PIVKA-II correlates with INR but not protein C or protein S concentrations in cord blood among newborns.

BACKGROUND: Protein induced by vitamin K absence (PIVKA)-II, inactive precursor of prothrombin, is elevated in vitamin K (VK) deficiency. Our aims were to find the prevalence of VK deficiency in neonates, assess the utility of international normalized ratio (INR) as a screening tool, and explore the relationship between PIVKA-II, activated partial thromboplastin time (aPTT) and VK dependent anticoagulants. METHODS: INR, aPTT, PIVKA-II, and proteins C and S activities were measured in neonatal cord blood prior to VK administration. RESULTS: We found 45% of neonates had subclinical VK deficiency based on PIVKA-II levels and 7% based on INR. Receiver operating characteristic (ROC) analysis assessed the utility of INR in detecting >4 ng/mL of PIVKA-II and ROC of the area under the curve was 0.70 (95% CI 0.46-0.92, p = 0.07). Proteins C and S activities were normal for age and did not correlate with PIVKA-II [(r = 0.40, p = 0.14) and (r = 0.29, p = 0.29), respectively]. There was no association between aPTT and PIVKA-II (p = 0.83). CONCLUSION: PIVKA-II seems to be a sensitive indicator of mild VK deficiency. Further studies are needed to investigate the lack of relationship between PIVKA-II and functional protein C or S levels.

Pancreatic islet function in nondiabetic and diabetic BB rats.

A decreased acute insulin response to glucose in islet cell antibody positive humans predicts diabetes. Because the dominant mechanism leading to decreased in vivo acute insulin response to glucose remains unclear, perifused islets were examined before and after diabetes onset in BB rats to assess the role of glucose sensitivity on insulin secretion in individual islets. Islets from normal WF rats, diabetes-prone rats without inflamed islets, diabetes-prone rats with inflamed islets, and diabetic rats were studied at 2.0, 8.3, and 16.7 mM glucose. Immunoreactive insulin from WF islets at 16.7 mM glucose was 0.15 +/- 0.02 ng.0-7 min-1 x islet-1 for the first phase and 1.00 +/- 0.05 ng.7-20 min-1 x islet-1 for the second phase of biphasic secretion, compared with basal secretion of 0.10 +/- 0.03 ng.20 min-1 x islet-1 at 2 mM glucose. Diabetes-prone noninflamed islets showed a 0.20 +/- 0.03 ng first-phase secretion, a 1.32 +/- 0.13 ng second-phase secretion after 16.7 mM glucose, and 0.093 +/- 0.02 ng.20 min-1 x islet-1 at 2 mM glucose, indicating no intrinsic BB rat strain secretion abnormality. Diabetes-prone inflamed islets had secretions of 0.35 +/- 0.02 ng during the first phase (P < 0.05 vs. WF) and 1.78 +/- 0.29 ng during the second phase (P < 0.05 vs. WF) after 16.7 mM glucose, with 0.24 +/- 0.08 ng.20 min-1 x islet-1 at 2 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage-mediated islet cell cytotoxicity in BB rats.

Islet cell killing mediated by natural killer cells and T-lymphocytes in diabetes-prone (DP) and diabetic BB rats has been described, but other killing mechanisms may also be involved. Histopathologic studies suggest that macrophages are the first immune cells to infiltrate islets. To determine if macrophages are the first cells mediating islet damage, macrophage-mediated cytotoxicity was evaluated in BB rats of different ages. Splenic macrophages isolated from DP rats at 33, 100, 120, and 140 days of age showed no enhanced islet killing compared with diabetes-resistant rats. Killing at diabetes onset (121 +/- 14 days) was markedly increased (43 +/- 9.3%) compared with age-matched diabetes-resistant controls (19 +/- 8.3%, P less than .001). Islet inflammation was monitored at all time points. At 120 and 140 days of age, 9 of 11 (82%) DP rats had insulitis, and cytotoxicity was increased in 6 of 11 (55%) rats, which is similar to the number of DP rats that progress to diabetes. At 100 days, 3 of 6 (50%) DP rats again showed diabetic levels of killing, even in the absence of insulitis. These data indicate that 1) islet inflammation is dissociated from clinical diabetes onset, 2) splenic macrophages may have islet-killing potential before islet inflammation, 3) macrophage-mediated islet killing is elevated in all animals immediately after diabetes onset, and 4) macrophages, in addition to natural killer cells and T-lymphocytes, are responsible for cell-mediated islet destruction and thus are candidates for the first cellular effector to result in islet killing.

In situ islet T cell receptor variable region gene usage in the nonobese diabetic mouse.

Several features of the genetics and immunopathology of diabetes in the nonobese diabetic (NOD) mouse, which spontaneously develops type I diabetes, are shared with the human disease. Immunohistochemical studies support the concept that T lymphocytes are the major components of inflammatory cells in the pancreatic islets and these cells may play a critical role in the destruction of the beta cells leading to diabetes. Therefore, we examined whether particular TCR-beta variable region genes were utilized by in situ islet T cells at different stages (4 - 5, 7, 14 - 15 and 16 weeks of age) of the disease process. Dot-blot hybridization was performed using RNA prepared from isolated islets, thymus, spleen, peripheral blood leukocytes and axillary lymph nodes of 10 to 15 mice pooled for each data point. Ten different TCR V-beta probes were used for the analyses. Limited usage of islet V-beta genes was observed only at the early prediabetic stage (4 - 5 weeks old) of the disease. At later stages of the disease (7 - 16 weeks old), no preferential usage of TCR genes was observed in the islets compared to those of peripheral lymphoid organs. These data suggest that only certain types of T cells bearing particular TCR V-beta genes may be responsible for initiating and perpetuating infiltration of immune cells into the islets and these particular T cells are only identified at the very early stages of the autoimmune process.

Effect of neurotropin on the binding of high molecular weight kininogen and Hageman factor to human umbilical vein endothelial cells and the autoactivation of bound Hageman factor.

Bradykinin is generated by activation of the plasma kallikrein-kinin (K-K) cascade and contributes to the symptoms of allergic reactions and the perception of pain. Neurotropin is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus, which is widely used clinically in Japan as an effective agent for these disorders. Factor XII (FXII) and high molecular weight kininogen (HK), two critical constituents of the plasma K-K cascade, bind to endothelial cells, and bound FXII is autoactivated in the presence of zinc ions. We have investigated the effects of Neurotropin on the interactions of FXII and HK with endothelial cells. Neurotropin inhibited the binding of both proteins to cultured human umbilical vein endothelial cells (HUVEC) and inhibited autoactivation of FXII upon HUVEC in a concentration-dependent manner. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and, consequently, the formation of bradykinin.

Quantitative and functional analyses of spleen and in situ islet immune cells before and after diabetes onset in the NOD mouse.

Cytofluorometric analysis using specific monoclonal antibodies directed against the T cell antigens Thy-1.2, CD4, CD8, CD4V beta(8.1 + 8.2 + 8.3), and the antigen Mac-1 expressed by mature macrophages and NK cells were used to characterize and quantify the phenotypes of (1) unfractionated and Percoll gradient fractionated in situ islet immune cells isolated from prediabetic and diabetic female NOD mouse spleens. We found in prediabetic female mice that the majority (approximately 70%) of the in situ islet immune cells were Thy-1.2 positive T cells. CD4 positive T cells (approximately 40%) were the most abundant phenotype together with double negative T cells (approximately 20%). The percentage of CD8 positive T cells were approximately 10%, and only approximately 4% of the immune cells were Mac-1 positive. The percentages of CD4V beta (8.1 + 8.2 + 8.3) positive and double negative T cells in diabetic spleens were significantly higher in comparison to prediabetic spleens. In C57B1/6J control nondiabetic mice the percentage of double negative T cells in the spleens was significantly 4-fold lower when compared to diabetic NOD spleens. The specific cytolytic activity mediated by in situ islet immune cells against 51Cr-labeled dispersed syngeneic single-cell islet cells at an effector to target ratio of 20 was twenty- to thirty-fold higher than that mediated by prediabetic splenic lymphoid cells. It is concluded that prediabetic NOD mouse in situ islet immune cells are mostly CD4 positive and double negative T cells, and that CD4 and CD8 positive T cells in the intra-islet infiltrate warrants further evaluation as potential effector T cells in target beta-cell destruction.

How should polypoid lesions of the gallbladder be treated in the era of laparoscopic cholecystectomy?

BACKGROUND: Definitive criteria for choosing the most appropriate treatment for each type of polypoid lesion of the gallbladder (PLG) have yet to be established. METHODS: The shapes, sizes, echo patterns, and echogenicities of PLGs that had been evaluated by means of ultrasonography in 72 patients who had undergone resective surgery were analyzed retrospectively to elucidate the ultrasonic characteristics of polypoid cancers and to establish criteria for selecting the most suitable treatment such as laparoscopic cholecystectomy for each type of PLG. RESULTS: Histologic examinations showed cholesterol polyps in 47 patients, adenomas in 8, cancers in 16, and an inflammatory polyp in 1. The diameters of 61% of the benign PLGs were less than 10 mm, whereas those of 88% of the cancers were more than 10 mm; 80% of the former were pedunculated and 56% of the latter were sessile. Seven of eight early-stage cancers had diameters less than 18 mm, whereas those of all eight more advanced cancers were greater than 18 mm. Five of the eight early-stage cancers were pedunculated, and six of the eight more advanced cancers were sessile. Cholecystectomy with or without full-thickness dissection were main surgical procedures used to resect benign PLGs and early-stage cancers, whereas cholecystectomy with partial liver resection was used for more advanced cancers. Laparoscopic cholecystectomy was performed in the recent 34 patients, four of whom had early-stage cancers. CONCLUSIONS: A PLG with a diameter of less than 18 mm is a potential early-stage cancer and therefore can be resected by laparoscopic cholecystectomy with full-thickness dissection. However, when cancer invades the subserosal layer or beyond, a second-look operation is necessary. A PLG with a diameter of greater than 18 mm may be an advanced cancer and should be removed by using cholecystectomy with partial liver resection or a more extended procedure with lymph node dissection.

Appraisal of intraoperative ultrasonography during laparoscopic cholecystectomy.

BACKGROUND: The usefulness of intraoperative ultrasonography during laparoscopic cholecystectomy (LC) has yet to be evaluated fully. METHODS: In 50 patients who underwent LC, the intraoperative ultrasonography findings were compared with those of preoperative ultrasonography, intraoperative cholangiography, and histology, and then its usefulness for examining anatomic relationships in the hepatoduodenal ligament, detecting bile duct stones, diagnosing gallbladder polyps and abnormally thickened walls, and determining the propriety of LC was appraised. RESULTS: The preoperative ultrasonography diagnoses were gallstones in 38 patients, polyps in 10, and cancer and adenomyomatosis in one each. In four patients endoscopic retrograde cholangiography showed bile duct stones. In all 50 patients intraoperative ultrasonography was useful for examining the anatomic relationships between the bile duct and vessels, such as the portal vein and hepatic artery, and showing the presence or absence of bile duct stones. On the basis of the intraoperative ultrasonography findings, gallstones were diagnosed in 38 patients, in five of whom bile duct stones were shown clearly, cholesterol polyps in eight, early-stage cancer or adenoma in two, and adenomyomatosis in two, and subsequently LC was performed. Histologic diagnoses of cholesterol polyps were made in eight of ten patients with polyps, and intramucosal cancer and an inflammatory polyp in one each. In one patient with a preoperative diagnosis of cancer the apparently elevated flat lesion was found to be partial thickening of the gallbladder wall, which was diagnosed as adenomyomatosis, and LC was chosen as the operative procedure. CONCLUSIONS: Intraoperative ultrasonography during LC is useful for detecting bile duct stones, diagnosing gallbladder polyps and abnormally thickened walls, and deciding whether LC is adequate for resection of the gallbladder.

Isolation and function of human and pig islets.

This report confirms reproducible methods to isolate and assess viability and function of pig and human islets. We processed 10 pig and five human pancreata. The pancreata were digested by a modification of Ricordi's automated method for islet isolation. The number of islet equivalents (150 microns diameter islets) was 335,190 +/- 79,345 islets per pig pancreas (5,146 +/- 1,274 islets/g pig pancreas) and 323,630 +/- 147,810 per human pancreas (6,252 +/- 2,572 islets/g human pancreas). The majority of islets were in the range of 50-200 microns diameter, and 20% of the islet population had a size distribution of 200 microns diameter in both porcine and human models. The purity of the final preparations exceeded 90%. The secretory response of perifused islets showed a biphasic insulin release pattern in both species. Perifused fresh pig islets released 2.5 pmol/L islet-1 min-1 at 2.0 mM glucose and 6.2 pmol/L islet-1 min-1 at 16.7 mM glucose. After 7 days culture at 37 degrees C, human islets released 1.32 pmol/L islet-1 min-1 at 2.0 mM and 12.24 pmol/L islet-1 min-1 at 16.7 mM. These results indicate that this procedure is useful to obtain pure, large, and functional islets from pig and human pancreata.

[Improving the anti-tumor activity of 5-fluorouracil by methotrexate].

The first clinical application of biochemical modulation (BCM) of 5-fluorouracil (5-FU) was the sequential MTX/5-FU regimen proposed in 1977 by Bertino for the treatment of colorectal cancer. In Japan, sequential MTX/5-FU therapy was mainly used as a new method of treating gastric cancer, and attracted a great deal of attention because it proved effective in many cases of advanced gastric cancer that had been unresponsive to the previous chemotherapy, particularly scirrhous gastric cancer with poor prognosis. Its therapeutic efficacy varied according to histologic type, it was effective in cases of peritoneal dissemination and disseminated intravascular coagulopathy (DIC), it was associated with fewer adverse effects, and it was a multidrug chemotherapy based on a clear rationale. With sequential MTX/5-FU therapy as a starting point, fundamental studies of BCM and its clinical applications have expanded rapidly in Japan. This paper provides an outline of sequential MTX/5-FU therapy from the aspects of its mechanism of action, indications, therapeutic efficacy, relevance to adjuvant therapy, counter-measures to adverse effects, and emergence of resistance to the drugs involved. The high therapeutic efficacy of this therapy in certain histologic types is also discussed, and its combined use with other forms of BCM, as in triple BCM (LV/5-FU + CDDP/5-FU + MTX/5-FU), is introduced.

[Chemotherapy of gastric cancer].

At present curative resection is the only radical treatment for gastric cancer, although recently developed combination chemotherapy shows increased activity in treating locally advanced and metastatic disease. Among several combination regimens, those based on biochemical modulation, such as sequential methotrexate/5-fluorouracil or low-dose CDDP/5-fluorouracil, are thought to provide increased efficacy with decreasing adverse reactions in unresectable gastric cancer. Therefore, a prospective randomized clinical trial comparing the prognosis of patients receiving adjuvant multi-agent chemotherapy with those treated only surgically should be pursued for estimation of the clinical benefit of these agents.

Activation of hepatic microsomal glutathione S-transferase of rats by a glutathione depletor, diethylmaleate.

The effect of glutathione depletor diethylmaleate on rat hepatic glutathione S-transferase and glutathione peroxidase was studied in vivo and in vitro. When diethylmaleate (600 mg/kg) was given i.p. to rats, liver glutathione was depleted within 2 h and recovered to the control level 5 h after diethylmaleate treatment. Both glutathione S-transferase and peroxidase activities in microsomes, not in cytosol, were markedly increased during glutathione depletion and only glutathione S-transferase activity remained at high levels after recovery of the glutathione content. The increase in microsomal glutathione S-transferase and peroxidase activities with concomitant exhaustion of glutathione was also observed by perfusion of the isolated liver with diethylmaleate (10 mM). When liver microsomes were incubated with diethylmaleate in vitro at 37 degrees C, glutathione S-transferase, but not peroxidase, activity was increased; the increase was not reversed by dithiothreitol. These results indicate that diethylmaleate activates microsomal glutathione S-transferase by direct reaction to the enzyme during glutathione depletion and suggest that glutathione S-transferase activity and glutathione peroxidase activity in the microsomal enzyme may be differently regulated.

Quantitative phenotypic and functional analyses of islet immune cells before and after diabetes onset in the BB rat.

Inflammatory cells invading islets are thought to be mediators of islet destruction in spontaneous autoimmune diabetes mellitus. Thus methods were developed to isolate and characterize in situ islet inflammatory cells from 75-95-day-old prediabetic and diabetic BB rats. Islet inflammatory cells were structurally examined using single- and double-colour flow cytometry. Functional studies consisted of cytolytic assays using normal rat islet target cells and in situ islet or spleen effector cells. Structural data reveal natural killer cells to be the major cell population (70%) of total immune cells present in inflamed islets during prediabetes. At diabetes onset, the natural killer cell population remained at a high level (47%), but an increasing population of T cells (40%) was noted also. Analyses of T-cell subsets before and after diabetes onset revealed CD4+ T cells as predominant (50-55% of total T cells) with double-negative (CD4-CD8-) T cells (25-30%) and CD8+ T cells (15-20%) also present in significant quantities. Activated T cells accounted only for a minority of T cells (< 3%). Functional studies indicate that in situ islet-derived cytolytic effector cells are more potent killers (ten-fold) of normal islet target cells than are splenic effector cells. These data suggest that in situ islet inflammatory cells (a) can be quantitatively studied both structurally and functionally; (b) express structural phenotypes differing substantially from splenic mononuclear cell populations; (c) are considerably more cytolytic than splenic effectors; and (d) should prove informative in determining the most significant autoimmune functional events prior to and during islet beta-cell destruction.

Alterations in respiratory function and hemodynamics during laparoscopic cholecystectomy under pneumoperitoneum.

Respiratory function and hemodynamics were studied during laparoscopic cholecystectomy (LC) under a 10 mmHg pneumoperitoneum (PP) by carbon dioxide insufflation. Blood-gas analysis and measurement of cardiac function by using a Swan-Ganz catheter were performed. Creatinine clearance rate was measured preoperatively and intraoperatively. Compared with values obtained before the institution of PP, blood-gas analysis showed a significant increase in PCO2 (P < 0.01), and a significant decrease in pH (P < 0.01) and base excess (P < 0.05) during PP. With respect to cardiac function, there was no significant change in cardiac output, pulmonary arterial pressure, and pulmonary arterial wedge pressure. Intraoperative creatinine clearance rate (Ccr) was decreased in 29 of 48 cases, increased in 18 cases, and unchanged in 1 case, resulting in no significant difference overall between the values measured preoperatively and intraoperatively. However, in eight individual cases, the Ccr was found to have decreased significantly. Although alterations in respiratory function were observed, LC at 10 mmHg PP did not cause any crucial problems in respiratory or cardiac function. It should be kept in mind, however, that renal blood flow may decrease in some cases even at intraabdominal pressures under 10 mmHg.

Effects of preoperative chemotherapy on DNA ploidy patterns, cell cycle, and histological findings in gastric and colonic cancer patients.

The effects of preoperative chemotherapy on gastric and colonic cancers have yet to be evaluated fully. In this study, its effects were assessed by studying DNA ploidy patterns, cell cycles, and histological findings in such patients. Thirty-nine patients with gastric or colonic cancer were given preoperative chemotherapy with UFT (an admixture of tegafur and uracil). Biopsy specimens for analysis were obtained before chemotherapy through a gastroscope or colonoscope and after chemotherapy from resected tumors. The DNA ploidy patterns and cell cycles were evaluated using a flow cytometer and the tissues were examined histologically. The DNA ploidy pattern was diploid (D) in 12 gastric and 13 colonic cancer patients and aneuploid (A) in 10 and 4 patients, respectively. After chemotherapy, the pattern changed in nine gastric (A-->D: 7, D-->A: 2) and six colonic cancer patients (A-->D: 3, D-->A: 3) and was unchanged in the remaining patients. Cell cyclic analysis showed decreased G1- and increased S-phase fractions in 10 of 12 patients with gastric and 6 of 10 patients with colonic cancer. Histologically, decreased tumor cellularity, increased fibrosis, and/or cytological changes were observed in both cancers after chemotherapy. Gastric and colonic cancers in which the DNA ploidy pattern changed from aneuploid to diploid, G1- decreased and S-phase increased, and/or histological changes were observed, were considered to have responded to preoperative UFT administration.