W. L. Cleveland

A new preprocessing approach for cell recognition

In this paper, we describe a novel strategy for combining fishers linear discriminant (FLD) preprocessing with a feedforward neural network to classify cultured cells in bright field images. This technique was applied to various experimental scenarios utilizing different imaging environments, and the results were compared with those for the traditional principal component analysis (PCA) preprocessing. Our FLD preprocessing was shown to be more effective than PCA due in large part to the fact that FLD maximizes the ratio of between-class to within-class scatter. The new cell recognition algorithm with FLD preprocessing improves accuracy while the speed is suitable for practical applications.

Auto‐Anti‐Idiotype: A Basis for Autoimmunity and a Strategy for Anti‐Receptor Antibodies

As frequently seems to be the ease, a series of serendipitous findings has shaped the overall objectives of research being carried oul in our laboratory. The first was the discovery of a highly potent agonist of the nicotinic acetylcholine receptor (AChR). tran.s-i.y -bis[«-trimethy!ammonio)-methyl]azobenzene (BisQ) (Bartels ct al. 1971. Wassermann et al. 1979) at a time when we were interested In photoregulating AChR activity (Erlanger 1976, Erlanger et al. 1982, 1983). Then, some casual hypothesizing and simple immunochemical experiments followed and we were thrust into the complex world of idiotypy. anti-idiotypy and autoimmunity, from which we will probably never escape alive. The objectives of our research can be characterized as follows: 1. To study the biological and biochemical properties of anti-idiotypic antibodies in relation to the etiology of such autoimmune diseases as myasthenia gravis (MG) and Graves disease. 2. To Investigate the full potential of an auto-anti-idiotypic strategy for the preparation of anti-receptor antibodies. With respect to the first objective, we will present data in support of the view that products of a functioning anti-idiotype network can cause an autoimmune disease, specifically the experimental form of myasthenia gravis (MG). It will be

The auto-anti-idiotypic route to antireceptor antibodies.

Data will be presented in support of the view that the products of a functioning anti-idiotype network can cause an autoimmune disease, specifically, the experimental form of myasthenia gravis (MG). We will describe how the disease can be induced in the rabbit by active immunization with an ant i l iga~~d’-~ and in the mouse by passive administration of hybridoma cells that secrete a monoclonal anti-idiotypic antibody specific for the acetylcholine binding site of the nicotinic acetylcholine receptor (AChR).

In vitro release of soluble CD23 by human lymphocytes in ragweed-sensitive versus nonatopic patients following stimulation with ragweed antigen E

BACKGROUND Soluble CD23 is the proteolytic cleavage product of the low affinity receptor (FcERII). Functions of CD23 and its soluble products may include upregulation of IgE production and stimulation of B lymphocyte growth. METHODS Soluble CD23 was quantitated in supernatants of lymphocytes from nine ragweed-sensitive and eight nonatopic subjects stimulated in vitro by antigen E (amb Al), crude ragweed extract, and pokeweed mitogen (PWM). RESULTS Although PWM stimulation produced no significant difference between groups, sCD23 release was significantly elevated in the cells of nonatopic patients stimulated with antigen E and crude ragweed extract (P less than .05). CONCLUSIONS This finding supports the concept of separate pathways of activation by antigen and mitogen for sCD23 release and suggests ragweed-sensitive and nonatopic patients have fundamental differences in the response of sCD23 release to ragweed antigen stimulation.

CD4 but not CD8 is comodulated with the T-cell antigen receptor (TCR) after activation of a CD4+ CD8+ human leukemia line with staphylococcal enterotoxin.

Recent studies support the possibility of an interaction between CD4/8 and the TCR complex. To determine if there is specificity in this interaction, we have studied the comodulation of CD4/8 with the CD3/TCR complex on a CD4+ CD8+ human leukemic T-cell line stimulated with staphylococcal enterotoxin A (SEA) bound to Raji cells. FACS analysis revealed that CD3 and the TCR were modulated from the surface. CD4 and not CD8 was comodulated with the T-cell receptor complex, supporting the existence of a docking site on the TCR with selectivity for CD4 or CD8 but not both. Fewer than 45 SEA molecules per presenting cell led to detectable comodulation. The ratio of %CD4/%TCR modulation varied with both time and the amount of SEA used for stimulation. ConA or PHA induced modulation of CD3 but, unlike SEA, failed to induce IL-2 secretion, suggesting multiple pathways and states of T-cell activation. Our findings also suggest that some human T leukemic lines can respond to antigen.

A general method for studying the secretion of macromolecules by single cells: I. Detection of immunoglobulin-secreting cells

Abstract A general method for detecting single cells secreting macromolecules has been developed and applied to the detection of mouse cells secreting antibodies. Secreted Ig molecules were precipitated in the immediate vicinity of an active cell with rabbit anti-mouse Ig antibody. Rapid removal of excess antibody not incorporated into immunoprecipitates was achieved with an electrophoresis technique. The immuno-precipitate surrounding the active cell was then stained with sheep anti-rabbit Ig antibody labeled with horseradish peroxidase. The use of enzyme markers has made the procedure much more convenient and rapid than previous precipitin assays for cell secretion that used radioiodine for detecting immunoprecipitates. Moreover, the availability of several enzyme markers makes possible the detection of cells secreting more than one molecular species. Experiments were also run in which cells producing antibodies specific for horseradish peroxidase (HRP) could be identified among the population of cells producing other immunoglobulins. Presumably, HRP-labeled antigens could be used to identify cells producing other specific antibodies. The generality of this procedure suggests that it may be useful for detecting single T-cells releasing regulatory molecules, since specific antisera are already available for several of these molecules.

Antibodies to Acetylcholine, Adenosine and Glucocorticoid Receptors by an Auto-Anti-Idiotypic Route

We will present data in support of the view that products of a functioning anti-idiotype network can cause an autoimmune disease, specifically the experimental form of myasthenia gravis (MG). It will be demonstrated that the disease can be induced in the rabbit by active immunization with an anti- ligand (19,20,52) and in the mouse by passive administration of hybridoma cells that secrete a monoclonal anti-idiotypic antibody specific for the binding site of the nicotinic acetylcholine receptor (AChR). We will also describe how an auto-anti-idiotypic procedure can produce monoclonal antibodies to receptors other than AChR, in particular the glucocorticoid receptor of rat liver cytosol and the adenosine receptors. With respect to the former, we will report on a monoclonal antibody that cross-reacts with the B-chain of insulin.

Anti-Receptor Antibodies by the Auto-Anti-Idiotypic Route

A procedure was developed for preparing antibodies (Ab’s) to the acetylcholine receptor (AChR) on the supposition that, regardless of functional differences, macromolecules of the same specificity will show binding-site homologies. Ab’s prepared in rabbits to a structurally constrained agonist of AChR (BisQ) mimicked the binding specificity of AChR in its activated state; agonist binding affinities parallelled biological activity. Rabbits immunized with a specifically purified preparation of anti-BisQ produced anti-idiotypic (anti-Id) Ab’s that cross-reacted with determinants on AChR preparations from Torpedo californica, Electrophorus electricus and rat muscle. Moreover, some rabbits showed signs of transient experimental myasthenia gravis, manifesting circulating AChR Ab’s.

A general method for studying the secretion of macromolecules by single cells. II. A simplified procedure with enhanced sensitivity.

Abstract A simple, sensitive precipitin-type single-cell secretion assay is described and applied to the study of immunoglobulin-secreting cells. Evidence is presented that its efficiency is comparable to that achieved with reverse hemolytic plaque techniques. Also described is a modification of the simplified procedure which possesses substantially increased sensitivity. Enhanced sensitivity is achieved through the use of monoclonal rheumatoid factors which preferentially react with rabbit or human immunoglobulins that are incorporated into immune complexes as a result of interaction with antigen. Addition of rheumatoid factor to the agarose-cell mixture leads to additional crosslinking of immune complexes that form around active cells, thereby increasing the probability of forming a detectable precipitate. The application of this procedure to the detection of cells producing T-cell products is also discussed.