How to dissect the plasticity of antigen-specific immune response: a tissue perspective.

Generation of antigen-specific humoral responses following vaccination or infection requires the maturation and function of highly specialized immune cells in secondary lymphoid organs (SLO), such as lymph nodes or tonsils. Factors that orchestrate the dynamics of these cells are still poorly understood. Currently, experimental approaches that enable a detailed description of the function of the immune system in SLO have been mainly developed and optimized in animal models. Conversely, methodological approaches in humans are mainly based on the use of blood-associated material because of the challenging access to tissues. Indeed, only few studies in humans were able to provide a discrete description of the complex network of cytokines, chemokines and lymphocytes acting in tissues after antigenic challenge. Furthermore, even fewer data are currently available on the interaction occurring within the complex micro-architecture of the SLO. This information is crucial in order to design particular vaccination strategies, especially for patients affected by chronic and immune compromising medical conditions who are under-vaccinated or who respond poorly to immunizations. Analysis of immune cells in different human tissues by high-throughput technologies, able to obtain data ranging from gene signature to protein expression and cell phenotypes, is needed to dissect the peculiarity of each immune cell in a definite human tissue. The main aim of this review is to provide an in-depth description of the current available methodologies, proven evidence and future perspectives in the analysis of immune mechanisms following immunization or infections in SLO.

Clinical importance of antibodies against platelet activating factor in antiphospholipid syndrome manifestations.

BACKGROUND: We assessed whether antibodies against platelet activating factor (PAF) are related to the presence of antiphospholipid syndrome (APS) clinical manifestations, in particular thrombosis, in patients with connective tissue diseases. MATERIALS AND METHODS: Anti-PAF, anticardiolipin (aCL), antibeta2 glycoprotein I (antibeta2GPI) and antiphosphatidylcholine (anti-PC) antibodies were determined in 52 patients with APS, 29 patients with systemic lupus erythematosus (SLE) aCL but without APS, 30 patients with SLE without aCL, and 30 patients with scleroderma. A new enzyme-linked immunosorbent assay (ELISA) was developed for determining anti-PAF antibodies in a bovine serum-free fashion. RESULTS: The ELISA showed high specificity. Homologous inhibition experiments showed 60-70% inhibition. Anti-PAF antibodies were found in 18/52 APS patients, 10/29 SLE/aCL+ patients, 9/30 SLE/aCL- patients and 3/30 scleroderma patients. Anti-PAF antibodies were significantly associated with anti-PC antibodies (odds ratio [OR] 12. 7, P < 0.01), and there was a modest association with immunoglobulin G (IgG) aCL (OR 3.1, P > 0.10), but not with IgM aCL or antibeta2GPI. Three SLE/aCL+ patients and five SLE/aCL- patients had clinical manifestations characteristic of APS. All these patients had anti-PAF antibodies, while none had high titres of aCL or antibeta2GPI antibodies and only one had anti-PC antibodies. Among the combined APS and SLE groups, the presence of anti-PAF antibodies was significantly associated with clinical manifestations which are characteristic of APS (OR 2.6, P = 0.02). The effect was independent of IgG aCL and antibeta2GPI antibodies. CONCLUSIONS: Anti-PAF antibodies are common in APS and SLE and comprise an independent factor for the development of thrombosis. Several patients experiencing thromboses have anti-PAF antibodies without other antiphospholipid specificities.

No evidence of epitope spreading after immunization with the major Sm epitope P-P-G-M-R-P-P anchored to sequential oligopeptide carriers (SOCs).

The sequence Pro-Pro-Gly-Meth-Arg-Pro-Pro (PPGMRPP) is the major B-cell epitope of the Sm autoantigen. The aim of the present study was to evaluate the immune response against the native forms of Sm and U1RNP and immune mediated tissue injury after immunization with the sequence PPGMRPP anchored in five copies to a new type helicoid sequential oligopeptide carrier (SOC) formed by the repetitive Lys-Aib-Gly moiety, [(PPGMRPP)(5)SOC(5)]. Rabbits (n=3) were immunized with 0.5 mg of (PPGMRPP)(5)SOC(5)in complete Freud's adjuvant and boosted at days 26, 53, 99; control rabbits were immunized with the PPGMRPP alone (n=3), phosphate buffered saline (PBS) (n=1), SOC(5)alone (n=1), a peptide at aminoacid (aa) position 158-177 of myelin basic protein (MBP aa 158-147) (n=1) and three La/SSB autoantigen B-cell epitopes (n=3). Antibodies to (PPGMRPP)(5)SOC(5)were determined by enzyme linked immunosorbent assay (ELISA); precipitating anti-Sm and anti-U1RNP antibodies were detected by RNA precipitation and western blot on HeLa total cellular and nuclear extract and 12s sucrose gradient fraction of rat liver extracts. High titres of anti-(PPGMRPP)(5)SOC(5)antibodies not recognizing the native forms of Sm or U1RNP antigens were detected in the (PPGMRPP)(5)SOC(5)immunized but not in the control animals. The sera of two (PPGMRPP)(5)SOC(5)immunized but not of the control rabbits recognized a 67 kDa protein in HeLa nuclear extract, distinct from the 70 kDa U1RNP antigen. Diffuse and segmental increase of mesangeal matrix and cells, crescent formation, segmental glomerular necrosis, rarely massive subendothelial deposits occluding the lumen and C3 complement component in the mesangeal area were seen in the kidneys of one (PPGMRPP)(5)SOC(5)immunized, but not of the remaining animals. In conclusion, the immune response induced by (PPGMRPP)(5)SOC(5)was specific for the immunizing epitope but not for the native forms of Sm and U1RNP antigens, but it was associated with immune mediated kidney injury.

Anti-phospholipid antibodies in HIV infection and SLE with or without anti-phospholipid syndrome: comparisons of phospholipid specificity, avidity and reactivity with beta2-GPI.

Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent assay (ELISA) methods. Sera from 30 patients with systemic lupus erythematosus (SLE), but without features of anti-phospholipid syndrome (APS) (SLE/non APS), six with SLE and secondary APS, (SLE/APS) and 11 with primary APS (PAPS) were also evaluated as controls. The resistance of the aPL antibody binding to dissociating agents was evaluated by treating the ELISA wells, after serum incubation with 2 M urea or 0.6 M NaCl for 10 min. An anti-beta2-glycoprotein-I (beta2-GPI) ELISA was used to assess serum reactivity against beta2-GPI, a plasma protein considered as the true antigen of aCL antibodies occurring in APS and SLE patients. The prevalence of aCL, aPS, aPI and aPC antibodies in HIV-1 infection was 36%, 56%, 34% and 43% respectively, which was comparable to that found in SLE/APS and PAPS patients and significantly higher than that observed in SLE/non-APS patients. Anti-beta2-GPI antibodies occurred in 5% of HIV-1 infected vs. 17% in SLE/non-APS (P=0.11), 50% in SLE/APS (P=0.009) and 70% in PAPS patients (P=0.0014). A significant decrease of aPL binding after urea and NaCl treatment was observed in the sera of HIV-1-infected, compared to that of APS patients, indicating that aPL antibodies from HIV-1 infected individuals have low resistance to dissociating agents. In conclusion, aPL antibodies (1) occur in HIV-1 infection; (2) tend to recognize various phospholipids but not beta2-GPI; and (3) are of low resistance to dissociating agents-a finding probably reflecting low antibody avidity. Finally, these, like the autoimmune-type aCL antibodies, tend to recognize the oxidized CL-a finding probably indicating autoantibody generation as a result of neoepitope formation by oxidized PLs.

Determination of tumor necrosis factor-alpha (TNF-alpha) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay.

OBJECTIVES: To develop a highly sensitive enzyme amplified lanthanide luminescence (EALL) immunoassay for tumor necrosis factor-alpha (TNF-alpha). METHODS: The method is based on the use of two monoclonal antibodies against TNF-alpha, one "capture" antibody and one labeled with biotin, in a "sandwich type" assay format. Alkaline phosphatase (ALP) conjugated to an antibiotin-polyclonal antibody is used as the enzyme label. ALP cleaves phosphate from diflunisal phosphate (DIFP) to produce diflunisal (DIF). The detection system is based on the combination of enzymatic amplification introduced by ALP and the formation of a highly fluorescent terbium complex that can be monitored by time resolved or conventional fluorimetry. RESULTS: By using 50 microL of sample, the dynamic range of the assay extends up to 2000 ng/L of TNF-alpha, with a detection limit of 1 ng/L, within-run CVs ranging from 3 to 15% and recoveries of 97 +/- 2%. By using 100 microL of sample the dynamic range of the assay extends up to 1000 ng/L of TNF-alpha with a detection limit of 0.2 ng/L, recoveries of 94 +/- 13%, within-run CVs ranging from 2 to 6.5% and between-run CVs ranging from 5 to 15%, in a total incubation time of 3h. No interference by the presence of other cytokines (IL-1beta IL-2, IL-4, IL-6, IFN-gamma) or by rheumatoid factors has been observed. The results obtained by the proposed method and by a commercially available kit (Medgenix TNF-alpha EASIA) correlated well (n = 26, r = 0.934). CONCLUSION: The proposed method is highly sensitive, simple and rapid and can reliably measure TNF-alpha in the ng range in biological specimens.

A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies.

A sensitive, highly reproducible, solid-phase enzyme immunoassay (ELISA), was developed in order to investigate whether the synthetic heptapeptide PPGMRPP-a major epitope of the Sm autoantigen-anchored in five copies to a sequential oligopeptide carrier (SOC), [(PPGMRPP)5-SOC5] is a suitable antigenic substrate to identify anti-Sm/antibodies. Sera with different autoantibody specificities [45 anti-Sm, 40 anti-U1RNP, 40 anti-Ro (SSA)/La(SSB) positive, 21 Antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens (ANA + /ENA - ) and 75 normal human sera, ANA negative] and 75 sera from patients with rheumatoid arthritis (RA) were tested for anti-(PPGMRPP)5-(SOC)5 reactivity in order to evaluate the specificity and sensitivity of the method to detect anti-Sm antibodies. RNA immunoprecipitation assays for the detection of anti-Sm and anti-U1RNP antibodies and counter immunoelectrophoresis (CIE) for the detection of anti-Ro(SSA) and anti-La(SSB) antibodies were used as reference techniques. The sensitivity of the method was 98% and the specificity was 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values were 82% and 86%, respectively. In a comparison of the above assay with an ELISA, using Sm/U1RNP purified complex as immobilized antigen it was shown that the sensitivity of the anti-Sm/U1RNP ELISA in detecting anti-snRNPs was 74%; in addition sera with anti-Sm antibodies gave higher binding in the anti-(PPGMRPP)5-(SOC)5 ELISA compared with anti-Sm/U1RNP ELISA. Intra- and inter-assay precision was measured on four sera with reactivities extending into a wide range of absorbance values showed that the intra-assay coefficient of variation (CV%) ranged from 2.7 to 6 and the inter-assay CV% ranged from 9 to 14.5. These results indicate that the PPGMRPP peptide anchored to a pentameric SOC as a carrier is a suitable antigen for detecting anti-Sm antibodies and that the above ELISA is a rapid, reproducible and valuable screening method to test anti-Sm/U1RNP reactivities.

Antibodies to beta 2-glycoprotein-I: urea resistance, binding specificity, and association with thrombosis.

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-beta 2-glycoprotein I (anti-beta 2GPI) antibodies using standard anticardiolipin (aCL) and anti-beta 2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-beta 2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-beta 2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D(o)) corresponding to the same optical density was defined as residual activity (RA = 100 D/D(o)). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120-1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA > or = 30%. Anti-beta 2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA > or = 30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-beta 2GPI negative and three non-APS anti-beta 2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when beta 2GPI was incubated with CL in the ELISA plates; thus some anti-beta 2GPI negative sera from APS patients recognized the CL/beta 2GPI complex, rather than CL or beta 2GPI alone. In conclusion, anti-beta 2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/beta 2GPI complex and not CL or beta 2GPI. Antibodies to either beta 2GPI or the CL/beta 2GPI complex derived from APS sera present a high resistance to urea. Anti-beta 2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease.

Autoantibodies to human recombinant erythropoietin in patients with systemic lupus erythematosus: correlation with anemia.

OBJECTIVE: To investigate the existence of circulating autoantibodies to erythropoietin (EPO) in sera from patients with systemic lupus erythematosus (SLE), and to correlate their presence with anemia and clinical activity. METHODS: Ninety-two consecutive patients with SLE, 80 patients with rheumatoid arthritis, and 42 normal individuals were studied. The patients with SLE were categorized into 3 groups according to hemoglobin (Hgb) level: group A (45 patients with Hgb > 12 gm/dl), group B (26 patients with Hgb 10.1-12 gm/dl), and group C (21 patients with Hgb < or = 10 gm/dl). In all patients with SLE, the disease activity was evaluated using the European Consensus Lupus Activity Measurement scale. Antibodies to EPO were detected using an enzyme-linked immunosorbent assay and purified recombinant human EPO as antigen. The specificity of the method was evaluated with homologous and cross-reactive inhibition assays. RESULTS: Antibodies to EPO were found in 15.2% of the SLE patient sera. The distribution of these antibodies among the 3 groups of SLE patients was as follows: 8.8% (4 of 45) from group A, 15.4% (4 of 26) from group B, and 28.6% (6 of 21) from group C. The prevalence of antibodies to EPO in patients with severe anemia (group C) was statistically significantly higher compared with patients without anemia (chi(2) = 4.31, P < 0.05). Patients with antibodies to EPO had higher disease activity scores (P < 0.005) and lower levels of the C4 component of complement (P < 0.05) compared with patients without antibodies to EPO. CONCLUSION: In this study, the presence of antibodies to EPO in the sera of SLE patients is demonstrated for the first time. The presence of these antibodies is associated with severe anemia and active disease.