Dick L. Robbins

Characterization of sperm protein 17 in human somatic and neoplastic tissue

Sperm protein 17 (Sp17) is a highly antigenic, testes-specific protein whose known function is to bind sperm to the zona pellucida. However, the Sp17 gene has been recently detected in normal non-testes tissues and malignant neoplasias. As the role of Sp17 in non-testes tissues is unknown, the characterization of the Sp17 gene in highly proliferating tissues may provide further insight into the regulation and alternative function of Sp17. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify the Sp17-1 transcript in multiple normal human tissues and cancer cell lines. Similarly, the Sp17-2 gene was examined by PCR. In addition, Northern and Western blot analyses were used to detect Sp17 mRNA and protein expression. The Sp17-1a and Sp17-1b transcripts were amplified from cancer cell lines. Similarly, an Sp17-2 transcript was also detected in cancer cell lines. Furthermore, Northern blot analysis revealed Sp17 mRNA expression in all cancer cell lines examined. However, Sp17 protein expression was not detected. The differential detection of the Sp17 transcripts in cancer cell lines as compared to normal non-testes tissues, suggests a potential pathogenic role for Sp17 in diseased cells. Moreover, the Sp17-2 transcript may be a marker for highly proliferating cells. Collectively, these data implicate Sp17 as a cancer testis antigen.

Characterization and Epitope Mapping of Human Monoclonal Antibodies to PDC-E2, the Immunodominant Autoantigen of Primary Biliary Cirrhosis

Further to define the epitopes of PDC-E2, the major autoantigen in primary biliary cirrhosis (PBC), we have developed and characterized five human monoclonal antibodies. These antibodies were derived by fusing a regional hepatic lymph node from a patient with PBC with the mouse human heterohybrid cell line F3B6. Previous studies of epitope mapping of PDC-E2 have relied on whole sera and have suggested that the immunodominant epitope lies within the inner lipoyl domain of the molecule. However, selective absorption studies using whole sera and a series of overlapping recombinant peptides of PDC-E2 have suggested that the epitope may also include a large conformational component. Moreover, several laboratories have suggested that autoantibodies against the 2-oxo acids dehydrogenase autoantigens are cross-reactive. The five monoclonal antibodies generated included three IgG2a and two IgM antibodies and were studied for antigen specificity using recombinant PDC-E2, recombinant BCKD-E2, histone, dsDNA, IgG (Fc), collagen and a recombinant irrelevant liver specific control, the F alloantigen. The antibodies were also used to probe blots of human, bovine, mouse and rat mitochondria. Finally, fine specificity was studied by selective ELISA and absorption against overlapping expressing fragments of PDC-E2. All five monoclonals, but none of the other mitochondrial autoantigens were specific for PDC-E2. In fact, although affinity purified antibodies to PDC-E2 from patients with PBC cross-reacted with protein X, the human monoclonals did not, suggesting that protein X contains an epitope distinct from that found on PDC-E2. Additionally, all three IgG2 monoclonals recognized distinct epitopes within the inner lipoyl domain of PDC-E2.

IGG3 Reactive Rheumatoid Factor in Rheumatoid: Arthritis: Etiologic and Pathogenic: Considerations

Rheumatoid factor (RF) is a polyclonal autoantibody directed against the Fc portion of IgG. Although the role of RF in patients with rheumatoid arthritis (RA) is unclear, immune complexes that form between RF and IgG can activate the classical complement (C) pathway, leading to pathogenic outcomes involving inflammatory events and tissue damage. The specificity of serum RF and RF produced by rheumatoid synovial cells (RSC) is different. Serum RF has specificity for rabbit IgG and human IgG subclasses IgG1, 2, and 4, but binds poorly to IgG3. The affinity of serum RF for IgG Fc is low, having an association constant of 10(4)-10(5) M-1. RSC RF, however, has specificity for human IgG and high avidity for IgG3. Because of this greater specificity and avidity for IgG3, and because RSC RF may be pathogenically more important than serum RF, an important role for IgG3-reactive RF in RA may exist. Binding of RF to IgG may be dependent on the allotype and glycosylation of IgG. Infectious agents present in RA patients may directly or indirectly induce the production of certain RF. In this communication, we review and expand on several observations examining the role of IgG3-reactive RF in RA including: 1) binding differences between RF derived from RSC and serum; 2) glycosylation characteristics of IgG and its interaction with RF; 3) apparent allotype dependent binding of IgG3-reactive RF; and 4) possible relationship between infectious agents and the production of IgG3-reactive RF. Taken together, these observations suggest an important role for IgG3-reactive RF in better understanding the etiology and pathogenesis of RA.

Giant cell arteritis associated with mononeuritis multiplex and complement-activating 19S IgM rheumatoid factor☆

Giant cell arteritis is a necrotizing granulomatous arteritis of large arteries, especially the aorta and its branches. Mononeuritis multiplex is a peripheral sensorimotor neuropathy usually producing foot or wrist drop, commonly associated with necrotizing arteritis of small and medium-sized arteries. Rheumatoid vasculitis is an example of the latter type of arteritis associated with high-titer 19S IgM rheumatoid factor typically occurring in patients with long-standing erosive rheumatoid arthritis. This report describes a 71-year-old man with biopsy-proved giant cell arteritis, mononeuritis (foot drop) multiplex, and high-titer complement-activating rheumatoid factor without rheumatoid arthritis. Possible pathogenic relationships are discussed.

Developmental Considerations of Sperm Protein 17 Gene Expression in Rheumatoid Arthritis Synoviocytes

Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17s structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations.

Genomic organization of an intron-containing sperm protein 17 gene (Sp17-1) and an intronless pseudogene (Sp17-2) in humans: a new model

Sp17 was initially thought to be a sperm specific protein involved in the interaction of the spermatozoon with the oocytes surrounding extracellular glycoprotein matrix. Recent reports, however, indicate that Sp17 expression is neither testis-specific nor is it exclusively used for binding to the zona pellucida of the oocyte. In this study, we provide comprehensive characterization of the genomic structure of Sp17. We identified an intron-containing gene (Sp17-1) containing five exonic and four intronic sequences. Analysis of Sp17 transcripts using rapid amplification of DNA complementary to RNA (cDNA) ends (RACE) and polymerase chain reaction (PCR) techniques showed the presence of alternative polyadenylation resulting in the production of varying lengths of mRNAs as well as the usage of different transcriptional start sites. Moreover, an earlier description of the human Sp17 mRNA describing a splice variant could not be confirmed. Comparison to mouse Sp17 gene organization demonstrated a high degree of conservation, suggesting selective evolutionary pressure for this protein to retain a conserved gene architecture. Additionally, we identified a second gene (Sp17-2), whose most striking characteristic was the complete absence of introns. This Sp17-2 gene has likely arisen by reverse transcription (RT) of a spliced Sp17-1 mRNA with subsequent integration into the human genome. Its open reading frame (ORF) is interrupted by stop codons, giving rise to a pseudogene. Furthermore, Southern blot analysis of human genomic DNA indicated the possibility of additional Sp17 species within the human genome.

Immunologic mechanisms in common rheumatologic diseases.

Rheumatoid arthritis and seronegative spondyloarthropathies are rheumatologic diseases that likely are caused by inflammatory reactions occurring in genetically predisposed individuals mounting an immune response to an antigen. Understanding the immunopathology of these diseases provides insight into their etiology, pathogenesis, and a rationale for therapies targeting immune component interactions. Although the antigen in rheumatoid arthritis is not known, several bacterial antigens have been associated with seronegative spondyloarthropathies. These antigens result in an interaction between human leukocyte antigen-B27 restricted CD8 positive T lymphocytes and the antigen presenting cell, producing an inflammatory response. Rheumatoid factors are autoantibodies directed against the fragment crystallizable portion of the immunoglobulin G. Rheumatoid factor immunoglobulin G immune complexes contribute to the inflammatory events in the rheumatoid joint, and may play an important role in antigen presentation. A novel antigen capture enzyme linked immunosorbent assay was developed that mimicked B cell surface expressed rheumatoid factor. Conversely, a direct binding enzyme linked immunosorbent assay mimicked secreted rheumatoid factor. Comparison of rheumatoid binding enzyme linked immunosorbent assays showed that the physical state of rheumatoid factor can affect binding characteristics. The state of glycosylation of immunoglobulin G may contribute to its antigenic structure. These physical characteristics may be important in rheumatoid factors pathogenic role in rheumatoid arthritis.

Allotype-dependent stimulation of peripheral blood and synovial lymphocytes by IgG3 in rheumatoid arthritis.

The immunopathologic process of rheumatoid arthritis (RA) is primarily expressed in the synovium where rheumatoid factor (RF) synthesis is concentrated. We hypothesized that RF synthesized by rheumatoid synovial cells (RSC) may be driven via a T cell-mediated immune response developed against IgG3 epitopes. To identify and characterize specific RSC RF epitopes and T cell antigens, two 28 amino acid peptides homologous with the C-terminus of IgG1 (P1) and IgG3 [G3m(5)] (P3) were synthesized and used in RF-binding studies and lymphocyte proliferation assays. Our results indicate that (i) the C-terminus of the CH3 domain contains epitopes for IgG3-reactive RSC RF; (ii) IgG3-reactive RSC RF binds primarily to IgG3 [G3m(5)]; (iii) P3 stimulated proliferation of T lymphocytes from both RA peripheral blood and RSC; and (iv) RF production was enhanced by P3 in selected RA cell cultures. These observations suggest that the C-terminus of IgG3 allotype G3m(5) may be important in T cell activation and RF production in RA.

Determination of the affinity of monoclonal 19 S IgM rheumatoid factor for IgG by modified immunoassay (ELISA)

In rheumatoid arthritis (RA), the pathogenicity of IgM rheumatoid factor (RF), an autoantibody whose antigen is IgG, is still unclear although RF-IgG complexes appear to be important mediators of immune injury. The polyclonality of RF in RA makes it difficult to characterize certain qualitative properties such as specificity and affinity which may be very important in determining pathogenicity. Monoclonal IgM RF can be used to circumvent this problem. Monoclonal RF secreting cells can be produced via hybridizations with RA B lymphocytes fused with mouse or human myeloma cell lines. Another source of monoclonal RF is the sera of patients with Waldenströms macroglobulinemia (WM). One particular WM IgM RF (Kas) was chosen for our experiments to measure affinities and specificities to eight different monoclonal IgGs (three IgG1s, three IgG3s, one IgG2, and one IgG4). 19 S IgM RF, a pentavalent molecule, was mildly reduced with DTT to make 7 S univalent fragments (7 S IgM RF). 7 S IgM RF was incubated with each of the different IgGs at several different concentrations. These mixtures were allowed to come to equilibrium. An aliquot was then used to determine the amount of free 7 S IgM RF by ELISA. By plotting the reciprocal of the fraction of bound RF versus the reciprocal of the concentration of free antigen at equilibrium, different affinities were determined. The results of these determinations compare favorably with published IgM RF affinities determined by more traditional methods. This method can also be used with proteolytic digest fragments of IgG and short synthetic peptides of the IgG molecule to better locate the antigen binding site. The technique may also help us to determine whether there are select clones of RSC producing RF with different affinities that could complex to a particular type of IgG which, in vivo, could produce greater inflammatory tissue damage. Furthermore, this methodology should be useful in the study of other autoimmune diseases characterized by pathogenic autoantibodies of differing affinities.

Isolation and purification of anticardiolipin antibody from plasma of a patient with antiphospholipid syndrome: induced generation of platelet thromboxane A2 synthesis

Antiphospholipid antibodies, particularly anticardiolipin antibodies (aCL) are autoantibodies frequently detected in the serum of patients with systemic lupus erythematosus (SLE) and the primary antiphospholipid antibody syndrome (PAPS). These patients commonly suffer from thrombosis, recurrent fetal loss and thrombocytopenia. Since platelet aggregation is pivotal in the genesis of thrombosis, we tested the hypothesis that perturbation of platelet membrane by aCL/beta 2-glycoprotein (aCL/beta 2GP) complex could trigger the biosynthesis of TXA2, a proaggregatory metabolite of AA. The preincubation of 14C-arachidonic acid (14C-AA)-labeled platelet pellets (14C-PP) from normal individuals with aCL alone followed by incubation with thrombin, resulted in a moderate increase in platelet thromboxane B2 (14C-TXB2) biosynthesis when compared to controls (without aCL). Similar incubations with beta 2GP-I alone resulted in negligible 14C-TXB2 biosynthesis. In contrast, the preincubations of normal 14C-PP with aCL/beta 2GP-I complex resulted in marked thrombin-induced TXB2 biosynthesis, underscoring the requirement of beta 2GP-I in aCL-induced platelet TXB2 biosynthesis. Taken together, these results are consistent with the view that aCL/beta 2GP-I platelet interactions do play a role, at least in part, in platelet hyperactivity and thrombosis in antiphospholipid antibody syndrome.