It has been repeatedly shown that a subset of CD4+ T cells that constitutively express CD25 on their surface plays a role in the maintenance of self-tolerance. They may directly or indirectly affect the development of autoimmunity in susceptible mice and humans. In this study, we examine the relationship between the percentage of peripheral CD4+CD25+ T cells and the state of disease in spontaneous models of autoimmune disease. We found that both BWF1 and SNF1 mice that spontaneously develop a lupus-like disease have inherently lower percentage of the CD4+CD25+ T cells in their CD4 repertoire compared with normal Balb/c and DBA/1 mice. The percentage of CD4+CD25+ T cells was found to be increased in both normal and lupus-prone mice as they reached 7 to 8 months of age. However, mice with an autoimmune background differed from mice on a normal background in that the number of CD4+CD25+ T cells never reached 5% of the CD4 population. The lower number of the CD4+CD25+ T cells in autoimmune mice was restored to the level seen in normal mice following administration of histone peptide H471 or OVA(323-339) peptide in the absence of adjuvant intranasally but not intradermally. As such transmucosal treatment may ameliorate disease, we conclude that a deficiency in the CD4+CD25+ T cell pool contributes to a susceptibility to develop spontaneous lupus disease.

Further mapping of the mouse T-cell factor specific for poly(Try,Glu)-polyD-LAla--polyLys is reported. It is shown to be a product of the I-A subregion of the H-2 complex by the use of antisera either raised specifically against or made specific, by absorption, for different regions of the H-2 complex. The factor cooperates across allogeneic barriers, e.g., when factor produced by one strian is combined with bone marrow cells of other H-2 incompatible strains.

Aims: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. Methods and Results: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient -MSC 96%, InterQuartile Range -IQR 3AE0%, n ¼ 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n ¼ 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2AE8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3AE3% n ¼ 6). Although all patients (n ¼ 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. Conclusions: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. Significance and Impact of the Study: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.

Objective Previous randomized controlled trials for treatment of rheumatoid arthritis (RA) with acid‐soluble chicken and bovine type II collagen (CII) have produced conflicting results. This randomized, double‐blind, controlled trial examined the therapeutic effect of bovine CII tablets in RA. Methods CII tablets were prepared by adsorption onto a lactose base. Patients with a duration of RA of ≥2 years and who had failed treatment with at least 1 slow‐acting drug were recruited, provided that they had active arthritis. Patients were randomly assigned to receive either 0.05 mg, 0.5 mg, or 5 mg of CII or placebo daily for 6 months. All slow‐acting drugs were stopped at least 4 weeks before starting CII, although prednisolone was permitted at dosages <10 mg/day. Clinical assessments were performed at screening and at 0, 1, 4, 8, 12, 16, 20, and 24 weeks of treatment. Results Fifty‐five patients were recruited. Initially, there were no significant differences in mean Disease Activity Scores between groups. At 24 weeks, there was a significant difference (P = 0.041, by Kruskal‐Wallis analysis of variance); the major components of this difference were attributable to relatively large decreases in the 0.5 mg CII group (19% of initial values) and to minimal decreases in patients receiving placebo (3% of initial values). Twenty patients had American College of Rheumatology 20% responses; 11 of these were in the 0.5 mg CII group and 3 were in each of the other groups, a significant difference (χ2 = 14.6, P = 0.002). There was no significant difference in any clinical measure between the placebo, 0.05 mg CII, and 5 mg CII groups. There were no side effects associated with CII treatment. Conclusion Treatment with 0.5 mg/day of bovine CII is well tolerated and produces small, but significant, disease improvement in RA. However, the therapeutic window is narrow. The difference between our results and those of other trials may relate to the dose, species, and formulation of the CII.

SUMMARYExtracellular calreticulin (CRT) as well as anti-CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in`epitope spreading' to other autoantigens such as the Ro/SS-A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti-CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjo Ègren's syndrome. Approximately 40% of all SLE patients were positive for anti-CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1±289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N-terminal half of the protein in 69% of the SLE sera from active disease patients, while the Cdomain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P-domains. Sera from both healthy and disease controls and primary Sjo Ègren's syndrome patients were non-reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N-terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.