Indoleamine 2,3 dioxygenase contributes to transferable tolerance in rat red blood cell inducible model of experimental autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is caused by autoantibodies against red blood cell (RBC) surface antigens that render RBC susceptible to Fc-mediated phagocytosis and complement-mediated lysis. Experimental AIHA can be induced by injection of rat RBC to naive mice, but a lymphocyte-mediated regulatory mechanism eventually suppresses the production of autoantibodies specific for mouse RBC. Critically, this tolerogenic response can be transferred to naive mice by splenocytes from the rat RBC-immunized mouse. Here we investigate whether indoleamine 2,3 dioxygenase (IDO) or the initiators of IDO cascade, including the cytotoxic T lymphocyte antigen (CTLA)-4 receptor and its soluble isoform, contribute to this tolerogenic mechanism. Splenocytes from experimental AIHA mice were transferred adoptively to naive mice under the cover of anti-CTLA-4, anti-soluble CTLA-4 antibodies or IDO inhibitor 1-methyl tryptophan (1-MT). Recipient mice were immunized with rat RBC and levels of antibody against self-RBC and rat-RBC were monitored. Our results indicate that transfer of tolerance to naive recipients is dependent upon IDO-mediated immunosuppression, as mice receiving previously tolerized splenocytes under the cover of 1-MT were refractory to tolerance and developed haemolytic disease upon further challenge with rat RBC. Initiators of IDO activity, CTLA-4 or soluble CTLA-4 did not mediate this tolerogenic process but, on their blockade, boosted antigen-specific effector immune responses.

Identification of candidate endothelial cell autoantigens in systemic lupus erythematosus using a molecular cloning strategy: a role for ribosomal P protein P0 as an endothelial cell autoantigen.

OBJECTIVE: To attempt to characterize the diversity and nature of antigens recognized by anti-endothelial cell antibodies (AECA) in patients with systemic lupus erythematosus (SLE) using a molecular cloning strategy. METHODS: AECA in sera of 15 SLE patients were measured by ELISA and Western blot analysis was used to examine the diversity of autoantigen targets in two clinically active patients. A human umbilical vein endothelial cell cDNA expression library was immunoscreened with sera from these two patients to identify their autoantigen targets. An anti-ribosomal P peptide antibody ELISA was used to assess the clinical significance of anti-ribosomal P protein antibodies in the sera of one patient. RESULTS: Significantly higher AECA levels were found in five patients with active disease and nephritis than in five patients with clinically inactive disease. Sera from two clinically active patients were found to recognize distinct spectra of autoantigens. The candidate autoantigens that were identified included (1) endothelial cell-specific plasminogen activator inhibitor; (2) the classical lupus antigen, i.e. ribosomal P protein P0; and (3) proteins never before described as putative autoantigens in SLE, including ribosomal protein L6, elongation factor 1alpha, adenyl cyclase-associated protein, DNA replication licensing factor, profilin II and the novel proteins HEAPLA 1 and HEAPLA 2 (human endothelial associated putative lupus autoantigens 1 and 2). In one patient, antibodies against ribosomal P protein P0 were predominant and levels of these antibodies correlated with total AECA levels, anti-DNA antibody titres, overall clinical score and renal disease in a longitudinal study. CONCLUSIONS: A panel of candidate endothelial autoantigens in SLE, which includes previously described autoantigens and novel targets, has been identified by a molecular cloning strategy. This novel molecular approach could also be applied to the identification of autoantigens in other autoimmune vascular diseases.

Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization.

Extracellular 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 Sjögren'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 C-domain 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 Sjögren'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.

Immunogenic properties of an anti-DNA antibody-derived peptide, 88H.64-80: location of a dominant idiotope defined by T and B cells.

Immunization of normal (BALB/cxNZW)F1 H-2(dxu) mice with peptide 88H. 64-80 derived from the framework (FR) 3 VH region sequence of anti-DNA mAb, V-88, induces the production of IgG anti-peptide antibodies which cross-react specifically with the parent mAb. However, immunization of these normal mice with peptide 88H.64-80 sometimes provokes increased production of anti-dsDNA antibodies. A set of alanine substitute homologues of peptide 88H.64-80 were made to identify the amino acid residues that contribute to the antigenic status of the peptide. Peptide 88H.64-80 contained an antibody epitope at the carboxyl terminus of the peptide, while substitution of particular residues throughout the peptide had a significant inhibitory effect on T cell stimulation. Finally, subclass analysis of IgG anti-88H.64-80 peptide antibodies revealed a close correlation between the production of IgG2a anti-peptide antibodies (associated with a TH1 T cell response) and the production of IgG anti-dsDNA antibodies, but there was no correlation with any other antibody subclass. Despite the ability of peptide 88H.64-80 to provoke both the production of anti-dsDNA antibodies as well as anti-V region antibodies, the sequence of this peptide differs by only one amino acid residue from a number of murine germline gene-encoded homologues. Peptide 88H.64-80 probably represents an epitope whose appearance correlates with the level of expression of the VH genes that carry its sequence, and as such is characteristic of cross-reactive idiotypes associated with pathology.

Molecular modeling of an anti-DNA autoantibody (V-88) and mapping of its V region epitopes recognized by heterologous and autoimmune antibodies.

Anti-DNA autoantibodies are a characteristic feature of human systemic lupus erythematosus (SLE) and lupus diseases in the mouse. V-88 is an IgG1/kappa ssDNA-binding Ab, derived from a lupus mouse, that bears a cross-species, cross-reactive Id (CRI) that has been implicated in the pathogenesis of both human and murine disease. A linear epitope map of V-88 has been determined with anti-idiotypic antisera obtained from rabbits, and candidate sequences for the idiotopes of the CRI have been proposed. We now report the modeling of the three-dimensional structure of the V regions of Ab V-88, to map the location of these idiotopes. The V region framework structure was derived from those of crystallographically determined Ab structures, and the complementarity determining region (CDR) structures were based upon the set of canonical structures adopted by these loop regions in Abs of known structure. One of the idiotopes is an extensive, highly accessible epitope consisting of framework regions spatially adjacent to CDR2 in the heavy chain. Epitopes recognized by an anti-idiotypic rabbit antiserum were compared with those recognized by autoimmune sera from SLE-prone mice, and common features were identified. By analogy with the crystal structure of an anti-DNA Ab BV04-01 complexed with a trinucleotide, the modeled structure also suggests a mode of binding of ssDNA to V-88. The location of the candidate CRI, although within the framework region of VH, is such that it could influence Ag specificity.

Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation.

Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.

Natural antibodies that react with V-region peptide epitopes of DNA-binding antibodies are made by mice with systemic lupus erythematosus as disease develops.

Cross-reactive idiotypes (CRI) have been detected on anti-DNA autoantibodies associated with lesions typical of systemic lupus erythematosus. In order to analyse the antigenic make up of idiotypes on anti-DNA monoclonal antibodies (mAb) V-88 (IgG1 kappa) and F-423 (IgG3 kappa), derived respectively from an adult (NZB x NZW)F1 and a fetal MRL/Mp-lpr/lpr mouse, a set of overlapping hexapeptides representing the VH and VL regions of mAb V-88 and F-423 were synthesized and reacted with a range of sera in pepscan enzyme-linked immunosorbent assays (ELISA) taken from normal and lupus mouse strains. Serum pools were collected both from normal BALB/c and lupus MRL/Mp-lpr/lpr and (NZB x NZW)F1 mice at 10, 20 and 30 weeks of age and analysed for the presence of spontaneously produced anti-V-region peptide IgM and IgG antibodies. IgM antibodies from both the lupus mice reacted with the same V-region epitopes, and although some epitopes mapped to similar locations in the two mAb, the maps for V-88 and F-423 were not identical. In MRL/Mp-lpr/lpr mice, as lupus disease progressed there was a switch from IgM antibodies to IgG anti-peptide antibodies whose specificity for the peptide antigens coincided with but was better defined than that of the IgM antibodies. The identified idiotopes were located in both complementary determining regions (CDR) and framework region (FR) regions, indicating that some contribute to CRI shared by other related antibodies, while others were unique to either mAb V-88 or F-423. In conclusion, we have dissected and identified a mosaic of antibody V-region idiotopes that contribute to the idiotype of an anti-DNA autoantibody and against which autoantibodies are made naturally in lupus disease.

Mucosal tolerance and suppression of collagen-induced arthritis (CIA) induced by nasal inhalation of synthetic peptide 184-198 of bovine type II collagen (CII) expressing a dominant T cell epitope.

The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RTlu haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184-198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine-specific, but cross-reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence was also autoantigenic. Use of independently synthesized and purified 184-198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA: this was found to delay the onset of disease, reduce mean disease severity, shift the anti-CII antibody response to favour antibodies of the IgG1, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184-198 peptide. The dominant 184-198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre-treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen.

Primary sequence and location of the idiotopes of V-88, a DNA-binding monoclonal autoantibody, determined by idiotope scanning with synthetic peptides on pins.

In this study, the primary sequence and location of the idiotopes of monoclonal antibody (mAb) V-88 have been examined. V-88 was derived from an adult (NZB x NZW)F1 mouse, has been partially defined previously with polyclonal anti-idiotype antisera, and is a member of the 16/6 idiotype (Id) family. From the inferred primary amino acid sequence of the antibody, sets of hexapeptides, overlapping by five residues, were synthesized on pins and used to scan the expression of epitopes (idiotopes) in the V regions of the light and heavy chains. A heterologous rabbit antiserum raised against the native antibody V-88, and absorbed to make it idiotype specific, was found to react with eight major epitopes distributed between the VH and VL regions. Half of these determinants mapped to the complementarity determining regions, with the others in framework sequences. Thus, the idiotype of antibody V-88 comprises, at least in part, continuous linear idiotopes in both hypervariable and framework areas. The process of absorbing the anti-idiotype antiserum on normal mouse immunoglobulin removed much of the background antibody activity against V region peptides, but left the activity against the dominant idiotopes. The sequence of a major idiotope, VATISG, in the FW2/CDR2 VH region is homologous to sequences of human antibodies that express the 16/6 idiotype, suggesting that Id.16/6 is at least in part defined by this region of the antibody. The same VH area is also homologous to sequences in bacterial and mammalian heat-shock proteins (hsp60-65). Thus there may be a functional link through idiotype connections, especially those involving Id.16/6, between anti-bacterial responses and production of autoantibodies, and some bacterial antigens may function indirectly as superantigens for B cells.

Development of the retinal tapetum lucidum of the walleye (Stizostedion vitreum vitreum).

The development of the retinal tapetum lucidum within the cells of the retinal pigment epithelium (RPE) has been investigated by both light and electron microscopy in the walleye (Stizostedion vitreum vitreum) in specimens ranging in total length from 25-140 mm. In addition changes in the arrangement of the photoreceptors (both rods and cones) in both light and dark-adaptation have also been studied. At 25 mm no evidence of a tapetum is present. At about 30 mm it makes its initial appearance as granular bodies formed within the apical smooth endoplasmic reticulum (SER) cisternae of the RPE cells in the superior temporal fundus. The developing tapetum then spreads peripherally and continues to thicken in existing areas. By 90 mm it is well established throughout the fundus but always appears better developed in the superior fundus. By 125-140 mm it is essentially adult in appearance. At 60-70 mm the rods and cones begin to form bundles producing macroreceptors of 20-30 photoreceptors. In dark-adaptation the rod bundles are retracted and have one or more cone cells centrally located in each bundle, with the bundles separated from one another by melanosomes. Initially when no tapetal material is present, post-larval walleye are positively phototactic and feed on zooplankton. In the adult condition when a tapetum lucidum and large macroreceptors are present, the walleye is negatively phototactic and feeds almost exclusively on larger organisms such as other fish.

A unique closed abduction-external rotation ankle fracture.

A unique closed abduction-external rotation ankle fracture with a combined medial malleolar fracture and avulsion of the deltoid ligament is presented. Thorough literature search has not revealed a similar case. This report is well documented with an operative photograph, radiographs, and a 20-month followup. A mechanical basis for such an injury pattern is described. The magnitude and complexity of the injuring forces applied to the ankle region in road trauma may lead--as in this presentation--to unusual injury combinations.