Retinal organoids from human-induced pluripotent stem cells: From studying retinal dystrophies to early diagnosis of Alzheimer's and Parkinson's disease.

Human-induced pluripotent stem cells (hiPSCs) have provided new methods to study neurodegenerative diseases. In addition to their wide application in neuronal disorders, hiPSCs technology can also encompass specific conditions, such as inherited retinal dystrophies. The possibility of evaluating alterations related to retinal disorders in 3D organoids increases the truthfulness of in vitro models. Moreover, both Alzheimer's (AD) and Parkinson's disease (PD) have been described as causing early retinal alterations, generating beta-amyloid protein accumulation, or affecting dopaminergic amacrine cells. This review addresses recent advances and future perspectives obtained from in vitro modeling of retinal diseases, focusing on retinitis pigmentosa (RP). Additionally, we depicted the possibility of evaluating changes related to AD and PD in retinal organoids obtained from potential patients long before the onset of the disease, constituting a valuable tool in early diagnosis. With this, we pointed out prospects in the study of retinal dystrophies and early diagnosis of AD and PD.

The light-dependent pseudo-capacitive charging of conjugated polymer nanoparticles coupled with the depolarization of the neuronal membrane.

The mechanism underlying visual restoration in blind animal models of retinitis pigmentosa using a liquid retina prosthesis based on semiconductive polymeric nanoparticles is still being debated. Through the application of mathematical models and specific experiments, we developed a coherent understanding of abiotic/biotic coupling, capturing the essential mechanism of photostimulation responsible for nanoparticle-induced retina activation. Our modeling is based on the solution of drift-diffusion and Poisson-Nernst-Planck models in the multi-physics neuron-cleft-nanoparticle-extracellular space domain, accounting for the electro-chemical motion of all the relevant species following photoexcitation. Modeling was coupled with electron microscopy to estimate the size of the neuron-nanoparticle cleft and electrophysiology on retina explants acutely or chronically injected with nanoparticles. Overall, we present a consistent picture of electrostatic depolarization of the bipolar cell driven by the pseudo-capacitive charging of the nanoparticle. We demonstrate that the highly resistive cleft composition, due to filling by adhesion/extracellular matrix proteins, is a crucial ingredient for establishing functional electrostatic coupling. Additionally, we show that the photo-chemical generation of reactive oxygen species (ROS) becomes relevant only at very high light intensities, far exceeding the physiological ones, in agreement with the lack of phototoxicity shown in vivo.

Multicellular 3D Models to Study Tumour-Stroma Interactions.

Two-dimensional (2D) cell cultures have been the standard for many different applications, ranging from basic research to stem cell and cancer research to regenerative medicine, for most of the past century. Hence, almost all of our knowledge about fundamental biological processes has been provided by primary and established cell lines cultured in 2D monolayer. However, cells in tissues and organs do not exist as single entities, and life in multicellular organisms relies on the coordination of several cellular activities, which depend on cell-cell communication across different cell types and tissues. In addition, cells are embedded within a complex non-cellular structure known as the extracellular matrix (ECM), which anchors them in a three-dimensional (3D) formation. Likewise, tumour cells interact with their surrounding matrix and tissue, and the physical and biochemical properties of this microenvironment regulate cancer differentiation, proliferation, invasion, and metastasis. 2D models are unable to mimic the complex and dynamic interactions of the tumour microenvironment (TME) and ignore spatial cell-ECM and cell-cell interactions. Thus, multicellular 3D models are excellent tools to recapitulate in vitro the spatial dimension, cellular heterogeneity, and molecular networks of the TME. This review summarizes the biological significance of the cell-ECM and cell-cell interactions in the onset and progression of tumours and focuses on the requirement for these interactions to build up representative in vitro models for the study of the pathophysiology of cancer and for the design of more clinically relevant treatments.

Photochemistry of Organic Retinal Prostheses.

Organic devices are attracting considerable attention as prostheses for the recovery of retinal light sensitivity lost to retinal degenerative disease. The biotic/abiotic interface created when light-sensitive polymers and living tissues are placed in contact allows excitation of a response in blind laboratory rats exposed to visual stimuli. Although polymer retinal prostheses have proved to be efficient, their working mechanism is far from being fully understood. In this review article, we discuss the results of the studies conducted on these kinds of polymer devices and compare them with the data found in the literature for inorganic retinal prostheses, where the working mechanisms are better comprehended. This comparison, which tries to set some reference values and figures of merit, is intended for use as a starting point to determine the direction for further investigation.

A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness.

The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.

Heterogeneous distribution of exocytotic microdomains in adrenal chromaffin cells resolved by high-density diamond ultra-microelectrode arrays.

Here we describe the ability of a high-density diamond microelectrode array targeted to resolve multi-site detection of fast exocytotic events from single cells. The array consists of nine boron-doped nanocrystalline diamond ultra-microelectrodes (9-Ch NCD-UMEA) radially distributed within a circular area of the dimensions of a single cell. The device can be operated in voltammetric or chronoamperometric configuration. Sensitivity to catecholamines, tested by dose-response calibrations, set the lowest detectable concentration of adrenaline to ∼5 µm. Catecholamine release from bovine or mouse chromaffin cells could be triggered by electrical stimulation or external KCl-enriched solutions. Spikes detected from the cell apex using carbon fibre microelectrodes showed an excellent correspondence with events measured at the bottom of the cell by the 9-Ch NCD-UMEA, confirming the ability of the array to resolve single quantal secretory events. Subcellular localization of exocytosis was provided by assigning each quantal event to one of the nine channels based on its location. The resulting mapping highlights the heterogeneous distribution of secretory activity in cell microdomains of 12-27 µm2. In bovine chromaffin cells, secretion was highly heterogeneous with zones of high and medium activity in 54% of the cell surface and zones of low or no activity in the remainder. The 'non-active' ('silent') zones covered 24% of the total and persisted for 6-8 min, indicating stable location. The 9-Ch NCD-UMEA therefore appears suitable for investigating the microdomain organization of neurosecretion with high spatial resolution.

A pH-sensitive closed-loop nanomachine to control hyperexcitability at the single neuron level.

Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E2GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E2GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy.

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

Platinum Nanozymes Counteract Photoreceptor Degeneration and Retina Inflammation in a Light-Damage Model of Age-Related Macular Degeneration.

Degeneration of photoreceptors in age-related macular degeneration (AMD) is associated with oxidative stress due to the intense aerobic metabolism of rods and cones that if not properly counterbalanced by endogenous antioxidant mechanisms can precipitate photoreceptor degeneration. In spite of being a priority eye disease for its high incidence in the elderly, no effective treatments for AMD exist. While systemic administration of antioxidants has been unsuccessful in slowing down degeneration, locally administered rare-earth nanoparticles were shown to be effective in preventing retinal photo-oxidative damage. However, because of inherent problems of dispersion in biological media, limited antioxidant power, and short lifetimes, these NPs are still confined to the preclinical stage. Here we propose platinum nanoparticles (PtNPs), potent antioxidant nanozymes, as a therapeutic tool for AMD. PtNPs exhibit high catalytic activity at minimal concentrations and protect primary neurons against oxidative insults and the ensuing apoptosis. We tested the efficacy of intravitreally injected PtNPs in preventing or mitigating light damage produced in dark-reared albino Sprague-Dawley rats by in vivo electroretinography (ERG) and ex vivo retina morphology and electrophysiology. We found that both preventive and postlesional treatments with PtNPs increased the amplitude of ERG responses to light stimuli. Ex vivo recordings demonstrated the selective preservation of ON retinal ganglion cell responses to light stimulation in lesioned retinas treated with PtNPs. PtNPs administered after light damage significantly preserved the number of photoreceptors and inhibited the inflammatory response to degeneration, while the preventive treatment had a milder effect. The data indicate that PtNPs can effectively break the vicious cycle linking oxidative stress, degeneration, and inflammation by exerting antioxidant and anti-inflammatory actions. The increased photoreceptor survival and visual performances in degenerated retinas, together with their high biocompatibility, make PtNPs a potential strategy to cure AMD.

Core-Shell Architecture in Poly(3-hexylthiophene) Nanoparticles: Tuning of the Photophysical Properties for Enhanced Neuronal Photostimulation.

This study shows that entirely thiophene-based core@shell nanoparticles, in which the shell is made of the oxidized form of the core polymer (P3HT@PTDOx NPs), result in a type II interface at the particle surface. This enables the development of advanced photon nanotransducers with unique chemical-physical and biofunctional properties due to the core@shell nanoarchitecture. We demonstrate that P3HT@PTDOx NPs present a different spatial localization of the excitation energy with respect to the nonoxidized NPs, showing a prevalence of surface states as a result of a different alignment of the HOMO/LUMO energy levels between the core and shell. This allows for the efficient photostimulation of retinal neurons. Indeed, thanks to the stronger and longer-lived charge separation, P3HT@PTDOx NPs, administered subretinally in degenerate retinas from the blind Royal College of Surgeons rats, are more effective in photostimulation of inner retinal neurons than the gold standard P3HT NPs.

[Predictors of clinical response in patients with ulcerative colitis treated with granulocyte-monocyte apheresis: analysis of the apheresis registry data]. / Predittori di risposta clinica in pazienti affetti da colite ulcerosa trattati con granulocito-monocito aferesi: analisi dei dati del registro aferesi.

We analyzed predictors of clinical response after a cycle of granulocytemonocyte apheresis in 173 patients with ulcerative colitis. Hemoglobin levels independently predicted good clinical outcome.

Dental Prosthesis in Esophagus: A Right Cervicotomic Approach.

Foreign body ingestion in the upper digestive tract is a relatively common emergency. Less than 1% have to be treated surgically. We report the case of a 68-year-old man who ingested a dental prosthesis, probably during a seizure, and thus unknowingly, and presented two days later to the emergency department complaining of a mild dysphagia. A chest radiograph showed the presence of a removable dental prosthesis in the upper esophageal tract. The patient was brought to the operating room where a multidisciplinary equipe was assembled. Two attempts of retrieval with a flexible and a rigid endoscope failed because the removable dental prosthesis was stuck in the right pyriform sinus. Therefore, the surgeon performed an uncommon right cervicotomy and retrieved the foreign body through a right-side esophagotomy. The surgical approach depends on the nature and location of the foreign body. Urgent treatment is required whenever the patient develops dyspnea or dysphagia because of the high risk of inhalation and asphyxia. Removal of any esophageal foreign body has to be performed within 12-24 h. Repeated attempts to retrieve large dental prosthesis using an endoscope may result in esophageal perforation therefore when such risk of complication is too high, a surgical approach becomes inevitable. In our opinion, surgery remains the extrema ratio after a failed endoscopic retrieval attempt but can be lifesaving despite high risk of complications.

Development of a novel index to discriminate bulge from mass on small-bowel capsule endoscopy.

BACKGROUND: Submucosal malignant masses (SMMs) and innocent bulges look similar on small-bowel capsule endoscopy (SBCE). In a previous observational study, 4 criteria associated with innocent bulges were recognized. OBJECTIVE: To devise and validate an index based on these criteria (smooth, protruding lesion index on capsule endoscopy [SPICE]) to discriminate SMMs from innocent bulges. DESIGN: Single-center, prospective study. SETTING: General hospital in Busto Arsizio, Italy. PATIENTS: This study involved 25 of 424 consecutive SBCEs performed on as many patients having SBCE findings of smooth, round, protruding lesions. INTERVENTION: Patients' evaluation up to the final diagnosis. At study entry, a short video clip of the lesion was obtained and deidentified for blind SPICE calculation. MAIN OUTCOME MEASUREMENTS: SPICE accuracy, using the final diagnosis of each patient as the criterion standard. RESULTS: Six patients had SMMs (4 GI stromal tumors, 2 neuroendocrine tumors), and 19 had innocent bulges. SPICE scores ranged from 0 to 4; they discriminated SMMs from innocent bulges (P = .002). A SPICE value >2 had 83.3% sensitivity and 89.4% specificity, and the area under the curve was 0.90 (95% confidence interval, 0.72-0.98; P < .001) for the detection of SMMs. LIMITATIONS: Single-center study; small sample size; no invasive ascertainment in 36% of patients. CONCLUSION: SPICE is easy to calculate and useful for distinguishing SMMs from innocent bulges. An index >2 is predictive of SMM.

The Italian Registry of Therapeutic Apheresis: granulocyte-monocyte apheresis in the treatment of inflammatory bowel disease. A multicentric study.

Leukocytes are thought to play an important role in the pathogenesis of inflammatory bowel diseases; granulocyte-monocyte adsorptive (GMA) apheresis, an extracorporeal technique aimed at removing activated circulating leukocytes from the blood, may represent a safe and effective therapeutic tool in these patients. The Italian Registry of Therapeutic Apheresis performed an observational, multicentric study involving 24 Gastroenterology Units. In this study, laboratory data and clinical outcomes of 230 patients (148 males, mean age 43.5 years) affected with ulcerative colitis (UC, n = 194) or Crohn's disease (CD, n = 36) who underwent one or more cycles of GMA were analyzed. Each cycle consisted of five GMA treatments. The patients were followed up for a mean of 8.7 (min. 3 to max. 12) months. At 3 months, positive outcome was achieved in 77.7% of UC patients (72.0% remission, 5.7% clinical response) and 61.3% of CD patients (54.8% remission, 6.5% clinical response). The cumulative proportion of positive outcome at 12 months was 87.1% for UC patients (83.7% remission, 3.4% clinical response) and 77.4% for CD patients (74.2% remission, 3.2% clinical response). No single clinical or laboratory parameter among those analyzed (age, sex, disease characteristics, history of smoking, medication history, baseline values of clinical activity index (CAI)/Crohn's disease activity index (CDAI), hemoglobin, white blood cells count, and erythrocyte sedimentation rate) was independently associated with clinical outcome. The procedure was well tolerated with no significant adverse effects registered.

Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces.

Poly(3-hexylthiophene) (P3HT) is a hole-conducting polymer that has been intensively used to develop organic optoelectronic devices (e.g., organic solar cells). Recently, P3HT films and nanoparticles have also been used to restore the photosensitivity of retinal neurons. The template-assisted electrochemical synthesis of polymer nanowires advantageously combines polymerization and polymer nanostructuring into one, relatively simple, procedure. However, obtaining P3HT nanowires through this procedure was rarely investigated. Therefore, this study aimed to investigate the template-assisted electrochemical synthesis of P3HT nanowires doped with tetrabutylammonium hexafluorophosphate (TBAHFP) and their biocompatibility with primary neurons. We show that template-assisted electrochemical synthesis can relatively easily turn 3-hexylthiophene (3HT) into longer (e.g., 17 ± 3 µm) or shorter (e.g., 1.5 ± 0.4 µm) P3HT nanowires with an average diameter of 196 ± 55 nm (determined by the used template). The nanowires produce measurable photocurrents following illumination. Finally, we show that primary cortical neurons can be grown onto P3HT nanowires drop-casted on a glass substrate without relevant changes in their viability and electrophysiological properties, indicating that P3HT nanowires obtained by template-assisted electrochemical synthesis represent a promising neuronal interface for photostimulation.

Membrane Environment Enables Ultrafast Isomerization of Amphiphilic Azobenzene.

The non-covalent affinity of photoresponsive molecules to biotargets represents an attractive tool for achieving effective cell photo-stimulation. Here, an amphiphilic azobenzene that preferentially dwells within the plasma membrane is studied. In particular, its isomerization dynamics in different media is investigated. It is found that in molecular aggregates formed in water, the isomerization reaction is hindered, while radiative deactivation is favored. However, once protected by a lipid shell, the photochromic molecule reacquires its ultrafast photoisomerization capacity. This behavior is explained considering collective excited states that may form in aggregates, locking the conformational dynamics and redistributing the oscillator strength. By applying the pump probe technique in different media, an isomerization time in the order of 10 ps is identified and the deactivation in the aggregate in water is also characterized. Finally, it is demonstrated that the reversible modulation of membrane potential of HEK293 cells via illumination with visible light can be indeed related to the recovered trans→cis photoreaction in lipid membrane. These data fully account for the recently reported experiments in neurons, showing that the amphiphilic azobenzenes, once partitioned in the cell membrane, are effective light actuators for the modification of the electrical state of the membrane.

Subretinally injected semiconducting polymer nanoparticles rescue vision in a rat model of retinal dystrophy.

Inherited retinal dystrophies and late-stage age-related macular degeneration, for which treatments remain limited, are among the most prevalent causes of legal blindness. Retinal prostheses have been developed to stimulate the inner retinal network; however, lack of sensitivity and resolution, and the need for wiring or external cameras, have limited their application. Here we show that conjugated polymer nanoparticles (P3HT NPs) mediate light-evoked stimulation of retinal neurons and persistently rescue visual functions when subretinally injected in a rat model of retinitis pigmentosa. P3HT NPs spread out over the entire subretinal space and promote light-dependent activation of spared inner retinal neurons, recovering subcortical, cortical and behavioural visual responses in the absence of trophic effects or retinal inflammation. By conferring sustained light sensitivity to degenerate retinas after a single injection, and with the potential for high spatial resolution, P3HT NPs provide a new avenue in retinal prosthetics with potential applications not only in retinitis pigmentosa, but also in age-related macular degeneration.

Neuronal firing modulation by a membrane-targeted photoswitch.

Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. In this respect, azobenzene-based photoswitches are promising nanoscale tools for neuronal photostimulation. Here we engineered a light-sensitive azobenzene compound (Ziapin2) that stably partitions into the plasma membrane and causes its thinning through trans-dimerization in the dark, resulting in an increased membrane capacitance at steady state. We demonstrated that in neurons loaded with the compound, millisecond pulses of visible light induce a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. These effects are persistent and can be evoked in vivo up to 7 days, proving the potential of Ziapin2 for the modulation of membrane capacitance in the millisecond timescale, without directly affecting ion channels or local temperature.

CARD15 gene variants and risk of reoperation in Crohn's disease patients.

OBJECTIVES: Several studies have investigated, with conflicting results, the risk factors for reoperation in Crohn's disease (CD) patients. CARD15 gene variants have been identified as a major genetic risk factor for CD patients and associated with ileal disease, stenosis, and risk of surgery. However, data regarding the association between these variants and the need for reoperation are very few and conflicting. This study evaluated the risk factors of reoperation, including CARD15 gene variants. METHODS: A total of 253 consecutive CD patients, recruited in four Italian tertiary-care inflammatory bowel disease (IBD) referral centers, who had submitted to surgery for CD, were included in the study. Clinical characteristics of CD patients, time and main indications for surgery, type of operation, postoperative therapy, and time to second surgery were recorded. CARD15 gene variants were determined by DNA sequencing analysis in each center. Factors related to surgical recurrence, including CARD15 variants, were estimated by Cox proportional hazard regression. RESULTS: In all, 89 patients (35.1%) showed at least one surgical recurrence. Reoperation was significantly correlated with stenosis as indications at initial surgery only. CARD15 variants were found in 36.0% of patients, but did not correlate significantly with the demographic and clinical characteristics of the patients, rate of first surgical recurrence, and time to second operation. CARD15 variants did not significantly affect the reoperation rate, irrespective of indications for surgery. CONCLUSIONS: Reoperation for CD is correlated with stenosis at initial surgery, but not with CARD15 gene variants. This finding does not justify more aggressive prophylactic therapy on the basis of CARD15 genotype.

Maintenance treatment with azathioprine in ulcerative colitis: outcome and predictive factors after drug withdrawal.

OBJECTIVES: Whether the duration of maintenance treatment with azathioprine (AZA) affects the outcome of ulcerative colitis (UC) is unclear. We investigated clinical outcomes and any predictive factors after withdrawal of AZA in UC. METHODS: In this multicenter observational retrospective study, 127 Italian UC patients, who were in steroid-free remission at the time of withdrawal of AZA, were followed-up for a median of 55 months or until relapse. The frequency of clinical relapse or colectomy after AZA withdrawal was analyzed according to demographic, clinical, and endoscopic variables. RESULTS: After drug withdrawal, a third of the patients relapsed within 12 months, half within 2 years and two-thirds within 5 years. After multivariable analysis, predictors of relapse after drug withdrawal were lack of sustained remission during AZA maintenance (hazard ratio, HR 2.350, confidence interval, CI 95% 1.434-3.852; P=0.001), extensive colitis (HR 1.793, CI 95% 1.064-3.023, P=0.028 vs. left-sided colitis; HR 2.024, CI 95% 1.103-3.717, P=0.023 vs. distal colitis), and treatment duration, with short treatments (3-6 months) more disadvantaged than >48-month treatments (HR 2.783, CI 95% 1.267-6.114, P=0.008). Concomitant aminosalicylates were the only predictors of sustained remission during AZA therapy (P=0.009). The overall colectomy rate was 10%. Predictors of colectomy were drug-related toxicity as the cause of AZA withdrawal (P=0.041), no post-AZA drug therapy (P=0.031), and treatment duration (P<0.0005). CONCLUSIONS: Discontinuation of AZA while UC is in remission is associated with a high relapse rate. Disease extent, lack of sustained remission during AZA, and discontinuation due to toxicity could stratify relapse risk. Concomitant aminosalicylates were advantageous. Prospective randomized controlled trials are needed to confirm whether treatment duration is inversely associated with outcome.