Marius Teodorescu

B cell depletion therapy in systemic lupus erythaematosus: relationships among serum B lymphocyte stimulator levels, autoantibody profile and clinical response

Objective: To assess the relationships between serum B lymphocyte stimulator (BLyS) levels, autoantibody profile and clinical response in patients with systemic lupus erythaematosus (SLE) following rituximab-based B cell depletion therapy (BCDT). Methods: A total of 25 patients with active refractory SLE were followed for ⩾1 year following BCDT. Disease activity was assessed using the British Isles Lupus Assessment Group (BILAG) system, and serum levels of BLyS and autoantibodies to dsDNA and extractable nuclear antigens (ENA) measured by ELISA. Serum immunoglobulins and anti-dsDNA antibodies were assessed for expression of the 9G4 idiotope (indicating VH4–34 germline gene origin). Results: Following BCDT, all patients depleted in the peripheral blood and improved clinically for ⩾3 months. Pre-BCDT BLyS levels were quantifiable (median 1.9 ng/ml) in 18/25 patients and rose in most patients at 3 months post-BCDT (median 4.15 ng/ml). Nine patients, all with quantifiable pre-BCDT serum BLyS, experienced a disease flare within 1 year. This group of patients was more likely to harbour anti-Ro/SSA antibodies (odds ratio 1.76; p = 0.06) with higher serum levels (p = 0.0027; Mann–Whitney U test). Serum levels of anti-ribonucleoprotein (RNP)/Sm were also higher in this group (p<0.05). Expression of VH4–34 by serum immunoglobulins and anti-dsDNA antibodies had no predictive value for the length of clinical response. Conclusions: Patients with SLE with an expanded autoantibody profile and raised BLyS levels at baseline had shorter clinical responses to BCDT. This may reflect a greater propensity to, and degree of, epitope spreading in such patients and suggests that treatment regimens beyond BCDT may be necessary to induce long-lasting clinical remissions in these individuals.

Anti–topoisomerase I (Anti–Scl‐70) antibodies in patients with systemic lupus erythematosus

OBJECTIVE To investigate the presence and clinical significance of anti-Scl-70 antibodies in patients with systemic lupus erythematosus (SLE). METHODS Levels of antibodies against Scl-70 were determined by a commercial clinical enzyme-linked immunosorbent assay (ELISA) during routine evaluation. Results were verified by an additional ELISA with a characterized bovine Scl-70, by ELISA with a recombinant human topoisomerase I, by Western blot, and by double diffusion in agar gel. Disease activity was estimated retrospectively by the Systemic Lupus Activity Measure (SLAM). RESULTS Of 128 consecutive SLE patients, 25% were positive for anti-Scl-70 antibody; this antibody activity was cognate in nature. No SLE patient could be classified as also having systemic sclerosis. The levels of anti-Scl-70 were significantly correlated with the SLAM score for the entire cohort (r = 0.563, P < 0.001) and for 7 individual patients with multiple longitudinal measurements (r = 0.755-0.951, P < 0.001; n = 6) (r = 0.378, P < 0.05; n = 1). A significant correlation was also found between the levels of anti-Scl-70 and anti-double-stranded DNA antibodies (r = 0.558, P < 0.001). Patients with anti-Scl-70 had significantly higher risk of pulmonary hypertension (P < 0.01) and renal involvement (P < 0.001) than patients without this antibody. CONCLUSION Anti-Scl-70 antibody is present in a significant subset of patients with SLE. For this subset, it offers a good correlate of disease activity and suggests increased risk for pulmonary hypertension and nephritis.

Value of isolated IgA anti-β2 -glycoprotein I positivity in the diagnosis of the antiphospholipid syndrome.

OBJECTIVE To examine the prevalence of isolated IgA anti-β2 -glycoprotein I (anti-β2 GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2 GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti-β2 GPI in a mouse model of thrombosis. METHODS Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti-β2 GPI titers and binding to domain IV/V of β2 GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti-β2 GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. RESULTS A total of 198 patients were found to be positive for IgA anti-β2 GPI isotype, and 57 patients were positive exclusively for IgA anti-β2 GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2 GPI-positive serum samples reacted with domain IV/V of anti-β2 GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2 GPI positivity was associated with an increased risk of arterial thrombosis (P < 0.001), venous thrombosis (P = 0.015), and all thrombosis (P < 0.001). The association between isolated IgA anti-β2 GPI and arterial thrombosis (P = 0.0003) and all thrombosis (P = 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2 GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls. CONCLUSION Isolated IgA anti-β2 GPI-positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid tests are negative and APS is suspected is recommended. IgA anti-β2 GPI antibodies directed to domain IV/V of β2 GPI represent an important subgroup of clinically relevant antiphospholipids.

Relationship Between Rheumatoid Factor Isotypes and IgG Anti-Cyclic Citrullinated Peptide Antibodies

Objective. To validate in a general patient population (GPP) the clinical value of measuring rheumatoid factor (RF) isotypes in relationship to IgG anti-cyclic citrullinated peptide (CCP) antibodies (CCP2 and CCP3). Methods. Serum samples were obtained as follows: 1021 GPP, for whom RF was ordered for diagnosis, 137 with rheumatoid arthritis (RA), 100 healthy blood donors (HBD), and 50 with systemic lupus erythematosus. Turbidimetry and ELISA were utilized for RF screening, and individual RF isotypes and IgG anti-CCP antibodies were measured by ELISA; RF IgG was measured after pepsin digestion. Results. We validated the generally accepted 90%–98% positive predictive value (PPV) and about 68% sensitivity of the anti-CCP2 test on our diagnosed cohorts as 96% (95% CI 89–99) and 65% (95% CI 56–73), respectively. The 282 RF IgM+ specimens identified in the GPP were subdivided into 3 subsets: (1) 83 as RF IgM+ IgG+ IgA+ with 63% (95% CI 51–73) anti-CCP2+ (i.e., sensitivity similar to the RA cohort); (2) 50 as RF IgM+ IgG− IgA+ with significantly fewer anti-CCP2+ (22%; 95% CI 12–36); and (3) about half as IgM+ IgG− IgA− with just 3% (95% CI 1–8) anti-CCP2+, i.e., not significantly different from the 1% (95% CI 0–5) in HBD. Thus, the chance for a specimen in the GPP to be anti-CCP2+ (i.e., to come from an RA patient) was increased by 7- and 21-fold, respectively, by identifying RF IgA and IgG in addition to IgM. About one-third of anti-CCP− RA patients in our cohort were RF IgM+ IgG+ IgA+, reflected as 3.4% in the anti-CCP2− GPP. The agreement between anti-CCP2 and anti-CCP3 was significantly higher for RF+ RA and GPP patients, 86% (95% CI 78–93) and 83% (95% CI 73–91), respectively, than for the RF− RA (27%; 95% CI 6–61), RF− GPP (4%; 95% CI 0–19), and non-RA controls. Anti-CCP2 but not anti-CCP3 significantly distinguished the HBD from the GPP (95% CI). Conclusion. Measurement of the 3 isotypes of RF may increase by 7- to 21-fold the chance of making the serologic diagnosis of RA; a testing algorithm is proposed. The anti-CCP antibody response appears significantly less peptide-specific in the presence of IgM RF than in its absence.

New classification of the shared epitope in rheumatoid arthritis: impact on the production of various anti-citrullinated protein antibodies

OBJECTIVE HLA-DR [shared epitope (SE)] alleles have recently been re-classified into S1, S2, S3P and S3D groups. S2 and S3P have been associated with increased risk for RA. We assessed the impact of S1, S2, S3P and S3D alleles on anti-citrullinated protein antibody (ACPA) production. Instead of comparing allele-carriers to non-carriers, we studied each allele group individually, using the X/X (non-SE) genotype as reference. METHODS Serum and genomic DNA samples of 91 RA patients and 78 healthy controls were obtained. Various ACPAs and IgM RF were determined by ELISA. HLA-DRB1 genotyping and subtyping was performed by PCR. HLA-DRB1 alleles were re-classified as described above. Correlations between SE and ACPAs were determined. RESULTS Not only S2 and S3P, but, to a lesser extent, S1 and S3D alleles also predisposed to anti-cyclic citrullinated peptide (CCP) production (P < 0.0001, P = 0.004, P = 0.01 and P = 0.027, respectively), with the following hierarchy of association: S2+S3P > S1+S3D > X/X. Similar associations were observed for anti-citrullinated vimentin. Anti-citrullinated fibrinogen (CF) exerted a different association pattern with the strongest correlation with S1 alleles [odds ratio (OR) 16.00; P = 0.05]. In addition, HLA-DRB1*15 alleles may represent a special predisposing effect for anti-CF antibody production. Finally, in this study, RF production was associated only with the HLA-DRB1*0401 SE allele (P = 0.04). CONCLUSIONS Our approach of comparing individual S allele carriers with X/X genotype patients allowed us to perform unequivocal analyses and demonstrate new associations. Thus, novel subgroups of RA could be identified with potential relevance for prognosis and therapy.

Enhancement of human MLR by very low concentrations of lipopolysaccharide and blocking of this enhancement by polymyxin B

Addition of 50 micrograms LPS/ml to as little as 5 pg of LPS/ml to the allogeneic human mixed leukocyte reaction (MLR) enhanced [3H]thymidine incorporation 2-4-fold. The same concentrations of LPS had no effect on thymidine incorporation in cultures without the allogeneic cells. The Limulus amebocyte lysate (LAL) test we used was sensitive only to 15 pg LPS/ml indicating that the MLR is more sensitive to LPS than the LAL test. A commercially available RPMI 1640 culture medium used by many investigators was found to contain concentrations of LPS that would significantly enhance the MLR. Polymyxin B blocked the MLR-enhancing effect of LPS while having no inhibitory or stimulating effect itself. We concluded that low concentrations of LPS are helpful in revealing MLR incompatibilities by enhancing the lymphocyte responses and that the addition of polymyxin B is essential in the study of factors affecting the MLR by eliminating any LPS contribution.

Clinical value of anti-ssDNA (denatured DNA) autoantibody test: beauty is in the eyes of the beholder

Abstract The recent guidelines for the clinical use of antinuclear antibody tests issued by a Committee of the College of American Pathologists (CAP), suggest widespread misunderstanding of the clinical value of testing for the level of anti-ssDNA (single-stranded DNA or total DNA) IgG antibodies. This misunderstanding may stem from misconceptions about the manner in which clinicians use, in clinical context, immunoassays that have results expressed on a wide numerical scale. When the anti-ssDNA antibody (Ab) test is used for the differential diagnosis of new patients suspected of an inflammatory rheumatic disease the clinical sensitivity for systemic lupus erythematosus (SLE) is close to 100%, and the specificity about 85%. Since anti-dsDNA (double stranded DNA) is present in only about 65% of new SLE patients, an abnormal anti-ssDNA Ab test in the remaining 35% provides the clinician with a valuable clue to search for other criteria for SLE. Contrary to a widely held belief that anti-ss-DNA Ab occurs frequently in patients with rheumatoid arthritis (RA), anti-ssDNA is present only in 10–15% of this patient population and then at relatively low levels. There is evidence that anti-ssDNA, like anti-dsDNA, is involved in the pathogenesis of lupus nephritis. Moreover, the increase in anti-ssDNA Ab level appeared to be the best predictor of forthcoming increase in anti-dsDNA and SLE flare. In a context of symptoms different from those of rheumatic diseases, anti-ssDNA antibodies may be elevated in a relatively high proportion of patients with any of several diseases; leukemia, preeclampsia, chronic hepatitis, renal complications of diabetes, and some inflammatory neurological diseases. In conclusion, anti-ssDNA helps to rule out SLE, helps in the diagnosis of SLE when anti-dsDNA is not present and is useful in follow-up of SLE patients. Like most other quantifiable laboratory indicators of abnormality, anti-ssDNA Ab test is also useful in clinical context for the diagnosis and prognosis of several other condition.

Specific binding of IL-8 to rabbit α-macroglobulin modulates IL-8 function in the lung

Abstract:Objective and Design: The purpose of this study was to compare chemotactic activity of IL-8 alone with that of IL-8 reacted with rabbit α-macroglobulins (αM) in vivo.¶Methods: Initially the binding of recombinant human IL-8 (rhIL-8) to rabbit αM was studied. 125I-labeled rhIL-8 was incubated with αM, and electrophoresed on native 5% gels or SDS-polyacrylamide 4-20% gradient gels. Next, rhIL-8 or rhIL-8 bound to αM was administered via an endotracheal tube to rabbits lungs.¶Treatment: An endotracheal tube was wedged into a segment of the lobe of each lung, and a sample instilled through the tube into this segment. After 4 h the lungs were lavaged.¶Results: rhIL-8 bound to αM retained its full chemotactic activity in vitro but exhibited a diminished ability to induce the influx of neutrophils into the rabbit lung.¶Conclusions: The data suggest that αM may facilitate IL-8 clearance from the lung.

Effect of circulating immune complexes on the binding of rheumatoid factor to histones

OBJECTIVE To determine whether the reaction of rheumatoid factor (RF) with solid phase histone is due to the simultaneous presence of circulating immune complexes (CICs) or aggregated IgG. METHODS Serum samples from 56 patients with seropositive rheumatoid arthritis (RA) and 50 random blood bank donors were used. Binding of immunoglobulins to histone was determined by enzyme linked immunosorbent assay (ELISA) and by western blots. Aggregated IgG was obtained by heating at 61oC for 30 minutes. RESULTS Among the RA sera tested by ELISA, 54% were positive for histone binding by IgM, IgG, or IgA and 20% by IgM only. Heating of normal sera caused a significant enhancement in the binding of IgG to histone (p<0.001). This binding had a non-cognate behaviour—that is, it was destroyed by pepsin treatment of serum and was not significantly inhibited by competition with free histone. The same behaviour was seen for IgM, IgG, and IgA binding from RA sera. However, cognate IgG antibody binding to histone was inhibited by free histone and was resistant to pepsin digestion. Addition of heat aggregated IgG to RA sera or pretreatment of histone with aggregated IgG caused a significant increase in IgM binding to histone. CONCLUSION IgM, IgG, and IgA RF bind to solid phase histone as a result of attachment to histone of immune complexes or aggregated IgG and not as a result of a cognate reaction with histone.

Complement-fixing properties of antinuclear antibodies distinguish drug-induced lupus from systemic lupus erythematosus.

The immunofluorescence antinuclear antibody (ANA) test has been widely used to monitor autoimmune disease, but its value for diagnostic purposes is compromised by low specificity and high prevalence in disease-free individuals. The capacity of autoantibodies to fix serum complement proteins when bound to antigen is an important effector function because this property is associated with acute and chronic inflammatory processes. The current study evaluates the complement-fixing properties of antinuclear antibodies (CANA) in three well-defined and clinically-related patient groups: systemic lupus erythematosus (SLE), drug-induced lupus (DIL) and drug-induced autoimmunity (DIA). Of 20 patients diagnosed with SLE, 90% displayed complement-fixing ANA while this feature was present in only two of 18 patients with DIL and no patients with DIA without associated disease even though the mean ANA titres were similar among these patient groups. CANA was significantly correlated with anti-Sm activity. Because SLE but not DIL or DIA can be a life-threatening disease associated with complement consumption in vivo, these results demonstrate that measurement of CANA is a diagnostically useful tool and may have immunopathologic implications.