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Background: The aim of this study was to compare four renal access techniques in percutaneous nephrolithotomy (PCNL)., Methods: A total of 437 patients who underwent PCNL at our center from January 2015 to December 2019 were included in the analysis. Telescopic metallic coaxial dilation (TMD) was used in 146 patients, single step balloon dilation (BD) in 98 patients, one-shot dilation with 30F Amplatz (OS 30F) in 106 patients, and one-shot dilation with 16F Amplatz (OS 16F) in 87 patients. Primary endpoints were perioperative outcomes and complications of the procedures., Results: Similar baseline characteristics were observed in the four groups. Fluoroscopy time was significantly shorter in OS 30F and OS 16F groups (P<0.0001). The drop in hemoglobin level was not significantly different between TMD and BD groups, but it was significantly lower in OS 16F group versus the OS 30F group and lower in OS 30F group versus the BD Group (P<0.0001). Despite this, the rate of blood transfusion was similar across groups (P=0.837). Moreover, a smaller tract was associated with reduced postoperative morbidity including time to nephrostomy removal (P=0.001), hospital stay (P<0.0001), VAS scale (P<0.0001). There were no significant differences in postoperative complications (P=0.683), and Clavien-Dindo grade ≥3 complication rates (P=0.486) among the groups. Stone-free rates and number of auxiliary procedures required to achieve stone-free status were also similar among all groups (P=0.964 and 0.988, respectively). Multinomial logistic regression analysis showed that BMI (P=0.002), stone size (P=0.002) and previous PCNL (P=0.038) were predictive factors associated with the choice of OS 16 approach., Conclusions: Different dilation methods are equally effective and safe to use in a PCNL procedure for kidney stone treatment, allowing similar stone free rates and risk of complications. The OS dilation techniques seem to allow a shorter X-ray exposure time, which might be beneficial for both patients and operators. The use of a 16 F dilator can reduce the postoperative morbidity. Risk of sepsis should be always kept in mind.

Background: Evidence about the clinical benefits of Hyperbaric Oxygen Therapy (HBOT) in patients with Fournier's Gangrene (FG) is controversial and inconclusive. We aimed to compare the mortality related to FG between patients undergoing surgical debridement and/or standard antibiotic therapy alone or in combination with HBOT., Methods: We performed a retrospective multi-institutional observational case-control study. All patients admitted with diagnosis of FG from June 2009 to June 2019 were included into the study. Patients received surgical debridement and/or standard antibiotic therapy alone or in combination with HBOT. Factors associated with FG related mortality were assessed with uni-and multivariate analyses. The main outcome measure was FG related mortality., Results: A total of 161 patients with diagnosis of FG were identified. Mean FG Severity Index was 8.6±4.5. All patients had broad-spectrum parenteral antibiotic therapy. An aggressive debridement was performed in 139 (86.3%) patients. A total of 72 patients (44.7%) underwent HBOT. Mortality due to FG was observed in 32 (36.0%) of patients who do not underwent HBOT and in 14 (19.4%) of patients who underwent HBOT (P=0.01). At the multivariate analysis, surgical debridement and HBOT were independent predictors of lower mortality while higher FG Severity Index was independent predictor of higher mortality., Conclusions: HBOT and surgical debridement are independent predictors of reduced FG related mortality.

One of the most common malignancies in men is prostate cancer, for which androgen deprivation is the standard therapy. However, prostate cancer cells become insensitive to anti-androgen treatment and proceed to a castration-resistant state with limited therapeutic options. Therefore, besides the androgen deprivation approach, novel biomarkers are urgently required for specific targeting in this deadly disease. Recently, germline or somatic mutations in the homologous recombination (HR) DNA repair genes have been identified in at least 20-25% of metastatic castration-resistant prostate cancers (mCRPC). Defects in genes involved in HR DNA repair can sensitize cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors, a class of drugs already approved by the Food and Drug Administration (FDA) for breast and ovarian cancer carrying germline mutations in BRCA1/2 genes. For advanced prostate cancer carrying Breast cancer1/2 (BRCA1/2) or ataxia telengiectasia mutated ( ATM ) mutations, preclinical studies and clinical trials support the use of PARP-inhibitors, which received breakthrough therapy designation by the FDA. Based on these assumptions, several trials including DNA damage response and repair (DDR) targeting have been launched and are ongoing for prostate cancer. Here, we review the state-of-the-art potential biomarkers that could be predictive of cancer cell synthetic lethality with PARP inhibitors. The identification of key molecules that are affected in prostate cancer could be assayed in future clinical studies to better stratify prostate cancer patients who might benefit from target therapy.

Background: Novel therapeutic strategies are urgently needed for the treatment of metastatic Urothelial Bladder Cancer. DNA damaging repair (DDR) targeting has been introduced in cinical trials for bladder cancer patients that carry alterations in homologous DNA repair genes, letting to envisage susceptibility to the Poly (adenosine diphosphate [ADP]) ribose polymerase (PARP) inhibitors., Main Body: PARP inhibition, by amplifying the DNA damage, augments the mutational burden and promotes the immune priming of the tumor by increasing the neoantigen exposure and determining upregulation of programmed death ligand 1 (PD-L1) expression. Thus, the combination of PARP-inhibition and the PD/PD-L1 targeting may represent a compelling strategy to treat bladder cancer and has been introduced in recent clinical trials. The targeting of DDR has been also used in combination with epigenetic drugs able to modulate the expression of genes involved in DDR, and also able to act as immunomodulator agents suggesting their use in combination with immune-checkpoint inhibitors., Conclusion: In conclusion, it may be envisaged the combination of three classes of drugs to treat bladder cancer, by targeting the DDR process in a tumor context of DDR defect, together with epigenetic agents and immune-checkpoint inhibitors, whose association may amplify the effects and reduce the doses and the toxicity of each single drug.

Background: The muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy. Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells., Methods: The J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis., Results: P5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels., Conclusions: In high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.

Background: To date, no study has presented results of photodynamic diagnosis (PDD) cystoscopy compared with white-light cystoscopy (WLC) in daily practice. The aim of the present study is to evaluate the diagnostic accuracy of hexylaminolevulinate hydrochloride (Hexvix(®)) PDD cystoscopy compared with standard WLC used in daily practice., Methods: An observational, open-label, comparative, controlled (within patient), multicenter study was carried out on 96 consecutive patients with suspected or confirmed bladder cancer. All patients had standard WLC followed by blue-light cystoscopy (BLC). Positive lesions detected using WLC and BLC were recorded. Biopsies/resection of each positive lesion were taken after the bladder was inspected. Sensitivity, specificity, positive predictive value, and negative predictive value with each method were calculated., Results: Overall, 234 suspicious lesions were detected; 108 (46.2%) were histologically confirmed to be bladder tumors/carcinoma in situ (CIS). The sensitivity of BLC biopsies was significantly higher than for WLC technique (99.1 vs 76.8%; p < 0.00001). The relative sensitivity of BLC versus WLC was 1.289, showing superiority of BLC of 28.9%. The specificity of BLC biopsies was not significantly different compared with WLC (36.5 vs 30.2%). Positive predictive value for BLC- and WLC-guided biopsies was 54.9 and 50.9%, respectively. Negative predictive value per biopsy for BLC- and WLC-guided biopsies was 97.4 and 64.8%, respectively. BLC and WLC reached the correct diagnosis in 97.9 and 88.5% of patients, respectively. This difference was statistically significant (p = 0.0265). The lack of a random biopsy protocol was the major limitation of the study., Conclusions: Hexvix(®) PDD cystoscopy used in daily practice enhances the diagnostic accuracy of standard cystoscopy with higher negative predictive value, potentially permitting an improvement in patient prognosis.

Introduction: Chronic benign prostate diseases are very common and certainly feature significantly in urological practice.The treatment of chronic benign prostate diseases is a common problem in clinical practice: few studies have been conducted in routine clinical practice to evaluate the efficacy of the treatments for this clinical condition. The objective of this study was to evaluate the efficacy of an extract of Serenoa repens (Permixon) in the treatment of lower urinary tract symptoms (LUTS) in patients with chronic benign prostate diseases with associated inflammation, also taking into consideration the influence of treatment on sexual function and, therefore, on patients' quality of life., Materials and Methods: All the 591 eligible subjects were evaluated on entering the study; after a screening visit, including medical history, physical examination, physical examination and digital rectal examination (DRE) and laboratory tests, the patients underwent uroflowmetry. The subjects under investigation were also asked to complete the IPSS, NIH-CPSI and IIEF-5 questionnaires, for the purpose of evaluating urinary symptoms and erectile function in relation to sexual activity in the previous 6 months., Results: The analysis of the uroflowmetry results showed that treatment with extract of Serenoa repens distinctly improves bladder voiding and lower urinary tract symptoms, as highlighted also by the improvement in the scores for the IPSS and NIH-CPSI questionnaires which serve as a basis for evaluating the urinary symptoms of patients with prostatic hyperplasia and chronic prostatitis respectively. The results also suggest that using an extract of Serenoa repens for 6 months in patients with chronic benign prostate diseases gives rise to an improvement in erectile function, as demonstrated by the increase in the scores for the IIEF-5 questionnaire after 6 months of treatment., Conclusions: The results of this study demonstrate how treatment for 6 months with an extract of Serenoa repens in routine clinical practice gives rise to a statistically significant improvement in Qmax values and in the IPSS, NHI-CPSI and IIEF-5 questionnaire scores, resulting not only in an improvement in urinary symptoms but also in an overall improvement in patients' quality of life.

Inflammatory bowel disease (IBD) is a chronic inflammatory state of the GI tract of unknown etiology. Classically, tissue injury in IBD is thought to be primarily mediated by Th1 cells in Crohn's disease or Th2 cells in ulcerative colitis. The discoveries of new subsets of T-helper cells, especially Th17 cells, have revolutionized our understanding of the disease immunopathology. Th17 cells seem to affect both innate and adaptive immune responses by the release of regulatory cytokines. Understanding the role of Th17 cells in IBD pathogenesis and targeting their regulatory cytokines may provide potential therapeutic approaches for the treatment of IBD in the future.

Aim: Clostridium difficile infection (CDI) is the leading infective cause of antibiotic associated diarrhea. The principal objective of this study was to assess the knowledge and awareness of internal medicine (IM) residents regarding the epidemiology, clinical recognition, diagnosis and management of CDI., Methods: A 20-question survey was distributed to 90 IM residents in all three years of their post graduate training in a university-based program. The survey instrument assessed the resident's knowledge of the current epidemiological trend, clinical recognition and presentation, diagnosis and management of CDI., Results: Forty two out of 90 (48%) residents completed the questionnaire. Only 10/42 (23.8%) of the residents recommended the gold standard investigation for diagnosing CDI. The majority of residents 29/42 (69%) were not aware of the existence of CDI in the outpatient setting and would not test for CDI. Only 50% of the residents were aware of the worse outcome of CDI in inflammatory bowel disease patients and only 12/42 (28.6%) would appropriately risk stratify and treat patients. Almost all of the residents (97.6%) knew about the appropriate time to consult surgery. There was no significant difference in the awareness with respect to the year of training (interns vs. residents), their career choices (primary care vs. fellowship) nor did the knowledge correlate with the United States medical licensing examination (USMLE) scores., Conclusion: IM residents had suboptimal knowledge of many aspects of the common problem of CDI. Educational efforts should be directed at IM residents, many of whom plan careers as primary care/hospitalists, who will encounter patients with CDI.

Objectives: PCA3 is a prostate specific non-coding mRNA that is significantly overexpressed in prostate cancer tissue. Urinary PCA3 levels have been associated with prostate cancer grade suggesting a significant role in the diagnosis of prostate cancer. We measured urinary PCA3 score in 925 subjects from several areas of Italy assessing in 114 the association of urinary PCA3 score with the results of prostate biopsy., Material and Methods: First-catch urine samples were collected after digital rectal examination (DRE). PCA3 and PSA mRNA levels were measured using Trascription-mediated PCR amplification. The PCA3 score was calculated as the ratio of PCA3 and PSA mRNA (PCA3 mRNA/PSA mRNA x 1000) and the cut off was set at 35., Results: A total of 925 PCA3 tests were performed from December 2008 to January 2010. The rate of informative PCA3 test was 99%, with 915 subjects showing a valid PCA3 score value: 443 patients (48.42%) presented a PCA3 score >/= 35 (cut-off) whereas the remaining 472 patients (51.58%) presented a PCA3 score lower the cut-off limit (< 35). Of the 443 patients with PCA3 score >/= 35, 105 (23.70%) underwent biopsy or rebiopsy. We found that 27 patients (25.71%) had no tumour at biopsy, 37 (35.24%) had HGPIN or ASAP and 41 (39.05%) had a cancer. Moreover, including the additonal 9 patients with PCA3 < 35, who underwent biopsy post PCA3 results, our data indicate that patients with negative biopsy (n = 31) show lower PCA3 score (mean = 54.9) compared with patients with positive biopsy (n = 45) (mean = 141.6) (p = 0.000183; two-tailed t-student test). The mean PCA3 score (79.6)for the patients diagnosed with HGPIN/ASAP at biopsy (n = 38) was intermediate between patients with negative and positive biopsy., Conclusions: Our results indicate that the PCA3 score is a valid tool for prostate cancer detection and its role in making better biopsy decisions. This marker consents to discriminate patients who have to undergo biopsy from patients who only need be actively surveilled: Quantitative PCA3 score is correlated with the probability of a positive result at biopsy.

The heat-stable enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. (125)I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant (125)I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that (125)I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.

Purpose: Radical prostatectomy often results in erectile dysfunction because of lesions to the erectile nerves. In this study we evaluated histomorphological alterations in cavernous smooth muscle and collagen content after radical prostatectomy., Materials and Methods: A total of 19 patients between 57 and 69 years old with prostate adenocarcinoma and normal erectile function, as reported and validated by RigiScan (UroHealth Systems, Laguna Niguel, California) testing, underwent corpora cavernosa biopsy in the operating room before radical prostatectomy, and 2 and 12 months after surgery. No patient underwent hormone therapy before or after surgery and none was diabetic. Elastic fibers (manual counting), muscle specific actin (immunostaining) and collagen content (computerized morphometric imaging) were measured in the 3 biopsies., Results: In all cases the first postoperative histological assessment revealed some disorganization. Trabecular elastic fibers (p <0.0003) and smooth muscle fibers were decreased and collagen content was significantly increased (p <0.0003) compared with preoperative biopsies. One year after surgery elastic fibers (p <0.0003) and smooth muscle fibers were decreased and collagen content was significantly increased (p <0.0003) compared with the first postoperative biopsy. Moreover, organized collagen and trabecular protocollagen deposits were increased., Conclusions: Progressive fibrosis in the corpora cavernosa after radical prostatectomy probably results from denervation and/or an ischemic process, which is caused in turn by the ligation of anomalous pudendal artery branches or of venous plexuses that drain to or from the corpora cavernosa. Fibrosis and the subsequent loss in elasticity and function of erectile tissue probably together cause erectile dysfunction.

The aim of this study was to determine the effects of olestra and sorbitol consumption on three accepted objective measures of diarrhea (stool output >250 g/day, liquid/watery stools, bowel movement frequency >3/day), and how stool composition influences reports of common gastrointestinal symptoms. A double-blind, placebo-controlled study compared the effects of sorbitol (40 g/day in candy), a poorly absorbed sugar-alcohol with known osmotic effects, with those of olestra (20 or 40 g/day in potato chips), a nonabsorbed fat, on objective measures of stool composition and GI symptoms. Sixty-six subjects resided on a metabolic ward for 12 days: 2 days lead-in, 4 days baseline, 6 days treatment. Sorbitol 40 g/day resulted in loose/liquid stools within 1-3 h of consumption. In contrast, olestra resulted in a dose-responsive stool softening effect after 2-4 days of consumption. Subjects reported "diarrhea" when mean stool apparent viscosity (peak force (PF), g) decreased from a perceived "normal" (mean +/- SE, 1355 +/- 224 g PF; firm stool) to loose (260 +/- 68 g PF) stool. Mean apparent viscosity of stool during treatment: placebo, 1363 +/- 280 g (firm); olestra 20 g/day 743 +/- 65 g (soft); olestra 40 g/day, 563 +/- 105 g (soft); and sorbitol 40 g/day, 249 +/- 53 g (loose). Of the 1098 stool samples collected, 38% (419/1098) were rated by subjects as "diarrhea," yet only 2% of treatment days (all in the sorbitol treatment group) met commonly accepted criteria for a clinical diarrhea. Sorbitol, but not olestra, increased the severity of abdominal cramping, urgency and nausea compared to placebo. Olestra consumption, at levels far in excess of normal snacking conditions, resulted in a gradual stool softening effect after several days of consumption, did not meet any of the three objective measures of diarrhea, and did not increase GI symptoms. Sorbitol consumption, at only 80% of the dose requiring a "laxative effect" information label, resulted in rapid onset loose/liquid stools and a significant increase in abdominal cramping, urgency and nausea. Overall, subjects categorized stool as "diarrhea" when stool decreased from their perceived "normal," but the vast majority of these reports were not associated with clinically significant diarrhea., (Copyright 2000 Academic Press.)

It is now widely accepted that H. pylori plays an important role in gastritis and PUD, and that eradication leads to a marked decrease in ulcer recurrence. H. pylori alone is not sufficient to cause ulcer disease. There is a complex interaction with acid production and mucosal protection. Treatment that is directed only at reducing acid production heals ulcers and is safe, but is temporary at best and does not address the underlying etiology. Currently treatment for H. pylori can only be recommended for patients with PUD confirmed to have H. pylori infection (Table 1). As further studies are done and more understanding is gained, these recommendations may be expanded. More research is needed with prospective studies to determine if eradication reduces the risk of gastric cancer. More research is also needed on shorter courses of treatment with fewer side effects, on the effectiveness of immunization against H. pylori, and on other innovative forms of treatment.

Assessment of presence of metastatic disease (m.d.) in bladder cancer (b.c.) can represent a main problem as influencing the appropriate therapeutic policy (mostly the indication to radical surgery). Evaluation of the real cost-effectiveness ratio of radiographic and radionuclide diagnostic work-up induced us to retrospectively review historical data about our b.c. patients (pts). From March 1988 to June 1991, 76 not consecutive pts with histologically proven bladder cancer were included in this study. 5 Pts were staged as T1, 25 as T2, 18 as T3a, 23 as T3b, 5 as T4. 2 Pts were graded as G1, 27 as G2, 44 as G3, 3 as Gx. Age varied from 39 to 89 years (average: 62.3). 79 Pts underwent the "basic work-up" (including chest plain film, bone and liver scans) and at least one follow-up control. 266 chest plain films, 22 chest x-ray tomograms, 2 chest CT scans, 27 bone x-ray tomograms, 231 bone scans, 240 liver scans, 17 liver ultrasonographies were totally realized. All pts underwent at least an abdomen-pelvic CT, but related results are not considered in the study. Fine needle aspiration cytologic biopsies were realized in selected cases; also these results are not selectively reported here. Together with cytologic positive results, only progression of m.d. was considered as its definite presence. Conventional x-ray examination (plain film integrated by x-ray tomograms of "suspicious" findings) resulted sufficiently complete and accurate to reveal chest m.d. Concerning skeletal diagnostic survey, only 6 pts (26%) out of 23 pts with "positive" bone scans really resulted affected by m.d.(ABSTRACT TRUNCATED AT 250 WORDS)

The AA. report the results obtained on patients suffering from surface bladder cancer [Ta-T1 (G1-G2)] treated with alpha 2b interferon with the aim to elongate the relapse period. The results turned out to be very satisfactory as concerns the tolerability of the drug as well as the actual improvement in relapse onset. Moreover, the AA. could notice the grading steadiness or decrease, as to neglistic relapses.

The aim of this study has been to assess the prevalence of autonomic neuropathy among non-insulin dependent diabetic patients, and to determine whether a correlation could be found between autonomic impairment and the following: age, sex, duration of diabetes, body mass and metabolic control. Two hundred and twenty one non-insulin dependent patients were submitted to four cardiovascular tests: heart rate response to deep breathing and to standing up; blood pressure response to handgrip and to standing up. Sixty six percent of patients showed at least one abnormal cardiovascular response. The prevalence of autonomic impairment was higher in non-insulin dependent than in insulin-dependent diabetics. Patients were grouped according to the extent of autonomic impairment: absent (33.5%), early (27.6%), definite (3.6%), severe (4.5%). An atypical pattern (abnormality of blood pressure responses in absence of a definite abnormality of heart rate responses) was found in 30.8% of patients. Heart rate responses correlated significantly with age (p less than 0.001). No correlation between test results and duration was found in the multivariate analysis. The tests' results did not correlate with metabolic control or body mass index. Patients with symptoms of autonomic neuropathy had values for heart rate response to deep breathing and to standing significantly lower than those without (p less than 0.05).

Fifteen infants (age 3-28 wk) suffered from severe diarrhea with acute dehydration and poor growth. Persistent watery stools and suboptimal nutrition necessitated central venous alimentation with prolonged hospitalization. Repeated stool and small intestinal fluid cultures yielded the classical enteropathogenic Escherichia coli serotype 0119:B14. In all patients, biopsy of the jejunum or rectal mucosa, or both, showed moderate to severe damage, irregular atrophy of surface epithelium, and subnuclear vacuolization of crypt epithelium. Ultrastructural studies revealed bacteria adherent to mucosal cells with flattening of microvilli, loss of the cellular terminal web, and cupping of the plasma membrane around individual bacteria. Heavily colonized cells had marked intracellular damage. Assays for heat-labile, heat-stable, and vero cell toxins were negative for these Escherichia coli isolates. Oral neomycin and nutritional support resulted in clearing of Escherichia coli 0119:B14 from stool and small bowel with improvement in histologic characteristics. Damage to enterocytes and villi by adherent nontoxigenic Escherichia coli 0119:B14 results in protracted diarrhea in infants.

Escherichia coli heat-stable enterotoxin (STa) induces intestinal secretion by binding to enterocyte receptors and activating the guanylate cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system. The intermediate steps between binding of STa and secretion are poorly understood, due in part to the lack of a convenient system to study the effects of STa at the cellular level. To establish such a model, we investigated the binding of 125I-STa, STa activation of guanylate cyclase, and STa-induced increase in cGMP production in a well-characterized human colonic cell line, T84. Binding was specific, linear with cell number, and time, temperature and pH dependent, and reversible. ST may also be internalized by these cells. Addition of unlabeled STa competitively inhibited binding of 125I-STa. These parameters closely resemble those described in intact rat enterocytes and cell-free membrane preparations. STa stimulated guanylate cyclase and cGMP production in a dose-related manner. The similar dose-response relationships for binding, guanylate cyclase stimulation by STa, and cGMP production suggest that the guanylate cyclase-cGMP system is coupled to ST occupancy of specific receptors. These data, together with the fact that STa induces chloride secretion from T84 cells suggest that T84 cells are a suitable and convenient system to study the cellular mechanism of action of STa.

We purified and sequenced the heat-stable enterotoxin produced by Citrobacter freundii. The toxin was detected during purification by reaction with monoclonal antibody to Escherichia coli heat-stable enterotoxin. The C. freundii toxin amino acid sequence was identical to that of the 18-amino-acid heat-stable enterotoxin (STa) produced by toxigenic E. coli.

In an effort to develop new approaches to the study and control of infectious diarrhea, we prepared murine monoclonal antibodies to the Escherichia coli heat-stable enterotoxin (STa). The toxin was purified from E. coli culture media and conjugated to bovine serum albumin. The STa-bovine serum albumin conjugate was used to immunize BALB/c mice, and the immune spleen cells from these mice were fused with SP2/0 myeloma cells. Resultant hybridomas were screened in an enzyme-linked immunosorbent assay protocol against 500 ng of STa-bovine serum albumin bound to microtiter wells as the solid-phase antigen. Five stable clones were selected and grown further in ascites fluid, which demonstrated anti-STa activity at dilutions of up to 1:500,000 in the enzyme-linked immunosorbent assay for heat-stable enterotoxin. In a competitive enzyme-linked immunosorbent assay format, the antibodies recognized several human and porcine strains of STa to various extents, but did not recognize E. coli heat-labile toxin, cholera toxin, or staphylococcal enterotoxin B. The antibodies were all able to bind lactoperoxidase-labeled [125I]STa, and antibody 20B3 was also able to dissociate [125I]STa bound to toxin receptors on rat jejunal villous cells. Preincubation of STa with antibodies 20B3 or 20F5 led to a concentration-dependent neutralization of toxin activity in a suckling mouse intestinal secretion assay. These antibodies are likely to provide new tools for the continued study of STa structure-function relationships and may lead to improved diagnosis and treatment of E. coli-induced infectious diarrhea.