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Luminal hydrolases as host defense against invasive amebiasis
April 2000
serum calcium and 25(OH)D concentrations below normal, these abnormalities were more prevalent in patients who had a bowel resection. Cortical osteoporosis was also overall more prevalent than the expected trabecular osteoporosis with CS use. Our findings suggest that in patients with Crohn's disease with reasonably well controlled disease activity, osteoporosis is not prevalent despite CS use. It is possible that in this disorder factors related to its pathogenesis may protect against undue suppression of bone formation by CS and playa more dominant role in the development of any bone loss observed.Our data further suggest that patients with Crohn's disease who underwent ileum resection should be carefully screened for osteoporosis. 5246 LUMINAL HYDROLASES AS HOST DEFENSE AGAINST INVASIVE AMEBIASIS. Easwaran P. Variyam, Prema Gogate, Case Western Reserve Univ Sch of Medicine, Cleveland, OH; Veterans Affairs Med Ctr, Cleveland, OH. Entamoeba histolytica (EH) causes considerable morbidity and mortality from colitis or liver abscess. But, in most infected individuals, it survives as a commensal in the colon lumen. Virulence of EH strains to experimental animals is well correlated to degree of their binding of concanavalin A (con A). Factors that protect against invasive amebiasis are poorly understood. A subset of normal colonic bacteria produces a variety of extracellular glycosidases that can modify soluble and cell surface glycoconjugates. We determined whether enteric bacterial glycosidases or luminal proteases (of host or microbial origin) modify con A binding of EH. Methods: Bacterial glycosidases were prepared as previously described (Gastroenterology 1981; 81:75l)from anaerobic human fecal culture supernates (containing mostly glycosidases) and fecal extracts (containing glycosidases and pancreatic and microbial proteases). EH strain 200:NIH was grown in Diamond's TYI-S-33 medium, alone or supplemented with fecal culture supernate, pancreatic proteases (trypsin 25p,g/ml and oc-chymotrypsin 50 p,g/ml)or both, or with fecal extracts (N=3). Trophozoites harvested from 72 h cultures were washed in ice cold phosphate buffered saline, pH 7.4, and were treated with 0.125 p,g/ml of fluorescein-labelled con A, a cone. at which trophozoites were uniformly stained on the surface. Fluorescence was graded qualitatively on a scale of 0 to 3+ and, in one experiment, by flow cytometry. Results: Untreated trophozoites were uniformly stained with con A on the surface with a grade of 3+. Neither fecal culture supernates, nor pancreatic proteases, alone modified con A binding. However, fecal extracts, or a combination of fecal culture supernates and pancreatic proteases decreased con A binding to a grade of 0 to +. On flow cytometry, the peak channel of fluorescence decreased from 171 to 148 when treated with fecal extract, and to 145 when treated with a combination of fecal culture supernate and pancreatic proteases. Thus, a combination of bacterial glycosidases and luminal proteases decreased con A binding of EH, which is a recognized virulence marker of EH. A similar combination of hydrolases has previously been shown to degrade the key glycoprotein on EH that mediates its binding to epithelial cells and to mucin and to decrease EH adherence to epithelial cells (Gut 1996; 39:521). Modification of surface characteristics ofEH by luminal hydrolases may be a host defense mechanism against invasive amebiasis. 5247 MICROSATELLITE INSTABILITY (MSI) AND IMMUNOHISTOCHEMICAL ANALYSIS OF ENDOMETRIAL CANCERS AS A TOOL TO IDENTIFY FAMILY MEMBERS AT RISK FOR HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC). Hans F. Vasen, Wiljo J. Leeuw, Jan-Willem Diersen, Juul Th Wijnen, Gemma G. Kenter, Hans Morreau, Dept of Gastroenterology, LUMC, Leiden, Netherlands; Lab of Pathology, Leiden Univ Med Ctr, Leiden, Netherlands; Dept of Pathology, Leiden Univ Med Ctr, Leiden, Netherlands; Lab of Anthropogenetics, Leiden, Netherlands; Dept of Gynaecology, Leiden Univ Med Ctr, Leiden, Netherlands. Background: HNPCC is an autosomal dominant disease caused by a mutation in one of the mismatch repair genes (MLH I, MSH2 and MSH6) and is characterized by an excess of cancers of the colorectum, endometrium and other sites. Defects in these genes lead to microsatellite instability in the tumours associated with HNPCC. MSI-analysis of colorectal cancer is recommended as a first screening tool in families suspected of HNPCC . A more direct method to identify the responsible MMR gene mutation is staining of the gene products by immunohistochemical analysis. The aims of the present study were to study MSI and protein staining of MLHI, MSH2 and MSH6 in endometrial cancers from patients with an identified mutation, Patients and methods: Families with a known MMR gene mutation were derived from the Dutch HNPCC Registry. We analyzed 40 MSI markers including the NCI reference set of 5 markers in 23 endometrial cancers. Immunohistochemical staining was performed using antibodies against MLHl, MSH2 and MSH6. Results: In the group of MLHI and MSH2 mutation carriers, all endometrial cancers demonstrated a MSI-high phenotype (according to the NCI criteria) while in the group of MSH6 mutation carriers, only 36% of the endometrial cancers were MSIhigh. However, all endometrial cancers from the MSH6 mutation carrier group exhibited instability in mononucleotide repeat markers. The immunohistochemical analysis using antibodies against MLHl, MSH2 and MSH6 showed a loss of staining of these proteins, concordant with the gene mutation involved. In addition in the tumors from MSH2 mutation carriers a reduced staining of the MSH6 protein was observed while vice
AGAA1139
versa in tumors of MSH6 mutation carriers a reduced staining of MSH2 was found. Conclusion: Our study showed that the results of MSI analysis of endometrial cancer indicate whether a MMR gene is mutated. Using immunohistochemical analysis it is also possible to predict which one of the MMR gene is mutated. These relative cheap (compared to mutation analysis) techniques allow the identification of relatives at risk for HNPCC. 5248 NOS-2 IS INDUCED IN POUCHITIS. Palvi O. Vento, Tuula Kiviluoto, Paivi Karkkainen, Heikki 1. Jarvinen, Eero Kivilaakso, Seppo Soinila, Dept of Surg Helsinki Univ Cent Hosp, Helsinki, Finland; Dept of Pathology Univ of Helsinki, Helsinki, Finland; Dept of Surg, Helsinki Univ Hosp, Helsinki, Finland; Dept of Surg Helsinki Univ Hosp, Helsinki, Finland; Dept of Biomed Univ of Helsinki, Helsinki, Finland, Background. NO synthase (NOS) is expressed in ulcerative colitis (UC). The aim was to examine NO production induced in pouchitis, its variation in different clinical forms of pouchitis and its correlation with histopathological changes of pouchitis. Methods. Biopsies were taken from pouch and from ileum before pouch reconstruction. The diagnosis of pouchitis was based on clinical, endoscopic and histological criteria. Patients were divided in five different groups:1. ileum from UC patients(N=8), 2. nopouchitis (N= 15), 3.chronic asymptomatic pouchitis (N =8), 4.chronic active pouchitis (N= 11),5. acute pouchitis (N= 11). NOS-2 immunoreactivity and acute inflammation were graded from 0 to 3 by investigators unaware of the clinical or histological data. Results. In 6 out of 8 specimens of group 1, no NOS-2 immunoreactivity was observed. Two specimens showed an occasional villus profile with some reactivity in the epithelium. In group 2, several specimens lacked NOS-2 reactivity, while in others some villi or crypts with NOS-2 immunoreactive epithelium were seen. In group 3, areas of NOS-2 immunoreactive epithelium were consistently observed in most specimens. In group 4, only one specimen lacked NOS-2 immunoreactivity, all others regularly showed moderate to extensive epithelial NOS-2 staining. Group 5 showed NOS-2 staining pattern similar to that of group 4, there being unexceptionally moderate or abundant epithelial NOS-2 staining. NOS-2 immunoreactivity scores were 0.25 :!: 0.16, 0.67 :!: 0.19, 1.19 :!: 0.40,2.0 :!: 0.23, and 2.18 :!: 0.12 in groups 1-5, respectively. The values of both group I and 2 differed significantly from those of groups 4 and 5 (p
hydrophobicity distalcolon (0) penneabillty mannitol (nmol/mg tissue) dextran (pmol/mg tissue)
control
PEG
mannitol
senna
48±2
61±2'
48±3
43±1'
26±O1
1.9±O.1'
28±O,3
46±O,4'
15,1±O,9
10,6±O.5'
138±14
211±1.7'
serum calcium and 25(OH)D concentrations below normal, these abnormalities were more prevalent in patients who had a bowel resection. Cortical osteoporosis was also overall more prevalent than the expected trabecular osteoporosis with CS use. Our findings suggest that in patients with Crohn's disease with reasonably well controlled disease activity, osteoporosis is not prevalent despite CS use. It is possible that in this disorder factors related to its pathogenesis may protect against undue suppression of bone formation by CS and playa more dominant role in the development of any bone loss observed.Our data further suggest that patients with Crohn's disease who underwent ileum resection should be carefully screened for osteoporosis. 5246 LUMINAL HYDROLASES AS HOST DEFENSE AGAINST INVASIVE AMEBIASIS. Easwaran P. Variyam, Prema Gogate, Case Western Reserve Univ Sch of Medicine, Cleveland, OH; Veterans Affairs Med Ctr, Cleveland, OH. Entamoeba histolytica (EH) causes considerable morbidity and mortality from colitis or liver abscess. But, in most infected individuals, it survives as a commensal in the colon lumen. Virulence of EH strains to experimental animals is well correlated to degree of their binding of concanavalin A (con A). Factors that protect against invasive amebiasis are poorly understood. A subset of normal colonic bacteria produces a variety of extracellular glycosidases that can modify soluble and cell surface glycoconjugates. We determined whether enteric bacterial glycosidases or luminal proteases (of host or microbial origin) modify con A binding of EH. Methods: Bacterial glycosidases were prepared as previously described (Gastroenterology 1981; 81:75l)from anaerobic human fecal culture supernates (containing mostly glycosidases) and fecal extracts (containing glycosidases and pancreatic and microbial proteases). EH strain 200:NIH was grown in Diamond's TYI-S-33 medium, alone or supplemented with fecal culture supernate, pancreatic proteases (trypsin 25p,g/ml and oc-chymotrypsin 50 p,g/ml)or both, or with fecal extracts (N=3). Trophozoites harvested from 72 h cultures were washed in ice cold phosphate buffered saline, pH 7.4, and were treated with 0.125 p,g/ml of fluorescein-labelled con A, a cone. at which trophozoites were uniformly stained on the surface. Fluorescence was graded qualitatively on a scale of 0 to 3+ and, in one experiment, by flow cytometry. Results: Untreated trophozoites were uniformly stained with con A on the surface with a grade of 3+. Neither fecal culture supernates, nor pancreatic proteases, alone modified con A binding. However, fecal extracts, or a combination of fecal culture supernates and pancreatic proteases decreased con A binding to a grade of 0 to +. On flow cytometry, the peak channel of fluorescence decreased from 171 to 148 when treated with fecal extract, and to 145 when treated with a combination of fecal culture supernate and pancreatic proteases. Thus, a combination of bacterial glycosidases and luminal proteases decreased con A binding of EH, which is a recognized virulence marker of EH. A similar combination of hydrolases has previously been shown to degrade the key glycoprotein on EH that mediates its binding to epithelial cells and to mucin and to decrease EH adherence to epithelial cells (Gut 1996; 39:521). Modification of surface characteristics ofEH by luminal hydrolases may be a host defense mechanism against invasive amebiasis. 5247 MICROSATELLITE INSTABILITY (MSI) AND IMMUNOHISTOCHEMICAL ANALYSIS OF ENDOMETRIAL CANCERS AS A TOOL TO IDENTIFY FAMILY MEMBERS AT RISK FOR HEREDITARY NONPOLYPOSIS COLORECTAL CANCER (HNPCC). Hans F. Vasen, Wiljo J. Leeuw, Jan-Willem Diersen, Juul Th Wijnen, Gemma G. Kenter, Hans Morreau, Dept of Gastroenterology, LUMC, Leiden, Netherlands; Lab of Pathology, Leiden Univ Med Ctr, Leiden, Netherlands; Dept of Pathology, Leiden Univ Med Ctr, Leiden, Netherlands; Lab of Anthropogenetics, Leiden, Netherlands; Dept of Gynaecology, Leiden Univ Med Ctr, Leiden, Netherlands. Background: HNPCC is an autosomal dominant disease caused by a mutation in one of the mismatch repair genes (MLH I, MSH2 and MSH6) and is characterized by an excess of cancers of the colorectum, endometrium and other sites. Defects in these genes lead to microsatellite instability in the tumours associated with HNPCC. MSI-analysis of colorectal cancer is recommended as a first screening tool in families suspected of HNPCC . A more direct method to identify the responsible MMR gene mutation is staining of the gene products by immunohistochemical analysis. The aims of the present study were to study MSI and protein staining of MLHI, MSH2 and MSH6 in endometrial cancers from patients with an identified mutation, Patients and methods: Families with a known MMR gene mutation were derived from the Dutch HNPCC Registry. We analyzed 40 MSI markers including the NCI reference set of 5 markers in 23 endometrial cancers. Immunohistochemical staining was performed using antibodies against MLHl, MSH2 and MSH6. Results: In the group of MLHI and MSH2 mutation carriers, all endometrial cancers demonstrated a MSI-high phenotype (according to the NCI criteria) while in the group of MSH6 mutation carriers, only 36% of the endometrial cancers were MSIhigh. However, all endometrial cancers from the MSH6 mutation carrier group exhibited instability in mononucleotide repeat markers. The immunohistochemical analysis using antibodies against MLHl, MSH2 and MSH6 showed a loss of staining of these proteins, concordant with the gene mutation involved. In addition in the tumors from MSH2 mutation carriers a reduced staining of the MSH6 protein was observed while vice
AGAA1139
versa in tumors of MSH6 mutation carriers a reduced staining of MSH2 was found. Conclusion: Our study showed that the results of MSI analysis of endometrial cancer indicate whether a MMR gene is mutated. Using immunohistochemical analysis it is also possible to predict which one of the MMR gene is mutated. These relative cheap (compared to mutation analysis) techniques allow the identification of relatives at risk for HNPCC. 5248 NOS-2 IS INDUCED IN POUCHITIS. Palvi O. Vento, Tuula Kiviluoto, Paivi Karkkainen, Heikki 1. Jarvinen, Eero Kivilaakso, Seppo Soinila, Dept of Surg Helsinki Univ Cent Hosp, Helsinki, Finland; Dept of Pathology Univ of Helsinki, Helsinki, Finland; Dept of Surg, Helsinki Univ Hosp, Helsinki, Finland; Dept of Surg Helsinki Univ Hosp, Helsinki, Finland; Dept of Biomed Univ of Helsinki, Helsinki, Finland, Background. NO synthase (NOS) is expressed in ulcerative colitis (UC). The aim was to examine NO production induced in pouchitis, its variation in different clinical forms of pouchitis and its correlation with histopathological changes of pouchitis. Methods. Biopsies were taken from pouch and from ileum before pouch reconstruction. The diagnosis of pouchitis was based on clinical, endoscopic and histological criteria. Patients were divided in five different groups:1. ileum from UC patients(N=8), 2. nopouchitis (N= 15), 3.chronic asymptomatic pouchitis (N =8), 4.chronic active pouchitis (N= 11),5. acute pouchitis (N= 11). NOS-2 immunoreactivity and acute inflammation were graded from 0 to 3 by investigators unaware of the clinical or histological data. Results. In 6 out of 8 specimens of group 1, no NOS-2 immunoreactivity was observed. Two specimens showed an occasional villus profile with some reactivity in the epithelium. In group 2, several specimens lacked NOS-2 reactivity, while in others some villi or crypts with NOS-2 immunoreactive epithelium were seen. In group 3, areas of NOS-2 immunoreactive epithelium were consistently observed in most specimens. In group 4, only one specimen lacked NOS-2 immunoreactivity, all others regularly showed moderate to extensive epithelial NOS-2 staining. Group 5 showed NOS-2 staining pattern similar to that of group 4, there being unexceptionally moderate or abundant epithelial NOS-2 staining. NOS-2 immunoreactivity scores were 0.25 :!: 0.16, 0.67 :!: 0.19, 1.19 :!: 0.40,2.0 :!: 0.23, and 2.18 :!: 0.12 in groups 1-5, respectively. The values of both group I and 2 differed significantly from those of groups 4 and 5 (p
hydrophobicity distalcolon (0) penneabillty mannitol (nmol/mg tissue) dextran (pmol/mg tissue)
control
PEG
mannitol
senna
48±2
61±2'
48±3
43±1'
26±O1
1.9±O.1'
28±O,3
46±O,4'
15,1±O,9
10,6±O.5'
138±14
211±1.7'