Reactivity of T cells from seronegative patients with myasthenia gravis to T cell epitopes of the human acetylcholine receptor.

Seronegative (SN) patients with myasthenia gravis (MG) have clinical and electrophysiologic features similar to those of seropositive (SP) patients, and they respond to the same therapeutic measures. However, because SN patients lack detectable (by standard radioimmunoassays) serum antibodies to acetylcholine receptor (AChR), which are considered to have a crucial role in MG, the pathophysiologic basis for the disease is not clear. We therefore compared the ability of peripheral blood lymphocytes (PBL) of SN patients (11) and SP patients (39) to respond to myasthenogenic T cell epitopes of human AChR. We tested two aspects that relate to T-cell immunity: 1) T cell responses to myasthenogenic peptides by proliferation and IL-2 production, and 2) the ability of antigen-presenting cells to bind these T-cell epitopes. T cells of SN patients did not differ from those of SP patients in their ability to respond and to bind the two human AChR-derived myasthenogenic peptides. This supports the belief that most SN patients indeed suffer from an autoimmune disease directed against the AChR. The presence of T-cell immunity in the absence of antibodies may emphasize the importance of AChR-specific T cells in MG.

Binding of peptides of the human acetylcholine receptor alpha-subunit to HLA class II of patients with myasthenia gravis.

MG is an autoimmune disease in which T cells specific to T-cell epitopes of the human acetylcholine receptor play a role. We have identified two peptides, p195-212 and p259-271, of the human acetylcholine receptor alpha-subunit, to which PBLs of MG patients responded by proliferation. Nevertheless, proliferation assays are relatively complicated to perform and might be affected by medications taken by the patients. Therefore, we tested the possibility of using a different assay to determine recognition of these peptides by MG patients. Thus, we performed a direct binding assay using biotinylated peptides and APCs from peripheral blood of MG patients and healthy controls. With this assay we detected the binding of the two peptides to the surface of intact APCs of both MG patients and control donors. Moreover, the presentation of peptide p259-271 by individuals with MG was significantly higher than that observed in healthy subjects. The peptides were specifically bound to HLA class II determinants on the APCs, as shown by inhibition with antibodies to the HLA class II haplotypes of the individuals investigated. Moreover, the binding of these peptides was in correlation with their ability to induce specific proliferative responses of peripheral blood T cells of these patients. The ability to screen for potentially pathogenic epitopes in each patient is of importance for the future design of specific inhibitory analogues that might be used to treat MG.

Photochemically-activated electrodes: application in design of reversible immunosensors and antibody patterned interfaces.

Antigen monolayers assembled onto Au electrodes associated with a quartz crystal act as electrochemical or microgravimetric quartz-crystal-microbalance (QCM) sensing interfaces for the complementary antibody. Electrochemical analysis of the antibody (Ab) is based on the insulation of the antigen monolayer electrode by the associated Ab towards a redox probe in the electrolyte solution. Ferrocene-modified glucose oxidase (Fc-GOx) and glucose are employed as redox probes for the amperometric transduction of the Ab association to the electrode. Bioelectrocatalyzed oxidation of glucose provides an electrochemical route to amplify the antigen-Ab complex formation. Electrochemical analysis of the dinitrophenyl antibody, DNP-Ab, by a dinitrophenyl-lysine monolayer electrode is presented. QCM analysis of the Ab is based on the frequency changes of the quartz crystal resulting from the association of the Ab to the crystal assembly. This method is discussed with the analysis of the fluorescein antibody, Flc-Ab, using a fluorescein monolayer-modified quartz crystal. A novel method to tailor reversible immunosensor devices by the application of photoisomerizable antigen monolayers on electrodes is presented. The antigen is modified by photoactive units exhibiting reversible photoisomerizable properties. In one photoisomer state, the antigen exhibits affinity for the Ab and enables its electrochemical or QCM analysis. Photoisomerization to the complementary state perturbs the antigen structure and the monolayer lacks affinity for the Ab. This enables the washing-off of the Ab and the regeneration of the actively sensing interface by a second illumination process that restores the antigen monolayer-modified surface. This method is exemplified by the development of a reversible DNP-Ab sensing electrode. N-Mercaptobutyl dinitrospiropyran was assembled as a photoisomerizable monolayer on a Au electrode. The dinitrospiropyran monolayer, SP-state, exhibits affinity for the DNP-Ab and enables the amperometric detection of the Ab using Fc-GOx and glucose as redox probe. The complementary photoisomerized protonated dinitromerocyanine monolayer, MRH(+)-state, lacks affinity for the DNP-Ab. By photoisomerization of the DNP-Ab associated with the SP-monolayer electrode to the MRH(+)-monolayer state, the DNP-Ab is washed-off, and by a second illumination process, the MRH(+)-monolayer is re-isomerized to the SP-monolayer assembly, which is the active interface for further analysis of the DNP-Ab. Cyclic amperometric detection of the DNP-Ab by the photoisomerizable dinitrospiropyran monolayer is demonstrated. The association of the DNP-Ab to the SP-monolayer electrode and the dissociation of the Ab from the MRH(+)-monolayer electrode are confirmed by QCM experiments using a dinitrospiropyran monolayer-modified quartz crystal. The insulating features of an antigen-Ab complex on a conductive surface and the photochemically controlled association of an antibody to a photoisomerizable monolayer assembled onto the surface were used to develop means for micropatterning of surfaces by the antibody. A dinitrospiropyran antigen monolayer was assembled onto conductive ITO glass. A DNP-Ab solution was used as 'ink solution' to pattern the surface. The Ab-pattern was imaged by electrochemical copper deposition onto the Ab-lacking surface domains. The dinitrospiropyran monolayer assembled onto ITO or Pyrex glass surfaces was employed as an active interface for the photolithographic patterning of the surface with the DNP-Ab. (ABSTRACT TRUNCATED)

Down-regulation of hepatic peripheral-type benzodiazepine receptors caused by acute lead intoxication.

In the present study we investigated the influence of acute lead poisoning upon the expression of benzodiazepine receptors. In addition, we examined if administration of PK 11195, an isoquinoline carboxamide derivative, to lead-poisoned rats could modulate the changes in receptor binding properties achieved by lead alone. Lead poisoning was ascertained by determination of urine delta-aminolevulinic acid levels and lead levels in rat livers. Scatchard analysis of saturation curves of [3H]PK 11195 binding to liver membranes of rats treated with lead alone or with both lead and PK 11195 showed and approximately two-fold decrease in receptor density in comparison with control groups. Peripheral benzodiazepine receptor density in kidneys and adrenals of poisoned rats was not changed by lead intoxication per se or by coadministration of PK 11195. Scatchard analysis of saturation curves of [3H]Ro 15-1788 binding in rat cerebral cortex tissue showed no difference in the receptor density between the various groups. The Kd values of all organs were in the nanomolar range (1-4 nM). We conclude that PK 11195 is not a protective agent of hepatic peripheral benzodiazepine receptors in lead intoxication. Moreover, it causes over-accumulation of lead in hepatocytes in an unknown mechanism of action.

Different roles of D-amino acids in immune phenomena.

Peptides and polypeptides either fully or partially built of D-amino acids interest researchers because of their advantages over all L peptides and polypeptides. In exploiting these characteristics, one should realize that the resulting molecules are nonetheless not inert, but rather may induce a unique immune response, which is hardly cross-reactive with the L-enantiomer. Moreover, cross-reaction between the L- and the D-sequences is limited also at the T cell level, probably due to different sterical conformations of the MHC-antigen-T cell receptor complexes formed. Polypeptides built exclusively of D-amino acids lead to antibody formation only at a relatively low concentration, otherwise they provoke immunological paralysis. The specificity of the immune response toward peptides containing D-amino acid residues is exquisite, and often D-amino acids play a dominant role in defining the specificity. Polypeptides composed exclusively of D-amino acids are thymus-independent antigens. Nevertheless, it is possible to prepare against them highly specific T cell hybridomas. In future plans for synthetic vaccines against infectious or autoimmune diseases, the inclusion of D-amino acids may be an advantage in terms of both specificity and efficacy, the latter because of longer persistence in an undigested form because they resist enzymatic degradation.

Processing requirements of two acetylcholine receptor derived peptides for binding to antigen presenting cells and stimulation of murine T cell lines.

We have previously identified two myasthenogenic T cell epitopes of the human acetylcholine receptor (AChR) alpha subunit, peptides p195-212 and p259-271. These peptides were the immunodominant T cell epitopes of AChR in SJL and BALB/c mice respectively, and only cryptic in C3H.SW strain. In order to find out whether these mouse strains differ in their requirements for processing of the same T cell epitopes, we used p195-212 specific T cell lines from SJL, TCSJL195-212, and C3H.SW, TCSW195-212, mice, and p259-271 specific T cell lines from BALB/c, TCBALB/c259-271, and C3H.SW, TCSW259-271, mice. The peptide-specific proliferative responses of the lines TCSW195-212 and TCSW259-271, originated from strains in which these peptides are cryptic epitopes, were inhibited significantly in the presence of several inhibitors of proteases or glutaraldehyde-fixed antigen presenting cells (APC). In contrast, the proliferative responses of the lines TCSJL195-212 and TCBALB/c259-271, established from strains in which these peptides are immunodominant, were only slightly affected by the above inhibitors or by fixation of the APC. Using a direct binding assay of biotinylated peptides to live intact APC, we showed that peptides p195-212 and p259-271 preserved their binding capacity to APC of SJL and BALB/c mice respectively when processing was inhibited. Thus, the AChR peptides that represent cryptic T cell epitopes have to be processed before they can be recognized by T cells, whereas no further processing is necessary for APC presentation and T cell stimulation when these peptides are immunodominant epitopes.

Direct binding of a synthetic multichain polypeptide to class II major histocompatibility complex molecules on antigen-presenting cells and stimulation of a specific T-cell line require processing of the polypeptide.

T-cell activation involves the recognition of foreign antigens as a complex with self-major histocompatibility complex (MHC) proteins on the surface of antigen-presenting cells (APC). Protein antigens usually require uptake by the APC and processing that results in the generation of peptide fragments. The branched synthetic polypeptide (Tyr, Glu)-Ala--Lys was chosen as a model antigen to follow the processing requirements, leading to T-cell activation. It has been demonstrated, by using fixed APC and various inhibitors of proteases, that (Tyr, Glu)-Ala--Lys has to be processed to stimulate a (Tyr, Glu)-Ala--Lys-specific T-cell line of C3H.SW (H-2b) origin to proliferate. To determine whether processing of (Tyr,Glu)-Ala--Lys is required to allow its association with the MHC class II molecules, biotin was covalently attached to it. Binding of the biotinylated (Tyr,Glu)-Ala--Lys to MHC class II gene products on the surface of intact normal APC was directly detected by phycoerythrin-streptavidin. The specificity of the binding was confirmed by its inhibition with anti-I-Ab antibodies as well as with excess of nonlabeled (Tyr,Glu)-Ala--Lys. Furthermore, introducing several inhibitors of proteases to the binding assay, we could substantiate that the proteolysis of (Tyr,Glu)-Ala--Lys is required to allow association of the resulting peptidyl T-cell epitopes with the MHC class II molecules themselves. The presence of the biotin moiety in the resulting peptides suggests that the T-cell epitopes of (Tyr,Glu)-Ala--Lys contain the N-terminal portion of the side chains of the branched polypeptide. An apparent Kd of 8.05 x 10(-8) M was determined, and optimal binding was detected after 10 hr of incubation with the antigen. The latter phenomenon is not due to slow uptake, since uptake of (Tyr,Glu)-Ala--Lys occurs mainly during the first 30 min of incubation, but rather reflects the events of processing that precede MHC interaction.

MHC class II-mediated T cell response to DNA.

Anti-DNA antibodies are elevated in several autoimmune diseases, including systemic lupus erythematosus. Yet, DNA was not shown to be presented by molecules of the major histocompatibility complex (MHC), nor was it reported to specifically stimulate T cells in vivo, although these steps are essential for antibody production. We now demonstrate DNA-specific T cell activation, which involves presentation of DNA by MHC class II molecules. T cells, isolated from lymph nodes of mice immunized with a murine monoclonal anti-anti-DNA antibody, proliferated in response to DNA. Moreover, presentation of DNA by murine antigen-presenting cells could be inhibited with an isotype-specific anti-Ia antibody, and with peptides restricted by the same H-2 haplotype, suggesting that it is MHC class II-mediated. These results indicate that DNA can play a direct role in the regulation of T cells and in autoimmune processes.

Left, but not right, one-lung ventilation causes hypoxemia during endoscopic transthoracic sympathectomy.

OBJECTIVE: To describe the respiratory and cardiovascular effects of one-lung ventilation, using a double-lumen tube, during endoscopic transthoracic sympathectomy. DESIGN: A prospective clinical study. SETTING: A university-affiliated medical center. PARTICIPANTS: Nineteen adult patients (10 men, 1 woman) between 16 and 35 years of age, ASA (American Society of Anesthesiologists) physical status I and II, participated in the study. INTERVENTIONS: Endoscopic transthoracic sympathectomy was performed under general anesthesia, using a double-lumen endobronchial tube, after induction of artificial pneumothorax plus insufflation of CO2 into the operated chest. Via radial artery cannulae, one to three arterial blood gas samples were taken during two-lung ventilation before surgery, at each one-lung ventilation, in most cases during the period of two-lung ventilation when switching between the operated sides, and after surgery. MEASUREMENTS AND MAIN RESULTS: Comparisons were performed using the Wilcoxon matched-pairs single-ranks test. Left-lung ventilation and right-chest operation caused profound decrease of arterial oxygen partial pressure (PaO2), compared with two-lung ventilation before surgery (70.7%, P > 0.0003) and compared with PaO2 at two-lung ventilation during and after surgery (decrease of 80.1% and 75.3%, respectively; P > 0.001 and < 0.005, respectively). Right-lung ventilation and left-chest operation did not cause hypoxemia. Arterial CO2 partial pressure, pH, and bicarbonate, as well as hemodynamic parameters, did not change from baseline values throughout surgery. CONCLUSIONS: Pulse oximetry and repeated blood gas measurements are needed during endoscopic transthoracic sympathectomy in order to detect and treat hypoxemic events, which may jeopardize the patient's life.

A computational fluid dynamic model of antigen-antibody surface adsorption on a piezoelectric immunosensor.

A novel computational fluid dynamic model describing the antigen-antibody binding on an electrode surface is presented. It was assumed that the adsorption rate of the antibody sample is dependent upon the flow field in the vicinity of the electrode. Numerical solution of the steady flow in a two-dimensional triangular cell using the Navier-Stokes equations was carried out for predicting mass adsorption on the surface of the crystal. The relationships between the mass adsorbed over the area surface of the electrode, the kinetics of the binding process, and the flow field were determined. The effect of the inlet conditions (location, velocity magnitude, and direction) on the time constant of the mass adsorption process was investigated. It was found that the time constant was decreased by moving the inlet near the edge of the crystal or increasing the normal to the boundary component of the velocity. These changes may significantly reduce the time needed to conduct the test.

Piezoelectric immunosensors for urine specimens of Chlamydia trachomatis employing quartz crystal microbalance microgravimetric analyses.

The assembly of a biosensor for Chlamydia trachomatis based on the microgravimetric quartz crystal microbalance (QCM) analysis of the bacteria association to an antibody-functionalized electrode is described. The sensing interfaces consist of a primary cystamine monolayer assembled onto Au electrodes associated with the quartz crystal. The monolayer is further modified with sulfosuccinylimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB) and the goat IgG-anti-mouse IgG Fc-specific Ab or the fragmented F(ab')2 anti-mouse IgG Ab that act as sublayers for the association of the sensor-active anti-C. trachomatis LPS-Ab. Bacteria in the concentration range from 260 ng.mL-1 to 7.8 micrograms.mL-1 are sensed by the functionalized crystals. The association of C. trachomatis to the sensing interface can be confirmed and amplified via interaction of the crystal with various anti-C. trachomatis antibodies. Urine-pretreated functionalized quartz crystals are applied in the analysis of C. trachomatis in urine samples. The sensitivity limits of the electrodes for sensing the bacteria in urine samples corresponds to approximately 260 ng.mL-1. The functionalized crystals assembled via association of anti-C. trachomatis LPS-Ab to the fragmented F(ab')2 anti-mouse IgG Ab reveal long-term stability upon storage at 4 degrees C.

Dichotomy between the T and the B cell epitopes of the synthetic polypeptide (T,G)-A--L.

Studies with the well-characterized, synthetic, random-multichain polypeptide poly(LTyr,LGlu)-poly(DLAla)-poly(LLys) (T,G)-A-L) led to the discovery of determinant-specific genetic control of the immune response, as well as to other immunological phenomena. Moreover, the tetrapeptide TyrTyrGluGlu built on the same backbone ("(T-T-G-G)-A--L") was found to represent its major B cell epitope. We have recently shown that for interaction with major histocompatibility complex class II molecules and stimulation of T cells, (T,G)-A--L requires proteolytic processing and the resulting T cell epitopes are close to the N termini of the branched polymer's side chains. Thus, we were interested to elucidate the major T cell epitope of (T,G)-A--L, by using the ordered polypeptides (T-T-G-G)-A--L and (T-G-T-G)-A--L, in which only the two internal amino acids of the tetrapeptide attached to the side chains are switched. We established T cell lines to these antigens, and found that the ordered analog (T-T-G-G-)-A--L, which was defined as the B cell epitope of (T,G)-A--L, did not represent its T cell epitope, whereas (T-G-T-G)-A--L, to which only a minor anti-(T,G)-A--L Ab response was directed, was found to be its major T cell epitope. In addition, there was no cross-reaction between (T-G-T-G)-A--L and (T-T-G-G)-A--L at the T cell level, similar to the lack of cross-reaction of their antibodies. Analysis of the repertoire of the T cell receptors used by these lines revealed that the (T,G)-A--L and the (T-T-G-G)-A--L specific T cell lines were not restricted in their V alpha and V beta TCR usage, whereas the (T-G-T-G)-A--L-specific line was restricted by both V alpha and V beta T cell receptor gene products. This difference might be due to the thymus-independent characteristics previously described for the latter antigen.

Production of colony-stimulating factor 1 by T cells: possible involvement in their interaction with antigen-presenting cells.

Colony-stimulating factor 1 (CSF-1) is required for the growth and differentiation of mononuclear phagocytes, and is also involved in modulating various activities in mature cells. We report herein that T-cell lines produce 4.6 and 1.5 kb mRNA species of CSF-1, and express the CSF-1 protein on their outer membranes, as determined by immunofluorescence staining with anti-CSF-1 antibodies. The CSF-1 protein is biologically active. Interested by the possible immunoregulatory function of CSF-1, we assessed its effect in an assay of antigen presentation to the T cell lines. We found that anti-CSF-1 antibodies inhibited T-cell stimulation. Moreover, soluble CSF-1 could not overcome this inhibition, but exerted a significant inhibitory activity on the interaction between T cells and antigen-presenting cells leading to T-cell activation and proliferation in vitro. Based on these observations we propose that T-cell CSF-1 may be involved in the interaction of these cells with CSF-1 receptor bearing antigen-presenting cells.

A comparison between arterial- and venous-sampled activated clotting time measurements.

In order to compare activated clotting time (ACT) sampled from an arterial (heparin-flushed) line with the control, a venous (heparin-free) line, arterial and venous ACT values were assessed before and after cardiopulmonary bypass in 150 patients while undergoing open-heart surgery. ACT was measured by Hemochron 801 automatic analyzer. Baseline arterial ACT was significantly higher than baseline venous ACT (14%; p < 0.001, using one-way analysis of variance and Bonferroni multiple comparisons test). The differences between the values of arterial and venous ACT after protamine reversal, between arterial ACT at baseline and after protamine reversal, and between venous ACT at baseline and after protamine reversal were not statistically significant. We conclude that arterial-sampled ACT measurement is suitable and reliable for monitoring heparin reversal by protamine after cardiopulmonary bypass.