Ennio Giardina

Overexpression of interleukin-23, but not interleukin-17, as an immunologic signature of subclinical intestinal inflammation in ankylosing spondylitis

OBJECTIVE Subclinical gut inflammation is common in spondylarthritis, but the immunologic abnormalities underlying this process are undefined. Perturbation of the interleukin-23 (IL-23)/Th17 axis has emerged as a fundamental trigger of chronic inflammation. This study was undertaken to investigate the expression and tissue distribution of IL-23/Th17-related molecules in Crohns disease (CD) and in subclinical gut inflammation in ankylosing spondylitis (AS). METHODS Quantitative gene expression analysis of Th1/Th2 and IL-23/Th17 responses was performed in intestinal biopsy samples obtained from 12 patients with CD, 15 patients with AS, and 13 controls. IL-23 tissue distribution and identification of IL-23-producing cells were evaluated by immunohistochemistry. RESULTS We demonstrated a strong and significant up-regulation of IL-23p19 transcripts in the terminal ileum in patients with AS and patients with CD. IL-23 was abundantly produced by infiltrating monocyte-like cells in inflamed mucosa from AS and CD patients. Notably, we also identified Paneth cells as a major source of IL-23 in patients with AS, patients with CD, and normal controls. Unlike CD, in AS patients, IL-23 was not associated with up-regulation of IL-17 and the IL-17-inducing cytokines IL-6 and IL-1beta. Finally, while the Th1-related cytokines interferon-gamma, IL-12p35, and IL-27p28 were overexpressed only in CD patients, IL-4, IL-5, and STAT-6 were also significantly increased in AS patients. CONCLUSION Our findings indicate that overexpression of IL-23, but not IL-17, is a pivotal feature of subclinical gut inflammation in AS. Identification of resident Paneth cells as a pivotal source of IL-23 in physiologic and pathologic conditions strongly suggests that IL-23 is a master regulator of gut mucosal immunity, providing a pathophysiologic significance to the reported association between IL-23 receptor polymorphisms and intestinal inflammation.

IgA- and insulin-containing (C3-fixing) circulating immune complexes in diabetes mellitus.

Sera of patients with type 1 and type 2 diabetes were examined for IgA- and insulin-containing immune complexes (IgA-ICs, ICs-insulin) using a solid-phase anti-C3 enzyme immunoassay. IgM-ICs and IgG-ICs were also investigated. IgA-ICs were detected in 6 of 26 type 1 diabetics, in 9 of 25 insulin-treated type 2 diabetics, and in 8 of 34 type 2 diabetics on oral hypoglycemic agents, but only in 2 sex- and age-matched controls. ICs-insulin was detected in 9 of 25 type 1 diabetics and in 1 of 19 insulin-treated type 2 diabetics, irrespective of the time of insulin treatment. ICs-insulin did not appear to be related to the presence of microangiopathy. IgA-ICs were found to be associated with the presence of microangiopathy, suggesting that they may play a role in the pathogenesis of the late diabetic complications.

Glucose-induced loss of glycosyl-phosphatidylinositol-anchored membrane regulators of complement activation (CD59, CD55) by in vitro cultured human umbilical vein endothelial cells

Aims/hypothesis. This study examines whether increased glucose concentrations are responsible for a decreased expression of membrane regulators of complement activation molecules. The effect of high glucose in determining an increase in membrane attack complex deposition on endothelial cells was also investigated. Methods. Endothelial cells were isolated from umbilical cord tissue, cultured in the presence of increased concentrations of glucose, and the expression of CD46, CD55, and CD59 was detected by ELISA (enzyme-linked immunosorbent assay) and by flow cytometry. Glucose-treated endothelial cells were also incubated with antiendothelial cell antibodies and fresh complement to assess the amount of membrane attack complex formation. Results. High concentrations of glucose decreased the expression of CD59 and CD55 by endothelial cells in a time-dependent and glucose concentration-dependent manner without affecting CD46 expression. High concentrations of soluble CD59 were found in the supernatants of cells treated with high glucose. The decrease in CD59 expression induced by high glucose concentrations was reversed by coincubation of cells with a calcium channel blocking agent (Verapamil). All of these effects were not reproduced by osmotic control media. Cells treated with concentrations of high glucose were more susceptible to complement activation and membrane attack complex formation after exposure to antiendothelial cell antibodies. Conclusion/Interpretation. We speculate that hyperglycaemia could directly contribute to a loss of CD59 and CD55 molecules through a calcium-dependent phosphoinositol-specific phospholipase C activation and subsequent regulation of cell wall expression of GPI-anchored proteins. This phenomenon could facilitate the activation of a complement pathway and could play a part in the aetiology of endothelial dysfunction in diabetes. [Diabetologia (2000) 43: 1039–1047]

Phenotype and functional changes of Vγ9/Vδ2 T lymphocytes in Behçet's disease and the effect of infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity

IntroductionInfliximab is a chimeric monoclonal antibody against tumor necrosis factor alpha (TNF-α) that has been introduced recently for Behçets disease (BD) patients who were resistant to standard treatment. The aim of this study was to analyse the functional changes of Vγ9/Vδ2 T lymphocytes in both active and inactive disease and the effect of infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity.MethodsWe investigated 1) cell expansion, 2) expression of TNFRII receptor, 3) perforin and gamma interferon (IFN) content, 4) release of granzyme A (GrA) and 5) phenotype changes, in vitro and in vivo, in Vγ9/Vδ2 T lymphocytes by means of fluorescence-activated cell sorter analysis of lymphocyte cultures from patients with active and inactive BD and healthy subjects.ResultsCell expansion, expression of TNFRII, perforin and gamma IFN content and release of granzyme A were significantly higher in active patients. In vitro and ex vivo treatment with infliximab resulted in a significant reduction of all parameters together with changes in the phenotype of Vγ9/Vδ2 T cells.ConclusionsAll together these data indicate that infliximab is capable of interfering with Vγ9/Vδ2 T cell function in BD and although cell culture models cannot reliably predict all potential effects of the drug in vivo, our results present the possibility that this drug may find use in a range of immunological disorders, characterized by dysregulated cell-mediated immunity.

IgG anti‐endothelial cell antibodies (AECA) in type I diabetes mellitus; induction of adhesion molecule expression in cultured endothelial cells

AECA were detected in 25 of 71 patients with type 1 diabetes mellitus and in two of 33 healthy subjects. Patients with diabetes of < 1 year duration and those with long‐standing disease had the highest levels of these antibodies. Inhibition studies suggest that at least part of the AECA reactivity is due to cross‐reactive anti‐ssDNA antibodies. AECA‐positive sera were able to increase intercellular adhesion molecule‐1 (ICAM‐1) and E‐selectin on human umbilical vein endothelial cells (HUVEC). Increased binding of polymorphonuclear (PMN) cells was also found to accompany raised E‐selectin expression. Soluble ICAM‐1 and E‐selectin were also found to be increased in the sera of AECA‐positive patients. An effect of AECA on endothelial cell function is suggested in diabetes mellitus.

Detection of anti-myeloperoxidase antibodies in the serum of patients with type 1 diabetes mellitus

Anti-myeloperoxidase (anti-MPO) antibodies were detected in 34 of 88 (38%) patients with type 1 diabetes mellitus but in only 3 of 55 (5.7%) healthy subjects and in 4 of 20 patients with autoimmune disease. Specificity of anti-MPO antibodies was assessed by MPO inhibition studies. No relationship was found between the occurrence of anti-MPO and anti-thyroperoxidase antibodies. Levels of soluble intercellular adhesion molecule 1 (ICAM-1) were found to be higher in anti-MPO antibodypositive (n=28, 508±126 ng/ml) than in anti-MPO antibody-negative (n=58, 438±140 ng/ml;P<0.05) patients. A state of chronic neutrophil activation has been described in diabetes mellitus. As anti-MPO antibodies can stimulate neutrophils to damage endothelial cells in systemic vasculitis, this suggests that a similar mechanism may be operative in the development of diabetic angiopathy.

Cross-Reactivity of Anti-ssDNA Antibodies With Heparan Sulfate in Patients With Type I Diabetes Mellitus

Anti-single-stranded–DNA antibodies cross-reactive with heparan sulfate were detected in serums of patients with type I (insulin-dependent) diabetes mellitus. The results suggested that heparan sulfate, the major glycosaminoglycan constituent of the glomerular basement membrane, may serve as a target antigen in vivo for cross-reactive anti-DNA antibodies. These polyreactive antibodies, directed toward repeating negatively charged units, may neutralize the heparan sulfate–associated polyanionic sites in the glomerulus, leading to an abnormal permeability of anionic plasma proteins.

Anti-single-stranded DNA antibody in the sera of patients with type 2 diabetes mellitus

Abstract Anti-single-stranded(ss)DNA antibodies were searched for by enzyme-linked immunosorbent assay (ELISA) in the serum of 202 outpatients with non-insulin-dependent diabetes mellitus and 135 healthy subjects to investigate their prevalence in the serum of patients with type 2 diabetes and their relationship with the presence of vascular complications. Of the 202 patients 128 had vascular complications. Anti-ssDNA antibodies were observed to be significantly more frequent in the serum of patients with vascular complications (33.6%) and in particular in patients with overt nephropathy (50%) than in patients without complications (6.7%) or controls (6.7%). Anti-ssDNA antibodies have been previously described in patients with type 1 diabetes before clinical evidence of vascular disease, and their cross-reactivity with a variety of anionic biological molecules or cells, i.e. platelets and endothelial cells, assessed. It seems not unreasonable that these autoantibodies detected in patients with type 2 diabetes could be of importance in the pathogenesis or progression of angiopathy.

Circulating Immune Complexes and Platelet Thromboxane Synthesis in Patients with Insulin-dependent (Type I) Diabetes Mellitus

Platelets from diabetic subjects with circulating immune complexes (CIC) synthesized greater amounts of thromboxane than did platelets from CIC-negative patients or controls. In view of the known action of CIC on platelet function, a relationship between these two factors may be suggested in the initiation and progression of microangiopathy in diabetes.

Two-site ELISA for quantification of the terminal C5b-9 complement complex in plasma: Use of monoclonal and polyclonal antibodies against a neoantigen of the complex

A quantitative ELISA procedure using monoclonal and polyclonal antibodies against neoantigens of the terminal C5b-9 complement complex has been developed. The ELISA was demonstrated to be both sensitive and reproducible. The normal range for C5b-9 determinations, defined as 2.5-97.5% interval of the values obtained in 76 healthy blood donors, was 3.12-10.3 AU/ml. The presence of rheumatoid factor did not affect the determination of C5b-9 as demonstrated by immunoabsorption studies.