Gene polymorphisms and serological markers of patients with active Crohn's disease in a clinical trial of antisense to ICAM-1.

Serological profiles for anti-Saccharomyces cerevisiae antibodies (ASCA)/ perinuclear antineutrophil cytoplasmic antibodies (pANCA) and gene polymorphisms in tumour necrosis factor (TNF)-alpha and intercellular adhesion molecule-1 (ICAM-1) are associated with occurrence and/or outcome in Crohn's disease. The aim of the study was to characterize the ASCA/pANCA profile, soluble ICAM-1 expression and single nucleotide gene polymorphisms (SNPs) in TNF-alpha and ICAM-1 genes. Crohn's patients with moderate disease activity were enrolled in a clinical trial of Alicaforsen (ISIS 2302). Peripheral blood samples were collected prospectively for serum studies and for potential analysis of gene polymorphisms. A multivariate analysis was performed to compare treatment effect with the biomarkers studied. Serological testing for ASCA/pANCA was obtained for 257 patients at baseline: 37% were ASCA(+)/pANCA(-) (Crohn's pattern), 9% had both markers, 15% were ASCA(-)/pANCA(+) and 39% had neither marker. When the data were analysed by multiple regression analysis, a trend was found within the Alicaforsen-treated groups for greater rates of remission in the ASCA(+)/pANCA(-) subgroup versus all other serological profiles (25 versus 14%, P = 0.068), but not versus the placebo remission rate (18.8%). Gene polymorphisms were assessed in 64 patients, 21 from the placebo group. ICAM-1 assessment revealed no over-representation. However, three unique TNF-alpha SNPs were identified that correlated significantly with remission; sites 290 (P = 0.0253), -2735 (P = 0.0317) and -3090 (P = 0.0067). Although the overall clinical trial was negative, we have identified a trend towards clinical remission with Alicaforsen therapy in a subgroup of patients with Crohn's disease expressing ASCA(+)/pANCA(-). Furthermore, we have identified three TNF-alpha SNPs that may also predict a positive therapeutic outcome.

The clinical importance of proton pump inhibitor pharmacokinetics.

Achieving the optimal clinical response for patients with upper gastrointestinal peptic disease is important. This response depends on the pathology treated as well as on the choice of proton pump inhibitor. Here, we identify factors in specific disease therapy and proton pump inhibitor (PPI) pharmacokinetic and pharmacodynamic characteristics that help us achieve this goal. These include differences in PPI bioavailability and acid-suppressive effects. Available data indicate that PPIs appear to have similar potency on a milligram basis, and that omeprazole and lansoprazole are more frequently double dosed than pantoprazole. The lower propensity for double dosing with pantoprazole may also result in lower medication acquisition costs and a reduction in physician visits due to ineffective therapy with the standard dosing of these other agents.

Dose ranging pharmacokinetic trial of high-dose alicaforsen (intercellular adhesion molecule-1 antisense oligodeoxynucleotide) (ISIS 2302) in active Crohn's disease.

BACKGROUND AND AIMS: To evaluate the safety, pharmacokinetics and clinical efficacy of the intercellular adhesion molecule-1 antisense phosphorothioate oligonucleotide alicaforsen (ISIS 2302) at 250-350 mg in Crohn's disease. METHODS: : Patients (> 50 kg) with active Crohn's disease (Crohn's disease activity index > or = 220) were assigned by gender, randomly, to two alicaforsen treatment groups: 300 or 350 mg, infused intravenously three times a week for 4 weeks. All patients weighing 36-50 kg received 250 mg of alicaforsen. Background aminosalicylates, antibiotics, immunosuppressives and corticosteroids were permitted, but tumour necrosis factor-alphainhibitors were prohibited. The primary end-point was clinical remission (Crohn's disease activity index < or = 150). RESULTS: Twenty-two patients were enrolled with a mean baseline Crohn's disease activity index of 304. Steroids were used by 27%, 5-aminosalicylic acid by 68% and immunosuppressives by 27%; 23% had previously received infliximab. Five subjects withdrew after one to three infusions for infusion-related symptoms. Nine patients (41%) experienced clinical remission. Fifty-three per cent of the evaluable subjects receiving more than three infusions experienced remission (18% at week 8; 29% at week 12). The overall response, using a minimum decrease of 70 in the Crohn's disease activity index, was 41-47% for the evaluable group, at weeks 8 and 12. The median duration of remission was 14 weeks. Plasma pharmacokinetic results showed overlapping levels (Cmax, AUC) for the three doses. The infusion-related reaction profile consisted of fever, chills, headache, nausea, emesis or arthralgias, typically occurring 2-4 h after completion of the first infusion. Reactions were less frequent in patients receiving background corticosteroids. The 2-4-h transient post-infusion partial thromboplastin time prolongation values, a class effect of phosphorothioate oligonucleotides, were 18, 21 and 23 s for 250, 300 and 350 mg, respectively. CONCLUSIONS: Alicaforsen (ISIS 2302), at fixed doses of 300 and 350 mg, achieved the desired drug exposure and may be an effective therapy for Crohn's disease. Infusion-related reactions were observed less frequently in patients on corticosteroids, and with decreasing frequency with continued treatment.

Amelioration of chronic and spontaneous intestinal inflammation with an antisense oligonucleotide (ISIS 9125) to intracellular adhesion molecule-1 in the HLA-B27/beta2 microglobulin transgenic rat model.

Adhesion molecules are known to be an important part of leukocyte migration and extravasation in both homeostatic and inflammatory conditions. Intracellular adhesion molecule-1 (ICAM-1 or CD54) is constitutively expressed on endothelial cells and is up-regulated during acute and chronic inflammation. We investigated the efficacy and consequences of interfering with CD54 after administration of an antisense oligonucleotide to ICAM-1 (CD54) in the transgenic HLA-B27/beta2 microglobulin rat model. One hundred percent of the HLA-B27 transgene + animals will spontaneously develop chronic inflammation (some more severely than others) in the gastric mucosa, cecum, and colon. We carried out two studies, i.p. injection and rectal administration of antisense. Following i.p. and rectal treatment, there were significant decreases in colonic mucosal wall thickness, histologic inflammation, CD54 expression in the colon and peripheral blood, and the percentage of colon weight per end body weight. Furthermore, decreased expression of CD49d, CD18, and tumor necrosis factor-alpha was observed in antisense treated rats. Therefore, the HLA-B27 transgenic model of spontaneous and chronic inflammatory bowel disease, which has increased expression of adhesion molecules, responds to both routes of administration of ICAM-1 antisense oligonucleotides. These studies support the regulatory role of adhesion molecules in chronic intestinal inflammation, the need for an understanding of how the route of drug delivery can alter the dose and area affected, and finally the role of antisense oligonucleotides as a therapeutic modality in chronic spontaneous inflammatory bowel diseases.

Double blind, placebo controlled trial of the remission inducing and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn's disease.

BACKGROUND AND AIMS: To evaluate the safety and efficacy of the intercellular adhesion molecule 1 (ICAM-1) antisense phosphorothioate oligonucleotide alicaforsen (ISIS 2302) in Crohn's disease. METHODS: Active (Crohn's disease activity index (CDAI) 200-350), steroid dependent (prednisone 10-40 mg) Crohn's patients were randomised into three treatment groups: placebo versus ISIS 2302 (2 mg/kg intravenously three times a week) for two or four weeks. Patients were treated in months 1 and 3, with steroid withdrawal attempted by week 10. The primary end point (steroid free remission) was a CDAI <150 off steroids at the end of week 14. RESULTS: A total of 299 patients were enrolled, with a mean baseline CDAI of 276 and steroid dose of 23 mg/day. Rates of steroid free remission were equivalent for the two and four week ISIS 2302 groups (20.2% and 21.2%) and the placebo group (18.8%). At week 14, steroid withdrawal was successful in more ISIS 2302 patients compared with placebo treated patients (78% v 64%; p=0.032). Steroid free remission was highly correlated with exposure (p=0.0064). Other clinical responses were correlated with exposure, with significant results versus placebo being observed in the highest area under the curve subgroup. CDAI scores decreased by 136 (112) at week 14 versus 52 (107) for placebo (p=0.027) and inflammatory bowel disease score questionnaire improved by 43 (31) versus 15 (36) for placebo (p=0.027). CONCLUSIONS: Although the primary outcomes failed to demonstrate efficacy, pharmacodynamic modelling suggests that alicaforsen (ISIS 2302) may be an effective therapy for steroid dependent Crohn's disease.

The concept and application of antisense oligonucleotides.

Since the identification of the double-stranded DNA helix by Watson and Crick in 1953, the knowledge of nucleotide structure and function has been an important potential tool in the study and therapy of disease. There is recent clinical evidence that antisense oligonucleotides may be important therapeutic compounds in the clinical therapy of a range of diseases, including infection (viruses and bacteria), oncology, and inflammation. Our laboratory-based understanding of antisense oligonucleotide activity has provided a foundation for their use in several human diseases. Potentially relevant applications include inflammatory bowel disease therapy, psoriasis, transplantation, rheumatoid arthritis, cytomegalovirus retinitis, hepatitis C, and solid tumor therapy. Here we will outline these applications as well as our ongoing clinical trials for Crohn's disease.

Alterations in protein kinase C isoforms in experimentally-induced colitis in the rat.

BACKGROUND: Although protein kinase C (PKC) isoenzymes have been implicated as mediators for multiple physiological processes, PKC also mediates cellular and intestinal mucosal injury. We have investigated the expression of the isoenzymes, PKCalpha, PKCdelta, PKCepsilon and PKCzeta in colonic mucosal tissue from TNBS-treated and HLA-B27 spontaneous colitis animals. METHODS: Colonic mucosal samples were taken at various times (2 h-14 d) after instillation of TNBS (75 mg/kg in 50% ethanol) or from HLA-B27 rats at 16-18 weeks of age. Tissues were homogenized and separated into membrane and cytosolic fractions by centrifugation. PKC activity was measured radioenzymatically. PKC protein for isoforms alpha, delta, epsilon and delta was assessed by Western blot while corresponding mRNA was analyzed by RT-PCR. RESULTS: PKC activity increased in cytosolic and membrane fractions by 1d after TNBS and returned to normal by d3. PKCalpha protein was translocated from cytosol to membrane by 2 h after TNBS followed by down-regulation until d3. Increases in PKCdelta, PKCepsilon and PKCzeta protein occurred initially in membrane fractions as early as 2 h after TNBS. Increases in cytosolic protein occurred at later times after induction of colitis. Protein levels for all isoenzymes remained increased up to 7d after TNBS. RT-PCR revealed that mRNA for PKCalpha decreased while PKC mRNA increased correspondingly with their respective protein levels. In HLA-B27 rats, protein levels for all isoforms were less than was detected in normal colonic tissue. CONCLUSIONS: The early increase in gene expression and protein levels for PKCalpha and zeta suggest that these isozymes may play roles in an acute model of colitis induced by TNBS. In contrast, the increase in PKCdelta and epsilon protein was not associated with mRNA changes suggesting that these isozymes are not similarly regulated in the inflamed colonic mucosa. In a chronic model of experimental colonic inflammation (HLA-B27), all of these isoforms appeared to be down-regulated.

Critical review of acid suppression in nonvariceal, acute, uppergastrointestinal bleeding.

Nonvariceal, upper gastrointestinal (GI) bleeding is a very common source of morbidity and mortality. The concept of ulcer clot dissolution being facilitated by a low gastric pH has allowed us to better understand the pathophysiology of nonvariceal upper GI bleeding. Placebo-controlled trials have shown the benefit of oral proton pump inhibitor administration in contrast to H(2) receptor antagonists. Furthermore, our recent experience with intravenous proton pump inhibitors has reinforced these observations.

Preclinical and clinical pharmacology of antisense oligonucleotides.

Antisense oligonucleotides are short (typically 15-20 bases in length pieces of synthetically manufactured, chemically modified DNA or RNA. They are designed to interact by Watson-Crick base pairing with mRNA transcripts encoding proteins of interest, and by virtue of this interaction inhibit the expression of the protein encoded in the mRNA. Since their first proposed use in 1978, antisense oligonucleotides have become come widely used as tools to modulate gene expression in tissue culture. The great specificity that these compounds exhibited in vitro has also led them to be viewed as potentially therapeutically useful. This interest has resulted in the progression of (to date) a dozen compounds into human clinical trials for a variety of indications ranging from cancer to inflammatory diseases. Here, we will review some of the progress that has been made with antisense pharmacology, both in vitro and in vivo, as well as describe the current status of this class of compound in clinical trials.

Bowel permeability and CD45RO expression on circulating CD20+ B cells in patients with ankylosing spondylitis and their relatives.

OBJECTIVE: Ankylosing spondylitis (AS) is reportedly associated with subclinical endoscopic gut inflammation in up to 57% of patients. Studies of bowel permeability, however, have not consistently revealed abnormalities in these patients. CD20+CD45RO+ expression is associated with increased antigen exposure, and previous work has shown increased expression in this B cell isoform in patients with Crohn's disease and their relatives, correlating with intestinal permeability abnormalities. We sought to re-examine intestinal permeability in patients with AS and their relatives, and relate any observed alterations in permeability with evidence of increased antigen presentation as assessed by the number of circulating B cells that were CD45RO positive. METHODS: We studied small intestinal and gastric permeability by measurement of excretion of lactulose, mannitol, and sucrose in 60 patients with AS and 24 of their first-degree relatives. We also studied expression of CD20+CD45RO+ by flow cytometry in these patients. RESULTS: Both patients and first-degree relatives had significantly increased small intestinal, but not gastric, permeability compared to controls. Among patients, current users of nonsteroidal anti-inflammatory drugs (NSAID) had significantly increased small intestinal permeability compared to nonusers, but relatives not using NSAID also had increased permeability. CD20+CD45RO+ expression was increased in one-third of patients but did not correlate with permeability abnormalities. CONCLUSION: Patients with AS have altered small intestinal, but not gastric, permeability. NSAID use cannot explain all the abnormality. Bowel permeability abnormalities, possibly genetically determined, may antedate development of bowel or joint symptoms. Increased CD20+CD45RO+ expression suggests increased antigen exposure, which may be related to previous or current intestinal permeability abnormalities.

Making sense of antisense.

Since the identification of the DNA double-stranded helix, the gene as a target of therapy and, moreover, the use of DNA as a drug have been possibilities. 'Antisense' is used by some living organisms, specifically viruses, to control gene replication. Only recently, the use of antisense DNA as a mechanism to control human gene translation has been appreciated. A recent report on the use of systemically administered oligonucleotides in human Crohn's disease is reviewed. DNA antisense oligonucleotides offer a technology capable of unique use at the laboratory bench as well as for highly specific therapeutic drugs. The conceptualization and possible future directions of these exciting compounds are reviewed.

A placebo-controlled trial of ICAM-1 antisense oligonucleotide in the treatment of Crohn's disease.

BACKGROUND & AIMS: Intercellular adhesion molecule 1 (ICAM-1) plays an important role in the trafficking and activation of leukocytes and is up-regulated in inflamed mucosa in Crohn's disease. ISIS 2302 is an antisense phosphorothioate oligodeoxynucleotide that inhibits ICAM-1 expression. The aim of this study was to obtain preliminary assessment of tolerability, pharmacology, and efficacy of ISIS 2302 in Crohn's disease. METHODS: Twenty patients with active, steroid-treated Crohn's disease were randomized (3:1, ISIS 2302 to placebo) to receive over 26 days 13 intravenous infusions of ISIS 2302 (0.5, 1, or 2 mg/kg) or saline placebo in a double-blinded study. The patients were followed up for 6 months. RESULTS: At the end of treatment. 47% (7 of 15) of ISIS 2302-treated and 20% (1 of 5) of the placebo-treated patients were in remission (Crohn's Disease Activity Index [CDAI] < 150). At the end of month 6, 5 of these 7 ISIS 2302-treated remitters were still in remission, and a 6th patient had a CDAI of 156. Corticosteroid usage was significantly lower (P = 0.0001) in the ISIS 2302-treated patients. These findings were corroborated by significant increases in beta7 and alpha d bearing CD3+ peripheral blood lymphocytes and by decreases in intestinal mucosal ICAM-1 expression during the treatment period. CONCLUSIONS: ISIS 2302 seems to be a well-tolerated and promising therapy for steroid-treated Crohn's disease.

New biotechnological therapies for Crohn's disease: where are we now?

Recent advances in mucosal immunology have provoked recent interest in the application of biodrugs to Crohn's disease intestinal inflammation. Our understanding of the roles of cytokines, adhesion molecules, cell trafficking and cellular immune mechanisms of disease has lead to a number of recent clinical trials. There has been simultaneous research by a number of groups using several animal models of gut inflammation. Better animal models and in particular, the use of gene knock-outs and transgenics has benefitted our understanding of the inflammatory components of Crohn's. Examples of early cellular biodrugs included anti-CD4 monoclonal antibodies. The recent FDA approval of infliximab was preceded by knowledge that levels of tumour necrosis factor (TNF)-alpha are suppressed in the clinical therapy of Crohn's. A recent study of interleukin (IL)-10 was not as favourably reported. Equivocal data has not supported the use of IL-10 for Crohn's disease therapy at this time. An ongoing multicentre phase III study of antisense to ICAM-1 for Crohn's disease is nearing completion and review. The newer monoclonal antibodies to enter into clinical studies of Crohn's disease therapy include those targeting the adhesion molecules beta7 and alpha4beta7. Numerous other biodrugs are in planning stages or early clinical studies. The coagulation pathway is another target that has been identified. This range of compounds will produce mixed efficacy in clinical studies and winners and losers will result. Moreover, combination therapy using 2 or more of these and other pre-existing compounds may prove clinically beneficial. Tailoring of pharmaceutical use to specific patients may also be expected, as we understand more about the subclasses of Crohn's disease.

Inhibition by rapamycin of P-glycoprotein 170-mediated export from normal lymphocytes.

P-glycoprotein 170 encoded by the MDR-1 gene mediates export of substrates including some immunosuppressive drugs. Rapamycin was compared to cyclosporine A for its ability to inhibit P-glycoprotein on normal human peripheral blood mononuclear cells (PBMC). Rhodamine 123 dye efflux measures P-glycoprotein activity and inhibition of P-glycoprotein results in dye retention. Normal CD4+, CD8+ and B cells include a substantial subset with cyclosporine A-sensitive rhodamine efflux. Rh123 dye efflux is also inhibited by rapamycin at comparable drug levels used in transplant models. CsA is approximately 100-fold more effective on inhibition of PBMC P-gp than is RAPA. P-glycoprotein inhibition of ex vivo lymphocytes with three multi-drug resistant T-cell lines showed susceptibility of P-glycoprotein to rapamycin dependent on the cell type. Compared to cyclosporine A, the reduced ability of rapamycin to inhibit P-glycoprotein reflects a reduced avidity in its binding to P-glycoprotein and perhaps increased access to the cell interior. The increased efficiency of RAPA as an immunosuppressive may in part be a result of its relatively low avidity for P-glycoprotein. The authors speculate that interactions with P-glycoprotein may partially modulate the immunosuppressive effects of rapamycin.

CD45RO expression on circulating CD19+ B cells in Crohn's disease correlates with intestinal permeability.

BACKGROUND/AIMS: Increased intestinal permeability is observed in Crohn's disease and a subset of first-degree relatives. An alteration in isoform expression of the common leukocyte antigen (CD45) is also found in a significant fraction of patients. Because this alteration may be a measure of antigen exposure, the hypothesis of the study was that this alteration would be observed in both patients and relatives of patients with Crohn's disease and that this would correlate with increased intestinal permeability. METHODS: Lactulose and mannitol permeability were defined in healthy controls, patients with Crohn's disease, and their first-degree relatives. Simultaneously, peripheral blood was assayed using flow cytometry for CD45RO expression on CD19+ B cells. RESULTS: A subset of relatives had significantly increased permeability, as did the majority of patients with Crohn's disease. A small fraction of peripheral B cells from controls expressed the CD45 isoform (< 6%). This fraction was significantly increased for patients with Crohn's disease and their relatives. Relatives with no clinical evidence of Crohn's disease were only found to have increased CD45RO expression if they had increased permeability. CONCLUSIONS: Individuals at risk for developing Crohn's disease include a subset with increased intestinal permeability. These people have an associated phenotypic alteration of circulating B cells that is not observed in controls or relatives with normal intestinal permeability.

Crohn's disease, ulcerative colitis, and normal intestinal lymphocytes express integrins in dissimilar patterns.

BACKGROUND/AIMS: The integrin family of adhesion molecules on intestinal lamina propria mononuclear cells (LPMNC) was studied using fluorescence-activated cell cytometry. These molecules are implicated in extravascular cell migration and are important regulators of disease. METHODS: Using fluorescence-activated cell cytometry, B- and T-cell subsets in the intestines of 10 normal patients, 11 patients with Crohn's disease, and 8 patients with ulcerative colitis were stained with monoclonal antibodies to a panel of integrins. RESULTS: Expression of alpha integrins on CD3+ T cells and CD19+ B cells was different in normal and inflammatory bowel disease LPMNC. Ulcerative colitis T cells expressed less beta 1 and alpha 4 and significantly more alpha 2 and alpha 6. There was a difference in alpha 4 and beta 1 expression between LPMNC B cells from Crohn's disease and normal intestines. Sixteen percent of CD19+ LPMNC B cells from Crohn's and 19% of ulcerative colitis LPMNC expressed alpha 2. Crohn's and ulcerative colitis CD19+ LPMNC B cells expressed more alpha 5 integrin than normal specimens. CD3+ T cells and CD19+ B cells expressed alpha 6 only in ulcerative colitis. Ulcerative colitis and Crohn's disease CD19+ LPMNC expressed less alpha 4, consistent with their reciprocal increases of alpha 5 and alpha 2. A difference in beta 7 (Peyer's patch specific) antigen was observed between inflammatory bowel disease and normal LPMNC for both CD3+ and CD19+ LPMNC. CONCLUSIONS: These findings identify the differences of lymphocyte homing capability in inflammatory bowel disease and normal intestine.

Analysis of peripheral blood lymphocyte populations and immune function from children exposed to cyclosporine or to azathioprine in utero.

We have analyzed PBL from 7 children exposed to CsA in utero and 4 children exposed to AZA in utero. Expression of CD3, CD4, CD8, and CD20 were normal for both groups of children; however, significant differences were detected in the expression of CD45RA, CD45R0, and CD29. While CsA-exposed children had higher density of CD45RA, and a higher proportion of CD45RA+R0- T cells, than did unexposed children, those exposed to AZA alone had decreased CD45RA+ and a large increase in CD45RA-R0+ T cells. It appears the exposure to CsA slightly delays T cell development, whereas exposure to AZA, without concomitant exposure to CsA, accelerates development to that of an adult. The effects of CsA abrogated the effects of AZA when both were present during pregnancy. The expression of CD29, the beta 1-integrin, on T cells has been linked to enhanced ability to respond to recall antigens and to home to sites of infection. Among children exposed to CsA, T cells from cord blood and a 5-month-old infant have a normal CD29 profile. However from 1 to 6 years of age the proportion of T cells expressing a high density of CD29 is significantly lower (4-fold) than that of T cells from unexposed children. Because these children have no outward signs of immunodeficiency, we postulate that this low proportion is still sufficient for normal immune responsiveness. The distribution of CD29 on T cells was different for the 3 study groups. Among CsA-exposed children, although the proportion of CD29hi T cells was much reduced, all were CD45RA+, as was also the case for unexposed children. In contrast, among children exposed only to AZA, the majority of CD29hi T cells were CD45R0+. Serological testing indicated that immunoglobulin and complement levels, as well as seroconversion in response to vaccination, were normal among CsA-exposed children, with no detectable autoantibodies to cellular or tissue components, including parietal cells. Unlike T cell development in inbred rodents, the immune system in humans appears to be remarkably resilient, and successfully adapts to the presence of CsA during its early developmental stages. This work suggests that the presence of CsA throughout pregnancy has only a minimal effect on fetal immune development and appears to have less impact on T cells than does exposure to AZA only. We conclude that children exposed to CsA in utero are not likely to be at risk of immunodeficiency or autoimmunity.

Expression of CD45RO on circulating CD19+ B-cells in Crohn's disease.

Crohn's disease is an immunoregulatory disorder of the intestine that can be associated with systemic manifestations. This study analysed B-cell differentiation antigens to identify B-cell subpopulations unique to patients with Crohn's disease. CD45 isoform expression was used as an indicator of B-cell differentiation stage. This work shows that B-cells in blood and gut of patients with Crohn's disease are at an advanced stage of differentiation based on their unusual presentation of transitional (RA+ RO+) and late stage (RO+)CD45 isoforms on lamina propria lymphocytes, whereas normal intestinal lamina propria lymphocytes B-cells express primarily CD45RA. Crohn's disease patients had heightened expression of the CD45RO isoform on CD19+ lamina propria lymphocytes, and was found in a statistically significant proportion of Crohn's peripheral blood mononuclear cells (PBMC) where CD19+ PBMC had an expression pattern affecting an unexpectedly high proportion of these differentiated or late stage CD45RO+ B-cells. The expression of CD45RO varied greatly among CD19+ PBMC from patients with Crohn's disease, so multiple regression analysis was performed between these CD45 isoforms and several clinical parameters. After grouping high and low CD45RO expression on CD19+ B-cells, a significant statistical difference was found between high Crohn's disease activity index (CDAI) and low CDAI Crohn's disease patients respectively.

Activated CD19+ B cell lamina propria lymphocytes in ulcerative colitis.

Although isotype differences in lamina propria lymphocyte (LPL) immunoglobulin production has been recognized between normal, ulcerative colitis (UC) and Crohn's patients, differences in B cell activation between these conditions has not been described. Using flow cytometry, we studied B cell LPL activation using the CD71 (transferrin receptor), CD25 (interleukin-2 receptor) and 4F2 (early B and T cell activation marker) monoclonal antibodies. CD19+ B cells from patients with UC had relatively increased expression of CD71, CD25 and 4F2 compared with patients with Crohn's disease or normal mucosa. This finding corresponds to an increased proportion of transitional (activated) B cells as defined by the co-expression of CD45RA and CD45RO found in UC LPL compared with normal or Crohn's patient LPL. Such data may identify an important link between the cellular state of activation of UC LPL B cells and the differences in immunoglobulin production between the inflammatory bowel diseases and normal intestine. Such findings may have further diagnostic or pathophysiologic importance in the study of these diseases. This work also provides further support for the CD45 transition of CD19 B cells from the high to low molecular weight isoform.