Selective removal of antigen-complexed IgG from cat plasma by adsorption onto a protein A-silica matrix.

The binding of normal cat IgG, heat-aggregated cat IgG and specific immune complexes (IC) containing cat IgG to a silica matrix containing covalently bound Staphylococcus aureus protein A was evaluated. The amounts of serum relative to protein A-silica, the flow rates and the perfusion times were representative of those existing when protein A-silica columns are used for therapeutic extracorporeal immunoadsorption of IgG and IC from humans and animals. When cat IgG was present in a large excess, approximately one molecule was bound to the matrix per molecule of solid-phase protein A with a KA of 1.5 X 10(6) 1/mol. Aggregated and immune complexed IgG bound to the matrix with relatively higher affinity. IC prepared in vitro between the purified envelope glycoprotein of the feline leukemia virus (FeLV gp70) and affinity-purified cat antibodies bound to the matrix even though normal IgG was present in greater than 10,000-fold excess. Once bound, IC were not eluted from columns upon further perfusion with normal serum. However, bound IgG was eluted from columns by further perfusion of normal serum or IC. IC were at least five-fold more efficient than normal IgG in exerting this effect. The results suggest that protein A-silica columns can be used for preferential removal of IC from plasma in a clinical or experimental setting.

Differential expression of transforming growth factor beta 1 and interleukin 10 in progressing and regressing areas of primary melanoma.

The coexistence of tumor specific immunity with a progressing tumor remains a major paradox of tumor immunology. This enigma is most evident in partially regressing melanomas, where efficient eradication of tumor cells is closely linked to uncontrolled tumor growth. Mechanisms involved in this differential susceptibility of tumor cells to the host immune response may include altered production of immunosuppressive cytokines, i.e., transforming growth factor (TGF) beta or interleukin (IL) 10. Since only limited amounts of tissue samples are available from primary tumors, a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was established which allowed to estimate the amount of cytokine mRNA expressed in a small number of melanoma cells segregated by indirect immunomagnetic isolation. Thereby, we determined the expression of TGF-beta 1 and IL-10 mRNA in melanoma cells obtained from regressing and progressing areas of 9 primary tumors. TGF-beta 1 mRNA could be detected in all undiluted samples from progressing areas and in 7 samples from regression zones. Titration of the sample revealed that in 6 cases TGF-beta 1 mRNA could be detected at a significant higher titer in progressing than in regressing areas. IL-10 mRNA was present in 8 samples obtained from progressing and in 7 samples from regressing tumor areas. In 6 tumors IL-10 mRNA was detectable at a higher titer in the progression zones. Specificity of the PCR amplification was confirmed with a series of restriction enzyme digestions of the resulting PCR product. Based on these findings the hypothesis that immunosuppressive cytokines, such as TGF-beta 1 or IL-10, represent important factors for the melanoma cells to escape immune surveillance is supported.

Reduction in platelet-binding immunoglobulins and improvement in platelet counts in patients with HIV-associated idiopathic thrombocytopenia purpura (ITP) following extracorporeal immunoadsorption of plasma over staphylococcal protein A-silica.

Platelet-directed antibodies and circulating immune complexes (CIC) were removed from plasma of patients with human immunodeficiency virus (HIV) infection and idiopathic thrombocytopenic purpura (ITP) by extracorporeal immunoadsorption using columns of Staphylococcal protein A-silica (Prosorba columns). In addition, stimulation of a broadly cross-reactive anti-F(ab')2 antibody response was observed. These antibodies also appeared to play a role in the additional removal of platelet-directed immunoglobulins (Igs) and CIC from plasma. Removal of these components from plasma was associated with diminishing levels of antibodies and CIC on patient platelets and significant increases in platelet counts. Extracorporeal immunoadsorption of IgG and CIC from plasma is a beneficial new treatment modality for HIV-associated ITP.