Patent Application: US-57639600-A

Abstract:
interstitial cystitis is a chronic bladder disease for which the exact etiology is unknown and for which there is no reliably effective treatment . however , it is known that the bladder epithelium is often abnormal in ic . we discovered that normal , adult , human bladder epithelial cells are inhibited from proliferating by an anti - proliferative factor present in ic urine specimens . inhibited proliferation may cause epithelial abnormalities characteristic of ic such as ulcerations and multiple tears in the bladder epithelium . we further discovered that levels of heparin binding — epidermal growth factor - like growth factor , a factor known be important for epithelial cell proliferation and wound healing in other tissues , are abnormally low in the urine of patients suffering from ic as compared to asymptomatic controls or patients with acute bacterial cystitis . the invention herein is directed to the use of urine levels of hb - egf as a diagnostic marker for ic .

Description:
interstitial cystitis ( ic ) is a chronic bladder disease for which the etiology is unknown and for which there is no effective and reliable therapy . the bladder epithelium is often abnormal in ic . therefore , we reasoned that the levels of epithelial growth factors such as heparin - binding epidermal growth factor - like growth factor ( hb - egf ) might be important for bladder epithelial proliferation . elisas were used to determine levels of heparin binding epidermal growth factor - like growth factor ( hb - egf ) as well as other growth factors in urine specimens from women with ic , asymptomatic control women without bladder disease , and women with acute , self - limited bladder epithelial damage from bacterial cystitis . the levels of the other growth factors assayed in urine from ic patients proved to be slightly elevated when compared to urine from normal and bacterial cystitis controls ( see fig2 - 4 ). however , urine hb - egf levels were specifically and significantly decreased in ic patients as compared to asymptomatic controls or patients with bacterial cystitis , whether expressed as concentration ( amount per volume of urine ) or the amount relative to creatinine in each specimen ( see fig1 ). these findings indicate that complex changes in the levels of urine growth factors are associated with ic , including significant and specific decreases in hb - egf levels in the urine of ic patients . with the above information in hand , we proceeded to determine whether the measurement of hb - egf levels in urine could be used as a diagnostic for ic . an optimal sensitivity and specificity of 0 . 84 and 0 . 82 , respectively , can be achieved at a cut - off value of 2 . 9 ng of hb - egf per ml of urine , making urine hb - egf measurements useful for a diagnostic assay for ic . ic patients were referred by physicians , the national institute of diabetes and digestive and kidney diseases ( niddk ), and the interstitial cystitis association . all ic patients had previously undergone diagnostic cystoscopy , and fulfilled the niddk diagnostic criteria for ic 28 . for preliminary studies performed at the university of maryland school of medicine , urine was collected from the ic patients at least three months following the most recent know bacterial urinary tract infection and one month following the last antibiotic use . age -, race -, and sex - matched controls were - volunteers with no history of ic or other urological disease . each control patient was required to have no symptom of urinary tract infection or antibiotic use for the last month . urine specimens collected at the university of pennsylvania for additional studies were obtained during routine office visits for management of ic . patients with acute bacterial cystitis were identified at the university of maryland school of medicine and the university of maryland — college park by the presence of bacterium (& gt ; 10 3 bacteria / ml with single type of bacterium isolated ) plus pyuria in combination with appropriate symptoms . twelve ( 12 ) of the fifteen ( 15 ) patients has & gt ; 10 5 bacteria / ml . all participants were at least 18 years old and enrolled in accordance with guidelines of the institutional review boards at the university of maryland school of medicine , the university of maryland , college park , and the university of pennsylvania . urine was collected by the clean catch method in which each patient wiped the labial area with 10 % povidone iodine / titratable iodine 1 % solution [ clinidine , guilford , conn . ], then collected a midstream urine into a sterile container . specimens obtained at the university of maryland for preliminary studies ( ic patients ; age -, race -, and sex - matched controls ; and bacterial cystitis patients ) were initially kept at 4 degrees c ., then transported to the laboratory where cellular debris was removed by the low speed centrifugation at 4 degrees c . specimens obtained at the university of pennsylvania ( from ic patients only ) and the university of maryland , college park ( from bacterial cystitis patients only ) for confirmatory studies were frozen at − 20 degrees c . for up to 4 weeks , then transported to the university of maryland school of medicine on ice . all specimens were subsequently aliquoted under sterile conditions , and stored at − 80 degrees c . until used . to assay for the levels of hb - egf in urine , each well of a 96 well immulon ii plate ( dynatech laboratories , chantilly , va .) was coated with 200 λ urine at 4 degrees c . overnight . following 5 washes with phohsphate buffer the plates were blocked with 5 % fetal bovine serum / 1 mm edta / 0 . 05 % tween 20 in pbs . anti - hb - egf antibody ( 1 μg / ml ) ( r & amp ; d systems , minneapolis , minn .) was added and the plates were incubated for 2 hours at 37 degrees c . following an additional 5 washes , biotinylated anti - goat igg / avidin d horseradish peroxidase was added and plates were incubated for 1 . 5 hours at room temperature , washed , and developed with abts [ 2 , 2 ′- azino - bis -( 3 - ethylbenzothiazoline - 6 - sulfonic )] substrate . absorbance was read at 405 nm . for determination of egf levels , urine from ic patients and controls was diluted 1 : 200 - 1 : 300 in rd5e diluent and pipetted into wells precoated with monoclonal anti - egf antibody , according to the manufacturer &# 39 ; s instructions ( r & amp ; d systems , minneapolis , minn .). following incubation at room temperature for 4 hours , plates were washed with phosphate buffered saline ( pbs ) and incubated further with hrp - linked polyclonal anti - egf , washed , and developed with tetramethylbenzidine ( tmb ) substrate ; development was stopped with 0 . 2 m h 2 so 4 . total igf1 levels were also measured by elisa ( diagnostic systems laboratories , webster , tx .). urine for these determinations was concentrated 30 - fold by lyophilization and reconstitution in ethanolic hcl in accordance with published recommendations 29 . after 30 minutes incubation at room temperature , samples were centrifuged at 10 , 000 rpm for 3 minutes to remove insoluble material , and supernatant neutralized to ph 7 with neutralization buffer . neutralized , extracted samples were added to wells along with anti - igf hrp - conjugate , and plates were incubated for 2 hours at room temperature . following washes , plates were developed with tmb chromogen solution ; development was stopped with 0 . 2 m h 2 so 4 . for determination of igfbp3 levels , undiluted urine specimens were added to wells precoated with polyclonal anti - igfbp3 , then incubated at room temperature for 2 hours . following washes , another polyclonal , hrp - labeled anti - igfbp3 antibody was added to the wells , and the plates were further incubated , washed , and developed with tmb substrate ; development was stopped with 0 . 2 m h 2 so 4 . for each protein measured , linear absorbance vs . concentration curves were prepared from results with known standard concentrations of recombinant growth factor or growth factor binding protein , and urine sample egf , igf1 , igfbp3 and hb - egf concentrations were plotted . ( see fig1 - 4 ). urinary creatinine was measured by the jaffe method , using picric acid , as previously described 30 . data were then expressed as both the amount of each growth factor or binding protein present per volume of urine or per milligram of urine creatinine . the latter allows the values to be normalized to kidney function ( excretion rate ), thereby eliminating variables due to volume produced ( excretion volume ). for the preliminary studies , comparisons of mean difference in hb - egf levels in urine specimens from ic patients vs . age -, race - and sex - matched controls were performed using a two - way analysis of variance , with age and case - control status as the two factors . for the confirmatory studies with larger sample populations of women with ic , asymptomatic control women , and women with bacterial cystitis , comparisons of mean difference in growth factor levels were performed using a two - tailed analysis of covariance with age as the covariate . logistic regression analysis was performed with case or control status serving as the dependent variable and the amount of hb - egp serving as the independent variable . both hb - egf concentration per milliliter of urine and hb - egf concentration per mg of urine creatinine were analyzed . sensitivity and specificity were derived from the logistic regression model , and the sensitivity and specificity determined for various cutoff values . we determined the levels of hb - egf in urine specimens from women with ic , asymptomatic control women , and women with bacterial cystitis . the quantity of inmmunoreactive hb - egf in the urine of ic patients was markedly decreased as compared to asymptomatic controls , reaching significance at the level of p & lt ; 0 . 001 in both the preliminary analysis ( in which age -, race - and sex - matched asymptomatic controls were used ) and the subsequent larger analysis ( in which women with ic , asymptomatic women , and women with bacterial cystitis were studied ). as shown in fig1 a , the concentration of hb - egf was strikingly lower in ic patient specimens ( 1 . 53 ± 0 . 16 ng / ml ) as compared to asymptomatic controls ( 6 . 33 ± 0 . 82 ng / ml , p & lt ; 0 . 001 ) or patients with bacterial cystitis ( 5 . 15 ± 0 . 98 ng / ml p & lt ; 0 . 001 ), with 37 of 50 ic patients ( 74 %) having levels below 2 ng / ml . the levels of hb - egf were also significantly lower in ic specimens than in urine from either control group when data were expressed per milligram of urine creatinine ( p & lt ; 0 . 001 and p = 0 . 028 , respectively ) ( fig1 b ). sensitivity and specificity measurements ( fig5 ) were derived from the logistic regression analysis , and a sensitivity of 84 % and a specificity of 82 % were achievable at a cutoff value of 2 . 9 ng hb - egf per ml urine . ( a similar analysis of ng hb - egf per mg urine creatinine indicated lower achievable sensitivity of 72 % with a specificity of 75 %). if a cutoff value of 5 . 0 ng hb - egf per ml urine was used , 98 % sensitivity was achievable with a specificity of 59 %. these findings indicate that measuring the concentration of urine hb - egf per ml urine is useful for the diagnosis of ic , either as a single assay with a cutoff of 2 . 9 ng / ml or as a screening assay with a cutoff of 5 . 0 ng / ml . the positive predictive value of the assay at the 2 . 9 ng / ml cut - off point is 72 %; the negative predictive value is 91 %. although differences in urine levels of other growth factors ( such as egf , igf1 ) or binding proteins ( such as igfbp3 ) could also be demonstrated between ic patients and controls , none of these was as sensitive or specific for the diagnosis of ic as differences in urine levels of hb - egf . with respect to the data shown in fig2 studies indicated a trend toward higher mean concentrations of immunoreactive egf in ic specimens ( 16 . 13 ± 1 . 52 ng / ml ) as compared to asymptomatic controls ( 8 . 02 ± 0 . 90 ng / ml ) or patients with bacterial cystitis ( 6 . 99 ± 1 . 31 ng / ml ) ( p & lt ; 0 . 001 for both comparisons ) ( see fig2 a ). similar results were obtained when the amount of egf was expressed per milligram of urine creatinine ( p = 0 . 001 for a comparison of ic and bacterial cystitis patients ) ( see fig2 b ). with respect to fig3 quantities of immunoreactive igf1 in the urine were measured because of the recognized importance of both igf1 and igf2 for bladder epithelial cell proliferation in vitro . a significant increase in urine igf1 levels was evident in ic patients ( 24 . 70 ± 1 . 83 pg / ml ) as compared to asymptomatic controls ( 12 . 11 ± 1 . 08 pg / ml , p & lt ; 0 . 001 ) or specimens from bacterial cystitis patients ( 14 . 97 ± 3 . 58 pg / ml , p = 0 . 01 ) ( see fig3 a ). this finding was similarly true if the amount of urine igf1 was expressed per milligram of urine creatinine ( p & lt ; 0 . 001 and p = 0 . 001 , respectively ) ( see fig3 b ). with respect to fig4 the activity of the igf &# 39 ; s is modified by igfbp &# 39 ; s . igfbp &# 39 ; s or their peptides ( generated by specific proteases ) can have their own direct stimulatory or inhibitory effects on epithelial cells via igfbp receptors . we chose to measure igfbp3 as one of the predominant igfbp &# 39 ; s in urine . our studies indicated that the concentration of igfbp3 was significantly higher in the urine of ic patients ( 13 . 42 ± 2 . 09 ng / ml ) as compared to asymptomatic controls ( 5 . 47 ± 0 . 71 ng / ml , p = 0 . 001 ) ( see fig4 a ). this finding was so true when data were expressed per milligram of urine ceatinine ( 20 . 09 ± 3 . 03 ng / ml vs . 6 . 81 ± 1 . 11 ng / ml , p & lt ; 0 . 001 ) ( see fig4 b ). however , the difference in concentration of urine igfbp3 between ic and bacterial cystitis patients did not achieve statistical significance ( 13 . 42 ± 2 . 09 ng / ml vs . 11 . 02 ± 1 . 54 ng / ml , p = 0 . 55 ) ( see fig4 a ). when expressed per mg of urine creatinine , the difference was statistically significant ( 20 . 09 ± 3 . 03 ng / ml for ic patients vs . 8 . 73 ± 1 . 73 ng / ml for bacterial cystitis patients , p = 0 . 004 ) ( see fig4 b ). the ratio of igf1 to igfbp3 was also calculated for ic patients and their controls . although there was a trend toward a lower ratio in the ic patients &# 39 ; urine than in urine from asymptomatic controls , the difference in igf1 : igfbp3 between the two groups did not reach statistical significance ( p = 0 . 09 ). the limited data that exist for bladder epithelial cell growth suggest that replication and differentiation are influenced by growth factors and regulatory proteins of growth factors . of greatest interest as potential stimulators of bladder epithelial cell replication is hb - egf , which is produced by bladder epithelial cells and can stimulate their growth in vitro 26 , 27 . hb - egf is produced by both kidney and bladder epithelial cells 20 , 24 , 26 . hb - egf is capable of autocrine and / or paracrine activity , having effects on the cell of origin as well as neighboring or distant cells within the urinary tract . hb - egf was specifically decreased in the urine of ic patients as compared to both asymptomatic controls and patients with bacterial cystitis . this decrease could also occur as a result of other inherent abnormalities in ic that secondarily affect hb - egf synthesis which may or may not be causally related to the disease process . because hb - egf is produced by bladder epithelial cells , it is conceivable that urine levels of this growth factor may be secondarily decreased as a result of thinning and denudation of epithelial cells as seen in ic . furthermore , epithelial cell surface glycosaminoglycans , which are commonly decreased in ic 6 , can influence binding to the hb - egf receptor 31 and could therefore influence hb - egf production secondarily . however , hb - egf has been shown to be important for replication of a variety of epithelial cells including hepatocyte , keratinocytes , gastric epithelial cells , and uterine epithelial cells , and is known to stimulate bladder epithelial replication in vitro 18 - 20 , 26 , 27 ; it is therefore possible that decreased synthesis of hb - egf by epithelial or other bladder cells contributes to the pathogenesis of ic by impairing normal bladder epithelial regeneration . ic is currently diagnosed by cystoscopy . although various markers have been suggested for ic , none has yet been shown to be clinically useful . the differences in mean urine concentrations of hb - egf between ic patients and controls were greater than one standard deviation , suggesting that the concentration of hb - egp is useful as a diagnostic marker for ic . determination of assay sensitivity and specificity at different cut - off values confirmed the usefulness of this assay . 1 . hanno , p . m ., staskin , d . r ., krane , r . j ., and wein , a . j ., eds . interstitial cystitis . london : springer - 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gallego , g ., and thomas , k . a . acidic fibroblast growth factor accelerates dermal wound healing . growth factors 7 : 1 , 1992 . 14 . antoniades , h . n ., galanopoulos , t ., neville - golden , j ., kiritsky , c . p ., and lynch , s . e . expression of growth factor and receptor mrnas in skin epithelial cells following acute cutaneous injury . am . j . pathol . 142 : 1099 , 1993 . 15 . werner , s ., peters , k . g ., longaker , m . t ., fuller - pace , f ., banda , m . j ., and williams , l . t . large induction of keratinocyte growth factor expression in the dermis during wound healing . proc . natl . acad . sci . usa 89 : 6896 , 1992 . 16 . nusrat , a ., parkos , c . a ., bacarra . a . e ., godowski , p . j ., delp - archer , c ., rosen , e . m ., and madara , j . l . hepatocyte growth factor / scatter factor effects on epithelia . j . clin . invest . 93 : 2056 , 1994 . 17 . behrens , m . t ., corbin , a . l ., and hise , m . k . epidermal growth factor receptor regulation in rat kidney : two models of renal growth . am . j . physiol . 257 : f1059 , 1989 . 18 . mccarthy , d . w ., downing , m . t ., brigstock , d . r ., luquette , m . h ., brown , k . d ., abad , m . s ., and besner , g . e . production of heparin - binding epidermal growth factor - like growth factor ( hb - egf ) at sites of thermal injury in pediatric patients . j . invest . dermatol . 106 : 49 , 1996 . 19 . marikovsky , m ., breuing , k ., liu , p . y ., eriksson , e ., higashiyama , s ., farber , p ., abraham , j ., and klagsbrun , m . appearance of heparin - binding egf - like growth factor in wound fluid as a response to injury . proc . natl . acad . sci usa 90 : 3889 , 1993 . 20 . homma , t ., sakai , m ., cheng , h . f ., yasuda , t ., coffey , r . j ., jr ., and harris , r . c . induction of heparin - binding epidernal growth factor - like growth factor mrna in rat kidney after acute injury . j . clin . invest . 96 : 1018 , 1995 . 21 . de boer , w . i ., rebel , j . m . j ., vermey , m ., de jong , a . a . w ., and van der kwast , t . h . characterization of distinct functions for growth factors in murine transitional epithelial cells in primary organotypic culture . exp . cell res . 214 : 510 , 1994 . 22 . jorgensen , p . e ., hilchey , s . d ., nexo , e ., poulsen , s . s ., and quilly , c . p . urinary epidermal growth factor is excreted from the rat isolated perfused kidney in the absence of plasma . j . endocrinol . 139 : 227 , 1993 . 23 . southgate , j ., hutton , a . r ., thomas , d . f . m ., and trejdosiewicz , l . k . nornal human urothelial cells in vitro : proliferation and induction of stratification . lab . invest . 71 : 583 , 1994 . 24 . chin , e . and bondy , c . insulin - like growth factor system gene expression in the human kidney . j . clin . endocrinol . metab . 75 : 962 , 1992 . 25 . jones , j . i . and clemmons , d . r . insulin - like growth factors and their binding proteins : biological actions . endocrine rev . 16 : 3 , 1995 . 26 . freeman , m . r ., schneck , f . x ., soker , s ., raab , c ., tobin , m ., yoo , j ., klagsbrun , m ., and atala , a . human urothelial cells secrete and are regulated by heparin - binding epidermal growth factor - like growth factor ( hb - egf ). proc . am . urol . assoc . 153 : 316a , 1995 . 27 . tobin , m . s ., freeman , m . r ., schneck , f . x ., klagsbrun , m ., and atala , a . growth factor biology of human urothelial cells grown under serum - free conditions . proc . am . urol . assoc . 153 : 406a , 1995 . 28 . division of kidney , urologic , and hematologic diseases ( dkuhd ) of the national institute of diabetes and digestive and kidney diseases ( niddk ). diagnostic criteria for research studies ( interstitial cystitis ). am . j . kidney dis . 13 : 353 , 1989 . 29 . third international symposium on insulin - like growth factors . valid measurements of total igf concentrations in biological fluids . endocrinology 136 : 816 , 1995 . 30 . schirmeister , j ., willman , h ., and kiefer , h . plasmakreatinin alf grober indikator der nierenfunktion . dtsch . med . wschr . 89 : 1018 , 1964 . 31 . shishido , y ., sharma , k . d ., higashiyama , s ., klagsbrun , m ., mekada , e . heparin - like molecules on the cell surface potentiate binding of diptheria toxin to the diphtheria toxin receptor / membrane - anchored heparin - binding epidernal growth factor - like growth factor . j . biol . chem . 270 : 29578 , 1995 . all references cited herein are incorporated by reference in their entirety . the examples provided herein are for illustrative purposes only , and are in no way intended to limit the scope of the present invention . while the invention has been described in detail , and with reference to specific embodiments thereof , it will be apparent to one with ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof .