Neurocognitive deficits and neuroimaging abnormalities are prevalent in children with lupus: clinical and research experiences at a US pediatric institution.

Neurocognitive impairments and neuroimaging abnormalities are frequently observed in adults with systemic lupus erythematosus. There is a paucity of similar data in childhood-onset disease. We hypothesized that neurocognitive and neuroimaging abnormalities would be prevalent in children undergoing neuropsychological evaluations. We reviewed patient neurocognitive evaluations performed at a large United States pediatric institution during the period 2001 to 2008. Records were retrieved from 24 children referred to neuropsychology due to clinical indications. Data from 15 children enrolled in a prospective structure-function association study were also analyzed. Subjects were predominantly African-American and Hispanic adolescent girls of average intelligence. aPL positivity and aspirin use was prevalent. Neurocognitive impairment was designated in 70.8% of retrospective, and 46.7% of prospective cohort patients. Deficits were seen at times of wellness, without previous neuropsychiatric lupus, and early in disease courses. Scores >1.5 standard deviations below published age-matched norms were common in tests of executive functioning, visual memory and visual-spatial planning. Features of depression were seen in 33.3% of the children in the retrospective cohort (clinical referrals). Cerebral and cerebellar volume loss was observed in a majority of blinded prospective cohort research magnetic resonance images (73.3% and 67.7% respectively). White matter hyperintensities were observed in retrospective and prospective cohort magnetic resonance images (36.6% and 46.7% respectively). Larger prospective studies that elucidate structure-function associations in children with systemic lupus erythematosus are planned.

Identification of new SLE-associated genes with a two-step Bayesian study design.

In our earlier study, we utilized a Bayesian design to probe the association of approximately 1000 genes (approximately 10,000 single-nucleotide polymorphisms (SNPs)) with systemic lupus erythematosus (SLE) on a moderate number of trios of parents and children with SLE. Two genes associated with SLE, with a multitest-corrected false discovery rate (FDR) of <0.05, were identified, and a number of noteworthy genes with FDR of <0.8 were also found, pointing out a future direction for the study. In this report, using a large population of controls and adult- or childhood-onset SLE cases, we have extended the earlier investigation to explore the SLE association of 10 of these noteworthy genes (109 SNPs). We have found that seven of these genes exhibit a significant (FDR<0.05) association with SLE, both confirming some genes that have earlier been found to be associated with SLE (PTPN22 and IRF5) and presenting novel findings of genes (KLRG1, interleukin-16, protein tyrosine phosphatase receptor type T, toll-like receptor (TLR)8 and CASP10), which have not been reported earlier. The results signify that the two-step candidate pathway design is an efficient way to study the genetic foundations of complex diseases. Furthermore, the novel genes identified in this study point to new directions in both the diagnosis and the eventual treatment of this debilitating disease.

Neutrophil and monocyte cell surface p150,95 has iC3b-receptor (CR4) activity resembling CR3.

Previous investigations of p150,95 (CD11c), the third member of the CD18 membrane glycoprotein family that includes CR3 (Mac-1 or CD11b) and LFA-1 (CD11a), had demonstrated that solubilized p150,95 bound to iC3b-agarose in a manner similar to isolated CR3. The current study showed that membrane surface p150,95 also expressed iC3b-receptor activity and was probably the same as the neutrophil receptor for iC3b- or C3dg-coated erythrocytes (EC3bi or EC3dg) that had been previously designated CR4. Normal neutrophil and macrophage CR4-dependent EC3bi rosettes were inhibited by monoclonal anti-p150,95, and cells from a patient with CD18 deficiency did not form CR4-dependent EC3bi rosettes. With neutrophils that bore large amounts of CR1 and CR3 and little p150,95, EC3bi were found primarily via CR1 and CR3, and demonstration of p150,95-dependent rosettes required large amounts of fixed iC3b, low-ionic strength buffer, and antibody blockade of CR1 and CR3. By contrast, culture-derived macrophages expressed eight times more p150,95 than did monocytes and EC3bi were bound to both p150,95 and CR3 when EC3bi bore small amounts of fixed iC3b and assays were carried out in isotonic buffer. Comparison of the amounts of CR1, CR3, and CR4 in various tissues by immunoperoxidase staining revealed that CR4 was the most abundant C3 receptor molecule on tissue macrophages, and suggested that CR4 might be involved in clearance of C3-opsonized particles or immune complexes.

Importance of an 85 kDa membrane glycoprotein for a variety of cell-cell interactions.

The membrane molecule termed "7F7-antigen" has been found to be involved in several examples of cell-cell interactions. This 85 kDa glycoprotein with a protein core of about 55 kDa contains N-linked and O-linked carbohydrates. It has an isoelectric point of 8.0-8.5 and is expressed on 20% of peripheral blood mononuclear cells, 35% of peripheral blood B-cells, follicular dendritic cells and vascular endothelium. It is also expressed on activated T-cells and its expression on B-cells, fibroblasts and monocytes increases after treatment with PWM, interferon-gamma and after three days culture, respectively. The MAb 7F7 used to define this antigen inhibits the initiation of T-cell proliferation induced by anti-CD3, PHA, ConA and (weakly) allogenic stimulator cells, but does not affect the growth of IL-2 dependent T-cells and does not interfere with the killing of PHA-blasts by allogenic IL-2 dependent T-cells. 7F7 also inhibits the binding of C3-coated sheep erythrocytes to B-cells, the PMA-induced aggregation of U937 and the binding of activated T-cells to fibroblasts. The 7F7-antigen is expressed on some non-Hodgkin lymphomas of B-cell differentiation, particularly those with follicular structure, but not on Burkitt's lymphoma, ALL or carcinomas of various tissues. It is, however, found on fibrous tissue surrounding infiltrating carcinoma cells. The expression of a melanoma antigen, P3.58, which was shown to be identical to 7F7-antigen correlates with stage and spread of invasive melanoma. It was concluded that the 7F7-antigen, which is probably related to a previously described adherence molecule (ICAM-1), is of biological importance for the initiation of T-cell responses. With the possible exception of melanoma its expression on neoplastic cells in vivo is unlikely to be of importance for the spread of malignant disease.

Reduction of cortisol levels after single intra-articular and intramuscular steroid injection.

BACKGROUND: This study was undertaken to determine the influence of single intra-articular or intra muscular injections of methylprednisolone acetate on the hypothalamic-pituitary-adrenal axis. PATIENTS AND METHODS: Twenty-one patients with rheumatic disease who had never been treated with systemic glucocorticoids and had not received local injections of these agents for the preceding 2 months, were given 40 mg of methylprednisolone acetate. Group I (11 patients) received one intra-articular injection into the knee, and Group II (10 patients) received the same dose intramuscularly. RESULTS: In Group I, serum cortisol levels were significantly decreased 24 hours after injection (228.2 +/- 8.7 nmol/L versus 193 +/- 16.3 nmol/L; P < 0.05). Serum cortisol levels were decreased in 9 of the 11 patients, by an average of 21.5%. Two patients' levels were below 138 nmol/L, which is considered to be the lower limit of normal range. Serum cortisol levels were below normal range in 3 patients 72 hours after intra-articular steroid injection. In Group II, serum cortisol levels were significantly decreased at 72 hours after injection (239.6 +/- 10.3 nmol/L versus 175.6 +/- 21.4 nmol/L; P < 0.01). Three patients' levels were below normal. By 72 hours postinjection, serum cortisol concentrations in 9 of 10 patients were decreased by an average of 31% compared to preinjection values. CONCLUSION: The present study suggests that decreased adrenocortical secretion, as reflected in depressed cortisol levels, can result from a single, low-dose, intra-articular or intramuscular injection of depot corticosteroids.

Enhancement of protein S anticoagulant function by beta2-glycoprotein I, a major target antigen of antiphospholipid antibodies: beta2-glycoprotein I interferes with binding of protein S to its plasma inhibitor, C4b-binding protein.

Thrombosis in the antiphospholipid syndrome has been associated with acquired deficiency of the anticoagulant protein S. We sought evidence that beta2-glycoprotein I, a major target antigen for antiphospholipid antibodies, is involved in regulation of protein S activity. Incubation of purified protein S or plasma with beta2-glycoprotein I reversed functional modulation of protein S by its plasma inhibitor, the C4b-binding protein. In a plasma-free ELISA, beta2-glycoprotein I prevented the binding of protein S and C4b-binding protein when preincubated with immobilized protein S but not when similarly preincubated with C4b-binding protein. beta2-glycoprotein I in fluid phase interfered with precipitation of protein S by sepharose-bound C4b-binding protein. Effects of beta2-glycoprotein I on protein S function were inhibited by one of four monoclonal anti-beta2-glycoprotein 1 antibodies. These data suggest that beta2-glycoprotein I helps maintain adequate plasma levels of circulating free, active protein S. Antiphospholipid (anti-beta2-glycoprotein I) antibodies might cause sporadic thrombosis, at least in part, by impairing this novel regulatory mechanism.

Dyslipoproteinemia in the course of active rheumatoid arthritis.

Concentrations of serum lipids and serum very low-density lipoproteins and low-density lipoproteins (VLDL+LDL, originally called beta lipoproteins) were measured and agarose gel electrophoresis of serum lipoproteins was performed in 69 patients with active rheumatoid arthritis (RA), 40 patients with psoriatic arthritis (PA), 21 patients with osteoarthritis (OA), and 65 healthy blood donors. These lipid parameters were also compared in 21 RA and 40 PA patients during periods of severe disease activity (SA) versus minimal disease activity (MA). RA patients had significantly decreased concentrations of total serum lipids, total serum cholesterol, cholesterol in LDL, and cholesterol in high-density lipoproteins (HDL) compared with healthy blood donors. RA patients with SA had significantly decreased cholesterol in LDL and HDL compared with patients with MA. As the disease activity decreased, RA patients had normalization of almost all serum lipid concentrations. Electrophoresis of serum lipoproteins showed heterogeneous patterns in RA patients. Patients with PA also had some evidence of dyslipoproteinemia. Serum lipids changed with disease activity in PA patients in a manner similar to that in RA patients. These data show that patients with RA and PA have a dyslipoproteinemia that is related to disease activity.

Cholesterol crystals in synovial and bursal fluid.

Cholesterol crystals were found in two patients with classic rheumatoid arthritis (RA). In one patient, cholesterol crystals were found in synovial fluid from both shoulder joints, and in the second they were in an olecranon bursa. To examine the possible systemic etiology of cholesterol crystals in synovial and bursal fluid, lipid concentrations and the presence of serum antilipoprotein antibodies were measured. Antilipoprotein antibodies were not found. The concentration of lipid and lipoproteins, as well as the normal pattern of lipoprotein on agarose gel, eliminates the possibility of hyperlipoproteinemia. Results seemed to exclude a systemic etiology for the formation of cholesterol crystals in synovial and bursal fluid in the RA patients. It appears that several local factors such as defective drainage, local destruction, increased permeability of synovial membrane, and intraarticular (bursal) bleeding are possible etiologies.

Antilipoprotein antibodies in rheumatoid arthritis.

Antibodies against very low-density lipoproteins and low-density lipoproteins (aLA) were found in 26 of 69 (38%) patients with active rheumatoid arthritis (RA) but not in any control subjects (ie, 40 patients with psoriatic arthritis, 21 patients with osteoarthritis, and 65 healthy blood donors). In 21 RA patients (30%), lipoproteins were found in the dissociated components of circulating immune complexes. RA patients with aLA had significantly decreased cholesterol levels in all lipoprotein fractions and total serum lipids, while serum triglycerides were significantly increased compared with RA patients without aLA. Anticardiolipin antibodies as measured by the Venereal Disease Research Laboratory test were not found in any subject in this study. These findings suggest a possible autoimmune origin of dyslipoproteinemia in some patients with active RA.

Pediatric rheumatic diseases.

The rheumatic diseases of childhood are a relatively common and extraordinarily diverse group of illnesses; nevertheless, they are at least distantly related by similarities of immunodysregulation. These pathophysiologic relationships are reflected in affected children in similarities of historical, physical, and laboratory data as well as therapeutic intervention.

The antiphospholipid syndrome: immunologic and clinical aspects. Clinical spectrum and treatment.

Antiphospholipid antibodies (aPL) have been associated with clinical conditions that involve arterial or venous thrombotic events and pregnancy morbidity including fetal loss and preeclampsia. These antibodies are detected by various functional tests for the lupus anticoagulant, the anticardiolipin ELISA, the anti-beta2-glycoprotein I ELISA, or ELISA tests for other aPL. The pathogenic mechanisms are poorly understood. A "2 hit" hypothesis has been entertained in which there is underlying vascular (endothelial) damage, and in the presence of an aPL, a thrombotic complication emerges. Although the role of immunologic processes and autoimmunity appears important, immunosuppressive therapy has not proven very effective. Treatment options are limited to antiplatelet therapy (primarily for arterial events) and anticoagulation (with coumadin, heparin, or low molecular weight heparins) because of lack of understanding of the inciting factors and the pathogenesis of the process.

Identification of a spontaneously shed fragment of B cell complement receptor type two (CR2) containing the C3d-binding site.

CR2 is a 140-kilodalton glycoprotein expressed on B lymphocytes which binds to both C3d and Epstein-Barr virus (EBV). The present study identified a 72-kilodalton C3d-binding protein (gp72) in the spent culture media of Raji B lymphoblastoid cells as a spontaneously shed fragment of the 140-kilodalton CR2 molecule. Both polyclonal and monoclonal antibodies (AB) were used in several assay systems to detect antigenic determinants shared between the gp72 fragment and CR2. Rabbit antibodies to either intact CR2 or gp72 blocked C3d receptor activity, and this inhibitory activity was removed by absorption of anti-CR2 with purified gp72.OKB7, a monoclonal anti-CR2 AB that blocks both C3d and EBV binding to CR2, reacted specifically with both CR2 and gp72, whereas both anti-B2 and HB-5 monoclonal anti-CR2 AB, that block neither of these receptor sites, were unreactive with gp72. The data suggested that the gp72 fragment was not present as such in intact cells, but rather was a product of cells generated by proteolysis of CR2. Thus, intrinsically labeled gp72 was isolated from Raji cell media by affinity chromatography on OKB7-Sepharose, but only intact CR2 was isolated from the Raji cell fraction solubilized in the presence of protease inhibitors. Several lines of evidence suggested that gp72 was not a second type of C3d receptor that was distinct from CR2. First, Raji cells expressed nearly identical amounts of OKB7 and HB-5 epitopes when analyzed by flow cytometry or radioimmune assay, excluding the possibility that B cells expressed OKB7 antigens in both CR2 and a distinct HB-5-negative C3d receptor. Second, all Raji cell surface C3d receptor activity was associated with HB-5-reactive CR2 molecules. We conclude that gp72 represents a spontaneously shed proteolytic fragment of CR2 that contains the C3d-binding site and the closely associated OKB7 epitope.

In vitro correlates of the L. casei animal model of Kawasaki disease.

The induction of coronary arteritis in mice by Lactobacillus casei cell wall (CW) is thought to represent an animal model of Kawasaki disease. Treatment of vascular endothelial cells (EC) in vitro with supernatants from CW stimulated human mononuclear cells (MNC) enhanced adherence of human polymorphonuclear leukocyte (PMN) to human EC, and EC expression of intercellular adhesion molecule-1 (ICAM-1) but not HLA-DR. Supernatants contained high concentrations of tumor necrosis factor-alpha (TNF-alpha) and PMN adherence correlated directly with the concentration of TNF-alpha. Intravenous human gamma globulin (IVGG) preparations did not block the effect of cytokine containing MNC supernates upon EC, ICAM-1 expression by EC, or PMN adherence to prestimulated EC. However, both EC ICAM-1 expression and enhanced PMN adherence to EC by CW induced MNC supernatants were blocked by anti-TNF-alpha treatment. The initial coronary inflammatory reaction in the mouse model appears to involve PMN adherence to vascular EC that have been activated by TNF-alpha released by MNC after stimulation with CW.

Lymphocyte subpopulation alterations induced by intravenous megadose pulse methylprednisolone.

Lymphocyte subpopulations from 9 children with different rheumatic diseases were studied before and 5, 24 and 48 h after high dose intravenous methylprednisolone. Leu-1 (+) cells, Leu-2a (+) cells, Leu-3a (+) cells and surface immunoglobulin (+) cells were counted. A panlymphopenia occurred 5 h after infusion, but numbers of all cell types were normal by 48 h. B cells were less affected than T cells. Leu-3a (+) cells were relatively more decreased than the other populations at 5 h and took longer to return to prepulse levels. This suggests a selective difference in distribution of lymphocyte subpopulations after pulse methylprednisolone infusion.

Cell surface proteins reacting with activated complement components.

The biologic activities mediated by the products of complement activation include cellular, bacterial, and viral lysis, inflammation, phagocytosis, and immunoregulation. These responses are achieved through the interaction of the activated forms of several of the complement proteins with cell membrane proteins. This report reviews aspects of the structure, ligand specificity, and function of the various complement receptors with particular emphasis on those receptors which bind to the activated fragments of C3. In addition, we briefly summarize the surface proteins on foreign particles that bind C3 and their possible role in the pathogenesis of these organisms.

Morbidity associated with long-term methotrexate therapy in juvenile rheumatoid arthritis.

To evaluate the adverse effects associated with long-term methotrexate (MTX) therapy in children with juvenile rheumatoid arthritis, we conducted a retrospective review of 62 patients with polyarticular juvenile rheumatoid arthritis, treated from 84 to 296 weeks with MTX weekly. Pulmonary function testing was performed before MTX therapy on 46 patients older than 6 years of age; 26 patients had serial pulmonary function testing, and no abnormalities were detected. In all 62 patients, liver function (alanine aminotransferase and aspartate aminotransferase activity) was monitored every 3 months. Transient liver function abnormalities developed in nine patients during treatment. Twelve patients underwent percutaneous liver biopsies after receiving 815 to 2980 mg of MTX; none had fibrosis or cirrhosis. Macrocytic anemia developed in one child receiving simultaneous long-term trimethoprim-sulfamethoxazole therapy and resolved after the trimethoprim-sulfamethoxazole was discontinued. No stomatitis or rashes were observed. Six patients were able to discontinue MTX therapy when their disease remitted; 56 continue MTX therapy. No child permanently discontinued MTX therapy because of an adverse effect. These data suggest that MTX may be better tolerated in children with juvenile rheumatoid arthritis than in adults with rheumatoid arthritis.

Structural requirements for C3d,g/Epstein-Barr virus receptor (CR2/CD21) ligand binding, internalization, and viral infection.

The structure of CR2, the human C3d,g/EBV receptor (CR2/CD21) consists of 15 or 16 60-70 amino acid repeats called short consensus repeats (SCRs) followed by a transmembrane and a 34-amino acid intracytoplasmic domain. Functions of CR2 include binding the human complement component C3d,g when it is covalently attached to targets or cross-linked in the fluid phase. In addition, CR2 binds the Epstein-Barr virus (EBV) and mediates internalization of EBV and subsequent infection of cells. In order to explore functional roles of the repetitive extracytoplasmic SCR structure and the intracytoplasmic domain of CR2, we have created truncated CR2 (rCR2) mutants bearing serial deletions of extracytoplasmic SCRs and also the intracytoplasmic tail. We then stably transfected these rCR2 mutants into two cell lines, murine fibroblast L cells and human erythroleukemic K562 cells. Phenotypic analysis of these expressed mutants revealed that 1) The C3d,g- and EBV-binding sites are found in the two amino-terminal SCRs of CR2, 2) expression of SCRs 3 and 4 is further required for high affinity binding to soluble cross-linked C3d,g, 3) the intracytoplasmic domain of CR2 is not required for binding C3d,g or EBV but is necessary for internalization of cross-linked C3d,g as well as for EBV infection of cells, 4) monoclonal anti-CR2 antibodies with similar activities react with single widely separated epitopes, and 5) no functional roles can yet be clearly assigned to SCRs 5-15, as rCR2 mutants not containing these SCRs show no major differences from wild-type rCR2 in binding or internalizing cross-linked C3d,g or mediating EBV binding and infection.

gp140, a C3b-binding membrane component of lymphocytes, is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4).

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.