Cyclosporine overcomes cold preservation/reperfusion injury of liver graft: Chemokine release and liver ultrastructure

There is a growing body of evidence that the cytokine, tumor necrosis factor-α (TNF-ga), plays an important role in the development of hepatic ischemia/reperfusion injury. We found that the immunosuppressants, cyclosporine-A (CsA), azathioprine, and FK506, have protective effects on such injury. The purpose of the present study was to elucidate mechanisms involved in these beneficial effects of the immunosuppressant, CsA, on liver injury following cold preservation and transplantation, with special reference to the suppression of TNF-α release. Rat livers were stored in Euro-Collins solution (EC) at 4°C for 6h and orthotopically transplanted. The animals allotted to two groups: group A (untreated controls) and group B (CsA pretreatment of recipients). CsA (10 mg/kg, p.o.) was given for 3 consecutive days preoperatively. CsA pretreatment of the recipients significantly improved the 2-week survival rate (0/6 for group A, 3/6 for group B;P<0.05) and this was associated with a significant decrease in serum TNF-α levels 2h posttransplantation (group A, 69.8±15.7 pg/ml; group B, 22.8±6.8; mean±SEM;n=12 each;P<0.05) and amelioration of sinusoidal endothelial injury, assessed by electron microscopy. Plasma endotoxin levels following reperfusion of the grafts were not altered by the CsA therapy. Morphologically, CsA pretreatment of the recipients did not alter activation of Kupffer cells. CsA pretreatment of the recipient aids in preventing cold preservation/reperfusion injury of the liver graft, possibly by modulating effects of TNF-α.     

Synergistic effect of 1,25-dihydroxyvitamin D3 and retinoic acid in inducing U937 cell differentiation.

We examined the effects of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), and its synthetic analogue, 22-oxa-1,25-(OH)2D3, on differentiation of U937 cells by studying the cellular growth, surface marker expression and cytosolic free Ca2+ concentration ([Ca2+]i). RA inhibited cellular growth but did not induce expression of Mo2 (CD14), a monocyte/macrophage specific surface marker. To the contrary, 1,25-(OH)2D3 did not inhibit cellular growth, but increased CD 14-positive cells. Simultaneous addition of 1,25-(OH)2D3 and RA had no additive effect on cellular growth inhibition or CD14 expression. With regard to [Ca2+]i, however, 5 days' incubation with either of them increased the basal [Ca2+]i level and induced U937 cells to respond to formyl-methionyl-leucyl-phenylalanine (FMLP). When the cells were incubated with both 10(-6) M RA and 10(-8) M 1,25-(OH)2D3, basal [Ca2+]i was higher and FMLP caused a greater increase in [Ca2+]i than when only RA or 1,25-(OH)2D3 was added. These data suggest that RA and 1,25-(OH)2D3 induce monocytoid differentiation in U937 cells through different pathways and act synergistically in the differentiation process. The 22-oxa-1,25-(OH)2D3 induced CD14 expression, basal [Ca2+]i increase and [Ca2+]i response to FMLP, but did not cause cellular growth inhibition in U937 cells, and in these points, 22-oxa-1,25-(OH)2D3 exhibited no significantly different effects from 1,25-(OH)2D3. Thus, 22-oxa-1,25-(OH)2D3 has the same potent activity as 1,25-(OH)2D3 in inducing differentiation of U937 cells.     

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.     

Active oxygen species generated by monocytes and polymorphonuclear cells in Crohn's disease

Chemiluminescence (CL) analysis of monocytes and polymorphonuclear cells (PMNs) was performed on 13 patients with Crohn's disease (CD) and 10 healthy volunteers. The percentages of monocyte populations in mononuclear cells obtained from the patients with CD were greater than those from the healthy volunteers, but the numbers of PMNs were not different between the two groups. The peak level of phorbol myristate acetate (PMA)-induced CL activity generated by diluted whole blood from the patients with CD was more significantly elevated than that from the healthy volunteers, whereas the peak levels of opsonized zymosan-induced CL activity did not differ between the two groups. In monocytes, the peak levels of both PMA- and opsonized zymosan-induced CL activity were significantly higher in the patients with CD than in the healthy volunteers. CL in PMNs, however, showed no significant difference between CD and controls. It is suggested that monocytes of CD have a large capacity to generate active oxygen species. The present study suggests that excessive active oxygen species released by monocytes and perhaps macrophages may play an important role in formation of the intestinal lesions in CD.This work was supported by the Grant of Tokuteishitsukan from the Japanese Ministry of Welfare and Health.     

In vitro reactivity and in vivo biodistribution of the monoclonal antibody A7 using human gastric carcinoma cell lines.

The monoclonal antibody (MAb) A7 has been used to treat patients with colorectal or pancreatic carcinoma with encouraging results. We therefore determined if MAb A7 would also react with gastric carcinoma cell lines. MAb A7 reacted with seven of eight gastric carcinoma cell lines tested. The intensity of the reaction, measured by flow cytometry, was equal to that of WiDr (colon) and HPC-YS (pancreas) cell lines. In nude mice bearing xenografts of the MAb A7-reactive gastric cancer line MKN45, the percentage injected dose of MAb A7 per g of tumour tissue on day 7 was 9.79; this value was 77% of that on day 1. The in vivo tumour-to-blood ratio of MAb A7 was 2.77 on day 7. Therefore, MAb A7 has long-term retention at binding sites as well as a high probability, high intensity and high specificity of reactivity against gastric cancer, which make it an ideal drug carrier for immunotargeted chemotherapy and immunodiagnosis.     

Clinical influence of vagotomy on postoperative acute phase response

BACKGROUND: Although there is increasing evidence suggesting that the vagus nerve functions as a connector between the nervous and immune systems in animals, little is known about the role of the vagus nerve in postoperative acute phase response in humans. MATERIALS AND METHODS: The extent of fever and acute phase protein response and the production of inflammatory cytokine during the early postoperative period were compared among the patients who had undergone total gastrectomy including truncal vagotomy (n = 13), those having distal gastrectomy with division of vagal branches (n = 14), and the patients with vagal nerve preserving gastrectomy (n = 12). RESULTS: There was no significant difference in serum levels of C-reactive protein, alpha-1-antirypsin, and interleukin-6 among the three groups. Also, postoperative maximum body temperature was similar. CONCLUSIONS: Vagotomy did not influence acute phase response after gastric cancer surgery. A multipathway mechanism for acute phase response including the induction of fever is suggested.     

Discrete breakpoint mapping and shortest region of overlap of chromosome arm 1q gain and 1p loss in human hepatocellular carcinoma detected by semiquantitative microsatellite analysis

Recurrent chromosomal gain at 1q is one of the most common features of human hepatocellular carcinoma (HCC), but how the gain at 1q contributes to hepatocarcinogenesis is still unclear. To identify the target genes, precise determination of the shortest region of overlap (SRO) and of breakpoints is necessary. Similarly, the role of loss at 1p, which is also a major cytogenetic aberration in HCC, needs to be determined. Fifty HCCs were examined with the aid of 59 microsatellite markers distributed throughout both arms of chromosome 1. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Alleles showing imbalance were subjected to multiplex PCR, using a retained allele as an internal control, to determine whether the imbalance was the result of chromosomal gain or loss. The SRO of the gains was defined as D1S2878-D1S2619 (1q23.-q25.3, 16.9 Mb), which involved 36 cases (72%). Gains in the number of copies of certain oncogenes within this region seemed to be critical for the pathogenesis of HCC. In contrast, the centromeric breakpoints of these gains varied, but they tended to occur mainly in the pericentromeric region (26 of 50 cases, 52%). Rearrangement of specific genes associated with the gains is unlikely. On the other hand, the SRO of deletion was defined as D1S2893-D1S450 (1p36.32-p36.22, 5.1 Mb). Four known putative tumor-suppressor genes (TP73, RIZ1, NBL1/DAN, and CDKN2C) were outside the SRO, suggesting the presence of other candidate genes with critical roles in hepatocarcinogenesis.     

Overexpression of p53, c-erbB-2 and epidermal growth factor receptor in human breast carcinomas

Overexpression of p53 protein, epidermal growth factor receptor (EGF-R), and c-erbB-2 protein was assessed by immunohistochemical staining of formalin-fixed, paraffin-embedded tissue from 64 invasive breast tumors. The correlation between abnormal expression of each protein and various disease parameters, including lymph node metastasis and histopathologic type and grade was analyzed. Despite the previous proposal, no significant correlation was found between lymph node metastases and overexpression of each gene in the primary tumors. In addition, some metastatic lesions did not always exhibit overexpression, even if it was evident in the primary tumors. Overexpression of c-erbB-2 protein correlated well with Bloom's histological grading. p53 expression was detected most often in tumors with hyperchromatism and more frequent mitosis. Overexpression of c-erbB-2 protein occurred more frequently in p53-positive tumors. The results indicate that abnormal expression of p53 protein causes genetic instability in the early stage of tumor development, resulting in subsequent overexpression of other oncogenes.