Kunihiko Koyama

Effect of food-restriction stress on immune response in mice

Daily 23-h food deprivation for 1-5 days induced gastric ulcers and atrophic changes of the spleen and thymus, accompanied by a rise in plasma cortisol and catecholamine levels in mice. It also modulated several immune cell functions in the spleen including a drop in the B cell population but no change in the mitogen response of the B cells, an increase in T cell population but no change in the L3T4/Lyt2 ratio and an early increase in natural killer activity and O2- production by macrophages. These effects are thought to correlate to the increase in stress-associated humoral factors and this may partly result from stress induced by food restriction.

Increased Superoxide Anion Production and Glutathione Peroxidase Activity in Peritoneal Macrophages from Autoimmune-Prone MRL/Mp-lpr/lpr Mice

We studied the release of superoxide anion (O-2) in peritoneal macrophages from autoimmuneprone MRL/Mp-lpr/lpr (MRL/l) mice. Compared to resident peritoneal macrophages from control MRL/Mp-+/+ (MRL/n) mice, macrophages from MRL/l mice exhibited an age-related increase of spontaneous and PMA-induced O-2 secretion in association with the development of the autoimmune process. Analysis of the kinetic parameters of NADPH oxidase in macrophages revealed that MRL/l macrophages were in a primed state, as shown by the decreased Km value of the oxidase for NADPH. Furthermore, we studied several key enzymes for their ability to scavenge the oxygen radicals in the macrophages. Among the enzymes studied, only glutathione peroxidase (GSH Px) activity was increased in peritoneal macrophages from MRL/l mice and this change was closely correlated with the increase in O-2 production. Thus, GSH Px activity in macrophages seems to play an important role in macrophage functions under increased oxidative stress.

Expression of the cell surface antigen detected by the monoclonal antibody A7 in pancreatic carcinoma cell lines.

In a previous study, we used a murine monoclonal antibody, A7, against human colon carcinoma as a drug-carrier to treat colorectal cancer.1 In the present study, we found that MAb A7 also reacted immunohistochemically with 73% of human pancreatic carcinoma cell lines, with the A7 antigen mainly being detected on the cell surface. However, the A7 antigen was found in only 9% of the spent media of these human pancreatic carcinoma cell lines by ELISA. On the other hand, the positive incidence of CA19-9, POA, ferritin, CEA, DU-PAN-2 and SLX in those spent media was 100%, 64%, 64%, 55%, 55% and 36%, respectively. These results suggest that the A7 antigen may only rarely be shed into the sera of pancreatic cancer patients, in which case MAb A7 could be a suitable drug-carrier in targeting chemotherapy for pancreatic cancer patients.

The Role of Oxygen Radicals in the Pathogenesis of Gastric Mucosal Lesions Induced in Mice by Feeding-Restriction Stress

To investigate the role of oxygen radicals in the pathogenesis of gastric mucosal lesions induced by psychological stress, we exposed 3-mo-old C3H mice to feeding-restriction stress for 1 to 5 days. Serial changes in the activities of antioxidant enzymes in the gastric mucosa, together with O2- production by macrophages, were measured. The stress increased the plasma cortisol level and started to produce acute gastric lesions (AGL) on the 2nd day. Before the development of AGL, the activity of superoxide dismutase in the gastric mucosa had already decreased and the ability of O2- production by macrophages was enhanced from the 1st day. This suggests that oxygen radicals play some role in the development of AGL induced by the stress.

Characterization of Variant γ-Glutamyl Transpeptidase Produced by Pancreatocholangiocarcinoma Cell Lines in a Protein-Free, Chemically Defined Medium

Four pancreatocholangiocarcinoma cell lines (HPC-Y1, HPC-YT, MIA PaCa-2, and HChol-Y1) were established to propagate in a protein-free, chemically defined medium, High γ-glutamyl transpeptidase (GGTP) activities were showed in their spent media (designated as the secreted GGTP). Their GGTP activities in the spent media were 125, 85, 110, and 153 IU/L/mg of lyophilized spent media, whereas GGTP activities extracted from their cancer cell lines with bromelain were 105, 37, 86, and 112 IU/L/l × l06 cells, respectively. The chemical characteristics of the GGTPs in the spent media from these cell lines resembled one of the GGTPs, sialic acid-rich GGTP, extracted from normal human pancreas with bromelain treatment as follows: the GGTPs secreted from the cancer cell lines bound to an anion exchange column moved fast on electrophoresis and then showed decreased electrophoretic mobility with neuraminidase treatment, showed a high affinity for concanavalin A and lentil lectin columns, and had an acidic isoelectric point. However, the elution patterns of erythroagglutinating phytohemagglutinin (E-PHA) column chromatography and thermostability tests demonstrated clear differences between the carcinoma GGTPs both in the spent media and cell lines and the sialic acid-rich GGTP of normal pancreas, namely the carcinoma GGTPs treated with neuraminidase showed affinity to E-PHA columns, and, in addition, the GGTPs in the spent media showed an apparent heat resistance at 56°C. These findings indicate that the carcinoma GGTPs have a different oligosaccharide structure from that in normal pancreatic GGTPs.

Studies on age-related functional changes in regulatory T cells and B cells involved in the autoantibody production of MRL/MpJ − + / + mice

Age-related changes in anti-DNA autoantibody production of MRL/MpJ- +/+ mice were investigated. In lipopolysaccharide (LPS)-stimulated cultures, spleen cells of the mice showed an age-related, marked increase in the ability to produce IgG class of the autoantibody after the age of 12 months, while they showed a tendency to decrease with age in the production of IgM class of the autoantibody. Serum levels of anti-DNA autoantibodies rose markedly in the IgG autoantibody but not in the IgM autoantibody after 12 months of age, which is well consistent with the observation in the LPS-stimulated cultures. T cell-depleted spleen cells, however, showed only a small increase with age in the IgG autoantibody productive ability. These results suggest that the age-associated increase in the IgG autoantibody production in the mice is under T-cell control. Age-associated changes in suppressor capacity in spleen cells of the mice were also investigated. Suppressive activity of the cells stimulated by 2-day incubation with concanavalin A (Con A) showed a clear increase as the donor age advanced, when assayed on the LPS-stimulated anti-DNA autoantibody production in vitro. The results indicate that, in MRL/MpJ-+/+ mice, suppressor capacity does not decline with age and is not related as a cause to the autoantibody production.

Effect of aging on immunological memory in gastrointestinal tract induced by sheep red blood cells in mice

SummaryTolerance induction by oral administration of sheep red blood cells (SRBC) was investigated in young and aged mice. Two-month old C3H/He mice receiving oral administration of 8xl09 SRBC a day for two weeks became tolerant to a subsequent SRBC challenge in the systemic immune system. In contrast, older C3H/He mice aged about 1 year old receiving the same treatment did not become tolerant but resisted tolerance induction and produced a prominent IgG memory in the systemic immune system. Autoimmune-prone NZB mice showed a similar resistance to tolerance induction even at 3 months of age. To investigate a possible role of the liver in the oral tolerance induction, young (2-month old) C3H/He mice received an injection of lxl08 SRBC via portal vein. The mice thus treated, were not tolerized at all but immunized.The results suggest that gut-associated local immune system play a key role in the induction of the oral tolerance and that the tolerance inducing function of the local immune system declines with aging. Antigens in the gut including denatured self antigens may immunize the systemic immune system of aged animals.

Effect of aging on induction of oral tolerance by intragastric administration of sheep red blood cells in mice

Fed SRBC induced oral tolerance in young C3H mice but not in the aged. An injection of SRBC via portal vein did not tolerize C3H mice. Gut associated local immune system seems to play a key role in induction of oral tolerance and the tolerance inducing function declines with aging. Mucosal immune system plays important roles in preventing invasion by viruses, bacteria, and other parasites. Oral injection of live virus generates local and systemic immunity (1). However, fed antigen often induces oral tolerance (2). Systemic immune system in mice shows age-associated resistance to tolerance induction by tolerogen via parenteral route (3). Here, we studied oral tolerance induction by SRBC in the systemic immune system of young and aged mice.