Altered expression of various receptors on T cells in young and old mice after mitogenic stimulation: a flow cytometric analysis.

To understand mechanism underlying the age-related impairment of T cell functions, changes in the expression of cell surface receptors were examined in T cells after mitogenic stimulation by flow cytometry and results were compared between young and old mice. Before stimulation, no significant difference was observed in the density of TCR alpha beta, CD3, CD122 (IL-2R beta), IL-2R gamma, CD28 and CD95 (Fas) of T cells between young and old mice. As for CD25(IL-2R alpha) and CTLA-4, relative intensity and percentage of positive cells were higher in old than in young mice, although the levels were low compared with those after stimulation. After mitogenic stimulation, increased expression of the density was observed in CD25, IL-2R beta, IL-2R gamma, CD28, CTLA-4 and CD95 and the magnitude of increase was more pronounced in T cells from young than those from old mice. The expression of TCR alpha beta and CD3 decreased after mitogenic stimulation, and the degree of expression quickly recovered to the initial level in young mice, but not in old mice. Lower expression of TCR, IL-2 receptors and co-stimulatory molecules in T cells from old mice could be responsible for the impaired proliferation after mitogenic stimulation.

Effect of restraint stress on immune system and experimental B16 melanoma metastasis in aged mice.

An overnight restraint stress was given to young and old mice and its effect was examined in terms of the number and function of T cells and natural killer (NK) cells in spleen and patterns of lung metastasis of B16 melanoma cells. A great decrease was observed in the number and proliferative activity of splenic T cells in old mice after the stress. The decrease in young mice was rather temporary with a quick recovery. The number of NK cells in spleen was not different between young and old mice before giving the stress, but a significant decrease was observed in the old after the stress. NK activity was always much lower in old than in young throughout the experiment. The pattern of metastasis of B16 melanoma cells was different between young and old mice. Metastatic colonies in lungs were larger in number and bigger in size in young mice than in old mice. After the stress, the number increased and the size unchanged in old mice, while the size increased and the number remained unchanged in young mice. It was shown that the same restraint stress resulted in a more serious influence on the immune cells in old than in young mice and gave rise to a differential effect on the pattern of tumor metastasis between young and old mice.

Impairment of signal transduction in T cells from old mice.

T cells from old mice showed impaired proliferative response to antigenic stimulation. To understand the mechanism underlying the age-related impairment of T cell functions, the signal transduction pathway was examined and compared between T cells from young and old mice, and between T cell clones established from a young and old mouse. The age-related changes in T cells were as follows: (1) reduction in the expression and the activation of protein tyrosine kinases associated with T cell receptor (TCR) after antigenic stimulation; (2) reduced phosphorylation of phospholipase C gamma 1 (PLC gamma 1); (3) reduced production of second messengers such as inositoltrisphosphate (IP3) and diacylglycerol (DAG); and (4) reduced influx of Ca2+ ion. Thus, a T cell clone established from an old mouse showed impaired proliferation by stimulation with anti-CD3 antibody, but was fully activated to the level of a T cell clone from a young mouse by stimulation with phorbol acetate myristate (PMA) plus ionomycin (INM). However, splenic T cells freshly prepared from old mice did not show full recovery by the same treatment. The results indicate that one major blockade in the signal transduction of T cells from old mice is present in the pathway just after TCR, but besides this, the blockade is also present in multiple sites down-stream, which can not be bypassed by stimulation with PMA plus INM.

Vitamin E enhances the immune functions of young but not old mice under restraint stress.

Young and old C57BL/6 male mice were given a diet containing a high dose of vitamin E (VE treatment) and its effect on the immune system was examined before and after the exposure to restraint stress. The VE treatment per se gave rise to a slight increase of splenic T cells in percentage and a significant enhancement of Con A response of spleen cells in young, but not in old mice. The VE treatment also resulted in the enhancement of production of IL-2 and IFNgamma in young, but not in old mice. Restraint stress led to thymic involution in both young and old mice. This thymic involution was not ameliorated by the VE treatment. Percentage of splenic T cells and their mitogenic response decreased just after the stress, but soon rebounded over the control level. The VE treatment further enhanced the recovery after the stress in young mice, but on the contrary suppressed the recovery in old mice. The results in the present study suggested that the VE treatment was effective in the prevention of immunological decline of young mice before and after the exposure to the stress. On the other hand, such a preventive effect was not observed in old mice that were already in the depressed state of immunological functions.

Age-related alteration of cytokine production profile by T cell subsets in mice: a flow cytometric study.

Spleen cells from young and old C57BL/6 mice were stimulated with a combination of anti-CD3 and anti-CD28 antibodies, and the profile of cytokine production was examined by two different methods; the concentrations of cytokines as measured by ELISA, and identification of cytokine-positive cells by flow cytometry. The ELISA method revealed that IL-2 production by spleen cells after stimulation was significantly lower in the old mice compared to the young mice. while IFN-gamma production was the reverse. The flow cytometric analysis showed that the percentage of IL-2 positive cells in spleen cells after the stimulation was significantly lower in the older mice than in the young mice, and vice versa for the percentage of IFN-gamma-positive cells. Regarding the T cell subsets, CD4+ T cells were a major source of IL-2 in both the young and old mice. IL-2-positive cells in both CD4+ and CD8+ T cells showed a significant decrease with age. On the contrary, CDX T cells were the major source of IFN-gamma. An age-related increase of IFN-gamma positive cells was observed in both CD4+ and CD8+ T cells. CD4 T cells were the major source of IL-4, and the percentage of IL-4-positive CD4+ T cells also increased with age, although the level of IL-4 production was modest in C57BL/6 mice compared with IL-2 and IFN-gamma. Such age-related changes of cytokine production are presumed to play an important role in the alteration of immunological capacity with age.

DNA methylation status of hMLH1, p16(INK4a), and CDH1 is not associated with mRNA expression levels of DNA methyltransferase and DNA demethylase in gastric carcinomas.

DNA methyltransferase and DNA demethylase are enzymes potentially affecting promoter methylation status. We examined levels of DNA methyltransferase (DNMT1, DNMT3a, DNMT3b) and DNA demethylase (MBD2) mRNA expression by semi-quantitative RT-PCR. In addition, we examined promoter methylation status of hMLH1, p16(INK4a), and CDH1 by methylation-specific PCR since all three of these genes are reported to be hypermethylated in gastric carcinoma. MBD2 appeared to be down-regulated in neoplasms. The levels of DNMT1, DNMT3a, DNMT3b, and MBD2 mRNA expression were not associated with either tumor stage or histologic type. Promoter hypermethylation of hMLH1, p16(INK4a), and CDH1 was detected in 5/20 (25%), 8/20 (40%) and 8/20 (40%) of gastric carcinomas, respectively. There was no clear relation between DNA methylation status of hMLH1, p16(INK4a), and CDH1 and the mRNA expression levels of DNMT1, DNMT3a, DNMT3b or MBD2. We divided the examined cases into two groups according to the number of hypermethylated genes. Cases with more than two hypermethylated genes comprised a hypermethylation group, and cases with no hypermethylation comprised a non-hypermethylation group. We found no group association for levels of DNMT1, DNMT3a, DNMT3b, and MBD2 mRNA expression. Our results suggest that the mRNA expression levels for pro-methylating (DNMT1, DNMT3a, DNMT3b) and anti-methylating (MBD2) enzymes is not a critical determinate of tumor-specific promoter hypermethylation of hMLH1, p(16INK4a), or CDH1 in gastric carcinoma.

Successful surgical resection of solid cystic tumor of the pancreas with multiple liver metastases and a tumor thrombus in the portal vein.

We report a case of solid cystic tumor of the pancreas with widespread liver metastases and a tumor thrombus in the portal vein. The patient was a 43-year-old woman. She was referred because of an upper abdominal mass and weight loss. Computed tomography disclosed a 10-cm cystic and calcified mass in the body and tail of the pancreas and multiple masses in the liver. She underwent a distal pancreatectomy with splenectomy, extended right lobectomy, and partial resection of the liver. All the tumors were completely resected despite the presence of 20 liver metastases. Histopathological studies showed a tumor thrombus in the intrahepatic portal vein. The patient is well without any signs of recurrence 8 months after the operation. Aggressive surgical resection is considered to yield a good outcome for solid cystic tumor with liver metastases and tumor thrombus of the portal vein.

Expression of Bub1 gene correlates with tumor proliferating activity in human gastric carcinomas.

Bub1 plays an important role at the spindle assembly checkpoint to prevent cell cycle progression following spindle damage. We examined the expression of human Bub1 mRNA in 20 gastric carcinoma tissues and corresponding nonneoplastic mucosas by reverse transcriptase-polymerase chain reaction and analyzed the relation with proliferative activity monitored by the expression of proliferating cell nuclear antigen (PCNA) on Western blotting as well as Ki-67 labeling index by immunohistochemistry. Increased expression of Bub1 mRNA was detected in 8 (40%) of the gastric carcinomas in comparison with their nonneoplastic counterparts, while 4 (20%) expressed Bub1 at lower levels. The expression of Bub1 mRNA was confirmed by in situ hybridization. The expression levels of Bub1 mRNA were well correlated with the levels of PCNA protein in 16 (80%) gastric carcinoma cases. The examination of Ki-67 labeling indices proved the close correlation between the expression levels of Bub1 and proliferating activity. These findings suggest that mRNA expression of human Bub1 gene is closely associated with the tumor-proliferating activity. Since genetic alterations of human Bub1 rarely occur in gastrointestinal cancers, the functional machinery of Bub1 to prevent cell cycle progression into anaphase might be well preserved in gastric carcinomas even with high proliferative activity.