David G. Haegert

Thyroid-associated ophthalmopathy : a model for the association of organ-specific autoimmune disorders

The development of a characteristic ophthalmopathy is a feature of autoimmune diseases of the thyroid. The link between the conditions has not yet been discovered, but here Jack Wall and colleagues develop the theory that an autoimmune response to a 64 kDa antigen expressed on both thyroid and eye muscle membranes is responsible for this thyroid-associated ophthalmopathy.

Technical improvements in the mixed antiglobulin rosetting reaction with consequent demonstration of high numbers of immunoglobulin-bearing lymphocytes in viable preparations of human peripheral blood

A modification of the mixed antiglobulin rosetting reaction (MARR) to improve sensitivity as a test for immunoglobulin (Ig)-bearing human blood lymphocytes is described. A mean 5.7%, lymphocytes Ig-positive by the MARR when rosettes were formed in medium containing 0.2% bovine serum albumin (BSA), increased to 20% when rosettes were formed in 5% BSA or by incubating the lymphocytes or indicator erythocytes with Vibrio cholerae neuraminidase before rosetting. Under these various rosetting conditions the MARR is as sensitive as the direct antiglobulin rosetting reaction (DARR). Further, with the MARR, false positive rosette formation due to unusual antimembrane antibodies can be excluded during the mixed antiglobulin rosetting procedure by use of blocking controls. Substitution of F(ab)2 antiglobulin for IgG anti-gamma, anti-alpha and anti-mu did not reduce the number of lymphocytes demonstrable with the MARR, indicating that the MARR does not react with adsorbed Ig molecules on lymphocytes. Summation of the number of sheep erythrocyte (E) rosetting lymphocytes and mixed antiglobulin rosetting lymphocytes approximated 100%, yet in T-enriched preparations a maximum of 4% of lymphocytes were Ig-positive by the MARR, suggesting that null lymphocytes which have been reported to be E-negative and immunofluorescence-negative are B lymphocytes with surface Ig determinants.

The mixed antiglobulin rosetting reaction (MARR) and the direct antiglobulin rosetting reaction (DARR): Sensitive tests for demonstration of immunoglobulin-bearing B lymphocytes

The technology of the mixed antiglobulin rosetting (MARR) and the direct antiglobulin rosetting reaction (DARR) is reviewed. These tests have been found to be more sensitive than the standard direct immunofluorescence (DIF) method for delineation of Ig-bearing B lymphocytes and to have practical advantages over DIF. Further, with these rosette tests, an important B lymphocyte population has been identified which is Ig-negative by DIF and functionally quite different from typical DIF-positive B lymphocytes but which expresses a low density of surface membrane Ig that is detectable by the MARR and the DARR.

Demonstration of IgM receptors on human peripheral blood monocytes using a direct plaque forming cell (PFC) assay

Abstract Human peripheral blood mononuclear cells were depleted of lymphocytes to obtain cell preparations enriched for monocytes; in preparations from seven donors the mean percentage of monocytes was 95%. Based on the observation that the binding of IgM cytophilic anti-sheep erythrocyte (SRBC) antibodies to monocytes is temperature dependent, a plaque forming cell (PFC) assay was developed and compared with a rosette test in delineation of IgM-receptor bearing monocytes. Whereas a mean of 2% of freshly prepared monocytes were IgM-receptor positive by rosettes, a mean of 34% formed direct plaques. If monocytes were VCN-treated then passively sensitized with cytophilic IgM anti-SRBC antibodies, means of 57% and 87% formed rosettes and direct plaques respectively; direct comparisons showed that the PFC assay was significantly more sensitive than the comparable rosette tests on both freshly prepared and VCN-treated monocytes. The specificity of plaque formation by VCN-treated monocytes was established in inhibition experiments in which inclusion of IgM but not IgG molecules in the test medium inhibited plaque formation. These results indicate that not only are IgM receptors present on a significant proportion of freshly prepared peripheral blood monocytes but also that some receptors are hidden in the monocytes membranes and can be revealed by VCN-treatment. The PFC assay was shown to have practical advantages over the comparable rosette test in demonstrating IgM-receptor bearing human peripheral blood monocytes.

Demonstration of T4, T8, M1 and B7 determinants on human T cells with a Rosette test: implications for the assay specificity of monoclonal antibodies

Under optimal test conditions significantly more freshly isolated human T cells reacted with OKT4, OKT8, OKM1 and OKB7 monoclonal antibodies (Mabs) in the indirect antiglobulin rosetting reaction (IARR) than by indirect immunofluorescence. Rabbit erythrocytes (E) coated with anti-mouse immunoglobulin were more sensitive indicator cells in the IARR than similarly coated sheep E. Treatment of T cells with neuraminidase further enhanced T cell reactivity in the IARR with each Mab so that an average of 60% or more of T cells were T4+, T8+ and M1+ and at least 40% had the T4+ T8+ phenotype. The various findings suggest that the rosette assay detects determinants on T cells that are expressed below the detection threshold of immunofluorescence. Moreover, these findings indicate that the cellular specificities of a particular Mab may change when one assay system is substituted for another or when the protocol of a particular assay is altered.