Low Antigen Concentrations Research Articles

Effect of prolonged exposure to low antigen concentration for sensitization

The local lymph node assay (LLNA) is an assay in mice to identify potential allergens. Compounds that do not induce a stimulation index (SI)≥3 are not considered sensitizers. Of the chemicals that do, the SI of 3 is used as a benchmark, and indicates the sensitizing potency of a chemical. Compared to the exposure duration of the LLNA (3 days), real life exposure often lasts for months or years. We therefore investigated whether prolonged exposure to sensitizers at concentrations that do not induce a SI≥3 in the LLNA, were able to surpass this threshold. Mice were treated for 2 months at 7-day intervals with a range of concentrations of the known allergens ethyl-p-aminobenzoate (benzocaine, BENZ), 2,4-dinitrochlorobenzene (DNCB), and tetramethyl thiuram disulfide (TMTD). Both proliferative activity and cytokine production were established at day 60. Neither BENZ nor TMTD showed a significant increase in the proliferation rate compared to vehicle controls. Only DNCB at concentrations originally above the EC 3 a significant increase in proliferation was seen after prolonged exposure. No significant effect on IFN-γ and IL-4 production was observed for all three compounds compared. These findings indicate that for classification of sensitizers the shorter exposure period employed in the standard LLNA is sufficient, and longer periods of exposure have no bearing on this classification.

Toward optimized antibody microarrays: a comparison of current microarray support materials

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.

Functional consequences of FcɛRIα up-regulation by IgE in human basophils

These studies examine the functional changes that occur after up-regulation of FcepsilonRIalpha by immunoglobulin E (IgE) for human basophils. Basophils were cultured with and without IgE antibody (PS myeloma IgE or anti-gp120-specific IgE) for 1 week and challenged with anti-IgE, anti-FcepsilonRIalpha, or antigen for histamine and IL-4 secretion. There were no statistically significant changes in their response to anti-IgE or anti-receptor antibodies, as compared with controls incubated for the same period, whereas receptor expression increased an average of 4-fold. There was increased responsiveness to antigenic challenge, most notably at suboptimal concentrations of antigen (gp120 peptide-ovalbumin conjugate). For a 6-fold difference in cell surface density of gp120-specific IgE, there was a 2.2-fold change in antigen potency or 3-fold increases in histamine release at lower antigen concentrations. Similar results were found for secretion of IL-4. Basophil sensitivity, which is a measure of the density of antigen-specific IgE required for 50% of maximal secretion, was used to determine whether up-regulation of FcepsilonRIalpha was coordinated with up-regulation of other components of the IgE-signaling pathway. The results indicated up-regulation of FcepsilonRI is not always accompanied by changes that allow sensitivity to be maintained. These results indicate that functional up-regulation does occur but that its magnitude may be modulated because not all components of the signaling pathway are up-regulated in a balanced manner.

Mechanism of Nanoparticle-Enhanced Turbidimetric Assays Applying Nanoparticles of Different Size and Immunoreactivity

The mechanism of a novel assay technique for nanoparticle-enhanced immunoturbidimetric assays is investigated. In a mixture of latex particles of two different sizes, coated with antibodies of different affinities, the aggregation behavior is monitored, which correlates with the antigen concentration. At low antigen concentrations, only the bigger latex particles coated with the high-reactivity antibody aggregate, whereas at higher antigen concentrations, the smaller latices coated with a lower reactivity antibody follow up in the aggregation process. This is shown for an immunoassay (C-reactive protein) by theoretical considerations based on a diffusion-controlled reaction and by transmission electron microscopy, analytical ultracentrifugation, and static light scattering as complementary qualitative and quantitative analytical techniques.

Factor XIII A-subunit concentration predicts outcome in stroke subjects and vascular outcome in healthy, middle-aged men.

There is growing evidence for a role of factor XIII (FXIII) in vascular disease. FXIII measures were determined in (i) a nested case-control study from the Second Northwick Park Heart Study of 63 men with myocardial infarction (MI) and 124 age-matched controls and (ii) in a case-control study of 475 subjects with acute stroke and 461 controls followed up for 54 months for mortality. In both studies, measures of FXIII A- and B-subunit antigen, FXIII activity and prothrombin fragments (F1 + 2) were made. An in vitro model was used to investigate the effects of thrombin activity on FXIII A- and B-subunit antigen levels. In study 1, patients clinically free of coronary artery disease who later developed MI had lower adjusted FXIII A-subunit levels at recruitment (129.2%vs 113.3%, P = 0.007). In study 2, stroke patients with large vessel disease had lower A-subunit antigen levels (102.1%vs 127.2%, P < 0.001), but higher F1 + 2 levels (0.941%vs 0.753%, P < 0.05), than subjects with small vessel disease. Levels of FXIII A-subunit (100%vs 117%, P < 0.0001) were lower and F1 + 2 higher (1.020%vs 0.702%, P < 0.0001) in stroke patients who had died compared with those still alive at the end of the follow-up period. Low concentrations of FXIII A-subunit antigen predicted vascular outcome in otherwise healthy subjects and relate to both size of infarct and poor post-stroke survival in patients with acute ischaemic stroke. Low in vitro concentrations of FXIII A-subunit antigen wererelated to increased thrombin generation and, thus, increased risk of thrombotic events.

CD8 lethargy in the absence of CD4 help.

CD4 T cell help was proposed to have a pivotal role in orienting CD8 T cell responses to antigen stimulation. By activating antigen-presenting cells (APC), CD4 cells would induce their expression of costimulatory molecules, the "signal two" required to induce full CD8 activation, preventing CD8 tolerance. Recent data on this subject is contradictory, as the absence of help did not always result in CD8 tolerance. These differences were attributed either to the presence of residual CD4 help or, respectively to the type of antigen stimulation, the peptide affinity, the CTL frequencies, and/or the strength of the response. We therefore reassessed the role of CD4 help in CD8 responses using a system where CD4 cells are absent and APC not activated. This system can be manipulated to induce CD8 tolerance (at high antigen concentrations) or CD8 memory (at low antigen concentrations). We found that the presence of CD4 help did not prevent tolerance induction. On the other hand, the absence of CD4 help did not induce CD8 tolerance, but rather led to differentiation stage intermediate between naive/memory/tolerant cells that we call "lethargy". These findings indicate that role of CD4 help in CD8 responses does not follow a simple on-off rule, as previously suggested. They also reveal that the "tolerance versus memory" dichotomy fails to account for all possible states/properties of antigen-experienced CD8 cells. Depending on the priming conditions, other intermediate stages of differentiation may occur.

Flow cytometry used for the analysis of calcium signaling induced by antigen-specific T-cell activation.

In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry. We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels ([Ca(2+)](i)) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals. The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or with efficient APC in the presence of low antigen concentration resulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustained the increase of intracellular calcium levels. In line with the strong and sustained calcium signals, the activation of the calcium-dependent transcription factors NF-AT, AP-1, and NF-kappaB was induced. Flow cytometric methods, feasible for the rapid and flexible analysis of calcium signaling upon antigen-specific T-cell activation, were established. Kinetics of the increase of mean fluorescence intensity reflected the calcium response of the total cell population whereas statistical analysis of fluorescence intensity at selected time points provided information on the activation state of single cells.

The Alpha Dx POINT-OF-NEED System

The Alpha Dx POINT-OF-NEED System

Role of B cells as antigen-presenting cells in vivo revisited: antigen-specific B cells are essential for T cell expansion in lymph nodes and for systemic T cell responses to low antigen concentrations.

Studies in B cell-deficient mice generated by continuous injection of anti-mu antibodies (muSM) showed that T cell priming in lymph nodes was dependent on antigen presentation by B cells. This concept has recently become controversial since a wide range, from complete deficiency to near normal T cell responses, was reported in studies carried out with B cell-deficient mice generated by gene disruption (muMT). In this study we show that in the absence of B cells, T cell responses are greatly reduced in all the available muMT mouse strains although responses in muMT of the C57BL/6 background (which were used for most studies with muMT) were much more variable and could reach up to 42% of control. In contrast, T cell responses in muMT --> F(1) bone marrow chimeras which have the same phenotype as muMT were totally impaired, suggesting a principle difference between mice developing without B cells (muMT mice) and muSM which are made B cell deficient only after birth. Normal T cell priming was completely restored by reconstitution of muMT and muMT --> F(1) mice with syngeneic B cells. Interestingly, only B cell populations containing antigen-specific B cells were capable of reconstituting T cell responses. Monoclonal B cells taken from Ig transgenic mice could not reconstitute responses to an irrelevant antigen. We also found that B cells were also required for systemic T cell priming when antigen concentrations were limiting but were not required for priming (for T cell help) when mice were immunized with a high antigen dose.

Enhancement of Antigen Driven Lymphocyte Proliferation Secondary to GP41-Induced B7 Expression on Adherent Monocytes

HIV-1 viral proteins are known to have immune regulatory effects. The interplay between these molecules and the host immune cells is complex. In this study the immune regulatory effects of gp41 on lymphocyte proliferation were evaluated as a function of the state of the monocyte. It is shown that monocyte adherence to tissue culture plates prevents suppression of lymphocyte proliferation to recall antigen in the presence of gp41. In addition, gp41 can enhance proliferation to low concentrations of Casta antigen when PBL are permitted time to adhere. It is shown that these effects are in part mediated through enhanced expression of the costimulatory molecules B7 and CD40. Cyclosporin A was not able to fully abrogate gp41-enhanced proliferation, indicating participation of a calcium-independent pathway. In addition, concentrations of anti-IL2 receptor antibody sufficient to inhibit maximal proliferation to antigen did not fully inhibit PBL proliferation to antigen that is augmented with gp41. Taken together these results suggest that modification of the monocyte state of activation or differentiation could mediate a response to gp41 that is immune enhancing.

Different levels of T-cell receptor triggering induce distinct functions in hepatitis B and hepatitis C virus-specific human CD4(+) T-cell clones.

CD4(+) T cells play a major role in the host defense against viruses and intracellular microbes. During the natural course of such an infection, specific CD4(+) T cells are exposed to a wide range of antigen concentrations depending on the body compartment and the stage of disease. While epitope variants trigger only subsets of T-cell effector functions, the response of virus-specific CD4(+) T cells to various concentrations of the wild-type antigen has not been systematically studied. We stimulated hepatitis B virus core- and hepatitis C virus NS3-specific CD4(+) T-cell clones which had been isolated from patients with acute hepatitis during viral clearance with a wide range of specific antigen concentrations and determined the phenotypic changes and the induction of T-cell effector functions in relation to T-cell receptor internalization. A low antigen concentration induced the expression of T-cell activation markers and adhesion molecules in CD4(+) T-cell clones in the absence of cytokine secretion and proliferation. The expression of CD25, HLA-DR, CD69, and intercellular cell adhesion molecule 1 increased as soon as T-cell receptor internalization became detectable. A 30- to 100-fold-higher antigen concentration, corresponding to the internalization of 20 to 30% of T-cell receptor molecules, however, was required for the induction of proliferation as well as for gamma interferon and interleukin-4 secretion. These data indicate that virus-specific CD4(+) T cells can respond to specific antigen in a graded manner depending on the antigen concentration, which may have implications for a coordinate regulation of specific CD4(+) T-cell responses.

B cells acquire antigen from target cells after synapse formation.

Soluble antigen binds to the B-cell antigen receptor and is internalized for subsequent processing and the presentation of antigen-derived peptides to T cells. Many antigens are not soluble, however, but are integral components of membrane; furthermore, soluble antigens will usually be encountered in vivo in a membrane-anchored form, tethered by Fc or complement receptors. Here we show that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigens from the target. B-cell antigen receptor accumulates at the synapse, segregated from the CD45 co-receptor which is excluded from the synapse, and there is a corresponding polarization of cytoplasmic effectors in the B cell. B-cell antigen receptor mediates the gathering of antigen into the synapse and its subsequent acquisition, thereby potentiating antigen processing and presentation to T cells with high efficacy. Synapse formation and antigen acquisition will probably enhance the activation of B cells at low antigen concentration, allow context-dependent antigen recognition and enhance the linking of B- and T-cell epitopes.

A DYNAMICAL MODEL OF B–T CELL REGULATION

We present a minimal regulatory model for the dynamics of the humoral immune response of two lymphocytes populations (B and T helper) interacting with a specific antigen pool (bacterium). Stability analysis reveals that the system accounts for the occurrence of multiple steady states in the absence as well as in the presence of the antigen population. The model exhibits (i) a state of immune memory, (ii) one state with high antigen and low helper concentration (disease), and (iii) one state with low antigen and high helper concentration (tolerance). The latter state allows oscillatory behavior. Injection of high antigen doses as well as minimal changes of structural parameters provoke the system to jump from the state of disease to the state of tolerance. This is reminiscent of therapies where the patient is treated with allergen, immuno-suppressants or drugs.

CA27.29: a valuable marker for breast cancer management. A confirmatory multicentric study on 603 cases

Recently, a fully automated method has become commercially available to measure the MUC-1-associated antigen CA27.29. The present investigation was performed in order to compare CA27.29 and CA15.3 in a wide series of patients affected with breast cancer. Overall, 603 cases with breast cancer and 194 healthy controls were investigated. Patients were enrolled in 4 institutions, while assays were performed in one laboratory. CA27.29 was measured by the ACS:180 BR assay (Bayer Diagnostics) and CA15.3 by the AxSYM (Abbott Laboratories). An excellent correlation was found between the results obtained by the two methods. The two markers showed comparable results in healthy controls, with higher levels in post-menopausal than in pre-menopausal subjects. The markers were significantly higher in primary breast cancer than in controls. The areas under the receiver operating characteristics (ROC) curves of the two tests were comparable, but CA27.29 showed better sensitivity in cases with low antigen concentrations (below the cut-off point). Accordingly, when comparing each test in different stage categories, significance levels of the differences were higher for CA27.29 than for CA15.3 for all T categories versus healthy controls, for pT1 versus pT2, for all N categories versus healthy controls and for node-negative versus N1-3 patients. From the results of the present study, that has been performed on samples taken at diagnosis and prior to any treatment from the widest series of patients with primary breast cancer reported so far, we can draw the following conclusions: CA27.29 provides comparable results to CA15.3; CA27.29 seems more sensitive than CA15.3 to limited variations of tumour extension; however, it cannot help clinicians in distinguishing stage I patients from stage II patients. However, from the point of view of clinical decision making, CA27.29 provides comparable results to CA15.3. CA27.29 is therefore suitable for routine use in the management of patients with breast cancer.

Single cell analysis of cytokine expression kinetics by human CD4+ T-cell clones during activation or tolerance induction.

Exposure to optimal peptide antigen concentrations induces human CD4+ T-cell clones to proliferate and secrete various cytokines. Higher (> 10-fold optimal) antigen concentrations cause long-term proliferative unresponsiveness, which can be reversed by exogenous interleukin-2 (IL-2). We call this condition 'tolerance'. We used intracellular cytokine staining and flow cytometric analysis to investigate the kinetics of interferon-gamma, tumour necrosis factor-alpha, IL-4 and IL-5 production during the initial phase of tolerance induction. Single cell analysis of interferon-gamma and IL-4 or IL-5 coexpression showed functional heterogeneity of cloned human CD4+ T cells. Superstimulation with phorbol 12-myristate 13-acetate and ionomycin (PI) revealed enhanced responsiveness shortly after tolerizing treatment, followed by reduced responsiveness. Both tolerized and activated T cells had similarly reduced cytokine responses when further stimulated with antigen during the following 48 hr, with limited enhancement following additional stimulation with PI. We conclude that cytokine induction is normally followed by a refractory phase, but that the expression of cytokines is enhanced in the initial phase of tolerance induction.

Mechanisms of allergen-specific immunotherapy.

Mechanisms of allergen-specific immunotherapy.

Serological analysis of specific IgA to Chlamydia pneumoniae: increased sensitivity of IgA antibody detection using prolonged incubation and high antigen concentration.

The microimmunofluorescence technique (MIF) is recognized as the only test hitherto allowing discrimination between different Chlamydia species and is considered to be the reference method for serology. This method was developed for the detection of IgG and IgM antibodies only. We investigated the effects of some test parameters on the ability of MIF to detect Chlamydia pneumoniae IgA. These parameters were the time needed for binding of serum IgA to C. pneumoniae antigen and the effect of antigen concentration on the outcome of IgA antibody testing. It was found that the most sensitive MIF tests for the detection of serum IgA antibodies were those in which an overnight incubation of sera with antigen slides containing high concentrations of chlamydial elementary bodies was employed. The number of patients with chronic infections found to have elevated IgA titers was increased by 25% using longer incubation times for the antibody-antigen reaction. Thirty-two sera from patients with coronary artery disease and confirmed chronic C. pneumoniae infection were used to compare antigen slides with low and high concentrations of elementary bodies with respect to IgA levels; 31/32 patients were found to have specific IgA antibodies to C. pneumoniae using the high antigen concentration, as opposed to only 22/32 patients using the low antigen concentration.

Use of D-dimer assays in the diagnosis of venous thrombosis.

In the course of fibrin formation, the D-domains of adjacent fibrin molecules within the fibrin polymer are covalently linked by factor XIIIa, leading to the formation of a D-domain dimer. Proteolysis of this cross-linked fibrin generates fibrin fragments D-dimer and E as terminal products. Fragment D-dimer therefore is an indicator for the proteolysis of cross-linked fibrin, whereas the monomeric fragment D can stem from fibrinogen and non-cross-linked fibrin. Various monoclonal antibodies have been prepared that distinguish between fragments D-dimer and D and allow the detection of fibrin derivatives in the presence of fibrinogen. These anti-D-dimer-antibodies have been shown to react with fragment D-dimer, but also detect dimeric D-domains within larger fibrin compounds, including cross-linked fibrin complexes generated in an early phase of coagulation activation. Assay systems for D-dimer antigen therefore may uncover intravascular clot formation early, by detection of fibrin complexes, and after completion of clot formation, by the detection of proteolytic fragments released from the particulate clot. Various trials have shown that low concentrations of D-dimer antigen in the blood exclude recent venous thrombosis or pulmonary embolism. Elevated levels may be caused by venous thrombotic disease, but also by a variety of other conditions, leading to intra- or extravascular fibrin formation. Assay systems include manual immunoagglutination assays, immunofiltration assays, microtiter plate enzyme-linked immunosorbent assay (ELISA) systems, automated ELISA systems, and latex-enhanced photometric immunoassays. According to clinical studies, D-dimer assays may be the "first line" of technical screening in symptomatic outpatients with suspected venous thrombosis or pulmonary embolism, but further prospective management trials, and improved standardization of assay systems, are needed for the validation of this approach.

Fc receptors for IgG and antigen presentation on MHC class I and class II molecules

Antigens internalized through specific membrane receptors are presented to helper CD4+T cells at antigen concentrations 103to 104fold lower than antigens internalized by fluid phase. B lymphocyte antigen receptors, mannose receptors and receptors for the Fc region of immunoglobulins, promote both internalization and efficient presentation at low antigen concentrations. Thus, binding to specific membrane receptors concentrate antigens on antigen presenting cells and mediates efficient uptake. Is this `quantitative' concentration of antigens on antigen presenting cells the end of the story? Or may `quality', i.e. selective intracellular antigen targeting, somehow influence the efficiency or specificity of MHC class I and class II-restricted antigen presentation?

Immunology: Ignorance or Tolerance in T-cell--Mediated Autoimmunity

Autoimmunity to antigens in pancreatic islets contributes to type 1 diabetes and thus control of this process has been of particular interest. Both tolerance (a process in which reactive CD8+ T cells are removed by apoptosis) or ignorance (a process in which autoreactive T cells do not respond to antigens in the islet) can occur. However, it has been unclear why some antigens induce one process but other antigens produce the other. Kurts et al. studied the effects of ovalbumin expressed in the islets of transgenic mice. They found that the concentration of the antigen determined whether tolerance or ignorance was observed. In low concentrations, ovalbumin was not detected by CD8+ T cells. However, if larger amounts of ovalbumin were expressed, cross-presentation (in which CD8+ T cells are activated in the lymph nodes by presentation of antigen from non-lymphoid tissues) occurred and resulted in deletion of CD8+ T cells reactive with ovalbumin. These two alternative mechanisms may allow an organism to be protected from autoimmunity in response to low or high concentrations of antigen. Kurts, C., Sutherland, R.M., Davey, G., Li, M., Lew, A.M., Blanas, E., Carbone, F.R., Miller, J.F.A.P., and Heath, W.R. (1999) CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose Proc. Natl. Acad. Sci. U.S.A. 96 : 12703-12707. [Abstract] [Full Text]