Reappraisal of classification of distal cholangiocarcinoma based on tumour depth.

BACKGROUND: In the eighth edition of the AJCC cancer staging classification, the T system for distal cholangiocarcinoma (DCC) has been revised from a layer-based to a depth-based approach. The aim of this study was to propose an optimal T classification using a measured depth in resectable DCC. METHODS: Patients who underwent pancreatoduodenectomy for DCC at 32 hospitals between 2001 and 2010 were included. The distance between the level of the naive bile duct and the deepest cancer cells was measured as depth of invasion (DOI). Invasive cancer foci were measured as invasive tumour thickness (ITT). Log rank χ2 scores were used to determine the cut-off points, and concordance index (C-index) to assess the survival discrimination of each T system. RESULTS: Among 404 patients, DOI was measurable in 182 (45·0 per cent) and ITT was measurable in all patients, with median values of 2·3 and 5·6 mm respectively. ITT showed a positive correlation with DOI (rS = 0·854, P < 0·001), and the cut-off points for prognosis were 1, 5 and 10 mm. Median survival time was shorter with increased ITT: 12·4 years for ITT below 1 mm, 5·2 years for ITT at least 1 mm but less than 5 mm, 3·0 years for ITT at least 5 mm but less than 10 mm, and 1·5 years for ITT 10 mm or more (P < 0·001). This classification exhibited more favourable prognostic discrimination than the T systems of the seventh and eighth editions of the AJCC (C-index 0·646, 0·622 and 0·624 respectively). CONCLUSION: ITT is an accurate approach for depth assessment in DCC. The four-tier ITT classification with cut-off points of 1, 5 and 10 mm seems to be a better T system than those in the seventh and eighth editions of the AJCC classification.

Pancytopenia in tuberous sclerosis.

A 46-year-old woman with edema and pancytopenia was referred for further evaluation. She was diagnosed as tuberous sclerosis with clinical manifestations such as facial adenoma sebaceous, ungual and periungual fibroma, subependymal nodules and renal angiomyolipoma. Her edema seemed due to hypercardiac function induced by massive anemia. X-ray revealed extraordinary thickening of the cortex of long bones of the extremities as well as patchy osteosclerotic findings in vertebra, suggesting that hematopoietic space was significantly reduced. Pancytopenia improved after splenectomy. Histological examination revealed several intrasplenic hemangiomas but its relationship to hypersplenism was not clear. It seemed that her massive pancytopenia was induced by a combination of hypersplenism and significant reduction in hematopoetic space. In tuberous sclerosis, various systemic complications sometimes induce severe hematological abnormalities. According to previous literatures, the present case of tuberous sclerosis manifested the most outstanding hematological complications.

[A case of internal hernia through a defect in the falciform ligament treated with laparoscopic surgery].

A 22-year-old male was admitted to our hospital with abrupt onset of upper abdominal pain. Abdominal US and CT revealed dilatation of the small intestine between the abdominal wall and a lateral segment of the liver. After a diagnosis of an internal hernia through a defect in the falciform ligament, emergency surgery was performed. Laparoscopic investigation showed incarceration of the small intestine in a defect of the falciform ligament. After releasing an incarceration, the hernia orifice was opened to prevent relapse. He was discharged on the 4th postoperative day. Internal hernia through a defect in the falciform ligament is extremely rare, with six reported cases including our own in Japan. Characteristic images of abdominal US and CT enable preoperative diagnosis of this condition. Surgery should be performed at an early stage after onset. In patients with no prior history of surgery, laparoscopic techniques may be useful.

Characterization of insulin-dependent diabetes mellitus induced by a new variant (DK-27) of encephalomyocarditis virus in DBA/2 mice.

A murine diabetes mellitus induced with a new diabetogenic variant (DK-27) which we isolated from the M variant of the encephalomyocarditis (EMC) virus was characterized. Male DBA/2 mice (9.5 weeks old) were infected with various infectious doses of DK-27 intraperitoneally. Blood glucose and insulin levels were examined in association with the viral replication. Pancreatic pathology and hormone contents and stable hemoglobin A1c (St-A1c) levels were also examined on the final day of observation (35 days of post-infection). In infected mice, blood glucose levels rapidly elevated at 72 hr, slightly decreased between 7 and 10 days and finally became sustained hyperglycemia. On the other hand, blood insulin levels elevated at 48 hr, promptly decreased, and subsequently became sustained hypoinsulinemia. Viral replication in pancreases reached the highest titers at 48 hr and rapidly disappeared with all infectious doses used. Pancreatic insulin contents in infected mice were not detectable, and glucagon contents were not affected. In pathological examination, atrophy of islets and marked diminution of B-cells were observed, and A-cells occupied the major part of an infected islet. St-A1c levels reflected lasting hyperglycemia. These findings show that DK-27 causes insulin-dependent diabetes mellitus by the specific and direct destruction of pancreatic B-cells in susceptible mice. Such a diabetic model mouse will be useful for therapeutic studies.

Acceleration of diabetes in young NOD mice with peritoneal macrophages.

To elucidate the roles of macrophages in the pathogenesis of NOD murine diabetes, peritoneal macrophages from NOD mice were injected into young NOD mice. We used 12 to 20 week-old NOD mice of both sexes as donors, and sex-matched 2-week-old NOD mice as recipients. Cyclophosphamide (CY), 200 mg/kg, was intraperitoneally injected into the donors. Two weeks later, peritoneal exudate cells (PEC) were collected from the diabetic donors. Macrophage-rich fractions (MRF) were collected by adherence. Then PEC(5-8 x 10(6)) or MRF(3-7 x 10(6)) were transferred, intraperitoneally, to the recipients. Two weeks later, some of the recipients were killed in order to perform immunofluorescent analysis of splenocytes and to assess pancreatic histology. Mac 1 positive splenocytes were increased in PEC- and in MRF-injected recipient mice. Insulitis was seen in PEC- and MRF-injected mice, but not in controls. Some of the recipients were injected with CY, 200 mg/kg, intraperitoneally, at two weeks post cell transfer. Two weeks after CY injection, the animals were examined for the presence of diabetes. The incidences of diabetes were 67% in PEC-injected mice, 40% in the MRF-injected group, and 3% in the controls. These results suggest that peritoneal macrophages accelerate the disease process in NOD mice.

Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection.

It has been reported that lactate dehydrogenase virus (LDV) selectively infects a subpopulation of macrophages, thereby affecting the immune system. We studied the effects of LDV infection on the development of diabetes in non-obese diabetic (NOD) mice. Five-week-old female NOD mice were infected with LDV (10(8) ID50/mouse) and observed until 23 weeks of age. None of the 21-LDV-infected mice developed diabetes, whereas 10/14 (71.4%) uninfected mice did. Although the subpopulations of T cells and the percentage of Mac1-positive cells in the NOD murine spleen and the number of harvested peritoneal macrophages were unaffected by LDV infection, the proportions of Ia-positive peritoneal macrophages were significantly decreased in LDV-infected compared with uninfected mice (1.1 +/- 0.2%, 6.5 +/- 2.9%; P < 0.01). In LDV-infected NOD mice, insulitis of the same grade as that seen in uninfected NOD mice was observed. In another experiment, 3, 5, 10 or 16-week-old female NOD mice were infected with LDV. None of the mice infected with LDV at 3, 5 or 10 weeks of age developed diabetes and only one of six infected at 16 weeks of age did. These findings indicate that LDV infection suppresses the development of diabetes in female NOD mice by reducing the capacity of Ia-positive macrophages, and suggest that the development of human type 1 diabetes may be suppressed by certain viral infections.

The suppressive effect of cyclophosphamide on low-dose streptozotocin-induced diabetes in mice.

We found that low-dose streptozotocin (SZ)-induced diabetes in mice is suppressed by cyclophosphamide (CY) and studied the mechanisms of this suppression. Male CD-1 mice were injected intraperitoneally with SZ (40 mg/kg) for five consecutive days (group SZ). In addition, CY (200 mg/kg) was injected on the first day (group CY-1) or on days 1 and 5 (group CY-2). In group SZ, all mice developed diabetes within nine days after the first injection of SZ. Group CY-1 did not develop hyperglycemia for 14 days and group CY-2 did not develop it throughout the experimental period (40 days). Insulitis was suppressed slightly in group CY-1 and completely in group CY-2. The number of spleen cells did not change in group SZ compared with that before cyclophosphamide treatment. They decreased significantly on days 3 and 7 in both groups CY-1 and CY-2 (p less than 0.01 or p less than 0.05), but then increased significantly on days 14 and/or 21 (p less than 0.01 or p less than 0.05) compared with those before cyclophosphamide treatment. The Thy 1.2- and L3T4-positive cells increased significantly in group SZ but they decreased on days 3 and 7 in both groups CY-1 and CY-2 (p less than 0.01) compared with the controls. These results suggest that (1) this large reduction of lymphocytes at the beginning of SZ treatment may lead to the prevention of hyperglycemia and insulitis, and (2) T-cells, especially L3T4-positive cells may play an important role in the pathogenesis of this type of diabetes.

Insulin autoantibodies in mouse models of insulin-dependent diabetes.

To clarify the significance of insulin autoantibodies (IAA) in insulin-dependent diabetes mellitus, we measured the IAA longitudinally in non-obese diabetic (NOD) mice, and in high-dose streptozotocin-induced diabetes (high-SZ) and EMC virus-induced diabetes (EMC) in mice, and compared the data with the occurrence of insulitis. The IAA were detected by the polyethylene glycol (PEG) method using 125I-(Tyr A14) human insulin. The IAA were found in 38% of NOD mice and correlated with the occurrence of insulitis. The prevalence of IAA was 0% before the appearance of insulitis, 80% at 12-14 weeks of age and 30% after 20 weeks of age in female NOD mice. In male NOD mice, IAA were found in 45% at 12-14 weeks of age and 20% after 20 weeks. In high-SZ mice, IAA were detected in several mice while insulitis was not present. In EMC-virus induced diabetic mice, IAA and lymphocytic infiltration into the islets were detected 4-14 days after EMC virus infection. These results suggest that (a) IAA are markers for islet autoimmunity in NOD mice, (b) the presence of IAA does not always reflect insulitis, (c) the presence of IAA is not sufficient for the development of overt diabetes and (d) the appearance of IAA may reflect a difference of the immune response genotype.

Insulin autoantibodies in non-obese diabetic (NOD) mice and streptozotocin-induced diabetic mice.

Similar to Insulin-dependent diabetes mellitus (IDDM) in man, diabetes in the non-obese diabetic (NOD) mouse and that induced by low-dose of multiple injections of streptozotocin (low-dose SZ) develop in conjunction with the presence of insulitis. We measured insulin autoantibodies (IAA) in NOD and low-dose SZ mice and compared the levels with mice given a single diabetogenic dose of streptozotocin (high-dose SZ) as well as control CD-1 mice. The mean insulin binding in female NOD mice was 3.08 +/- 1.49 (mean + SD)% and that in male NOD mice was 2.86 +/- 3.70%, as compared with 1.10 +/- 0.35% in the control CD-1 mice (p less than 0.01, p less than 0.05). Sera from low-dose SZ and high-dose SZ mice showed 1.07 +/- 0.23% and 0.93 +/- 0.45% of IAA which did not differ from controls. The number of mice with IAA above the mean + 2SD value of CD-1 mice were 8/9 female NOD mice and 9/20 male NOD mice. Insulitis was found in all NOD and low-dose SZ mice but not in any high-dose SZ mice and control CD-1 mice. These results suggest that (a) IAA are markers for islet autoimmunity in the NOD mouse, (b) presence of IAA does not reflect insulitis, and (c) the appearance of IAA may reflect a difference of the immune response genotype.