Increased epithelial cell proliferation in the ileal pouch mucosa of patients with familial adenomatous polyposis To eliminate the risk of colorectal cancer in patients with familial adenomatous polyposis -LRB- FAP -RRB- , reconstructive proctocolectomy is performed . Although most colonic mucosa is resected during the ileal pouch anal anastomosis , adenomas and carcinomas may develop in the pouch . This may be caused by altered cell kinetics due to intraluminal changes in the pouch . In 32 patients with FAP , biopsy specimens from the mucosa of the pouch and also of the afferent ileal loop were taken . Tissue sections were immunohistochemically processed with the monoclonal antibodies M30 and MIB-1 to assess apoptotic and proliferative indices , respectively . Cell proliferation was also assessed by a modified sign test . There were no significant differences in apoptotic rates between the mucosa of the pouch and the mucosa of the afferent ileal loop . However , cell proliferation was significantly higher in the mucosa of the pouch vs afferent ileal loop , both by using the quantitative -LRB- 68.3 % vs 61.6 % , p = 0.001 -RRB- and semiquantitative methods -LRB- p < 0.05 -RRB- . Our newly developed semiquantitative approach outperformed previously described methods . The higher cell proliferation in the pouch as compared to the afferent ileal loop may contribute to the increased risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopic surveillance . Introduction Familial adenomatous polyposis -LRB- FAP -RRB- is an autosomal-dominant disease with an estimated prevalence of 1 in 5,000 to 1 in 7,500 -LSB- 5 -RSB- . It is characterized by the development of hundreds of adenomas in the large intestine . Without surgical intervention , virtually all patients will develop colorectal cancer at relatively young age . To eliminate the risk of colorectal cancer , a restorative proctocolectomy with ileal pouch anal anastomosis -LRB- IPAA -RRB- is accepted as one of the surgical treatments of choice in these patients . In the IPAA procedure , almost all colonic mucosa is resected , thus reducing cancer risk significantly . However in patients with FAP , adenomas are also present in the upper gastrointestinal tract ; approximately 5 % of the patients having gastric , 60 to 90 % duodenal -LSB- 4 -RSB- , 50 % jejunal -LSB- 14 -RSB- , and 9 to 25 % ileal adenomas -LSB- 14 , 23 , 34 -RSB- . Occurrence of ileal adenomas is of special interest because the terminal ileum , which is used to construct the pouch , may already contain adenomas . There is accumulating evidence that adenomas develop in the pouch . The reported incidence after a follow-up of at least 5 years is 8 to 60 % , increasing up to 75 % in certain subgroups -LSB- 14 , 26 , 33 , 38 -RSB- , which is much higher as compared to the reported incidence of 9 -- 25 % in the preoperative ileum of patients with FAP -LSB- 14 , 23 , 34 -RSB- . Therefore , it seems that in the ileal pouch of patients with FAP , the development of adenomas is accelerated . At present , the malignant potential of the pouch adenomas is unclear . However , 13 patients with a carcinoma in the pouch have been recently described in the literature -LSB- 2 , 3 , 7 , 9 , 13 , 24 , 25 , 29 , 35 -- 37 -RSB- . Stasis in the pouch causes a change in the luminal content , which is in close contact with the ileal mucosa . There is an increase in the concentration of short-chain fatty acids to colonic levels -LSB- 10 -RSB- , an increase in anaerobic bacterial counts resulting in a more colonic type of flora , and an increased deconjugation and dehydroxylation of bile acids by anaerobic bacteria -LSB- 20 , 21 -RSB- . The intestinal flora could play a role in initiation and promotion of colon cancer by activation of various classes of carcinogens , such as nitrosamines , polycyclic aromatic hydrocarbons , and glycosides -LSB- 17 -RSB- . A direct link between intestinal bacterial flora and tumor promotion in the APCMin / + mouse model for FAP has already been established -LSB- 22 -RSB- . Therefore , we hypothesize that the intraluminal ecological changes in the pouch are responsible for enhanced tumor formation in the mucosa of the pouch . Alteration of cell kinetics in the mucosa , including a lower rate of apoptosis and a higher rate of cell proliferation , is associated with tumor initiation and promotion . In colorectal cancer , the balance between apoptosis , cell proliferation , and cell shedding is disturbed , and such changes may favor the development of adenomas -LSB- 30 -RSB- . Up to now , no data are available on changes in proliferation and apoptosis rates of the ileal mucosa of the pouch after IPAA in patients with FAP . The aim of this study is to investigate changes in apoptosis and cell proliferation rates , occurring in the mucosa of the pouch of patients with FAP , in comparison with the ileum of the afferent loop . The results may contribute to a better understanding of the enhanced adenoma formation in the pouch compared to normal ileum . Materials and methods Patients and tissues The study was approved by the regional medical ethical commission , and informed consent was obtained from all patients . Patients with FAP and an IPAA , who were under surveillance in the Radboud University Nijmegen Medical Centre or regional affiliated hospitals , were invited to participate in this study . Thirty-two patients with FAP were included . The diagnosis FAP was based on either a clinical presentation of at least 100 colonic adenomas or a mutation in the APC gene . Data concerning the surgical procedures were obtained from medical records . From each patient , mucosal biopsy specimens of both the pouch and the afferent ileal loop were obtained during a regular surveillance endoscopy , in the period January 2002 until April 2004 . Patients were fasted overnight . On the day of examination , patients were encouraged to drink liberally . No laxatives or cathartic enemas were given . To clear the pouch of fecal ruminants , two 250-ml water enemas were given before the endoscopy . The endoscopy was performed with an Olympus GIF-1T140 video endoscope . From January 2002 until August 2003 , a 2.8-mm diameter biopsy forceps -LRB- FB 13K-1 Olympus , Tokyo , Japan -RRB- was used , and from September 2003 until April 2004 , a 3.0-mm diameter biopsy forceps -LRB- B102-C1-30.160 MedWork/Treier Endoscopie GA , Beromünster , Switzerland -RRB- was used . The afferent loop was introduced up to 20 cm proximal of the pouch . The mucosa was sprayed with 1 % indigo carmine dye -LRB- Laboratoires SERB , Paris -RRB- at 1:1 dilution with water , where after photographs were taken to evaluate number and size of adenomas present . For pathological examination , at least four biopsies were taken at random locations from the afferent ileal loop -LRB- 10 to 20 cm proximal from the pouch -RRB- , four biopsies from the pouch mucosa -LRB- at least 5 cm proximal from the anal verge -RRB- , and four biopsies from adenomas if present . The biopsies from adenomas were used only for pathological evaluation , i.e. , to exclude serious dysplasia in pouch adenomas , and were not used for research purposes . The biopsies were stretched on filter paper to maintain correct orientation of crypts , fixated in formalin , and embedded in paraffin . Immunohistochemistry Cell proliferation and apoptosis were measured in the stretched formalin-fixed nonadenomatous tissue . The crypt cell proliferation activity was assessed after staining with the monoclonal antibody MIB-1 -LRB- Dako A/S , Glostrup , Denmark -RRB- , which recognizes the Ki-67 nuclear antigen of dividing cells in formalin-fixed paraffin-embedded tissue -LSB- 8 -RSB- . Apoptosis was assessed by staining with the monoclonal antibody M30 -LRB- Roche Diagnostics , Mannheim , Germany -RRB- , which recognizes cleaved cytokeratin 18 . Immunoreactivity of M30 is confined to the cytoplasm of apoptotic epithelial cells and is expressed during early apoptosis -LSB- 18 -RSB- . Tissue sections of 4-μm thickness were cut from paraffin blocks , mounted on electrostatic slides -LRB- Super Frost Plus , Menzel-Gläser , Germany -RRB- , and dried overnight , followed by drying in a stove at 50 °C for 15 min . Sections were put in xylol for 10 min and taken from xylol through 100 % alcohol to water . After deparaffinization , endogenous peroxidase was blocked by treatment with 3 % hydrogen peroxide in phosphate-buffered saline -LRB- PBS -RRB- for 30 min . Pretreatment was performed by heating the tissue sections in citrate buffer -LRB- 10 mmol/l , pH 6.0 -RRB- at 180-W power in a microwave oven for 10 min . After cooling at room temperature for 1.5 h , sections were rinsed with PBS . Then , 20 % normal horse serum -LRB- Vector Laboratories , Burlingame , CA , USA -RRB- was applied for 10 min . The sections were then incubated overnight at 4 °C with either the mouse monoclonal antibodies MIB-1 at 1:1,000 dilution or M30 at 1:100 dilution . Thereafter , the sections were rinsed in PBS for 10 min and incubated with biotinylated horse anti-mouse IgG -LRB- Vector -RRB- for 30 min . After rinsing for 10 min in PBS , incubation with ` avidin and biotinylated horseradish peroxidase macromolecular complex ' -LRB- Vector -RRB- was performed for 45 min . Finally , 5 mg/10 ml diaminobenzidine was used as chromogen , and the sections were put in 0.5 % CuSO4/0 .9 % NaCl for 5 min to intensify the staining . Mayer hematoxylin counterstaining was applied . Sections were dehydrated through 100 % alcohol , cleared in xylol , and coverslipped with permount -LRB- Fisher Scientific , Fiar Lawn , NJ , USA -RRB- . Tissue sections of rectal carcinoma were used as positive controls . Evaluation of immunostaining results Investigators were blinded for the origin of the tissue sections , regarding patient and biopsy location . For evaluation of M30 staining , tissue sections were examined by light microscopy . M30 positivity was identified as brown cytoplasmic staining . M30-positive cells were marked by a first investigator -LRB- BvH -RRB- and reevaluated by an expert pathologist -LRB- IN -RRB- . In all cases , the complete section was evaluated , and all M30-positive epithelial cells were counted . The apoptotic index was expressed as the number of M30-positive cells per tissue area in square millimeters . Tissue area was assessed by using a Zeiss KS400 computer-aided system . In each MIB-1-immunostained tissue section , crypts whose entire length could be visualized were photographed under × 400 magnification using a Zeiss KS400 computer-aided system . Crypts were excluded when they did not reach the muscularis mucosae or had multilayered bases . MIB-1 positivity was identified as brown nuclear staining . The number of MIB-1-positive epithelial cells and the total number of epithelial cells in up to five crypts per tissue section were counted from screen . The labeling index for each crypt was given by the ratio of MIB-1-positive cells and the total number of crypt epithelial cells and is expressed as percentage of total . For each patient , the labeling indices of pouch and afferent ileal loop were expressed as means of three to five counted crypts . If less than three crypts could be photographed for either pouch or ileal loop , the patient was excluded from analysis . The photographed crypts of five randomly selected patients were counted twice by one investigator -LRB- BvH -RRB- to determine intraobserver variability . Although great effort was made to obtain well-orientated mucosal crypts when using the quantitative method , however , not all biopsies reached the criteria mentioned above , and therefore , could not be examined . This problem mainly occurred in the biopsies taken from the pouch and might be due to friability of the pouch mucosa . We therefore developed a new semiquantitative scoring system . A representative part of the biopsies showing several complete crypt/villous axes was photographed under × 100 magnification . The photographs were judged pair-wise -LRB- pouch vs afferent loop -RRB- during which the investigators had to choose from four possible outcomes ; one of the two locations showed most MIB-1 positivity . MIB-1 positivity did not differ or no judgement could be made . Judgement was based on relative length of the area of positive cells and the relative size of the stem cell compartment . Five investigators , two pathologists -LRB- IN , HvK -RRB- , two gastroenterologists -LRB- PF , FN -RRB- , and one junior investigator -LRB- BvH -RRB- independently compared the paired photographs of biopsies of pouch and afferent ileal loop of all patients . When three or more observers agreed in their judgement , this judgement was denoted as consensus judgement . If this criterion was not met , no consensus was reached . If the quality of the tissue sections was poor , no judgment was made . One investigator -LRB- BvH -RRB- judged the whole series twice for evaluation of intraobserver reliability . Statistical analyses Values for apoptosis and cell proliferation in the quantitative study were not expected to be normally distributed ; therefore , they were presented as median and range . The Wilcoxon matched-pairs signed-ranks test was used to compare the paired observations in the apoptosis staining and the paired mean labeling indices in the cell proliferation study . Consensus judgements on the semiquantitative assessment of cell proliferation , favoring either pouch or ileal afferent loop , were compared with a Sign test . To evaluate the reliability of this semiquantitative method to assess cell proliferation , Cohen 's kappa was calculated for the first and second series of judgements by the prime investigator to determine intraobserver reliability . Also , for each pair of investigators , a Cohen 's kappa was calculated . The mean Cohen 's kappa was taken as value for interobserver reliability . Consensus judgments were compared to the difference in mean labeling index between pouch and ileal afferent loop for each evaluable patient . A p value less than 0.05 was considered as significant -LRB- SPSS for Windows 11.0.1 , 2001 -RRB- . Results Patient characteristics Patient characteristics are given in Table 1 . The median age of the 32 patients -LRB- 19 men , 13 women -RRB- included in the study was 32 -LRB- range 16 -- 72 -RRB- years . Table 1Patient characteristics ApoptosisCell proliferationNumber of patients studied3220Male/Female19/1312 / 8Median age in years -LRB- range -RRB- 32 -LRB- 16 -- 72 -RRB- 29 -LRB- 16 -- 62 -RRB- Median age at surgery in years -LRB- range -RRB- 24 -LRB- 10 -- 55 -RRB- 20 -LRB- 10 -- 52 -RRB- Median age pouch in months -LRB- range -RRB- 96 -LRB- 9 -- 216 -RRB- 105 -LRB- 9 -- 216 -RRB- IPAA : hand-sewn/double-stapled / unknown9/21/25 / 14/1Carcinoma at surgery40Patients with adenomas at biopsy : yes/no24/815 / 5For quantitative cell proliferation analysis , 12 patients had to be excluded from analysis -LRB- right column -RRB- due to absence of sufficient evaluable crypts -LRB- see under `` Materials and methods '' -RRB- . Twenty-three patients were operated in the Radboud University Nijmegen Medical Centre . The median age at the time of reconstructive colectomy was 24 -LRB- range 10 -- 55 -RRB- years . A mucosectomy with hand-sewn IPAA was performed in 9 patients , and a double-stapled IPAA was performed in 21 patients . For two patients , the information about the performed technical procedure could not be retrieved . At the moment of colectomy , four patients had a colorectal adenocarcinoma localized in the rectum , sigmoid , hepatic flexure , or appendix , respectively . At the time of endoscopy , the median age of the pouch was 96 -LRB- range 9 -- 216 -RRB- months . The medication used was loperamide by 18 patients , psyllium fibres by 3 patients , and iron , metoclopramide , colestyramine , omeprazole , sulindac , tramadol , tamoxifen , gosereline , nifedipine , metoprolol , furosemide , or losartan each by 1 patient . Thirteen patients were not on medication 3 months before endoscopy . One patient used sulindac , a nonspecific cyclooxygenase inhibitor , which is thought to influence cell proliferation and especially apoptosis -LSB- 15 , 27 -RSB- . Exclusion of this patient from analyses had no effect on the results . Histological examination revealed pouch adenomas in 24 patients -LRB- 75 % -RRB- . Apoptosis In both pouch and afferent ileal loop , M30-positive cells were predominantly detected in the mucosal villi -LRB- Fig. 1 -RRB- . Fig. 1Apoptotic epithelial cells -LRB- encircled -RRB- using M30 immunohistochemistry . Original magnification × 200 -LRB- left -RRB- and × 400 -LRB- right -RRB- In the 32 pairs of biopsies from pouch and afferent ileal tissue investigated , the median absolute number of apoptotic cells in the pouch mucosa was two per tissue section -LRB- range 0 -- 9 -RRB- , which was identical to the values in the afferent ileal loop -LRB- 2 , range 0 -- 19 -RRB- . The median apoptotic index -LRB- expressed as number of M30-positive cells per mm2 tissue section area -RRB- did not differ between pouch -LRB- median 0.4 / mm2 , range 0 -- 2.9 -RRB- and afferent ileal tissue -LRB- median 0.3 , range 0 -- 2.7 ; see Fig. 2 -RRB- . Fig. 2Box-Whisker plots of apoptosis expressed as number of M30-positive epithelial cells per mm2 tissue section area of pouch and afferent ileum of 32 patients with FAP . Difference between groups was not significant . Values are given as median -LRB- fat line -RRB- , lower to upper quartile -LRB- green box -RRB- , and minimum and maximum values -LRB- error bars -RRB- Cell proliferation Quantitative comparison The intraobserver reliability for counting of the photographed crypts -LRB- Fig. 3 -RRB- was rs = 0.855 , p = 0.002 . Fig. 3Detection of proliferating crypt cells with MIB-1 immunohistochemistry . Brown stained nuclei are positive . Nonproliferative crypt stem cells are visualized at the base of the crypt . Original magnification × 400 In 12 pouch tissue sections , less than three crypts were available for counting , and these samples were therefore excluded from analysis . For the same reason , two afferent ileal tissue sections were excluded . In the 20 pairs of pouch and afferent ileum tissue sections left for comparison , median labeling index -LRB- expressed as percentage of MIB-1-positive epithelial crypt cells -RRB- was significantly higher in the pouch as compared to the afferent ileum -LRB- median 68.3 % , range 52.9 -- 79.6 % vs median 61.6 % , range 38.0 -- 73.9 % ; Wilcoxon signed-ranks test , p = 0.001 ; Fig. 4 -RRB- . Fig. 4Box-Whisker plots of cell proliferation expressed as median labeling index -LRB- MIB-1-positive crypt cells per total number of crypt cells -RRB- of 3 -- 5 crypts in tissue sections of pouch and afferent ileum of 20 patients with FAP . Difference between groups was significant -LRB- p = 0.001 -RRB- . Values are given as median -LRB- fat line -RRB- , lower to upper quartile -LRB- red box -RRB- , and minimum and maximum values -LRB- error bars -RRB- Semiquantitative comparison The results of the semiquantitative comparison of the photographed tissue sections from the pouch and afferent ileum mucosae -LRB- Fig. 5 -RRB- are visualized in Fig. 6 . In 15 patients -LRB- 47 % -RRB- , the proliferation was higher in the pouch ; in 7 patients -LRB- 22 % -RRB- , there was no difference ; in 5 patients -LRB- 16 % -RRB- , proliferation was higher in the afferent ileal loop ; in 2 patients -LRB- 6 % -RRB- , no consensus could be achieved ; and in 3 patients -LRB- 9 % -RRB- , no judgement was possible due to poor quality of the tissue sections . These differences were significant -LRB- p < 0.05 -RRB- . Fig. 5Photographs used for semiquantitative comparison of cell proliferation in MIB-1 immunohistochemically stained tissue sections of pouch -LRB- left -RRB- and afferent ileal mucosae -LRB- right ; original magnification × 100 -RRB- Fig. 6Histogram of semiquantitative comparison ; 0 ileal mucosa shows more cell proliferation than pouch mucosa , 1 no difference in cell proliferation between pouch and ileal mucosae , 2 pouch mucosa shows more cell proliferation than ileal mucosa , 3 no judgement possible , 4 no consensus could be reached Intraobserver reliability was κ = 0.87 . Cohen 's kappa for each pair of observers ranged from 0.26 to 0.59 with a mean Cohen 's kappa of 0.38 , which corresponds with `` fair agreement '' following Byrt 's guidelines -LSB- 6 -RSB- . Thus , interobserver reliability was acceptable . In cases in which a labeling index could be assigned to both pouch and afferent ileal loop , consensus judgment was available in 18 cases -LRB- Fig. 7 -RRB- . In all cases in which the semiquantitative analyses showed more proliferation in the pouch -LRB- n = 11 -RRB- , this was confirmed by the quantitative method ; the median value of the difference in proliferation between pouch and ileum was 0.107 -LRB- range 0.032 -- 0.175 -RRB- . In the five cases where no difference was observed with the semiquantitative approach , the median value of the difference in proliferation between pouch and ileum was 0.023 -LRB- range − 0.096 to 0.120 -RRB- . There were only three cases in which the semiquantitative method showed more proliferation in the afferent loop , and the median value of the difference in proliferation between pouch and ileum in these cases was 0.098 -LRB- range − 0.021 to 0.262 -RRB- . The Spearman 's correlation coefficient between the semiquantitative and the quantitative methods was 0.273 -LRB- p = 0.244 -RRB- . Fig. 7Semiquantitative comparison vs difference in mean labeling index of pouch minus afferent ileal mucosa ; 0 ileal mucosa shows more cell proliferation compared to the pouch mucosa , 1 no difference in cell proliferation between pouch and ileal mucosae , 2 pouch mucosa shows more cell proliferation compared to the ileal mucosa Discussion Although both the pouch and its afferent loop contain the same preexisting ileal mucosa , adenomas occur more frequently in the pouch of patients with FAP than in the afferent ileal loop , suggesting an accelerated adenoma formation in the pouch -LSB- 14 , 26 , 33 , 38 -RSB- . To investigate the role of cell kinetics as a possible explanation for this observation , apoptosis and cell proliferation rates in the mucosa of the pouch were compared with those of the afferent ileal loop from the same patient to eliminate bias caused by interindividual differences . Cell proliferation was significantly higher in the pouch mucosa in comparison to mucosa of the afferent ileal loop . No significant difference in apoptosis was found in the mucosa of the pouch and afferent ileal loop . A low amount of apoptotic cells in the pouch as well as in the ileal mucosa was found . This might be caused by the APC mutation-induced apoptotic resistance -LSB- 16 -RSB- . Only a trend but no significant difference in apoptosis was found . Taking into account the broad range of apoptotic rates as found in our study samples , a significant difference might be found when a larger group of patients could be studied . There are no clear guidelines for estimation of ileal proliferation . For estimation of colonic proliferation , an accepted method is to count proliferating cells in five colonic crypts . Although most biopsy specimens were stretched and orientated directly after endoscopy , only a lower number of crypts could be counted completely along the longitudinal axis in most patients . For this reason , we accepted three full crypts as the minimal number to assess proliferation . Using this criterion , tissue sections from 12 patients still could not be used to determine the cell proliferation and had to be excluded from the study . Especially in the tissue sections of the pouch , this problem was evident and is possibly caused by a higher fragility of this tissue . In the remaining 20 pairs of tissue sections left for comparison , we found a significantly higher median labeling index in the pouch compared to the ileal afferent loop . To overcome the problem of the relative low number of assessable crypts , another quicker but less quantitative method was applied in which five investigators compared photographs of tissue sections of pouch and ileal mucosae . In accordance with the results of the first method , we found significantly higher cell proliferation in the mucosa of the pouch compared to that of the ileal mucosa . Although no significant correlation could be found in a case-by-case comparison between both methods , in all cases in which the semiquantitative analyses showed more proliferation in the pouch , this was confirmed by the quantitative analyses of crypts . Furthermore , the inter - and intraobserver variability was good . In addition , the semiquantitative method is far less time-consuming and can therefore give a relatively fast and easy impression of eventual differences in cell proliferation . Evaluation of this method in a larger study may further demonstrate its value . Data on cell proliferation in ileal mucosa in patients with FAP are limited . Previously , de Silva et al. -LSB- 11 -RSB- reported a labeling index of 19.8 % in afferent ileal loop mucosa of patients with FAP or ulcerative colitis -LRB- UC -RRB- , far lower than the 61.6 % that was found here . Their labeling index for cell proliferation in ileal pouch mucosa -LRB- 33.6 % -RRB- was also much lower than in the present study . Only the labeling index of 51.7 % as found by them in pouches with pouchitis nears the values we obtained . However , the majority of patients included in the study of de Silva et al. were patients with UC -LRB- 23 of the 26 patients -RRB- , so direct comparison with our results therefore seems inappropriate . In addition , Goldberg et al. -LSB- 12 -RSB- reported a median labeling index of 34.9 % in the ileal pouch of patients with FAP , which is much lower than the 68.3 % we found . However , their labeling indices were based on a minimum of three counted crypts , and if this number could not be reached , halves of crypts were included in the analyses . Moreover , their study group consisted of only 5 patients with FAP , whereas in the present study , 3 to 5 whole crypts of 20 patients were counted . Several studies indicated that cell proliferation of normal-appearing colorectal mucosa of patients with adenomas or carcinomas was 19 to 86 % higher compared to colorectal mucosa of healthy persons -LSB- 1 , 28 , 30 , 32 -RSB- . These findings strongly suggest an association between the presence of adenomas or carcinomas and an increased mucosal cell proliferation . However , the difference in cell proliferation between pouch and afferent ileal mucosae of 6.7 % as found in this study is less pronounced in comparison to the above referred findings . A possible explanation for this relatively small difference in cell proliferation between pouch and ileal mucosae in our study may be that intestinal epithelial cell proliferation is already very high in patients with FAP -LSB- 19 , 31 -RSB- , and these high cell proliferation rates make a further increase less pronounced . The higher proliferation found in the pouch mucosa in comparison to mucosa of the afferent ileal loop can only be explained by intraluminal changes that occur after construction of the pouch . Whether changes in bacterial flora , bile acid composition , short-chain fatty acids , or other compounds are responsible for this finding remains unclear , but a better understanding of this process is necessary to find a possible treatment for this group of patients . In conclusion , the increased cell proliferation in the ileal pouch mucosa compared to the mucosa of the afferent ileal loop may contribute to the enhanced risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopical surveillance of the pouch in these patients . In addition , cell proliferation can be used as an early endpoint marker in chemopreventive studies in these patients . The applied new method for semiquantitative evaluation of cell proliferation in immunohistochemically stained tissue sections seems promising , as it offers a relatively fast and easy means of assessment .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O

familial adenomatous polyposis
restorative proctocolectomy
cell proliferation


The role of GlnD in ammonia assimilation in Mycobacterium tuberculosis Summary Introduction Mycobacterium tuberculosis is responsible for the largest number of human deaths from a single bacterial agent . Nearly two million people die from tuberculosis each year and more than eight million are newly infected .1 A better understanding of the basic metabolism of this pathogen could lead to new strategies for eradication . Although progress has been made in understanding some of the nutritional requirements of this organism both in vitro and in vivo , in particular its carbon source acquisition ,2,3 little is known about nitrogen metabolism . M. tuberculosis has four enzymes with glutamine synthetase -LRB- GS -RRB- activity -LRB- GlnA1-4 -RRB- .4,5 Of these , GlnA1 , GlnA3 and GlnA4 synthesise l-glutamine , whereas GlnA2 synthesises the d-glutamine and d-isoglutamine required for cell wall biosynthesis .5 The major GS , GlnA1 , is expressed to a high level and is exported .6 The role of the secreted enzyme is not clear , but it has been suggested that it may play a role in pH modulation , although the biosynthetic reaction does require a source of ATP . GS catalyses the production of glutamine from glutamate and ammonia . Since this reaction requires ATP , it needs to be strictly regulated in the presence of excess ammonia to conserve both energy and glutamate pools in the cell . GS activity can be controlled by several mechanisms , including feedback inhibition , transcriptional control of gene expression and by post-translational modification . In Escherichia coli a regulatory cascade of three proteins , GlnD , PII and GlnE , is involved in the latter mechanism -LRB- Fig. 1 -RRB- . GlnD is a uridylyl transferase which modifies the PII protein . PII in turn controls the activity of GlnE . GlnE is an adenylyl transferase which controls the interconversion of GS and GS-AMP . Transfer of the AMP moiety to the GS enzyme reduces its glutamine synthetic activity . GlnE is also able to deadenylylate GS with the predominant reaction being determined by interaction with the PII protein ; PII promotes the adenylylation reaction , whereas PII-UMP promotes the deadenylylation reaction . In this way the cells can rapidly control GS biosynthetic activity in response to ammonia availability . An M. tuberculosis GlnA1 mutant is auxotrophic for glutamine , and attenuated in macrophages and guinea pigs ,7 suggesting that the assimilation of ammonia via this pathway is required in vivo . We have previously shown that , in contrast to other bacteria including closely related organisms such as Streptomyces coelicolor , GlnE is an essential gene in M. tuberculosis .8 Thus the control of GS activity seems critical to normal growth . Here we show that glnD is not essential and that although it has an effect on GS activity in the cells , it is not required for virulence . Materials and methods Culture M. tuberculosis H37Rv -LRB- ATCC25618 -RRB- was grown in Middlebrook 7H9 plus 10 % v/v OADC supplement -LRB- Becton Dickinson -RRB- and 0.05 % w/v Tween 80 , Middlebrook 7H10 agar with 10 % v/v OADC supplement or TSM media -LRB- 1.5 g/L K2HPO4 , 0.5 g/L KH2PO4 , 0.5 g/L MgSO4 , 0.5 mg/L CaCl2 , 0.1 mg/L ZnSO4 , 0.1 mg/L CuSO4 and 50 mg/L ferric chloride -RRB- supplemented with 10 % v/v OADC and 0.05 % w/v Tween 80.9 Working pH 7.2 . For TSM-high ammonia , 30 mM -LRB- NH4 -RRB- 2SO4 was added ; for TSM-low ammonia , 0.1 mM -LRB- NH4 -RRB- 2SO4 was added , l-amino acids -LRB- alanine asparagine , glutamine and glutamate -RRB- were added to 3 mM . Growth curves were obtained in 12 mm diameter borosilicate tubes with 4 -- 5 ml media and stirring at 250 rpm with an 8 mm flea . Hygromycin was used at 100 μg/ml and kanamycin at 20 μg/ml . Quantitative RT-PCR Probes and primers were designed for quantitative PCR for sigA -LRB- endogenous control -RRB- , amt , glnB and glnD using the software Primer Express -LRB- Table 1 -RRB- . cDNA was synthesised from RNA using RT and random hexamer primers using AMV reverse transcriptase . PCR was carried out in a Taqman 7900 using a standard PCR master mix . For sigA , amt , glnB and glnD , the primer pairs were SigA-R and SigA-F , Amt-R and Amt-F , GlnB-R and GlnB-F , and GlnD-F and GlnD-R , respectively , and the probes used were SigA-T , Amt-T , GlnB-T and GlnD-T . The primer and probe concentrations were first optimised . The optimal primer concentration was 300 nM for all four genes , the probe concentration was 100 nM for sigA and glnB , 125 nm for glnD and 200 nm for amt . In order to measure relative gene expression levels , standard curves for each primer-probe set were generated using genomic DNA . CT values were converted into the equivalent of ng using the standard curve . Control reactions without RT were used to confirm that there was no significant contaminating genomic DNA present . CT values for genomic DNA were converted to ng and subtracted from the plus RT values . In order to standardise the samples to ensure that equal amounts of cDNA were used , each value was standardised to sigA to generate unit-less values . At least three independent RNA samples were assayed in triplicate for each gene . Construction of glnD mutant We used our previous method for generating delivery vectors with a marker cassette .10 The delivery vector was constructed by amplifying two regions flanking glnD such that an in-frame deletion was engineered and cloning them into p1NIL . Primer pairs gap7 CACAACGGATACCACAAC and gap8 CGTCAATGCTGTTGCTGC , and gap5 CAAGACCTGGGGAGACGC and gap6 CAGTTTGTCGGTGCCCTC were used to amplify the upstream and downstream regions and the PCR products were cloned into pGEM EasyT -LRB- Promega -RRB- . The two regions were then excised as KpnI-EcoRI and EcoRI -- HindIII fragments respectively and cloned into the KpnI-HindIII sites of p1NIL thereby deleting 1.7 kbp of the glnD gene . The marker cassette from pGOAL19 -LRB- hyg , lacZ , sacB -RRB- was introduced as a PacI fragment to generate the final delivery vector pKOD3 . A deletion mutant was constructed according to our previous method .10 Briefly , we treated the vector DNA with UV and electroporated M. tuberculosis to generate a single cross-over strain .11 This strain was streaked out without antibiotics and double cross-overs selected and screened on 2 % w/v sucrose and 50 μg/ml X-gal . White colonies were patch tested for hygromycin and kanamycin sensitivity and then screened by PCR for the deletion gene . Potential mutants were confirmed by Southern blotting using XhoI digested genomic DNA and hybridising to a probe derived from the upstream flanking region . GS assays Cell-free extracts were generated using the MiniBeadBeater .12 Culture filtrates were prepared using 0.2 μM filters and the filtrates were concentrated using Centricon 20 units -LRB- Amicon -RRB- . Total GS activity was assayed using the transferase assay .13 GS activity is given in nmol per minute per mg of total protein . Virulence assays THP-1 cells were maintained in culture , treated with PMA to induce differentiation , washed and then infected as described .14 A total of 5 × 105 macrophages were infected at MOIs of 1:50 and 1:5 bacteria to macrophage . Extra-cellular bacteria were removed by washing several times . Determination of the initial inoculum was assessed by plating serial dilutions and the number of intra-cellular bacteria was monitored over 7 days . Mice were infected with approximately 106 viable mycobacteria in 200 μl of pyrogen-free saline via a lateral tail vein . Where appropriate , infected mice were killed by cervical dislocation in accordance with humane endpoint protocols under the Animals Scientific Procedures Act , 1986 -LRB- UK -RRB- . Results Expression of glnD and nitrogen regulation We are interested in the regulatory cascade that controls the activity of GS by post-translational modification . We previously demonstrated that GlnE , an adenylyl transferase which modifies GS , is essential in M. tuberculosis .8,15 The activity of GlnE is modified by the PII protein -LRB- encoded by GlnK or GlnB -RRB- and PII is controlled by the GlnD protein -LRB- Fig. 1 -RRB- . In order to gain a better understanding of this pathway , we extended our work to look at the other members of the cascade . The glnD gene is arranged in an apparent operon with two other genes , amt and glnB -LRB- Fig. 3A -RRB- . The start and stop codons of amt and glnB overlap , but there is a 60 bp gap between glnB and glnD which could theoretically contain a promoter . We wanted to determine if the genes in this region are controlled by ammonia availability . We used RT-quantitative PCR to look at the expression levels of the three genes relative to sigA -LRB- Fig. 2 -RRB- . Amt and glnB were up-regulated three-fold and glnD was up-regulated two-fold in low-ammonia medium as compared to high-ammonia medium . The lesser induction of glnD is not unexpected as it is often seen with genes that are at the 3 ′ end of the operon . Alternatively , glnD could be independently expressed from a promoter located in the intergenic region . Construction of glnD mutant In order to characterise the role of GlnD in the regulatory cascade which controls GS activity , we constructed a deletion mutant . An in-frame deletion of the gene was made in the vector p1NIL and the gene cassette from pGOAL19 containing the hyg , lacZ , sacB genes was inserted . A two-step homologous recombination process was used to generate the mutant .10 The in-frame deletion and expected genotype was confirmed by Southern blotting -LRB- Fig. 3 -RRB- . Out of 32 double cross-overs screened , 12 were mutants . One strain -LRB- Tame 69 -RRB- was selected for further study . We analysed the ability of the glnDΔ strain to grow in various nitrogen sources , since it has been shown that mutations in the control of GS activity can lead to deleterious effects on growth . We measured growth of the deletion mutant Tame 69 with various nitrogen sources . The mutant was able to use all the nitrogen sources and there was no difference in the growth rates from the wild-type strain -LRB- data not shown -RRB- .9 Thus the mutant is not compromised in its ability to utilise any of these substrates . Control of total GS activity In other bacteria , GlnD is involved in the post-translational modification cascade which ultimately controls GS activity by modulating its adenylylation state . It has also been shown to play a role in regulation of nitrogen-controlled genes in Corynebacteria .16 Therefore , we investigated total GS activity of the mutant grown with ammonia or glutamate as nitrogen sources -LRB- Fig. 4 -RRB- to determine if deletion of glnD affected the activity of GS . The majority of GS is found in the culture filtrate in M. tuberculosis6 and our data confirmed that we had higher activity in this fraction than in the cell-free extracts for the wild-type strain . Interestingly , we found that total GS activity was higher in ammonia-rich conditions -LRB- 30 mM ammonium sulphate -RRB- as compared to either low ammonia -LRB- 0.1 mM -RRB- or 7H9 -LRB- glutamate -RRB- in the wild-type strain . This was in agreement with RT-qPCR data for glnA1 which showed increased expression in high-ammonia medium -LRB- Pashley and Parish , unpublished data -RRB- , although glnA2 expression was unchanged .9 There was no significant difference in the total GS activity between the mutant and wild-type in the cell-free extracts . However , in culture filtrates the situation was very different . We found a large reduction in total GS activity in the mutant strain grown in all media except low ammonia -LRB- where the level of activity was at its lowest -RRB- . This was particularly pronounced in ammonia-rich conditions , where the wild-type activity was at its highest . Virulence in macrophages and SCID mice M. tuberculosis glnA1 mutants are glutamine auxotrophs and showed a reduced ability to multiply in macrophages and guinea pigs .7 Since extra-cellular GS activity was reduced in the glnD mutant , we determined whether GlnD played any role in intra-cellular survival . We used two measures of this : -LRB- 1 -RRB- the ability of the mutant to grow in macrophages and -LRB- 2 -RRB- its ability to cause disease in mice . For the macrophage infection assay we used the human macrophage-like THP-1 cell line . Infections were carried out at two different MOI -LRB- Fig. 5 -RRB- . The results showed that there was no difference between the wild-type and mutant strain , indicating that disruption of GlnD function had no effect on intra-cellular survival . We also tested virulence in the SCID mouse model to detect any strong attenuation profile -LRB- Fig. 5 -RRB- . Again , the mutant strain behaved just as the wild-type and was not attenuated . Discussion We have shown that expression of glnB and amt is controlled in response to ammonia levels . The up-regulation of these genes in low-ammonia conditions is in agreement with previous findings in other related bacteria . For example , in Corynebacterium glutamicum , these genes are transcribed as an operon under the control of nitrogen availability ; the operon is switched off in nitrogen-rich media and is turned on during nitrogen starvation .17,18 Our previous results indicate that at least one member of the GS regulatory cascade is essential , as we have been unable to construct GlnE mutants in M. tuberculosis .8,15 Our current hypothesis is that GlnE is required to inactivate GlnA1 because the latter is expressed at such a high level . If it were all enzymatically active , the intra-cellular levels of glutamate and/or ATP would quickly be depleted . In agreement with this we have shown that the adenylylation function is essential , but the deadenylylation function is not -LRB- Pashley et al. , unpublished -RRB- . Deletion of GlnD would result in a lack of uridylyation of the PII protein . Since unmodified PII promotes the adenylylation activity of GlnE , inactivation of GS should still occur in the mutant and therefore deletion of GlnD is possible . However , our hypothesis predicts that PII is likely to be essential since it directly modulates GlnE activity . This concurs with our preliminary evidence that we are unable to make a glnB deletion mutant , in contrast to the relative ease of constructing a glnD mutant -LRB- Pashley and Parish , unpublished data -RRB- . Although the glnD mutant was viable , there were substantial changes in the level of GS activity . Normally , a large amount of GS is exported from the cell during growth .6 Export of GS has been linked to high levels of expression , rather than any specific export mechanism .19 If glnD deletion leads to reduced expression of glnA1 , this would explain why only extra-cellular levels are depleted . The observation that total GS activity in the culture filtrates was markedly reduced in the glnD mutant is an intriguing one . It is possible that overall expression of GlnA1 is markedly reduced and therefore little of it will be exported to the outside . Alternatively , GlnA1 production could be completely abolished . The enzyme assay we used does not distinguish between the four M. tuberculosis GS enzymes , so GS activity measured could arise from GlnA2-4 . However , this seems unlikely , since it has already been demonstrated that deletion of the glnA1 gene leads to glutamine auxotrophy , implying that all the biosynthetic activity in the cell is GlnA1 .7 It is likely that the GlnD regulatory cascade only controls activity of GlnA1 , although we can not rule out the possibility that GlnA3 activity would also be modulated . However , GlnA2 and GlnA4 will not be controlled by this cascade -LRB- since they can not be modified by GlnE -RRB- . Thus GS activity arising from enzymes other than GlnA1 can not be rule out . Further work to determine the levels of expression of each GS would help to further elucidate the mechanism of control of GS activity in the whole cell . However , the fact that GlnA1 is the major GS in M. tuberculosis confirms the importance of this post-transcriptional regulatory control system . Although extra-cellular GS was markedly reduced in the glnD mutant , the strain was still fully virulent in macrophages and SCID mice . This was a surprising observation , since it has already been demonstrated that GlnA1 is required for virulence .7 Our data indicate that GS is not required at high levels for virulence in the SCID mouse and that the presence of the lower level of intra-cellular GS is sufficient for bacterial survival . It is possible that a defect may be seen in the glnD mutant under conditions in which intra-cellular multiplication is seen or in activated , rather than resting macrophages . However , since we have not measured extra-cellular GS directly during infection , we can not exclude the possibility that extra-cellular GlnA1 is found in the glnD mutant in vivo . Therefore , the attenuation of GlnA1 mutants most likely results from their auxotrophic nature and the lack of availability of glutamine in the host environment . In conclusion , we have shown that glnD is not an essential gene , but that it is required for the normal expression and activity of GS in culture filtrates of M. tuberculosis . Future work to address the question of how deletion of glnD results in altered expression of GS by determining the mechanism of regulation for ammonia-regulated genes is underway .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

nitrogen metabolism
glutamine synthetase


Antibody therapy in renal cell carcinoma The treatment of metastasized renal cell carcinoma -LRB- RCC -RRB- still represents a formidable challenge , despite the development of small molecule , tyrosine kinase inhibitors -LRB- TKI -RRB- that have made a major impact on the disease . Although the percentage of patients achieving a partial response or stabilization of disease has been impressive , these effects are mostly non-durable . Additionally , drug-related side effects can be quite severe . Alternative treatment modalities might be monoclonal antibodies -LRB- mAbs -RRB- . mAbs against RCC-associated antigens have been developed and have shown promise . Additionally , current efforts focus on Bevacizumab that recognizes vascular endothelial growth factor -LRB- VEGF -RRB- . VEGF overexpression in RCC provides the opportunity to inhibit this proangiogenic pathway . Also with Bevacizumab , promising results have been obtained , particularly in combination with other treatment modalities . It is likely that mAbs , either as single agents or in combination with other agents , may become useful additions to the armamentarium to diagnose and treat RCC . Introduction The hypothesis by Ehrlich in early 1900s that malignant cells express unique structures that can be used to guide cytotoxic therapy to tumors -LSB- 1 -RSB- followed by the development of the hybridoma technique by Kohler and Milstein almost 70 years later -LSB- 2 -RSB- has led to the development of anti-cancer reagents with unique characteristics . One of the most distinguishing factors is the possibility to select monoclonal antibodies -LRB- mAbs -RRB- recognizing target molecules with very restricted expression in normal tissues . To date , tumor-specific antigens -LRB- antigens expressed on all tumor cells of a particular tumor type not expressed by normal cells -RRB- have not been identified . The members of the so-called cancer-testis family do exhibit highly tissue-restricted expression , but are considered promising target molecules for cancer vaccines , less for antibody therapy , particularly in view of the extreme intra - and inter-tumor heterogeneity -LSB- 3 -RSB- . Similar to other malignancies , monoclonal antibodies -LRB- mAbs -RRB- targeting renal cell carcinoma -LRB- RCC -RRB- associated molecules were developed without understanding the molecular events underlying RCC -LSB- 4 -- 8 -RSB- . The increased understanding of molecular events important in the carcinogenesis of RCC led to the recognition that these aberrations can be used to target RCC . Specifically , aberrant von Hippel-Lindau -LRB- VHL -RRB- gene expression has been identified as a general event in clear cell RCC -LRB- ccRCC -RRB- -LSB- 9 -RSB- , which represents 80 -- 85 % of localized cases and 90 -- 95 % of metastatic RCC -LRB- mRCC -RRB- . The loss of a functional VHL gene product leads to accumulation of the transcription factor HIF-1α that is an obligatory element for the transcription of several genes . This includes vascular endothelial growth factor -LRB- VEGF -RRB- and carbonic anhydrase 9 -LRB- CA9 -RRB- , targets for which most clinical experience with mAbs in RCC has been generated -LRB- Bevacizumab and G250 , respectively -RRB- . The rationale and effects of Bevacizumab and G250-directed therapy are fundamentally different : Bevacizumab treatment leads to VEGF-depletion and consequently to diminished neovascularization followed by tumor cell death , mainly due to loss of vascularization . In contrast , G250 treatment targets the cell surface of RCC cells where it must exert toxic effects . Both approaches have advantages and disadvantages . Bevacizumab treatment has the advantage that VEGF depletion can be achieved in the circulation , and homing to all tumor vessels is not necessary . However , other regulatory pathways can also lead to neovascularization and small , non-vascularized tumor loci will not be affected . G250 treatment has the advantage that RCC cells can be targeted , irrespective of tumor size . However , in view of the generally poor perfusion rate and high interstitial fluid pressure in RCC , deep penetration of tumors may be difficult . Also , since G250-binding alone does not confer a lytic signal to RCC cells , tumor cell kill requires effector cells or coupling of G250 to toxic agents . Bevacizumab Bevacizumab is a humanized mAb against VEGF that binds and neutralizes all of the major isoforms of VEGF -LSB- 10 -RSB- . This prevents VEGF from interacting with its receptors and activation of downstream signaling pathways . This mode of action is thought to lead to regression of existing microvasculature , normalization of mature vasculature , and inhibition of the production of new vasculature -LSB- 11 -RSB- . Whether all these effects are true for RCC is unclear at the moment . Significant protein dose levels are needed to maintain sufficiently high Bevacizumab levels to trap VEFG , the target of Bevacizumab . The first Bevacizumab trial in metastatic RCC -LRB- mRCC -RRB- patients addressed whether Bevacizumab treatment could lengthen the time to progression of disease and the response rate -LSB- 12 -RSB- . Survival was a secondary end point . In this randomized phase II trial , 116 patients with metastatic , refractory clear cell RCC were randomized to placebo , low-dose -LRB- 3 mg/kg -RRB- Bevacizumab , or high-dose -LRB- 10 mg/kg -RRB- Bevacizumab given intravenously every 2 weeks . All patients had prior disease progression while on systemic treatment ; the vast majority had received prior interleukine-2 . Patients with disease progression on placebo crossed over to receive low-dose Bevacizumab . Bevacizumab treatment resulted in a significant prolongation of the time to progression of disease in the high-dose antibody group -LRB- 4.8 months as compared with 2.5 months -RRB- . Possibly , the low-protein dose was inadequate to sufficiently deplete circulating VEGF levels in-between injections , explaining the poor outcome in this group . The study was inadequately powered to show a significant difference in overall survival between groups . Based on this encouraging result , Bevacizumab has been combined with other treatment modalities to augment the therapeutic index . Bevacizumab in combination The AVOREN trial investigated the effects of standard therapy of interferon alfa-2a plus placebo or interferon alfa-2a plus Bevacizumab , administered every 2 weeks at a dose of 10 mg/kg -LSB- 13 -RSB- . In this randomized , double blind phase III trial , 649 patients with first-line mRCC were enrolled . The primary analysis endpoint was assessment of improvement in progression-free survival -LRB- PFS -RRB- , defined as the length of time the tumor did not grow or patient death did not occur . Other endpoints of the study included overall survival , time to progression , time to treatment failure , overall response rate , and safety profile . The addition of Bevacizumab to IFN-α2a significantly increased PFS -LRB- 10.2 vs. 5.4 months -RRB- and objective tumor response rate -LRB- 30.6 vs. 12.4 % ; P < 0.0001 -RRB- . Additionally , the combination treatment showed a trend toward improved overall survival -LRB- P = 0.0670 -RRB- , which leads to the conclusion that the combination of Bevacizumab with IFN-α2a is superior to either of the single treatment regimens in mRCC . The working mechanism explaining the superiority of this combination treatment has not been defined yet , but most likely the superiority is the net effect of the reduction of the immunosuppressive effects due to decreased VEGF levels combined with the immunomodulatory effects of IFN . Considering that the epidermal growth factor receptor -LRB- EGFR -RRB- is also overexpressed in RCC , a multicenter , phase II study evaluated the addition of erlotinib -LRB- Tarceva -RRB- , an EGFR inhibitor , to Bevacizumab in metastatic RCC patients -LSB- 14 -RSB- . Treatment consisted of 10 mg/kg Bevacizumab given intravenously every 2 weeks and 150 mg erlotinib given orally each day . With 15 -LRB- 25 % -RRB- patients showing objective responses , and an additional 36 patients -LRB- 61 % -RRB- with stable disease after 8 weeks of treatment , a randomized phase II trial was performed evaluating Bevacizumab + placebo versus Bevacizumab + erlotinib . Disappointingly , identical response rates and PFS rates for the two arms were observed -LSB- 15 -RSB- , and it is doubtful that EGFR-targeting is of any benefit . The effect of Bevacizumab and low-dose interleukine-2 -LRB- IL-2 -RRB- in mRCC was evaluated in a phase II trial in previously untreated , good and intermediate risk , mRCC patients . Patients received 8-week cycles of IL-2 -LRB- 250,000 U/kg per day s.c. Day 1 -- 5 during week 1 and 125,000 U/kg per day s.c. Day 1 -- 5 during weeks 2 -- 6 , followed by a 2 week break -RRB- , and Bevacizumab 10 mg/kg was administered i.v. every 2 weeks starting on day-14 . With 16 of the planned 35 patients enrolled , and 11 evaluable patients for response , 1 partial response -LRB- PR -RRB- and 3 stable disease -LRB- SD -RRB- lasting > 3 months were observed -LSB- 16 -RSB- . All patients with SD demonstrated some degree of tumor shrinkage . Similar to the working mechanism of the Bevacizumab/IFN combination , the anti-tumor effects are possibly the result of the reduction of the immunosuppressive effects due to decreased VEGF levels combined with the general immune activating effects of IL-2 . Interestingly , treated patients demonstrated an increase in the number of regulatory T cells without effect on DC activation . Larger , randomized studies will be necessary to address the value of this combination treatment . In a phase I trial , Bevacizumab has also been combined with sunitinib -LRB- Sutent ® -RRB- , a tyrosine kinase inhibitor , with the hypothesis that this combination may increase antitumor efficacy by maximizing inhibition of the VEGF pathway . The Bevacizumab dose was kept constant -LRB- 10 mg/kg -RRB- while the sunitinib dose was escalated starting at 25 mg -LRB- escalation with 12.5 mg increments -RRB- . Of 13 patients evaluated for best response , 4 had partial responses , 7 had stable disease , and 2 had PD -LSB- 17 -RSB- . Similarly , the combination of Bevacizumab and the mTOR inhibitor CCI-779 -LRB- Temsirolimus ® -RRB- has been investigated . Patients received 25 mg/week Temsirolimus and 5 or 10 mg/kg Bevacizumab . In 12 evaluable patients , 7 PR and 3 SD were observed -LSB- 18 -RSB- . The encouraging results certainly deserve further testing of these combinations in phase II trials . Monoclonal antibody G250 G250 is a mAb against CA9 , a molecule which is ubiquitously expressed in ccRCC -LSB- 19 -RSB- . CA9 expression in non-ccRCC has also been documented , and there , it is most likely a reflection of -LRB- sustained -RRB- hypoxia -LSB- 20 -RSB- . Clinical efforts with mAb G250 in RCC have focused on radioimmunotherapy and passive immunotherapy . This mAb was described as a mAb recognizing an RCC-associated antigen , absent in normal kidney and homogeneously expressed in most RCC -LSB- 4 -RSB- , most notably clear cell RCC -LSB- 19 -RSB- . In 2000 , the G250 antigen molecule was identified and shown to be CA9 -LSB- 21 -RSB- . The molecular characterization allowed transcriptional regulation studies that revealed a strict dependence of G250 expression on HIF-1α -LSB- 22 -RSB- . Thus , the molecular mechanism responsible for CAIX expression in ccRCC is similar to VEGF , namely due to non-functional VHL protein leading to HIF-1α accumulation and gene expression . The first clinical trials with mAbG250 were already performed and published before the molecular characterization of G250 antigen was achieved . The combined data from the immunohistochemical tissue distribution , animal experiments and ex vivo perfusion of tumor bearing kidneys had provided sufficient evidence to initiate a biopsy-based phase I protein dose escalation trial with murine mAbG250 . The rationale of G250-directed therapy obviously differs from Bevacizumab : Bevacizumab treatment leads to VEGF-depletion and consequently to diminished neovascularization whereas G250 treatment targets RCC cells directly . This first mAbG250 clinical trial demonstrated various pivotal aspects : most notably , virtually no uptake in other tissues resulting in excellent tumor visualization , and very high tumor uptake -LSB- 23 -RSB- . The G250 antibody uptake that was observed was up to 10-fold higher than any other mAb uptake in solid tumors , which led to the design of a phase I/II radioimmunotherapy -LRB- RIT -RRB- trial with murine mAbG250 . RIT led to stabilization of disease in 17 of 33 patients , with tumor shrinkage observed in two patients . Transient liver toxicity was observed , quite likely the result of mAbG250 liver uptake , although there was no correlation between the amount of 131I administered or hepatic absorbed radiation dose and the extent and nature of hepatic toxicity -LSB- 24 -RSB- . Because the murine G250 antibody was highly immunogenic , restricting multiple injections , mAbG250 was chimerized . The results of the phase I protein dose escalation trial with chimeric G250 -LRB- cG250 -RRB- basically duplicated the results from the murine G250 trial : virtually no uptake in other tissues resulting in excellent tumor visualization , and very high tumor uptake . The half-life of the antibody was extended , as was to be expected , but , more importantly , the chimerized from of G250 was almost immunosilent -LSB- 25 -RSB- . Thus , multiple injections became possible . Various phase I and phase II trials have been performed with cG250 aimed at therapeutic intervention . Based on the very high uptake levels , several RIT trials were performed . In the first phase I trial with 131I-cG250 , one patient showed a partial response -LRB- > 9 months -RRB- -LSB- 26 -RSB- which set the stage for phase II RIT trials in metastatic RCC patients . RIT studies with single high dose 131I-G250 , rapid fractionated dose 131I-G250 -LSB- 27 -RSB- , and sequential high dose 131I-G250 -LSB- 28 -RSB- have resulted in only occasional therapeutic responses , although dosimetric analyses suggest that tumor-sterilizing levels can be reached . Even two sequential high-dose treatments with 131I-G250 did not result in objective responses , but in stabilization of previously progressive disease in a few patients . RIT with G250 has been accompanied by bone marrow toxicity similar to mAb RIT in other tumor types and considering the minimal benefit , 131I-based RIT with cG250 have been abandoned . Since RCC is a radiotherapy resistant tumor , possibly even higher radiation doses are necessary to achieve tumor-sterilizing levels . Current G250 RIT efforts are directed to 177-lutetium and 90-yttrium labeled G250 . It is hypothesized that the use of more powerful radionuclides that are also better retained in the tumor cells may lead to clinical responses . Animal experiments have demonstrated the superiority of 177Lu - and 90Y-labeled G250 over 131I-G250 -LSB- 29 -RSB- . Importantly , stabilization of previously progressive disease has been observed in almost all 177Lu-G250 treated patients , although the maximum tolerable 177Lu dose has not been achieved . Dosimetric analyses of the first patients treated with 177Lu-G250 suggest that indeed tumor-sterilizing levels may be achieved . Figure 1 illustrates targeting of 177Lu-G250 in a patient with metastatic renal cancer . Fig. 1Anterior -LRB- left -RRB- and posterior -LRB- right -RRB- whole body scans acquired 7 days post-injection of patient injected with177Lu-cG250 . Please note high uptake in both pulmonary lesions and in contralateral kidney lesions . Uptake in liver is due to the conjugation methodology and is not related to G250 antigen expression In view of the obvious tumor-specific accumulation of cG250 , passive immunotherapy of RCC patients has also been studied extensively . In vitro mAbG250 can elicit antibody-dependent cellular cytotoxicity -LRB- ADCC -RRB- , which can be enhanced by low dose IL-2 -LSB- 30 -RSB- . Various -LRB- non-randomized -RRB- clinical trials have now been completed with cG250 alone , and in combination with IL2 or interferon -LSB- 31 , 32 -RSB- . Thus far , these treatments appear to lead to extended survival time . The apparent clinical benefit appears to be quite substantial with a documented median survival of 22 months in patients with metastatic RCC who have progressive disease at study entry . Nevertheless , it is difficult to judge the value of this treatment . Clearly , randomized trials are necessary to unequivocally demonstrate whether passive immunotherapy with cG250 is of benefit for metastatic RCC patients . The largest trial , which is currently ongoing , is the adjuvant ARISER trial -LRB- adjuvant Rencarex immunotherapy phase III trial to study efficacy in nonmetastatic renal cell carcinoma -RRB- . In this phase III randomized , double blind , placebo-controlled trial , patients with ECOG performance status of 0 with completely resected primary clear cell RCC and no evidence of remaining local or distant disease , are treated . The study is designed to detect a significant difference between the two treatment arms with respect to disease-free survival ; patients will be followed-up long-term to determine overall survival statistics . Recently , the potential utility of mAbG250 as a diagnostic imaging agent was investigated -LSB- 33 -RSB- . The excellent imaging capability had been noted in almost all patients , but this line of research was not pursued mainly because detection of suspect renal masses and occult metastatic RCC was not deemed advantageous . Additionally , treatment modalities for metastasized RCC were poor , and , therefore , efforts focused on treatment . However , with a steady increase of incidentally discovered renal masses and new therapeutic modalities becoming available , imaging might become of importance to distinguish more potentially malignant tumours from less aggressive variants . In the first prospective clinical trial with 124I-labeled cG250 , a very high specificity and sensitivity to identify ccRCC in patients with suspect renal masses was demonstrated , a clear indication of the potential clinical utility . Whether this imaging modality can be used to follow therapy effects remains to be determined . In conclusion , it is reasonable to assume that Bevacizumab and G250 monoclonal antibodies either as single agents or in combination with other agents may become useful additions to the armamentarium to diagnose and treat -LRB- cc -RRB- RCC . Several trials evaluating the combination of G250 or Bevacizumab with registered RCC treatments are currently in progress and will further define the role of these mAbs in RCC .

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O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O 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O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O B O O O O O O O O O O O O O O O B O

monoclonal antibody
renal
rcc
vegf
g250
caix
therapy