Cellular immune responses of hydatid patients to Echinococcus granulosus antigens.

The reactivity of peripheral blood mononuclear cells (PBMC) from 40 hydatid patients to hydatid fluid (HF) and to two hydatid fractions (pH5PPT and pH5SUP) was evaluated by the incorporation of 3H-methylthymidine into DNA. Maximal responses were detected using 200 micrograms/ml protein after 7-9 days incubation. The three antigen preparations were inducers of PBMC proliferation, with a good correlation (r2 = 0.87) of the responses induced by HF and by the fraction pH5PPT, which contains the two major hydatid antigens (5 and B). Lymphocytes from healthy donors and non-hydatid patients showed no response to these antigens. Neither direct nor inverse correlation was found between the results of the serological tests and of the PBMC proliferation assays. The majority of the patients (75%) responded in serological and in cellular tests. Of the remaining patients, six showed high antibody response associated with a negative PBMC proliferation assay and conversely four seronegative patients were found to respond positively in the PBMC proliferation assay. No relationship of the pattern of immune responsiveness to the patient's clinical forms could be established. Use of the PBMC proliferation assay with hydatid antigens appears rational in those patients which are low antibody producers, but the test is still not to be considered applicable for routine diagnosis.     

Caveats for the use of surface-adsorbed protein antigen to test the specificity of antibodies.

Rabbit antisera against apo-cytochrome c, which was prepared by removal of the covalently bound heme prosthetic group from yeast iso-1 cytochrome c, were tested for reactivity against native yeast iso-1-cytochrome c. When the antigen was adsorbed to a microtiter plate in a conventional enzyme-linked immunosorbent assay (ELISA), the antisera were unable to distinguish between their cognate antigen apo-cytochrome c, a random coil protein, and native cytochrome c, a small globular protein of remarkable conformational stability in solution. However, when the assay was conducted under conditions where antigen and antibody were free to associate in solution, that is in a solution-phase radioimmunoassay (RIA), the antisera were highly specific for apo-cytochrome c. Similarly, antibodies induced by native cytochrome c and discriminating strongly between native and apo-cytochrome c in a solution-phase RIA, did not distinguish between native and apo-cytochrome c in a solid-phase ELISA. This discrepancy of results obtained by different immuno assay procedures clearly indicates that adsorption to plastic alters the antigenic structure of even a conformationally stable protein such as cytochrome c. A conventional solid-phase ELISA strongly selects for those antibodies that recognize the unfolded antigen. The results presented warrant serious thoughts about previous reports on anti-peptide antibodies reacting with native whole protein molecules, as tested by those ELISA procedures that have the protein antigen adsorbed to plastic.     

Clinical efficacy of a new CD28‐targeting antagonist of T cell co‐stimulation in a non‐human primate model of collagen‐induced arthritis

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co‐stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen‐induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA‐4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C‐reactive protein (CRP) and interleukin (IL)‐6 and anti‐collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII‐induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.     

Allergic arthritis induced by cationic proteins: role of molecular weight.

Previous studies have shown that chronic murine allergic arthritis can only be induced with cationized BSA, related to excellent retention of the cationic antigen in the joint. We now investigate the impact of size of cationic proteins on their potential to induce this form of arthritis. After intra-articular injection, antigen retention is much enhanced with high molecular weight cationized proteins, like albumin or immunoglobulin, compared to small-sized proteins like myoglobulin and lysozyme. Consequently, severe chronic arthritis was only found with the former ones. The role of size is further substantiated with poly-L-lysine-coupled lysozyme. This derivative shows excellent retention in vivo and causes a chronic destructive arthritis in preimmunized mice, in contrast to the poor arthritis seen with native cationic lysozyme. Control experiments made it clear that antigen retention is the most important denominator and that differences in chronicity are not related to gross variations in T-cell reactivity. Retention studies in vitro revealed that the potential to bind to joint structures is similar for the various proteins, suggesting that in vivo conditions determine size-related differences in antigen clearance. Our data indicate that cationicity per se does not make a protein a proper arthritogen.     

Autoantigen BiP and autoimmune diseases

Immunoglobulin Binding Protein (BiP) is a member of heat shock protein 70 famaily, and is also known as an autoantigen in rheumatoid arthritis (RA) patients. Serum anti-BiP antibody is detected up to 60% of RA patients, and recent reports demonstrated that serum anti-BiP antibody is also detected in systemic lupus erythematosus patients. Notably, anti-citrullinated BiP antibody is revealed as another member of anti-citrullinated protein/peptide antibodies (ACPAs). Since ACPAs are supposed to be closely associated with RA pathogenesis, immune responses to citrullinated BiP could play an important role in RA. Indeed, immunization of citrullinated BiP exacerbated inflammatory arthritis in mice. Moreover, T cell responses to BiP were reported in human RA and mice models. In mice models, native BiP administration induced IL-4 and IL-10 producing CD4(+) T cells and regulated inflammatory arthritis. In this way, immune responses to BiP are various, and dysregulation of the balances between pro-inflammatory and regulatory responses to BiP could lead to the autoimmune responses and diseases.     

Prevention of collagen-induced arthritis (CIA) by treatment with polyethylene glycol-conjugated type II collagen; distinct tolerogenic property of the conjugated collagen from the native one

Administration of a soluble protein into animals prior to challenge immunization induces immunological tolerance which is specific for the protein. In addition, chemical modification of proteins with polyethylene glycol (PEG) has been reported to convert the immunogenic proteins to become tolerogenic. However, differences in tolerogenic properties between PEG-modified proteins and the native counterparts have never been analysed. The ability of PEG-conjugated type II collagen (PEG-CII) to attenuate CIA, an animal model for rheumatoid arthritis, was compared with the native unconjugated CII. Groups of DBA/1 J mice were treated weekly with i.p. injections with PEG-CII, native CII, or vehicle alone for 3 weeks, before they were challenged with CII in adjuvants. The induction of tolerance was confirmed in both PEG-CII- and CII-pretreated mice when suppression of lymph node T cell proliferation in response to CII was noted. The degrees of suppression of T cell proliferation were comparable between the two pretreated groups. However, induction of arthritis and production of IgG anti-CII antibody were more markedly suppressed in PEG-CII-pretreated mice than in native CII-pretreated mice, although the severity of arthritis and antibody levels in the latter group were also lower than in control mice. IgG2a and IgG2b antibody levels were equally suppressed in the two pretreated groups, whereas the IgG1 level was significantly lower in the PEG-CII-pretreated group than in the native CII-pretreated group. The results provide the first evidence that attachment of PEG to CII renders the protein more tolerogenic.     

异质性胞核核糖核蛋白A2基因的克隆、表达、纯化及其临床应用

目的：构建含异质性胞核核糖核蛋白A2（hnRNP A2) cDNA片段的基因克隆，制备并纯化重组蛋白hnRNP A2，用以检测抗hnRNP A2/RA33的抗体，方法，从人外周血单个核细胞提取细胞总RNA，应用RT－PCR方法扩增hnRNP A2 cDNA，将其克隆于pUC-T1质粒中，测定其序列。构建高效表达载体pET28a-hnRNP A2，转化大肠杆菌BL21（DE3）plys S并进行诱导表达，将含目的蛋白的组分经金属螯合树脂亲和层析柱进行蛋白纯化。以该纯化蛋白为包被抗原，ELISA方法检测类风湿关节炎（RA）97例，系统性红斑狼疮（SLE）50例，混合性结缔组织病（ECTD）8例，其他关节炎29例，其他弥漫性结缔组织病（CTD）99例，结果：构建hnRNP A2重组表达载体并在大肠杆菌中获得hnRNP A2高效表达；在RA，SLE，MCTD，其他关节炎，其他CTD患者中抗hnRNP A2/RA33抗体阳性率分别为36.8%,24%,75%,3.75%,10.10%，在RA中特异性为86．67％，结论：以纯化的基因重组蛋白hnRNP A2为抗原检测hnRNP A2/RA33抗体是早期诊断RA的可靠方法。     

Cytokine mRNA levels in antigen-stimulated peripheral blood mononuclear cells

Levels of cytokine mRNA coding for granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), interleukin (IL) 2, IL 1 beta, IL 4 or IL 6 have been measured by Northern blot analysis after antigen stimulation. As source for RNA we used peripheral blood mononuclear cells (PBMC) from donors which showed a proliferative response after tetanus toxoid or Candida albicans stimulation. For comparison PBMC were also stimulated with lectins and anti-CD3 antibody. With some variations among donors, antigens clearly induced measurable levels of IFN-gamma, GM-CSF and IL 2 mRNA. Increased levels for IL 6 were also detected after antigen stimulation. In contrast to polyclonal T cell stimuli, antigens showed delayed kinetics of mRNA steady-state levels and resembled in this respect more closely the stimulation with pokeweed mitogen. Thus, cytokine mRNA levels may be assessed in unfractionated PBMC after antigen stimulation. The two tested antigens also clearly show a cytokine pattern distinct from that induced in polyclonal stimulations such as anti-CD3.     

Induction of strong hepatitis B virus (HBV) specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection.

Humanized BALB/c mice (termed trimera mice) conditioned by lethal total body irradiation and bone marrow transplantation from SCID mice have been described to support rapid engraftment of human peripheral blood mononuclear cells (PBMC) and the induction of strong B and T cell responses after immunization in vivo. Moreover, these mice can be infected with the hepatitis B and C viruses (HBV, HCV). The current study employed this model to study therapeutic vaccination approaches against the HBV. Thus, strong primary Th cell responses against the HBV core (HBc) and the Borrelia burgdorferi control antigen were induced by transfer of antigen-loaded dendritic cells together with autologous PBMC from HBV-naive donors as well as by vaccination with high doses of antigen or a DNA plasmid encoding for HBcAg. Moreover, primary peptide-specific CTL responses against the immunodominant epitope HBc(18 - 27) were induced by HBc particle or DNA vaccination of chimera engrafted with HBV-naive PBMC. Finally, strong HBc-specific Th cell and antibody responses were induced by HBc or DNA vaccination of mice reconstituted with PBMC from a chronic HBV patient. Thus, since HBc represents the immunodominant antigen in self-limited HBV infection, HBc particles or DNA vectors are good candidates for therapeutic vaccination, that will be further studied in our model and clinical studies.     

人肝再生增强因子单克隆抗体的制备:以非纯化的大肠杆菌表达抗原筛选杂交瘤

目的 :利用杂交瘤技术、以非纯化的大肠杆菌表达重组蛋白作为筛选抗原 ,制备小鼠抗人肝再生增强因子(hALR)的单克隆抗体。方法 :用实验室纯化的重组hALR 硫氧环蛋白融合蛋白免疫BALB c小鼠 ;小鼠脾细胞与SP2 0骨髓瘤细胞融合后经HAT选择培养基筛选杂交瘤 ;以重组质粒pQE30 hALR与空质粒pQE30在大肠杆菌中诱导表达后的细菌裂解产物作为筛选抗原和对照抗原 ,用ELISA方法筛选能分泌抗hALR单克隆抗体的阳性杂交瘤细胞克隆 ;进一步以ELISA方法和免疫印迹方法检测该杂交瘤细胞产生的抗体对真核细胞表达的重组hALR及人体血清中天然hALR的反应性。结果 :成功筛选出一株能稳定分泌抗hALR单克隆抗体的杂交瘤细胞 ;其产生的抗体能对真核细胞表达的重组hALR及人体血清中天然的hALR发生特异的抗原抗体反应。结论 :大肠杆菌表达的重组蛋白在以空质粒表达产物作为对照下 ,不经过任何纯化步骤也能够用于单克隆抗体制备中的杂交瘤筛选 ;hALR单克隆抗体为深入研究hALR提供了研究手段。     

Administration of anti-type II collagen antibody sustains footpad swelling of mice caused by a delayed-type hypersensitivity reaction and induces severe arthritis

Delayed-type hypersensitivity (DTH) is an immune reaction induced by antigen. In the mice footpads at which DTH is elicited, transient swellings which usually peaks at 24-48 h after the antigen challenge are observed. We found that the footpad swellings of mice are sustained for at least 7 days after the antigen challenge if the mice were injected with anti-type II collagen monoclonal antibody (anti-CII MoAb) before the antigen challenge. A histological section of the swelled hindpaw revealed that severe joint inflammation and bone destruction was induced. These features were not observed in the footpads of the DTH-induced mice. Analysis of the inflammatory reaction induced by both the DTH and the anti-CII MoAb injection, here named as DTH arthritis, revealed the following: (1) DTH arthritis is elicited in an antigen-specific manner; and (2) the development of DTH arthritis is mediated by antigen-specific T cells, especially CD4+ T cells.     

Confirmation of a false-positive result associated with a competition inhibition assay used for detecting antibodies to a protein epitope of Mycobacterium leprae.

A competitive antibody-binding assay (CABA) was developed to detect antibodies in infected armadillos and leprosy patients which compete with an M. leprae-specific 125I monoclonal antibody IIIE9 for the species-specific M. leprae-IIIE9 epitope on the 65-kD protein. The results suggest armadillos and leprosy patients produce antibodies that inhibit the binding of 125I-IIIE9 monoclonal antibody to the IIIE9 epitope on crude, native 65-kD protein preparations. When purified, recombinant 65-kD protein was substituted for crude antigen, there was no evidence in the CABA of antibody to the IIIE9 epitope. False-positive results, possibly induced by steric hindrance, are likely to be associated with CABA which incorporate crude cell wall extracts as solid-phase antigen.     

Presence of human T-cell responses to the Mycobacterium leprae 45-kilodalton antigen reflects infection with or exposure to M. leprae

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-gamma) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-gamma secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-gamma secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test.     

Altered (Oxidized) C1q Induces a Rheumatoid Arthritis- like Destructive and Chronic Inflammation in Joint Structures in Arthritis-Susceptible Rats

Previous studies have identified an altered C1q molecule in synovial fluids from the joints of rheumatoid arthritis patients. We therefore immunized arthritis-susceptible Lewis 1A.AVN rats with either native C1q (C1q nat), altered (oxidized) C1q (C1q ox), or type II collagen (CII, induces arthritis in these animals), in order to induce arthritis. Unlike C1q nat, both CII and C1q ox were able to induce swelling and erythema of joints consistent with an arthritis-like inflammatory reaction. Histopathological evaluation of individual joint sections revealed synovitis, bursitis and tendovaginitis, massive joint destruction, and severe pannus formation. In a time-course study, no differences in onset of arthritis or pathology were observed between C1q ox-induced arthritis and that induced by CII. High titers of antibodies recognizing CII, but not C1q (native or oxidized), were detected in rats immunized with CII. In contrast C1q ox, but not C1q nat, induced antibodies reactive with both C1q and CII. Antibodies from C1q ox-immunized animals contained an antibody subset that reacted with C1q but not CII and a subset that reacted with CII but not C1q, implying that induction of an immune response to CII does not require CII. These data support the hypothesis that C1q may provide one of the early antigens involved in induction of arthritis, before CII becomes available as antigen.     

Autoimmunity Versus Allo- and Xeno-Reactivity in SCID Mice

The ability of SCID mice to accept xenografts has been exploited to study the survival, function and potential of peripheral blood mononuclear cells (PBMC) from patients with autoimmune disorders to produce tissue injury in the mouse. Studies performed with PBMC obtained from patients with organ specific and multisystem autoimmune diseases indicate that human PBMC survive in SCID mice for several months, produce IgG and autoantibodies with the same specificities as are found in the donor. Tissue injury is not generally observed in the SCID mouse recipient. SCID mice have also been partially reconstituted with bone marrow from BB (diabetic) and MRL (lupus) mice. SCID mice injected with both spleen cells from mice with collagen induced arthritis together with native bovine collagen developed more severe arthritis than the donors. SCID mice have therefore proven to be a useful resource to study autoimmunity. In both xeno- and allografts of mature lymphocytes, graft versus host reactions occur. Further studies will be necessary to improve donor cell survival without aggravating graft versus host disease.     

Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes

We describe here a novel method for measuring in vitro antibody secretion from the tissue culture of human B lymphocytes in peripheral blood mononuclear cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzyme-linked immunosorbent assay (ELISA) titers of the antibody secreted in the cell supernatant were determined. The validation results demonstrated that human PBMC remained viable and continued to secrete antibodies (total immunoglobulin A [IgA] and IgG) for up to 4 days of incubation at 37 degrees C with 5% CO(2) in cell cultures. The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue culture and correlated negatively with the storage time of the whole blood at room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific antibody response after oral vaccination with a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Postimmunization PBMC secreted antibodies to cholera toxin in the cell supernatants. Antibody production did not require any in vitro antigen stimulation. In the ALS assay, antigen-specific antibody titers of prevaccination samples were barely detectable, whereas serum antitoxin ELISA titers in background of prevaccine samples were significantly higher than the ALS titers. We conclude that, without any in vitro antigen stimulation after vaccination, PBMC secrete antibodies into the supernatants in the ALS assay. This assay can quantitatively measure the antigen-specific antibody production from the PBMC culture in postvaccination blood samples.     

A novel, helminth-derived immunostimulant enhances human recall responses to hepatitis C virus and tetanus toxoid and is dependent on CD56+ cells for its action

We have described previously an immunostimulant derived from Onchocerca volvulus, the helminth parasite that causes onchocerciasis. Recombinant O. volvulus activation-associated secreted protein-1 (rOv-ASP-1) was a potent adjuvant for antibody and cellular responses to protein, polypeptide and small peptide antigens. Our aims were to determine whether rOv-ASP-1 is immunostimulatory for human peripheral blood mononuclear cells (PBMC) and, if so, whether it could augment cellular responses against human pathogen antigens in vitro. Cytokines from rOv-ASP-1-stimulated human PBMC were measured by a fluorescence activated cell sorter-based multiplex assay. Recall responses of normal healthy donor (NHD) and chronic hepatitis C virus (c-HCV)-infected patient PBMC to tetanus toxoid (TT) or HCV core (HCVco) antigen, respectively, were measured by interferon-gamma enzyme-linked immunospot assays. Interferon-gamma was the predominant cytokine induced by rOv-ASP-1. 77.3% of NHD anti-TT and 88.9% of c-HCV anti-HCVco responses were enhanced by rOv-ASP-1. The immunostimulant effect was dependent upon contact between CD56+ and CD56- fractions of PBMC. We have described a helminth-derived protein that can act as an immunostimulant for human recall responses in vitro to TT and, perhaps more importantly, HCV antigens in patients with chronic HCV infection. Our longer-term goal would be to boost anti-viral responses in chronic infections such as HCV.     

Development of a Novel In Vitro Assay (ALS Assay) for Evaluation of Vaccine-Induced Antibody Secretion from Circulating Mucosal Lymphocytes

We describe here a novel method for measuring in vitro antibody secretion from the tissue culture of human B lymphocytes in peripheral blood mononuclear cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzyme-linked immunosorbent assay (ELISA) titers of the antibody secreted in the cell supernatant were determined. The validation results demonstrated that human PBMC remained viable and continued to secrete antibodies (total immunoglobulin A [IgA] and IgG) for up to 4 days of incubation at 37°C with 5% CO₂ in cell cultures. The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue culture and correlated negatively with the storage time of the whole blood at room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific antibody response after oral vaccination with a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Postimmunization PBMC secreted antibodies to cholera toxin in the cell supernatants. Antibody production did not require any in vitro antigen stimulation. In the ALS assay, antigen-specific antibody titers of prevaccination samples were barely detectable, whereas serum antitoxin ELISA titers in background of prevaccine samples were significantly higher than the ALS titers. We conclude that, without any in vitro antigen stimulation after vaccination, PBMC secrete antibodies into the supernatants in the ALS assay. This assay can quantitatively measure the antigen-specific antibody production from the PBMC culture in postvaccination blood samples.     

Comparison of anti-arthritic properties of leflunomide with methotrexate and FK506: Effect on T cell activation-induced inflammatory cytokine production in vitro and rat adjuvant-induced arthritis

OBJECTIVE AND DESIGN: To examine the effect of leflunomide (LEF) on T cell activation-induced inflammatory cytokine production in human peripheral blood mononuclear cells (PBMC) and rat established adjuvant-induced arthritis (AIA), and compare these effects with methotrexate (MTX) and FK506 (tacrolimus), focusing on improvement of joint function in AIA. METHODS: Human PBMC were cultured with immobilized anti-CD3/CD28 monoclonal antibody to produce tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6. The active metabolite of LEF was used in in vitro study. AIA was induced in female Lewis rats. Paw swelling and grip strength were measured as indicators of arthritis and joint function, respectively. Rats were therapeutically administered LEF (3.2-32 mg/kg) from days 15-24 by oral administration. RESULTS: LEF inhibited anti-CD3/CD28 induced production of TNF-alpha, IL-1beta and IL-6, with IC50 values of 27, 21 and 21 microg/ml, respectively. LEF also suppressed mouse bone marrow cell MTT conversion, with an IC50 value of 15 microg/ml. LEF significantly inhibited paw swelling and loss of grip strength in established AIA at 10 and 32 mg/kg. The inhibition of paw swelling and grip strength loss by LEF was more potent than MTX. However, maximum recovery of grip strength loss by LEF (23.5%) was less potent compared to that with FK506 (57.8%). CONCLUSIONS: LEF inhibited anti-CD3/CD28 induced inflammatory cytokine production in human PBMC at concentrations showing deleterious effects on bone marrow cell proliferation. LEF is superior to MXT in improving arthritis and joint function in established AIA, but is inferior to FK506 in recovering joint function, probably due to its anti-proliferative actions.     

A molecular model of antigen retrieval using a peptide array

Even though antigen retrieval is highly denaturing, it paradoxically restores immunoreactivity after formalin fixation. It is unclear how this happens. We address this question using a peptide array to model formalin fixation and antigen retrieval. The peptides are linear stretches based on the native protein sequence, containing antibody epitopes of HER-2, estrogen receptor, progesterone receptor, and Ki-67. Of the 7 peptides, 6 retain their immunoreactivity after formalin fixation. However, if formalin fixation is performed in the presence of an irrelevant protein, immunoreactivity is abrogated, regardless of the peptides' amino acid composition. Fixation of an external protein around the antibody epitope prevents antibody binding. Antigen retrieval restores immunoreactivity. These findings demonstrate that native protein conformation is not relevant during antigen retrieval. Moreover, the loss and recovery of immunoreactivity associated with fixation and antigen retrieval, respectively, can be accounted for completely with a model of steric interference by adjacent proteins.