Nora E. Riley

MYCOPHENOLATE MOFETIL IMPAIRS HEALING OF LEFT-SIDED COLON ANASTOMOSES 1

Introduction. Inadequate healing and consequent leakage from bowelanastomoses are a significant cause of postoperative morbidity and mortality.Immunosuppressive drugs are known to disturb healing processes and to impairthe mechanical stability of bowel anastomosis. Mycophenolate mofetil (MMF) isan immunosuppressive agent that selectively inhibits the proliferation of Tand B lymphocytes and has been shown to be effective in preventing allograftrejection after organ transplantation. Adverse effects are few; however,nothing is known in regard to possible adverse effects of MMF administrationon the healing of bowel anastomosis. The aim of the present study was toevaluate the effect of systemic MMF administration on the healing of colonanastomoses inrats. Methods. Rats underwent laparotomy, division of the left colon, andsigmoidostomy. MMF (25 mg/kg) or vehicle was administered intraperitoneally intwo groups (n=21 per group) 3 days before surgery and then once dailyuntil euthanization (7 animals per group; 2, 4, and 6 days after surgery).Bursting pressure measurements, histologic evaluation, morphometric analysis,mucin and collagen staining, and BrdU immunohistochemistry of the anastomoticsite were performed. Furthermore, matrix protein expression at the anastomoticsite was determined by collagen I and fibronectin Westernblots. Results. Administration of MMF significantly decreased anastomoticbursting pressure postoperatively. Accordingly, histology, mucin staining, andBrdU immunohistochemistry and measurements of the colonic crypt depth showedmore extended inflammation, a significantly lower proliferation rate, and asignificantly thinned mucosal layer in the MMF-treated groups when compared tocontrol animals, whereas matrix synthesis at the anastomotic site was notdifferent. Conclusions. The administration of the immunosuppressive agent MMFsignificantly impairs healing and mechanical stability of colon anastomosis inrats during the early postoperative period. MMF act to disturb host reparativeprocesses mainly by impairment of reparative colonic epithelium proliferationand less by a disturbance of matrixsynthesis.

The mechanism of cytokeratin aggresome formation: the role of mutant ubiquitin (UBB+1)

Aggresome formation in cells involves the failure of the ubiquitin-proteasome pathway to dispose of proteins destined for degradation by the 26S proteasome. UBB(+1) is present in Mallory bodies in alcoholic liver disease and in aggresomes formed in Alzheimers desease. The present investigation focuses on the role that UBB(+1) plays in cytokeratin aggresome formation in Mallory bodies (MBs) in vitro. Immunoprecipitation with a monoclonal antibody to cytokeratin-8 (CK-8) was used. The immunoprecipitate was incubated for 24 h in the presence of different constituents involved in aggresome formation including ubiquitin, UBB(+1), the proteasome inhibitor PS341, an ATP generating energy source, a deubiquitinating enzyme inhibitor, a purified proteasome fraction, and an E(1-3) conjugating enzyme fraction. MB-like protein aggregates formed in the presence of ubiquitin, plus UBB(+1) or PS341. These aggregates stained positively for CK-8. UBB(+1), and a proteasome subunit Tbp7, as demonstrated on Western blots. A second approach was used to form MBs in vitro in cultured hepatocytes transfected with UBB(+1) protein using Chariot. The cells were double stained using CK-8 and ubiquitin antibodies. The two proteins colocalized in MB-like aggregates. The results support the possibility that aggresome formation is a complex multifactor process, which is favored by inhibition of the proteasome and by the presence of UBB(+1).

Microtubules are required for cytokeratin aggresome (Mallory body) formation in hepatocytes: an in vitro study

Mallory bodies are cytokeratin-ubiquitin aggresomes that form in hepatocytes in many different chronic liver diseases. One of the key components in aggresome formation, not yet investigated in Mallory body formation, is the role of microtubules. An in vitro tissue culture assay is required to test for microtubule involvement in Mallory body formation so that Mallory body formation can be observed in the presence or absence of microtubule-disrupting agents. In this report, a new model of in vitro Mallory body formation was developed, which uses cultured hepatocytes isolated from drug-primed mice. When hepatocytes were incubated in the presence of antimicrotubule agents, they failed to form Mallory bodies. It is concluded that intact microtubules are required for Mallory body formation.

Expression of insulin-like growth factor binding proteins and collagen in experimental colitis in rats.

Objectives and design Crohns disease is complicated by smooth muscle hyperplasia and stricture formation. Insulin-like growth factor (IGF)-1 and insulin-like growth factor binding proteins (IGFBPs) may be involved in stimulating intestinal smooth muscle growth and collagen synthesis. Therefore, we investigated the expression of IGFBPs, collagen and collagenase activity in rat colitis and the effects of IGF-1 on IGFBP and collagen expression in rat colonic smooth muscle cells. Methods Animals were sacrificed during a 4-week time course of 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. RNA from the animals’ colons was blotted and hybridized with collagen-1 and IGFBP mRNA probes. Tissue proteins were screened for IGFBPs by Western ligand blotting. Collagenase activity was measured by zymography. Rat colonic smooth muscle cells in primary culture were incubated with IGF-1 then collagen-1, and IGFBP mRNAs and proteins were measured. Results In the rat tissue, IGFBP-3 mRNA and protein were increased 2 h after induction of colitis. IGFBP-4 mRNA was elevated after 2 h and IGFBP-4 protein after 4 h. IGFBP-5 mRNA was upregulated after 2 h with a peak at 12 h. IGFBP-5 protein was upregulated after 1 h and reached a peak at 3 days. Collagen-1 mRNA was increased after 5 days. Collagenase levels were decreased after 1 h and returned to normal by 28 days. In rat colonic smooth muscle cells, IGF-1 increased collagen-1 and IGFBP-5 expression. Conclusion We demonstrated an upregulation of IGFBP and collagen expression and a downregulation of collagenase in rat colitis. In colonic smooth muscle cells, we found an upregulation of collagen-1 and IGFBP-5 following IGF-1 incubation. These results suggest an important role of IGF-1 in the collagen synthesis in colitis, mediated by IGFBPs.