Expanded TCRß CDR3 clonotypes distinguish Crohn's disease and ulcerative colitis patients.

We aimed to determine whether the TCR repertoires of Crohn's disease (CD) patients contain highly prevalent disease-specific T-cell clonotypes reflective of the characteristic and highly shared aberrant serum antibody reactivity to gut commensal flagellin antigens. The CD4 TCRß CDR3 sequence repertoires from active CD (n = 20) and ulcerative colitis (UC) (n = 10) patients were significantly more diverse, and individual sequences over-represented, compared to healthy controls (HC) (n = 97). While a very small number of expanded public CDR3 sequences are highly shared between active CD and UC, the majority of significantly expanded TCRß CDR3 clonotypes are private to CD and UC patients with equivalent prevalence among IBD patients. Further defining TCR clonotypes by Vß-CDR3 linkage showed significant differences in the TCR repertoires between UC and CD. Flagellin antigen exposure induced expansion of several TCRß CDR3 sequences in CD4 cells from a flagellin-seropositive subject including sequences highly shared by or relatively private to CD (and UC) patients. These data suggest that flagellin-reactivity contributes to the expansion of a small number of CD4 clonotypes but does not support flagellin antigens as predominantly driving CD4 cell proliferation in CD. Disease-specific expanded TCRß CDR3 clonotypes characterize CD and UC and the shared exposure to the gamut of gut microbial antigens.

Human gastric epithelial cells contribute to gastric immune regulation by providing retinoic acid to dendritic cells.

Despite the high prevalence of chronic gastritis caused by Helicobacter pylori, the gastric mucosa has received little investigative attention as a unique immune environment. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in the small intestinal mucosa, also contributes to gastric immune regulation. We report that human gastric tissue contains high levels of the RA precursor molecule retinol (ROL), and that gastric epithelial cells express both RA biosynthesis genes and RA response genes, indicative of active RA biosynthesis. Moreover, primary gastric epithelial cells cultured in the presence of ROL synthesized RA in vitro and induced RA biosynthesis in co-cultured monocytes through an RA-dependent mechanism, suggesting that gastric epithelial cells may also confer the ability to generate RA on gastric dendritic cells (DCs). Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In H. pylori-infected gastric mucosa, gastric RA biosynthesis gene expression was severely disrupted, which may lead to reduced RA signaling and thus contribute to disease progression. Collectively, our results support a critical role for RA in human gastric immune regulation.

The microbiota, the immune system and the allograft.

The microbiota represents the complex collections of microbial communities that colonize a host. In health, the microbiota is essential for metabolism, protection against pathogens and maturation of the immune system. In return, the immune system determines the composition of the microbiota. Altered microbial composition (dysbiosis) has been correlated with a number of diseases in humans. The tight reciprocal immune/microbial interactions complicate determining whether dysbiosis is a cause and/or a consequence of immune dysregulation and disease initiation or progression. However, a number of studies in germ-free and antibiotic-treated animal models support causal roles for intestinal bacteria in disease susceptibility. The role of the microbiota in transplant recipients is only starting to be investigated and its study is further complicated by putative contributions of both recipient and donor microbiota. Moreover, both flora may be affected directly or indirectly by immunosuppressive drugs and antimicrobial prophylaxis taken by transplant patients, as well as by inflammatory processes secondary to ischemia/reperfusion and allorecognition, and the underlying cause of end-organ failure. Whether the ensuing dysbiosis affects alloresponses and whether therapies aimed at correcting dysbiosis should be considered in transplant patients constitutes an exciting new field of research.

Human primary gastric dendritic cells induce a Th1 response to H. pylori.

Adaptive CD4 T-cell responses are important in the pathogenesis of chronic Helicobacter pylori gastritis. However, the gastric antigen-presenting cells that induce these responses have not yet been identified. Here we show that dendritic cells (DCs) are present in the gastric mucosa of healthy subjects and are more prevalent and more activated in the gastric mucosa of H. pylori-infected subjects. H. pylori induced gastric DCs isolated from noninfected subjects to express increased levels of CD11c, CD86 and CD83, and to secrete proinflammatory cytokines, particularly interleukin (IL)-6 and IL-8. Importantly, gastric DCs pulsed with live H. pylori, but not control DCs, mediated T-cell secretion of interferon-gamma. The ability of H. pylori to induce gastric DC maturation and stimulate gastric DC activation of Th1 cells implicates gastric DCs as initiators of the immune response to H. pylori.

Cycling of gut mucosal CD4+ T cells decreases after prolonged anti-retroviral therapy and is associated with plasma LPS levels.

The gut mucosa is an important site of HIV immunopathogenesis with severe depletion of CD4+ T cells occurring during acute infection. The effect of prolonged anti-retroviral therapy (ART) on cycling and restoration of T lymphocytes in the gut remains unclear. Colon and terminal ileal biopsies and peripheral blood samples were collected from viremic, untreated, HIV-infected participants, patients treated with prolonged ART (>5 years), and uninfected controls and analyzed by flow cytometry. In the gut, the proportion of cycling T cells decreased and the number of CD4+ T cells normalized in treated patients in parallel with beta 7 expression on CD4+ T cells in blood. Cycling of gut T cells in viremic patients was associated with increased plasma LPS levels, but not colonic HIV-RNA. These data suggest that gut T-cell activation and microbial translocation may be interconnected whereas prolonged ART may decrease activation and restore gut CD4+ T cells.

Successful granulocyte-colony stimulating factor treatment of Crohn's disease is associated with the appearance of circulating interleukin-10-producing T cells and increased lamina propria plasmacytoid dendritic cells.

Granulocyte-colony stimulating factor (G-CSF) has proved to be a successful therapy for some patients with Crohn's disease. Given the known ability of G-CSF to exert anti-T helper 1 effects and to induce interleukin (IL)-10-secreting regulatory T cells, we studied whether clinical benefit from G-CSF therapy in active Crohn's disease was associated with decreased inflammatory cytokine production and/or increased regulatory responses. Crohn's patients were treated with G-CSF (5 microg/kg/day subcutaneously) for 4 weeks and changes in cell phenotype, cytokine production and dendritic cell subsets were measured in the peripheral blood and colonic mucosal biopsies using flow cytometry, enzyme-linked immunosorbent assay and immunocytochemistry. Crohn's patients who achieved a clinical response or remission based on the decrease in the Crohn's disease activity index differed from non-responding patients in several important ways: at the end of treatment, responding patients had significantly more CD4(+) memory T cells producing IL-10 in the peripheral blood; they also had a greatly enhanced CD123(+) plasmacytoid dendritic cell infiltration of the lamina propria. Interferon-gamma production capacity was not changed significantly except in non-responders, where it increased. These data show that clinical benefit from G-CSF treatment in Crohn's disease is accompanied by significant induction of IL-10 secreting T cells as well as increases in plasmacytoid dendritic cells in the lamina propria of the inflamed gut mucosa.

Peptide YY administration decreases brain aluminum in the Ts65Dn Down syndrome mouse model.

We have previously reported the Ts65Dn (Ts) mouse has impaired intestinal absorptive function and amino acid metabolism. Peptide YY (PYY) has enhanced glucose absorption in mice and turkeys. Other studies have reported that persons with Down syndrome have increased intestinal absorption of aluminum. Alzheimer's-like lesions have been reported in Ts mice. Trial 1 of this study examined brain Al concentrations, plasma metabolites and intestinal metabolism of 40 control and 40 Ts mice administered 300microg PYY/kg body weight or 0.9% saline for 3d. Trial 2 examined nutrient digestibility of 12 C and 12 Ts given PYY or saline for 14d. In Trial 1, PYY lowered (p<0.05) the brain Al pool (mg/g FBW) in both C and Ts mice by 80% compared to saline. Ts mice had increased plasma NH3 (329 vs. 269 microM, p<0.05), decreased plasma glucose (7.4 vs. 8.4 mM, p<0.01), elevated apparent energetic efficiency of jejunal glucose uptake (p<0.01) and elevated brain Al pool (0.41 vs. 0.12 microg, p=0.06) compared to C mice. In Trial 2, PYY increased small intestinal density (mg/cm) 12% in both genotypes (p<0.05), but did not alter nutrient digestibility. Brain Al accretion and hyperammonemia are proposed risk factors for Alzheimer's disease (AD). Ts mice and PYY appear to be suitable models for the study of metabolic and neurological anomalies in Down syndrome and AD.

Peptide YY Y1 receptor activates mitogen-activated protein kinase and proliferation in gut epithelial cells via the epidermal growth factor receptor.

The G-protein-coupled peptide YY (PYY)/neuropeptide Y Y1 receptor (Y1R) subtype is highly expressed in the proliferative zone of human colonic crypt epithelial cells but biochemical and biological support for growth effects have been lacking. Using a model gut epithelial cell system, we have stably expressed the human Y1R in IEC-6 cells and show that the Y1R does couple to mitogen-activated protein kinase (MAPK) phosphorylation and cell growth. This pathway uses pertussis-toxin-sensitive G-proteins and betagamma subunits, inhibited by co-transfected alpha-transducin. The Src-family tyrosine kinase inhibitor PP1, as well as specific inhibition of the epidermal growth factor receptor tyrosine kinase (EGFR TK) by PD153035, also blocks PYY stimulation of MAPK. This pathway further requires protein kinase C with EGFR TK inhibition blocking PYY-induced protein kinase Cepsilon (PKCepsilon) translocation to the cell membrane. Finally, we show that PYY stimulates growth in Y1R-expressing gut epithelial cells that is dependent on EGFR TK activity. These results demonstrate a novel pathway involving G(i)/G(o) protein, EGFR and PKC to activate MAPK. Further, they support a role for PYY and the Y1R in regulating growth in human colonic epithelium.

Primary structures of PYY, [Pro(34)]PYY, and PYY-(3-36) confer different conformations and receptor selectivity.

We synthesized PYY-(1-36) (nonselective between Y(1) and Y(2) receptor subtype agonists), [Pro(34)]PYY (selective for Y(1)), and PYY-(3-36) (selective for Y(2)) to determine whether solution conformation plays a role in receptor subtype selectivity. The three peptides exhibited the expected specificities in displacing labeled PYY-(1-36) from cells transfected with Y(1) receptors (dissociation constants = 0.42, 0.21, and 1,050 nM, respectively) and from cells transfected with Y(2) receptors (dissociation constants = 0.03, 710, and 0.11 nM, respectively) for PYY-(1-36), [Pro(34)]PYY, and PYY-(3-36). Sedimentation equilibrium analyses revealed that the three PYY analogs were 80-90% monomer at the concentrations used for the subsequent circular dichroism (CD) and (1)H-nuclear magnetic resonance (NMR) studies. CD analysis measured helicities for PYY-(1-36), [Pro(34)]PYY, and PYY-(3-36) of 42%, 31%, and 24%, suggesting distinct differences in secondary structure. The backbone (1)H-NMR resonances of the three peptides further substantiated marked conformational differences. These patterns support the hypothesis that Y(1) and Y(2) receptor subtype binding affinities depend on the secondary and tertiary solution state structures of PYY and its analogs.

Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway.

The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a(-/-) mice, which lack AT(1A) receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT(1)) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT(1) receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT(1) signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT(1) receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.

Peptide YY/neuropeptide Y Y1 receptor expression in the epithelium and mucosal nerves of the human colon.

OBJECTIVES: Peptide YY is an abundant distal gut hormone which regulates secretion, motility, and possibly epithelial proliferation in the gut. Though messenger RNA for the peptide YY Y1 receptor subtype occurs in the basal colonic crypts of humans, peptide YY receptors themselves have not been clearly localized within the adult human gastrointestinal tract. Using an antiserum directed against the C-terminus of the Y1 receptor we determined the actual extent of Y1 receptor protein expression in the human colon in order to identify areas targeted for peptide YY effects and suggest additional physiological roles for PYY in the human gut. RESULTS: Y1 receptor protein expression was seen throughout the colonic epithelium along its basolateral aspect. There was an unexpected dense distribution of Y1 receptor immunoreactivity in varicose fibers within the mucosa. Staining was also noted in nerve fibers of the muscularis mucosae, in the submucous and myenteric plexuses, and in nerves in the muscularis propria. CONCLUSIONS: Widespread distribution of Y1 receptors in the colonic epithelium and mucosal nerve fibers suggests diverse regulatory roles for peptide YY in modulating epithelial function as well as secretomotor reflexes in response to lumenal peptide YY-release signals.

Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2.

Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.

Peptide YY inhibits vasopressin-stimulated chloride secretion in inner medullary collecting duct cells.

mIMCD-k2 cells are derived from the inner medullary collecting duct of a mouse and exhibit electrogenic sodium absorption and cAMP- and vasopressin (AVP)-stimulated electrogenic chloride secretion [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; and N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The purpose of the present study was to determine how peptide YY (PYY) affects electrogenic Na+ and Cl- current in mIMCD-k2 cells. Short-circuit currents (Isc) were measured across monolayers of mIMCD-k2 cells mounted in Ussing-type chambers. PYY did not alter baseline Isc, nor did it alter Isc in chloride-free conditions, indicating no effect on electrogenic sodium transport. Baseline chloride current in these cells is low; therefore, chloride short-circuit current (IClsc) was stimulated with AVP (10 nM) added to the basolateral surface and 10 microM amiloride added to the apical surface. Although apical applications of PYY had no effect, basolateral application of PYY caused attenuation of IClsc, with the maximal inhibitory dose (100 nM) causing 52 +/- 1.3% inhibition (IC50 = 0.11 nM). Inhibition by PYY of IClsc is mediated through the Y2 receptor subtype, as PYY-(3-36) was the only PYY analog tested that caused inhibition and was equipotent to PYY. Inhibition by PYY of IClsc was abolished following incubation with pertussis toxin. We also show that PYY inhibits AVP-stimulated cAMP accumulation, with a maximal inhibitory dose (100 nM) causing a 38% +/- 6% inhibition (IC50 = 0.16 nM), comparable to inhibition by PYY of IClsc. We conclude that PYY acts through either Gi or Go to inhibit adenylate cyclase activity, leading to a decrease in AVP-stimulated chloride current.

The neuropeptide Y/peptide YY Y1 receptor is coupled to MAP kinase via PKC and Ras in CHO cells.

The neuropeptide Y/peptide YY (PYY) Y1 receptor subtype mediates proliferative responses. This report identifies effector molecules which mediate mitogen-activated protein kinase (MAPK) phosphorylation by Y1 receptor activation in transfected CHO cells. Pertussis toxin pretreatment abolishes this effect, indicating involvement of Gi or G(o) proteins. Inhibition of protein kinase C (PKC) also blocks PYY-induced MAPK phosphorylation. Additionally in this cell model PYY causes an increase in GTP binding to Ras protein, and cotransfection of dominant negative constructs for Ras and Raf blocks PYY effects on MAPK. These data suggest a novel mechanism for Y1 receptor coupling to MAPK, which is at once pertussis toxin-sensitive as well as PKC- and Ras-dependent.

Peptide YY receptor distribution and subtype in the kidney: effect on renal hemodynamics and function in rats.

This study characterizes the location and subtype of peptide YY (PYY) receptors in rat and rabbit kidney and the effect of PYY on renal function and renal hemodynamics in rats. Receptor autoradiography performed on kidney sections revealed a dense concentration of specific high-affinity binding sites [dissociation constant (Kd) = 0.7 +/- 0.1 nM] in the papilla of the rat, as well as cortical and papillary binding in the rabbit (papilla, Kd = 1.6 +/- 0.6 nM) and some medullary binding in both species. In the rat papilla, neuropeptide Y (NPY) and the Y1 agonist [Leu31,Pro34]NPY competed with PYY for binding (Kd = 1.1 +/- 0.4 nM and 1.6 +/- 0.5 nM, respectively), but NPY-(13-36) (Y2 agonist) and pancreatic polypeptide (PP, Y4 agonist) were without effect, demonstrating that the PYY receptor in the rat papilla is of the Y1 subtype. In the rabbit papilla, NPY and NPY-(13-36) competed with PYY (Kd = 0.5 +/- 0.1 and 3.1 +/- 0.6 nM, respectively), but [Leu31,Pro34]NPY and PP were without effect, evidence that the PYY receptor in the rabbit papilla is of the Y2 subtype. Infusion of PYY into rats (47 pmol x kg(-1) x min[-1]) increased mean arterial pressure (103 +/- 6 to 123 +/- 8 mmHg) and decreased renal plasma flow (13 +/- 1.8 to 8.4 +/- 2.1 ml/min) but produced no significant change in glomerular filtration rate or sodium excretion. Injection of PYY or angiotensin II directly into the renal artery caused a dose-related vasoconstriction, which was less intense but of longer duration for PYY than for angiotensin II. These results show that receptors for PYY are widely distributed in the kidney and that exogenously administered PYY causes renal vasoconstriction and may influence renal sodium excretion.

Retinoic acid is a negative regulator of the neuropeptide Y/peptide YY Y1 receptor gene in SK-N-MC cells.

To identify effectors of neuropeptide Y Y1 receptor gene expression predicted by putative regulatory elements in its 5' flanking region, we examined the effects of several transcriptionally active agents on Y1 receptor mRNA levels and 125I-peptide YY binding capacity in SK-N-MC cells. Phorbol ester caused a rapid, transient 2.6-fold increase in Y1R mRNA levels. However, all trans-retinoic acid caused a rapid, sustained decrease in Y1 receptor mRNA levels (to 25% of control). Cycloheximide pretreatment did not attenuate the maximal inhibitory effect of retinoic acid, but it prolonged the time to achieve maximal efficacy. The retinoic acid effect was secondary to both a significant decrease in Y1 receptor mRNA stability and a decreased Y1 receptor gene transcription rate. Y1 receptor binding capacity was also significantly decreased after retinoic acid treatment (368 +/- 25 vs. 496 +/- 28 fmol/mg of protein for control). These data support a role for retinoic acid as an important agent regulating Y1 receptor gene expression and mediating Y1 receptor down-regulation.

Total parenteral nutrition alters NPY/PYY receptor levels in the rat brain.

The regulation of appetite and satiety is complex and may involve peptide mediators such as cholecystokinin (CCK) and neuropeptide Y (NPY). Studies have indicated that calories administered enterally and parenterally impact on feeding, and possibly via the release of such mediators. Recent data from our laboratory have shown that total parenteral nutrition (TPN) reduces sham feeding in dogs by 50%. We hypothesized that TPN may alter feeding via an NPY-mediated mechanism. To test our hypothesis, we examined the effect of continuous administration of TPN on NPY receptor levels in the rat brain. Rats were surgically prepared with intravenous catheters. After 72 h of TPN infusion, the rats were anesthesized with sodium pentobarbital and their brains were removed. Neuropeptide Y receptor density was assessed by autoradiography in the paraventricular nucleus, olfactory cortex, dentate gyrus, and thalamus. These results were compared to the control group receiving intravenous saline. A third group receiving enteral nutrition was examined as well. Neuropeptide Y receptor numbers were significantly increased in the paraventricular nucleus of rats receiving TPN compared to the groups receiving intravenous saline or enteral nutrition. We conclude that continuous parenteral nutrition significantly increases NPY receptor density in the rat brain suggesting that TPN may impact feeding via the regulation of NPY receptor-mediated effects.

Distribution of binding sites for thromboxane A2 in the mouse kidney.

Thromboxane A2 (TxA2) is a potent vasoconstrictor eicosanoid that has been implicated in the pathogenesis of both human and experimental renal diseases. The biological actions of TxA2 in the kidney are mediated through specific cell-surface receptors. In this report, we characterize the distribution of thromboxane receptors (TxR) within the normal mouse kidney by receptor autoradiography. With the iodinated TxR agonist [125I]BOP, TxA2 binding sites were detected throughout the kidney. Competitive inhibition curves of whole kidney binding demonstrated a half-maximal inhibitory concentration of 6.5 nM. When Scatchard analysis was performed on anatomically discrete regions, the [125I]BOP binding in the medulla was best fit by a one-site model, with a dissociation constant (Kd) of 8.2 +/- 2.2 nM. In contrast, [125I]BOP binding in the cortex was better described by a two-site model, with estimated Kd of 262 +/- 16 pM for a higher affinity site and 16.9 +/- 1.3 nM for a lower affinity site. These sites do not appear to represent receptor isoforms that arise from alternative splicing of mRNA. The lower affinity binding sites may represent a novel TxR or an alternative affinity state for the previously characterized high-affinity binding site.

Localization and characterization of neuropeptide Y/peptide YY receptors in the brain of the smooth dogfish (Mustelis canis).

Multiple receptor subtypes specific for the neuropeptide Y (NPY)/peptide Y (PYY) family of peptides exist in mammals, but little is known about the distribution of this receptor family in other vertebrates. Saturable binding sites for 125I-labeled porcine PYY were localized in frozen sections of the brain of the smooth dogfish (Mustelis canis) by radioligand binding and autoradiography. Saturable 125I-porcine PYY binding sites were distributed widely in the cerebral hemispheres, optic lobes, hypothalamus, cerebellum and hindbrain. Binding was saturable, specific for PYY and related peptides, and of high affinity (Kd = 2.53 nM). The specificity of the binding site was analyzed by performing competitive inhibition experiments with nonradioactive PYY, NPY, and [Leu31, Pro34]-NPY and NPY13-36, synthetic peptide analogs specific for the mammalian Y1 and Y2 receptor subtypes, respectively. Saturable 125I-porcine PYY binding sites in all regions of the dogfish brain closely resembled the mammalian Y1 NPY receptor subtype in specificity for these substances. There was no evidence for expression of multiple receptor subtypes. We conclude that a single receptor specific for the NPY/PYY family of peptides is widely expressed in the smooth dogfish brain and that this receptor closely resembles the mammalian Y1 receptor subtype, suggesting that the Y1 receptor is the ancestral receptor in this family.

Diagnostic utility of K-ras mutational analysis on bile obtained by endoscopic retrograde cholangiopancreatography.

BACKGROUND: Because K-ras oncogene mutations are present in more than 90% of pancreaticobiliary tumors, we tested prospectively whether K-ras mutational analysis of bile samples obtained during ERCP was superior to conventional bile cytology for diagnosis of malignancy. METHODS: Bile samples from 20 patients undergoing ERCP for evaluation of jaundice were examined by both cytologic study and the polymerase chain reaction for the presence of K-ras oncogene mutations. RESULTS: Polymerase chain reaction products were amplified from the bile of 8 of 12 patients with malignancy and 3 of 8 with benign disease; K-ras oncogene mutations were present in 4 of 8 polymerase chain reaction products from malignant samples but absent in all 3 from benign samples. No cytologic results were positive for malignancy. CONCLUSIONS: Bile obtained during ERCP can yield positive results in K-ras mutational analysis, even when results of conventional bile cytology are negative. In this study, K-ras mutational analysis had a sensitivity of 33% (4 of 12), a specificity of 100%, and a positive predictive value of 100% for diagnosis of malignancy.