer than is the general population, although the incidence of neoplasms in general is considered lower in Japan than in Western countries. The cause of this increased risk associated with either transplantation or geography has not yet been established. METHOD: The incidences and sites of malignant neoplasms were analyzed in 285 kidney recipients, who had been followed up for 3007 patient-years. The relationship between immunosuppressive states, the numbers of CD4-positive T lymphocytes, and the presence of malignant neoplasms was studied retrospectively. RESULTS: Eighteen malignant neoplasms were found in 17 of the 285 patients (6%). The malignancies developed in these patients an average of++ 1 26.5 months after transplantation. The incidence was only 3.9% at 10 years, increasing to 13.9% at 20 years. No difference in the time-course incidence was found between azathioprine-based and cyclosporin-based immunosuppressive regimens. The malignancies developed in the digestive organs in more than half of the patients, and were mainly in the liver, colon and rectum, and stomach, with a relatively low incidence of skin cancer and lymphoma. There was only one case of Epstein-Barr virus genome found in 5 specimens that were tested. Concerning the immunosuppressive state, CD4-positive T lymphocyte counts were not related directly with malignancies in our series. CONCLUSION: The cumulative incidence of malignancy increased markedly in the second posttransplant decade. The site of cancers in kidney recipients mirrors that of general Japanese malignancies. Our results revealed neither the cause nor predictor for malignancies in kidney transplant patients.

Increased urinary excretion of bilirubin metabolites in association with hyperbilirubinemia after esophagectomy.

This study was conducted to investigate the rationale for postoperative hyperbilirubinemia after major surgery. The serum bilirubin values and urinary excretion of bilirubin metabolites (BM) were monitored in 11 patients who underwent esophagectomy via right thoracotomy for esophageal cancer. Both the serum bilirubin values and the urinary excretion of BM increased postoperatively in all patients. The maximum serum bilirubin level in four patients with septic complications, two of whom developed pneumonia and two, anastomotic leakage (group A), was significantly higher at 5.25+/-4.16 mg/dl than in the remaining patients without septic complications (group B), at 2.11+/-0.07 mg/dl. The peak value of urinary BM was 99.5+/-88.2 micromol/day in group A and 23.5 +/-26.7 micromol/day in group B. The correlation between the level of serum bilirubin and urinary BM excretion was found to be significant. Thus, the metabolism of bilirubin was increased by extensive surgical stress and septic insult.

Involvement of interleukin-6 in activation of lysosomal cathepsin and atrophy of muscle fibers induced by intramuscular injection of turpentine oil in mice.

Serum IL-6 level increased after the injection of turpentine oil into the right gastrocnemius muscle in mice. The mRNA level of IL-6 was highest in the injected muscle at 12 h after injection, but was not identified in the opposite muscle. The activities of cathepsins B and B+L started to elevate after 12 h in the injected muscle and markedly increased after day 3. Likewise, the mRNA levels of cathepsins B and L markedly increased from day 1 to day 5 in the injected muscle. However, a very mild increase was also observed in the opposite muscle. Immunohistochemical staining of cathepsins B and L exhibited positive reactions as fine granules in myofibers at 12 h and strong positive reactions in the infiltrating macrophages at 3 days. Atrophy of myofibers type 1 and 2 was evident in a time-dependent manner in the injected muscle. Treatment with rat anti-mouse IL-6 receptor monoclonal antibody inhibited the increase in cathepsin activities in the injected muscle. We conclude that IL-6 produced in the inflamed muscle is involved in the process of muscle degeneration, especially through the activation of lysosomal cathepsins.

[A case report of Vp3 hepatocellular carcinoma responding to multidisciplinary treatment].

We report a 60-year-old man who had hepatocellular carcinoma with tumor thrombus in the main portal vein and left portal branch (VP3HCC). He was treated with transarterial chemo-embolization, surgical resection and intra-arterial infusion chemotherapy. He is now surviving without any sign of recurrence for 12 months after the initial therapy, even though the prognosis of VP3HCC is poor. This is a case in which the effect of multidisciplinary treatment was indicated.

Gene expression of albumin and liver-specific nuclear transcription factors in liver of protein-deprived rats.

Protein-energy malnutrition causes hypoalbuminemia. Recent work has suggested that this may be partly due to decreased transcription of the albumin gene. This study examined the role of cis-acting and transacting elements of the albumin gene during protein deprivation. Male 7-wk-old Donryu rats were fed a protein-free diet (0% casein diet) for 10 d or given restricted (pair-fed control) or free access (freely fed control) to a 25% casein diet. Serum albumin concentrations were significantly lower in the protein-deprived rats (29 +/- 1 g/L) than in the pair-fed controls (42 +/- 3 g/L) or the freely fed controls (45 +/- 3 g/L). The albumin mRNA level was also significantly lower in livers of protein-deprived rats (36% of pair-fed control). However, gel mobility shift analysis using liver nuclear extracts did not show any significant difference between the protein-deprived rats and the pair-fed controls in the binding activity to the B and D sites of the albumin promoter. Furthermore, gel mobility shift-Western blot analysis showed no significant difference between the two groups in the protein levels of nuclear transcription factors binding to the D sites. The amounts of mRNA of hepatocyte nuclear factor-1 binding to the B site were not significantly different between these two groups. These results suggest that the proximal promoter region may not play a major role in the down-regulation of the albumin gene during protein deprivation.

CYP1A1, CYP2E1 and GSTM1 polymorphisms are not associated with susceptibility to squamous-cell carcinoma of the esophagus.

We investigated the genetic polymorphisms of CYP1A1, CYP2E1 and GSTM1 in Japanese esophageal cancer patients (n = 53) with a histological diagnosis of squamous-cell carcinoma, to determine whether susceptibility to esophageal cancer is associated with these polymorphisms. There were no significant differences in the frequency distribution of any one of the 3 polymorphisms between esophageal cancer patients and 132 healthy Japanese controls. The genotype distributions in tobacco smokers or alcohol drinkers were also quite similar for male patients and male controls. The age at onset of esophageal cancer was also similar for patients with any genotype of the 3 polymorphisms. We conclude that the 3 polymorphisms are unlikely to be associated with esophageal cancer susceptibility.

Anti-interleukin-6 receptor antibody prevents muscle atrophy in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-dependent proteolytic pathways.

Progression of skeletal muscle atrophy is one of the characteristic features in cancer patients. Interleukin-6 (IL-6) has been reported to be responsible for the loss of lean body mass during cancer cachexia in colon-26 adenocarcinoma (C-26)-bearing mice. This study was carried out to elucidate the intracellular proteolytic pathways operating in skeletal muscle in C-26-bearing mice, and to examine the effect of anti IL-6 receptor antibody on muscle atrophy. On day 17 after tumor inoculation, the gastrocnemius muscle weight of C-26-bearing mice had significantly decreased to 69% of that of the pair-fed control mice. This weight loss occurred in association with increases in the mRNA levels of cathepsins B and L, poly-ubiquitin (Ub) and the subunits of proteasomes in the muscles. Furthermore, enzymatic activity of cathepsin B+L in the muscles also increased to 119% of the control. The administration of anti-murine IL-6 receptor antibody to C-26-bearing mice reduced the weight loss of the gastrocnemius muscles to 84% of that of the control mice, whose enzymatic activity of cathepsin B+L and mRNA levels of cathepsin L and poly-Ub were significantly suppressed compared with those of the C-26-bearing mice. Our data indicate that both the lysosomal cathepsin pathway and the ATP-dependent proteolytic pathway might be involved in the muscle atrophy of C-26-bearing mice. The results also suggest that anti IL-6 receptor antibody could be a potential therapeutic agent against muscle atrophy in cancer cachexia by inhibiting these proteolytic systems.

Temperature-dependent enhancement of proteolysis in C2C12 myotubes in association with the activation of 26S proteasome.

The effect of temperature on protein metabolism of C2C12 myotubes was investigated in order to estimate the potential effect of fever on muscle catabolism. The half-life of long-lived proteins in C2C12 myotubes was significantly (13%) shorter when incubated at 40 degrees C than at 37 degrees. The activities of cathepsins B and L were not significantly different at 37 and 40 degrees C, nor were the levels of the protein and mRNA of the two cathepsins. In contrast, the chymotrypsin-like activity of 26S proteasome was elevated by 53% at 40 degrees C, compared to that at 37 degrees C, although it was not associated with an increase in the levels of the protein and mRNA of proteasome subunits. mRNA levels of calpain and ubiquitin were not affected by temperature. It is concluded that temperature-dependent enhancement of proteolysis in C2C12 myotubes is associated with an increase in 26S proteasome activity.

A total parenteral nutrition solution supplemented with a nucleoside and nucleotide mixture sustains intestinal integrity, but does not stimulate intestinal function after massive bowel resection in rats.

The effects of supplementing a total parenteral nutrition solution with a nucleoside and nucleotide mixture on mucosal adaptive processes after massive bowel resection were studied. Male Wistar rats (n=30) underwent 80% small intestine resection, were randomized into two groups and received either standard total parenteral nutrition (TPN) or TPN supplemented with a nucleoside and nucleotide mixture (2.5 mL.kg-1.d-1). An additional five rats, fed a nonpurified diet and not resected, were used as controls. After 4 or 7 d, rats were killed and samples were collected for mucosal indices and intestinal enzymatic activities (disaccharidases and diamine oxidase). After massive small bowel resection and TPN, residual jejunal mucosal wet weights, villus heights, protein and RNA contents on d 4 and 7, and total wet weights and DNA contents on d 7 were significantly lower than in the control group. Administration of the nucleoside and nucleotide mixture resulted in significantly higher residual jejunal total and mucosal weights, proteins, DNA, RNA contents, and the ratio of proliferating cell nuclear antigen positive cells per crypt than did the standard TPN solution on d 7. However, disaccharidase and diamine oxidase activities were not affected by supplementation with the nucleoside and nucleotide mixture. Our data suggest the supplementation of a nucleoside and nucleotide mixture to a TPN solution can attenuate the initial mucosal atrophy and improve intestinal cell turnover after massive bowel resection, but the supplementation has little effect on enterocyte enzymatic activities.

Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice.

The muscles of IL-6 transgenic mice suffer from atrophy. Experiments were carried out on these transgenic mice to elucidate activation of proteolytic systems in the gastrocnemius muscles and blockage of this activation by treatment with the anti-mouse IL-6 receptor (mIL-6R) antibody. Muscle atrophy observed in 16-wk-old transgenic mice was completely blocked by treatment with the mIL-6R antibody. In association with muscle atrophy, enzymatic activities and mRNA levels of cathepsins (B and L) and mRNA levels of ubiquitins (poly- and mono-ubiquitins) increased, whereas the mRNA level of muscle-specific calpain (calpain 3) decreased. All these changes were completely eliminated by treatment with the mIL-6R antibody. This IL-6 receptor antibody could, therefore, be effective against muscle wasting in sepsis and cancer cachexia, where IL-6 plays an important role.

Modulation of thermogenic response to parenteral amino acid infusion in surgical stress.

Thermogenic response to parenteral infusion of amino acid mixtures in rats undergoing surgical stress was investigated with the aid of a small animal indirect calorimeter. Male Wistar rats (n = 12) were laparotomized and received intestinal abrasion along with construction of a parenteral route. After 48 h, nutrient-induced thermogenesis (NIT) produced by amino acid mixtures was examined and compared with that of control rats (n = 12) without surgical stress. The NIT values of the leucine-enriched solution were greater than those of the control solution in both rats with and without surgical stress. The NIT generated by the leucine-enriched solution in rats with surgical stress was significantly higher than that in rats without. NIT values for 10 different kinds of single amino acid solutions with identical concentrations was then examined in 60 rats. The NIT values of the leucine and the glycine solutions were relatively higher than those of other solutions. However, when puromycin was injected intraperitoneally into 12 rats just before measurement of the NIT in response to amino acid mixtures, no significant differences were found in NIT values between the mixtures. In summary, surgical stress increased the thermogenic response to the leucine-enriched solution, indicating that utilization of leucine may be augmented under surgical stress. In fact, leucine itself may be a thermogenic amino acid. Inhibition of protein synthesis prevented the increase in thermogenic response induced by the leucine-enriched solution. We conclude that thermogenic responses to parenteral amino acid mixtures may differ depending not only upon the compositions of amino acids but also upon the host conditions.

Role of interleukin-6 in skeletal muscle protein breakdown and cathepsin activity in vivo.

In order to elucidate the acute and chronic effects of interleukin-6 (IL-6) on muscle protein degradation, the weight of skeletal muscles and the activities of lysosomal cathepsins (B and L) in the muscles were examined in two animal models. Two intraperitoneal injections of recombinant human IL-6 into rats did neither significantly affect the cathepsin activities in the soleus and the extensor digitorum longus muscles nor the weight of these muscles. On the other hand, the gastrocnemius muscles of the IL-6 transgenic mice underwent severe atrophy accompanied by a marked increase in cathepsin activities. We conclude that IL-6 mediates muscle protein degradation with enhancing lysosomal cathepsin activity, and that these muscle reactions are mandated by chronic exposure to a high level of IL-6.

Interleukin-6 induces proteolysis by activating intracellular proteases (cathepsins B and L, proteasome) in C2C12 myotubes.

1. A cell culture system of C2C12 myotubes was established as a model of the muscle. With the aid of this model, the half-lives of intracellular proteins as well as the activities and mRNA levels of proteasomes (26S and 20S) and cathepsins (B, L, and H) were examined in the presence of various amounts of cytokines. 2. It was found that 100 units/ml recombinant human interleukin-6 somewhat shortened the half-life of long-lived proteins to 23.79 +/- 1.55 h (control: 25.60 +/- 1.87 h). When 1% fetal bovine serum contained in the culture medium was replaced by 0.5 mg/ml bovine serum albumin, interleukin-6 was more effective since 10 units/ml of interleukin-6 shortened the half-life to 19.09 +/- 2.87 h (control: 22.26 +/- 321 h). Interleukin-6 (100 units/ml) increased the activity of 26S proteasome by 31.5%, of cathepsin B by 53.5% and of cathepsin B+L by 21.3%. These increases occurred in association with an increase in their transcription. 3. On the other hand, 1000 units/ml of recombinant human tumour necrosis factor alpha prolonged the half-life of long-lived proteins while reducing the protease activities of 20S proteasome (-27.1%), cathepsins B (-64.6%) and B+L (-54.9%). 4. These results suggest that interleukin-6 induces degradation of long-lived intracellular proteins by activating both the non-lysosomal (proteasomes) and lysosomal (cathepsins) proteolytic pathways. It is therefore concluded that interleukin-6 is a candidate for a proteolysis-inducing factor in myotubes and may play an important role in the progression of muscle degradation in systemic inflammatory responses induced by sepsis or severe injury.

Changes of proteasomes and cathepsins activities and their expression during differentiation of C2C12 myoblasts.

C2C12 myoblasts fuse to form multinucleated myotubes and express muscle specific proteins during differentiation. To elucidate developmental regulation of intracellular proteolytic systems, enzymatic activities, protein and mRNA levels of proteasomes (20S and 26S) and lysosomal cathepsins (B, L, and H) were examined. Myoblasts were differentiated fully to myotubes 6 days after starting differentiation. In this developmental process, the 26S proteasome activity decreased, while the 20S proteasome activity increased. Expression of proteasome subunits of 20S (RC2, RC8) and regulatory components of 26S (S4, S7) was down-regulated, though total protein levels of proteasomes showed no remarkable changes. On the other hand, enzymatic activities of cathepsins B and B + L increased in association with an increase of their transcriptional and translational levels. Expression of their specific endogenous inhibitor, cystatin beta, also increased. Maturation of the lysosomal proteolytic system was tightly linked to the differentiation process. These results suggested that signals for differentiation of myoblasts mediate a change of intracellular proteolytic systems, involving up-regulation of lysosomal cathepsins and down-regulation of proteasomes.

Interrelation of intracellular proteases with total parenteral nutrition-induced gut mucosal atrophy and increase of mucosal macromolecular transmission in rats.

Total parenteral nutrition (TPN) is known to induce mucosal atrophy and to increase macromolecular transmission of the small intestine. The potential participation of various proteases in that process was investigated. Male Wistar rats were randomly divided into two groups: the TPN group (n = 11) received a standard TPN (250 kcal/kg per day, 1.78 g nitrogen/kg per day) and the FED group (n = 10) received a standard rat food for 1 week. This was followed by an examination of gut macromolecular transmission of fluorescein isothiocyanate dextran 70,000 (FITC-dextran) after intragastric injection and of the activities of gut mucosal cathepsins B, H, and L and of proteasome. Mucosal wet weight and protein content decreased significantly by TPN for 1 week. In both groups, the activities of all proteases in the ileum were significantly greater than in the jejunum. In the TPN group, cathepsin L and H activities in the ileum, and cathepsin B activity in both the jejunum and the ileum, were greater than those in the FED group. The portal concentration of FITC-dextran was higher than arterial and venous concentrations in the both groups. In the TPN group, the portal FITC-dextran concentration increased significantly compared with the FED group. In conclusion, active proteolysis is not associated with TPN-induced mucosal atrophy. Cathepsins activities in the ileum increase as a result of TPN. Interrelationship is implicated between increase of lysosomal protease activity and the deterioration of the intestinal barrier function, which permits macromolecular transmission.