Yair Molad

Colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils.

Since colchicine-sensitive microtubules regulate the expression and topography of surface glycoproteins on a variety of cells, we sought evidence that colchicine interferes with neutrophil-endothelial interactions by altering the number and/or distribution of selectins on endothelial cells and neutrophils. Extremely low, prophylactic, concentrations of colchicine (IC50 = 3 nM) eliminated the E-selectin-mediated increment in endothelial adhesiveness for neutrophils in response to IL-1 (P < 0.001) or TNF alpha (P < 0.001) by changing the distribution, but not the number, of E-selectin molecules on the surface of the endothelial cells. Colchicine inhibited stimulated endothelial adhesiveness via its effects on microtubules since vinblastine, an agent which perturbs microtubule function by other mechanisms, diminished adhesiveness whereas the photoinactivated colchicine derivative gamma-lumicolchicine was inactive. Colchicine had no effect on cell viability. At higher, therapeutic, concentrations colchicine (IC50 = 300 nM, P < 0.001) also diminished the expression of L-selectin on the surface of neutrophils (but not lymphocytes) without affecting expression of the beta 2-integrin CD11b/CD18. In confirmation, L-selectin expression was strikingly reduced (relative to CD11b/CD18 expression) on neutrophils from two individuals who had ingested therapeutic doses of colchicine. These results suggest that colchicine may exert its prophylactic effects on cytokine-provoked inflammation by diminishing the qualitative expression of E-selectin on endothelium, and its therapeutic effects by diminishing the quantitative expression of L-selectin on neutrophils.

Cytokine Polymorphisms and Histologic Expression in Autopsy Studies: Contribution of TNF-α and TGF-β1 to the Pathogenesis of Autoimmune-Associated Congenital Heart Block

Although Abs to SSA/Ro-SSB/La are necessary for the development of congenital heart block (CHB), the low frequency suggests that fetal factors are contributory. Because CHB involves a cascade from inflammation to scarring, polymorphisms of the TNF-α promoter region and codons 10 and 25 of the TGF-β gene were evaluated in 88 children (40 CHB, 17 rash, 31 unaffected siblings) and 74 mothers from the Research Registry for Neonatal Lupus (NL). Cytokine expression was assessed in autopsy material from two fetuses with CHB. Significantly increased frequency of the −308A (high-producer) allele of TNF-α was observed in all NL groups compared with controls. In contrast, the TGF-β polymorphism Leu10 (associated with increased fibrosis) was significantly higher in CHB children (genotypic frequency 60%, allelic frequency 78%) than unaffected offspring (genotypic frequency 29%, p = 0.016; allelic frequency 56%, p = 0.011) and controls, while there were no significant differences between controls and other NL groups. For the TGF-β polymorphism, Arg25, there were no significant differences between NL groups and controls. In fetal CHB hearts, protein expression of TGF-β, but not TNF-α, was demonstrated in septal regions, extracellularly in the fibrous matrix, and intracellularly in macrophage infiltrates. Age-matched fetal hearts from voluntary terminations expressed neither cytokine. TNF-α may be one of several factors that amplify susceptibility; however, the genetic studies, backed by the histological data, more convincingly link TGF-β to the pathogenesis of CHB. This profibrosing cytokine and its secretion/activation circuitry may provide a novel direction for evaluating fetal factors in the development of a robust animal model of CHB as well as therapeutic strategies in humans.

Treatment of lupus patients with a tolerogenic peptide, hCDR1 (Edratide): immunomodulation of gene expression.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulation of cytokines, apoptosis, and B- and T-cell functions. The tolerogenic peptide, hCDR1 (Edratide), ameliorated the clinical manifestations of murine lupus via down-regulation of pro-inflammatory cytokines and apoptosis, up-regulation of the immunosuppressive cytokine TGF-beta, and the induction of regulatory T-cells. In the present study, gene expression was determined in peripheral blood mononuclear cells of 9 lupus patients that were treated for 26 weeks with either hCDR1 (five patients), or placebo (four patients). Disease activity was assessed by SLEDAI-2K and the BILAG scores. Treatment with hCDR1 significantly down-regulated the mRNA expression of the pathogenic cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-10, of BLyS (B-lymphocyte stimulator) and of the pro-apoptotic molecules caspase-3 and caspase-8. In contrast, the treatment up-regulated in vivo gene expression of both TGF-beta and FoxP3. Furthermore, hCDR1 treatment resulted in a significant decrease in SLEDAI-2K (from 8.0+/-2.45 to 4.4+/-1.67; P=0.02) and BILAG (from 8.2+/-2.7 to 3.6+/-2.9; P=0.03) scores. Thus, the tolerogenic peptide hCDR1, immunomodulates, in vivo, the expression of genes that play a role in SLE, consequently restoring the global immune dysregulation of lupus patients. Hence, hCDR1 has a potential role as a novel disease-specific treatment for lupus patients.

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A 3 adenosine receptor expression

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.

Seasons of the Year and Activity of SLE and Behçet's Disease

Patients with systemic lupus erythematosus (SLE) were evaluated using a telephone questionnaire on the activity of various disease manifestations during the seasons of the past year. The results were compared to those of patients with Behcets disease (BD), using the same questionnaires, and analyzed in relation to the mean temperature, humidity, barometric pressure, and ultraviolet radiation (UVR) in the patients location, obtained from the official Israeli Meteorological Service. It was found that SLE patients had a tendency towards winter worsening of clinical manifestations, shown as increased incidence of joint pains, weakness, fatigue, Raynauds phenomenon, and rash, as well as increased number of hospital admissions, sick leaves, and need to raise the dose of medications. The symptoms of patients with BD were not correlated to seasons of the year, except for increased joint pains in autumn and spring. We suggest that UVR accumulation might cause exacerbations in SLE patients several months after prolonged exposure to sunlight in the summer.

Serum cobalamin and transcobalamin levels in systemic lupus erythematosus

PURPOSE The purpose of this study was to assay serum cobalamin levels in patients with systemic lupus erythematosus (SLE) as there are few case reports on the association of pernicious anemia and SLE. PATIENTS AND METHODS Serum cobalamin levels were assayed in 43 female SLE patients by a radio-dilution assay using purified intrinsic factor. RESULTS Cobalamin levels were found to be significantly lower in the SLE group compared with a normal control group, eight of whom (18.6%) had serum cobalamin levels equal to or lower than 180 pg/mL (mean: 129.25 +/- 40.05 pg/mL). None of the SLE patients had been found to have pernicious anemia. The transcobalamin II level and unsaturated vitamin B12 binding capacity, but not the cobalamin level, were positively correlated with SLE activity. CONCLUSION Our results may indicate a subtle cobalamin deficiency in SLE patients without pernicious anemia.

Connective tissue disorders: systemic lupus erythematosus, Sjögren's syndrome, and scleroderma.

Connective tissue disorders are systemic, autoimmune, multiorgan diseases in which the central and peripheral nervous systems are frequently involved. The objective of this chapter is to describe the neurological manifestations of three of the most common systemic autoimmune disorders: systemic lupus erythematosus (SLE), scleroderma, and Sjögrens syndrome (SS). In SLE the neuropsychiatric manifestations involve mainly the central nervous system (CNS), including cognitive dysfunction, headache, psychosis and mood changes, seizures, cerebrovascular disease, and myelopathy. Peripheral nervous system (PNS) manifestations are less common and include polyneuropathies as well as mononeuropathies and acute inflammatory demyelinating polyneuropathy. Antiphospholipid syndrome (APLS) is relatively common and should be searched for whenever focal neurological symptoms occur. In scleroderma the PNS is more commonly involved; symptoms include polyneuropathies, entrapment neuropathies, and mononeuropathies (mostly cranial neuropathies or mononeuritis multiplex). Additionally autonomic involvement occurs and myopathies are relatively common. In SS the PNS is similarly involved with several types of polyneuropathies, mononeuropathies, and autonomic dysfunction. Also common are myelopathies and aseptic meningitides. These and other, less common manifestations, as well as the diagnostic procedures and the therapeutic approaches, will be dealt with in this chapter.

Association of HLA-B5 with Clinical Expression and Severity of Behcet's Disease in Israel.

There were 55 Israeli patients with Behcets disease (BD) included in a study conducted to determine the correlation between HLA-B5 and clinical manifestations and severity of the disease. The systemic manifestations of BD were analyzed in relation to HLA typing, and a systemic severity score for BD was calculated according to potential morbidity and mortality associated with various clinical features. Of the 55 patients, 42 (76.4%) were sephardic Jews, 2 (3.6%) were ashkenazi Jews, and 11 (20.0%) were Israeli Arabs. There were 39 (70.9%) HLA-B5 positive patients; they had a significantly higher incidence of thrombophlebitis and a lower rate of erythema nodosum. The HLA-B5-positive patients were significantly older at disease onset, and their severity score tended to be higher, although not statistically significant.The results of our study imply that HLA-B5 in Israeli patients is associated with specific clinical features, especially more vascular disease, and may be associated with a more severe course of BD. This is of general interest because American and North European patients also have less HLA-B5 and less severe disease. (J Clin Rheumatol 1999;5:137-140).

Neutrophil Adhesion Molecule Expression in Familial Mediterranean Fever: Discordance between the Intravascular Regulation of β2 Integrin and L-Selectin Expression in Acute Attack

Background To determine the surface expression of neutrophil β2 integrin (CD11b/CD18) and L-selectin (LS) adhesion molecules in patients with familial Mediterranean fever (FMF) and to investigate the in vitro regulation of their expression in response to chemoattractant stimuli. Methods Neutrophil surface expression of CD11b and LS molecules was analyzed by flow cytometry in anticoagulated whole blood drawn from FMF patients and normal controls, and the in vitro regulation of these molecules induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) was assayed. Results Patients during acute FMF attacks showed a statistically significant increased neutrophil surface CD11b compared with normal controls (mean fluorescence intensity: 22.8 ± 13.7 vs 12.8 ± 10.41, respectively; p = .03). There was no difference in LS expression between the groups. Neutrophils of FMF patients regulate CD11b and LS expression induced by chemoattractant (FMLP) stimulation to a degree similar to that in controls. Conclusions β2 Integrin is up-regulated during an acute attack of FMF in dissociation with LS expression, suggesting a unique nonchemoattractant-mediated neutrophil activation.

A central role of plasmin in cardiac injury initiated by fetal exposure to maternal anti-Ro autoantibodies

OBJECTIVE Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating β2-glycoprotein I (β2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.