Epitope analysis of the Goodpasture antigen using a resonant mirror biosensor

We have used a new technique for studying molecular interactions—a resonant mirror biosensor—to identify B cell epitopes within the Goodpasture antigen, which has recently been identified as the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1). Recombinant antigen (r-α3) was immobilized onto the sensing surface of a sample cuvette, and the binding of patients’ autoantibodies or a MoAb to the Goodpasture antigen was followed in real time. All patients’ sera bound r-α3 in this system, while control sera did not bind. A MoAb inhibited the binding of all patients’ autoantibodies to r-α3, from 27% to 90% (mean inhibition 60%), and patients’ sera cross-inhibited the binding of each other to the antigen. Binding was inhibited by pre-incubation of autoantibody with both native sheep α3(IV)NC1 and purified human α3(IV)NC1 monomers. Inhibition experiments using soluble overlapping peptides from human α3(IV)NC1 identified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that there is very limited heterogeneity in the autoantibody response in Goodpasture’s disease. The resonant mirror biosensor can be successfully used to monitor antibody–antigen binding using polyclonal sera, and to map epitopes on autoantigens.     

The non-collagenous domains of the alpha 3 and 4 chains of type IV collagen and their relationship to the Goodpasture antigen

The Goodpasture antigen is the target recognized by anti-glomerular basement membrane (GBM) antibodies in anti-GBM disease or Goodpasture's syndrome. This structure is present in all normal GBM, but when serum containing anti-GBM antibodies is used to examine renal tissue from most males with classical Alport's syndrome, the Goodpasture antigen appears to be missing. The nature of the Goodpasture antigen is uncertain although it has been putatively and controversially localized to the non-collagenous domain of a novel type IV collagen chain (alpha 3) by one group, and a short peptide sequence has been published (M2). We have performed several experiments to determine whether M2 represents the Goodpasture antigen and we have also studied the corresponding sequence of the alpha 4 chain of type IV collagen (M3). Firstly, we demonstrated by polymerase chain reaction (PCR) amplification using specific priming oligonucleotides that mRNAs corresponding to M2 and M3 were found within the kidney and that the published sequences were correct. When heterologous antibodies were raised against M2 and M3 these bound specifically to GBM in an ELISA based on collagenase-digested basement membrane and this binding could be inhibited by incubation with collagenase-digested GBM but not with ovalbumin. On further examination of the target molecules using Western blots, the anti-M2 antibody bound to a single high molecular weight band of collagen-digested GBM in contrast to the anti-M3 antibody that bound to the same bands as Goodpasture serum. We then established ELISAs for anti-M2 and anti-M3 activity using the peptides M2 and M3. While rabbit anti-M2 and M3 antibodies bound specifically to their respective peptides in these ELISAs, there was no binding of three high titre Goodpasture's syndrome sera or two sera from Alport's syndrome patients with inhibitable anti-GBM antibody post-renal transplant. We have shown that the sequences of M2 and M3 correspond to proteins present within the collagenase-resistant part of the GBM, suggesting that these do represent parts of novel type IV collagen chains. However, sera containing anti-GBM antibodies did not bind to either peptide in solid-phase ELISAs, and these antibodies may recognize a different peptide sequence, features of the tertiary structure of these peptides or interactions between collagen chains.     

Presence of glomerular basement membrane (GBM) antibodies in HIV− patients with Pneumocystis carinii pneumonia

Following the unexpected finding of antibodies to GBM in a patient with Pneumocystis carinii pneumonia in the absence of kidney abnormalities, the presence of anti-GBM antibodies was analysed in 14 patients with pulmonary P. carinii infection who did not have clinical evidence of autoimmune glomerulonephritis. Patients were divided into three groups: HIV⁻ with P. carinii pneumonia (n = 4), HIV⁺ with P. carinii pneumonia (n = 5) and HIV⁻ carriers of P. carinii without pneumonia (n = 5). As control groups, HIV⁻ patients with community-acquired non-P. carinii pneumonia (n = 6) and healthy individuals (n = 16) were included. Anti-GBM antibodies, studied with a quantitative enzyme immunoassay (EIA) for anti-α3 chain of collagen IV antibodies, were detected in three out of the four HIV⁻ patients with P. carinii pneumonia, but not in any individuals of the other categories. These results suggest that P. carinii alveolar injury or the host response to the organism could affect the basal membrane Goodpasture antigen or a similar antigen, and induces anti-GBM antibody production in HIV⁻ patients, and support the hypothesis that, at least in some cases, Goodpasture's syndrome could be triggered by an alveolar lesion induced by a P. carinii pneumonia.     

Characterization of human helper and suppressor factors with special reference to HLA-DR determinants

Human helper and suppressor cells can be induced entirely in vitro, by culturing lymphocytes with a small or large dose of antigen for 4 days in a Marbrook—Diener system. The corresponding helper or suppressor factor (vitro) can then be released by a second pulse of antigen for 1 day. However, if sensitization has taken place in vivo by a naturally encountered antigen, then helper or suppressor factor (vivo/vitro) can be released by a single pulse of antigen for 1 day from putative in vivo-induced helper or suppressor cells. Helper factor_vitro was compared with helper factor_vivo/vitro, stimulated by a streptococcal protein antigen derived from Streptococcus mutans. The results of affinity chromatography suggest that both helper factors have an antigen-specific binding site and bind to monoclonal antibodies directed against HLA-DR, MT, β₂M and μ chain and a `constant' part on helper factor (HF) determinants. The HF<sub>vivo/vitro</sub> unlike HF<sub>vitro</sub> may reflect in vivo-sensitization and thus assay putative in vivo-sensitized antigen specific helper cells. Similarly, suppressor factor <sub>vivo/vitro</sub> might be a measure of in vivo-induced suppressor cells. Like HF, suppressor factor (SF) has an antigen-specific binding site, HLA-DR, MT and β<sub>2</sub>M determinants and a `constant' portion of SF. The DR determinants were further characterized by immunoadsorption, using monoclonal antibodies to the α- and β-chain of DR antigen. The results suggest that helper and suppressor factor_vivo/vitro express similar Ia determinants. Both expressed an α-chain determinant, recognized by one of the two anti-α-chain antibodies (TAL/1B5), and a β-chain non-polymorphic determinant recognized by one of the four anti-β chain antibodies used (DA6.231). However, both HF and SF derived from DRw6- but not from DR4-typed lymphocytes expressed MT1 (DRw6,1,2) or MT2 (DRw6,3,5,8) determinants. As DR and MT determinants can be expressed on the same molecule, it is possible that both are involved in helper and suppressor activities.     

Mechanisms of binding of cutaneous lymphocyte-associated antigen-positive and αeβ7-positive lymphocytes to oral and skin keratinocytes

Intraepithelial lymphocytes (IEL) utilize the integrin αeβ7 on their surface to bind to E-cadherin on epithelial cells in the gut and breast. In oral mucosa and skin IEL express αeβ7 and the cutaneous lymphocyte-associated antigen (CLA) but the mechanisms of adhesion of these subsets to keratinocytes are unknown. Levels of αeβ7 and CLA were up-regulated on peripheral blood lymphocytes (PBL) by transforming growth factor-β (TGF-β) and interleukin-12 (IL-12), respectively, and both groups of lymphocytes adhered onto oral and skin keratinocytes. Adhesion of IL-12-activated PBL was totally abolished by anti-lymphocyte-associated function antigen type 1 (anti-LFA-1) antibodies but was unaffected by anti-αeβ7 antibodies indicating that adhesion of the CLA-positive subset is mediated via LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Adhesion of TGF-β-activated PBL to E-cadherin-positive oral and skin keratinocytes was partially inhibited by anti-αeβ7 antibodies but was unaffected by the blocking antibody E4.6 against E-cadherin which detects the binding site for αeβ7-positive lymphocytes in breast and gut epithelium. TGF-β-activated PBL also bound to an E-cadherin-negative oral keratinocyte cell line and adhesion was inhibited by anti-αeβ7 antibodies. These results strongly suggest that in oral epithelium and epidermis αeβ7-positive lymphocytes do not bind to E-cadherin and there may be a novel second ligand for the αeβ7 integrin.     

Autoimmune haemolytic anaemias. III. Preparation and examination of specific antisera against complement components and products, and their use in serological studies

The preparation and examination of agglutinating antisera, specific for β1E, β1A, α2D and the /β1C/ determinant of the β1C molecule are described. With these reagents it could be established that anticomplement serum does not contain antibodies against the B determinant of the β1C molecule, while antiserum prepared with β1C globulin does. Further, the complement factors present on red cells, the complement coating of which was effected in various ways, were studied.

Whereas red cells incubated in vitro with haemolytic iso- or auto-antibodies are agglutinated by anti-β1E, anti-β1A and anti-α2D sera, the cells of patients with autoimmune haemolytic anaemia of any of the serologically defined groups of this disease, only reacted with anti-α2D. The possible significance of these findings is discussed.

     

Characterization of conformation independent antigenic determinants in the triple-helical part of calf and rat collagen

About 20 per cent of the antibodies in rabbit antisera to native calf or rat collagen exhibited affinity for denatured rabbit collagen and could be isolated by immunoadsorption. Such antibodies reacted with the unfolded α1-chain as well as with the α2-chain of collagen. Inhibition experiments suggested that the two kinds of polypeptide chains are not completely equivalent in their antigenic determinants. These determinants were not significantly influenced by a treatment of native collagen with pronase, a procedure known to remove short, non-helical sequences at both ends of the molecule. The results suggested that the antigenic determinants are conformation independent. They are, however, mainly located in the middle region of collagen, having a rather complex conformational structure.

Cyanogen bromide cleavage of collagen did not impair the serologic activity of these determinants but with one exception none of the individual cyanogen bromide peptides possessed the full activity of the entire α-chain. However, most of the peptides could be agglutinated by the antibodies when put onto tanned red cells. Inhibition studies of these agglutination reactions clearly demonstrated that virtually all of the peptides carry unique antigenic determinants, which occasionally are shared by a few other peptides. Additional evidence for heterogeneity was obtained by further cleavage of the cyanogen bromide peptides with proteases. The minimum number of antigenic determinants thus estimated in calf collagen was nine. Evidence is provided that their structure in most cases does not correspond to sequences of the type Gly-Pro-X.

     

Expression of human–Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG)

The nicotinic AChR, a pentamer composed of α₂βγ(or ε)δ subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the α-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse–Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the α-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human α+Torpedoβγδ AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the α-subunit. Interestingly, the anti-α-subunit antibodies predominated in low titre (0.6–7.4 nm) but not in high titre (10–386 nm) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-α-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the α-subunit.     

Human peripheral blood T lymphocytes express α-, μ- and F(ab')2-related determinants in their surface membranes

Whereas a mean of 81% of freshly isolated human T cells bound purified chicken anti-F(ab')₂ antibodies to their surface membranes, 14 and 2% bound chicken anti-μ and chicken anti-α antibody preparations respectively as demonstrated by the mixed antiglobulin rosetting reaction (MARR). Neuraminidase treatment of T cells significantly increased their reactivity in the MARR so that an average of 98, 39 and 40% bound anti-F(ab')₂ anti-μ and anti-α reagents respectively. When using anti-F(ab')₂ antibodies, inclusion of Fabγ in the rosetting medium caused greater than 90% inhibition of the MARR whereas Fcγ produced only 25% inhibition; this indicates that the determinants seen by anti-F(ab')₂ antibodies are not carbohydrate in nature since Fabγ and Fcγ fragments prepared from a human IgG myeloma express identical carbohydrate moieties. To discover whether the various immunoglobulin (Ig) related T cell molecules play a role in antigen recognition, each antiglobulin reagent was assessed for its capacity to inhibit T cell binding of a selected antigen, i.e. fluorescein-labelled keyhole limpet haemocyanin. Whereas the frequency of antigen-binding lymphocytes (ABL) varied from 8·1 to 15·1 per 10³ cells, fewer than 1 in 10³ cells were Ig-positive with a rabbit F(ab')₂ anti-light chain reagent. Thus virtually all ABL were T cells. Each antiglobulin reagent produced 50% or greater inhibition of antigen binding thereby suggesting that F(ab')₂-, α-, and μ-related determinants are in close proximity to, or part of, the T cell antigen receptor. More substantial evidence for a direct association of Ig-related surface determinants and the T cell antigen receptor is provided by the finding that under capping conditions, modulation of surface F(ab')₂-related determinants by anti-F(ab')₂ antibodies reduced antigen binding. Further, that anti-F(ab')₂ antibodies induced F(ab')₂-, α- and μ-related determinants to co-cap suggests that F(ab')₂-related determinants are stably associated with α- and/or μ-related determinants in the T cell membrane and are part of the same Ig-related molecule(s).     

Expression of functional Toll-like receptors by salivary gland epithelial cells: increased mRNA expression in cells derived from patients with primary Sjögren's syndrome

Alpha-fodrin, an intracellular organ-specific cytoskeleton protein, was identified recently as an autoantigen associated with Sicca- and Sjögren''s syndrome (SS). Identification of the antigenic determinants of α-fodrin is a prerequisite to developing highly sensitive and specific anti-α-fodrin antibodies, which provides potential means for the diagnosis of primary Sjögren''s syndrome (pSS) in patients. Based on the structure and predicted antigenic sites of α-fodrin protein with 560 amino acids (α-fodrin 560), we prepared a set of overlapping recombinant protein fragments covering antigenic epitopes and synthesized a set of peptides derived from the α-fodrin protein. These recombinant proteins and synthesized peptides were subjected to screening with pSS patients sera, respectively. The peptide with the strongest immunoreactivity was used as antigenic peptide to define further the role of anti-α-fodrin-peptide antibodies in the sera of 135 patients with pSS, 48 patients with systemic lupus erythematosus (SLE), 88 patients with rheumatoid arthritis (RA) and 83 normal controls. Our data showed that the N-terminal peptide of amino acids 46–59 (N46) of α-fodrin 560 was the epitope with strongest antigenicity. The prevalences of anti-N46 peptide antibodies (α-N46PA) in patients with pSS, SLE, RA and normal controls were 78.5%, 10.4%, 21.6% and 6.0%, respectively. The sensitivity and specificity of the autoantibodies in pSS were 78.5% and 86.8%, respectively. These results suggest the α-N46PA which shows highest sensitivity and specificity is of significance to develop an effective diagnostic approach for pSS.     

Classification of Blood-Group Antibodies as β2M or γ Globulin

Thirty selected blood-group antibodies (excluding anti-A and anti-B) have been classified as β₂M (19S γ) globulin, γ (7S γ) globulin or mixtures, using the following three methods: fractionation on a DEAE-cellulose column; indirect anti-globulin tests, using specific anti-β₂M-globulin and anti-γ-globulin sera; and treatment with 2-mercapto-ethanol. With only minor exceptions, results obtained with the three methods were in agreement.Most blood-group antibodies within the Le, MNSs and P systems appear to be `naturally occurring'' and these were found to be β₂M globulin. Blood-group antibodies within the Rh, K and Jk systems, which had arisen after an antigenic stimulus, were usually γ globulin but were occasionally β₂M globulin.Antibodies composed of β₂M globulin usually behave as agglutinins but may behave as incomplete antibodies (e.g. some examples of anti-Jk^a); conversely, antibodies composed of γ globulin usually behave as incomplete antibodies but may behave as agglutinins (e.g. an example of anti-M).The ability to bind complement seems to be related more to the blood-group specificity of the particular antibody than to its molecular size. For example, anti-Jk^a, when composed either of γ or β₂M globulin, seems invariably to bind complement, whereas potent anti-M or anti-Rh, whether composed of γ or β₂M globulin, do not bind complement.     

Activated α4 Integrins are Preferentially Expressed on Immature Thymocytes and Activated T Cells

We have identified a novel mAb, SG31, which recognizes the mouse integrin α4 subunit. Unlike the epitopes recognized by other anti-α4 antibodies, the SG31 epitope is expressed on subpopulations of thymocytes and peripheral T cells. After manganese ion, but not phorbol myristic acetate activation, the epitope is induced and expressed on the majority of peripheral T cells. These data suggest that the SG31 epitope is an activation epitope and that manganese ions activate α4 integrins by inducing a conformational change. Comparative flow cytometric analyses showed that the SG31 epitope as well as the epitope detected by other anti-α4 antibodies is expressed on all B lineage cells. In the T lineage, expression of the α4 integrins is down-regulated during thymocyte development. Although mature thymocytes still express the α4 integrins, they lose almost entirely the activation epitope recognized by SG31. In contrast, the most immature thymocytes express high levels of this epitope. In the periphery, SG31 epitope is expressed mostly by activated T cells, in contrast to the overall population of T cells that express the α4 integrins at homogenous levels. These results suggest that the activation of the α4 integrins is parallel to that of T cells.     

VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii To Prevent Toxoplasmic Encephalitis

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4⁺ and CD8⁺ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.     

The Relation of the Level of Serum Anti-TF, -Tn and -Alpha-Gal IgG to Survival in Gastrointestinal Cancer Patients

Objective: To evaluate the relation of the level of serum anti-TF, -Tn and -αGal carbohydrate antibodies to survival in gastrointestinal cancer patients.Methods: The level of anti-TF (Thomsen-Friedenreich antigen), -Tn and -αGal IgG was analysed in the serum of patients with gastric (n = 83) and colorectal (n = 51) cancers in the long-term follow-up, using ELISA with polyacrylamide glycoconjugates. To evaluate overall survival and the risk of death, the Kaplan-Meier method and the Cox proportional hazards model were used in the univariate analysis of patients groups.Results: A significantly better survival was observed: (1) in patients with an increased level of anti-TF antibodies (all, stage III, T2-4, N1-2 and G3; P = 0.004-0.038, HR = 0.16-0.46); and (2) in patients with an increased level of anti-Tn antibodies (G1-2 tumors; P = 0.034-0.042, HR = 0.34-0.47). A significantly worse survival was observed in gastrointestinal, gastric and colorectal groups with an increased level of serum anti-αGal antibodies. This association depended on the patho-morphology of tumors (all, stages I-II, III, T2-4, N0, N1-2 and G1-2; P = 0.006-0.048, HR = 1.99-2.33). In the combined assessment of the anti-TF and -αGal antibodies level of the whole gastrointestinal group (n = 53), P = 0.002, HR = 0.25, 95% CI 0.094-0.655. In the follow-up, the survival time was shorter in patients whose level of anti-αGal antibodies rose (P = 0.009-0.040, HR = 2.18-4.27). The level of anti-TF antibodies inversely correlated with neutrophil/lymphocyte ratio (NLR, r = - 0.401, P = 0.004, n = 49). Patients with a higher level of anti-αGal antibodies and NLR values demonstrated a significantly worse survival (P = 0.009, HR = 2.98, n = 48).Conclusions: The preoperative levels of anti-TF, -Tn and -αGal antibodies and their dynamics are of prognostic significance. The method for the determination of circulating anti-carbohydrate antibodies may be a useful supplement in clinical outcome assessment.     

Characteristics of the Vδ2 CDR3 Sequence of Peripheral γδ T Cells in Patients with Pulmonary Tuberculosis and Identification of a New Tuberculosis-Related Antigen Peptide

Antigen-specific γδ T cells may play an important role in the immune response to Mycobacterium tuberculosis. However, little is known about the characteristics of the length distribution of the δ2-chain complementarity determining region 3 (δ2 CDR3) of the γδ T-cell receptor (TCR) in patients with active pulmonary tuberculosis (TB) on a large scale. In addition, M. tuberculosis-activated γδ T cells potentially inhibit intracellular mycobacterial growth, but phosphoantigen-activated γδ T cells do not. Only a few M. tuberculosis-related antigen peptides or proteins that are recognized by γδ TCR have been identified. Twenty-four healthy donors (HDs) and 27 TB patients were included in the present study. The gene-scanning technique found that the δ2 CDR3 length distribution patterns of γδ TCR in TB patients were perturbed, and each pattern included different predominant CDR3 sequences. The predominant δ2 CDR3 sequences of γδ TCRs, which originated from TB patients and HD γδ T cells that were stimulated by M. tuberculosis heat resistance antigen (Mtb-HAg), were used as probes to screen peptides recognized by γδ TCR using a phage display library. We identified four peptides that bound to the predominant δ2 CDR3 fragments and showed homology to M. tuberculosis genes in a BLAST search. Notably, one peptide was related to M. tuberculosis H37Rv (QHIPKPP), and this fragment was confirmed as a ligand for the γδ TCR. Two fragments, Ag1 and Ag2, activated γδ T cells from HD or TB patients. In summary, the δ2 CDR3 lineage of TB patients apparently drifts, and the predominant δ2 CDR3 sequence that recognizes M. tuberculosis may exhibit specificity. The identified M. tuberculosis-related antigen peptides may be used as vaccines or adjuvants for protective immunity against M. tuberculosis.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

Proportion of human colostral immunoglobulin-A molecules containing the secretory determinant

Concentrations of total colostral IgA were compared with concentrations remaining after precipitation with antibodies to secretory piece (SP). The anti-SP was obtained by filtering anti-colostral IgA through an immunosorbent column with firmly attached human serum globulins. IgA concentrations were measured by radial immunodiffusion with anti-α-chain serum.

The proportions of SP-containing IgA molecules were: in whole (defatted) colostrum, at least 68 per cent; in the polymeric IgA fraction, at least 88 per cent; in the monomeric IgA fraction, 43 per cent. Free SP was also detected in colostrum. The non-identity of SP with several known components of colostrum was ascertained by blocking experiments.

     

Profile of the regions on the α-chain of human acetylcholine receptor recognized by autoantibodies in myasthenia gravis

Eighteen synthetic overlapping peptides encompassing the entire extracellular part (residues α 1–210) of the α-chain of human acetylcholine receptor (AChR) and a 19th peptide (residues α 262–276) corresponding to an extracellular connection between two transmembrane regions were prepared and used for the measurement, by solid-phase radioimmunoassay, of the binding of autoantibodies in plasma from myasthenia gravis (MG) patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides. The regions recognized resided predominantly within the areas α 10–30, α 111–145 and α 175–198 and, less frequently, region α 45–77. Differences in the recognition profile of the peptides from patient to patient indicated that the autoantibody responses were under genetic control. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 myasthenia sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases. The mapping of the continuous antigenic regions recognized by autoantibodies on the α-chain of human AChR has permitted a comparison of the regions recognized by autoantibodies and autoimmune T-cells from the same donor. It also provided a peptide-based direct antibody binding method for diagnosis of MG.     

Altered peptide ligands of myelin basic protein (MBP87–99) conjugated to reduced mannan modulate immune responses in mice

Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87–99 (MBP_87–99), an immunodominant peptide epitope identified in MS. Mutations of residues K⁹¹ and P⁹⁶, known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R⁹¹, A⁹⁶]MBP_87–99 and [A⁹¹, A⁹⁶]MBP_87–99. Immunization of mice with these altered peptide ligands emulsified in complete Freund’s adjuvant induced both interferon-γ (IFN-γ) and interleukin-4 (IL-4) responses compared with only IFN-γ responses induced to the native MBP_87–99 peptide. It was of interest that [R⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan induced 70% less IFN-γ compared with the native MBP_87–99 peptide. However, [A⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan did not induce IFN-γ-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A⁹¹, A⁹⁶]MBP_87–99 peptide conjugated to reduced mannan did not cross-react with the native MBP_87–99 peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-A^s, novel interactions were noted. It is clear that the double-mutant peptide analogue [A⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS.     

An Extracellular Protease of Streptococcus gordonii Hydrolyzes Type IV Collagen and Collagen Analogues

Streptococcus gordonii is a frequent cause of infective bacterial endocarditis, but its mechanisms of virulence are not well defined. In this study, streptococcal proteases were recovered from spent chemically defined medium (CDM) and fractionated by ammonium sulfate precipitation and by ion-exchange and gel filtration column chromatography. Three proteases were distinguished by their different solubilities in ammonium sulfate and their specificities for synthetic peptides. One of the enzymes cleaved collagen analogs Gly-Pro 4-methoxy-β-naphthylamide, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), and p-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (pZ-peptide) and was released from the streptococci while complexed to peptidoglycan fragments. Treatment of this protease with mutanolysin reduced its 180- to 200-kDa mass to 98 kDa without loss of enzymatic activity. The purified protease cleaved bovine gelatin, human placental type IV collagen, and the Aα chain of fibrinogen but not albumin, fibronectin, laminin, or myosin. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. Maximum production of the 98-kDa protease occurred during growth of S. gordonii CH1 in CDM containing 0.075% total amino acids at pH 7.0 with minimal aeration. Higher initial concentrations of amino acids prevented the release of the protease without reducing cell-associated enzyme levels, and the addition of an amino acid mixture to an actively secreting culture stopped further enzyme release. The purified protease was stored frozen at −20°C for several months or heated at 50°C for 10 min without loss of activity. These data indicate that S. gordonii produces an extracellular gelatinase/type IV collagenase during growth in medium containing minimal concentrations of free amino acids. Thus, the extracellular enzyme is a potential virulence factor in the amino acid-stringent, thrombotic, valvular lesions of bacterial endocarditis.