Role of Cervicovaginal Antibody in the Pathogenesis of

Transcription

1 INFECTION AND IMMUNITY, July 198, p /8/7-76/7$2./ Vol. 29, No. 1 Role of Cervicovginl Antibody in the Pthogenesis of Recurrent Urinry Trct Infection in Women LINDA M. KURDYDYK,' KENNETH KELLY,2 GODFREY K. M. HARDING,'3* PATRICIA MIRWALDT,' LINDA THOMPSON,' FREDERICK J. BUCKWOLD,' 3 AND ALLAN R. RONALD' 3 Deprtments ofmedicl Microbiology,' Medicine,3 nd Immunology,2 University ofmnitob, Winnipeg, Mnitob R3E OZ3, Cnd Introitl coloniztion with Enterobctericee is considered to be one of the principl predisposing fctors to recurrent urinry trct infections (UTI) in dult femles. One proposed mechnism llowing introitl coloniztion in these ptients is the bsence of locl cervicovginl ntibody. To test this hypothesis, we exmined cervicovginl wshings from 22 ptients with history of recurrent UTI nd 29 norml controls with no history of UTI for specific locl ntibody by using indirect immunofluorescence. No significnt difference in ntibody ws found in these popultions. Fourteen percent (3/22) of the ptients hd ntibody to their introitl Escherichi coli nd 34% (1/29) of the controls hd ntibody to their fecl E. coli. Consequently, sensitive rdioimmunossy technique ws developed to detect cervicovginl ntibody. A solid phse ws prepred by coupling to Sephrose 4B pool of eight serogroups ofe. coli which re frequently implicted in UTI. Seril dilutions of cervicovginl wshings were rected with the solid phse, nd the bsorbed nti-e. coli ntibodies were detected by the uptke of "MI-lbeled nti-humn immunoglobulin G (IgG) or nti-humn IgA. The ntibody levels were quntitted by interpoltion on stndrd curve prepred by using imnunospecificlly purified humn nti-e. coli ntibodies. IgG nd IgA levels were mesured in wshings from 1 colonized ptients, 13 noncolonized ptients, nd 12 controls. There were no significnt differences in IgG nd IgA levels in cervicovginl wshings mong the three groups. In these studies, introitl coloniztion ws not relted to cervicovginl ntibody. It is generlly ccepted tht recurrent urinry trct infections (UTI) originte from the predominnt fecl Enterobctericee which colonize the vginl introitus nd scend the urethr into the bldder (5, 13). Stmey et l. (12) hve shown tht women susceptible to recurrent urinry infection more frequently crried Enterobctericee on the vginl introitus thn selected controls who hd never experienced urinry infection. We hve lso shown tht norml, helthy femles rrely colonize their distl urethrs with Enterobctericee nd tht the vginl introitus, fourchette, cutneous perineum, nd the distl urethr hve similr microbil flor (7). Susceptibility to introitl coloniztion is considered to be one of the principl fctors predisposing individuls to recurrent UTI. A number of fctors considered to medite introitl coloniztion hve been investigted in recent yers. Stmey et l. nd Stmey nd Timothy (11, 12) demonstrted tht the ph of the vginl introitus nd the glycogen nd potssium concentrtions in the vginl fluid were similr in control nd ptient groups. Additionl studies from the sme group showed no signifi- 76 cnt difference in the frequency of the common indigenous bcteri between the ptient nd control groups (2). Stmey nd Howell (9) used bcteril gglutintion to study the role of cervicovginl ntibody nd did not find ny significnt difference between ptients nd controls. In subsequent investigtion employing n indirect immunofluorescence technique, Stmey et l. (14) were ble to demonstrte difference in locl cervicovginl ntibody between ptients nd controls. Studies with Neisseri gonorrhoee (15) nd orl streptococci (16) hve demonstrted tht locl ntibody cn prevent bcteril dherence to epithelil cell surfces. It hs been hypothesized tht ptients with recurrent UTI my hve diminished or bsent locl ntibody, fcilitting the dherence of Enterobctericee to vginl epithelil cells nd promoting coloniztion, which in turn predisposes to recurrent UTI (2). To further investigte this hypothesis, we ssyed cervicovginl wshings from ptients with recurrent UTI nd from controls with no history of recurrent UTI for specific ntibody by using n indirect immunofluorescence proce-

2 VOL. 29, 198 dure, nd subsequently, more sensitive nd quntittive rdioimmunossy. MATERIALS AND METHODS Study popultion. The ptient group consisted of 29 femles who hd t lest two symptomtic UTI within the preceding 12 months. Twenty-two ptients were colonized with Enterobctericee t the time of collection of cervicovginl wshings. The men ge of the ptients ws 35.7, with rnge of 21 to 66 yers. Intrvenous urogrms were norml in ll but one of 26 ptients. This ptient hd minor clycel distortion. Twenty-six ptients were premenopusl, one hd hd hysterectomy, nd two hd juvenile-onset dibetes mellitus. Thirty-one helthy, nonpregnnt femle volunteers, with no history of UTI, were selected s controls. None ws colonized with Enterobctericee t the time of entry nd during the study. The men ge of the control subjects ws 37.8 yers, with rnge of 19 to 57 yers. Thirty control women were premenopusl, nd two hd hd hysterectomies. Collection nd processing of specimens. Periurethrl, vginl, nd rectl swbs were obtined from ech prticipnt in the dorsl lithotomy position nd then trnsported to the lbortory in 1 ml of phosphte-buffered sline (PBS), ph 7.2. Specimens were not collected during menses or during ntimicrobil therpy. After blending in Vortex mixer,.1- nd.1-ml portions were inoculted onto split sheep blood gr nd McConkey gr pltes. Orgnisms were isolted, quntitted, nd identified by stndrd bcteriologicl methods. The collection nd processing of cervicovginl wshings nd performnce of the indirect immunofluorescence procedure were crried out essentilly s described by Stmey et l. (14). Cervicovginl wshings were collected s 5-ml distilled wter wsh of the cervix nd vgin. This ws ccomplished by inserting speculum into the vgin, then plcing rubber ctheter in the mid-vgin, nd gently injecting nd withdrwing 5 ml of sterile distilled wter in syringe. Specimens were llowed to stnd t 4C for 2 to 3 h, nd the superntnt ws then filtered through.45-gm Nlgene filter. The smples were then lyophilized nd stored t room temperture. Before testing, the specimen ws reconstituted with 1. ml of sterile distilled wter. Cervicovginl wshings to be tested for ntibody by rdioimmunossy were centrifuged t 6, x g for 1 min, nd the superntnt ws stored t -2'C. Indirect immunofluorescence. The controls' rectl Enterobctericee nd the ptients' predominnt introitl Enterobctericee were suspended to concentrtion of 1' colony-forming units (CFU) per ml of PBS. Twenty-microliter portions of the suspensions were plced on clen glss slides, ir dried, nd het fixed. The fixed orgnisms were rected with 2 1d of the individuls' cervicovginl wshings, nd the slides were incubted in moist drk chmber t 37C for 3 min, wshed twice with PBS, ir dried, nd treted with 2 pl of 1:5 dilution of fluorescein-conjugted horse nti-humn gmm globulin (Roboz, Surgicl Instrument Co., Wshington, D.C.). After incubtion t 37C for 3 min, the slides were wshed twice with CERVICOVAGINAL ANTIBODY AND UTI PBS, ir dried, mounted, nd immeditely exmined for fluorescence under oil immersion with x1,25 mgnifiction (Leitz Wetzlr Ortholux II, Ernst Leitz, Cnd, Ltd., Midlnd, Ontrio). Bcteri outlined with n even fluorescent border were recorded s positive (fluorescence subjectively grded s 1+, 2+, or 3+). Specimens were considered positive for the presence of specific ntibody if >1+ fluorescence ws seen on the mjority of cells on the slide. As negtive control, ech orgnism ws rected with the fluorescein lbeled nti-humn gmm globulin without previous exposure to cervicovginl wshings. As positive control, known positive serum ws rected with its specific orgnism with ech run. Rdjoimmimossy. (i) lodintion. Monospecific rbbit ntibodies to humn immunoglobulin G (IgG) nd IgA (Cppel Lbortories Inc., Cochrnville, P.) were isolted by dsorption onto immunosorbents prepred by coupling humn IgG or IgA (Cppel Lbortories) to cynogen bromide-ctivted Sephrose 4B (Phrmci Fine Chemicls, Pisctwy, N.J.). After exhustive wshing of the immunosorbents with PBS, the bound ntibodies were eluted with.4 M glycine-hydrochloride buffer, ph 2.8. The ph of the elutes ws djusted to neutrlity, nd the preprtions were dilyzed ginst PBS, ph 7.4. The monospecificity of both immunospecificlly purified ntiimmunoglobulin ntibody preprtions ws confirmed by Ouchterlony nd immunoelectrophoretic nlyses ginst the purified IgG nd IgA preprtions nd whole humn serum. The purified nti-immunoglobulins were subsequently lbeled with iodine-125 (Rdiochemicl Center, Amershm, Englnd) by the method of Greenwood et l. (4). Approximtely 9 Itg of purified ntihumn IgG or IgA ws lbeled, using 16 jig of chlormine T nd 2. mci of The rection ws stopped by the ddition of 24 mg of sodium metbisulphite. Seprtion of "2I-lbeled rbbit nti-humn immunoglobulins from free "I ws crried out by gel filtrtion (Biogel P6, Bio-Rd Lbortories, Richmond, Clif.). Since the '25I-lbeled nti-immunoglobulin my lose some immunorectivity due to rdition "25I-lbeled nti-immu- dmge on prolonged storge, noglobulin ws freshly prepred monthly. The lbeled mteril ws subdivided into 1-ml portions to void repeted freezing nd thwing nd stored t -2'C. (ii) Solid phse. A pool of eight common urinry serogroups (1, 2, 4, 6, 7, 16, 18, nd 75) ws used s ntigens. The E. coli 2, 16, nd 18 strins were obtined from the Center for Disese Control, Atlnt, G. Types 1, 4, 6, 7, nd 75 were isolted from ptients' periurethrl nd rectl swbs. Cultures were grown t 37C for 18 h in brin hert infusion broth (Difco Lbortories, Detroit, Mich.). A lte log phse culture ws obtined by inoculting 5 ml of brin hert infusion broth with.1 ml of overnight cultures of ech serogroup nd incubting for 18 h. The culture ws hrvested by centrifugtion t 12, x g for 1 min, wshed twice with.5 M NHCO3-N2CO3 buffer (ph 9.7), nd resuspended to concentrtion of 18 CFU/ml of NHCO3-N2X3 buffer. The pooled serogroups were covlently bound to Sephrose 4B by the method of Stge nd Mnnik (8). 77

3 78 KURDYDYK ET AL. To determine the optimum mount of E. coli-sephrose 4B, we coupled incresing mounts of E. coli to Sephrose 4B in the rnge 16 to 18 CFU/ml, solid phse. Volumes of.2 ml of 1:1 suspension (vol/vol) of the solid phse in ssy buffer (.1% bovine serum lbumin,.5% Tween 2,.1% sodium zide,.5 M ethylenediminetetrcetic cid in PBS of ph 7.4) were rected with.2 ml of seril dilutions of positive serum smple (previously shown by immunofluorescence ssy to contin nti-e. coli ntibody) for 16 h t 22C, wshed three times with cold ssy buffer, nd then rected with n excess of '"I-lbeled rbbit nti-humn IgG for 16 h t 22C. The solid phses were wshed three times with cold ssy buffer before the bound rdioctivity ws mesured. There ws no significnt increse in bound rdioctivity in ntibody excess (low serum dilution) when the ntigen concentrtion ws incresed from 17 to 18 CFU/ml of Sephrose 4B (Fig. 1), indicting tht concentrtion of 17 CFU/ml of Sephrose 4B ws sufficient for mximl bsorption of nti-e. coli ntibody. The mount of lbeled nti-immunoglobulin to be used in excess of bound ntibody ws determined by recting.2-ml volumes of Sephrose 4B-E. coli (18 CFU/ ml of pcked gel) with.2 ml of 1:2, dilution of positive serum smple for 16 h t 22C nd wshing three times with cold ssy buffer. The wshed solid phses were then rected with.1-ml volumes of incresing mounts of '"I-lbeled rbbit nti-humn IgG (4. x 14 to 7.7 x 15 cpm) for 16 h t 22C, wshed three times with cold ssy buffer, nd mesured for bound rdioctivity. The counts bound to bsorbed ntibody incresed with the ddition of incresing mounts of lbeled nti-immunoglobulin, reching mximum fter ddition of 3. x 15 cpm (Fig. 2). Therefore, dding mounts in excess of 3 x 15 cpm should ensure ccurte quntittion of bsorbed nti-e. coli ntibody. Subsequently, in the rdioimmunossy the Sephrose 4B-E. coli used consisted of 18 CFU/ml of Sephrose. After incubtion with nti-e. coli ntibody or specimens under investigtion, '25"-lbeled nti-immunoglobulin ws dded in mounts of 4 x 15 cpm per.1 ml of buffer. The Sephrose 4B-E. coli suspension ws stored t 4C nd checked periodiclly for leching of orgnisms. If leching of orgnisms from the solid phse lollk O > 16. < z N 1 o ,536 PURIFIED ANTIBODY RECIPROCAL DILUTION FIG. 1. Estimtion of E. coli ntigen density in CFU/ml ofsephrose 4 B for quntittive bsorption of ntibody in humn serum. z 2 8- n.x A 4 INFECT CPM 251-ANTI-HUMAN IgG ADDED (x5 IMMUN. FIG. 2. Estimtion of the mount of '251-lbeled nti-humn IgG required to sturte nti-e. coli IgG ntibody bound to Sephrose 4 B-E. coli t 18 CFU/ ml ofgel. occurred on storge or due to mechnicl perturbtion of the suspension, the free E. coli ntigen would rect with ntibody in smples nd subsequently inhibit its binding to the Sephrose 4B-E. coli, resulting in low or negtive rdioimmunossy results with respect to ntibody content of the smple. This pplies lso to the stndrd purified nti-e. coli ntibody nd would 12- be detected by reduced uptke of '25I-lbeled ntiimmunoglobulin; hence the need to include the stndrd nti-e. coli ntibody with ech ssy. To remove ny free orgnisms, we repetedly filtered nd suspended Sephrose 4B-E. coli in ssy buffer on corse Buchner funnel nd finlly resuspended it in n equl volume of ssy buffer. (iii) Rdioimmunossy. Cervicovginl wshings were serilly diluted in ssy buffer, nd smples (.2 ml) of ech dilution were dded to duplicte glss culture tubes contining.2-ml volumes of 1:1 suspension (vol/vol) of Sephrose 4B-E. coli in ssy buffer. The tubes were incubted t room temperture (22) for 16 h with gentle shking. The solid phse ws sedimented by centrifugtion t 8 x g for 2 min, the superntnt ws removed by spirtion, nd the solid phse ws wshed three times with 1 ml of cold ssy buffer. I251-lbeled rbbit nti-humn IgG or IgA ws diluted in ssy buffer to concentrtion of 2. x 16 cpm per ml. Smples of.2 ml (4 x 15 cpm) of '251-lbeled nti-immunoglobulin were dded to ech tube. After the solid-phse ws incubted t 22C for 16 h with gentle shking, it ws wshed three times with cold ssy buffer, nd the bound rdioctivity ws counted (Beckmn gmm 8, Beckmn Instruments Inc., Fullerton, Clif.). Nonspecific binding of 125I-lbeled rbbit nti-humn immunoglobulin to the solid phse ws mesured by including controls which hd ssy buffer insted of smple dilutions dded to the Sephrose 4B-E. coli. Specificlly bound rdioctivity ws clculted with the following eqution: % rdioctivity bound = (B - C)/CT x 1, where B = lbeled nti-humn IgG or IgA bound to ntigen, C = nonspecific binding, nd CT = mount of lbeled nti-humn IgG or IgA dded to ech tube. (iv) Preprtion of stndrd curves. Purified nti-e. coli ntibodies were isolted from pooled humn ser by bsorption with Sephrose 4B to which n excess (>112 CFU/ml of pcked gel) of urinry E. coli serogroups hd been coupled. Seril dilutions of 8

4 VOL. 29, 198 the purified ntibody solution were rected with Sephrose 4B-E. coli nd the pproprite "ni-lbeled ntiimmunoglobulin in the rdioimmunossy, nd stndrd curves were prepred by plotting the percent rdioctivity bound ginst the reciprocl dilution of the purified ntibody preprtion. Since the specific ctivity of the "ni-lbeled ntiimmunoglobulin my differ with ech preprtion nd decreses with rdioctive decy nd since it is possible tht rdition dmge could reduce the immunorectivity of the "ni-lbeled ntibody, it is essentil with ech ssy of vginl wshings to include seril dilutions of the stble stndrd of purified nti-e. coli ntibodies. Thus, ntibody levels in specimens could be quntitted reltive to tht of the stndrd ntibody preprtion. Sttisticl methods. Fisher's exct test ws used for sttisticl comprison of the indirect immunofluorescence results, nd one-wy nlysis of vrince ws used for sttisticl comprison of the rdioimmunossy results. Fisher's exct test ws lso used for comprison of the rdioimmunossy nd immunofluorescence ssy results. RESULTS Indirect immunofluorescence. Cervicovginl wshings from 22 ptients with recurrent UTI nd whose introitus ws colonized with E. coli nd from 29 norml controls with no history of recurrent UTI nd whose introitus ws not colonized were tested by indirect immunofluorescence for the presence of specific ntibody. Three of the 22 ptients (14%) demonstrted ntibody to their introitl E. coli in cervicovginl wshings. Of the 29 control subjects, 1 (34%) hd detectble ntibody to their fecl E. coli in cervicovginl wshings. This difference ws not significnt (P =.16). Rdioimmunossy. Antibody levels in cervicovginl wshings were quntitted by interpoltion on stndrd curves. Figures 3 nd 4 show the stndrd curves used for quntitting IgG nd IgA ntibody, respectively. Ech point 6- X U 2\ PURIFIED ANTIBODY RECIPROCAL DILUTION FIG. 3. Stndrd curve for quntitting IgG ntibodies ginst E. coli urinry serogroups in cervicovginl wshings. CERVICOVAGINAL ANTIBODY AND UTI 4- z - ; 2- u - - I ,384 PURIFIED ANTIBODY RECIPROCAL DILUTION FIG. 4. Stndrd curve for quntitting IgA ntibodies ginst E. coli urinry serogroups in cervicovginl wshings. represents the men for the 1 replictes, nd the lines denote the stndrd devitions. In the ssy of ntibody levels in smples, bound rdioctivity ws plotted s function of smple dilution, the points were best fitted to the stndrd curve, nd ntibody levels were determined by interpoltion by using the scending portion of the curve where '251-lbeled nti-immunoglobulin is in excess. Six percent bound ws tken s the lower limit for ssigning positive result; the vlue (6%) represents rdioctivity nonspecificlly bound to the solid phse. Antibody in specimens ws quntitted reltive to the stndrd; reltive levels were reported s the percent bound rdioctivity x the reciprocl cervicovginl wshing dilution. IgG nd IgA ntibodies were quntitted in cervicovginl wshings from 1 femles with history of recurrent UTI nd whose introitus ws hevily colonized with E. coli (215 CFU/ ml of PBS); 13 noncolonized femles with history of recurrent UTI; nd 12 femles with no history of recurrent UTI who were not colonized. Immunoglobulin ntibody levels in specimens from the three groups re shown in Fig. 5. The men level of IgG ntibody ws 17. in cervicovginl wshings from controls, 16.7 from noncolonized ptients, nd 1.8 (P >.5) from colonized ptients. The men IgA ntibody levels were 9.9 for controls, 7.6 for noncolonized ptients, nd 3.9 for colonized ptients (P >.5). Therefore, there were no significnt differences in IgG nd IgA ntibody levels mong the three groups. In ddition, the men IgG ntibody levels were greter thn the men IgA ntibody levels in ech group. Comprison of rdioimmunossy nd immunofluorescence results. Cervicovginl wshings from 1 hevily colonized ptients, 1 noncolonized ptients, nd 8 controls were exmined for the presence of nti-e. coli ntibody by both indirect immunofluorescence nd rdioimmunossy. Since no significnt differ- 79

5 8 KURDYDYK ET AL. ences mong the three groups were found by rdioimmunossy either for IgG or IgA ntibody, the rdioimmunossy results for IgG nti-e. coli ntibody were compred with the immunofluorescence results. Specimens were considered positive by rdioimmunossy for ntibody ctivity if IgG ntibody levels were >6% (rdioctivity nonspecificlly bound). Results of the two methods re shown in Tble 1. More specimens were positive for nti- E. coli ntibody by rdioimmunossy (64.3%) thn by indirect immunofluorescence (17.9%); this difference ws significnt (P <.2). DISCUSSION Introitl coloniztion is considered to be one of the principl predisposing fctors to recurrent UTI (13). It is importnt to determine why femles with recurrent infections re more frequently colonized with Enterobctericee thn norml femles in order tht preventive nd V) >-j CX z LU 6- -j I IgG. x * IT x I*o 'A. hi - - -d I- *I--< x z1 o NZNr4Z R Z P4Z Z-IU ZU V FIG. 5. IgG nd IgA reltive ntibody levels ginst E. coli urinry serogroups in cervicovginl wshings from controls, noncolonized ptients, nd colonized ptients. TABLE 1. Comprison of rdioimmunzossy (RIA) nd immunofluorescence ssy (IFA) results Group RIA totl) (positive/ IFA totl) (positive/ Colonized ptients 6/1 /1 Noncolonized ptients 6/1 4/1 Controls 6/8 1/8 Totl percent positive 64 3% 17.i %v gfisher's exct test, P < INFECT. IMMUN. therpeutic mesures cn be initited. Locl ntibody hs been shown to prevent the dherence of streptococci nd N. gonorrhoee to epithelil cell surfces (15, 18). Reports of the possible role of cervicovginl ntibody in the pthogenesis of recurrent UTI hve ppered in the pst few yers. Bcteril gglutintion ws used by Stmey nd Howell (9) to mesure ntibody in cervicovginl wshings from ptients with recurrent UTI nd norml controls. Agglutintion ws performed by recting the ptients' introitl E. coli or the controls' predominnt rectl E. coli with seril dilutions of the pproprite wshing. No significnt differences in gglutintion titers between ptients nd controls were reported. Employing more sensitive technique, indirect immunofluorescence, Stmey et l. (14) subsequently reported tht cervicovginl ntibody ws inversely relted to introitl coloniztion. Cervicovginl ntibody to the predominnt fecl Enterobctericee ws found in 1 of 13 (77%) norml controls with no history of UTI. In contrst, 6 of 23 (26%) ptients with recurrent UTI hd ntibody to Enterobctericee colonizing the vginl vestibule. However, there were exceptions to this inverse reltionship; 26% of the colonized ptients studied hd ntibody in their cervicovginl wshings, nd ntibody ws not detected in specimens from 23% of the controls. In the current study, we used the sme indirect immunofluorescence technique nd found no significnt difference in the presence of specific locl ntibody in cervicovginl wshings between norml controls nd ptients with recurrent infections. In the control group, 1 of 29 (34%) controls demonstrted ntibody to their fecl E. coli in cervicovginl wshings. In contrst, 3 of 22 (14%) ptients demonstrted ntibody to their introitl E. coli. The pprent conflict in the results of these two studies cnnot be ttributed to differences in methods. However, it should be noted tht interprettion of fluorescence is subjective. While this study ws in progress, Tuttle et l. (16), using double ntibody rdioimmunossy, reported quntittive difference in the mount of IgA in the vginl fluid from girls with recurrent UTI nd from girls who hd never experienced UTI. Vginl IgA levels were significntly lower in specimens from ptients when compred to those of the controls. However, n overlp in IgA levels ws noted in the two groups. Their technique quntitted totl IgA without regrd to ntibody specificity. Antibodies which re ble to rect with the potentil colonizing or infecting orgnisms (E. coli) nd inhibit the dherence of the orgnism to the vginl introitus were not specificlly qunti-

6 VOL. 29, 198 CERVICOVAGINAL ANTIBODY AND UTI 81 tted. Therefore, their results do not convincingly demonstrte ny reltionship between specific ntibody nd the degree of coloniztion. In the current study, cervicovginl wshings from ptients with recurrent UTI who were hevily colonized, from noncolonized ptients with recurrent UTI, nd from norml noncolonized femles were tested for the presence of IgG nd IgA ntibody directed ginst E. coli by sensitive nd quntittive rdioimmunossy technique. No significnt quntittive differences in IgG nd IgA ntibody were demonstrted mong the three groups (Fig. 5). The men IgG nd IgA ntibody levels were greter in specimens from the control group compred to those from noncolonized ptients nd prticulrly those from colonized ptients. One might infer tht by incresing the popultion numbers, significnt difference in ntibody levels would be demonstrted. However, it must be emphsized tht there ws lrge degree of overlp in ntibody levels mong the three groups; nd, further, high levels of ntibody were found in specimens from hevily colonized ptients, nd low levels of ntibody were found in specimens from noncolonized ptients nd from controls. These results do not support the hypothesis tht cervicovginl ntibody is inversely relted to introitl coloniztion. Men IgG ntibody levels were greter thn men IgA ntibody levels in specimens from ll three groups. This grees with other studies (1, 3, 6, 9). The IgA/IgG ntibody rtio found in the cervicovginl wshings ws.48. This is considerbly higher thn the rtios of.2 to.16 reported in norml serum (17), suggesting tht the ntibody in cervicovginl wshings quntitted by the rdioimmunossy ws derived loclly from the vgin, cervix, or both. The rdioimmunossy ws compred with the immunofluorescence method for the detection of nti-e. coli ntibody in cervicovginl wshings from the three groups. More specimens were positive for nti-e. coli ntibody by rdioimmunossy thn by indirect immunofluorescence. This difference is significnt nd cn be ttributed to the greter sensitivity of the rdioimmunossy technique. In summry, this investigtion ws designed to study the role of cervicovginl ntibody in the pthogenesis of recurrent UTI. By indirect immunofluorescence, no significnt difference in specific locl cervicovginl ntibody ws demonstrted between ptients with recurrent UTI nd the norml controls with no history of UTI. Subsequently, with the more sensitive, specific, nd quntittive rdioimmunossy, no quntittive differences in IgG nd IgA ntibody were found in cervicovginl wshings from ptients nd controls. Both high nd low levels of ntibody were found in hevily colonized nd noncolonized ptients. Introitl coloniztion which predisposes to recurrent UTI ws not relted to presence or bsence of cervicovginl ntibody. However, these studies do not negte n importnt role for dherence in the pthogenesis of recurrent UTI. Adherence to the vginl epithelil cells which promotes introitl coloniztion my be the principl fctor which predisposes to infections. A number of fctors other thn cervicovginl ntibody medite dherence nd my be responsible for determining why some women re more susceptible to introitl coloniztion nd hence to recurrent UTI. ACKNOWLEDGMENT This work ws supported by grnt MA-5973 from the Medicl Reserch Council of Cnd. LITERATURE CITED 1. Chipperfield, E. J., nd B. A. Evns Effects of locl infection nd orl contrception on immunoglobulin levels in cervicl mucus. Infect. Immun. 11: Fowler, J. E., R. LH, nd T. A. Stmey Studies of introitl coloniztion in women with recurrent urinry infection. VII. The role of bcteril interference. J. Urol. 118: Govers, J., nd J. P. Girrd Some immunologicl properties of humn cervicl nd vginl secretions. Gynecol. Invest. 3: Greenwood, F. C., W. M. Hunter, nd J. S. 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