Diversity of amino acid signaling pathways on autophagy regulation: a novel pathway for arginine.

Autophagy is the intracellular bulk degradation process to eliminate damaged cellular machinery and to recycle building blocks, and is crucial for cell survival and cell death. Amino acids modulate autophagy in response to nutrient starvation and oxidative stress. We investigated the relevance of reactive oxygen species (ROS) production on the regulation of autophagy using amino acids, both as a mixture and individually, in rat hepatoma H4-II-E cells. Nutrient starvation elevated ROS production and stimulated autophagy. Treatment with complete (CAA), regulatory (RegAA) and non-regulatory (NonRegAA) amino acid mixtures showed significant suppression of ROS production, whereas only CAA and RegAA exhibited significant suppression of autophagy, suggesting a dissociation of the two responses. The effects of individual amino acids were examined. Leucine from RegAA decreased ROS production and suppressed autophagy. However, methionine and proline from RegAA and arginine, cystine and glutamic acid from NonRegAA suppressed autophagy with an opposite increase in ROS production. Other amino acids from the NonRegAA group showed stimulating effects on ROS production without an autophagic response. Arginine's effect on autophagy suppression was not blocked by rapamycin, indicating an mTOR-independent pathway. Inhibitor studies on arginine-regulated autophagy may indicate the involvement of NO pathway, which is independent from ROS and mTOR pathways.

Shortened blood coagulation times in genetically obese rats and diet-induced obese mice.

The aim of this study was to investigate blood coagulation times in genetically obese rats and diet-induced obese (DIO) mice in order to clarify the relationship between visceral obesity and blood coagulation. WBN/Kob-Lepr(fa) (fa/fa) rats, a genetically obese model, exhibited a significantly shorter activated partial thromboplastin time (aPTT) and prothrombin time (PT) than age-matched Wistar rats. C57BL/6J mice fed a high-fat diet (60%), a DIO model, exhibited significantly shorter aPTT, PT and thrombin time than lean mice fed a standard diet. Higher body weight, visceral fat weight and insulin resistance were also shared by fa/fa rats and DIO mice. These results suggest that visceral obesity is related to accelerated blood coagulation in addition to disrupted metabolism of glucose and lipids.

Correlation of age with distribution of periodontitis-related bacteria in Japanese dogs.

We analyzed the distribution of 11 periodontitis-related bacterial species in dental plaque collected from 176 Japanese dogs divided into young (less than 2 years of age), middle-aged (2-7 years of age) and elderly (more than 8 years of age) groups using a polymerase chain reaction method. Clinical examination revealed that no dogs in the young group were affected by periodontitis, whereas the rates for gingivitis and periodontitis were high in the middle-aged and elderly groups. In addition, the total numbers of bacterial species in the middle-aged and elderly groups were significantly greater than in the young group. Our findings suggest that age is an important factor associated with the distribution of periodontitis-related bacteria and periodontal conditions in dogs.

Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype.

Porphyromonas gulae is a gram-negative black-pigmented anaerobe which is known to be a pathogen for periodontitis in dogs. Approximately 41kDa filamentous appendages on the cell surface (FimA) encoded by the fimA gene are regarded as important factors associated with periodontitis. The fimA genotype was classified into two major types and strains in type B were shown to be more virulent than those in type A. In the present study, we characterized a strain with a novel fimA genotype and designated it as type C. The putative amino acid sequence was shown to be similar to the genotype IV fimA of Porphyromonas gingivalis, a major pathogen of human periodontitis. Analyses using an oral squamous cell carcinoma cell line derived from tongue primary lesions revealed that the type C strain inhibited proliferation and scratch closure more than genotype A and B strains. In addition, experiments using a mouse abscess model demonstrated that the type C strain could induce much higher systemic inflammation when compared with strains of the other genotypes. Furthermore, molecular analyses of oral swab specimens collected from dogs demonstrated that the detection frequencies of P. gulae and the genotype C in the periodontitis group were significantly higher than those in the periodontally healthy group. These results suggest that FimA of P. gulae is diverse with the virulence of genotype C strains the highest and that molecular identification of genotype C P. gulae could be a possible useful marker for identifying dogs at high risk of developing periodontitis.

Diversity of fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs.

Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.

Comparison of the blood coagulation profiles of ferrets and rats.

The aim of this study was to examine the blood coagulation profiles of ferrets and compare them with those of rats. The ferret activated partial thromboplastin time (aPTT) was slightly longer than the rat aPTT. In contrast, the ferret prothrombin time and thrombin time were profoundly shorter than the corresponding rat values. The fibrinogen level in ferret plasma was 2 times higher than that in rats. Heparin prolonged all blood coagulation times in a concentration-dependent manner in both ferret and rat plasma. A significantly (P<0.01) higher concentration of heparin was required to double the aPTT in ferrets than rats. These blood coagulation data for ferrets will be useful in experimental animal studies.

Duodenogastric reflux after choledochoduodenostomy: evaluation by technetium-99m scintigraphy.

BACKGROUND/AIMS: Persistence of dyspeptic symptoms after choledochoduodenostomy (CDD) is common. There is evidence that at least some of these symptoms may be attributed to duodenogastric reflux (DGR). The aim of the study was to quantify DGR after CDD. METHODOLOGY: A total of 6 patients who had undergone cholecystectomy with a standard side-to-end CDD for choledocholithiasis or Lemmel syndrome were studied by symptom evaluation, biliary scintigraphy and endoscopy at least 6 months after surgery. Duodenogastric reflux was quantified using continuous intravenous infusion of 99mTc-HIDA. RESULTS: The incidence of DGR after CDD was 67% compared to healthy control. In the majority of the patients the DGR was mild to moderate, but not with the clinical symptoms. CONCLUSIONS: 99mTc-HIDA scanning of the hepatobiliary system is a reasonable and reliable method for the quantitative evaluation of DGR. CDD is associated with a high incidence of DGR, but its occurrence does not produce significant clinical symptoms.

Role of sialyl Lewis X in liver metastasis in view of liver-associated immunity.

BACKGROUND/AIMS: As sialyl Lewis X is a lignad of the selectin family, it has been proposed that sialyl Lewis X-rich colon cancer cells metastasize to the liver by adhesion to selectins on hepatic endothelial cells. However, little is known about the interaction between sialyl Lewis X and hepatic immune cells. We evaluated the role of sialyl Lewis X in liver metastasis in view of liver-associated immunity. METHODOLOGY: RCN-9, a colonic cancer cell line derived from Fischer rats, and its subclone RCN-H4, which exhibited high metastatic potential to the liver, were used. In an attempt to investigate the underlying basis for the difference in hepatic metastasis formation, we assessed the susceptibility of both cell lines to lysis by hepatic sinusoidal lymphocytes in 51Cr-release assays, and the expression of a number of carbohydrate antigens by both cell lines by flow cytometry. RESULTS: Hepatic sinusoidal lymphocytes mainly consist of natural cytotoxic lymphocytes, including NKT cells. The H4 colonic cancer subclone showed decreased susceptibility to lysis by hepatic sinusoidal lymphocytes, as compared to the parent cell line. In addition, a significant increase of sialyl Lewis X expression was noted in the H4 subclone. Neuraminidase treatment of H4 cells increased their susceptibility to hepatic sinusoidal lymphocyte-mediated killing. Furthermore, rats inoculated with neuraminidase-treated H4 cells produced fewer metastatic nodules in the liver than those inoculated with untreated H4 cells. CONCLUSIONS: Sialyl Lewis X expression in tumor cells reduced their susceptibility to hepatic sinusoidal lymphocyte-mediated killing, and thus, may facilitate the ability of the tumor cells to metastasize to the liver.

A series of immune responses leading to the induction of T cell IL-12/IL-18 responsiveness in patients with relatively large tumor burdens.

The induction of interleukin-12 (IL-12) responsiveness in T cells depends on T cell receptor (TCR) triggering, and is regarded as a parameter of recently TCR-sensitized T cells. Here, we investigated whether IL-12 responsiveness could be detected in freshly prepared T cells from tumor-bearing patients, and if so whether such patients exhibited additional immunological parameters related to IL-12 responsiveness. CD4(+) and CD8(+) T cell populations from an appreciable proportion of tumor-bearing patients exhibited high levels of IL-12 responsiveness as evaluated by IL-12-stimulated interferon-gamma (IFN-gamma) production. T cell populations with high IL-12 responsiveness were observed in the group of patients with moderate to large tumor mass or tumor metastases rather than in patients with small tumors. The frequency of such a T cell population was also lower in post-surgery tumor-free patients, showing the correlation between IL-12 responsiveness and the presence of a certain extent of tumor burden. More importantly, a higher incidence of IL-12 responsiveness was observed in tumor-bearing patients exhibiting detectable plasma IL-12 levels, and correlated with IL-18 responsiveness. T cell IL-12 and IL-18 responsiveness is induced by TCR triggering and subsequent IL-12 stimulation respectively. Furthermore, TCR-triggered T cells stimulate antigen-presenting cells (APC) to produce IL-12. Therefore, the present observations suggest that an immune response loop from TCR sensitization to the induction of IL-12/IL-18 responsiveness via IL-12 production operates in tumor-bearing patients, particularly in those with relatively large tumor burdens.

Suppression of T-cell function in gastric cancer patients after total gastrectomy with splenectomy: implications of splenic autotransplantation.

Background. In Japan since the 1960s, patients with gastric cancer have routinely had splenectomy combined with gastrectomy to ensure that lymph node dissection is complete. However, the influence of splenectomy on long-term immunity is unclear.METHODS: Forty-nine gastric cancer patients who underwent total gastrectomy for cure with ( n = 25) and without ( n = 24) splenectomy were assessed for immunologic function, including the proportion of lymphocyte subsets, purified protein derivative from tuberculin (PPD) response, natural killer (NK) activity, and phytohemagglutinin (PHA) response.RESULTS: Peripheral T-cell mediated functions, e.g., PPD and PHA response, were significantly suppressed in patients who underwent gastrectomy with splenectomy compared with those who had gastrectomy alone. Decreased T-cell subsets (CD 3+, 4+, 8+) and increased NK cell subsets (CD 16+, 57+) were observed in patients who underwent splenectomy. Patients who did not undergo splenectomy had immunologic responses within the normal range.CONCLUSIONS: Splenectomy decreased T-cell mediated responses over the long term. As a potential means to co-rrect this T-cell dysfunction in patients with splenectomy, splenic autotransplantation should be considered in future research.