ABSTRACT
BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.
Subject(s)
Bacterial Proteins/metabolism , Clostridium histolyticum/enzymology , Cysteine Endopeptidases/metabolism , Endopeptidases/metabolism , Islets of Langerhans/enzymology , Tissue and Organ Harvesting/methods , Animals , Bacterial Proteins/genetics , Clostridium histolyticum/genetics , Cysteine Endopeptidases/genetics , Endopeptidases/genetics , Microbial Collagenase/isolation & purification , Microbial Collagenase/metabolism , Rats , Rats, Inbred Lew , Recombinant Proteins/metabolism , Thermolysin/metabolism , Time FactorsABSTRACT
PURPOSE: We investigated the effect and safety of chemotherapy for patients over eighty-year-old. OBJECT: Ten patients with advanced or recurrent pancreas or biliary tract cancer who were over 80 years old and administered gemcitabine hydrochloride (GEM), were named as "the oldest group", and compared with the control group. RESULT: The overall response rate was 22. 2% without a significant difference between those 2 groups. The mean duration of treatment and the mean period from the initial chemotherapy to death were 8. 0 and 12 months, respectively, without a significant difference. There was a higher incidence of initial reduction of GEM in the oldest group. There was no significant difference at the DI ratio (actual drug intensity/ideal drug intensity) between 2 groups. Side effects found were 5 cases of neutropenia, 4 cases of anemia and appetite loss greater than grade 3. The dropout ratio (50%) in the oldest group was significantly higher. There was no death related with chemotherapy. CONCLUSION: Chemotherapy can be used safely and usefully, if appropriately reduced from first administration, for a patient over eighty years old.
