Robert P. Searles

Impairment of the protein C anticoagulant pathway in a patient with systemic lupus erythematosus, anticardiolipin antibodies and thrombosis

We have identified an inhibitor of the protein C anticoagulant pathway in the plasma of a patient with systemic lupus erythematosus and a history of recurrent deep vein thrombosis, fetal wastage, and seizures. The patients plasma contained anticardiolipin antibodies as well as a weak lupus anticoagulant. Examination of this patients plasma revealed normal levels of protein C and protein S antigen, normal levels of functional protein C, as well as essentially normal levels of every blood coagulation factor. In a modified prothrombin time assay, the activated protein C-mediated prolongation of the clotting time observed in normal plasma was not observed in this patients plasma. Gel permeation chromatography of the patients plasma revealed that the inhibitory material was a high molecular weight protein that coeluted with the IgM peak. The inhibitor did not appear to circulate as a complex with protein C, since the inhibitor could easily be separated from protein C during fractionation procedures, and did not interfere with the activation of protein C in plasma as assessed by a functional amidolytic assay. Our findings suggest that the recurrent thrombotic episodes observed in this patient may have occurred as a result of the patients antiphospholipid antibody neutralizing specific phospholipids essential for the full expression of the anticoagulant activity of activated protein C.

Anti-Ia Reactivity in Sera from Patients with Systemic Lupus Erythematosus

Antileukocyte antibodies in sera from patients with systemic lupus erythematosus (SLE) were characterized by determining cross-reacting specificies with the antigens defined by OKT3, OKT4, OKT8, OKM1 and anti-Ia hybridoma antibodies (Abs). T cells were prepared by sheep erythrocyte (E) rosetting after removal of adherent cells. T cells, or non-T cells, were preincubated with SLE sera at 4 degrees C and then with monoclonal Abs. Binding by specific monoclonal Abs was assessed by two methods: rosetting with ox erythrocytes conjugated with goat anti-mouse IgG and also in the fluorescence-activated cell sorter using fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Using the rosetting method, we found that sera from SLE can block the binding of monoclonal mouse hybridoma anti-Ia Abs to T cells; the blocking of other monoclonal Abs was not consistent. Using fluorescence-activated cell sorter analysis, preincubation with SLE sera lowered the intensity of staining and total percentage of either T or non-T cells stained by monoclonal anti-Ia Abs. Blocking of anti-Ia Abs binding by SLE sera was not histocompatibility leukocyte antigen (HLA)-DR restricted and was not due to Fc receptor binding. These results indicated that antibodies in SLE sera react with structures contiguous to or identical with Ia determinants. Anti-Ia activities in SLE sera correlate with SLE disease activity. In addition, there was a significant negative correlation between anti-Ia blocking activity in the sera and the percentage of Ia-positive T cells in the blood of SLE patients. Antibodies in SLE sera with anti-Ia blocking activity may play an important role in immune dysregulation in SLE patients.

The presence of Ia antigen on human peripheral blood T cells and T-cell subsets: Analysis with monoclonal antibodies and the fluorescence-activated cell sorter

Abstract Peripheral blood T cells from normal donors, from newborns, and from patients with rheumatoid arthritis were analyzed for the presence of surface Ia antigens using monoclonal anti-Ia reagents and a fluorescence-activated cell sorter. Two out of four monoclonal anti-Ia antibodies detected Ia antigen on 2–22% (mean 7.4 ± 5.9%) of normal E rosette (+) cells. A subpopulation of E rosette (+) cells thus expresses an Ia molecule with antigenic determinants similar to the Ia on B cells. The staining is weak in comparison to the staining with anti-Ia of B cells, but could not be blocked by preincubation of the T cells with aggregated human IgG. Confirming a previous report by Yu et al. (D. T. Y. Yu, R. J. Winchester, S. M. Fu, A. Gibefsky, H. S. Ko, and H. G. Kunkel, J. Exp. Med. , 151 , 91, 1980) in rheumatoid arthritis more Ia-positive T cells were detected (mean 17.4 ± 12.5%) that also express a higher number of Ia molecules on their surface. On the contrary, the number of Ia(+) T cells was decreased in cord blood. Both helper T cells [OKT4(+)] and suppressor T cells [OKT8(+)] contain a subfraction that is Ia(+), as evidenced by additive staining and two-color immunofluorescence experiments. In RA, an increase in Ia(+) cytotoxic/suppressor cells is mainly responsible for the higher proportion of Ia(+) T cells. We have previously identified a subpopulation of T cells staining with both OKT8 and OKM1, a marker thought to be specific for cells of myelomonocytic lineage. We now report that this OKT8(+) OKM1(+) overlap population is almost entirely Ia(+) and therefore could represent an activated cell type. However, Ia antigen was also detected on OKT3(+) cells after removing OKM1(+) E rosette (+) cells.

Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis: evidence for activation of neutrophils and its mediation by a factor present in synovial fluid.

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O2−) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O2− generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O2− release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O2− release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O2− generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.

Cross--reactivity of antilymphocyte and antinuclear antibodies in systemic lupus erythematosus.

Abstract Both antinuclear antibody (ANA) and antilymphocyte antibody (ALA) are prevalent in systemic lupus erythematosus (SLE). This report presents data supporting cross-reactivity between these two antibodies. Differential absorption of SLE sera showed that chicken erythrocyte nuclei absorbed IgM ALA while intact human and chicken erythrocytes had no effect. RNase digestion of chicken nuclei fragments partially removed the ALA absorbing effect. Both IgM and IgG ANA were also absorbed by the chicken nuclei. Absorptions with intact human lymphocytes removed ALA activity and absorption with human mononuclear cells removed ANA activity. ALA and ANA were both found in PBS eluates from absorbed chicken nuclei, demonstrating specificity of nuclear absorption.

T lymphocyte interaction with immunoglobulin G antibody in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple immune disturbances whose mechanisms remain unclear. We examined the interaction of antilymphocyte antibodies with cultured normal T lymphocytes. T cells were prepared by E-rosetting after petri-dish removal of adherent cells and cultured for 2-7 d in the presence of SLE sera or normal human sera. Cultured T cells were washed and sonicated, and the amount of cell-associated IgG was quantitated by radioimmunoassay or enzyme-linked immunoassay (ELISA) methods. T cells cultured with 27 of 39 SLE sera showed marked increments of associated immunoglobulin G (IgG) although this was not observed with sera from mixed connective tissue disease patients containing high titers of ribonucleoprotein antibody or normal donors. The effective factors for IgG association in SLE sera were absorbed with normal peripheral blood lymphocytes or T cells. Anti-T cell IgG cytotoxic activity strongly correlated with T cell IgG association (P less than 0.01). T cell-associated IgG was not removed by stripping of cell membrane IgG from living cells by acid buffer treatment; indirect immunofluorescence of cells fixed after 2-4 d of culture revealed cytoplasmic IgG staining. IgG anti-T cell antibodies appeared to associate inside the cell membrane or to penetrate into the cytoplasm of cells. T cell Fc receptor blocking by heat-aggregated IgG or anti-beta 2-microglobulin antibody did not alter IgG cell association. Since pepsin-digested SLE sera showed no T cell association activity, whole IgG antibody molecules appeared to be necessary for interaction with cultured T cells. In addition, reduction and alkylation of active SLE sera completely nullified T cell reactivity. When normal T cells were cultured with SLE sera showing marked IgG T cell association, viability of cultured T cells decreased rapidly after 4 d, which suggests that IgG anti-T cell antibodies were associated with cell destruction. IgG cell-associating antilymphocyte antibodies present in SLE sera may cause T cell disturbances in vivo and may be related to the lymphocytopenia present in SLE patients.

A Case of Guillain-Barré Syndrome with Immunologic Abnormalities

Guillain-Barré syndrome and the nephrotic syndrome developed in a patient simultaneously. Analysis of renal biopsy by light, immunofluorescent, and electron microscopy showed lipoid nephrosis. T-cell lymphocytopenia, lymphocytotoxic antibodies, depressed lymphocyte mitogenesis, and anergy also complicated the illness. The immunologic abnormalities resolved when the polyradiculoneuritis and lipoid nephrosis remitted. This previously undescribed association illustrates the occurrence of two postulated cell-mediated autoimmune disorders despite the presence of depressed cell-mediated immunity.

Human Anti‐F(ab′)2 Antibodies and Pepsin Agglutinators React with Fv Determinants

Affinity‐purified human IgG anti‐tetanus antibody was subjected to papain and then pepsin digestion, and residual fragments retaining antibody activity were re‐isolated by adsorption and elution from Sepharose‐tetanus toxoid columns. Both Fab(and Fv fragments isolated by gel filtration showed strong reactivity with anti‐F(ab()2 antibodies. Failure of tetanus toxoid to completely block reactivity of anti‐F(ab()2 antibody with anti‐tetanus Fv fragments indicates that these antibodies react with framework antigens within variable antibody regions

Long-term treatment of rheumatoid arthritis comparing nabumetone with aspirin

This report summarizes the results of a 17-investigator multicenter six-month randomized double-blind parallel group study. The safety and efficacy of nabumetone 1,000 mg taken at bedtime was compared with that of aspirin 900 mg four times daily in the treatment of adult patients with active class II or III classical or definite rheumatoid arthritis. Two hundred sixty-four patients were entered into the study. Two hundred fifty-seven (126 nabumetone and 131 aspirin) patients were evaluable for safety. Two hundred thirty-four (113 nabumetone and 121 aspirin) patients were evaluable for efficacy. There was significant improvement in each of six clinical measurements of efficacy in both treatment groups and little difference between groups. The somewhat greater improvement in articular index and duration of morning stiffness in the nabumetone-treated group did not reach statistical significance. There was an equal percentage of patient withdrawal for lack of efficacy in each group. Overall, the rate of patient withdrawal due to adverse experiences was greater (p = 0.01) for aspirin-treated patients. These experiences were usually dispepsia, abdominal pain, and tinnitus. It was concluded that nabumetone was an effective anti-inflammatory drug in the treatment of rheumatoid arthritis with less toxicity than aspirin.

Systemic lupus erythematosus sera inhibit antigen presentation by macrophages to T cells

Several reports have demonstrated that systemic lupus erythematosus (SLE) patients have a decreased response to exogenous antigens both in vivo and in vitro. We examined the effects of SLE sera on macrophage (M phi) antigen-presenting functions. M phi from normal donors were pulsed with tetanus toxoid antigen in the presence of SLE or normal human serum (NHS), fixed in paraformaldehyde, and incubated with autologous T cells. Of 16 SLE sera tested, 11 inhibited the T-cell proliferative response (measured by [3H]thymidine uptake) compared to control NHS; mean percentage inhibition was 53 +/- 23%. This inhibition did not result from interference with antigen uptake by M phi and was found in both IgM and IgG fractions of the sera. There was a positive correlation between the amount of inhibition and the cytotoxic reactivity of the SLE sera against M phi as measured by Terasaki assay (r = 0.659, P less than 0.01). However, the presence and the amount of the inhibition did not correlate with serum immune complexes by Clq ELISA, serum anti-DR antibodies, or clinical disease activity of the SLE patients. We conclude that some SLE sera possess IgM and IgG antibodies reactive with M phi which affect M phi antigen-presenting functions, and might relate to decreased antigenic response in SLE patients.