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Laboratory tests play an important role in the diagnosis and differentiation of chronic inflammatory bowel diseases, enabling endoscopies to be avoided in many cases.
By Dr Jacqueline Gosink
Fecal calprotectin (FC) has in recent years become an established marker for distinguishing chronic inflammatory bowel diseases (CIBD) from functional gut disorders such as irritable bowel syndrome (IBS) and for monitoring the disease activity in CIBD patients. FC determination can be complemented by serological tests for disease-specific antibodies, which enable differentiation of the two main forms of CIBD, namely Crohn’s disease (CD) and ulcerative colitis (UC). New therapeutic drug monitoring assays measure the level of infliximab or adalimumab in patient blood and the presence of inhibitory antibodies against these drugs, allowing the medication and dosage to be tailored to the patient’s individual response.
CIBD
CIBD are autoimmune diseases which are characterized by inflammation of different regions of the gastrointestinal tract. UC affects predominantly the colon, while CD can affect any part of the gastrointestinal tract. The diseases are episodic with symptomatic phases (relapses) alternating with asymptomatic phases (remission). The severity of the symptoms and the duration of relapses vary from patient to patient. Diagnosis of CIBD is based a combination of clinical symptoms, endoscopy, histology, radiology and laboratory diagnostics. In order to obtain a definitive diagnosis the inflammatory status of the intestinal epithelium is investigated using invasive methods such as endoscopy and biopsy. These procedures are, however, costly and unpleasant for patients. Laboratory tests, especially measurement of FC and detection of disease-specific antibodies, provide valuable non-invasive support for CIBD diagnostics.
FC
When the intestinal tract is inflamed, neutrophile granulocytes migrate through the intestinal mucosa into the lumen and secrete calprotectin. The calprotectin accumulates in feces and is secreted with it. FC can therefore be used as a marker for inflammatory processes that affect only the gastrointestinal tract. Its concentration is proportional to the degree of inflammation. FC is more effective for CIBD diagnostics than general clinical indices and classical serological inflammation markers, such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) or leukocyte count.
Moreover, calprotectin is already produced at the start of the disease, making it a suitable marker for early diagnosis. Measurement of FC is especially suitable for differentiating CIBD from IBS. Only patients with elevated FC need to be referred for further invasive tests. FC is also a suitable surrogate marker for assessing the disease activity and for predicting disease recurrence after surgery. The risk of relapse is proportional to the measured FC value (Fig. 1).
Low FC concentrations are associated with long-lasting remission, while high values indicate ongoing or recurring inflammation requiring further investigation and intervention. Current international guidelines for CIBD diagnostics, for example from the European Crohn’s and Colitis Organisation [1], recommend measuring FC for differential diagnostics and also highlight the good correlation between the FC concentration and the disease activity. An FC concen-tration of less than 50 µg/g is considered inconspicuous and excludes an inflammatory cause of intestinal complaints with high certainty. Values between 50 µg/g and 120 µg/g lie in the borderline range and further monitoring of patients is indicated, for example by measuring FC again after two to three weeks. When the FC concentration is greater than 120 µg/g, the inflammatory status of the epithelium should be examined using imaging methods.
FC measurement
Calprotectin can be measured quickly and efficiently in stool samples using ELISA. The EUROIMMUN Calprotectin ELISA, for example, takes just 75 minutes and can be processed manually or automatically. It offers a broad measurement range of 1.9 to 2100 µg/g and correlates well with other FC assays. Pre-analytical sample preparation can be reduced to a minimum through the use of special stool dosage tubes, which enable extraction of a defined amount of stool in just one step.
In a study with stool samples from 47 clinical characterised patients with CIBD or IBS (Fig. 2), the Calprotectin ELISA from EUROIMMUN yielded a sensitivity of 94.1 % at a specificity of 95.5 % (excluding samples in the borderline range). Thus, there was a very high correlation between the FC level and the clinical diagnosis.
CIBD-associated antibodies
Serological determination of disease-specific antibodies provides additional support in CIBD diagnostics, enabling non-invasive differen-tiation between CD and UC (Table 1). Combined testing for all relevant antibodies is recommended to increase the diagnostic sensitivity [2].
Autoantibodies against acinus cells of exocrine pancreas are a reliable marker for CD. They have a high significance due to their organ specificity, disease association and frequently high serum concentration. The main target antigens of these autoantibodies are the proteoglycans CUZD1 (rPAg1) and GP2 (rPAg2) [3]. Antibodies against Saccharomyces cerevisiae (ASCA) enrich the diagnosis of CD by a further parameter. The presence of ASCA can indicate a more severe disease course of CD requiring aggressive immunotherapy.
Autoantibodies against intestinal goblet cells occur exclusively in UC. The target antigen of these autoantibodies has not yet been identified. Anti-neutrophile cytoplasm autoantibodies (ANCA) represent a further marker for UC. In the indirect immunofluorescence test (IIFT) they generate a perinuclear (pANCA) reaction on ethanol-fixed granulocytes, but no reaction on formalin-fixed granulocytes. This pattern contrasts with ANCA in vasculitis, which show a reaction on both substrates. The most important target antigen of ANCA in CIBD is DNA-bound lactoferrin.
Multiplex antibody detection
All relevant CIBD-associated antibodies can be determined in parallel by IIFT based on biochip mosaics. The EUROIMMUN CIBD Profile 3, for example, provides a combination of CUZD1- and GP2-transfected cells, control-transfected cells, intestinal goblet cells, ethanol-fixed granulocytes, lactoferrin-specific granulocytes, lactoferrin-depleted granulocytes, and fungal smears of S. cerevisiae (Fig. 3). The broad antibody analysis yields a high diagnostic rate for CD and UC.
Therapeutic drug monitoring
There is no cure for CIBD, and therapy focuses on reducing inflammation and relieving symptoms. Patients are frequently administered with tumour necrosis factor α (TNFα) inhibitors like infliximab or adalimumab. Due to different clinical responses to these biologics, the dosage must be optimized for each patient. By measuring the drug concentration in blood, the most suitable dose can be individually determined. Despite the good overall effectiveness of these drugs, a substantial number of patients form antibodies directed against the drugs, which can hamper the function of infliximab or adalimumab. Low drug levels can be an indication for the presence of these anti-drug antibodies (ADA). Regular monitoring of drug levels and determination of ADA enables fine tuning of the drug dosage, early switch to other therapeutics and prevention of side effects.
The concentration of infliximab or adalimu-mab in patient sera can be precisely deter-mined using MabTrack Level ELISAs (Sanquin Reagents, distributed by EUROIMMUN). As complementary tests, MabTrack Anti-Drug Antibody ELISAs enable specific detection of antibodies against infliximab or adalimumab. The ELISAs are easy to perform and can be fully automated. Thus, under or over treatment of CIBD can be recognised promptly and medication and dosage adjusted accordingly.
Perspectives
Reliable laboratory diagnostics for CIBD enable the number of endoscopies to be reduced by about two thirds, a benefit for both patients and healthcare budgets. FC determination represents a simple, non-invasive method to select patients for endoscopic clarification. This is especially relevant given the much higher prevalence of IBS (10-20 %) compared to CIBD (around 0.4 %). FC measurement also allows regular assessment of the disease activity without the need for endoscopy, enabling more efficient patient management. Multiplex antibody testing allows CD and UC to be differentiated. A clear distinction between the two diseases is important due to significant differences in treatment and prognosis. Therapeutic drug monitoring of patients on infliximab or adalimumab is ushering in a new era of personalized treatment for CIBD patients, whereby the medication and dosage can be optimised to the patient’s exact needs. Since infliximab and adalimumab are also used to treat other autoimmune diseases, especially rheumatoid arthritis and psoriasis, these assays will also play an important future role in other areas of medical diagnostics.
References
1. Magro F., et al. J Crohns Colitis 2017; 11(6): 649-670.
2. Komorowski L., et al. J Crohns Colitis 2013; 7(10): 780-790.
3. Homšak E., et al. Wien Klin Wochenschr 2010;
122 (Suppl 2): 19-25.
The author
Jacqueline Gosink, PhD
EUROIMMUN AG
Seekamp 31
23560 Lubeck
Germany
Figure 3. IIFT results. (a). Antibodies against CUZD1/GP2; (b). Antibodies against intestinal goblet cells; (c). Control-transfected cells; (d). pANCA; (e). Antibodies against DNA-bound lactoferrin; (f). Control lactoferrin-depleted granulocytes; g). Antibodies against S. cerevisiae.
Rheumatoid arthritis shows a prevalence of 0.5–1.6 % globally. The identification of biomarkers for early treatment response could aid in the fine tuning of therapy and therefore contribute to increased treatment efficacy and the timely use of biologicals when no response from disease-modifying anti-rheumatic drugs is observed. Our group has identified several biomarkers for early diagnosis and treatment response.
By F..L. Ochoa-González, J..C. Fernández-Ruiz, M..F. Romo-García and Dr J..E. Castañeda-Delgado
Introduction
Rheumatoid arthritis (RA) is a chronic disease of autoimmune etiology characterized by persistent inflammation of the synovial membrane, which lines the inner surface of capsules of synovial joints. The worldwide estimated prevalence is about 1 % of the adult population. It is more frequent in women with a ratio of 3:1. The cause of RA is unknown; however, genetic and environ-mental factors contribute to RA. Several genes have been associated with an increased risk of developing RA: mainly certain HLA class II antigens associated with the shared epitope that is responsible for antigen presentation to lymphocytes. Smoking and some causative microorganisms of oral diseases such as periodontitis and gingivitis (Porphyromonas gingivalis and Aggregati-bacter Actimomyctemcomitans) have also been associated with RA [1]. The relation-ship of genetic traits/environment and the link to inflammation and autoimmunity is being explored. In this regard post-translational modifications of proteins such as citrullination of arginine by peptidyl arginine deiminase (PAD; some of it mediated by PAD-like enzymes coming from oral pathogens) or carbamylation of lysine (mediated by cyanate from cigar smoke) contribute to breaking immunological tole-rance by creating neoepitopes of autologous proteins resulting in generation of auto-antibodies against modified peptides. An examples include anti-citrullinated protein antibodies (ACPAs), antibodies to the Fc part of IgG [rheumatoid factor (RF)], or autoantigens that cross-react with bacterial or viral antigens [2]. These autoantibodies contribute to the increased inflammatory response observed in RA patients.
The clinical manifestations of RA are mainly associated with symmetric inflammation of small and large joints, accompanied by morning stiffness. Patients with RA usually present multiple comorbidities as a result of chronic inflammation, the main ones being cardiovascular disease or pulmonary mani-festations. RA greatly affects the patient’s quality of life, as it interferes with physical function. In long-term disease without treatment, the accumulation of joint damage is irreversible and leads to disability at an early age without the possibility of recovering normal function. Therefore, it is of great importance to establish an early diagnosis; it has already been shown that beginning treatment prevents the progression of joint damage in up to 90 % of patients in the early stages of the disease.
There is no cure for RA, which is why the goal of treatment is to reach remission, defined as no disease activity and low disease activity with low risk to progression. Therefore, therapeutic approaches are based on drugs that interfere with signs and symptoms of RA, such as disease-modifying antirheumatic drugs (DMARDs). DMARDs are categorized into conventional synthetic (csDMARDs), targeted synthetic (tsDMARDs) and biologic (bDMARDs). The csDMARDs include sulfasalazine, leflunomide, hydroxychloroquine and methotrexate. The joint working group of the American College of Rheumatology and the European League Against Rheumatism (ACR-EULAR) recommends treating all new cases of RA as soon as possible using methotrexate combined with short-term glucocorticoids. It has been reported that a proportion of patients treated with either DMARDs or bDMARDs do not reach treatment target (reduction of DAS28 and disease activity). The presence of autoantibodies, joint damage and high disease activity are associated with rapid disease progression that can be slowed by adding bDMARDs [3]; therefore, better prognostic markers for treatment response are also needed.
According to current ACR-EULAR 2010 classification criteria (not diagnostic criteria), RA patients have joint pain and synovial inflammation, morning stiffness in the joints with duration of at least 30 minutes. There are several serological determinations that aid in the classification of these patients, such as the use of cyclic citrullinated peptides (CCPs) to detect ACPAs, as well as RF and erythrocyte sedimentation rate [4]. Nevertheless, the diagnostic tests, such as the CCP test, that have a high specificity (range: 90–96 %) have several caveats: (1) low sensitivity (range 67–83 %); and (2) when negative, RA cannot be discarded because of the possibility that the patient has seronegative RA [5]. This is also the case for RF, which shows a similar sensitivity but a lower specificity. Therefore, there is a need for diagnostic tools that could help to further clarify the diagnosis of RA. As stated above, early diagnosis and treatment remains one of the crucial points for the management of RA and thus prevention of loss of physical function; however, the auxiliary diagnostic tools remain insufficient to distinguish RA from other rheumatic diseases. It is for these reasons that our group has focused on the search for early biomarkers of disease and of treatment response.
MicroRNAs as diagnostic biomarkers
MicroRNAs (miRNAs) are small RNA molecules (approximately 21 bp long) that modulate transcription and translation. Without miRNAs all the genes that are transcribed into mRNA (messenger RNA) would be translated to proteins, but miRNAs regulate which mRNA will or will not be translated into proteins. One single miRNA can control the production of many proteins and therefore small changes in the abundance of an miRNA could result in bigger changes at the protein level. This is these molecules are very important and are indicators of what is happening inside the body before these changes can be observed as clinical symptoms.
Recently, using microarray technology, our group detected changes in the expression profile of several hundred mRNAs in patients with early RA whose symptoms at that moment were barely classifiable by a rheumatologist [6]. What was behind that drastic change? As miRNAs are the main regulators of mRNA, we hypothesized that miRNAs could be responsible. Therefore, we analysed this miRNA profile in patients with early RA (using the same technology) and we identified 97 miRNAs that were over-expressed in early RA [7]. It seemed (but more experiments are needed to confirm this) that only 97 miRNAs are responsible for regulating around 2000 mRNA and all this in the early phases of the disease and the start of arthritis, when symptoms can’t be clearly classified by the clinician. This discovery encouraged us to explore whether any of these miRNAs can serve as a biomarker for the detection of early RA. Thus we performed an analysis named receiver operator characteristic curve (ROC curve). This kind of graph shows how many patients a biomarker (in this case a miRNA) can classify correctly and how many incorrectly. From this analysis we found that miRNA mir-361-5p had a specificity of 82.61 and sensitivity of 81.25. The value for sensitivity is bigger than the one of CCP (as mentioned previously) meaning that probably mir-361-5p can help to correctly identify those with the disease (true positives). However, a wider study is needed to confirm such observations and take it to the clinical lab.
The interest in miRNAs as biomarkers is increasing not only because they are master regulators as we mentioned before, but also because they have much greater stability in several fluids types, such as saliva or serum [8], compared to mRNA and this facilitates detection. Currently, the detection of miRNAs is performed by PCR or massive sequencing, which are technologies that need special equipment and infrastructure. However, recently, research groups have started to work on novel ideas for miRNA detection, such as small electronic devices to quantify miRNAs from a drop of serum [9].
Circulating miRNAs as potential biomarkers of treatment response
miRNAs have been proposed by different authors as possible biomarkers of response to csDMARDs, bDMARDs and tsDMARDs in RA. The abnormal expression pattern of miRNAs reflects the underlying patho-physiological processes owing to its direct relationship with the inflammatory processes. Some miRNAs have been described as markers of response to anti-tumour necrosis factor alpha (TNFα) treatment in patients with RA, which is the case with hsa-miR-hsa-16-5p, hsa-miR-23-3p, hsa-miR125b-5p, hsa-miR-126-3p, hsa-miRN-146a-5p and hsa-miR-223-3p that are upregulated in patients who respond after therapy and show a reduction in inflammatory parameters [TNFα, interleukin-6 (IL-6), IL-17, RF and C-reactive protein (CRP)] [10, 11]. Even methotrexate treatment seemed to have an effect on the expression of hsa-miR-132-3p, hsa-miR-146a-5p and hsa-miR-155-5p, where good responders have a downregulation of these miRNAs [12] (Fig. 1).
Our research team evaluated the expression of serum miRNAs by flow cytometry (Firefly™ technology) in a cohort of patients receiving treatment with tofacitinib (TOFA, a tsDMARD) for 5 years as part of an open-label study. Treatment with TOFA does not affect miRNA expression directly; however, in patients experiencing RA flare-up we identified changes in two miRNAs: hsa-miR-432-5p was downregulated and hsa-miR-194-5p was upregulated. Our findings suggest that these miRNAs could be used as a biomarkers for relapse. By monitoring them, relapse could be predicted and prevented by allowing a return to a treatment scheme before the patient’s symptoms worsen. How-ever, more research is required, as it is the first time that these miRNAs have been found to be involved in the inflammatory response of patients with RA [13]. If these findings can be confirmed, these miRNAs could be useful biomarkers for prediction of therapy effectiveness as well as therapy monitoring and could, therefore, be a useful support tool for generating personalized treatment regimens for RA patients.
Perspectives
The miRNA biomarkers reported above await further validation in the clinical setting. The design of clinical studies for such validation should account for and analyse possible reallife situations, such as the similarity of symptoms of RA patients with other rheumatic diseases and confounding variables.
References
1. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011; 365: 2205–2219.
2. Cheng Z, Meade J, Mankia K, Emery P, Devine DA. Periodontal disease and periodontal bacteria as triggers for rheumatoid arthritis. Best Pract Res Clin Rheumatol 2017; 31: 19–30.
3. Calabrese LH, Calabrese C, Kirchner E. The 2015 American College of Rheumatology Guideline for the treatment of rheumatoid arthritis should include new standards for hepatitis B screening: comment on the article by Singh et al. Arthritis Care Res 2016; 68: 723–724.
4. Kay J, Upchurch KS. ACR/EULAR 2010 rheumatoid arthritis classification criteria. Rheumatology 2012; 51(Suppl 6): vi5–9.
5. Mathsson Alm L, Fountain DL, Cadwell KK, Madrigal AM, Gallo G, Poorafshar M. The performance of anti-cyclic citrullinated peptide assays in diagnosing rheumatoid arthritis: a systematic review and meta-analysis. Clin Exp Rheumatol 2018; 36: 144–152.
6. Macías-Segura N, Castañeda-Delgado JE, Bastian Y, Santiago-Algarra D, Castillo-Ortiz JD, Alemán-Navarro AL, Jaime-Sánchez E, Gomez-Moreno M, Saucedo-Toral CA, et al. Transcriptional signature associated with early rheumatoid arthritis and healthy individuals at high risk to develop the disease. PLoS One 2018; 13: e0194205.
7. Romo-García MF, Bastian Y, Zapata-Zuñiga M, Macías-Segura N, Castillo-Ortiz JD, Lara-Ramírez EE, Fernández-Ruiz JC, Berlanga-Taylor AJ, González-Amaro R, et al. Identification of putative miRNA biomarkers in early rheumatoid arthritis by genome-wide microarray profiling: A pilot study. Gene 2019; 720: 144081.
8. Huang W. MicroRNAs: biomarkers, diagnostics, and therapeutics. Methods Mol Biol 2017; 1617: 57–67.
9. Labib M, Khan N, Ghobadloo SM, Cheng J, Pezacki JP, Berezovski MV. Three-mode electrochemical sensing of ultralow microRNA levels. J Am Chem Soc 2013; 135: 3027–3038.
10. Castro-Villegas C, Pérez-Sánchez C, Escudero A, Filipescu I, Verdu M, Ruiz-Limón P, Aguirre MA, Jiménez-Gomez Y, Font P, Rodriguez-Ariza A, et al. Circulating miRNAs as potential biomarkers of therapy effectiveness
in rheumatoid arthritis patients treated with anti-TNFα. Arthritis Res Ther 2015; 17: 49.
11. Filková M, Aradi B, Senolt L, Ospelt C, Vettori S, Mann H, Filer A, Raza K, Buckley CD, et al. Association of circulating miR-223 and miR-16 with disease activity in patients with early rheumatoid arthritis. Ann Rheum Dis 2014; 73(10): 1898–1904.
12. Singh A, Patro PS, Aggarwal A. MicroRNA-132, miR-146a, and miR-155 as potential biomarkers of methotrexate response in patients with rheumatoid arthritis. Clin Rheumatol 2019; 38: 877–884.
13. Fernández-Ruiz JC, Ramos-Remus C, Sánchez-Corona J, Castillo-Ortiz JD, Castañeda-Sánchez JJ, Bastian Y, Romo-García MF, Ochoa-González F, Monsivais-Urenda AE, et al. Analysis of miRNA expression in patients with rheumatoid arthritis during remission and relapse after a 5-year trial of tofacitinib treatment. Int Immunopharmacol 2018; 63: 35–42.
The authors
Fatima de Lourdes Ochoa-González1,2 MSc, Julio Cesar Fernández-Ruiz2,3 MSc, Maria Fernanda Romo García2,3 MSc, Julio Enrique Castañeda-Delgado*4 PhD
1 Unidad Académica de Biología, Universidad Autónoma de Zacatecas, Zacatecas, México
2 Unidad de Investigación Biomédica de Zacatecas, Instituto Mexicano del Seguro Social, Zacatecas, México
3 Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luís Potosí, San Luís Potosí, México
4 Cátedras-CONACYT, Unidad de Investigación Biomédica de Zacatecas, Instituto Mexicano del Seguro Social, Zacatecas, México
*Corresponding author
E-mail: julioenrique_castaneda@yahoo.com.mx
Figure 1. Circulating microRNAs after treatment for rheumatoid arthritis (RA). Flow diagram showing the expression of some microRNAs after methotrexate (MTX), anti-TNFα and tofacitinib (TOFA) treatment. Red, upregulated microRNAs; blue, downregulated microRNAs; qPCR, quantitative PCR.
March 2024
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