Using protein-based motifs to stabilize peptides.

While the use of synthetically derived novel inhibitor peptides as a source of new therapeutics for medicine remains incredibly promising, there is a major problem with implementing this technology, as many synthetic peptides have proven to be unstable and are degraded by peptidases in the host cell. In this study, we have investigated methods by which peptides can be stabilized using protein-based motifs in order to prevent the action of peptidases. Using an in vivo approach our laboratory developed to screen for synthetic peptides which can inhibit the growth of Escherichia coli, we found that protecting the amino or carboxyl terminus of the peptides via fusion to the very stable Rop protein, or the incorporation of two proline residues, increased the frequency at which potent inhibitor peptides could be isolated. Using an in vitro degradation assay in which extracts from several different cell types were tested, we demonstrated that peptides stabilized with multiple proline residues were more resistant to degradation than peptides stabilized by amidation or acetylation, two approaches that are routinely utilized to improve the stability of peptide drugs.

Is interstitial cystitis an infectious disease?

Interstitial cystitis (IC) is a chronic bladder disorder of unknown aaetiology. Although several investigators have attempted to identify an infectious cause for IC, none has yet been found. Our own studies to search for an infectious cause discovered instead, a toxic factor in the urine of approximately 95% of IC patients that is made by and inhibits, the normal proliferation of bladder epithelial cells. Additional research is necessary to determine whether this factor is encoded by the eucaryotic cells themselves or an unidentified intracellular microorganism.

Concordance of interstitial cystitis in monozygotic and dizygotic twin pairs.

Our objective was to determine whether there is a greater concordance of interstitial cystitis (IC) among monozygotic than dizygotic twins. Members of the Interstitial Cystitis Association (ICA) who responded to a survey about first-degree family members with IC symptoms or confirmed IC were requested to identify themselves if they were 1 of a twin pair. Each twin respondent and co-twin were then evaluated via a questionnaire and acquisition of hydrodistention reports as to their meeting modified National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) criteria for IC. Of the co-twins of 8 monozygotic twin respondents, 2 had probable and 3 had confirmed IC compared with none of the co-twins of the 26 dizygotic twin respondents (including 15 female co-twins). There is a greater concordance of IC among monozygotic than dizygotic twin pairs suggesting a genetic susceptibility to IC.

Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis.

We previously determined that the urine of interstitial cystitis (IC) patients specifically contains a factor (antiproliferative factor [APF]) that inhibits primary bladder epithelial cell proliferation, and that it has significantly decreased levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and increased levels of epidermal growth factor (EGF) compared with urine from asymptomatic controls and patients with bacterial cystitis. We sought to confirm the specificity of these findings for IC using a larger patient population, including control patients with a variety of urogenital disorders. Clean catch urine specimens were collected from 219 symptomatic IC patients, 113 asymptomatic controls without bladder disease, and 211 patients with various urogenital diseases including acute bacterial cystitis, vulvovaginitis, chronic nonbacterial prostatitis, overactive bladder, hematuria, stress incontinence, neurogenic bladder, benign prostatic hyperplasia, bladder or pelvic pain without voiding symptoms, bladder cancer, prostate cancer, or miscellaneous diagnoses including anatomic disorders. APF activity was determined by (3)H-thymidine incorporation into primary normal adult human bladder epithelial cells. HB-EGF and EGF levels were determined by enzyme-linked immunosorbent assay. APF activity was present significantly more often in IC than control urine specimens (P <0.005 for IC vs any control group; sensitivity = 94%, specificity = 95%, P <10(-82) for IC vs all controls). HB-EGF levels were also significantly lower and EGF levels significantly higher in IC urine than in specimens from controls (P <10(-84) and P <10(-36), respectively). These findings confirm the utility of APF, HB-EGF, and EGF as markers for IC. Understanding the reasons for altered levels of these markers may lead to understanding the pathogenesis of this disorder.

Catheter-associated urinary tract infections.

Nosocomial urinary tract infection (UTI) is the most common infection acquired in both hospitals and nursing homes and is usually associated with catheterization. This infection would be even more common but for the use of the closed catheter system. Most modifications have not improved on the closed catheter itself. Even with meticulous care, this system will not prevent bacteriuria. After bacteriuria develops, the ability to limit its complications is minimal. Once a catheter is put in place, the clinician must keep two concepts in mind: keep the catheter system closed in order to postpone the onset of bacteriuria, and remove the catheter as soon as possible. If the catheter can be removed before bacteriuria develops, postponement becomes prevention.

Practice guidelines for the treatment of uncomplicated cystitis.

The Infectious Diseases Society of America has published guidelines for the treatment of uncomplicated cystitis. Recommendations are that for healthy, adult, nonpregnant women with bacterial cystitis, 3 days of trimethoprim/ sulfamethoxazole (TMP/SMZ) or trimethoprim alone is standard therapy in those regions where less than 10% to 20% of Escherichia coli that cause such infections is resistant to TMP/SMZ. In those regions where resistance is more than 10% to 20%, the committee recommended using an oral fluoroquinolone for 3 days, and that alternatives such as nitrofurantoin for 7 days or fosfomycin as single-dose therapy need more study. These recommendations were established in the late 1990s as resistance to TMP/SMZ among uropathogens was increasing in the United States, a phenomenon earlier observed in other parts of the world. Clinicians should be alert to patients infected with possibly resistant organisms, eg, patients who have recently been hospitalized or are receiving antibiotics.

Optimal electrocardiographic leads for detecting acute myocardial ischemia.

This study identifies the most sensitive electrocardiographic leads for monitoring ST-segment changes caused by acute coronary ischemia. The data set consisted of 120-lead electrocardiograms (ECGs) digitally recorded during balloon-inflation angioplasty in 3 groups of patients with single-vessel disease (left anterior descending [LAD], 32; right coronary artery [RCA], 36; left circumflex [LCx], 23). The ST deviation was measured in all recorded leads during baseline and ischemic states, and its difference between these 2 states (DeltaST) was calculated at 352 sites and plotted as DeltaST maps. The patients in each group were divided, by means of DeltaST criteria, into subgroups of "responders" and "nonresponders." Mean DeltaSTs for each group/subgroup were calculated and standardized by the corresponding standard deviation (SD); these values were plotted as mean DeltaST and t maps. Sites where extrema of DeltaST occurred most frequently were sought in bootstrap trials, performed in each group/subgroup. The results suggest that the optimal sites for the ischemia-sensitive leads are: V(3) (+) and just below V(8) (-) for LAD-related ischemia; the left iliac crest (+) and above V(3) at the third intercostal space (-) for RCA-related ischemia; and just below V(8) (+) and above V(2) at the third intercostal space (-) for LCx-related ischemia. Three "optimal" bipolar leads using these sites registered, in the responders from the LAD, RCA, and LCx groups, mean DeltaST (+/-SD) of 232 +/- 59, 245 +/- 96 and 158 +/- 91 microV, respectively; the corresponding t values were 15.14, 9.90, and 6.75. In the 12-lead ECG, only lead V(3) approached optimal DeltaST and t values for the LAD responders (187 +/- 61 microV; t = 11.75) and lead III for the RCA responders (191 +/- 76 microV; t = 9.73), but even these values were significantly suboptimal (P = 0.0011 and P = 0.0120, respectively). We found that the "optimal" bipolar leads can be derived, to an excellent approximation, from the 12 standard leads or from 3 EASI leads (with 3 electrodes at Frank's transverse level and 1 on the manubrium), by using precalculated regression coefficients. By means of bootstrap trials, we estimated the mean sensitivity (SE) and the mean positive predictive value (PPV) with which 3 "optimal" vessel-specific leads could identify ischemia related to the LAD, RCA, and LCx arteries, in the test set, as (SE/PPV) 94.7/92.8%, 78.7/80.9%, and 81.5/80.9%. A similar diagnostic performance can be achieved by vessel-specific leads derived from the 12-lead ECG (93.0/93.4%, 76.6/82.0%, and 82.7/77.1%) and, interestingly, from the EASI lead system (97.8/88.4%, 78.0/80.2%, and 76.8/83.2%). Thus, although the "optimal" bipolar leads for detecting ischemia related to each of the 3 coronary arteries were found to require sampling outside the 12-lead ECG, these leads can be derived from the full set of 12 standard leads or--for clinical monitoring applications--from the EASI lead system by using fewer electrodes at convenient locations.

Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production.

PURPOSE: The etiology of interstitial cystitis is unknown. We previously identified an interstitial cystitis urine factor, antiproliferative factor, that inhibits proliferation of bladder epithelial cells in vitro and complex changes in epithelial growth factor levels, including profound decreases in heparin-binding epidermal growth factor-like growth factor (HB-EGF). Bladder and renal pelvic catheterization of patients with interstitial cystitis indicated that the antiproliferative factor is made and/or activated in the distal ureter or bladder. Therefore, we determined whether bladder epithelial cells from interstitial cystitis cases produced the antiproliferative factor and whether purified antiproliferative factor could alter production of growth factors known to be abnormal in interstitial cystitis. MATERIALS AND METHODS: Antiproliferative factor activity was determined by 3H-thymidine incorporation into primary bladder epithelial cells. The antiproliferative factor was purified by size fractionation followed by sequential chromatography involving ion exchange, hydrophobic interaction and high performance liquid chromatography. HB-EGF, epidermal growth factor, insulin-like growth factor and insulin-like growth factor binding protein 3 levels were determined by enzyme-linked immunosorbent assay. RESULTS: Bladder epithelial cells from patients with interstitial cystitis produced a single antiproliferative factor with the same purification profile as that purified from interstitial cystitis urine. Purified antiproliferative factor specifically inhibited HB-EGF production by bladder epithelial cells in vitro, and the effect of interstitial cystitis urine or purified antiproliferative factor on bladder cell proliferation was inhibited by recombinant human HB-EGF in a dose dependent manner. Similar to urine HB-EGF, serum HB-EGF was also significantly lower in interstitial cystitis cases than in controls. CONCLUSIONS: Bladder epithelial abnormalities in interstitial cystitis may be caused by a negative autocrine growth factor that inhibits cell proliferation by down-regulating HB-EGF production. Furthermore, decreased levels of urine and serum HB-EGF indicate that interstitial cystitis may be a urinary tract manifestation of a systemic disorder.

Construction and characterization of a highly regulable expression vector, pLAC11, and its multipurpose derivatives, pLAC22 and pLAC33.

A number of different expression vectors have been developed to facilitate the regulated overproduction of proteins in Escherichia coli and related bacteria. Some of the more popular ones include pKK223-3, pKK233-2, pTrc99A, and the pET family of expression vectors. These vectors were designed to be regulable and can be grown under conditions that repress protein production or under conditions that induce protein production. Unfortunately, however, numerous researchers have found that these vectors produce significant amounts of protein even when grown under repressed conditions. We describe here a new expression vector, pLAC11, which was designed to be more regulable and thus more tightly repressible when grown under repressed conditions. The tight regulation of pLAC11 was achieved by utilizing the O3 auxiliary operator, CAP binding site, promoter, and O1 operator that occur in the wild-type lac control region. The pLAC11 vector can be used to conduct physiologically relevant studies in which the cloned gene is expressed at levels comparable to that obtainable from the chromosomal copy of the gene in question. In experiments in which a bacterial cell contained both a null allele in the chromosome and a second copy of the wild-type allele on pLAC11, we observed that cells grown under repressed conditions exhibited the null phenotype while cells grown under induced conditions exhibited the wild-type phenotype. Two multipurpose derivatives of pLAC11, pLAC22, and pLAC33 have also been constructed to fulfill different experimental needs.

Percutaneous sacral third nerve root neurostimulation improves symptoms and normalizes urinary HB-EGF levels and antiproliferative activity in patients with interstitial cystitis.

OBJECTIVES: A highly effective treatment for interstitial cystitis (IC) remains elusive. We determined whether sacral third nerve root (S3) percutaneous neurostimulation (PNS) might be effective in relieving symptoms of IC, as well as in normalizing urinary factors that are specifically altered in IC. METHODS: Six consecutive patients with symptoms and cystoscopic findings compatible with IC underwent 5 days of continuous S3 neurostimulation by way of leads placed percutaneously into the S3 foramen. Patients filled out voiding frequency diaries and pain and urgency questionnaires before PNS and at the end of PNS when the leads were removed. Urine specimens were collected at these two time points and measured for heparin-binding epidermal growth factor-like growth factor (HB-EGF) by enzyme-linked immunosorbent assay and for antiproliferative factor (APF) activity by (3)H-thymidine uptake by normal human bladder urothelial cells. RESULTS: S3 PNS significantly improved all measured parameters toward normal values. Voiding frequency decreased twofold from 23.1 +/- 4.6 to 10.6 +/- 4.0 voids daily during PNS (P = 0.0001). Pelvic pain on a scale of 1 to 10 decreased from 7.0 +/- 1.6 to 2.3 +/- 3.2 (P = 0.05). Urinary urgency on a scale of 1 to 10 decreased from 6.0 +/- 2.2 to 1.8 +/- 1.7 (P = 0. 02). Urinary HB-EGF concentration increased sevenfold from 1.5 +/- 2. 1 to 11.0 +/- 1.7 ng/mL (P <0.0001), and urinary APF activity decreased from -76.1% +/- 31% to -4.5% +/- 8.8% (P = 0.005). CONCLUSIONS: S3 PNS significantly decreased symptoms and normalized urinary HB-EGF and APF activity in patients with IC. These results suggest that permanent S3 PNS may be beneficial in treating IC.

Pilot study of sequential oral antibiotics for the treatment of interstitial cystitis.

PURPOSE: Interstitial cystitis is a chronic disease of unknown etiology characterized by bladder pain, urgency and frequency. Although a single microbe has not been implicated as a cause of interstitial cystitis, several groups noted various organisms in the urine of some women with interstitial cystitis and some patients reported that antibiotics decrease symptoms. Consequently we performed a prospective, randomized, double-blinded, placebo controlled pilot study of sequential oral antibiotics. MATERIALS AND METHODS: We randomized 50 patients with interstitial cystitis to receive 18 weeks of placebo or antibiotics, including rifampin plus a sequence of doxycycline, erythromycin, metronidazole, clindamycin, amoxicillin and ciprofloxacin for 3 weeks each. RESULTS: Intent to treat analysis demonstrated that 12 of 25 patients (48%) in the antibiotic and 6 of 25 (24%) in the placebo group reported overall improvement (p = 0.14), while 10 and 5, respectively, noticed improvement in pain and urgency (p = 0.22). In the antibiotic group 20 participants (80%) had adverse effects compared with 10 (40%) in the placebo group (p = 0.009). CONCLUSIONS: Our findings suggest that these antibiotics alone or in combination may sometimes be associated with decreased symptoms in some patients but they do not represent a major advance in therapy for interstitial cystitis.

The role of cytotoxic necrotizing factor-1 in colonization and tissue injury in a murine model of urinary tract infection.

Cytotoxic necrotizing factor-1 (CNF1) is commonly found in Escherichia coli isolates from patients with urinary tract infection (UTI). To determine whether CNF1 is an important UTI virulence factor we compared the ability of a clinical E. coli UTI isolate and a CNF1-negative mutant of that isolate to colonize and induce histological changes in the urinary tract in a murine model of ascending UTI. We found no evidence that the mutant strain was attenuated.

Diagnostic accuracy of derived versus standard 12-lead electrocardiograms.

To compare the diagnostic yield of electrocardiograms (ECGs) recorded by 12 standard leads with that of 12-lead ECGs derived from 3 bipolar EASI leads, we analyzed pertinent ECG data for 290 normal subjects and 497 patients who had had a prior myocardial infarction (MI); the latter group comprised 36 patients with a non-Q MI, 282 patients with a Q-wave MI, and 179 patients with a history of ventricular tachycardia (VT). We first estimated statistically an optimal set of coefficients for deriving the 12 standard leads from EASI leads and assessed this transformation in terms of goodness of fit. To gauge the diagnostic information content of the recorded vs. derived 12-lead ECGs, we performed successively two-group diagnostic classification--based on the Cardiac Infarction Injury Score (CIIS)--separating each of the patient subgroups from the normal group; the classification was repeated for 200 sets of patients selected randomly (with replacement), and the results were plotted as mean receiver operating characteristics. We found that derived 12-lead ECGs correlated well with the recorded ones, and reproduced faithfully the diagnostic features needed for the CIIS. When the CIIS was determined from features of the recorded standard 12 leads, its mean diagnostic performance (assessed in terms of area under the receiver operating characteristics curve) was 0.9004 for detecting non-Q MIs, 0.9546 for Q-wave MIs, and 0.9919 for MIs complicated by a history of VT. When, instead, features of derived 12 leads were used to determine the CIIS, diagnostic performance remained virtually unchanged (at 0.8905, 0.9531, and 0.9906, respectively). We conclude that, in our population, EASI-derived 12-lead ECGs contain nearly the same diagnostic information as standard 12-lead ECGs.

Bladder stretch alters urinary heparin-binding epidermal growth factor and antiproliferative factor in patients with interstitial cystitis.

PURPOSE: The etiology of interstitial cystitis is unknown. Urine from patients with interstitial cystitis has been shown to inhibit urothelial proliferation through a putative antiproliferative factor and to contain decreased levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared to controls. Stretch of detrusor smooth muscle cells is known to stimulate HB-EGF production. Because bladder hydrodistention sometimes alleviates the symptoms of interstitial cystitis, we determined whether the stretch stimulus of hydrodistention alters antiproliferative factor activity and/or HB-EGF in interstitial cystitis urine specimens. MATERIALS AND METHODS: Urine was collected immediately before, and 2 to 4 hours and 2 weeks after hydrodistention from 15 patients with symptoms and cystoscopic findings compatible with interstitial cystitis and 13 controls. Hydrodistention was performed with the subject under general or regional anesthesia and bladders were distended to 80 cm. water 3 times. Urinary HB-EGF was measured by enzyme-linked immunosorbent assay and urinary antiproliferative factor activity was determined by measuring 3H-thymidine uptake by normal human bladder urothelial cells. RESULTS: Hydrodistention significantly increased urinary HB-EGF in patients with interstitial cystitis toward normal control values (before distention p = 0.003, 2 weeks after distention p = 0.67). Urine antiproliferative factor activity decreased significantly after hydrodistention in patients with interstitial cystitis. However, antiproliferative factor activity in interstitial cystitis and control specimens was still statistically different 2 weeks after distention (before distention p = 0.0000004, 2 weeks after distention p = 0.04). CONCLUSIONS: Bladder stretch increased HB-EGF and conversely reduced antiproliferative factor activity in urine from patients with interstitial cystitis but not controls up to 2 weeks after distention. These results provide additional evidence for the possible role of antiproliferative factor and decreased HB-EGF in the pathophysiology of interstitial cystitis. To our knowledge this is also the first human study to show that in vivo bladder stretch can alter urinary factors that regulate cell growth.

Guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women. Infectious Diseases Society of America (IDSA).

This is part of the series of practice guidelines commissioned by the Infectious Diseases Society of America (IDSA) through its Practice Guidelines Committee. The purpose of this guideline is to provide assistance to clinicians in the diagnosis and treatment of two specific types of urinary tract infections (UTIs): uncomplicated, acute, symptomatic bacterial cystitis and acute pyelonephritis in women. The guideline does not contain recommendations for asymptomatic bacteriuria, complicated UTIs, Foley catheter-associated infections, UTIs in men or children, or prostatitis. The targeted providers are internists and family practitioners. The targeted groups are immunocompetent women. Criteria are specified for determining whether the inpatient or outpatient setting is appropriate for treatment. Differences from other guidelines written on this topic include use of laboratory criteria for diagnosis and approach to antimicrobial therapy. Panel members represented experts in adult infectious diseases and urology. The guidelines are evidence-based. A standard ranking system is used for the strength of the recommendation and the quality of the evidence cited in the literature reviewed. The document has been subjected to external review by peer reviewers as well as by the Practice Guidelines Committee and was approved by the IDSA Council, the sponsor and supporter of the guideline. The American Urologic Association and the European Society of Clinical Microbiology and Infectious Diseases have endorsed it. An executive summary and tables highlight the major recommendations. Performance measures are described to aid in monitoring compliance with the guideline. The guideline will be listed on the IDSA home page at http://www.idsociety.org It will be evaluated for updating in 2 years.

Serum immunoglobulin response and protection from homologous challenge by Proteus mirabilis in a mouse model of ascending urinary tract infection.

We tested the hypothesis that experimental Proteus mirabilis urinary tract infection in mice would protect against homologous bladder rechallenge. Despite production of serum immunoglobulin G (IgG) and IgM (median titers of 1:320 and 1:80, respectively), vaccinated (infected and antibiotic-cured) mice did not show a decrease in mortality upon rechallenge; the survivors experienced only modest protection from infection (mean log(10) number of CFU of P. mirabilis Nal(r) HI4320 per milliliter or gram in vaccinated mice versus sham-vaccinated mice: urine, 100-fold less [3.5 versus 5.5; P = 0.13]; bladder, 100-fold less [3.1 versus 5.1; P = 0.066]; kidneys, 40-fold less [2.7 versus 4.3; P = 0.016]). Western blots using protein from the wild-type strain and isogenic mutants demonstrated antibody responses to MR/P and PMF fimbriae and flagella. There was no correlation between serum IgG or IgM levels and protection from mortality or infection. There was a trend toward elevated serum IgA titers and protection from subsequent challenge (P >/= 0.09), although only a few mice developed significant serum IgA levels. We conclude that prior infection with P. mirabilis does not protect significantly against homologous challenge.

Antiproliferative activity is present in bladder but not renal pelvic urine from interstitial cystitis patients.

PURPOSE: To determine whether an antiproliferative urine factor that we previously discovered to be specific for urine from interstitial cystitis (IC) patients originated in the lower urinary tract or a more proximal site. MATERIALS AND METHODS: Sequential catheterized urine specimens were collected under sterile conditions from the bladder and renal pelvis of 20 IC patients and one control patient (with stress incontinence). Antiproliferative activity was determined by 3H-thymidine incorporation of primary normal adult bladder epithelial cells cultured with pH- and osmolality-corrected bladder or ureteral urine specimens; significant inhibition was defined as a change in 3H-thymidine incorporation greater than 2 standard deviations from the mean of control cells. RESULTS: Bladder urine specimens from 19 of 20 IC patients significantly inhibited 3H-thymidine incorporation as compared to cell medium alone (mean change for bladder specimens = -68.7+/-7.5%), while a renal pelvic specimen from only 1 of 20 IC patients inhibited proliferation significantly (mean change for renal pelvic specimens = 3.2+/-3.4%) (p<.001 by Fisher's exact test). The one inhibitory IC renal pelvic specimen inhibited by 31% while a bladder specimen obtained during the same procedure inhibited by 94%. In comparison, neither bladder nor renal pelvic urine from the control patient had inhibitory activity. CONCLUSIONS: The antiproliferative factor previously found in the urine of IC patients appears to be made and/or activated in the distal ureter or urinary bladder.

Effect of Escherichia coli cytotoxic necrotizing factor 1 on repair of human bladder cell monolayers in vitro.

We hypothesized that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) might impair migration or proliferation of bladder cells and could potentially interfere with repair of the bladder epithelium. Using experimentally wounded human T24 bladder epithelial cell monolayers as an in vitro model, we found that both the number of T24 cells and the maximum distance they migrated into wounded regions was significantly decreased by bacterial extracts containing E. coli CNF1.

A diagnostic in vitro urine assay for interstitial cystitis.

OBJECTIVES: A low molecular weight urine factor that inhibits the proliferation of normal bladder epithelial cells in vitro was previously shown to be present significantly more often in the urine of patients with interstitial cystitis (IC) than in the urine of asymptomatic age-, race-, and sex-matched control subjects. We sought to determine the specificity of this finding for IC by determining whether the urine of patients with other urogenital inflammatory disorders also contains a factor that inhibits bladder epithelial cell proliferation. METHODS: Urine was collected from women with IC, acute bacterial cystitis, or vulvovaginitis, as well as from asymptomatic control women. The proliferation of primary normal adult bladder epithelial cells was determined by measuring 3H-thymidine incorporation in vitro. RESULTS: Osmolality- and pH-corrected urine specimens from 50 (86%) of 58 women with IC significantly inhibited human bladder epithelial cell proliferation compared with 3 (8%) of 36 asymptomatic control women, 7 (12%) of 58 women with bacterial cystitis, and 0 (0%) of 12 women with vulvovaginitis (P < 0.001 for the comparison of mean percent change in 3H-thymidine incorporation with IC urine versus urine from each of the control groups). Optimal sensitivity and specificity values of 91.4% and 90.6%, respectively, were achievable at a cutoff of 25% inhibition of 3H-thymidine incorporation, using all three control groups. CONCLUSIONS: The measurement of urine antiproliferative activity may be a useful noninvasive means for diagnosing IC in women.

Polymerase chain reaction amplification of bacterial 16S rRNA genes from cold-cup biopsy forceps.

PURPOSE: In looking for a possible infectious cause for interstitial cystitis (IC), we previously determined that bladder tissue specimens from both IC patients and controls were uniformly positive by polymerase chain reaction assay (PCR) for bacterial 16S ribosomal RNA genes from various genera including Escherichia, Propionobacterium, Acinetobacter, and Salmonella. We therefore determined whether the biopsy forceps might be contaminated with bacterial DNA. MATERIALS AND METHODS: A total of 23 samples were obtained following disinfection of 6 cold-cup bladder biopsy forceps (2 to 5 specimens from each forceps over a period of 19 months). DNA was extracted from each sample, and PCR performed using nested primers from a highly conserved region of the bacterial 16S rRNA gene. Amplified DNA was purified and sequenced, and the sequences obtained were compared with bacterial rRNA gene sequences recorded in GenBank. RESULTS: Thirteen of 23 forceps specimens were positive by PCR for bacterial DNA, including at least one rinse from each of the 6 forceps. In comparison, none of 9 negative control specimens (sterile distilled water put into tubes and processed in the same manner as forceps rinses) had detectable bacterial DNA. Sequence data indicated the presence of a predominant organism in 12 of the 13 positive specimens, with >95% homology to DNA from several different genera of bacteria including Escherichia, Propionobacterium, Stenotrophomonas and Pseudomonas. CONCLUSIONS: These data indicate that reusable bladder biopsy forceps are frequently contaminated with bacterial DNA. Tissue specimens procured with such instruments therefore are inappropriate sources to look for the presence of bacterial pathogens by PCR.