Takao Saotome

Morphological and immunocytochemical identification of periacinar fibroblast-like cells derived from human pancreatic acini.

Fibroblast-like cells in the periacinar region may play an important role in periacinar fibrosis. In the present study, we isolated and cultured periacinar fibroblast-like cells (PFCs) derived from human pancreatic acini and examined the characteristics of human PFCs morphologically and immunocytochemically. Immunocytochemical study of human PFCs showed that they were positively stained with antibodies against type I collagen/procollagen, type III collagen/procollagen, fibronectin, prolyl hydroxylase β subunit, type IV collagen, laminin, α-smooth muscle actin, vimentin, and nonmuscle myosin. Electron microscopic study showed that human PFCs contained a number of microfilaments, forming dense bodies in the cytoplasm. These results indicated that human PFCs possess characteristics of myofibroblasts. Expression of α-smooth muscle actin, a marker of the myofibroblast-like phenotype, was increased with time in culture and was enhanced by treatment with transforming growth factor (TGF)-β1. Collagen synthesis in human PFCs was stimulated by TGF-β1 and the proliferation of human PFCs was stimulated by plateletderived growth factor. These findings suggest that PFCs from human pancreas seem to be involved in periacinar fibrosis.

A blockade of complement activation prevents rapid intestinal ischaemia-reperfusion injury by modulating mucosal mast cell degranulation in rats

We attempted to define the putative role of complement activation in association with mucosal mast cell (MMC) degranulation in the pathogenesis of rapid intestinal ischaemia‐reperfusion (I/R) injury. We prepared complement activity‐depleted rats by the administration of the anti‐complement agent K‐76COOH and the serine‐protease inhibitor FUT‐175. Autoperfused segments of the jejunum were exposed to 60 min of ischaemia, followed by reperfusion for various time periods, and the epithelial permeability was assessed by the 51Cr‐EDTA clearance rate. The number of MMC was immunohistochemically assessed. In control rats, the maximal increase in mucosal permeability was achieved by 30–45 min of reperfusion. This increase was significantly attenuated by the administration of either K‐76COONa alone or in combination with FUT‐175. In contrast, the administration of carboxypeptidase inhibitor (CPI), which prevents the inactivation of complement‐derived anaphylatoxins such as C5a, significantly enhanced the increase in I/R‐induced mucosal permeability. These findings were confirmed morphologically by light microscopy and scanning electron microscopy. In addition, the I/R‐induced mucosal injury was accompanied by a marked decrease in the number of MMC, and administration of K‐76COOH significantly inhibited this change. These results indicate that complement activation and the generation of complement‐derived anaphylatoxins are key events in I/R‐induced mucosal injury. It is likely that intestinal I/R‐induced mucosal injury may be partially mediated by MMC activation associated with the complement activation.

Supplement of a Chitosan and ascorbic acid mixture for Crohn’s disease: A pilot study

OBJECTIVE Although the pathogenesis of Crohns disease remains unclear, dietary fat is thought to exacerbate intestinal inflammation. Chitosan is a water-insoluble dietary fiber, and a chitosan and ascorbic acid mixture has been shown in rats to increase fecal fat excretion without affecting protein digestibility. However, it remains unclear whether a chitosan and ascorbic acid mixture is safe and effective for patients with Crohns disease. We designed a pilot trial to investigate the tolerability and amount of fat excretion after the oral administration of a chitosan and ascorbic mixture for inactive Crohns disease. METHODS Eleven outpatients were given seven tablets daily of a chitosan and ascorbic mixture (chitosan was given at 1.05 g/d) for 8 wk. Patients did not interrupt their respective therapies for Crohns disease. RESULTS The bowel movements of most patients increased slightly during the study. Nutritional and inflammatory markers in patients did not differ before and after treatment. The chitosan and ascorbic acid mixture significantly increased the fat concentration in the feces during treatment. CONCLUSIONS These results indicated that oral administration of a chitosan and ascorbic acid mixture in patients with Crohns disease is tolerable and increases fecal fat excretion without affecting disease activity.

Pro-inflammatory cytokine-induced matrix metalloproteinase-1 (MMP-1) secretion in human pancreatic periacinar myofibroblasts

Matrix metalloproteinases (MMPs) are the proteases involved in the degradation of the extracellular matrix. MMP-1 is thought to be one of the key enzymes in fibrolysis, a process closely related to tissue remodeling. In the present study, we investigated MMP-1 secretion from human pancreatic periacinar myofibroblasts in response to pro-inflammatory cytokines IL-1β and TNF-α. We also attempted to clarify the intracellular signaling pathways mediating the cytokine-induced MMP-1 secretion. MMP-1 secretion was measured by an enzyme-linked immunosorbent assay. MMP-1 molecules were analyzed by Western blotting. MMP-1 mRNA expression was evaluated by Northern blotting. IL-1β and TNF-α stimulated the MMP-1 secretion in a dose- and time- dependent manner. Ninety percent of MMP-1 was secreted as inactive form (pro-MMP-1). The effects of IL-1β and TNF-α were significantly inhibited by PD98059 MEK/ERK inhibitor). In contrast, SB203580 (p38 MAPK inhibitor), GF109203X (PKC inhibitor), and PDTC (NF-ĸB inhibitor) did not alter the MMP-1 secretion induced by IL-1β and TNF-α. These effects were also observed at the mRNA level. In conclusion, in human pancreatic periacinar myofibroblasts, MMP-1 secretion was regulated by the pro-inflammatory cytokines via the MEK/ERK cascade. Thus, human pancreatic periacinar myofibroblasts may play an important role in the remodeling of damaged pancreatic tissue in chronic pancreatitis via MMP-1 secretion.

Multicenter Study of Plasma Diafiltration in Patients With Acute Liver Failure

Plasma diafiltration (PDF) is a blood purification therapy in which simple plasma exchange (PE) is performed using a selective membrane plasma separator while the dialysate flows outside the hollow fibers. A prospective, multicenter study was undertaken to evaluate the changes in bilirubin, IL‐18, and cystatin C, as well as the 28‐day and 90‐day survival rates, with the use of PDF according to the level of severity as measured by the Model for End‐Stage Liver Disease (MELD) score. Twenty‐one patients with liver failure were studied: 10 patients had fulminant hepatitis and PDF therapies were performed 28 times; 11 had acute liver failure with the therapy performed 96 times. Levels of total bilirubin, IL‐18, and cystatin C decreased significantly after treatment. The 28‐day survival rate was 70.0% and that at 90 days was 16.7%. According to the severity of the MELD score, each of the results compared well with the use of Molecular Adsorbent Recirculating System or Prometheus therapy. In conclusion, PDF appears to be one of the most useful blood purification therapies for use in cases of acute liver failure in terms of medical economics and the removal of water‐soluble and albumin‐bound toxins.

Inflixmab as a possible treatment for the hemorrhagic type of Crohn’s disease

Acute lower gastrointestinal bleeding is a rare complication of Crohn’s disease (CD). Although anti-tumor necrosis factor-Α (TNF-Α, infliximab) therapy has been established for patients with inflammatory and fistulous CD, there has been little evidence on whether infliximab is effective for the hemorrhagic type of CD. We report a case of a 31-year-old man with CD who had recurrent sudden-onset bloody stool. After a second surgery, he visited our hospital because of bloody stool. Infusion of infliximab stopped the bleeding and promoted the healing of ulcers in the ileum and ileocolon anastomosis. We suggest that infliximab therapy should be tried to stop acute gastrointestinal bleeding in CD before there is a surgical emergency.

A case of severe acute pancreatitis treated with CTR-001 direct hemoperfusion for cytokine apheresis.

Abstract:  Severe acute pancreatitis is a clinical entity that can develop into multiple organ failure (MOF), and still has a poor prognosis. It is generally agreed that excessive humoral mediators such as pro‐inflammatory cytokines play important roles in the pathogenesis of organ failure in patients with severe acute pancreatitis (SAP). Furthermore, it has been reported that continuous hemodiafiltration (CHDF) can remove the excess humoral mediators during the hypercytokinemic state of systemic inflammatory response syndrome (SIRS). We experienced a case of severe acute pancreatitis induced by alcohol abuse, on whom we performed cytokine apheresis. The patient was a 46 year‐old male. He received 14 cytokine apheresis procedures, for about 4 hours in each session, using a CTR‐001 direct hemoperfusion (DHP) cartridge. His serum levels  of  pro‐inflammatory  cytokines  such  as  interleukin‐6 (IL‐6; 1649.1 ± 667.1–1257.1 ± 489.4 pg/mL, P = 0.013) decreased significantly after the CTR‐001 procedures. However tumor necrosis factor‐α (TNF‐α) (26.2 ± 1.7–24.3 ± 1.9 pg/mL, P = 0.087), IL‐1β (6.1 ± 2.9–3.49 ± 1.1 pg/mL, P = 0.477), IL‐8 (192.5 ± 33.4–229.5 ± 51.8 pg/mL, P = 0.754) and IL‐10 (14.4 ± 2.7–14.0 ± 1.9 pg/mL, P = 0.726) did not decrease statistically. Therefore, we conclude that in this case, cytokine apheresis using a CTR‐001 cartridge was effective for reducing the pro‐inflammatory cytokines during severe acute pancreatitis.

Effects of synthetic serine protease inhibitors on proliferation and collagen synthesis of human pancreatic periacinar fibroblast-like cells

Protease inhibitors are currently used as therapeutic agents for chronic pancreatitis in Japan. We previously reported that human pancreatic periacinar fibroblast-like cells (hPFCs) could be cultured from isolated pancreatic acini, and those are thought to play a crucial role in pancreatic fibrosis correlating with platelet-derived growth factor (PDGF) and transforming growth factor &bgr;1 (TGF-&bgr;1) (Pancreas 1997;14:373–82). The present study was designed to examine the effects of synthetic serine protease inhibitors (FOY-007 and FOY-305) on proliferation and collagen synthesis of hPFCs under cytokine stimulation. The cell proliferation and collagen synthesis were evaluated using assays of [3H]-thymidine incorporation and procollagen type I c-terminal peptide (PIP), and [14C]-proline incorporation to de novo synthesized collagen, respectively. The cell proliferation stimulated by PDGF was inhibited by the application of FOY-007 dose dependently (1–100 &mgr;M) and FOY-305 at 100 &mgr;M. FOY-007 attenuated the collagen synthesis and PIP production stimulated by TGF-&bgr;1 dose dependently, but FOY-305 inhibited only PIP production. Both protease inhibitors demonstrated no effect on the proliferation and collagen synthesis of hPFCs when they were not stimulated by PDGF or TGF-&bgr;1. Thus, serine protease inhibitors act on hPFCs to diminish the effects of PDGF on proliferation and the effects of TGF-&bgr;1 on collagen synthesis.

Epithelial expression of caveolin‐2, but not caveolin‐1, is enhanced in the inflamed mucosa of patients with ulcerative colitis

Caveolae are vesicular invaginations of the plasma membrane that act as a scaffold of the assembly of many classes of signaling molecules. Caveolins are the principal structural component of caveolae membranes, and three distinct forms of caveolins have been identified: caveolin-1, caveolin-2, and caveolin-3. In this study, we evaluated the changes in the caveolin-1 and caveolin-2 expression in the inflamed mucosa of patients with IBD. Tissue samples were obtained endoscopically from patients with ulcerative colitis (UC) (n = 18), Crohns disease (n = 10) and ischemic colitis (n = 8). Normal colorectal tissues were also obtained (n = 15). The caveolin expression was evaluated by standard immunohistochemical procedure. In normal colonic mucosa, caveolin-1 expression was detected in the smooth-muscle cells of the muscularis mucosae and the endothelial cells, but caveolin-2 expression was not detected. In the inflamed mucosa of patients with active UC, caveolin-2 expression was clearly detectable as small scattered foci on the luminal surfaces of epithelial cells, but caveolin-1 expression was similar to that in normal mucosa. Caveolin-2 expression increased in accordance with the disease activity of UC. This enhanced caveolin-2 expression was not detected in active Crohns disease or ischemic colitis. In conclusion, we demonstrated that the epithelial expression of caveolin-2 is markedly enhanced in the inflamed mucosa of patients with UC. It is likely that the enhanced caveolin-2 expression in patients with UC was associated with the altered signal transductions in the intestinal epithelial cells. Furthermore, our results suggest that there are differences in the phenotypic features of epithelial cells between UC and Crohns disease.

Unusual hypersensitivity to warfarin in a critically ill patient

A patient was admitted to the intensive care unit because of respiratory failure, and warfarin therapy was started at 2 mg/day for the treatment of pulmonary embolism, together with other medications. Despite the low dosage of warfarin, international normalized ratio (INR) was markedly elevated from 1·15 to 11·28 for only 4 days, and bleeding symptoms concurrently developed. Vitamin K2 was infused along with discontinuation of warfarin. One day later, the INR was found to have decreased, and bleeding was also improved. An objective causality assessment indicated a probable relationship between clotting abnormality and warfarin administration, although the degree of elevation of the INR was unusual in the light of the daily warfarin dose and duration of its exposure. Based on the clinical status of the patient, it was suspected that several conditions contributed to the abnormal hypersensitivity to warfarin. Contributory factors probably included pharmacokinetic interactions with co‐administrated drugs, vitamin K deficiency caused by decreased dietary intake, reduced gut bacterial production, impaired intestinal absorption and hepatic synthetic capacity, and increased consumption of clotting factors. In view of our experience in the present case, it should be stressed that close monitoring of coagulation capacity is necessary in critically ill patients in order to avoid fatal haemorrhage after initiating warfarin therapy regardless of the dosage.