Monoclonal Antibodies: Past, Present and Future.

Monoclonal antibodies (mAbs) are immunoglobulins designed to target a specific epitope on an antigen. Immunoglobulins of identical amino-acid sequence were originally produced by hybridomas grown in culture and, subsequently, by recombinant DNA technology using mammalian cell expression systems. The antigen-binding region of the mAb is formed by the variable domains of the heavy and light chains and contains the complementarity-determining region that imparts the high specificity for the target antigen. The pharmacokinetics of mAbs involves target-mediated and non-target-related factors that influence their disposition.Preclinical safety evaluation of mAbs differs substantially from that of small molecular (chemical) entities. Immunogenicity of mAbs has implications for their pharmacokinetics and safety. Early studies of mAbs in humans require careful consideration of the most suitable study population, route/s of administration, starting dose, study design and the potential difference in pharmacokinetics in healthy subjects compared to patients expressing the target antigen.Of the ever-increasing diversity of therapeutic indications for mAbs, we have concentrated on two that have proved dramatically successful. The contribution that mAbs have made to the treatment of inflammatory conditions, in particular arthritides and inflammatory bowel disease, has been nothing short of revolutionary. Their benefit has also been striking in the treatment of solid tumours and, most recently, as immunotherapy for a wide variety of cancers. Finally, we speculate on the future with various new approaches to the development of therapeutic antibodies.

A 5-week study of the pharmacokinetics and pharmacodynamics of LY2189265, a novel, long-acting glucagon-like peptide-1 analogue, in patients with type 2 diabetes.

AIM: To investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of LY2189265 (LY), a novel, long-acting glucagen-like peptide-1 analogue, administered once weekly to subjects with type 2 diabetes. METHODS: This was a placebo-controlled, parallel-group, subject- and investigator-blind study of LY in subjects (N = 43) with type 2 diabetes mellitus controlled with diet and exercise alone or with a single oral antidiabetic medication. Subjects taking metformin or thiazolidinediones continued on their therapy. Subjects receiving sulfonylurea, acarbose, repaglinide or nateglinide were switched to metformin prior to enrollment. Subjects received five once-weekly doses of 0.05, 0.3, 1, 3, 5 or 8 mg. Effects on glucose, insulin and C-peptide concentrations were determined during fasting and following standard test meals. The pharmacokinetics of LY and its effects on HBA1c, glucagon, body weight, gastric emptying and safety parameters were assessed. RESULTS: Once-weekly administration of LY significantly reduced (p < 0.01) fasting plasma glucose, 2-h post-test meal postprandial glucose and area under the curve (AUC) of glucose after test meals at doses ≥1 mg. These effects were seen after the first dose and were sustained through the weekly dosing cycle. Most doses produced statistically significant increases in insulin and C-peptide AUC when normalized for glucose AUC. Statistically significant reductions in HBA1c were observed for all dose groups except 0.3 mg. The most commonly reported adverse effects (AEs) were nausea (35 events), headache (20 events), vomiting (18 events) and diarrhoea (8 events). CONCLUSIONS: LY showed improvement in fasting and postprandial glycaemic parameters when administered once weekly in subjects with type 2 diabetes. The pharmacokinetics and safety profiles also support further investigation of this novel agent.

LY2189265, a long-acting glucagon-like peptide-1 analogue, showed a dose-dependent effect on insulin secretion in healthy subjects.

AIM: To assess the safety, tolerability, pharmacokinetics, pharmacodynamics and potential immunogenicity of single, escalating subcutaneous injections of a once-weekly glucagon-like peptide-1 analogue in healthy subjects. METHODS: This phase 1, three-period, crossover, double-blind, placebo-controlled study investigated single, escalating subcutaneous doses of LY2189265 (LY) ranging from 0.1 to 12 mg; approximately six subjects were randomized to each dose. Parameters of safety, including adverse events, were assessed. The pharmacokinetic profile was assessed over 14 days. Pharmacodynamic parameters (glucose and insulin concentrations) were measured following a step-glucose infusion (day 3) and as part of an oral glucose tolerance test (OGTT) (day 5). RESULTS: LY was generally well tolerated with some increase in gastrointestinal symptoms with escalating doses. There were small dose-dependent increases in pulse rate with doses ≥1.0 mg and diastolic blood pressure with doses ≥3.0 mg. The half-life of LY was approximately 90 h, with C(max) occurring between 24 and 48 h in most subjects. Evidence of increase in glucose-dependent insulin secretion and suppression of serum glucose excursions were observed during an OGTT at all doses compared to placebo; no episodes of hypoglycaemia occurred. No subjects developed antibodies to LY2189265. CONCLUSIONS: LY showed an acceptable safety profile and exhibited the expected glucagon-like peptide-1 pharmacological effects on glucose suppression and insulin secretion with a half-life that supports once-weekly dosing.

Non-invasive assessment of selective 5-HT(1B/1D)-receptor agonist-induced peripheral vascular effects in humans: comparison of different techniques.

OBJECTIVE: To compare the sensitivity of three non-invasive techniques for detecting serotonin (5-HT)(1B/1D)-receptor agonist-induced peripheral vascular effects in humans: the measurement of (1) systolic (SBP) and diastolic (DBP) blood pressures, (2) dorsal hand vein (DHV) diameter and (3) toe-arm systolic blood pressure gradient (DeltaSBP(toe-arm)). METHODS: A double-blind, placebo-controlled, three-way, cross-over study was performed in 12 healthy male volunteers. According to a randomly assigned allocation schedule, subjects were administered sumatriptan 3 mg, sumatriptan 6 mg or placebo subcutaneously. Measurements were performed at baseline, every 5 min for 30 min and at 40 min and 60 min after drug administration. SBP and DBP were recorded using a semi-automated oscillometric device. DHV diameter was measured using a linear variable differential transformer. DeltaSBP(toe-arm) was calculated after measuring toe and arm SBP with a strain-gauge technique. Sensitivity was evaluated with responsiveness statistics. RESULTS: Based on weighted mean and compared with placebo, sumatriptan 3 mg and 6 mg increased SBP by 3.3 mmHg ( P=0.023) and 6.4 mmHg ( P<0.001) and DBP by 5.0 mmHg ( P=0.006) and 7.5 mmHg ( P<0.001), respectively. Sumatriptan 3 mg and 6 mg decreased DHV diameter by 36% ( P=0.015) and 40% ( P=0.005), respectively. DeltaSBP(toe-arm) did not change. Peak changes were observed within 10-15 min after drug administration. The rank order of responsiveness was: BP > DHV diameter > DeltaSBP(toe-arm.) CONCLUSIONS: Clinically relevant doses of subcutaneous sumatriptan increased blood pressure and decreased DHV diameter without affecting DeltaSBP(toe-arm). The increase in blood pressure appeared to be dose dependent. Compared with DHV diameter and DeltaSBP(toe-arm), blood pressure measurement appeared to be the most sensitive technique for detecting selective 5-HT(1B/1D)-receptor agonist-induced peripheral vascular effects in humans.

Slowly inactivating sodium currents are reduced by exposure to oxidative stress.

Exposure of cardiac myocytes to oxidant stress has been implicated in the development of reperfusion arrhythmias. Studies on the effects of free radical generating systems on the fast sodium current have suggested an increase in a "window" current. The resulting increase in sodium influx has been hypothesized to cause an intracellular sodium load that stimulates Na+, Ca2+ exchange and promotes a Ca2+ overload. To test this proposal, the time course for effects of oxidative stress on a sodium current elicited with voltage ramps was investigated in feline ventricular myocytes. No window current was observed; instead, a slowly inactivating sodium current was generated at negative voltages near the sodium threshold potential. At room temperature there were no effects of a 30-min exposure to 1 mm H2O2 on this slowly inactivating sodium current. Likewise, there were no effects of either 1 mm H2O2 or 1.5 mm t-butyl hydroperoxide on fast sodium currents recorded at cool temperatures (12-15 degrees C). Experiments were repeated with t-butyl hydroperoxide at warm temperatures (30-33 degrees C), and the fast sodium current was reduced in magnitude and the reversal potential shifted to more negative voltages. These results demonstrate a temperature dependence for the loss of the fast sodium current during exposure to t-butyl hydroperoxide. Two exponentials were fit to the decaying phase of the fast sodium current and the slow time constant of inactivation was prolonged, suggesting delayed inactivation of the sodium current. Currents elicited with a steady-state inactivation protocol suggested development of a non-inactivating component during exposure to t-butyl hydroperoxide at warm temperatures. Direct evaluation of the slowly inactivating sodium current elicited by voltage ramps at warm temperatures (33-35 degrees C), and analysed as subtraction currents to remove background leak currents, showed a gradual reduction. It is concluded that the non-inactivating component identified during analysis of the fast sodium current was not the result of enhancement of either a slowly inactivating sodium current or a window current. Thus, an increase in sodium influx through voltage-dependent sodium channels does not occur during exposure to oxidative stress, and therefore, cannot induce an intracellular sodium load.

Protein kinase-dependent Cl- currents in feline ventricular myocytes.

A Cl- current (ICl) induced by isoproterenol (ISO) has been identified in isolated guinea pig ventricular myocytes. This ISO-induced ICl can be inhibited by propranolol and mimicked by forskolin (FSK), suggesting that beta-receptors, cAMP, and protein kinase A (PKA) are involved in regulating the involved Cl- channel. Because activation of protein kinase C (PKC) mediated via alpha-adrenergic receptor stimulation is also known to regulate several ion channels, the idea that activation of PKC also can induce ICl was investigated by using isolated feline ventricular myocytes and the whole-cell patch-clamp technique. We found that extracellularly applied phorbol 12-myristate 13-acetate (PMA) could activate ICl in feline ventricular cells. Control experiments indicated that in the absence of PMA or other interventions, the steady-state current-voltage relation of patches maintained for more than 40 minutes was unchanged over a voltage range from -100 to +80 mV. This suggests that the present findings are not complicated by the development over time after patching of a steady-state ICl, similar to the findings reported for canine atrial myocytes. When induced by PMA, ICl was noninactivating and outwardly rectifying; it reversed polarity at approximately the equilibrium potential for Cl- and was sensitive to the Cl- channel blocker 9-anthracene carboxylic acid. In contrast, PMA failed to induce ICl when either staurosporine or calphostin C was added to the patch pipette solution used to internally dialyze the myocytes. The kinetic properties of PMA- and FSK-induced ICl were similar. When supramaximal concentrations of both ISO (1 mumol/L) and PMA (6 mumol/L) were applied simultaneously, the size of the induced ICl was the same as that induced by the same concentrations of either agonist applied alone. In addition, maximal induction of ICl with PMA (6 mumol/L) prevented the effects of FSK (1 mumol/L, the concentration causing approximately 40% of the maximal response [approximately EC40]), yet the effects of simultaneously applied submaximal concentrations (eg, approximately EC25 to approximately EC40) of both 0.5 mumol/L PMA and 1 mumol/L FSK were roughly additive. The results suggest that (1) both PMA and ISO or FSK can induce ICl with approximately equal efficacy, (2) the PMA- and ISO- or FSK-induced ICls are similar, and (3) they all flow through the same set of Cl- channels, implying that channel phosphorylation via either PKA or PKC can activate this feline cardiac ICl.

Interactions of H2O2, EGTA and patch pipette recording methods in feline ventricular myocytes.

Free radical generating systems produce a sequence of changes in the action potentials of isolated cardiac myocytes. Efforts to understand the nature of the alterations in ionic currents have yielded mixed results. The use of patch-pipettes for voltage-clamp experiments and manipulation of pipette solution composition may contribute to the confusion. In this study, action potentials were recorded from feline ventricular myocytes during exposure to 100 microM H2O2. Action potential changes recorded with conventional, high resistance microelectrodes occurred in three stages as previously reported. The action potential changes recorded with patch-pipettes showed increases in action potential duration, notch and plateau potentials over time. These cells did not display after depolarizations or loss of excitability as seen with conventional microelectrodes. With the patch-pipette method, untreated control cells had time-dependent changes in the action potentials. When the pipette filling solution was modified by the addition of 5 mM EGTA, the H2O2-induced changes appeared reduced but not eliminated. The initial control values of action potential parameters before exposure to H2O2 varied with the presence or absence of EGTA. These data show both direct effects of EGTA and of time on action potential parameters independent of H2O2-exposure. These artifacts of the patch-pipette method and the composition of the pipette solutions affect the sequence of changes in the action potential during exposure to H2O2.

A 3,4-diaminopyridine-insensitive, Ca(2+)-independent transient outward K+ current in cardiac ventricular myocytes.

A previously unrecognized current that initially is not present and requires at least 25 min of intracellular access to develop can be found in approximately 75% of cardiac myocytes isolated from cat ventricle within 90 min after intracellular access is obtained with conventional suction patch pipette electrodes. We refer to this patch-duration-dependent (PDD) current as IK(PDD). IK(PDD) can be elicited with depolarizing test steps (Vt) ranging between -40 and +60 mV applied after a hyperpolarizing conditioning step to -140 mV for 200 ms from a holding potential of -40 mV. It shows an ohmic voltage dependence and appears to be an essentially pure K+ current. At Vt = 30 mV, the current is a time-dependent, transient current with a time to peak of 1.06 +/- 0.10 ms (n = 5) and a decay phase that can be fit to the sum of two decaying exponentials (tau f = 3.30 +/- 0.51 ms and tau s = 2.48 +/- 5.6 ms; n = 5). The voltage dependence of the steady-state inactivation can be fit to a single exponential Boltzmann distribution with a slope factor of 8.97 mV, and the voltage at which 50% of the channels are inactivated is -78 mV. The current can be blocked by 0.2 mM Ba2+ extracellularly applied or Cs+ intracellularly applied but is insensitive to 0.5 mM 3,4-diaminopyridine. These characteristics are unlike those for other known K+ currents. The lack of similarity between IK(PDD) and any currently documented cardiac K+ current suggests that IK(PDD) is either a previously undescribed K+ current or a modification of IK1 that makes it adopt an ohmic nature transiently, even in the presence of millimolar internal Mg2+.

Lack of myocardial lipid peroxidation during acute reperfusion injury in perfused guinea pig hearts.

OBJECTIVE: The aim was to test the hypothesis that free radical induced lipid peroxidation contributes to the arrhythmias and contractile changes associated with reperfusion. Evidence of oxidative stress in the membrane lipids was sought using four different approaches: (1) determination of lipid peroxidation metabolites; (2) direct detection of free radical formation using a lipophilic spin trap; (3) assessment of lipid peroxidation substrate; and (4) evaluation of antioxidant levels in the heart. METHODS: Electrical and contractile functions were monitored in isolated perfused guinea pig hearts subjected to ischaemia for 20 or 60 min followed by reperfusion for 1 or 20 min. The reperfused hearts were flushed with cold phosphate buffer with EDTA and the ventricles weighed and homogenised. Malonaldehyde content was determined using both a thiobarbituric acid spectrophotometric assay and a p-aminobenzoate spectrofluorescence assay. A lipophilic spin trap, PBN, was injected into guinea pigs 2 h before they were killed. Hearts containing spin traps were perfused for 5 min, then subjected to ischaemia followed by 1 min reperfusion. Lipid extracts containing PBN were examined using electron paramagnetic resonance spectroscopy. The content of polyunsaturated fatty acids and alpha tocopherol were determined by GLC and HPLC, respectively. RESULTS: Malonaldehyde content of the heart tissues did not increase during any combination of ischaemic and reperfusion episodes. The assays were effective at detecting raised malonaldehyde in hearts perfused with either 0.5 mM malonaldehyde standard or 0.5 mM cumene hydroperoxide. No spin adducts were found in either the extracted membrane lipids or the aqueous layer. Spin adducts were detected in myocardial lipids from animals exposed to non-lethal doses of gamma irradiation, confirming the presence of the spin trap in the heart tissue and the ability to detect spin adducts when formed. There were no differences in the percentage of total fatty acids that were polyunsaturated between reperfused hearts and continously perfused controls. Furthermore, alpha tocopherol content of the hearts exposed to ischaemia and reperfusion was not decreased relative to hearts perfused without an ischaemic episode. CONCLUSIONS: The results suggest that the early functional disturbances during reperfusion are not related to peroxidation of membrane lipids.

Mutagenicity of basic fractions derived from lamb and beef cooked by common household methods.

Mutagen production was examined in lamb and beef in relation to certain common household cooking methods. Mutagenicity was assessed, after extraction of the basic fraction of cooked meat samples, using Salmonella typhimurium strain TA1538 with added rat-liver S-9 homogenate. Little or no mutagenicity was found in barbecued lamb chops, in microwave-cooked lamb chops, sirloin steak, leg of lamb, or rolled beef loaf, in roasted leg of lamb or rolled beef loaf, in stewed blade steak or in boiled chuck steak. However, the basic fraction from well-done, edible fried or grilled meat contained mutagenic activity equivalent to approximately 30,000 TA1538 revertants/100 g cooked meat. It was found tht the mutagenic activity of grilled lamb chops, sirloin and rump steaks was directly related to the average surface temperatures attained during cooking. Use of butter as a frying medium was particularly associated with higher mutagenicity in meat samples. Fried meats (rump and fillet steaks) generally yielded higher mutagenic activity than did grilled meats (rump steak, lamb chops) at comparable temperatures of the cooking medium. Using similar cooking procedures, lamb did not differ markedly from beef in mutagenic activity.

Characterization of the electrophysiological effects of metoprolol on isolated feline ventricular myocytes.

Metoprolol is considered to be a class II antiarrhythmic agent that is highly specific for cardiac beta-1 adrenergic receptors, yet long-term administration can produce prolongation of the rate-corrected Q-T interval in humans. Action potentials and sodium (INa), "L"-type calcium (ICa) and transient outward (Ito) or inward rectifying potassium (IKl) currents were recorded from isolated cat ventricular myocytes using the whole-cell-patch technique to determine if metoprolol can directly affect cellular electrophysiological activity. External and pipette solutions, holding potentials and voltage-clamp protocols appropriate to isolate and examine INa, IKl, Ito and ICa were used. Metoprolol reversibly decreased both the duration and voltage of the action potential plateau but had no effect on upstroke velocity, the repolarization rate during phase 3 or the resting potential. Confirming previous reports suggesting that metoprolol appears to have little or no local anesthetic activity, INa was not affected by metoprolol at concentrations up to 100 microM during voltage-clamp pulses applied at less than 1 Hz when holding potential was negative to -110 mV. However, when trains of pulses to -10 mV from a holding potential of -110 mV were applied at 1 to 5 Hz, use-dependent inhibition of INa occurred, suggesting that 100 microM metoprolol may interact with inactivated Na channels to inhibit INa. Metoprolol (10 and 100 microM) also caused a concentration-dependent decrease in peak inward IKl elicited in response to hyperpolarizing from -40 mV to potentials negative to the IKl reversal potential. In addition, during strong hyperpolarizations (i.e., less than or equal to -150 mV) an inward (i.e., downward) droop in current was observed during the inactivation phase approximately 20 to 30 msec after pulse onset. Metoprolol (10 microM) also reduced peak Ito and ICa without altering the time courses of inactivation of either current or the level of the steady-state outward current elicited positive to -40 mV; steady-state ICa, on the other hand, was reduced. The sensitivity to block by metoprolol was: IKl greater than ICa greater than or equal to Ito greater than INa.

Effects of free radicals on the electrophysiological function of cardiac membranes.

Abnormal electrical activity in heart cells can result in irregular heart rhythms or arrhythmias. Any form of pathological or toxicological damage to the sarcolemmal membrane presents the risk of precipitating arrhythmias and compromise of the heart's function as a pump. An array of cardiovascular conditions from coronary artery disease and myocardial infarction to cardiomyopathies and hypertrophy, can induce arrhythmias. Many of these conditions recently have been linked to increases in free radical production. Early studies suggesting a role for free radicals in the abnormal function of ischemic and reperfused hearts use anti-free radical interventions to reduce arrhythmias. More recent works have taken advantage of different free radical-generating systems to show a reproducible sequence of changes in the cellular action potential; these data suggest changes in the transmembrane movement of ions through membrane channels. Biochemical evidence supports a possible involvement of ion exchange mechanisms in the cardiac sarcolemma. All the evidence indicate that free radical injury may have profound effects on the electrical function of myocardial cells.

Antibody responses in the liver and bile of rats injected with horse erythrocytes.

The intravenous injection of 1 ml of a 0.1% (or higher %) suspension of horse red blood cells (HRBC) into rats results in the appearance of high levels of agglutinating activity in bile as well as serum. Lower doses of HRBC induce reduced responses in both fluids, demonstrating that the biliary response is as dose-dependent as the serum response. The presence of IgM anti-HRBC antibody forming cells (AFC) in the liver in numbers equivalent to those detected in the spleen at day 6 of the study suggests them to be the most likely source of the biliary antibody. Also, the injection of HRBC into other parenteral sites (intramuscular [i.m.], intraperitoneal [i.p.] and intrathoracic cavity [i.t.]) results in a biliary response which is considered to originate from the IgM-, IgG- and IgA-anti-HRBC AFC detected in the liver, again in numbers approximating those detected in the spleen. Splenectomy of rats immunized i.p., i.m. and i.t. had only a minimal effect on the liver and biliary responses, indicating that AFC arising in peripheral lymphoid tissues can also relocate in liver. It is concluded that the liver is a major site of antibody synthesis following parenteral immunization leading to substantial levels of antibody being secreted into the gastrointestinal tract.

Abnormal electrical activity induced by free radical generating systems in isolated cardiocytes.

Oxygen free radicals may participate in a variety of pathological cardiac conditions which are associated with an increased incidence of arrhythmias. However, evidence that free radicals per se can alter the electrical function of the myocardium is not convincing. Physiological solutions containing 3 mM dihydroxyfumaric acid (DHF), a compound known to generate free radicals, were superfused over calcium-tolerant cells isolated from the adult canine ventricle. The time course for changes in transmembrane action potentials was monitored using conventional microelectrode techniques. Changes were observed which could be conveniently segregated into three stages. Initially during superfusion with DHF, the voltage of the action potential plateau became more positive and the action potential duration increased (stage 1). Continued superfusion was associated with the development of both early and delayed afterdepolarizations (stage 2), which occasionally produced triggered beats. Subsequently, some cells failed to repolarize beyond -40 mV following an action potential upstroke. In cells which maintained normal levels of resting membrane potential, early and delayed afterdepolarizations ceased concomitant with the development of an increasingly more negative plateau voltage. Action potential duration decreased and plateau potential "collapsed", eventually merging with the resting level of the membrane potential. Resting membrane potential then gradually depolarized to less than -40 mV and all cells became inexcitable within 6 to 20 min (stages 3). Exposure of cells to xanthine (2 mM): xanthine oxidase (0.01 U/ml), another system known to generate free radicals, produced similar results. Superfusion with DHF solutions containing either superoxide dismutase or catalase delayed the appearance and attenuated the development of the changes in the cardiocyte action potential. The results demonstrate that isolated cardiocytes exposed to free radical generating solutions can undergo changes in their electrophysiological activity that resemble those said to underlie disturbances of cardiac rate and rhythm in the clinical setting.

Monitoring the occurrence and duration of electroconvulsive fits.

During 100 routine psychiatric hospital electroconvulsive treatments the medical staff administering treatment experienced little difficulty in deciding that a fit had occurred. The judgement was based on simple clinical observation of a succession of clonic bodily movements, and high inter-rater agreement was found for the measurement of fit duration. The cuff technique was also used, but it tended to underestimate fit duration and is not recommended for routine ECT practice.

Antibody synthesis in the rat liver: an association between antibody-forming cells in the liver and biliary antibodies following intravenous injection of horse erythrocytes.

The transient appearance of IgM in the bile of rats injected intravenously with horse erythrocytes (HRBC) correlated with the occurrence of anti-HRBC IgM-secreting cells in the liver. Both responses were reduced in animals splenectomised at the time of immunisation. When rats were given 2 injections of HRBC spaced by 28 days, IgG, IgA and IgM antibody-forming cells (AFC) were detected in the liver along with IgM and non-IgM anti-HRBC antibody in bile. There was a 10-fold increase in the total numbers of mononuclear cells (MNC) retrievable from the liver at the height of the biliary antibody response, the majority of which were not AFC. These results suggest that antigen entering the spleen stimulates the release of a population of MNC which may localise in the liver and that a minor portion of these produces specific antibody which is secreted into bile.

Mucus colonization as a determinant of pathogenicity in intestinal infection by Campylobacter jejuni: a mouse cecal model.

Human isolates of the intestinal pathogen Campylobacter jejuni have been shown to colonize mucus on the outer surface and deep within the intestinal crypts of gnotobiotic or germfree mice. The cecal crypts are preferentially colonized. A model of mucus colonization by C. jejuni in the mouse cecum has been developed, using antibiotic- and magnesium sulfate-treated specific-pathogen-free animals. These spiral-shaped bacteria colonize the mucus in a similar manner to the normal spiral-shaped microbiota. No evidence of adhesion to the intestinal surface was found with a wide variety of microscopic techniques. The campylobacters were seen to be highly motile in living preparations of gut tissue and rapidly tracked along intestinal mucus. Just as many of the normal spiral-shaped bacteria of intestinal surfaces can achieve close association with the epithelium through mucus association and do not adhere to the surface, C. jejuni colonizes the intestinal mucosa via mucus colonization. Thus, a major determinant of pathogenicity in intestinal infection with C. jejuni is proposed to be an ability to colonize intestinal mucus. The possession of specific adhesins is unlikely to be a significant determinant of pathogenicity. Better understanding of the mechanism of mucus association and the properties of the bacterium that are responsible will provide a basis for the rational selection of preventative measures. The model of mucus association in adult antibiotic-treated mice provides an opportunity for colonization studies with variant organisms and immunization studies.