Involvement of MMP-7 in invasion of pancreatic cancer cells through activation of the EGFR mediated MEK-ERK signal transduction pathway.

AIMS: To clarify the involvement of matrix metalloproteinase-7 (MMP-7) in cell dissociation and the subsequent invasion of pancreatic cancer cells. METHODS: Western blotting, in vitro invasion assay, immunocytochemistry, and immunohistochemistry were performed in pancreatic cancer cell lines or pancreatic cancer tissue. RESULTS: The active form of the MMP-7 protein was expressed exclusively in the conditioned medium of dissociated (PC-1.0 and AsPC-1) pancreatic cancer cells, whereas proMMP-7 protein was only detected in the conditioned medium of non-dissociated (PC-1 and Capan-2) cells. Both intracellular and conditioned medium localised MMP-7 was greatly reduced by treatment with the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the mitogen activated protein kinase kinase (MEK) inhibitor U0126 in pancreatic cancer cells. MMP-7 treatment significantly induced the disruption of tight junction (TJ) structures and subsequent cell dissociation, and activation of the EGFR mediated MEK- ERK (extracellular signal regulated protein kinase) signalling pathway in the non-dissociated pancreatic cancer cells. Moreover, the strong in vitro invasiveness of dissociated cells was inhibited by AG1478 and U0126 treatment, whereas the weak invasiveness of non-dissociated cells was apparently induced by MMP-7 treatment. In addition, MMP-7 expression was stronger at the invasive front than at the centre of human pancreatic tumours. CONCLUSION: MMP-7 is involved in cell dissociation and the subsequent invasion of pancreatic cancer cells. It induces the disruption of TJ structures and forms a positive feedback loop with activation of the EGFR mediated MEK-ERK signalling pathway.

Altered cytokine response in rat acute pancreatitis complicated with endotoxemia.

We demonstrated that the dynamic aspects of cytokine production in rat acute pancreatitis, which was induced by cerulein and aggravated by subsequent lipopolysaccharide (LPS) injection. A priming effect by induction of mild pancreatitis with cerulein enhanced the subsequent cytokine production by LPS injection. Alternatively, after induction of severe pancreatitis with cerulein and LPS, cytokine production was markedly suppressed for > or = 90 hours. Production of interleukin-2 (IL-2) by splenocytes decreased, and mortality rate after cecal ligation and puncture (CLP) increased significantly after induction of severe acute pancreatitis. These results suggest that the suppression of a cytokine response in severe acute pancreatitis may alter the defense system and, as a result, increase mortality after CLP.

Elastase activity enhances the adhesion of neutrophil and cancer cells to vascular endothelial cells.

BACKGROUND: Elastase activity in cancer cells has been reported to promote their metastasis. Hence, we analyzed the influence of elastase activity of cancer cells on their responsive adhesion to vascular endothelial cells. MATERIALS AND METHODS: Human pancreatic (S2-007, S2-013, S2-020, S2-028) and colonic (COLO205) cancer cell lines were used. S2-007, S2-013, and S2-020 possess high elastase activity, whereas S2-028 and COLO205 have low elastase activity. Adhesive reactions of these cancer cells and neutrophils to TNFalpha-activated HUVEC were analyzed. Bound cells onto HUVEC were counted after incubation for 10 min. The effects of suppression of elastase activity by ZD8321, a potent elastase inhibitor, and supplementation of human neutrophil elastase (NE) on the adhesive reactions were also analyzed. In addition, E-selectin expression on HUVEC and concentrations of soluble E-selectin in the medium were measured. RESULTS: Adhesion of cells with high intracellular elastase activity to TNFalpha-activated HUVEC was suppressed by ZD8321. On the other hand, adhesion of cells with low elastase activity was enhanced by exogenous NE. Expression of E-selectin, a key molecule in leukocyte-endothelial cell interaction, on HUVEC was increased by NE. Soluble E-selectin concentration in the medium increased after the adhesive reaction between neutrophils and HUVEC. This increase was thought to be due to the shedding of cell surface E-selectin. Such responses were inhibited by ZD8321. CONCLUSION: Elastase activity has a biological function of stimulating both the E-selectin expression on HUVEC and the resultant adhesive reaction of cancer cells with them. Inhibition of elastase activity is a potent strategy for controlling cancer metastasis.

Tumor necrosis factor alpha acts on cultured human vascular endothelial cells to increase the adhesion of pancreatic cancer cells.

We studied the effect of tumor necrosis factor alpha (TNFalpha), one of the major inflammatory cytokines, on the adhesive reaction of pancreatic cancer cells to human umbilical vein endothelial cells (HUVECs) and on the hepatic metastasis of cancer cells in vivo. After TNFalpha stimulation, the expression of E-selectin, an adhesion molecule to neutrophils on HUVECs, increased. In addition, the adhesion of pancreatic cancer cells to HUVECs increased after TNFalpha stimulation, as was observed with neutrophils. The TNFalpha-induced adhesive response depended on the extent of sialyl Lewis(a) expression on cancer cells. The hepatic metastasis in vivo was often observed when cancer cells expressing a high amount of sialyl Lewis(a) were inoculated intrasplenically after increase in plasma TNFalpha concentration by lipopolysaccharide administration. Because sialyl Lewis(a) on cancer cells is a ligand for E-selectin on HUVECs, as sialyl Lewis(x) on neutrophils, TNFalpha upregulated the adhesive interaction between sialyl Lewis(a) on cancer cells and E-selectin on HUVECs. These results suggest that production of TNFalpha after surgical trauma may stimulate the hematogenic metastasis of cancer cells with a high sialyl Lewis(a) expression.

Increased concentrations of plasma IL-18 in patients with hepatic dysfunction after hepatectomy.

We investigated the dynamic aspects of circulatory IL-18 and other inflammatory cytokines in patients who underwent a hepatectomy. In patients with post-operative hepatic dysfunction, plasma concentrations of these cytokines increased, reflecting severe surgical trauma. IL-6, IL-10 and IFN-gamma increased in the early phase, while IL-18 increased in the later phase after 1 week. Interestingly, the increase in the plasma IL-18 concentration was correlated with that in serum bilirubin levels in hepatectomized patients. Hence, the decrease in the hepatic metabolism of IL-18 may cause the plasma accumulation of IL-18. This mechanism was confirmed using rat experiments. Intravenously administered human IL-18 was excreted into bile. Furthermore, the plasma clearance of human IL-18 was prolonged in bile duct-ligated rats. These results suggest that IL-18 is metabolized in the liver and excreted into bile, and an increase in plasma IL-18 in patients with hepatic dysfunction reflects the decreased metabolism in the liver.

Relationship between plasma cytokine concentration and multiple organ failure in patients with acute pancreatitis.

The dynamic aspects of circulating cytokines and cytokine modulators and their relationship with development of multiple organ failure (MOF) in patients with acute pancreatitis were analyzed. All cytokine and C-reactive protein levels in the circulation were higher than those in the MOF group. In particular, plasma concentrations of soluble tumor necrosis factor receptors (sTNF-RI and sTNF-RII) were significantly higher in patients with MOF than in those without even at admission. Furthermore, plasma concentrations of sTNF-Rs and interleukin-1 (IL-1) receptor antagonist (IL-1ra) were much higher than those of their counterparts, TNFalpha and IL-beta, respectively. These results suggest that the plasma concentrations of sTNF-Rs are useful predictors for the development of MOF, and actions of TNF-alpha and IL-1beta could be regulated by their modulators (soluble receptor and receptor antagonist, respectively) in the pathologic condition of severe acute pancreatitis.

[SIRS and CARS: discussion based on the pathologic condition of acute pancreatitis].

Relationship between SIRS and CARS in the pathologic condition of acute pancreatitis was discussed. SIRS promotes excessive inflammatory reaction and CARS induces the susceptibility to infection. Both conditions can develop into organ failure in acute pancreatitis. Hence, countermeasures for both conditions are mandatory in the care of patients with acute pancreatitis.

Anti-tumor effects of 25-hydroxycholesterol and low-dose recombinant tumor necrosis factor-alpha on rat liver tumorigenesis: modulated differentiation therapy for hepatocellular carcinoma.

Previously, we have demonstrated that treatment of rat AH136B ascites hepatoma cells with 25-hydroxycholesterol (25-OH) induces apoptosis. In this study, to elucidate whether modulated differentiation therapy may be useful in treating patients with hepatoma, we assayed the effects of 25-OH and/or tumor necrosis factor-alpha (TNF) on rat AH136B ascites hepatoma cells in vivo. Here we show that a high concentration of TNF (250 U/ml) mediated a stronger cytocidal effect than a low concentration of TNF (25 U/ml) in vitro. Flow cytometric DNA analysis also showed that treatment of cells with the high concentration of TNF (250 U/ml) increased the percentage of AH136B cells in the G0/G1 phase, while 25 U/ml of TNF did not cause any marked change in cell kinetics. In in vivo experiments, 25-OH (80 microg/rat) and TNF (500 U/rat) were administered after transplanting AH136B cells into Donryu rats intraperitoneally. We found that tumor development was completely inhibited by this treatment in 3 of 9 rats, and their 40-day survival rate was 44%; in contrast, rats administered 25-OH (80 microg/rat) alone or TNF (500 U/ml) alone developed peritonitis carcinomatosa, and died within 13 days of inoculation. These findings suggest that local combined treatment with 25-hydroxycholesterol and low-dose TNF can induce synergistic anti-tumor effects.

Suppression of rat liver tumorigenesis by 25-hydroxycholesterol and all-trans retinoic acid: differentiation therapy for hepatocellular carcinoma.

To determine whether differentiation therapy is useful in treating patients with hepatoma, we assayed the effects of 25-hydroxycholesterol (25-OH) and/or all-trans retinoic acid (ATRA) on rat AH136B ascites hepatoma cells. Flow cytometric DNA analysis showed that treatment of cells with 25-OH alone induced entry into the sub-G1 phase in a dose-dependent manner. While ATRA alone was ineffective, it enhanced the activity of 25-OH. Condensed and fragmented nuclei occurred mainly in cells treated with 25-OH (4 microg/ml). When cells treated with 25-OH (4 microg/ml), or 25-OH (4 microg/ml) + ATRA (1 microM) were transplanted into Donryu rats, we found that tumor development was completely inhibited; in contrast, rats administered the methanol-treated AH136B cells developed tumors. These findings suggest that AH136B cells in the sub-G1 phase can not recover tumorigenicity, and that the administration of a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, such as 25-OH, and ATRA may be effective in treating patients with hepatoma.

Pattern electroretinogram elicited by a dartboard pattern.

The receptive field size of retinal ganglion cells is closely related to their eccentricity from the fovea. To elicit larger pattern-reversal electroretinograms (P-ERGs), it may be useful to stimulate the retina with patterns having elements that parallel this change in receptive field size. We describe a dartboard pattern consisting of reversal elements that enlarge gradually from the central to the peripheral stimulus field. The utility of the dartboard pattern for eliciting P-ERG was investigated by comparing it with the conventional uniform checkerboard pattern, but with the other stimulus parameters remaining unchanged (96% contrast, 35.9 cd/m2 mean luminance, 3.3 rev/s temporal frequency, 15 degrees circular field). The dartboard pattern produced a significantly larger P50 amplitude than did the checkerboard pattern, while no difference in peak latency was found when 54 min of arc was used as a standard check size for the checkerboard pattern. The dartboard pattern for eliciting P-ERG should prove clinically useful.

Systemic inflammatory response syndrome and organ dysfunction following gastrointestinal surgery.

OBJECTIVES: Progression from systemic inflammatory response syndrome (SIRS) to sepsis, severe sepsis, and septic shock has been demonstrated in a variety of patients. However, the presence of SIRS alone was not helpful in predicting the development of multiple organ dysfunction syndrome (MODS) since SIRS includes many nonprogressive conditions. This study was conducted to investigate the clinical significance of SIRS in postoperative patients. DESIGN: Retrospective study. SETTING: The surgical department of a university hospital. PATIENTS: Two hundred ninety-two consecutive patients who received elective common gastrointestinal surgery (esophagectomy, pancreatoduodenectomy, hepatectomy, gastrectomy, colorectal resection, and laparoscopic cholecystectomy) between 1992 and 1995. INTERVENTIONS: Patients were analyzed for preoperative physiologic status, surgical stress parameters, and postoperative status of SIRS, complications, and end-organ dysfunction. MEASUREMENTS AND MAIN RESULTS: Duration of SIRS or positive criteria's number of SIRS after surgery significantly correlated with surgical stress parameters (blood loss/body weight and operation time) and peak serum C-reactive protein concentrations. SIRS that continued or reappeared after postoperative day 3 was an early sign of postoperative complications. SIRS continuing consecutively for 2 days after postoperative day 3 had a 70.6% positive predictive value and a 92.5% negative predictive value for postoperative complications. Septic complications and prolongation of SIRS were associated with MODS. Five of six patients who met the SIRS criteria for >30 days developed severe MODS, and three of them died. CONCLUSIONS: SIRS is a useful criterion for the recognition of postoperative complications and end-organ dysfunctions. Early recovery from SIRS may arrest the progression of organ dysfunction.

[Immunoglobulin decrease accompanying adverse reactions with bucillamine].

Forty-two RA patients treated with Bucillamine (B) were divided into two groups with (n = 26) and without (n = 16) adverse reactions, and IgG, M, A and total protein concentration x globulin ratio (Ig) before and after B were compared. IgG did not differ before B administration, but was significantly lower in the adverse reactions group after B, and in each group it was significantly lower after B treatment with a much greater decrease demonstrated in the adverse reactions group. IgA before B did not differ either between the two groups or between before and after B administration in the non-adverse reaction group, but was significantly lower only after B in the adverse reaction group. IgM did not differ either before or after B treatment between the two groups, and was significantly lower following B administration in both groups. Ig showed results similar to IgA. In the adverse reaction group, IgG, A, M and Ig recovered following suspension of administration. It is concluded that the adverse reaction brought on by B administration might occur with the decrease in immunoglobulin.

[Allergic granulomatosis and angiitis associated with intestinal perforation and eye movement disorder].

A perforation in the small intestine and one and a half syndrome were observed in 45-year-old male with allergic granulomatosis and angiitis (AGA). The patient was first admitted to our hospital in 1988 with complaints of bronchial asthma and an abnormal density on his chest X ray. The diagnosis of PIE syndrome was made based on marked peripheral blood eosinophilia. He was successfully treated with prednisolone and had been well on the maintenance therapy of small doses of it. In May 1990, peripheral neuropathy occurred in his bilateral lower legs and it was followed by nausea and vomiting. The laboratory findings on admission showed leukocytosis (19,300/mm3) with 43% eosinophilia and elevated blood level of IgE (3000 IU/ml). On the tenth day of admission, ileus symptom and subsequently a perforation of the small intestine were observed. The mesenterial specimens obtained during the emergency operation showed the findings compatible with AGA. Remission of AGA was induced by the treatment with 60 mg prednisolone and 100 mg cyclophosphamide per day following methylprednisolone pulse therapy. He was well on maintenance prednisolone until June 1991, when he suddenly developed one and a half syndrome. Although cranial CT scan and MRI showed no abnormal findings, cyclophosphamide therapy was resumed and prednisolone was increased because microangiopathy due to AGA was thought to be the cause of this central nervous system symptom. The one and a half syndrome improved with this therapy. This case suggests that the long term treatment with prednisolone and cyclophosphamide would be indicated in the severe AGA with close monitoring of the symptoms and peripheral blood eosinophilia.

[Linearity and nonlinearity of pattern ERG and pattern VECP].

To investigate the linearity and nonlinearity of both pattern electroretinogram (P-ERG) and pattern visually evoked cortical potential (P-VECP), a checkerboard pattern was presented to normal subjects with the reversal rate of transient and steady-state condition. Using a personal computer (BASIC) system, we made simulated waves (linear model) at 3 to 15 rev/s stimuli summated from an original wave evoked at 1 rev/s stimulation. Both simulated P-ERGs and P-VECPs were found to be different from the original ones, and the nonlinear component, which was interpreted as the difference between the original and simulated waves, was thought to increase with the increment of pattern reversal rate. However the 2 nd harmonic component of the original wave drastically decreased at above 7.5 rev/s stimulation. Thus, it is concluded that the nonlinearity of both P-ERG and P-VECP does not correlate with those 2 nd harmonic components.

Luminance-unbalanced pattern onset-offset electroretinogram and visual evoked cortical potential.

The effects of a luminance-unbalanced pattern onset-offset mode of stimulation on electroretinograms and visual evoked cortical potentials were investigated. With the use of originally devised software, only offset luminance was varied from 14.7 to 62.3 cd/m2. A vertical grating pattern (1.5 c/deg, 38.8 cd/m2 mean luminance, 0.95 contrast) was presented for 260 ms and was absent for 260 ms to normal subjects. With an increase in the luminance level of the offset pattern from the lowest level, the amplitude of the onset electroretinogram increased by degrees, while that of the offset electroretinogram gradually decreased. Conversely, onset visual evoked cortical potential responses decreased gradually, and offset visual evoked cortical potentials increased correspondingly. Furthermore, the spatial tuning of the onset pattern electroretinogram was already ambiguous, even when there was only a 4-cd/m2 difference between onset and offset pattern luminances. Thus, luminance control is indispensable for pattern onset-offset stimulation.

[Rapid diagnosis of gram-negative bacteriuria with endotoxin-specific chromogenic test].

Limulus amebocyte lysate test is a simple, yet sensitive laboratory method for detecting endotoxin. Although the specificity of the test was questioned, we recently solved this problem by removing a (1----3)-beta-D-glucan-sensitive factor G from the lysate (Endospecy, Seikagaku Kogyo Co., Ltd, Tokyo). In this study, we have established a method for the determination of endotoxin urine in using Endospecy. An aliquot of 0.2 ml of Endospecy dissolved in 4.4 ml of 0.1 mol/l Tris-HCl buffer (pH 8.0) was added to 5 microliter of urine sample. The mixture was incubated at 37 degrees C for 30 min. After diazo-coupling, absorbance was measured at 545 nm. The standard curve using Escherichia coli 0111: B4 endotoxin showed a good linearity. The addition-recovery test of various endotoxins showed nearly 100% recovery. Normal urine contained less than 60 pg/ml of endotoxin. Urine from patients with Gram-negative bacteriuria showed definitely high values, whereas urines infected either with Gram-positive bacteria or with fungi all showed nearly normal values. This method will be useful as a rapid and reliable test for the diagnosis of urinary tract infection with Gram-negative bacteria.

Concurrence of lymphoma type adult T-cell leukemia in three sisters.

Three sisters, ranging in age from 56 to 59 years, who developed lymphoma type adult T-cell leukemia (ATL) during a 19-month period are described. The patients were born in the Amakusa area of Kumamoto Prefecture, an area where the incidence of malignant lymphoma is high. The histologic diagnosis, made on the basis of the Lymphoma Study Group in Japan (LSG) classification of lymph nodes, was diffuse, medium-sized cell type in the elder sister, and mixed cell type in the middle and younger sisters. The patients and their elder brother had serum antibodies against ATL-associated antigens (ATLA).