Search

Malaltia de Crohn; Colitis ulcerosa; Vacunació Enfermedad de Crohn; Colitis ulcerosa; Vacunación Crohn's disease; Ulcerative colitis; Vaccination Patients with inflammatory bowel disease (IBD) may require different immunosuppressive treatments throughout their illness. It is essential to assess the immunization status of patients at diagnosis or, if this is not possible, at least before the beginning of immunosuppressive therapy and, subsequently, administering the appropriate vaccines. Therefore, the aim of this work is to establish clear and concise recommendations on vaccination in patients with IBD in the different settings of our clinical practice including vaccination in children, during pregnancy, breastfeeding or on trips. This consensus document emphasises the differences between inactivated and attenuated vaccines and the different degrees of immunosuppression and correlates them with the administration of both mandatory and optional vaccines recommended to our patients with IBD. Finally, as a summary, 17 recommendations are established based on the available scientific evidence and expert opinion. A multidisciplinary team with extensive experience in IBD and vaccination, made up of specialists in gastroenterology, paediatrics, nursing and pharmacy, has participated in the preparation of these recommendations of the Spanish Working Group on Crohn's Disease and Ulcerative Colitis. Los pacientes con enfermedad inflamatoria intestinal (EII) pueden requerir diferentes tratamientos inmunosupresores a lo largo del curso de su enfermedad. Por ello, es fundamental evaluar el estado de inmunización en el momento del diagnóstico o, si no es posible, siempre antes de iniciar un tratamiento inmunosupresor, y administrar las vacunas apropiadas. El objetivo del presente documento es establecer unas recomendaciones claras y concisas sobre la vacunación en pacientes con EII en diferentes escenarios de práctica clínica, incluyendo situaciones especiales como la vacunación en la edad pediátrica, el embarazo, la lactancia o en viajes al extranjero. Se presentan las diferencias entre vacunas inactivadas y atenuadas, los diferentes grados de inmunosupresión y su relación con las pautas de administración de las diferentes vacunas (tanto obligatorias como opcionales) recomendadas a los pacientes con EII. En el documento, se establecen 17 recomendaciones basadas en la evidencia científica disponible y opinión de expertos. En la elaboración de estas recomendaciones del Grupo Español de Trabajo en Enfermedad de Crohn y Colitis Ulcerosa ha participado un equipo multidisciplinar con amplia experiencia en EII y vacunación formado por especialistas de gastroenterología, pediatría, enfermería y farmacia.

[Abstract] Objectives: Mobile apps are useful tools in e-health and self-management strategies in disease monitoring. We evaluated the Harvey–Bradshaw index (HBI) mobile app self-administered by the patient to see if its results agreed with HBI in-clinic assessed by a physician. Methods: Patients were enrolled in a 4-month prospective study with clinical assessments at months 1 and 4. Patients completed mobile app HBI and within 48 h, HBI was performed by a physician (gold standard). HBI scores characterized Crohn's disease (CD) as remission

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders. This work was supported by Spanish Ministerio de Economía y Competitividad (MINECO, SAF2016-80535-R) and Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, PID2019-106658RB-I00), co-financed by European Development Regional Fund “A way to achieve Europe” ERDF, and Agència de Gestió d’Ajuts Universitaris i de Recerca from Generalitat de Catalunya (2017/SGR/1582). SC-L, MV-dA, CC, AL-P, and EC are recipients of fellowships from Spanish Ministerio de Educación, Cultura y Deporte (FPU15/02897), Spanish MINECO (BES-2014-069237 and BES-2017-082107), Chilean Agencia Nacional de Investigación y Desarrollo (2018-72190154), and European Community Seventh Framework Program (FP7/2007/2013; 229673), respectively. SC-L and JL are recipients of short-term fellowships from European Federation of Immunological Societies-Immunology Letters (EFIS-IL) and Erasmus+ from the European Union, respectively. The ENEIDA registry of GETECCU is supported by Biogen, Janssen, Takeda and Pfizer. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Background: Multiple studies have described the effectiveness of ustekinumab (UST) and vedolizumab (VDZ) in patients with Crohn’s disease (CD) failing anti- Tumor necrosis factors (TNFs), however, the effectiveness of VDZ or UST as a third-class biologic has not yet been described. Aims and Methods: In this retrospective multicenter cohort study, we aimed to investigate the effectiveness of VDZ and UST as a third-class biologic in patients with CD. Results: Two-hundred and four patients were included, 156/204 (76%) patients received VDZ as a second- and UST as a third-class therapy (group A), the remaining 48/204 (24%) patients received UST as a second- and VDZ as a third-class therapy (group B). At week 16–22, 87/156 (55.5%) patients and 27/48 (56.2%) in groups A and B, respectively, responded to treatment (p = 0.9), 41/156 (26.2%) and 15/48 (31.2%) were in clinical remission (p = 0.5). At week 52, 89/103 (86%) patients and 25/29 (86.2%) of the patients with available data had responded to third-class treatment in groups A and B, respectively (p = 0.9), 31/103 (30%) and 47/29 (24.1%) were in clinical remission (p = 0.5). Conclusion: Third-class biological therapy was effective in more than half of the patients with CD. No differences in effectiveness were detected between the use of VDZ and UST as a third-class agent.

[eng] Crohn’s disease [CD] is a chronic inflammatory disease of the intestinal tract with considerable heterogeneity among affected patients in terms of disease phenotype and therapeutic responses. Despite the increase in the number of drugs approved for the management of CD, a significant percentage of patients remain unresponsive or lose response over time to treatments, and eventually require surgery to control disease activity and/or complications. Nonetheless, in a fraction of these refractory patients, intestinal resection may not be possible due to disease location, extension or previous surgeries. For such patients, autologous hematopoietic stem cell transplantation [HSCT] represents a potential salvage therapy despite the risks associated with this procedure. Stem cell transplantation is an accepted therapy for hematological disorders, aplastic anemia and immunodeficiencies. In the context of autoimmune diseases, the serendipitous benefits of transplantation were initially reported in patients suffering from both immune-mediated diseases and hematological disorders. This led to trials that have shown the efficacy of autologous HSCT in treating an array of autoimmune diseases including refractory severe multiple sclerosis, systemic lupus erythematosus, juvenile idiopathic arthritis, rheumatoid arthritis and, more recently, CD. The benefit of HSCT in autoimmunity is thought to originate from the ability of intense immune depletion to eliminate auto-reactive cells regardless of their specificity. This would lead to de novo generation of immune cells that could re-establish tolerance, although no objective evidence of this ‘resetting’ has been reported thus far. To explore this hypothesis, we group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumor necrosis factor [TNF]-α were included for comparison. Severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T- cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content. HSCT dramatically renewed the pre-existing T-cell receptor [TCR] repertoire; however, persistent high-frequency TCR clones were detected in all patients in both blood and biopsy throughout the first year of follow-up. The number of persistent resident T cell clonotypes in tissue after treatment decreased in patients that achieve endoscopic remission. Furthermore, we found that low TCR peripheral diversity at baseline, and up to 1 year after HSCT, were associated with a lack of response to the immunoablative protocol. Peripheral blood immune remodeling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα., [spa] Se ha demostrado la eficacia del trasplante hematopoyético de células madre (THCM) en pacientes con enfermedad de Crohn severamente refractaria. La hipótesis aceptada es que el proceso de THCM elimina células auto-reactivas, devolviendo así la tolerancia. No obstante, no existen estudios específicos y exhaustivos sobre la reconstitución inmune en pacientes con enfermedad de Crohn tratados con THCM. En esta tesis, monitorizamos una cohorte de 18 pacientes con enfermedad de Crohn refractaria que recibieron trasplante autólogo de células madre hematopoyéticas, de los cuales el 50% consiguieron la remisión endoscópica sin tratamiento de mantenimiento. Para elucidar los mecanismos que determinan la eficacia, monitorizamos los cambios después de THCM en la composición de células inmunes tanto en sangre como en tejido intestinal. Como resultado, observamos que la ablación inmune producida durante el TCMH induce cambios dramáticos en las células T y B en sangre en todos los pacientes, independientemente de la eficacia del tratamiento. La remisión endoscópica en la semana 52 posterior al TCMH se asoció con cambios transcripcionales intestinales significativos. Una comparación de la transcripción en remisión con la de la remisión inducida por fármaco identificó genes comunes y únicos en la respuesta inducida por el TCMH. A través de un análisis de deconvolución de los datos del transcriptoma de la biopsia intestinal, mostramos que la respuesta al TCMH, pero no a fármaco inmuno- modulador, se asocia con una expansión de las células B naïve y una disminución en el contenido de células T memoria. Como se esperaba, la remisión endoscópica, en respuesta a tanto a TCMH como a fármaco, condujo a una reducción significativa en el contenido de neutrófilos intestinales y macrófagos M1 La remodelación inmune de la sangre periférica después del TCMH no predice la eficacia. No obstante, la depleción de las células T intestinales mantenida un año después del trasplante se asocia con la curación mucosa después del TCMH.

The quality of life of a significant proportion of IBD patients is poor as a result of persistent disease activity and repeated surgery, among others. Current treatments for Crohn’s disease are not able to neither prevent this serious impact nor improve the long term prognosis of a significant proportion of patients. Therefore, new therapeutic approaches are needed in order to modify the immune response of these patients. We hypothesize that administration of ex-vivo generated autologous tol-DCs to Crohn’s disease patients may arrest Th1 lymphocyte proliferation and therefore may restore specific tolerance against non-pathogenic antigens in the gut. The overall objective of this thesis was to generate and characterize tol-DCs for the purpose of implementing an autologous immunotherapy treatment for Crohn’s disease patients. In the first study, we described the generation of tol-DCs from healthy donors and Crohn’s disease patients by use of clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterized their functional properties. Our main findings demonstrated that the combination of dexamethasone with a specific cytokine cocktail yields clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. This characteristic tolerogenic profile is maintained when tol-DCs are activated using heat-inactivated Gram-negative bacteria as maturative stimulus. Whole microorganisms contain multiple PAMPs capable of stimulating DCs by different pathways. Our results clearly showed a strong inhibitory effect on DC phenotype, a robust inhibition of pro-inflammatory cytokines, increased IL-10 secretion, and inhibition of T-cell proliferation and Th1 induction. Interestingly, we showed that tol-DCs have reduced immunogenic capacity in autologous, allogeneic and antigen-specific T-cell responses. We further evaluated the ability of tol-DCs to induce CD4+ T-cell hypo-responsiveness. Our results demonstrated that T-cells or antigen-specific T-cells previously cultured with tol-DCs are anergic exhibiting a reduced capacity to proliferate as well as reduced IFN-gamma secretion when rechallenged with fully competent mDCs. With regard to tol-DCs clinical application, we importantly found that their tolerogenic properties remain stable after washing out dexamethasone and subsequent restimulation with LPS, CD40L or different Gram-negative enterobacteria strains. All these properties led us to conclude that this cell product is suitable to be tested in clinical trials of immune-based diseases such as Crohn’s disease. We further identified a positive biomarker for tol-DCs, MERTK receptor is highly expressed on clinical grade dexamethasone-induced human tol-DCs and contributes in their tolerogenic properties. Our results demonstrated that MERTK expression in human DCs is regulated by glucocorticoids and described a new function of this receptor in directly regulating T-cell response. Interestingly, our findings showed that neutralization of MERTK with monoclonal antibodies in allogeneic MLR cultures leads to increased T-cell proliferation and IFN-gamma production. The direct regulation of T-cell response was confirmed by the use of recombinant MERTK-Fc protein, used to mimic MERTK on DCs. Our results remarkably showed that MERTK-Fc suppresses naïve and antigen-specific memory Tcell proliferation and activation. These findings identified a new non-cell autonomous regulatory function of MERTK expressed on DCs. Additionally, we described that this regulation is mediated by the neutralization of MERTK soluble ligand PROS1. We also found that MERTK is expressed on T-cell surface and that PROS1 drives an autocrine pro-proliferative effect on these cells. In summary, the results of this work demonstrated that MERTK on DCs regulates T-cell activation and expansion through the competition for PROS1 interaction with MERTK in the T-cells. We showed that MERTK expression in human DCs has a key role in instructing adaptive immunity and identified MERTK as a potent suppressor of T-cell response. Therefore targeting MERTK may provide an interesting approach to effectively increase or suppress tolerance for the purpose of immunotherapy., Esta tesis doctoral estudia el proceso de generación de células dendríticas tolerogénicas en grado clínico, con el objetivo de establecer un protocolo destinado al tratamiento de la enfermedad de Crohn. El estudio realizado ha permitido la caracterización de dichas células y sus propiedades tolerogénicas, incluyendo la descripción novedosa de un marcador de células tolerogénicas y el estudio de sus propiedades funcionales relacionadas con la inducción de tolerancia.

Aim: To evaluate inflammatory activity in patients with Crohn's disease (CD) using technetium-99m-hexamethylpropyleneamine oxime (99mTc-HMPAO) granulocyte scintigraphy. Methods: Twenty patients (7 male and 13 female) with CD and five healthy volunteers were selected for 99mTc-HMPAO granulocyte scintigraphy. The Crohn's Disease Activity Index (CDAI), blood tests and C-reactive protein (CRP) of each patient were performed 7 d before the scintigraphic images. The leukocytes were labeled according to the International Society of Radiolabeled Blood Elements (ISORBE) consensus protocol and the scintigraphic images, including single photon emission computed tomography, were obtained 30 min and 2 h after injection of the radiolabeled leukocytes. Results: The labeling yield of the leukocytes with the lipophilic complex 99mTc-HMPAO was 55.0% +/- 10%. Six of the 20 patients (30%) presented congruent results for the three parameters investigated (CDAI, Scintigraphic Index and CRP). On the other hand, 14 patients (70%) did not show congruent results. There was no significant correlation between the indices analyzed according to the Spearman test (P > 0.05, n = 20). Conclusion: The results suggest that 99mTc-HMPAO-labeled leukocyte scintigraphy could be important for determining inflammatory activity in CD even in the absence of clinical symptoms.

Assessment of disease extension and activity is crucial to guide treatment in Crohn's disease. The objective of the current cross-sectional study was to determine the accuracy of MR for this assessment.50 patients with clinically active (n = 35) or inactive (n = 15) Crohn's disease underwent ileocolonoscopy (reference standard) and MR. T2-weighted and precontrast and postcontrast-enhanced T1-weighted sequences were acquired. Endoscopic activity was evaluated by CDEIS (Crohn's Disease Endoscopic Index of Severity); in addition endoscopic lesions were classified as absent, mild (inflammation without ulcers) or severe (presence of ulceration).The comparison of intestinal segments with absent, mild and severe inflammation demonstrated a progressive and significant (p0.001) increase in the following MR parameters: wall thickness, postcontrast wall signal intensity, relative contrast enhancement, presence of oedema, ulcers, pseudopolyps and lymph node enlargement. Independent predictors for CDEIS in a segment were wall thickness (p = 0.007), relative contrast enhancement (p = 0.01), presence of oedema (p = 0.02) and presence of ulcers at MR (p = 0.003). There was a significant correlation (r = 0.82, p0.001) between the CDEIS of the segment and the MR index calculated according to the logistic regression analysis coefficients. The MR index had a high accuracy for the detection of disease activity (area under the receiver operating characteristic (ROC) curve 0.891, sensitivity 0.81, specificity 0.89) and for the detection of ulcerative lesions (area under the ROC curve 0.978, sensitivity 0.95, specificity 0.91) in the colon and terminal ileum.The accuracy of MR for detecting disease activity and assessing severity brings about the possibility of using MR as an alternative to endoscopy in the evaluation of ileocolonic Crohn's disease.

La presencia de microorganismos patógenos en alimentos es uno de los problemas esenciales en salud pública, y las enfermedades producidas por los mismos es una de las causas más importantes de enfermedad. Por tanto, la aplicación de controles microbiológicos dentro de los programas de aseguramiento de la calidad es una premisa para minimizar el riesgo de infección de los consumidores. Los métodos microbiológicos clásicos requieren, en general, el uso de pre-enriquecimientos no-selectivos,enriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodosinmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es unatécnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento. En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado yevaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo. Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productoscárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados.En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario., The presence of pathogens in foods is among the most serious public health concerns, and the diseases produced by them are a major cause of morbidity. Consequently, the application of microbiological control within the quality assessment programs in the food industry is a premise to minimize the risk of infection for the consumer. Classical microbiological methods involve, in general, the use of a non-selective pre-enrichment, selective enrichment, isolation on selective media, and subsequent confirmation using morphological, biochemical and/or serological tests. Thus, they are laborious, time consuming and not always reliable (e.g. in viable but non-culturable VBNC forms). A number of alternative, rapid and sensitive methods for the detection, identification and quantification of foodborne pathogens have been developed to overcome these drawbacks. PCR has become the most popular microbiological diagnostic method, and recently, the introduction of a development of this technique, RTi-PCR, has produced a second revolution in the molecular diagnostic methodology in microbiology. RTi-PCR is highly sensitive and specific. Moreover, it allows accurate quantification of the bacterial target DNA. Main advantages of RTi-PCR for its application in diagnostic laboratories include quickness, simplicity, the closed-tube format that avoids risks of carryover contaminations and the possibility of high throughput and automation.In this work, specific, sensitive and reliable analytical methods based on molecular techniques (PCR and NASBA) were developed for the detection, identification and quantification of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and Salmonella spp.). Real-time PCR based methods were designed and optimised for each one of these target bacteria: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, and also a real-time PCR basedmethod previously described for Salmonella spp. was optimised and multicenter evaluated. In addition, an NASBA-based method was designed and optimised for the specific detection of M. avium subsp. paratuberculosis. The potential application of the NASBA technique for specific detection of viable M. avium subsp. paratuberculosis cells was also evaluated.All the amplification-based methods were 100 % specific and the sensitivity achieved proved to be fully suitable for further application in real food samples. Furthermore, specific pre-amplification procedures were developed and evaluated on meatproducts, seafood products, milk and water samples. Thus, fully specific and highly sensitive real-time PCR-based methods were developed for quantitative detection of L. monocytogenes on meat and meat products and on salmon and cold smoked salmon products; and for quantitative detection of M. avium subsp. paratuberculosis on water and milk samples. The M. avium subsp. paratuberculosis-specific real-time PCR-based method was also applied to evaluate the presence of this bacterium in the bowelof Crohn's disease patients using colonic biopsy specimens form affected and unaffected volunteers. In addition, fully specific and highly sensitive real-time NASBA-based methods were developed for detection of M. avium subsp. paratuberculosis on water and milk samples.In conclusion, this study reports selective and sensitive amplification-based assays for the quantitative detection of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and) and for a quick and unambiguously identification of Salmonella spp. The assays had an excellent relative accuracy compared to microbiological reference methods and can be used for quantification of genomic DNA and also cell suspensions. Besides, in combination with sample pre-amplification treatments,they work with high efficiency for the quantitative analysis of the target bacteria. Thus, they could be a useful strategy for a quick and sensitive detection of foodborne pathogens in food products and which should be a useful addition to the range of diagnostic tools available for the study of these pathogens.