Addition or removal of concomitant methotrexate alters adalimumab effectiveness in rheumatoid arthritis but not psoriatic arthritis.

Objective: Randomized trials have shown that concomitant methotrexate (MTX) augments the effectiveness of tumour necrosis factor (TNF) inhibitors in rheumatoid arthritis (RA), but its benefit in psoriatic arthritis (PsA) has not been demonstrated. The goal of this study was to examine whether the impact of concomitant MTX on therapeutic outcomes in patients with PsA was similar to its effects in RA. Methods: We used data from highly comparable and concurrent observational studies of patients with PsA (N = 1424) or RA (N = 3148) who initiated adalimumab therapy during routine clinical care. The 28-joint Disease Activity Score (DAS28) and patient-reported pain scores were evaluated in patients who received 24 months of continuous treatment with adalimumab monotherapy or adalimumab + MTX and in patients who initiated or stopped concomitant MTX during ongoing adalimumab therapy. Results: Twenty-four months of continuous treatment with adalimumab + MTX was superior to adalimumab monotherapy in RA patients, while no significant difference was observed in patients with PsA. RA patients who added MTX during the study showed significant individual improvements in DAS28 and pain scores at 6 months after the change in therapy, while those who removed MTX had slight increases in disease activity. In contrast, in patients with PsA, neither initiation nor removal of MTX during continuous adalimumab therapy had a significant effect on therapeutic outcomes. Conclusion: Addition of MTX to adalimumab confers further therapeutic benefit in patients with RA, but not in those with PsA, suggesting differences in MTX effects in these two patient populations. Clinicaltrials.gov NCT01078090, NCT01077258, NCT01111240.

Absence of specific alternatively spliced exon of CD44 in macrophages prevents colitis.

CD44 is a transmembrane molecule appearing in numerous isoforms generated by insertions of alternatively spliced variant exons (CD44v) and having various binding partners. CD44v7 on T cells was proposed to promote colitis by preventing T-cell apoptosis. Here we demonstrate that Cd44v7-deficient T cells - like Cd44 wild-type (Cd44WT) T cells - provoked disease in two different colitis models: the model induced by CD4+CD45RBhigh T-cell transfer into Rag2-deficient mice and a new model based on ovalbumin (OVA)-specific T-cell transfer into Rag-sufficient, OVA-challenged mice. In contrast, CD44v7 absence on macrophages in recipient mice prevented colitis. Prevention was associated with the downregulation of signal transducer and activator of transcription 3 (STAT3)-activating and Foxp3-counteracting interleukin-6 (IL-6), lower numbers of phospho-STAT3-containing lymphocytes, and higher Foxp3+ T-cell counts in the colon. Consequently, the protected colons showed lower IL-12, IL-1ß expression, and decreased interferon-γ levels. Importantly, stimulation of T cells by Cd44v7-deficient macrophages induced upregulation of Foxp3 in vitro, while cotransfer of Cd44WT macrophages into Cd44v7-deficient mice reduced Foxp3+ T-cell counts and caused colitis. Accordingly, the CD44v7 ligand osteopontin, whose levels were elevated in Crohn's disease, specifically induced IL-6 in human monocytes, a cytokine also increased in these patients. We suggest macrophage-specific targeting of the CD44v7 pathway as a novel therapeutic option for Crohn's disease.

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria.

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.

Cyclophosphamide pulse therapy followed by azathioprine or methotrexate induces long-term remission in patients with steroid-refractory Crohn's disease.

BACKGROUND: In patients with steroid-refractory Crohn's disease, the therapeutic goal is to achieve both rapid remission and maintenance of clinical response. AIM: To evaluate the long-term benefit in patients treated with cyclophosphamide pulse therapy and azathioprine or methotrexate, a combination shown to be effective in a recent pilot study. METHODS: Sixteen patients with acute steroid-refractory Crohn's disease participated in a prospective open-labelled uncontrolled pilot study between December 1998 and June 2003. All had a median number of 4 monthly pulses of intravenous cyclophosphamide (750 mg) and were followed until relapse of the disease. RESULTS: Thirteen of 16 patients (81%) achieved remission within 8 weeks after two pulses of cyclophosphamide in combination with azathioprine or methotrexate, with a Crohn's Disease Activity Index decrease from 294 to 111 (median). Remission sustained for 19 months (median, range: 1-45). Moreover, eight patients with pyoderma gangrenosum and erythema nodosum who responded to cyclophosphamide have maintained their remission for up to 30 months. CONCLUSIONS: In steroid refractory patients with Crohn's disease, cyclophosphamide is highly effective to induce remission. This uncontrolled study indicates that cyclophosphamide-induced remission is long-lasting under standard immunosuppressive therapy.

Critical comment: analyzing the effect of novel therapies on cytokine expression in inflammatory bowel disease: do cytokine levels reflect clinical response?

Over the past several years, research in the field of cytokine production and function has become indispensable to understand the immunopathology of chronic intestinal inflammation. Thereupon, clinical studies analyzing cytokine production have generated a tremendous amount of data. In patients with inflammatory bowel disease, several studies examined pro-inflammatory cytokines in gut tissue and plasma, but a clear interpretation of the results with respect to disease activity or therapeutic response has been hampered by patient- and sample-related pitfalls.

Short-term treatment with anti-CD44v7 antibody, but not CD44v4, restores the gut mucosa in established chronic dextran sulphate sodium (DSS)-induced colitis in mice.

Increased expression of CD44 variant isoforms have been shown on the inflammatory infiltrates in human and mouse colitis and blockade or deletion of CD44 isoforms inhibit experimental colitis. The objective of this study was to find out if short-term treatment of CD44 antibodies specific to CD44v7, but not to other variant isoforms, suppresses leucocyte-endothelial interaction in chronic dextran sodium sulphate (DSS)-induced colitis in mice. Chronic colitis was induced by oral administration of four cycles of 5% DSS in BALB/c mice. Expression of CD44 was investigated on isolated mononuclear cells of the gut immune system. In established colitis, mice were treated with antibodies against CD44v7 or CD44v4 three times in 7 days. Intravital microscopy was used to study leucocyte-endothelial interactions and leucocyte extravasation. As a marker of inflammatory infiltrates myeloperoxidase was quantified in gut tissue. CD44-induced apoptosis was determined by fluorescence staining of hypodiploidic cell nuclei. In chronic DSS-induced colitis both CD44 variant isoforms, v4 and v7 were significantly up-regulated on mononuclear cells. However, whereas anti-CD44v7 antibody treatment induced a marked restoration of the gut mucosa and significantly reduced endothelial sticking and extravasation of circulating leucocyte in vivo (P < 0.01), application of anti-CD44v4 or an isotype control antibody had no anti-inflammatory effect. A significant reduction of myeloperoxidase activity was detected after blockade of CD44v7, but not v4. Short-term treatment with anti-CD44v7 antibody blocks T cell extravasation and recruitment to the intestinal mucosa and cures established experimental colitis.

An interleukin 12 p40-IgG2b fusion protein abrogates T cell mediated inflammation: anti-inflammatory activity in Crohn's disease and experimental colitis in vivo.

BACKGROUND AND AIMS: Interleukin-12 (IL-12), a p35/p40 heterodimer, plays a pivotal role in the immune response in Crohn's disease (CD). Since IL-12 p40 dimers act as IL-12 antagonists, we assayed p40 dimer proteins to modulate chronic intestinal inflammation. METHODS: We generated a fusion protein consisting of the IL-12(p40) subunit fused to the constant region of IgG2b. IL-12(p40)-IgG2b was tested in a murine 2,4,6,-trinitrobenzene sulphonic acid (TNBS) colitis model and in lamina propria mononuclear cells (LPMNC) from patients with CD in vitro. RESULTS: Dimeric IL-12(p40)-IgG2b fusion protein bound specifically to the IL-12 receptor. In concentrations <10(-7) M, it acted as an IL-12 antagonist as it inhibited interferon gamma (IFN-gamma) secretion, suppressed proliferation, and increased apoptosis of LPMNC from patients with CD. However, in concentrations >10(-6) M, IL-12(p40)-IgG2b increased IFN-gamma secretion and lymphocyte proliferation thereby acting as an IL-12 agonist. In TNBS colitic mice, IL-12(p40)-IgG2b decreased mortality (10% v 68%), prevented body weight loss, reduced tumour necrosis factor alpha, and increased IL-10 secretion. CONCLUSIONS: The IL-12(p40)-IgG2b fusion protein has dichotomic properties as a specific IL-12 antagonist and selective repressor of mucosal inflammation at low concentration and as an IL-12 agonist at high concentration.

Downregulation of CD44v6 in colorectal carcinomas is associated with hypermethylation of the CD44 promoter region.

Overexpression of the cell adhesion protein CD44v6 has been demonstrated in colorectal cancer and other gastrointestinal tumors. While CD44v6 is upregulated in benign colorectal adenomas and well-differentiated colorectal cancer tissues, downregulation frequently occurs during disease progression. The mechanism of downregulation, however, is unknown. Therefore, we evaluated the methylation status of the CD44 promoter as a mechanism for decreased CD44v6 expression in advanced colorectal carcinomas. We demonstrated by methylation-sensitive restriction enzyme digestion that the CpG islands of the CD44 promoter were methylated in 6/21 (28%) of benign colorectal adenomas. Interestingly, in colorectal carcinomas the frequency of promoter methylation was significantly increased (10/19; 53%) compared to 7/21 (33%) in the corresponding normal mucosa. Methylation seems to be associated with a more advanced cancer stage, but the trend did not reach statistical significance. In colorectal carcinomas with CD44 promoter methylation CD44v6 mRNA was detected by reverse transcription-polymerase chain reaction in 3/10 carcinomas, whereas in tumors without CD44 promoter methylation CD44v6 expression was observed in 8/9 (P

The gut as an organ of immunology.

BACKGROUND: In normal conditions human gut mucosa is infiltrated with a large number of mononuclear cells due to continuous stimulation by luminal antigens. This state of "physiological" inflammation is tightly controlled, as several mucosal cells interact to maintain an appropriate local immune response. Moreover, gut-associated lymphoid tissue must constantly distinguish harmless antigens that are present in food and on commensal bacteria from pathogenic microbes. INTERVENTIONS AND RESEARCH: The oral administration of soluble protein antigens induces a state of systemic immunological unresponsiveness specific to the fed protein, termed oral tolerance. The two major mechanisms to explain oral tolerance are anergy/deletion of autoreactive lymphocytes and active suppression. Changes in the pathways of immune activation are detected in chronic intestinal inflammation, such as inflammatory bowel disease or celiac disease. CONCLUSION: An appreciation of the current knowledge of the gut immune system is of importance for understanding and development of new treatment modalities in chronic intestinal inflammation.

Safety and efficacy of intravenous pulse cyclophosphamide in acute steroid refractory inflammatory bowel disease.

BACKGROUND AND AIMS: One major problem in the management of steroid refractory attacks of patients with inflammatory bowel disease (IBD) is the establishment of a rapidly acting immunosuppressive regimen. Based on its well known efficacy in systemic vasculitis, intravenous cyclophosphamide pulse therapy was used in refractory IBD patients to evaluate both its efficacy and safety. METHODS: Between December 1998 and May 2001, seven patients (Crohn's disease, n=5; indeterminate colitis, n=1) with severe steroid refractory IBD (Crohn's disease activity index (CDAI) 264-479 points) received 4-6 cycles of monthly treatment with intravenous cyclophosphamide (750 mg) in a prospective uncontrolled pilot study. RESULTS: All patients improved after two intravenous pulses of cyclophosphamide and six of seven patients achieved complete remission (CDAI <150 points). One patient with Crohn's disease of the small and large bowel showed an impressive clinical response but did not enter into remission. Tapering to low dose steroids was possible in all responders. Remission was maintained in all patients for 18 months (median) but required a second course of intravenous pulse cyclophosphamide in one patient. The drug was well tolerated except for two episodes of candida oesophagitis. CONCLUSIONS: Intravenous pulse cyclophosphamide may be a safe and effective treatment in patients with severe IBD unresponsive to steroid treatment and merits evaluation in a controlled trial.

Increased expression of interleukin-12 receptor beta(2) on lamina propria mononuclear cells of patients with active Crohn's disease.

BACKGROUND AND AIMS: Since interleukin-12 is pathogenetically involved in Crohn's disease (CD) but not in ulcerative colitis (UC), expression and mechanisms of induction of interleukin-12 receptor (IL-12R) subunits beta(1) and beta(2) were analyzed in lamina propria mononuclear cells (LPMNC) of patients with CD and UC. PATIENTS AND METHODS: LPMNC from patients with CD ( n=17), UC ( n=14), and controls ( n=19) were isolated by standard techniques. IL-12R beta(1) and IL-12R beta(2) transcripts were semiquantified by RT-PCR, and expression of IL-12R beta(2) chain was characterized by flow cytometry. LPMNC were activated by cross-linking with anti-CD3 antibodies and B7-1 costimulation. RESULTS: IL-12R beta(1) and IL-12R beta(2) transcript concentrations were higher in inflamed specimens than in noninflamed segments of patients with CD but not in UC. Increased percentage of mucosal CD4(+)/IL-12R beta(2)(+) cells was observed in active CD, but not UC. In vitro stimulation of LPMNC with anti-CD3 antibodies resulted in an increase in IL-12R beta(1) transcripts irrespective of B7-1 mediated costimulation (84% and 95%, respectively). However, increased expression of IL-12R beta(2) mRNA (110%) was detected only after B7-1 costimulation. CONCLUSION: Our data indicate that increased mucosal expression of IL-12R beta(2) on LPMNC in CD but not in UC may be the result of B7-1 costimulation. Modulation or inhibition of IL-12R beta(2) expression on LPMNC could provide a selective therapeutic approach in CD.

The in vitro anti-inflammatory effects of recombinant anti-CD25 immunotoxin on lamina propria T cells of patients with inflammatory bowel disease are not sufficient to cure experimental colitis in mice.

BACKGROUND AND AIMS: In chronic inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis an aberrant mucosal immune regulation is observed accompanied by upregulation of proinflammatory cytokines. Lamina propria T cells of inflamed mucosa have an activated phenotype characterized by increased expression of surface markers such as CD25. We therefore determined the anti-inflammatory effect of a recombinant immunotoxin consisting of an anti-CD25 single chain variable fragment (scFv) fused to a deletion mutant of Pseudomonas exotoxin A [RFT5(scFv)ETA'] on isolated lamina propria lymphocytes of patients with IBD and in the murine model of trinitrobenzene sulfonic acid (TNBS) induced colitis. PATIENTS AND/METHODS: Lamina propria lymphocytes of 25 patients with IBD and 19 control patients were stimulated in absence or presence of RFT5(scFv)ETA'. Interferon-gamma production was determined in the supernatant by ELISA and the induction of apoptosis by flow cytometry after propidium iodide staining. BALB/c mice received TNBS intrarectally and were treated with RFT5(scFv)ETA'. RESULTS: In vitro the administration of RFT5(scFv)ETA' significantly reduced interferon-gamma production and increased apoptosis in lamina propria lymphocytes isolated of inflamed mucosa. However, this contrainflammatory regulation did not result in gain of weight or increased life span in experimental colitis in vivo. CONCLUSION: In addition to the downregulation of the proinflammatory cytokine in vitro, RFT5(scFv)ETA' induced neither a direct nor a bystander effect in an in vivo model of colitis. Therefore our data do not support potential therapeutic implications of targeting CD25 by RFT5(scFv)ETA' in chronic IBD.

Expression of CD44v6 has no prognostic value in patients with colorectal cancer.

BACKGROUND AND AIMS: In colorectal cancer patients' mortality is largely influenced by spreading of tumour cells from the primary tumour site and subsequent metastasis formation. CD44 is an adhesion molecule and represents a highly variable family of isoforms. The isoform CD44v6 has been associated with metastatic spread and poor prognosis in animal models and several human cancers. Results of immunohistological studies in primary colorectal cancer are mostly retrospective and contradictory. The aim of our prospective study was to assess the controversial role of CD44v6 as a prognostic factor in colorectal cancer. METHODS: In 93 patients we analysed tumour CD44v6 expression in prospectively sampled stage I-IV colorectal adenocarcinomas using RT-PCR and Southern blotting. The prognostic value of the CD44v6 expression was assessed using univariate and multivariate analysis. RESULTS: CD44v6 expression was found in 47 % of the cases. CD44v6 expression failed to show any association with the clinical or histological variables examined. CD44v6 expression did not correlate with survival in long-term follow-up. The most important prognostic factor in this cohort was tumour stage. CONCLUSIONS: Changes in CD446 expression level do not predict tumour spread or patient survival in colorectal cancer.

Decreased CD44v6 expression in lamina propria lymphocytes of patients with inflammatory bowel disease.

Splice variants of the glycoprotein CD44 are transiently expressed on lymphocytes during T cell activation. Increased expression of CD44v6 on peripheral blood lymphocytes (PBL) of patients with inflammatory bowel disease (IBD) was described recently. The aim of this study was therefore to characterize CD44v6 expression on CD4(+) lamina propria lymphocytes (LPL) of patients with active IBD in comparison to controls. CD44v6 expression on CD4(+) LPL (n = 19) of controls and patients with active IBD (Crohn's disease n = 14, ulcerative colitis n = 15) was analyzed by flow cytometry and compared to that on autologous PBL. Thereby, in vitro regulation of CD44v6 on LPL and PBL via CD3 and CD2 and the costimulatory signal B7-1 was examined. In addition, the role of protein kinase C (PKC) in CD44v6 expression was tested. CD44v6 expression was increased in CD4(+) LPL (median, 45%) compared to PBL (median, 38%). Surprisingly, in IBD CD44v6 was downregulated on CD4(+) lamina propria T cells, irrespective of their state of inflammation (median, 28%). CD44v6 expression on LPL was not upregulated upon CD3 activation alone but following costimulation with B7-1. However, CD2-mediated T cell activation sufficiently induced upregulation of CD44v6 on LPL and PBL. In our study, downregulation of CD44v6 on LPL of patients with IBD was not due to defective PKC activation. Taken together, these data indicate that decreased CD44v6 expression on LPL in IBD might be a feature of an inappropriate costimulatory signal in T cell activation.

Human biliary mucin binds to E-selectin: a possible role in modulation of inflammation.

E-selectin, expressed on endothelial cells, mediates adhesion of leukocytes and tumor cells to endothelium. CA19-9 (sialyl-Lewis(a)) and sialyl-Lewis(x) are specific ligands for E-selectin. We have recently shown that mucin-rich culture media from human gallbladder epithelial cells contains CA19-9. In this study, we have tested whether human biliary mucin binds to E-selectin. The ability of mucins to inhibit the adhesion of HL-60 cells to immobilized E-selectin was taken as an index for E-selectin binding. Gallbladder bile, hepatic bile, and culture medium from human gallbladder epithelial cells completely inhibited the adhesion of HL-60 cells to E-selectin. The mucin-rich fractions of human bile exhibited strong inhibition, whereas mucin-free fractions had little effect. In contrast to human bile samples, CA19-9-free medium from cultured dog gallbladder epithelial cells failed to inhibit HL-60 binding. Furthermore, after CA19-9 immunoaffinity chromatography, which selectively extracted CA19-9 from bile, bile samples showed poor inhibition of HL-60 adhesion to immobilized E-selectin. A good correlation was observed between E-selectin binding and CA 19-9 concentrations in bile. Our results show that human bile has E-selectin binding activity that is mediated by the CA19-9 side chain of biliary mucin.

Activation of beta(1) integrins mediates proliferation and inhibits apoptosis of intestinal CD4-positive lymphocytes.

A characteristic of lamina propria lymphocytes (LPL) is their low proliferative response to stimuli of the CD3 pathway. beta(1) integrins were expressed on LPL; however, their function is unknown. Therefore, we determined whether beta(1) integrins contribute to T cell responses by providing costimulatory signals. Integrins on CD4(+) LPL of controls and patients with inflammatory bowel disease were characterized by flow cytometry. Cells were stimulated by anti-CD3 or anti-CD2 antibodies either alone or in combination with a stimulatory beta(1) integrin antibody (12G10). Proliferation and apoptosis were measured by [(3)H]thymidine pulsing or flow cytometry. Cytokine mRNA and apoptosis-related transcripts were quantified by reverse transcriptase-PCR. We demonstrated that beta(1) integrin costimulation restored CD3-induced proliferation of CD4(+) LPL and reduced activation-induced apoptosis. Activation of beta(1) integrins by addition of 12G10 antibody to CD3-stimulated cells restored their capacity to express proinflammatory cytokine transcripts. Further, expression of the activated form of beta(1) integrins was significantly elevated on LPL from inflamed mucosa. These studies demonstrate that beta(1) integrin costimulation modulates the response of LPL after TCR stimulation. An increased expression of activated beta(1) integrins on LPL in intestinal inflammation may abolish their unresponsiveness to antigens and perpetuate the inflammatory process.

Stage-specific alternative cplicing of CD44 and alpha 6 beta 1 integrin in colorectal tumorigenesis.

Recent reports suggest that cancerogenesis induces changes in alternative processing of human genes. However, little is known about the regulation of alternative splicing during malignant transformation. Therefore, we examined changes in alternative splicing of two different adhesion molecules, alpha 6 beta 1 integrin and CD44, in multiple stages of colon tumorigenesis. Using semiquantitative RT-PCR it is shown that the alternatively spliced isoforms of both adhesion molecules, alpha 6A and -B and CD44v6, are significantly upregulated in colorectal adenoma (n = 20) compared to normal colon mucosa (n = 32) (P < 0.01). Although beta1 isoforms were expressed in almost all tissues, there was a significant increase in the intensity of gene expression of beta 1A compared to beta 1B (P <0.05) in adenoma tissue. Interestingly, CD44v6 and alpha 6 variant isoforms were downregulated in carcinoma tissue (n = 28) compared to adenoma. These results establish a link between neoplastic transformation and alternative splicing of cell adhesion molecules. Furthermore, these data suggest that colon epithelial cells carrying splice variants of adhesion molecules might acquire a selective growth advantage during early tumorigenesis.