Biomedical subjects
B Zweiman
Publications and source records attributed to B Zweiman.
Circulating T-cell subsets in Guillain-Barré syndrome.
We compared the distribution of circulating T-cell subsets within 2 weeks of onset of symptoms in 14 patients with acute Guillain-Barré syndrome (GBS) and 37 normal controls. The levels of OKT4+ (putative helper-inducer) cells was definitely abnormal (decreased) in 3/13 tested. The levels of OKT8+ (putative suppressor-cytotoxic) cells were elevated in 3 and decreased in 2 of the 14 tested. Abnormal OKT4/OKT8 ratios were detected in 5 (2 elevated and 3 decreased) patients. Four of the 5 GBS patients with abnormal OKT4+/OKT8+ ratios were studied sequentially at least 4 times over 1-10 months; there was a return towards a normal ratio in all. Serial studies in 3 other GBS patients showed consistently normal values. In comparison, sequential studies over 5-24 months in 7 normals showed no abnormal OKT4+/OKT8+ ratios. Thus, abnormalities in OKT4+/OKT8+ ratios appear to be a marker of systemic events during symptomatic phases of GBS. It is not as yet known whether this is related to the cause or is secondary to the clinical manifestations of GBS.
Accumulation of leukotriene C4 and histamine in human allergic skin reactions.
To determine whether lipoxygenase products of arachidonic acid metabolism are released in vivo during human allergic cutaneous reactions, we serially assayed chamber fluid placed over denuded skin sites for the presence of both C-6 peptide leukotrienes (e.g., LTC4, LTD4, and LTE4) and leukotriene B4 (LTB4), using radioimmune assay and HPLC separation, and compared it to histamine (assayed radioenzymatically) in 13 atopic and two nonatopic volunteers. Skin chamber sites challenged with ragweed or grass pollen antigen (250-750 protein nitrogen units/ml) for the first hour and phosphate-buffered saline (PBS) for the next 3 h were assayed hourly and compared to sites challenged with PBS alone. As assessed by HPLC, LTC4 composed greater than 85% of the C-6 peptide leukotriene released at any skin site, whereas little LTD4 or LTE4 was detected. LTC4 was present in significantly greater concentrations at antigen sites as compared to PBS-challenged sites throughout the 4-h period. Minimal concentrations of LTB4 were found throughout this time period and were not different at antigen or PBS sites. Histamine was present in significantly greater concentrations at antigen rather than PBS sites, but the pattern of release was different from that of LTC4. Peak histamine release invariably occurred during the first hour and decreased progressively thereafter, whereas the greatest amounts of LTC4 were detected during the 2nd to 4th hours. The amount of LTC4 accumulating at the site was dependent upon the dosage of antigen used in the epicutaneous challenge. We have demonstrated in this study that of the leukotrienes assessed LTC4 is released in the greatest quantity in situ during in vivo allergic cutaneous reactions and that it is present at such sites for at least 4 h after antigen challenge. Since intradermal injection of LTC4 in humans induces wheal and flare responses that persist for hours, our findings support the hypothesis that LTC4 is an important mediator of human allergic skin reactions.
Autoimmune aspects of myasthenia gravis.
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The role of myelin P2 protein in the production of experimental allergic neuritis.
Myelin P2 protein has been proposed as the primary antigen in whole myelin-induced experimental allergic neuritis (EAN). We investigated the neuritogenic properties of P2 by sensitizing Lewis rats with complete Freund's adjuvant (CFA) containing P2, P2 plus phosphatidyl serine, or whole myelin containing an equivalent amount of P2. Animals were examined using a battery of clinical, electrophysiological, immunological, and morphological methods. Myelin-immunized rats developed the characteristic features of EAN. P2-sensitized rats developed a similar but much less intense disorder. When rats were sensitized with P2 in the presence of phosphatidyl serine, however, they developed radiculoneuropathy that was indistinguishable from myelin-induced EAN. Inoculation with phosphatidyl serine plus complete Freund's adjuvant or complete Freund's adjuvant alone had no detectable effect on peripheral nerves. These studies demonstrate that sensitization of rats with a single myelin antigen, P2 protein, is sufficient to induce the clinical, electrophysiological, and neuropathological features of EAN.
Corticosteroid effects on circulating lymphocyte subset levels in normal humans.
The effects of in vivo corticosteroid administration on levels of lymphocyte subsets in normal humans require further definition. Using monoclonal antibodies, we carried out a double-blind, placebo-controlled study of oral and intravenous methylprednisolone in 10 normal volunteers. Four hours after a 7-day oral course of 0.5 mg/kg/day, there was modest lymphopenia but no significant selective alteration in lymphocyte subsets. In contrast, 4 hr after a single intravenous injection of 1.0 mg/kg, there was more pronounced lymphopenia (P less than 0.01), a selective, relative decrease in T4 cells (P less than 0.001), and a more modest decrease in the percentage of T3 cells. The possible mechanisms and implication of these changes are discussed.
Correlations of in vivo mediator release with late cutaneous allergic responses in humans. I. Kinetics of histamine release.
To evaluate the contribution of mast cell-derived mediators in the late cutaneous allergic response, the duration and quantity of antigen-induced histamine release was compared to the intensity of the antigen-induced skin reactions in atopic volunteers. Chambers containing either pollen extract or buffer were appended to denuded bases for 1 hr and were replaced hourly with buffer for 3 additional hr. These were compared to the extinction dilution skin test titer and to the mean diameters of the 20-minute wheal and induration at 6 and 8 hr after intradermal injection of antigen. Chamber-fluid histamine levels were significantly higher at antigen than at buffer sites throughout the 4 hr. The hourly histamine levels correlated with the size of the induration at 6 and 8 hr but not with the wheal size or skin test titer. We conclude that (1) histamine is released for at least 4 hr at skin sites of antigen challenge as a consequence of prolonged release either from individual or sequentially activated mast cells, and (2) the quantity of histamine released correlates with the intensity of the late-phase skin response. We hypothesize that histamine might be a marker for prolonged release from the mast cell of other mediators that are responsible for the late-phase response.
Increased antigen-induced local and systemic mediator release in rhinitis subjects with pulmonary symptoms in the pollen season.
In order to delineate parameters that might discriminate between allergic subjects who develop R or R-P symptoms during natural antigen exposure, 26 subjects allergic to grass or ragweed pollen were classified into R or R-P groups, and then the antigen sensitivity and degree of in vivo mediator release were compared. Antigen-skin sensitivity was quantitated by dilutional skin-test titration, and bronchial sensitivity was quantitated by the amount of inhaled antigen required to receive the FEV1 by 20%. Mediator release was determined by measuring the amount of histamine that was released into skin chambers during antigen incubation and the rise in plasma histamine and serum NCA during antigen-induced bronchospasm. Compared to the 13 R subjects, the 13 R-P subjects were: (1) more sensitive to antigen by both skin-test and inhalation challenge, (2) responded to inhalation of antigen with a greater fall in FEV1 and a greater rise in serum NCA and plasma histamine, and (3) released more histamine into skin chambers after antigen incubation. Even when R and R-P subjects were matched by comparing only subjects with equal skin sensitivity to antigen, greater increases in serum NCA and plasma histamine occurred after inhalation of antigen in the R-P subjects. These data are consistent with the hypothesis that allergic rhinitis subjects who develop pulmonary symptoms during natural pollen exposure are more sensitive to antigen and release more mediators in response to antigen administration. It is therefore possible that the degree of mediator release may be an important factor in determining the pattern of clinical responses to antigen exposure.
Studies on the mechanism of clinical tolerance in solar urticaria.
Tolerance to artificial ultraviolet radiation (UVR) was induced in three patients with solar urticaria by administering graded whole body exposures to long-wave ultraviolet radiation (UV-A, 320-400 nm) in a phototherapy cabinet. Plasma histamine levels, mast cell ultrastructure and cutaneous responses to intradermally injected codeine and histamine were examined before and after the induction of tolerance. No evidence of serum complement activation could be demonstrated following exposure of serum samples to UVR in vitro. These studies suggest that the state of tolerance is due neither to mediator (histamine) depletion nor to a systemic effect induced by UV-A but may be due to an increase in the mast cell degranulation threshold.
Effects of cromolyn on codeine-induced histamine release in vivo.
Cromolyn has been shown to inhibit histamine release from mast cells induced by various stimuli in vitro. However, the local effects of cromolyn on codeine-induced wheal and flare skin reactions are not well understood. Intradermal injection of codeine induced prominent whealing in almost all humans. We studied the effect of local cromolyn injection on codeine-induced skin reactions, histamine release, and ultramicroscopic changes in mast cells in 10 volunteers. The finding in this study showed that injection of a 2% cromolyn solution before or together with the codeine injection does not affect the subsequent skin reactions, histamine release and ultramicroscopic changes of mast cells.
Immune responses to myelin antigens in multiple sclerosis.
Multiple sclerosis is considered to be a putative immunopathologic disease and there has been considerable effort over the years to prove an autoimmune etiology for it. To date, the evidence is all indirect and there is no proof of either antibody and/or cell-mediated hypersensitivity to any single identifiable CNS constituent whether a constituent of normal CNS or specific to the CNS of MS patients.
Thymic B-cell activation in myasthenia gravis.
We studied secretion of immunoglobulin (Ig) by freshly isolated and pokeweed mitogen (PWM)-stimulated thymus cells and blood mononuclear cells in patients with myasthenia gravis (MG) and control subjects undergoing elective cardiac surgery. We used a protein A reverse hemolytic plaque assay to enumerate cells secreting IgG, IgM, and IgA (IgSC), and an ELISA assay for measuring IgG secreted into culture supernatants. We found that freshly isolated suspensions of MG thymus cells, compared with control thymus cells, contained increased numbers of cells that spontaneously secreted immunoglobulin. Thymus mononuclear cells from control as well as MG patients appeared capable of B-cell differentiation responses when stimulated by PWM. PWM-induced responses were greater in thymic than in autologous blood mononuclear cells in some MG patients and controls, although B cells were much less frequent in suspensions of thymic cells than blood cells. Thus, the thymus provides a favorable milieu for differentiation of its few B cells. In MG, the thymus may be a site of accentuated in vivo B-cell activation, as evidenced by increased numbers of resident IgSC.
In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood cells: role of suppressor T cells in normal subjects.
Peripheral blood mononuclear cells of 15 of 20 patients with generalized myasthenia gravis synthesized antibodies to acetylcholine receptor (AChR) when the cells were stimulated in vitro with pokeweed mitogen. In contrast, mononuclear cells of 1 of 16 normal subjects synthesized detectable AChR antibodies. Peripheral blood mononuclear cells of five normal subjects were studied before and after putative suppressor T cells (OKT8+) were removed by a fluorescent activated cell sorter. Depletion of OKT8+ cells did not result in production of AChR antibodies, but pokeweed mitogen-induced polyclonal IgG synthesis and activation of B cells to form immunoglobulin-secreting cells (reverse hemolytic plaque assay) were increased. Therefore, failure of blood mononuclear cells of normal subjects to synthesize detectable anti-AChR in response to pokeweed mitogen is not due to suppression by OKT8+ cells.
Tetanus toxoid reactive T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients.
Cerebrospinal fluid (CSF) lymphocytes of 6 multiple sclerosis (MS) patients were cultured with tetanus toxoid (TT) and irradiated autologous antigen presenting cells (APC) followed by propagation of the responding T-cells in interleukin-2 containing medium. TT-reactive cell lines were recovered from 4 of the 6 CSF samples, even though the patients had not been TT booster immunized in recent years. These findings suggest an active circulation of antigen reactive lymphocytes from the systemic immune compartment(s) into the CSF even without recent activation by booster immunization. Since immune reactions to TT are very unlikely to be pathogenic in MS, these findings also indicate that presence of CSF lymphocytes reactive to a particular antigen does not necessarily imply a causal role.
Recovery of myelin basic protein reactive T cells from spinal cords of Lewis rats with autoimmune encephalomyelitis.
Mononuclear cells were isolated from the spinal cords of 13 Lewis rats with clinical EAE induced by immunization with MBP in complete Freund's adjuvant. MBP-reactive lymphocytes were recovered from nine of these 13 SC-MNC populations by in vitro culture with MBP, followed by propagation of responding lymphocytes in IL 2-containing medium. T cell subset phenotype determination indicated a predominance of helper phenotype T cells in each of the two cultures examined. Clinical EAE was induced in naive Lewis rats by adoptive transfer of MBP-reactive lymphocytes recovered from three of these cultures.
The immunohistology of the thymus in myasthenia gravis.
We have investigated cell subpopulations in frozen sections of thymus tissue obtained from myasthenic (MG) and control subjects. With the use of an avidin-biotin immunoperoxidase system with monoclonal antibodies, the following cell surface antigens were studied on frozen sections (12 MG and 3 control thymus); T11, T4, T6, T8, IgM, IgD, and Ia. The pattern of T cell phenotypes in MG thymus is similar to that of normal control thymus when examined by immunohistologic techniques. MG cortical thymocytes are virtually all T11+, T4+, T8+, and T6+. In the medulla, at least 45% of thymocytes are T11+, with T4+ cells predominating over T8+ cells. Approximately 10% of medullary thymocytes are T6+. Scattered medullary cells expressing surface IgM and IgD are identified in both MG and normal thymuses. However, unlike the normal thymus, the MG thymus has numerous secondary follicles containing IgM- and IgD-bearing cells. This finding supports the hypothesis that the MG thymus microenvironment is aberrant. The Ia antigen is found in similar tissue section localization patterns in MG and control thymus. Ultramicroscopic studies show the Ia antigen predominantly on epithelial and interdigitating dendritic cells. By immunoperoxidase techniques, numerous keratin-positive cells are demonstrated in MG and control thymus. This suggests that thymic epithelial cells, like epithelial cells elsewhere, contain keratin. Because these data differ in degree from our previous findings in suspensions of MG thymocytes, this study emphasizes the importance of examining tissue sections as well as cell suspensions when one is studying lymphocyte surface markers.
Isolation of myelin basic protein-reactive T-cell lines from normal human blood.
T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.
IL-2 secretion by soluble antigen-reactive human T-cell clones.
Human tetanus toxoid specific T-cell lines and clones capable of producing IL-2 were established. IL-2 production occurred only when the antigen-specific T cells were cultured with both tetanus toxoid antigen and an autologous, irradiated adherent cell population. The T-cell lines and clones remained strictly dependent on exogenous IL-2 for proliferation at all other times. Phenotypic characterization with monoclonal antibodies recognizing T-cell subsets revealed that the antigen-specific lines and clones bore predominantly OKT3 and OKT4 markers with essentially no OKT8 positive cells present. T-cell clones which were demonstrated to secrete IL-2 activity could also partially deplete media of IL-2 if cultured in the absence of soluble antigen and irradiated adherent cells.