Range Of Antigen Concentrations Research Articles

Computational analysis of 4-1BB-induced NFκB signaling suggests improvements to CAR cell design

BackgroundChimeric antigen receptor (CAR)-expressing cells are a powerful modality of adoptive cell therapy against cancer. The potency of signaling events initiated upon antigen binding depends on the costimulatory domain within the structure of the CAR. One such costimulatory domain is 4-1BB, which affects cellular response via the NFκB pathway. However, the quantitative aspects of 4-1BB-induced NFκB signaling are not fully understood.MethodsWe developed an ordinary differential equation-based mathematical model representing canonical NFκB signaling activated by CD19scFv-4-1BB. After a global sensitivity analysis on model parameters, we ran Monte Carlo simulations of cell population-wide variability in NFκB signaling and quantified the mutual information between the extracellular signal and different levels of the NFκB signal transduction pathway.ResultsIn response to a wide range of antigen concentrations, the magnitude of the transient peak in NFκB nuclear concentration varies significantly, while the timing of this peak is relatively consistent. Global sensitivity analysis showed that the model is robust to variations in parameters, and thus, its quantitative predictions would remain applicable to a broad range of parameter values. The model predicts that overexpressing NEMO and disabling IKKβ deactivation can increase the mutual information between antigen levels and NFκB activation.ConclusionsOur modeling predictions provide actionable insights to guide CAR development. Particularly, we propose specific manipulations to the NFκB signal transduction pathway that can fine-tune the response of CD19scFv-4-1BB cells to the antigen concentrations they are likely to encounter.3kX61-hrxkM_LLAPo69ACnVideo

Square wave voltammetric approach to leptin immunosensing and optimization of driving parameters with chemometrics

Square wave voltammetry serves as an effective analytical means to evaluate antigen-antibody coupling at the solid-liquid interface. Herein, we describe 3-aminopropyltrimethoxysilane (APTMS) induced irreversible immobilization of anti-leptin to micellar gold nanoparticles (AuNPs). Antibodies (Abs) were orthogonally loaded on micellized AuNP assemblies via amino residual groups. The ratio of bound Ab molecules was determined by the Bradford assay. The AuNP/Ab layer modified electrodes with variable antibody surface coverage (∼400 ± 55–200 ± 30 Ab/NP) were analyzed in terms of change in backward, net current (Ip) components. The rate of antigen coupling was found to be consistent with the variation in antibody density as well as the binding affinity. The lowest detection limit was observed at the femtomolar level (0.25 fM/mL) over a wide range of antigen concentration (6.2 ng/mL to 0.12 fg/mL). The variables affecting the epitope-paratope interaction were further optimized using a chemometric approach and a response surface methodology (RSM).

A label-free electrochemical immunosensor based on encapsulated signal molecules in mesoporous silica-coated gold nanorods for ultrasensitive assay of procalcitonin

For immobilization and signal amplification of the probes, it is feasible and promising by using porous nanomaterials as nanocarriers. Herein, a novel label-free electrochemical immmunosensor was efficiently designed for ultrasensitive detection of procalcitonin (PCT). The immunosensor was prepared by using porous silica-coated gold nanorods (Au NRs@m-SiO2) to load electroactive dye thionine (Thi) on the electrode surface. Apart from the improved electrical conductivity, the porous feature highly increased the loading amount of Thi to boost the detection signals, while the good biocompatibility and protective microenvironment are beneficial to the largely improved stability for the target. For quantification of PCT, the developed immunosensor exhibited a good linear relationship in the antigen concentration range of 0.001–100 ng mL−1 with an ultra-low limit of detection (LOD, 0.39 pg mL−1, S/N = 3). Moreover, the built platform was successfully applied to such assay in human serum samples. The research provides some valuable guidelines for clinical screening and diagnosis of other biomarkers.

Electrical Double Layer Gated Field Effect Transistor Biosensors for the Quantitative Detection of Beta-Human Chorionic Gonadotropin

In this research, we have developed biosensors to detect beta human chorionic gonadotropin (beta-HCG) which is a hormone widely used to test for pregnancy and also serves as a biomarker for risk assessment of certain types of cancers. The biosensor methodology is based on extended gate design of electrical double layer (EDL) gated field effect transistor (FET). In this study, we fabricated a sensor array chip and functionalized the gold electrode surface with antibodies to capture the antigen, beta-HCG. Each sensor in the sensor array chip consists of two gold electrodes, one connected to the gate voltage supply and the other connected to the gate terminal of the FET, which is located on isolated PCB such that the test solution does not directly contact the transistor active region. A hand-held sensor measurement unit is fabricated which can apply the biasing voltages, measurement the resulting drain current signal and display the output results in LCD monitor. Fluorescence measurement technique is used to confirm and analyze the surface functionalization with antibodies to beta-HCG. The relation between beta-HCG antigen concentration and electrical signal is established by testing the sensor array chips using the hand-held device. The test samples are prepared by diluting purified stock proteins in 1X PBS, as it simulates the physiological conditions of human urine. Using our FET biosensor, we can directly detect beta-HCG in high ionic strength media like 1X PBS, without performing additional sample processing. Most pregnancy tests have a sensitivity of 25–50 IU/l (1.5-3 ng/ml), based on immunochromatography which is the most widely technique for detection of beta-HCG. Using our sensor methodology, we can obtain a sensor response curve with a dynamic range of antigen concentration between 0.03 to 30 ng/ml. As the antigen concentration approximately approaches 10 ng/ml, the current gain value is nearly saturated. The result shows that when beta-HCG antigen concentration increases, current gain value decreases. This sensing methodology can facilitate quantitative detection of beta-HCG in physiological conditions, whereas the current commercially available assays provide only qualitative test results. The sensor demonstrates a very wide dynamic range of detection, with a very low detection limit, which shows the potential of our sensor to detect pregnancies in very early stages. Furthermore, this sensor platform can enable the use of beta-HCG for risk stratification of cancers as well. The sensor can be batch produced and does not use additional labels/reagents or sample pre-processing techniques, which can greatly enhance the cost-effectiveness, comparable to the current commercialized beta-HCG assays.

Synthetic Antigen Gels as Practical Controls for Standardized and Quantitative Immunohistochemistry.

Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX-XXX, 2019).

Zika virus detection using antibody-immobilized disposable cover glass and AlGaN/GaN high electron mobility transistors

Zika virus detection was demonstrated using antibody-functionalized cover glasses externally connected to the gate electrode of an AlGaN/GaN high electron mobility transistor (HEMT). A pulsed bias voltage of 0.5 V was applied to an electrode on the region of the cover glass region functionalized with antibody, and the resulting changes of drain current of the HEMT were employed to determine the presence of Zika virus antigen concentration ranging from 0.1 to 100 ng/ml. The dynamic and static drain current changes as a function of Zika virus concentration were modeled with a spring-like elastic relaxation model and the Langmuir extension model, respectively. Excellent fits to the data were found with relaxation time constants of antibody and antigen molecules in the range of 11 μs and 0.66–24.4 μs, respectively, for the concentration range investigated. The ratio of antibody bound with antigen to the total available antibody on the functionalized contact window was in the range of 0.013–0.84 for the Zika antigen concentration range of 0.1–100 ng/ml. Since the HEMT is not exposed to the bio-solution, it can be used repeatedly. The functionalized glass is the only disposable part in the detection system, showing the potential of this approach for hand-held, low cost sensor packages for point-of-care applications.

A glassy carbon immunoelectrode modified with vanadium oxide nanobelts for ultrasensitive voltammetric determination of the core antigen of hepatitis C virus

The authors describe a voltammetric immunoassay for the hepatitis C virus core antigen. It is based on the use of vanadium oxide nanobelts that were used to modify a glassy carbon electrode (GCE). The nanobelts have a large specific surface area that can be heavily loaded with antibody against the hepatitis C virus core antigen. Following blocking with BSA and by using riboflavin as an electrochemical probe, the immuno GCE (best operated at a working voltage of −0.45 V vs. Ag/AgCl) has a linear response that covers the 10 fg·mL−1 to 100 ng·mL−1 core antigen concentration range and a limit of detection as low as 1.3 fg mL−1. Compared to the other redox probes, riboflavin is superior because it is oxidized in the negative potential range, where the number of interfering species for riboflavin is small. The method was successfully applied to the determination of hepatitis C virus core antigen in spiked serum. In our perception, the use of a V2O5 nanobelt film as a support for bioreceptors has a wide scope in that in that it may be extended to immunoassays for various other diagnostically significant analytes.

In Vitro Quantified Determination of β-Amyloid 42 Peptides, a Biomarker of Neuro-Degenerative Disorders, in PBS and Human Serum Using a Simple, Cost-Effective Thin Gold Film Biosensor.

A simple in vitro biosensor for the detection of β-amyloid 42 in phosphate-buffered saline (PBS) and undiluted human serum was fabricated and tested based on our platform sensor technology. The bio-recognition mechanism of this biosensor was based on the effect of the interaction between antibody and antigen of β-amyloid 42 to the redox couple probe of K4Fe(CN)6 and K3Fe(CN)6. Differential pulse voltammetry (DPV) served as the transduction mechanism measuring the current output derived from the redox coupling reaction. The biosensor was a three-electrode electrochemical system, and the working and counter electrodes were 50 nm thin gold film deposited by a sputtering technique. The reference electrode was a thick-film printed Ag/AgCl electrode. Laser ablation technique was used to define the size and structure of the biosensor. Cost-effective roll-to-roll manufacturing process was employed in the fabrication of the biosensor, making it simple and relatively inexpensive. Self-assembled monolayers (SAM) of 3-Mercaptopropionic acid (MPA) was employed to covalently immobilize the thiol group on the gold working electrode. A carbodiimide conjugation approach using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N–hydroxysuccinimide (NHS) was undertaken for cross-linking antibody of β-amyloid 42 to the carboxylic groups on one end of the MPA. The antibody concentration of β-amyloid 42 used was 18.75 µg/mL. The concentration range of β-amyloid 42 in this study was from 0.0675 µg/mL to 0.5 µg/mL for both PBS and undiluted human serum. DPV measurements showed excellent response in this antigen concentration range. Interference study of this biosensor was carried out in the presence of Tau protein antigen. Excellent specificity of this β-amyloid 42 biosensor was demonstrated without interference from other species, such as T-tau protein.

Solid sorbitan esters nanoparticles are efficient and low-cost vehicles for subunit vaccines: Proof of concept with Neisseria meningitidis protein Mip

In this study we present evidence of the vaccine potential of nanoparticles based on sorbitan esters (sorbitan monooleate, Span® 80), solid sorbitan esters nanoparticles (SSN) which are the lowest cost antigen nanovehicles proposed to date. We choose the Neisseria meningitidis macrophage infectivity potentiator (Mip) protein as a proof of concept target. The rMip-SSN nanosystems were evaluated for their physico-chemical properties, antigen loading and integrity as well as physical stability in various storage conditions. The immune response elicited by rMip-SSN was compared to that elicited by rMip-SSN with the conventional adjuvant Al(OH)3 or with rMip in saline. SSN were able to associate, at a wide range of antigen concentrations with 100% efficiency, preserving the association and integrity of the antigen after long-term storage. Antibodies elicited by rMip-SSN mediated deposition of complement factors on the surface of meningococcal isolates and protective bactericidal titres even after rMip-SSN had been stored for nine months lyophilized at 4 °C. The association of different antigens to SSN can be easily achieved by electrostatic interactions by mixing the preformed nanosystems with the antigens of choice. Thus we can envisage the use of these nanoplatforms for the development of low-cost immunization kits ready for use for flexible vaccination strategies.

TiO2 nanoparticles doped with Celestine Blue as a label in a sandwich immunoassay for the hepatitis C virus core antigen using a screen printed electrode

The authors describe an electrochemical immunoassay for the core antigen of hepatitis C virus (HCV). The method is based on the use of a screen-printed carbon electrode (SPCE) that was modified with a Nafion@TiO2 nanocomposite and loaded with secondary antibody (Ab2) to entrap Celestine Blue (CB). The material has architecture of the type CB/Ab2/Nafion@TiO2. A nanocomposite consisting of graphene, ionic liquid and fullerene was deposited on the SPCE first, and rhodium nanoparticles (RhNPs) were then deposited on the surface of modified electrode in order to immobilize primary antibody (Ab1). The antigen and CB/Ab2/Nafion@TiO2 were conjugated one by one to form a sandwich-type immunocomplex. The signal was obtained by differential pulse voltammetry whose intensity is related to the concentration of the antigen. The assay, if operated at a working voltage of typically 0.35 V (vs. Ag/AgCl) has a response that is linear in the 0.1 to 250 pg⋅mL−1 HCV core antigen concentration range, and the limit of detection is as low as 25 fg⋅mL−1. The assay was applied to the determination of the HCV core antigen in spiked human serum samples. In our perception, the method represents a promising platform for the detection of various antigens if appropriate antibodies are available.

Characterization of specimens for quality control of malaria rapid diagnostic tests: what is the best indicator for performance?

Malaria rapid diagnostic tests (RDTs) are now considered an acceptable alternative for parasite based diagnosis in the absence of adequate quality microscopy. Selection of the most appropriate RDT among the sheer number of tests available commercially has long been a challenge, raising the need to objectively assess and compare test performance. In order to facilitate RDT procurement and stimulate improvement of the quality of RDTs, methods were developed for the collection and characterization of geographically diverse, malaria infected clinical blood specimens to set up a specimen bank for the evaluation of malaria RDTs, as part of the WHO Malaria RDT Product Testing programme. Parasite positive samples were diluted to low (200 p/ul) and higher (2000 p/ul) parasite densities and used to provide comparative data on the performance of RDT lots submitted by manufacturers. Only samples containing a single species and producing consistent antigen quantification results were eligible for inclusion in the WHO Malaria Specimen Bank. At both 200 and 2000 p/μl, a large variation in antigen concentrations was found, especially for P. falciparum HRP2, and to a lesser extent for pLDH and aldolase (both in P. falciparum and P. vivax samples); a trait that was independent of geographical origin. Possible reasons for this include the duration of infection at the time of blood collection and principally, differences in antigen expression between different isolates and stages of parasites. It is critical that performance evaluation panels are standardised at a parasite density being not only clinically relevant but also being close to the test detection limit to detect differences in test performance. Given the significant fluctuation in antigen concentrations at a given parasitaemia, appropriate sample sizes are required to cover that range and challenge the RDTs at the low end of this range of antigen concentrations. Antigen concentrations that are relevant for improvements in target sensitivity for RDTs are still to be determined for samples with parasite densities below 200 p/μl.

Label-free aptamer sensor based on silicon microring resonators

Abstract We present a label-free DNA aptamer sensor based on silicon microring resonators for the detection of human immunoglobulin E (IgE) and human thrombin. An array of silicon resonators consisting of three sensor microrings and one reference microring was fabricated and its surface sensitivity, detection limit and repeatability were investigated by the deposition of poly (sodium-4-styrenesulfonate) (PSS) and poly (allylamine hydrochloride) (PAH) multilayers. The surface sensitivity and the limit of detection of the fabricated sensor ring are found to be 0.35 nm/ng mm−2 and 26 pg/mm2 in air and 0.24 nm/ng mm−2 and 38 pg/mm2 in water, respectively. We have demonstrated quantitative, sensitive and specific detection of human IgE and thrombin with aptamer modified silicon microring resonators over a broad range of antigen concentrations. The sensor array showed an experimental detection limit of 33 pM and 1.4 nM for IgE and thrombin, respectively. Multiplexing was also performed successfully with a mixed solution of IgE and thrombin and proved the sensor to be highly specific. We believe our silicon microring resonator technology in combination with aptamers offers a low cost solution for various point-of-care applications.

A Novel Immunogen to Modulate Cytokine Production and Promote Immune System Reconstitution in HIV–AIDS

This 'proof of concept' study was implemented in anticipation of identifying and testing a novel antigen of human origin as a potential immunogen in a paradigm that emphasizes immunomodulation and immune system reconstitution as requisites to the development of an effective human immunodeficiency virus (HIV)-acquired immune deficiency syndrome vaccine. Fifteen HIV-infected, highly active antiretroviral therapy (HAART) naive, otherwise healthy male seropositive patients were stratified by [CD4+] into 3 groups of 5 patients: group 1 >500/mm; group 2 > 250/mm but <500/mm; and group 3 < 250/mm. Five healthy male subjects were used as controls. Replicate peripheral blood mononuclear cell (PBMC) [H]thymidine uptake phytohemaglutinin-stimulated proliferation studies, and serum cytokine assays were carried out in the presence or absence of Kveim antigen at dilutions ranging from 0.001 to 100 μg/mL. Serum cytokines [interleukin-2 (IL-2), IL-4, IL-6, interferon gamma, and tumor necrosis factor alpha] were assayed using standardized methodology. Nonparametric statistical analyses and linear regression analysis were used to test for statistical significance and strength of associations. PBMCs harvested from HIV-infected patients and incubated, ex vivo, demonstrated reproducible, antigen concentration-dependent changes in cytokine production over a range of antigen concentrations (0.001-100 μg/mL) in contrast to antigen-naive PBMCs and controls. Significant correlations were demonstrated between antigen concentration and the amount of cytokines secreted. The magnitude of the cytokine response and the patterns of cytokine secretion were HIV group-specific and could be used to identify and distinguish between the 3 groups of HIV-infected subjects. A shift toward the production of type 1-like (Th1) cytokines characteristically seen in systemic sarcoidosis and associated with effective HAART was seen when patterns of cytokine secretion were compared between antigen exposed and antigen-naive PBMCs. PBMCs harvested from seropositive HIV-infected patients and exposed to the Kveim antigen have the following properties: (1) They demonstrate proliferation and exhibit an antigen concentration-dependent secretion of cytokines. The magnitude of the cytokine response can be used to identify and distinguish between groups of seropositive patients stratified by [CD4+]. (2) These PBMCs secrete cytokines in patterns suggestive of a shift to a type 1-like (Th1) response characteristic of HAART and sarcoidosis as opposed to the type 2-like (Th2) cytokine profile characteristic of HIV-acquired immune deficiency syndrome.

An Electrochemically Reduced Graphene Oxide-Based Electrochemical Immunosensing Platform for Ultrasensitive Antigen Detection

We present an electrochemically reduced graphene oxide (ERGO)-based electrochemical immunosensing platform for the ultrasensitive detection of an antigen by the sandwich enzyme-linked immunosorbent assay (ELISA) protocol. Graphene oxide (GO) sheets were initially deposited on the amine-terminated benzenediazonium-modified indiun tin oxide (ITO) surfaces through both electrostatic and π-π interactions between the modified surfaces and GO. This deposition was followed by the electrochemical reduction of graphene oxide (GO) for preparing ERGO-modified ITO surfaces. These surfaces were then coated with an N-acryloxysuccinimide-activated amphiphilic polymer, poly(BMA-r-PEGMA-r-NAS), through π-π stacking interactions between the benzene ring tethered to the polymer and ERGO. After covalent immobilization of a primary antibody on the polymer-modified surfaces, sandwich ELISA was carried out for the detection of an antigen by use of a horseradish peroxidase (HRP)-labeled secondary antibody. Under the optimized experimental conditions, the developed electrochemical immunosensor exhibited a linear response over a wide range of antigen concentrations with a very low limit of detection (ca. 100 fg/mL, which corresponds to ca. 700 aM). The high sensitivity of the electrochemical immunosensor may be attributed not only to the enhanced electrocatalytic activity owing to ERGO but also to the minimized background current owing to the reduced nonspecific binding of proteins.

Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).

Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.

The enormous diversity of the naive T cell repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. The low frequency of antigen-specific naive T cells, their high activation threshold, and the constrains of antigen-processing and presentation have hampered analysis of naive repertoires to complex protein antigens. In this study, libraries of polyclonally expanded naive T cells were used to determine frequency and antigen dose–response of human naive CD4+ T cells specific for a variety of antigens and to isolate antigen-specific T cell clones. In the naive repertoire, T cells specific for primary antigens, such as KLH and Bacillus anthracis protective antigen, and for recall antigens, such as tetanus toxoid, cytomegalovirus, and Mycobacterium tuberculosis purified protein derivative, were detected at frequencies ranging from 5 to 170 cells per 106 naive T cells. Antigen concentrations required for half-maximal response (EC50) varied over several orders of magnitude for different naive T cells. In contrast, in the memory repertoire, T cells specific for primary antigens were not detected, whereas T cells specific for recall antigens were detected at high frequencies and displayed EC50 values in the low range of antigen concentrations. The method described may find applications for evaluation of vaccine candidates, for testing antigenicity of therapeutic proteins, drugs, and chemicals, and for generation of antigen-specific T cell clones for adoptive cellular immunotherapy.

Rapid Polyelectrolyte‐Based Membrane Immunoassay for the Herbicide Butachlor

Oppositely charged water‐soluble polyelectrolytes were used in the developed membrane immunoenzyme assay for the herbicide butachlor. High‐affinity and rapid binding between polyanion polymethacrylate and polycation poly(N‐ethyl‐4‐vinylpyridinium) was applied to separate reacted and free immunoreactants. Competitive immunoassay format with peroxidase‐labeled antigen was realized. The insoluble colored product of the peroxidase reaction was formed by bound labeled immune complexes and was reflectometrically detected. The assay combines short duration (15 min), high sensitivity (0.03 g/mL) and availability for out‐of‐laboratory testing. Different image processing algorithms were used to determine the herbicide content. Low variation coefficients of the measurements in the proposed quantitative assay, namely 4.8–9.0% for the range of antigen concentrations from 0.1 to 3.0 ng/mL, are evidence of the assay effectiveness. Possibility to control the butachlor content in mineral, artesian, and drinking water was demonstrated.

Results from modeling of B-Cell receptors binding to antigen

In the late 80's, Dintzis et al. conducted an experiment which showed that T-Cell independent activation of B-cells needs high-valence antigen and happens only in a narrow range of antigen concentration. These experiments were believed to be explained by the “immunon” theory that requires that a minimum number of receptors need to be cross-linked to activate a cell. However, the immunon theory does not take into account receptor dynamics and cannot explain the lack of immune response at high antigen concentration or low antigen valence. We propose instead a simple, new mechanism for the T-Cell independent activation of B-Cell, which includes receptor endocytosis. Our model focuses on the fact that for the majority of antigens where the B-Cell is activated with T-Cell help, the kinetic parameters for binding, unbinding and endocytosis must be tuned so that there is an equilibrium between the number of receptors bound on the surface of the B-Cell and the number of antigen-bound receptors endocytosed. This equilibrium mechanism is probably generic and will also occur even when the B-Cell is activated by antigen without T-Cell help. By computer modeling, we show that if we accept this hypothesis of the requirement for equilibrium between the two mechanisms of binding and endocytosis, then we can explain both the valence cutoff and the low and high zone tolerance seen in the Dintzis experiment.

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.

In vitro antibody response to the tetrapeptide repeats of Plasmodium falciparum circumsporozoite protein by splenocytes from mice infected with P. yoelii yoelii.

The antibody response to the recombinant protein, R32tet32, which contained the repetitive sequence (NANP)n of Plasmodium falciparum CSP was determined in C57BL/6 mice during the course of nonlethal infection with Plasmodium yoelii 17X. Marked suppression of the IgG antibody response to R32tet32 occurred when mice were immunized at peak parasitemia (on day 16). In vitro antibody responses of spleen cells from acutely infected mice to R32tet32 were similarly suppressed. Stimulation of normal spleen cells cultured for 5 days with 100 ng/ml of R32tet32 gave an optimal IgG antibody response, but spleen cells from infected mice obtained at peak parasitemia failed to respond to a broad range of antigen concentrations. Cocultivation studies employing enriched lymphocyte populations from infected and uninfected C57BL/6 mice indicated that both T and B cells from infected mice were defective in their response to R32tet32. The response to the repetitive region was restored by the addition of recombinant mouse interleukin-2 (IL-2) at a dose of 50 U/ml to cultures of spleen cells from infected mice.