[Show abstract][Hide abstract] ABSTRACT: Infliximab is an anti-tumor necrosis factor (TNF) used for treatment of inflammatory bowel disease (IBD) as well as rheumatoid arthritis, psoriasis, and other inflammatory conditions. Antibodies to infliximab (ATI) develop in approximately 45% of infliximab-treated IBD patients and are correlated with loss of clinical response. Scarce data exist as to factors which predict infliximab immunogenicity.To investigate factors that may predict formation of antibodies to infliximab (ATI) and infliximab therapy failure an observational study of consecutive IBD patients treated with infliximab between 2009 and 2013 was performed. Trough levels of ATI were measured. Patients were monitored for disease activity using clinical activity indexes and were classified according to ATI formation and clinical response. All clinical and demographic parameters were analyzed for association with the designated outcomes.One hundred fifty-nine patients were included and 1505 sera were tested. On multivariate analysis, Jewish Ashkenazi ethnicity was protective against both development of ATI (odds ratio [OR] 0.35, 95% confidence interval [CI] 0.17-0.7, P = 0.005) and treatment failure (OR 0.29, 95% CI 0.13-0.66, P = 0.003). Concomitant immunomodulator therapy was also negatively associated with immunogenicity and infliximab therapy failure (OR 0.31, 95% CI 0.15-0.65, P = 0.002; OR 0.42 95% CI 0.18-0.99, p = 0.04, respectively), whereas episodic therapy was positively associated with both outcomes (OR 4.2 95% CI 1.07-16.1, p = 0.04, OR 4.45 95% CI 1.2-16.6, p = 0.026 respectively). All other variables, including IBD type, gender, weight, age, smoking status and disease duration, were not predictive of ATI formation or clinical failure. However, among Crohn's disease patients, a non-stricturing non-penetrating phenotype was protective against ATI formation (OR 0.4, 95% CI 0.14-0.96, p = 0.04). P = 0.04, respectively), whereas episodic/interrupted therapy was positively associated with both outcomes (OR 4.2, 95% CI 1.07-16.1, P = 0.04; OR 4.45, 95% CI 1.2-16.6, P = 0.026, respectively). All other variables, including IBD type, sex, weight, age, smoking status, and disease duration, were not predictive of ATI formation or clinical failure. However, among Crohn disease patients, a nonstricturing nonpenetrating phenotype was protective against ATI formation (OR 0.4, 95% CI 0.14-0.96, P = 0.04).Jewish Ashkenazi ethnicity is protective of ATI formation and infliximab therapy failure. These findings suggest a role for ethnicity, and implicitly for genetic predisposition, in modulating the risk of anti-TNF immunogenicity and treatment unresponsiveness.
Medicine 05/2015; 94(18):e673. DOI:10.1097/MD.0000000000000673 · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cross-immunogenicity of the recently approved infliximab-biosimilar Remsima (CT-P13) with the originator drug Remicade is still unknown.
Sera of patients with IBD with or without measurable anti-Remicade antibodies to infliximab (ATI) were tested for their cross-reactivity to two batches of Remsima. Experiments were repeated after deglycosylation of Remicade/Remsima, IgG purification, excipients' dialysis and monomer purification by size exclusion chromatography. Anti-Remicade antibodies were tested for their functional inhibition of TNF-α binding by Remsima/Remicade by competition assay. Cross-reactivity of anti-adalimumab antibodies with Remicade/Remsima was also investigated.
125 patients' and controls' sera were tested (median age 31 years, IQR 24.5-39.5). All 56 anti-Remicade ATI-negative controls (14 healthy individuals, 42 patients with IBD) were also negative for anti-Remsima ATI. All 69 positive anti-Remicade IBD sera were cross-reactive with Remsima. ATI titres against Remicade or Remsima were strongly correlated (r values between 0.92 and 0.99, p<0.001 for all experiments, Spearman's correlation test). The background ELISA signal for Remsima was slightly higher compared with Remicade in negative controls (1.25±0.6 µg/mL vs 0.76±0.5 µg/mL, respectively, p<0.001), and persisted after deglycosylation, dialysis or protein size filtration, but abolished by IgG purification and significantly diminished by monomer purification. Anti-Remicade ATIs of patients with IBD (n=10) exerted similar functional inhibition on Remsima or Remicade TNF-α binding capacity (p=NS for all inhibition curve points). Antibodies-to-adalimumab in adalimumab-treated patients with IBD (n=7) did not cross-react with either Remicade or Remsima.
Anti-Remicade antibodies in patients with IBD recognise and functionally inhibit Remsima to a similar degree, suggesting similar immunogenicity and shared immunodominant epitopes on these two infliximab agents. In contrast, anti-adalimumab antibodies do not cross-react with Remsima or Remicade.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
Gut 04/2015; DOI:10.1136/gutjnl-2015-309290 · 14.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the prevalence of double negative (DN) sera and the mechanisms responsible for DN status.
Sera of inflammatory bowel disease patients treated with infliximab (IFX) were tested for drug/antibodies to infliximab (ATI) trough levels and the proportion of DN results was compared between a commercially available double antigen ELISA (with labeled IFX as the detection antibody) and an anti-lambda ELISA (with anti-human lambda chain detection antibody). Repeat testing with lower than customary serum dilution (1:10) was performed. Patients with DN status were matched with IFX+/ATI- controls and were followed-up for subsequent development of non-transient ATI to investigate if DN status precedes ATI.
Of 67 sera obtained at time of loss of response, only 6/67 (9%) were DN by anti-lambda ELISA compared to 27/67 (40%) with double antigen ELISA (P < 0.001, Fisher's Exact test). Of the latter 27 sera, 22% were also DN by anti-lambda ELISA, whereas 44% were actually IFX positive (IFX+ATI-), 30% were ATI positive (IFX-ATI+) and 4% were double positive (IFX+ATI+). Re-testing using a 1:10 dilution converted most DN results into IFX+ and /or ATI+ status. Patients with DN status had shorter survival free of non-transient ATI compared with matched controls (log rank test, P < 0.001). In 9/30 (30%) of these patients, non transient ATI occurred before and after the event at which the DN serum was obtained, supporting the view that a DN result may represent a particular time-point along the two curves of ATI titer rise and infliximab drug level decline.
DN status may result from false negative detection of IFX or ATI by double antigen ELISA, suggesting a transitional state of low-level immunogenicity, rather than non-immunological clearance.
World Journal of Gastroenterology 02/2015; 21(6):1907-14. DOI:10.3748/wjg.v21.i6.1907 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The T cell cytokine IL-17 and the Th-17 pathway appear to have a role in the pathogenesis of inflammatory bowel diseases. IL-2 is a potent stimulator of lymphocyte proliferation and IL2/IL21 receptor polymorphisms have recently been associated with susceptibility to IBD.
To evaluate the expression of IL-17, IL-2 and TNFα in Crohn's disease (CD) patients with and without anti-TNFs.
Cytokine expression was evaluated by ELISA and intracellular staining of CD4+ T-cells from the peripheral blood and lamina propria of CD patients and of non-IBD controls. The results were stratified by disease activity and anti-TNF treatment.
IL2 expression was significantly elevated in CD patients not treated with anti-TNFs in comparison to healthy controls (19.6% vs. 33.3%, P = 0.03) and CD patients treated with anti-TNFs (20.4% vs. 33.3%, P = 0.02), and similar in infliximab-treated patients and controls. IL17 expression was similar in CD patients and controls, and was not affected by anti-TNF therapy. TNFα expression in patients with active CD was increased compared to controls (35.5% vs 25.7%, P < 0.005), and was significantly decreased in anti-TNF treated patients in comparison to CD patients without anti-TNFs (39.6% vs 26.2%, P = 0.01).
Expression of IL2 was significantly decreased in anti-TNF-treated CD patients in comparison to non-treated CD patients and controls. This novel finding may indicate a further mechanism of anti-TNF therapy in CD. Expression of IL17 was not influenced by presence of CD or anti-TNF therapy, which may partly explain the failure of recent clinical trials investigating anti-IL17 therapy in CD.
[Show abstract][Hide abstract] ABSTRACT: To elucidate the relative role of the immune system and intestinal epithelium in the ethiopatogenesis of Celiac disease (CD).
A patient with childhood CD who underwent allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia was followed for 5 years after resumption of gluten containing diet. Immunological memory to gliadin epitopes was assessed in the index patient and in 5 newly diagnosed CD patients by standard serology testing and by CFSE-based proliferation assays of peripheral blood CD4+ cells and of intestinal LPL towards gliadin-TTG antigens. Intestinal lymphocytes' origin was determined by combined immuno-histochemistry and fluorescent in-situ hybridiazation (FISH).
Over 5 years of follow-up after receiving BMT from a HLA-matched woman and cessation of gluten-free diet, the patient has remained well, with negative periodic antibodies assays and unremarkable serial duodenal biopsies. In vitro proliferation assays showed lack of a memory response of the patient's peripheral blood and lamina propria CD4+ T-cells towards TTG, gliadin or TTG-treated gliadin, whereas memory responses were evident in the newly diagnosed CD patients. Immuno-FISH of post-BMT duodenal mucosa showed that the chromosomal phenotype of all the epithelial cells was XY. In contrast, CD45+ lymphocytic lineage cells were all donor-derived XX cells, presumably originating in the transplanted bone marrow and re-populating the intestinal wall.
CD resolution following allogeneic BMT is associated with absent gliadin-specific memory response, and with a dichotomous lymphocyte-epithelial chimeric intestine. These observations suggest that the pathogenesis of CD is critically dependent upon the immune system rather than the epithelial compartment.
[Show abstract][Hide abstract] ABSTRACT: To characterise the temporal evolution of antibodies to infliximab (ATI).
Prospective observational study of infliximab-treated patients with inflammatory bowel disease between 2009 and 2012.
Trough levels of infliximab and ATI were measured before each infusion by anti-λ ELISA. Patients were monitored for disease activity by clinical activity indexes and for dose-intensification or infliximab cessation. The occurrence of transient ATI disappearing spontaneously without intervention was recorded separately.
125 patients were included (98 Crohn's disease, 27 ulcerative colitis, median follow-up 11.5±22 months) and 1119 sera were analysed for infliximab and ATI levels. Kaplan-Meier analysis showed that 42% of patients remained ATI-free by 4 years of treatment. Most (90%) of the patients who developed ATI did so within the first 12 months of therapy, whereas transient ATI were detected throughout the duration of infliximab therapy (p<0.001). ATI incidence was similar between patients who received infliximab previously (episodic/interrupted therapy patients, n=14) and scheduled-therapy patients (n=111). In the scheduled group, combination immunomodulator+infliximab resulted in longer ATI-free survival compared with monotherapy (p=0.003, logrank test). Survival free of clinical loss of response was enjoyed by 51% of patients, and serial measurements showed that ATI development often preceded the onset of clinical flare.
When followed prospectively, most patients who develop ATI do so within the first 12 months of therapy. This incidence is reduced by concomitant immunomodulator even in scheduled-therapy patients. In contrast, transient ATI, which are of little clinical significance, can appear haphazardly at any time during treatment. The onset of clinical loss of response may lag behind the appearance of anti-infliximab antibodies.
Gut 09/2013; 63(8). DOI:10.1136/gutjnl-2013-305259 · 14.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are few therapeutic options for patients with inflammatory bowel disease who lose response to infliximab because they produced antibodies against the drug. We performed a retrospective analysis to investigate whether administration of immune modulators to 5 patients who developed antibodies to infliximab (ATI) restored response to this drug; 3 patients were given azathioprine/6MP and 2 were given methotrexate. Concentrations of infliximab and ATIs, and anti-tumor necrosis factor (TNF) activity, were analyzed using ELISA-based competition assays of serum samples collected before and after patients were given the immunomodulator. In all patients, levels of ATIs gradually decreased and trough levels of infliximab increased; clinical responses were restored to all patients. In competition assays, immunomodulator-induced elimination of ATIs was associated with increased anti-TNF activity in serum. The addition of immunomodulators to therapy might be helpful to patients that have lost response to anti-TNF agents due to formation of anti-drug antibodies.
Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 10/2012; 11(4). DOI:10.1016/j.cgh.2012.10.020 · 7.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anti-infliximab antibodies (ATIs) are associated with lower serum infliximab (IFX) trough levels and diminished clinical response. The current most prevalent method for detection of ATI is a double-antigen (DA) enzyme-linked immunosorbent assay (ELISA) utilizing IFX for ligand and detection antibody. Serum IFX interferes with ATI measurement in this method. An alternative ELISA using antihuman lambda chain (AHLC) antibody for ATI detection may be less amenable to this interference. The aim of our study was to compare the performance of AHLC-ATI versus DA-ATI for prediction of clinical response and evaluate the clinical significance of positive ATI in the presence of detectable IFX levels in IFX-treated inflammatory bowel disease (IBD) patients.
In all, 63 patients' sera were analyzed for IFX levels and antibody levels by AHLC and DA. The results were compared with the clinical response to IFX. Percentage of patients with IFX+ATI+ status among IFX-treated patients and the clinical outcome of IFX+ATI+ patients were assessed.
ATIs were demonstrated in 22/63 (34.9%) and 18/63 (28.5%) sera of patients by AHLC and DA assay, respectively (P = 0.6). Detectable ATI and in IFX was detected in four patients (6.3%) by AHLC but not by DA assay. IFX+ATI+ status was documented in 8.7% of available sera and was associated with a trend for loss of response.
AHLC and DA ELISA are equally effective for ATI detection in patients with undetectable serum IFX. AHLC ELISA detects ATI in some patients with detectable serum IFX. This IFX+ATI+ status may be a harbinger of evolving loss of response to the drug.
[Show abstract][Hide abstract] ABSTRACT: Anti-drug antibodies can be elicited by infliximab and adalimumab, but the rate of their decay after therapy is stopped is unknown.
To investigate the decline of anti-drug antibody titre after anti-TNF cessation, and to evaluate the clinical utility of anti-drug antibody measurement before anti-TNF re-induction.
Inflammatory bowel disease (IBD) patients who stopped anti-TNF therapy and had measurable anti-drug antibodies were prospectively followed up by serial blood measurements of antibodies levels. The clinical outcome of a second cohort of patients who received re-induction by infliximab or adalimumab after a drug holiday >4 months was determined vis-à-vis their anti-drug antibodies status before re-induction.
The first cohort included 22 patients with anti-drug antibodies who were prospectively followed up after cessation of anti-TNF. Sixteen had antibodies-to-infliximab (ATI) and six had antibodies-to-adalimumab (ATA). ATI titres declined within 12 months to below detection levels in 13/16 infliximab-treated patients, whereas ATA titres became undetectable in only 2/6 adalimumab-treated patients (P = 0.04). The second cohort comprised 27 patients who resumed anti-TNFs (24 infliximab, 3 adalimumab). Of these, 3/5 patients with measurable anti-drug antibodies before re-induction experienced severe hypersensitivity reaction and/or nonresponse mandating drug-discontinuation, compared to 11/22 patients who were re-induced without measurable anti-drug antibodies (OR = 1.5, 95% CI 0.2-11, P = 0.7).
Antibodies to infliximab titres decline to undetectable levels within one year of cessation of infliximab in the majority of patients, whereas antibodies to adalimumab seem to persist longer after adalimumab discontinuation. Measuring antibodies to infliximab prior to infliximab re-induction is probably of little clinical utility, especially if more than a 12-month drug-holiday has elapsed.
[Show abstract][Hide abstract] ABSTRACT: Limited data suggest the absence of infliximab in breast milk, thereby implying the safety of this drug during breast-feeding. We aimed to re-evaluate the presence of infliximab in breast milk of nursing IBD patients.
Serum and breast milk were obtained post-partum from 3 breast-feeding patients with Crohn's disease before and after re-initiation of infliximab. ELISA assay was employed to measure infliximab level in maternal serum and in breast milk. The level of infliximab was also measured in breast milk of a control group of 8 nursing healthy mothers.
Infliximab was undetectable in breast milk prior to the first infusion and was also not measurable in 8 lactating women not exposed to infliximab. Infliximab levels in breast milk rose up to 101ng/ml within 2-3days of the infusion. These levels of infliximab in breast milk were roughly 1/200th of the level in blood.
In contrast with prior reports, infliximab can be detected in the breast milk of nursing mothers. The miniscule amounts of infliximab transferred in breast milk are unlikely to result in systemic immune-suppression of the infant. Nonetheless, local effects of this exposure on the neonates' intestine and potential immune sensitization or tolerization towards the drug can not be excluded and merit further investigations.
Journal of Crohn s and Colitis 12/2011; 5(6):555-8. DOI:10.1016/j.crohns.2011.05.006 · 6.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To localise the immunogenic part of infliximab and evaluate the clinical usefulness of measuring antibodies against infliximab fragments.
A specialised inflammatory bowel disease (IBD) centre in a tertiary hospital.
Serum was collected from patients with IBD and controls. Antibodies against whole infliximab (ATI) and against the digested Fc, F(ab')(2) and F(ab') fragments were measured by a specifically developed ELISA and by western blotting. A separate ELISA was used to determine infliximab levels in serum.
109 serum samples from 62 infliximab-treated patients were tested along with 64 control samples. Anti-F(ab')(2) antibodies were found in 28/42 (67%) samples with positive ATI, all from infliximab-exposed patients. Anti-F(ab')(2) antibodies were also present in 26 of the remaining 67 (39%) samples from exposed patients despite absent ATI. No specific anti-Fc antibodies were detected. Low trough infliximab level and high ATI level was found in 10/12 patients (83%) with complete loss of response to infliximab, but in only 5/14 patients (36%, p=0.02) who regained response to intensified infliximab regimen and in 2/24 patients (8%, p<0.001) in maintained remission while on 5 mg/kg/8 week infliximab treatment. Although Anti-F(ab')(2) antibodies showed similar test characteristics to ATI in patients losing response to infliximab, they were also detected in 61% of patients in maintained remission, thereby limiting their clinical usefulness. No cross reactivity to adalimumab was noted.
F(ab')(2) is the immunogenic fragment of infliximab. However, ATI level in serum--combined with measurement of trough infliximab level--is better correlated with the clinical response to infliximab or with its loss.
Gut 01/2011; 60(1):41-8. DOI:10.1136/gut.2009.201533 · 14.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the antiinflammatory effect of lidocaine in intestinal epithelial cells.
HT-29 and T-84 cells were grown in culture with and without TNF-alpha, lidocaine, aconitine and veratridine. The secretion of IL-8 and IP-10 was measured by ELISA. A cDNA microarray was used to assess gene expression. Real-time PCR was used to confirm the results. Western blots and a modified electromobility shift assay (EMSA) were used to assess NFkappaB activation.
Lidocaine inhibited spontaneous and TNF-alpha induced secretion of IL-8 and IP-10. The combination of veratridine or aconitine, voltage-gated sodium channels (VGSC) agonists that open VGSCs, with lidocaine did not alter the effect of lidocaine on cytokine secretion. Gene array analysis revealed that IkappaB transcription was induced by TNF-alpha and inhibited by lidocaine. IkappaB real-time PCR confirmed this observation. A Western blot analysis demonstrated that the degradation of IkappaB following TNF-alpha treatment was markedly inhibited by lidocaine. Lidocaine treatment resulted in decreased generation of phosphorylated IkappaB. A modified EMSA was complementary and demonstrated marked inhibition of NFkappaB nuclear binding.
Lidocaine inhibits IL-8 and IP-10 secretion from intestinal cells. This effect is mediated by inhibition of NFkappaB activation via decreased IkappaB phosphorylation and is not mediated by lidocaine's effect on VGSC.
[Show abstract][Hide abstract] ABSTRACT: The onset of the effect of thiopurines is delayed for several months. The aim of this study was to investigate immune mechanisms for this delay.
The effects of thiopurines on human peripheral blood T cells and on lamina propria lymphocytes were investigated for apoptosis induction by Annexin V/propidium iodide (PI) and for cytokine secretion by intracellular staining and ELISA assays. To investigate the mechanism of the effect of thiopurines in vivo, Balb/C mice were co-immunised with HEL/OVA (hen egg lysozyme/ovalbumin) antigens, and then repeatedly challenged by HEL only, while being treated by mercaptopurine or vehicle alone for either 4 or 20 weeks. The memory response of CD4+ splenocytes towards HEL/OVA was then determined by CFSE (carboxyfluorescein succinimidyl ester) dilution.
Thiopurines arrested the proliferation of stimulated T cells but did not enhance the apoptosis of either resting T cells or activated T cells until day 5 poststimulation. Despite the proliferation arrest, stimulated T cells successfully differentiated into effector cells, as evidenced by their capacity for proinflammatory cytokine secretion, potent adhesion and cytotoxicity. Prolonged mercaptopurine treatment of mice for 20 weeks selectively reduced the CD4+ memory response to a repeatedly encountered HEL antigen, but did not affect the T cell memory pool to the previously presented OVA antigen. A shorter, 4 weeks, treatment with mercaptopurine did not inhibit the memory response to either antigen.
T cells arrested from cycling by thiopurines can still differentiate into potent effector cells capable of propagating the inflammatory process. Thiopurine treatment results in depletion of antigen-specific memory T cells, but this effect is dependent upon repeated encounters with the antigen over a prolonged time course.
Gut 11/2008; 58(3):396-403. DOI:10.1136/gut.2008.157339 · 14.66 Impact Factor