The natural history of Crohn’s disease follows a path of progression from an inflammatory to a fibrostenosing disease, with most patients requiring surgical resection of fibrotic strictures. Potent anti-inflammatory therapies reduce inflammation but do not appear to alter the natural history of intestinal fibrosis. The aim of this study was to determine the relationship between intestinal inflammation and fibrogenesis and the impact of a very early “top-down” interventional approach on fibrosis in vivo.
In this study, we removed the inflammatory stimulus from the Salmonella typhimurium mouse model of intestinal fibrosis by eradicating the S. typhimurium infection with levofloxacin at sequential time points during the infection. We evaluated the effect of this elimination of the inflammatory stimulus on the natural history of inflammation and fibrosis as determined by gross pathology, histopathology, mRNA expression, and protein expression.
Fibrogenesis is preceded by inflammation. Delayed eradication of the inflammatory stimulus by antibiotic treatment represses inflammation without preventing fibrosis. Early intervention significantly ameliorates but does not completely prevent subsequent fibrosis.
This study demonstrates that intestinal fibrosis develops despite removal of an inflammatory stimulus and elimination of inflammation. Early intervention ameliorates but does not abolish subsequent fibrosis, suggesting that fibrosis, once initiated, is self-propagating, suggesting that a very early “top-down” interventional approach may have the most impact on fibrostenosing disease.
Helicobacter pylori infection is associated with a lower risk of chronic autoimmune diseases including IBD. H. pylori modulates the gastric immune response, decreasing the local inflammatory response to itself. In mice, chronic Salmonella typhimurium infection induces colitis similar to Crohn’s disease characterized by inflammation which progresses towards fibrosis. The aim of this study was to determine whether prior H. pylori infection acts at a distance to modulate the immune response of S. typhimurium-induced colitis.
Mice were infected with the mouse-adapted strain of H. pylori (SS1), followed by infection with S. typhimurium. The effect of H. pylori on colitis was determined by gross pathology, histopathology, cytokine response, and development of fibrosis in the cecum. Gastritis and systemic immune response was measured in response to infection.
H. pylori suppresses the Th17 response to S. typhimurium infection in the mouse cecum, but does not alter the Th2 or Treg response or the development of fibrosis. H. pylori infection induces IL-10 in the mesenteric lymph nodes, suggesting an extra-gastric mechanism for immunomodulation. H. pylori/S. typhimurium co-infection decreases inflammation in both the cecum and the stomach.
This study demonstrates a potential mechanism for the negative association between H. pylori and IBD in humans. H. pylori represses the lower gastrointestinal tract Th17 response to bacterially induced colitis via extra-gastric immunomodulatory effects, illustrating immunological crosstalk between the upper and lower gastrointestinal tract.
Inflammatory bowel disease; Helicobacter pylori; Salmonella typhimurium; colitis; gastritis
While the number of clinical trials performed yearly is increasing, the application of these results to individual patients is quite difficult. This article reviews key portions of the process of applying research results to clinical practice. The first step involves defining the study population and determining whether these patients are similar to the patients seen in clinical practice in terms of demographics, disease type, and disease severity. The dropout rate should be compared between the different study arms. Design aspects, including randomization and blinding, should be checked for signs of bias. When comparing studies, clinicians should be aware that the outcomes being studied may vary greatly from one study to another, and some outcomes are much more reliable and valuable than others. The definition of clinical response should also be scrutinized, as it may be too lenient. Surrogate outcomes should be viewed cautiously, and their use should be well justified. Clinicians should also note that statistical significance, as defined by a P-value cutoff, may be the result of a large sample size rather than a clinically significant difference. The treatment effect can be estimated by calculating the number needed to treat, which will demonstrate whether changes in clinical practice are worthwhile. Finally, this article discusses some common issues that can arise with figures.
Statistics; clinical trials; randomized controlled trials; outcomes; clinical research
Maintenance treatment in ulcerative colitis often fails to prevent flares and long term complications. The first key to maintenance is to use effective therapy, even when patients become asymptomatic. The second key is to communicate the importance of adherence to patients, and to help them achieve long term adherence. Simplified dosing schedules are of some benefit, but the bond between patient and doctor, and the patient's belief in the efficacy of the therapy are essential. Decreased co-pays (a fixed amount paid by patients seeking care that is not reimbursed my medical insurance) have been associated with increased adherence, and incentives for patients may be a cost-effective approach to improving adherence. While the most substantial data on the association between adherence and clinical outcomes is in 5-ASAs, non-adherence can also limit the efficacy of thiopurines and biologics. The third key to maintenance treatment is monitoring and maintaining control of inflammation. Decreased histologic and endoscopic damage to the colon has been associated with decreased risk of colon cancer. The most cost-effective way to monitor smoldering inflammation is not known, but endoscopy, structured symptom indices, and biomarkers may be valuable approaches. The fourth key to maintenance treatment is optimizing immunomodulator therapy with thiopurines, and possibly methotrexate in the future. The fifth key to maintenance treatment in ulcerative colitis is maintaining biologic efficacy by avoiding low trough levels and being vigilant for subclinical inflammation and symptom recurrence at the end of dose intervals. Combination therapy with immunomodulators improves trough levels in Crohn's, and may prove to have benefits for the maintenance of biologic efficacy in ulcerative colitis.
Adherence; Maintenance; Ulcerative colitis
Background and Aims
Allopurinol has been presented as a safe and effective adjunct to thiopurine therapy in inflammatory bowel disease (IBD). We aimed to determine the rate of infectious complications and clinical successes with combination thiopurine/allopurinol in IBD, and to identify which variables predict 6-thioguanine, 6-methylmercaptopurine, and white blood cell levels. Additionally we aimed to identify which variables predict complications.
A retrospective database search identified patients with inflammatory bowel disease on both thiopurines and allopurinol. Regression modeling was used to identify which variables predicted metabolite levels, white blood cell levels, and complications.
Twenty-seven subjects were found, with 20 treated intentionally and 7 inadvertently after a concurrent gout diagnosis. Thirteen of 20 patients had a major clinical improvement and 7 of 16 stopped steroids. Five infectious complications occurred. These included 2 cases of shingles, and one each of PCP, EBV, and viral meningitis. Significant predictors of metabolite levels included the dose of thiopurine and allopurinol, age, and BMI. Low white blood cell count levels were associated with increased doses, high BMI, and older age. Despite having only 5 events, there was a difference in absolute lymphocyte count between patients with and without infection (median 200 per mm3 vs 850 per mm3 respectively, p=0.0503)
Adjunctive allopurinol therapy in shunting patients produced major clinical improvement in 48% of patients. However, a surprising number of opportunistic infections have occurred. Low absolute lymphocyte count may be a previously unrecognized indicator of risk of opportunistic infections.
azathioprine; 6-mercaptopurine; allopurinol; inflammatory bowel disease; shunting
The study of intestinal microbiota has been revolutionized by the use of molecular methods, including terminal restriction fragment length polymorphism (T-RFLP) analysis. A number of microbiota studies of Crohn’s disease patients have examined samples from stool or from the neoterminal ileum with a standard biopsy forceps, which could be contaminated by colonic bacteria when the forceps passes through the colonoscope channel.
To determine whether sheathed biopsy forceps are able to obtain terminal ileal microbiota samples with less colonic bacterial contamination compared to unsheathed (standard) biopsy forceps.
Prospective randomized single center-study.
Patients and Methods
We obtained four (paired) biopsy specimens from adjacent locations in the terminal ileum using the sheathed and standard forceps of 27 consecutive subjects undergoing colonoscopy and characterized the microbiota using T-RFLP. We calculated the Bray Curtis similarity index (BCI) between samples (sheathed vs. unsheathed forceps) within patients and tested for significant differences across all patients.
There was not a significant difference in the microbial diversity of samples obtained using sheathed vs. unsheathed forceps. The difference in microbial diversity between patients was much greater than the variability within patients by proximal vs. distal site or by forceps type.
T-RFLP is based on PCR amplification, so it is not always sensitive to rare bacterial species.
Standard unsheathed forceps appear to be sufficient for microbiota sample collection from the terminal ileum.
Terminal Ileum; Terminal restriction fragment length polymorphism; Microbiota; Microbial diversity; Crohn’s disease
Radiologic imaging has been a critical aid in the diagnosis, assessment, and management of inflammatory bowel disease since the first description of the disease by Crohn in 1932. Newer techniques, including computed tomography enterography, magnetic resonance enterography, and ultrasound, have entered clinical use for assessing disease activity and complications, and have largely replaced classic barium studies. We review the clinical utility of these imaging modalities and examine several imaging research approaches that offer new insights into the tissue structure of inflammatory bowel disease that could eventually prove to have prognostic value in this setting.
Inflammatory bowel disease; radiography; abdominal magnetic resonance imaging tomography; radiograph computed ultrasonography; radiation
The accumulation of apoptosis-resistant fibroblasts within fibroblastic foci is a characteristic feature of idiopathic pulmonary fibrosis (IPF), but the mechanisms underlying apoptosis resistance remain unclear. A role for the inhibitor of apoptosis (IAP) protein family member X-linked inhibitor of apoptosis (XIAP) has been suggested by prior studies showing that (1) XIAP is localized to fibroblastic foci in IPF tissue and (2) prostaglandin E2 suppresses XIAP expression while increasing fibroblast susceptibility to apoptosis. Based on these observations, we hypothesized that XIAP would be regulated by the profibrotic mediators transforming growth factor (TGF)β-1 and endothelin (ET)-1 and that increased XIAP would contribute to apoptosis resistance in IPF fibroblasts. To address these hypotheses, we examined XIAP expression in normal and IPF fibroblasts at baseline and in normal fibroblasts after treatment with TGF-β1 or ET-1. The role of XIAP in the regulation of fibroblast susceptibility to Fas-mediated apoptosis was examined using functional XIAP antagonists and siRNA silencing. In concordance with prior reports, fibroblasts from IPF lung tissue had increased resistance to apoptosis compared with normal lung fibroblasts. Compared with normal fibroblasts, IPF fibroblasts had significantly but heterogeneously increased basal XIAP expression. Additionally, TGF-β1 and ET-1 induced XIAP protein expression in normal fibroblasts. Inhibition or silencing of XIAP enhanced the sensitivity of lung fibroblasts to Fas-mediated apoptosis without causing apoptosis in the absence of Fas activation. Collectively, these findings support a mechanistic role for XIAP in the apoptosis-resistant phenotype of IPF fibroblasts.
myofibroblast; idiopathic pulmonary fibrosis; inhibitor of apoptosis; lung fibrosis; apoptosis
A recent large-scale randomized controlled trial (RCT) demonstrated
that rectal indomethacin administration is effective in addition to
pancreatic stent placement (PSP) for preventing post-endoscopic retrograde
cholangiopancreatography (ERCP) pancreatitis (PEP) in high-risk cases. We
performed a post hoc analysis of this RCT to explore
whether rectal indomethacin can replace PSP in the prevention of PEP and to
estimate the potential cost savings of such an approach.
We retrospectively classified RCT subjects into four prevention
groups: (1) no prophylaxis, (2) PSP alone, (3) rectal indomethacin alone,
and (4) the combination of PSP and indomethacin. Multivariable logistic
regression was used to adjust for imbalances in the prevalence of risk
factors for PEP between the groups. Based on these adjusted PEP rates, we
conducted an economic analysis comparing the costs associated with PEP
prevention strategies employing rectal indomethacin alone, PSP alone, or the
combination of both.
After adjusting for risk using two different logistic regression
models, rectal indomethacin alone appeared to be more effective for
preventing PEP than no prophylaxis, PSP alone, and the combination of
indomethacin and PSP. Economic analysis revealed that indomethacin alone was
a cost-saving strategy in 96% of Monte Carlo trials. A prevention strategy
employing rectal indomethacin alone could save approximately $150 million
annually in the United States compared with a strategy of PSP alone, and $85
million compared with a strategy of indomethacin and PSP.
This hypothesis-generating study suggests that prophylactic rectal
indomethacin could replace PSP in patients undergoing high-risk ERCP,
potentially improving clinical outcomes and reducing healthcare costs. A RCT
comparing rectal indomethacin alone vs. indomethacin plus PSP is needed.
Although efficacy and effectiveness studies are both important when evaluating interventions, they serve distinct purposes and have different study designs. Unfortunately, the distinction between these two types of trials is often poorly understood. In this primer, we highlight several differences between these two types of trials including study design, patient populations, intervention design, data analysis, and result reporting.
Prediction research is becoming increasing popular; however, the differences between traditional explanatory research and prediction research are often poorly understood, resulting in a wide variation in the methodologic quality of prediction research. This primer describes the basic methods for conducting prediction research in gastroenterology and highlights differences between traditional explanatory research and predictive research.
Background and Methods
Crohn’s disease causes intestinal inflammation leading to intestinal fibrosis. Spironolactone is an anti-fibrotic medication commonly used in heart failure to reduce mortality. We examined whether spironolactone is anti-fibrotic in the context of intestinal inflammation. In vitro, spironolactone repressed fibrogenesis in TGFβ-stimulated human colonic myofibroblasts. However, spironolactone therapy significantly increased mortality in two rodent models of inflammation-induced intestinal fibrosis, suggesting spironolactone could be harmful during intestinal inflammation. Since IBD patients rarely receive spironolactone therapy, we examined whether spironolactone use was associated with mortality in a common cause of inflammatory colitis, Clostridium difficile infection (CDI).
Spironolactone use during CDI infection was associated with increased mortality in a retrospective cohort of 4008 inpatients (15.9% vs. 9.1%, n=390 deaths, p<0.0001). In patients without liver disease, the adjusted OR for inpatient mortality associated with 80 mg spironolactone was 1.99 (95% CI: 1.51 – 2.63) In contrast to the main effect of spironolactone mortality, multivariable modeling revealed a protective interaction between liver disease and spironolactone dose. The adjusted odds ratio for mortality after CDI was 1.96 (95% CI: 1.50 – 2.55) for patients without liver disease on spironolactone vs. 1.28 (95% CI: 0.82 – 2.00) for patients with liver disease on spironolactone, when compared to a reference group without liver disease or spironolactone use.
We propose that discontinuation of spironolactone in patients without liver disease during CDI could reduce hospital mortality by 2-fold, potentially reducing mortality from CDI by 35,000 patients annually across Europe and the US.
Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-β1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the “active” lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-β1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.
Myofibroblast; Idiopathic Pulmonary Fibrosis; Inhibitor of Apoptosis; Lung Fibrosis; Fas
Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and – independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.
Fibrosis; Rho-kinase; Inhibitor of Apoptosis; Mesenchymal Cell; Focal Adhesion Kinase; AKT
Preliminary research suggests that rectally administered nonsteroidal antiinflammatory drugs may reduce the incidence of pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP).
In this multicenter, randomized, placebo-controlled, double-blind clinical trial, we assigned patients at elevated risk for post-ERCP pancreatitis to receive a single dose of rectal indomethacin or placebo immediately after ERCP. Patients were determined to be at high risk on the basis of validated patient- and procedure-related risk factors. The primary outcome was post-ERCP pancreatitis, which was defined as new upper abdominal pain, an elevation in pancreatic enzymes to at least three times the upper limit of the normal range 24 hours after the procedure, and hospitalization for at least 2 nights.
A total of 602 patients were enrolled and completed follow-up. The majority of patients (82%) had a clinical suspicion of sphincter of Oddi dysfunction. Post-ERCP pancreatitis developed in 27 of 295 patients (9.2%) in the indomethacin group and in 52 of 307 patients (16.9%) in the placebo group (P = 0.005). Moderate-to-severe pancreatitis developed in 13 patients (4.4%) in the indomethacin group and in 27 patients (8.8%) in the placebo group (P = 0.03).
Among patients at high risk for post-ERCP pancreatitis, rectal indomethacin significantly reduced the incidence of the condition. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00820612.)
Murine noroviruses (MNV) are currently the most prevalent viruses infecting mouse research colonies. Concurrent infection of research mice with these viruses can dramatically alter the experimental outcome in some research models, but not others. In this report, we investigated the effect of MNV1 and MNV4 on a murine model of intestinal inflammation and fibrosis induced by Salmonella typhimurium infection in C57BL/6 mice. Subsequent co-infection of these mice with MNV1 or MNV4 did not lead to major changes in histopathology, the inflammatory response, or the fibrotic response. Thus, MNV does not substantially alter all gastrointestinal research models, highlighting the importance of investigating potential alterations in the research outcome by MNV on an individual basis. We hypothesize that this is particularly important in cases of research models that use immunocompromised mice, which could be more sensitive to MNV infection-induced changes.
murine norovirus; Salmonella typhimurium; animal model; fibrosis; large intestine; inflammatory bowel disease
It has been assumed that the symptoms measured in disease activity indices for ulcerative colitis reflect those symptoms that patients find useful in evaluating the severity of a disease flare. In this qualitative focus group study, we aimed to identify which symptoms are important to patients and to compare these symptoms with a comprehensive list of commonly measured symptoms to determine/evaluate whether the patient-reported important symptoms are represented in current disease activity indices for ulcerative colitis. Patients in this sample confirmed 15 symptoms but not 11 other symptoms found in common ulcerative colitis activity indices. Patients identified an additional 14 symptoms not included in commonly used ulcerative colitis activity indices, which they believed to be important in evaluating the onset or severity of an ulcerative colitis flare. Current indices capture only a portion of the clinical symptoms that are important to patients in an ulcerative colitis flare, and may neither accurately measure nor fully reflect patients’ experience of ulcerative colitis. These findings present an opportunity to develop better patient-centered measures of ulcerative colitis.
focus groups; inflammatory bowel disease; patient-reported outcomes; qualitative research; symptom domains; ulcerative colitis
Inflammation occurs in episodic flares in Crohn’s disease, which are part of the waxing and waning course of the disease. Healing between flares allows the intestine to reconstitute its epithelium, but this healing results in the deposition of fibrotic scar tissue as part of the healing process. Repeated cycles of flares and healing often lead to clinically significant fibrosis and stenosis of the intestine. Patients are treated empirically with steroids, with their many side effects, in the hope that they will respond. Many patients would be better treated with surgery if we could identify which patients truly have intestinal fibrosis. Ultrasound elasticity imaging (UEI) offers the potential to radically improve the diagnosis and management of local tissue elastic property, particularly intestinal fibrosis. This method allows complete characterization of local intestine tissue with high spatial resolution. The feasibility of UEI on Crohn’s disease is demonstrated by directly applying this technique to an animal model of inflammatory bowel disease (IBD). Five female Lewis rats (150 –180g) were prepared with phosphate buffered solution (PBS) as a control group and six were prepared with repeated intrarectal administration of trinitrobenzenesulfonic acid (TNBS) as a disease group. Preliminary strain measurements differentiate the diseased colons from the normal colons (p < 0.0002) and compared well with direct mechanical measurements and histology (p < 0.0005). UEI provides a simple and accurate assessment of local severity of fibrosis. The preliminary results on an animal model also suggest the feasibility of translating this imaging technique directly to human subjects for both diagnosis and monitoring.
Ultrasound elasticity imaging; Strain imaging; Nonlinear tissue elasticity; Crohn’s disease
Many patients with lower gastrointestinal bleeding do not have an identifiable source of bleeding at colonoscopy. A significant percentage of these patients will have recurrent bleeding. In many patients, the presence of multiple diverticula leads to a diagnosis of presumed diverticular bleeding. Current treatment options include therapeutic endoscopy, angiography, or surgical resection, all of which depend on the identification of the diverticular source of bleeding. This report describes a case of recurrent bleeding in an elderly patient with diverticula but no identifiable source treated successfully with barium impaction therapy. This therapeutic modality does not depend on the identification of the bleeding diverticular lesion and was well tolerated by our 86-year-old patient.
Lower gastrointestinal bleeding; Diverticula; Therapeutic barium enema; Diverticular hemorrhage; Hematochezia; Anemia
Colorectal cancer (CRC) is a feared complication of chronic ulcerative colitis (UC). Annual endoscopic surveillance is recommended to detect early neoplasia. 5-aminosalicylates (5-ASAs) may prevent some UC-associated CRC. Therefore, in patients prescribed 5-ASAs for maintenance of remission, annual surveillance might be overly burdensome and inefficient. We aimed to determine the ideal frequency of surveillance in patients with UC maintained on 5-ASAs.
We performed systematic reviews of the literature, and created a Markov computer model simulating a cohort of 35 year-old men with chronic UC, followed until age 90. Twenty-two strategies were modeled: Natural History (no 5-ASA or surveillance), surveillance without 5-ASA at intervals of 1 to 10 years, 5-ASA plus surveillance every 1 to 10 years, and 5-ASA alone. The primary outcome was the ideal interval of surveillance in the setting of 5-ASA maintenance, assuming a third-party payer was willing to pay $100,000 for each quality-adjusted life-year (QALY) gained.
In the Natural History strategy, the CRC incidence was 30%. Without 5-ASA, annual surveillance was the ideal strategy, preventing 89% of CRC and costing $69,100 per QALY gained compared to surveillance every 2 years. 5-ASA alone prevented 49% of CRC. In the setting of 5-ASA, surveillance every 3 years was ideal, preventing 87% of CRC. 5-ASA with surveillance every 2 years cost an additional 147,500 per QALY gained, and 5-ASA with annual surveillance cost nearly $1 million additional per QALY gained compared to every 2 years. In Monte Carlo simulations, surveillance every 2 years or less often was ideal in 95% of simulations.
If 5-ASA is efficacious chemoprevention for UC-associated CRC, endoscopic surveillance might be safely performed every 2 years or less often. Such practice could decrease burdens to patients and to endoscopic resources with a minimal decrease in quality-adjusted length of life, since 5-ASA with annual surveillance may cost nearly $1 million per additional QALY gained.
Chemoprevention; Colonic Neoplasms; Decision Support Techniques; Surveillance; Ulcerative Colitis