Interleukin-36α axis is modulated in patients with primary Sjögren's syndrome.

The aim of this study was to investigate the expression of the interleukin (IL)-36 axis in patients with primary Sjögren's syndrome (pSS). Blood and minor labial salivary glands (MSG) biopsies were obtained from 35 pSS and 20 non-Sjögren's syndrome patients (nSS) patients. Serum IL-36α was assayed by enzyme-linked immunosorbent assay (ELISA). IL-36α, IL-36R, IL-36RA, IL-38, IL-22, IL-17, IL-23p19 and expression in MSGs was assessed by reverse transcription-polymerase chain reaction (RT-PCR), and tissue IL-36α and IL-38 expression was also investigated by immunohistochemistry (IHC). αß and γδ T cells and CD68(+) cells isolated from MSGs were also studied by flow cytometry and confocal microscopy analysis. IL-36α was over-expressed significantly in the serum and in the salivary glands of pSS. Salivary gland IL-36α expression was correlated with the expression levels of IL-17, IL-22 and IL-23p19. IL-38, that acts as inhibitor of IL-36α, was also up-regulated in pSS. αß(+) CD3(+) T cells and CD68(+) cells were the major source of IL-36α in minor salivary glands of pSS. γδ T cells were not significantly expanded in the salivary glands of pSS but produced more IL-17, as their percentage correlated with the focus score. Higher expression of IL-36α and IL-36R was also demonstrated in γδ T cells isolated from pSS compared to controls. In this study we demonstrate that a significant increase in circulating and tissue levels of IL-36α occurs in pSS patients.

Analysis of IL-6, IL-10 and IL-17 genetic polymorphisms as risk factors for sepsis development in burned patients.

Infection risk, sepsis and mortality after severe burn are primarily determined by patient age, burn size, and depth. Whether genetic differences contribute to otherwise unexpected variability in outcomes is unknown. We sought to determine whether there was an association between IL-6, IL-10 and IL-17 polymorphisms with cytokine production and development of sepsis. We evaluated 71 patients with burns ≥15% TBSA and 109 healthy subjects. The genotypes of IL-6 (-174C/G), IL-10 (-819C/T and -1082A/G) and IL-17 (7488T/C) polymorphisms were identified applying polymerase chain reaction protocols. The cytokine levels in serum were determined with enzyme-linked immunoabsorbent assays. Our results demonstrated no significant differences in the genotype frequencies studied between burn patients and healthy subjects. No significant associations were found among IL-6 and IL-17F genotypes and the related cytokine serum levels. Only IL-10 promoter -1082GG genotype was related to an increased IL-10 production in burned patients. In addition, septic subjects bearing -1082G/G genotype have shown the highest and non-septic bearing -1082A/* genotypes the lowest IL-10 serum levels. All together these data seem to indicate that genetically determined individual difference in IL-10 production might influence the susceptibility to septic complications in burned patients and suggest that these markers might be useful in burned patient management.

Reduction of plasma granzyme A correlates with severity of sepsis in burn patients.

The risk of mortality is high in burn patients and correlates with age, burn area extent, and sepsis. Immunosuppression has been reported to occur after severe burn. Cytotoxic cells possess specialized granules containing perforin and a group of serine proteases (granzymes). Granzyme A is a serine protease constitutively expressed by gammadelta and NK cells, in agreement with their functional cytolytic potential. In vitro studies have shown that GrA may be released extracellularly during cytotoxic cell degranulation, indicating the activation of cytotoxic cells. The aim of our study was to determine plasma GrA activity in burned patients and to verify if decreased GrA levels were associated with poor prognosis. Specific GrA activity was tested in the plasma of burned and healthy subjects by esterase assay. Plasma GrA was significantly decreased in septic rather than in nonseptic burn patients and in healthy subjects (p < 0.05 and p < 0.001, respectively). At day 3 plasma GrA was significantly lower in nonsurvivor than in survivor septic patients (p < 0.05). The value of 91 mOD showed a sensitivity of 100% and a specificity of 84% in differentiating survivor from nonsurvivor septic patients. Because this is a retrospective study, Granzyme A is not a confirmed predictor of septic outcome after burn, but its determination could give useful information about the development and severity of sepsis.

Blocking TNF in vitro with infliximab determines the inhibition of expansion and interferon gamma production of Vgamma9/Vdelta2 T lymphocytes from patients with active rheumatoid arthritis. A role in the susceptibility to tuberculosis?

BACKGROUND: Side effects of TNF neutralisation - mostly infectious complications - were recognized, the most important being pulmonary tuberculosis infections. gamma/ d T cells contribute to protective immune response against mycobacterium tuberculosis. OBJECTIVES: The aim of the present study was to assess the expansion capacity of Vgamma9/Vdelta2 T cells from (tuberculin purified protein derivative (PPD) positive and PPD negative) patients with active rheumatoid arthritis (RA), and to examine the in vitro effect of infliximab on this lymphocyte subset. METHODS: 28 PPD negative RA patients were studied and compared with 14 PPD positive RA patients, 45 PPD-negative and 110 PPD-positive healthy volunteers. Cell separation, expansion in vitro of Vgamma9/Vdelta2 T lymphocytes (EF) and the expression of tumor necrosis factor receptor II and IFN-gamma content by Vgamma9/Vdelta2 T lymphocytes were studied before and after infliximab in vitro addiction. RESULTS: The EF from PPD positive subjects was higher than that from PPD negatives. Patients with RA have the highest levels. The addition of infliximab to the cultures from PPD-positive patients determined a significant inhibition of cell expansion and TNF RII expression and a significant decrease of IFN gamma content. CONCLUSION: In this study we have documented that gamma/ delta T lymphocytes from patients with PPD positive rheumatoid arthritis have a high capacity to respond in vitro to phosphoantingens with expansion TNF-RII expression and IFN gamma production that is inhibited by the exposure to infliximab. These results might be of relevance in view of the effect of TNF blocking on the pulmonary tuberculosis infection.

Circulating dendritic cells following burn.

Burns are associated with immune suppression and subsequent development of sepsis. Dendritic cells (DCs) are potent antigen-presenting cells that serve as a critical link between the innate and acquired immune systems, and are essential in coordinating the host response to pathogens. Using multicolour flow cytometry, the percentages of LIN(-) DR(+) CD11c(+) myeloid (mDC) and LIN(-) DR(+) CD123(+) plasmacytoid (pDC) subsets were determined in peripheral blood from 32 people (15 septic and 5 non-septic burn victims and 12 age- and gender-matched healthy controls, up to 20 days from injury). Analysis revealed significant reductions in circulating mDCs and pDCs in survivor as well as non-survivor septic cases compared with non-septic cases and controls (p<0.001). These findings suggest that deficiencies in mDCs and pDC subsets are related to sepsis following severe burn, and may contribute to immunosuppression among burn victims.

Serum Levels of Cortisol, Immunoglobulin, and C-reactive Protein in Burn Patients.

Thermal injury is known to induce alterations in the immune system, but the precise mechanisms have yet to be elucidated. It has been shown that thermal injury in more than 20% of the total body surface area (TBSA) leads to disturbances in the cortisol metabolism and the equilibrium of the hypothalamic-pituitary-adrenal axis. We investigated the temporal relationship between serum cortisol levels, C-reactive protein, and immunoglobulin levels in the post-burn period. Twenty-one adult burn patients (mean age, 52 ± 17 yrs) were included in the study (TBSA, 10-80%); nine developed sepsis and five died. The nonseptic group consisted of twelve patients. Thirty healthy blood donors served as controls. Our results suggest that increased cortisol and decreased immunoglobulin levels could be related to severe sepsis and clinical outcome.

Skin dendritic cells in burn patients.

The body's immunological response to burn injury has been a subject of great inquiry in recent years. Burn injury disturbs the immune system, resulting in a progressive suppression of the immune response that is thought to contribute to the development of sepsis. Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells.DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens, and their subsequent presentation on the cell surface is linked to major histocompatibility molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues, where they present antigen to T cells and induce an immune response. The purpose of this study was to determine the effects of burn injury on skin DCs in terms of percentage, HLA-DR, and Toll-like receptor-4 (TLR-4) expression. The skin DCs were isolated from burned skin and non-burned skin in the same patient at 7 days post-injury, and skin DCs were isolated from unburned healthy individuals as control. DCs from burned skin notably express low levels of HLA-DR and TLR-4 soon after cell isolation. In the post-burn period the ability of skin DCs to respond to bacterial stimuli is impaired. These changes in DC behaviour might contribute to the impaired host defences against bacteria during burn sepsis.

Pentoxifylline inhibits Vgamma9/Vdelta2 T lymphocyte activation of patients with active Behçets disease in vitro.

The aim of this study is to evaluate the in vitro effect of pentoxifylline (PTX) on T Vgamma9/Vdelta2 lymphocyte function in Behçets disease (BD). We investigated the effect of PTX on Vgamma9/Vdelta2 T cell expansion and expression of TNFRII receptor and perforin content before and after PTX addition by means of FACS analysis lymphocyte cultures from patients with active and inactive BD and healthy subjects. The addition of PTX at a concentration of 1 mg/ml determined a significant inhibition of cell expansion, a down regulation of TNF receptor expression and inhibited the PMA-induced degranulation of perforin. Taken together these data indicate that PTX is capable of interfering with Vgamma9/Vdelta2 T cell function in BD, and although cell culture models cannot reliably predict all of the potential effects of the drug in vivo, our results encourage the possibility that this drug may find use in a range of immunological disorder characterized by dysregulated cell-mediated immunity.

Decrease of circulating dendritic cells in burn patients.

Burn injury is associated with immune suppression and the subsequent development of sepsis. Severe burn injury is associated with depressed immune response, including a functional impairment of Th1 lymphocytes and natural killer cells and a decrease in interferon-a production. Dendritic cells (DCs) are potent antigen-presenting cells and play a key role in T cell activation; they are essential in coordinating the host response to pathogens. Using three-colour flow cytometry, we determined the percentage of lineage-negative LIN-DR+ DCs in burn patients and healthy subjects. The percentages of DCs were lower in the circulation of septic than in nonseptic patients and healthy subjects at all times examined (14 days) after burn injury. In contrast, the DC percentage in nonseptic patients was low at day 1, increased from day 3 to day 10, and reverted to normal levels at day 14. The data from the present study suggest that the DC percentage decreased early after burn injury. In addition, in the presence of severe sepsis, the DC percentage remained lower until day 14. This DC reduction may contribute to the immunosuppression observed after burn injury.

Staphylococcal Scalded Skin Syndrome: Criteria for Differential Diagnosis from Lyell's Syndrome. Two Cases in Adult Patients.

A review of the relative international literature of the last few years is followed by a description of two cases of staphylococcal scalded skin syndrome in adults. As in both cases the initial diagnosis was that of Lyell's syndrome, the main criteria for the differential diagnosis of the two pathologies are considered in order to permit specific and effective treatment.

The level of soluble Granzyme A is elevated in the plasma and in the Vgamma9/Vdelta2 T cell culture supernatants of patients with active Behçet's disease.

OBJECTIVE: Gramzyme A (GrA) is a serine proteinase with trypsin-like activity that is released extracellularly during the degranulation of cytotoxic cells. Among the cytotoxic cells, gamma/delta T cells participate in the early phases of the immune response and are known to express perforin and granzymes constitutively in agreement with their cytolytic pontential. METHODS: GrA activity was detected using the synthetic substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester in the plasma and supernatants of peripheral blood mononuclear cell cultured in the presence of Dimethylallyl pyrophosphate to obtain Vgamma9/Vdelta2 T cell expansion. RESULTS: Significantly high levels of GrA were found in the serum and supernatants of lymphocytes from patients with active Behçet's disease cultured in the presence of DMAPP. Levels were found to be significantly lower after remission. A positive correlation was observed between GrA levels in the supernatants and the Vgamma9/Vdelta2 T cell expansion factor. CONCLUSION: These results strongly suggest that Vgamma9/Vdelta2 T cells are active participants in the pathogenesis of the disease through their degranulation and granzyme release.

IVIG in APS pregnancy.

For more than two decades, the intravenous administration of high doses of IgG pooled from the plasma of healthy donors (immune globulin therapy, also known as 'IVIG') has benefited patients with a variety of autoimmune disorders. A potential therapeutic role of IVIG in the prevention of thrombosis and of miscarriages in antiphospholipid syndrome (APS) has been postulated. Multicenter randomized controlled trials attempted to define the role of IVIG in preventing pregnancy complications in APS indicate that simple anticoagulation could not be completely satisfactory, and certain patient subgroups might take advantage of IVIG therapy alone or in combination with heparin.

Impaired expression of erythrocyte glycosyl-phosphatidylinositol-anchored membrane CD59 in patients with psoriatic arthritis. Relation to terminal complement pathway activation.

OBJECTIVE: Complement-mediated injury is regulated by many factors; among these CD59 has been identified as a widely distributed glycoprotein that inhibits membrane C5b-9 (terminal complement component) formation. The aim of the study was to assess erythrocyte CD59 expression in patients with psoriatic arthritis in order to understand the role of CD59 in the pathogenesis. METHODS: Washed erythrocytes from 50 patients with psoriatic arthritis, 8 with cutaneous psoriasis and 24 healthy subjects were incubated with monoclonal anti-CD59 antibody followed by a second FITC conjugated antibody and fluorescence intensity analysed by FAC-Scan flow cytometer to assess their CD59 membrane expression. SC5b-9 levels were measured in the plasma by ELISA and results compared with CD59 values. Immune complexes, complement C3 and C4 and rheumatoid factor were also determined. RESULTS: Impaired expression of erythrocyte membrane-anchored CD59 was found in patients with psoriatic arthritis; the lowest levels were seen in active patients (p < 0.01). Increased SC5b-9 was seen in the plasma of patients with active disease. An inverse correlation was also found between plasma C5b-9 and the CD59 expression levels (r = -0.81, p < 0.001). CONCLUSION: The low CD59 expression on erythrocytes from patients with psoriatic arthritis may be an index of a low tissue CD59 expression. This impairment could facilitate the activation of complement pathway and increase the risk for arthritis. Membrane attack complex formation in deficient membrane bound CD59 may also exacerbate synovial cell injury and inflammation.

Humoral and cell mediated immune response to cow's milk proteins in Behçet's disease.

OBJECTIVE: To investigate the humoral and cellular immune response against cow's milk proteins in Behçet's disease and to distinguish any behaviour during active or inactive disease. METHODS: Peripheral blood mononuclear cells from 16 patients and from eight normal controls were cultured in the presence of phytohaemagglutinin (PHA), beta-casein, beta-lactoglobulin, or chicken egg albumin. Interferon gamma (IFNgamma) and interleukin 4 (IL4) were measured in the culture supernatants by enzyme linked immunosorbent assay (ELISA). Serum samples from 46 patients with Behçet's disease and from 37 healthy subjects were also studied for antibody detection. Antibodies to beta-casein, beta-lactoglobulin, and chicken egg albumin were determined by ELISA. RESULTS: High IFNgamma but not IL4 levels were found in the supernatants of lymphocytes from patients with active disease cultured in the presence of cow's milk proteins. Levels were comparable with those obtained in cultures stimulated with PHA. A significantly higher level of anti-beta-casein and anti-beta-lactoglobulin IgG and IgA antibodies was found in patients with active Behçet's disease. No relation was found between their occurrence and the age of the patients, the duration of disease, or the presence of gastrointestinal abnormalities. Antibodies to chicken albumin were detected at low levels and with a prevalence similar to that of healthy subjects. CONCLUSION: The results indicate that an active immune response occurs in Behçet's disease. This response involves an increased frequency of antibodies to cow's milk protein and a strong Th1 polarisation after exposure to these antigens. The occurrence of these abnormalities supports a putative role for cow's milk proteins immune response in the pathogenesis of Behçet's disease.

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.

Polymorphonuclear leukocyte myeloperoxidase levels in patients with Behçet's disease.

Myeloperoxidase was measured in the plasma and in the supernatants of polymorphonuclear granulocyte cultures from patients with Behçet's disease. High levels were found in both plasma and supernatants from patients with active disease. The addition of pentoxifylline to granulocyte cultures determined a significant decrease of myeloperoxidase levels in active patients only. Hyperactive neutrophils are present during the course of Behçet's disease and may be considered of importance in the pathogenesis of the vascular lesions.

Enhancement of endothelial cell E-selectin expression by sera from patients with active Behçet's disease: moderate correlation with anti-endothelial cell antibodies and serum myeloperoxidase levels.

We studied the in vitro E-selection expression of endothelial cells treated with sera from patients with Behçet's disease (BD) and factors (anti-endothelial cell antibodies, anti-neutrophil cytoplasmic antibodies, cytokines, and myeloperoxidase (MPO) that may contribute to adhesion molecule expression. A total of 21 patients with BD and 27 healthy controls were studied. In vitro E-selectin endothelial cell expression was investigated by ELISA after HUVEC incubation with sera or purified IgG from patients with BD and controls. Increased E-selectin expression was observed when endothelial cells were incubated with sera from patients with active disease or from patients with circulating anti-endothelial cell antibodies and high levels of plasma myeloperoxidase. Abnormalities of endothelial cell function have been suggested to play a role in the occurrence of vascular damage in BD. Our findings suggest that anti-endothelial cell antibodies and neutrophil hyperactivity, as inferred from the high plasma MPO levels in patients with active disease, may explain endothelial cell activation and neutrophil accumulation in vascular lesions.

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.

Anti-single-stranded DNA antibody in the sera of patients with type 2 diabetes mellitus. Relation to vascular complications.

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 auto-antibodies detected in patients with type 2 diabetes could be of importance in the pathogenesis or progression of angiopathy.