bluuebunny/crossref_metadata_2025 huggingface dataset:
High levels of IL-10 and determination of other cytokines and chemokines in HIV-associated haemophagocytic syndrome
Authors: Benveniste O , Dereuddre-Bosquet N , Clayette P , Leport C , Vildé J-L , Dormont D
Published: March 2003

Haemophagocytic syndrome (HPS) and HIV infection are both associated with cytokine network dysregulation. We therefore analysed plasma levels and mRNA synthesis in peripheral blood mononuclear cells (PBMC) of cytokines, chemokines and chemokine receptors in one HIV-infected patient with HPS. We compared the results with those for eight HIV-infected patients with similar CD4+ T cell counts (207/mm3versus controls: median 214/mm3) and plasma virus load (4·1 log copies/ml, versus controls: median 4·2 log copies/ml). The HPS patient had a lower viral DNA load in PBMC and higher plasma levels of interferon-gamma, IL-10, and macrophage inflammatory protein (MIP)-1β. No difference in plasma tumour necrosis factor-alpha (TNF-α), IL-6 and MIP-1α concentration was observed between the HPS patient and control patients. No difference was observed in TNF-α, IL-1β, IL-10, IL-4, MIP-1α, MIP-1β, RANTES, CXCR-4, and CCR-5 mRNA levels in PBMC, but IL-6 levels were higher in the HPS patient. Our results emphasize the role of IL-10 in the control of immune hyperactivation that is observed in HPS.
T lymphocyte lines isolated from atheromatous plaque contain cells capable of responding toChlamydiaantigens
Authors: Curry A J , Portig I , Goodall J C , Kirkpatrick P J , Gaston J S H
Published: March 2003

Chlamydia pneumoniae infection is associated with atherosclerosis and the organism has been identified in arterial lesions. To determine whether T lymphocyte-mediated immune responses to Chlamydia antigens within plaque could contribute to pathogenesis, we have derived T cell lines from atherosclerotic plaques of 32 patients. Culture with IL-2 alone proved insufficient for cellular activation and expansion, but additional stimulation with phytohaemagglutinin (PHA) or recall antigens allowed consistent establishment of T cell lines. Furthermore, in cultures of approx. 500 tissue fragments, Chlamydia organisms proved as effective as other recall antigens in producing outgrowth of arterial T cells (20–25% wells produced T cell lines). Testing the antigen responsiveness of T cell lines showed that those derived using Chlamydia organisms were more likely to respond to Chlamydia (5/29+) than those isolated using other stimuli (6/69+ for PHA; 5/57+ for PPD and tetanus toxoid (TT)). However, lines responsive to each of the recall antigens were observed. Using recombinant Chlamydia antigens, some Chlamydia-specific T cell lines were shown to respond to OMP2 and/or hsp60. Those recognizing Chlamydia hsp60 did not cross-react with human hsp60, but human hsp60-responsive lines were also observed. Thus, atherosclerotic plaque tissue contains a variety of memory T lymphocytes, and amongst these are cells capable of recognizing Chlamydia antigens. In a C. pneumoniae-infected plaque, such T cells may be activated by local antigen and could contribute to the inflammatory process in the arterial wall through CD40 ligand expression and cytokine secretion.
Eosinophil granule-derived major basic protein induces IL-8 expression in human intestinal myofibroblasts
Authors: Furuta G T , Ackerman S J , Varga J , Spiess A M , Wang M Y , Wershil B K
Published: March 2003

Eosinophil infiltration occurs in a variety of allergic and inflammatory diseases. The release of preformed mediators from eosinophils may contribute to inflammatory responses. We investigated the ability of eosinophiL-derived major basic protein and eosinophiL-derived neurotoxin to stimulate production of IL-8 from intestinal myofibroblasts. Intestinal myofibroblasts (18-Co cells) were incubated with major basic protein, eosinophiL-derived neurotoxin, or a synthetic analogue of major basic protein, poly- L-arginine. Immunoreactive IL-8 was measured by ELISA and IL-8 mRNA levels were analysed by Northern blot or reverse transcription-polymerase chain assay. Major basic protein induced IL-8 mRNA production and release of significant levels of IL-8 immunoreactive protein. By contrast, eosinophiL-derived neurotoxin stimulated little IL-8 release. The induction of IL-8 mRNA by poly-L-arginine was significantly inhibited by actinomycin D. These findings demonstrate a novel interaction between eosinophils and intestinal fibroblasts that may be involved in the pathogenesis of diseases associated with tissue eosinophilia.
Tuberculosis (TB) and HIV infection are independently associated with elevated serum concentrations of tumour necrosis factor receptor type 1 andβ2-microglobulin, respectively
Authors: Lawn S D , Rudolph D , Wiktor S , Coulibaly D , Ackah A , Lal R B
Published: March 2003

The aim of this study was to identify immune markers that are independently associated with HIV infection or TB in vivo. Using commercially available assays, we measured concentrations of five immune markers in sera from 175 out-patients attending medical clinics in Cote D'Ivoire and Ghana, West Africa. Patients were categorized into groups with TB only (TB+HIV−, n = 55), TB and HIV co-infection (TB+HIV+, n = 50), HIV infection only (TB−HIV+, n = 35), or neither infection (TB−HIV−, n = 35). TB+HIV+ and TB−HIV+ groups were matched for blood CD4+ lymphocyte count. Mean ±  s.d. concentrations of β2-microglobulin were similarly increased in both the TB−HIV+ (5·3 ± 2·1 μg/ml, P < 0·0001) and the TB+HIV+ (5·0 ± 1·5 μg/ml, P < 0·0001) groups compared with the TB−HIV− group (2·2 ± 1·8 μg/ml), but were only slightly increased in the TB+HIV− group (3·2 ± 1·8 μg/ml, P = 0·01). In contrast, mean serum concentrations of soluble tumour necrosis factor receptor type I (sTNF-RI) were similarly elevated in the TB+HIV− (1873 ± 799 pg/ml, P < 0·0001) and TB+HIV+ (1797 ± 571 pg/ml, P < 0·0001) groups compared with uninfected subjects (906 ± 613 pg/ml), but there was only a small increase in sTNF-RI in the TB−HIV+ group (1231 ± 165 pg/ml, P = 0·03). Both TB and HIV infection were associated with substantial elevation of serum concentrations of soluble CD8, soluble CD54, and sTNF-R type II. Analysis of additional samples from groups of TB+HIV− and TB+HIV+ patients receiving anti-TB treatment showed significant and equal reductions in mean serum sTNF-RI concentrations, but no significant change in mean β2-microglobulin. Thus, serum β2-microglobulin and sTNF-RI serve as relatively independent markers of HIV infection and TB, respectively, in studies of co-infected persons.
Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2
Authors: Seissler J , Schott M , Morgenthaler N G , Scherbaum W A
Published: March 2003

IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2β. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604–979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699–874 (45% positive) and the C-terminus depending on the presence of aa 931–979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621–628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933–979) and an IA-2-specific region between residues 771–932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887–932) and N-terminal regions (aa 604–771) which are conserved in IA-2 and IA-2β. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.
Point mutations in the promoter region of the CYBB gene leading to mild chronic granulomatous disease
Authors: Weening R S , De Boer M , Kuijpers T W , Neefjes V M E , Hack W W M , Roos D
Published: March 2003

Chronic granulomatous disease (CGD) is a clinical syndrome of recurrent bacterial and fungal infections caused by a rare disorder of phagocytic cells. In CGD, the phagocytes are unable to generate oxygen radicals after stimulation of these cells, due to a defect in the NADPH oxidase system. This NADPH oxidase is a multicomponent enzyme of at least four subunits, of which the β-subunit of cytochrome b558, gp91-phox, is encoded by an X-linked gene (called CYBB). We report here five patients from two families; in each family we found a different mutation in the promoter region of CYBB. Both mutations prevented the expression of gp91-phox in the patients' neutrophils and thus caused inability of these cells to generate oxygen radicals. However, the mutations left the gp91-phox expression and the function of the NADPH oxidase in the patients' eosinophils intact. The relatively mild course of the CGD in these patients can probably be attributed to the fact that the eosinophils have retained their oxidative capacity. Furthermore, our results indicate that neutrophils and eosinophils differ in their regulation of gp91-phox expression.
Opsonization of apoptotic neutrophils by anti-neutrophil cytoplasmic antibodies (ANCA) leads to enhanced uptake by macrophages and increased release of tumour necrosis factor-alpha (TNF-α)
Authors: Moosig F , Csernok E , Kumanovics G , Gross W L
Published: March 2003

Since proteinase 3 (PR3)-ANCA interact with PR3 on the surface of apoptotic polymorphonuclear neutrophils (PMN) and ingestion of apoptotic PMN is known to modulate macrophage inflammatory reactions, we raised the question whether PR3-ANCA-opsonized apoptotic PMN influence the uptake by macrophages and their state of activation. We therefor analysed the effects of PR3-ANCA-opsonized apoptotic PMN on the uptake process by enzymatic assay. We further investigated the production of TNF-α, IL-10, IL-12 and the secretion of lipid inflammatory mediators (TxB2, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)) by human monocyte-derived macrophages using FACS and ELISA methods. We show that PMN-opsonization by PR3-ANCA substantially enhances phagocytosis by macrophages and thereby triggers the production of TNF-α and TxB2. These in vitro findings indicate that PR3-ANCA opsonization of apoptotic PMN might be an important mechanism in the pathogenesis of Wegener's granulomatosis (WG), prompting macrophages to produce proinflammatory mediators. These mediators, mainly TNF-α, might prime further PMN leading to perpetuation of the known priming-dependent mechanisms of ANCA action.
Rearrangement of the human heavy chain variable region gene V3–23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region
Authors: Mageed R A , Harmer I J , Wynn S L , Moyes S P , Maziak B R , Brüggemann M , Mackworth-Young C G
Published: March 2003

To formulate a ‘logic’ for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ-line human V3–23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3–23 gene in combination with different mouse Vκ genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human μ-chains and mouse light chains, were multimeric in structure and expressed the human V3–23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non-CDR3 sequences intrinsic to the V3–23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.
Anti-subnucleosome reactivities in systemic lupus erythematosus (SLE) patients and their first-degree relatives
Authors: Mohan C , Liu F , Xie C , Williams Jr R C
Published: March 2003

Antibodies specific for dsDNA appear to have different genetic origins and pathogenic consequences, compared with histone/dsDNA-specific antibodies, in a recently described murine model. The purpose of this study was to examine if this is also true in human lupus. Sera from 40 SLE families (comprising 40 probands and 153 first-degree relatives), and 45 normal adult controls were assayed for the levels of anti-dsDNA, anti-H1/dsDNA, anti-H2A/H2B/dsDNA, and anti-H3/H4/dsDNA autoantibodies by ELISA. Both the probands and the first-degree relatives exhibited significantly increased levels of antinuclear antibodies (ANA) targeting the different subnucleosomal epitopes. Importantly, probands with anti-dsDNA antibodies had a significantly higher incidence of renal disease compared with those with just anti-H2A/H2B/dsDNA antibodies, in resonance with murine studies. The frequency of anti-dsDNA and anti-H2A/H2B/DNA ANA among the first-degree relatives was 11·8% and 18·3%, respectively. Surprisingly, whereas probands with anti-dsDNA ANA had families with several seropositive members, first-degree relatives of patients with anti-H2A/H2B/DNA ANA (but not anti-dsDNA ANA) were uniformly ANA-free. These findings suggest that anti-dsDNA ANA in lupus may not only have worse disease associations, they may also have very different genetic origins, compared with anti-H2A/H2B/DNA (or anti-nucleosome) ANA.
Haemodialysis monocytopenia: differential sequestration kinetics of CD14+CD16+ and CD14++ blood monocyte subsets
Authors: Nockher W A , Wiemer J , Scherberich J E
Published: March 2003

In peripheral blood the majority of circulating monocytes present a CD14highCD16− (CD14++) phenotype, while a subpopulation shows a CD14lowCD16+ (CD14+CD16+) surface expression. During haemodialysis (HD) using cellulosic membranes transient leukopenia occurs. In contrast, synthetic biocompatible membranes do not induce this effect. We compared the sequestration kinetics for the CD14+CD16+ and CD14++ monocyte subsets during haemodialysis using biocompatible dialysers. Significant monocytopenia, as measured by the leucocyte count, occurred only during the first 30 min. However, remarkable differences were observed between the different monocyte subsets. CD14++ monocyte numbers dropped to 77 ± 13% of the predialysis level after 15 min, increasing to ≥ 93% after 60 min. In contrast, the CD14+CD16+ subset decreased to 33 ± 15% at 30 min and remained suppressed for the course of dialysis (67 ± 11% at 240 min). Approximately 6 h after the end of HD the CD14+CD16+ cells returned to basal levels. Interestingly, the CD14+CD16+ monocytes did not show rebound monocytosis while a slight monocytosis of CD14++ monocytes was occasionally observed during HD. A decline in CD11c surface density paralleled the sequestration of CD14+CD16+ monocytes. Basal surface densities of important adhesion receptors differed significantly between the CD14+CD16+ and CD14++ subsets. In conclusion, during HD the CD14+CD16+ subset revealed different sequestration kinetics, with a more pronounced and longer disappearance from the blood circulation, compared with CD14++ monocytes. This sequestration kinetics may be due to a distinct surface expression of major adhesion receptors which facilitate leucocyte–leucocyte, as well as leucocyte–endothelial, interactions.
Immune complex processing in C1q-deficient mice
Authors: Nash J T , Taylor P R , Botto M , Norsworthy P J , Davies K A , Walport M J
Published: March 2003

Complement and Fcγ receptors are known to mediate the processing of immune complexes (IC), and abnormalities in these mechanisms may predispose to the development of lupus. We explored the processing of IC in mice deficient in complement component C1q. 125I-labelled IC comprising Hepatitis B surface antigen (HBsAg)/human anti-HBsAg (HBsAg/Ab) were injected intravenously and the sites of IC clearance determined by direct counting of organ uptake at various time points. The liver and spleen were the main sites of IC uptake in all mice. The splenic uptake of IC was significantly reduced in the C1q-deficient mice compared with the control mice. C1q-deficient mice also exhibited an initial accelerated hepatic uptake of IC similar to that seen in human subjects with hypocomplementaemia. The hepatic localization of IC at later time points was similar in both groups of mice. These data in mice are consistent with previous observations in humans that confirm that the classical pathway of complement plays an important role in the appropriate processing of IC in vivo.
Dipalmitoylphosphatidylcholine modulates inflammatory functions of monocytic cells independently of mitogen activated protein kinases
Authors: Tonks A , Morris R H K , Price A J , Thomas A W , Jones K P , Jackson S K
Published: March 2003

Phosphatidylcholine (PC) is the major phospholipid of pulmonary surfactant and it is hypothesized that PC and its subspecies modulate the functions of alveolar macrophages. The most abundant of these subspecies is dipalmitoylphosphatidylcholine (DPPC). This study was undertaken to determine the effect of PC on monocyte function using a human monocytic cell line, MonoMac-6 (MM6). This study showed that preincubation of MM6 cells with DPPC at 125 μg/ml for 2 h inhibited the oxidative response to either zymosan or phorbol-12-myristate-13-acetate (PMA) by 30% (P < 0·001). This inhibition with DPPC was independent of LPS priming. When DPPC was replaced with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) there was no inhibition and in contrast a significant increase in oxidant production was observed. We also demonstrated that total PC (tPC; a heterogeneous species of PC from egg) and DPPC but not PAPC significantly inhibited the release of TNF-α from MM6 cells (P < 0·05). DPPC did not inhibit phosphorylation of the mitogen activated protein kinases (MAPKs) p44/p42 or p38 in stimulated cells. Measurements of membrane fluidity with spin label EPR spectroscopy indicate that DPPC incorporation significantly alters the membrane fluidity of MM6 cells. These results suggest that DPPC, the major component of pulmonary surfactant, may play a role in modulating leucocyte inflammatory responses in the lung. This may in part be related to membrane effects but does not include alterations in p44/p42 or p38 MAPK signalling.
Age-related changes in membrane lipid composition, fluidity and respiratory burst in rat peritoneal neutrophils
Authors: Alvarez E , Ruiz-Gutiérrez V , Sobrino F , Santa-María C
Published: March 2003

The O·2−. production has been studied in rat peritoneal neutrophils of different age (3, 12 and 24 months), in order to analyse whether the neutrophil respiratory burst is modified with increasing age. To stimulate NADPH oxidase, the enzyme responsible for the respiratory burst, two stimuli that act in different way have been used: phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (N-FMLP). Production of O·2− decreased with age in neutrophils stimulated with N-FMLP (about 40%), but not in the stimulated with PMA. No difference in NADPH oxidase activity was found with age. The NADPH is supplied to the respiratory burst mainly by the pentose phosphate shunt. A progressive and significant decrease in the two most important enzymes of this route, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was detected as a function of age; in spite of this reduction, the NADPH produced by cells from old animals seems not limiting for the O·2− production. The N-FMLP-induced decrease in the O·2− production may be related to the age-dependent increase in the membrane fluidity observed. A decline in the cholesterol/phospholipid ratio and a rise in the total polyunsaturated fatty acids content were found, that correlated well with the increase in the membrane fluidity. The decrease (50%) of phosphatidylinositols in the 24-month-old animals may be also related to the age-impairment in the respiratory burst found after stimulation with N-FMLP. These studies suggest that the age-related alterations in neutrophil may result in diminished neutrophil function and increased susceptibility to infection in the ageing.
Elevated serum L-selectin levels and decreased L-selectin expression on CD8+ lymphocytes in systemic sclerosis
Authors: Shimada Y , Hasegawa M , Takehara K , Sato S
Published: March 2003

L-selectin is expressed on most circulating leucocytes and mediates leucocyte rolling on endothelium at sites of inflammation. Following rolling or activation of leucocytes, cell surface L-selectin is released as soluble L-selectin (sL-selectin). In the present study, we assessed serum levels of sL-selectin by ELISA and blood leucocyte L-selectin expression by flow cytometry in patients with systemic sclerosis (SSc). Serum levels of sL-selectin in patients with SSc (n = 51) were significantly higher than in normal controls (n = 30) while sL-selectin levels were similar for systemic lupus erythematosus patients (n = 20) and normal controls. Furthermore, SSc patients with elevated sL-selectin levels had inflammatory joint involvement, pitting scar/ulcers, and diffuse pigmentation more frequently than those with normal sL-selectin levels. The frequency of L-selectin+ population among CD8+ T cells was significantly decreased in SSc patients (n = 30) compared with normal controls (n = 20), while that among CD4+ T cells, B cells, monocytes, and neutrophils was similar for SSc patients and normal controls. These suggest that elevated sL-selectin levels and decreased frequency of L-selectin+ CD8+ T cells in SSc patients may be involved in inflammation associated with SSc.
Direct B/B–cell interactions in immunoglobulin synthesis
Authors: Shinozaki K , Yasui K , Agematsu K
Published: March 2003

The principal roles of B/B-cell interactions in immune response have not yet been established. We therefore investigated B/B-cell interactions in immunoglobulin synthesis via direct cell-to-cell contact, particularly in the tumour necrosis factor receptor (TNFR)/tumour necrosis factor (TNF) family. We prepared highly purified peripheral blood B cells and stimulated them with Epstein–Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCLs) as activated human B cells. The IgG production by B cells was increased by the addition of fixed LCLs in a dose-dependent manner in the presence of IL-10 plus IL-2. LCLs strongly expressed CD40 and CD70 on their surface, but marginal or no CD154, CD27, OX40 (CD134) and CD134 ligand. The enhancement of immunoglobulin production by LCLs was completely blocked by the initial addition of anti-CD70 blocking MoAb, but not by anti-CD154 or anti-CD134 ligand MoAb. The addition of LCLs also caused a reduction in CD27 expression on B cells, and this effect was completely blocked by anti-CD70 MoAb, indicating a direct B cell–LCL contact via CD27/CD70. LCLs markedly promoted B-cell differentiation into plasma cells in the presence of IL-10 plus IL-2. These findings demonstrate that direct interactions between B and B cells via CD27/CD70 induce immunoglobulin production by promoting the generation of plasma cells.
Characterization of conditions for the primary culture of human small intestinal epithelial cells
Authors: Aldhous M C , Shmakov A N , Bode J , Ghosh S
Published: March 2003

Intestinal epithelial cells (IECs) are important for many aspects of gut physiology and pathology. Different approaches have been tried for the primary culture of human IECs, with varying degrees of success, as apoptosis easily occurs. Our aim was to develop a method for primary culture of human IECs from biopsy material. IECs and Lamina propria (LP) cells were liberated from duodenal biopsies obtained from subjects undergoing routine endoscopy for clinical investigations, whose small bowel was macroscopically normal. IECs were cultured on collagen membranes in a 12-well tissue culture cluster, with LP cells and allogeneic Epstein-Barr Virus (EBV)-transformed B lymphocytes (allo-B cells) underneath, in the well. Cultured IECs were characterized by light and confocal microscopy. Cytokine levels in culture supernatants were measured by ELISA. Cells showed the columnar morphology of IECs, even after several days in culture. Best results were obtained from IECs cultured above both LP and allo-B cells. IECs did not form monolayers as do transformed epithelial cell lines, but they did preserve their original cell-cell contacts. Analysis of culture supernatants showed that IL-10 was produced by IECs initially, but IL-1ra was produced by LP cells in the underlying wells with increasing time in culture. Very little IL-1β was produced from any cultures. These results show that IECs can be isolated and maintained in primary culture for a short while, which could open new possibilities for research using patient material instead of cell lines.
Detection of ‘antiphospholipid’ antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies bindingβ2-glycoprotein I and prothrombin
Authors: Sheng Y , Hanly J G , Reddel S W , Kouts S , Guerin J , Koike T , Ichikawa K , Sturgess A , Krilis S A
Published: March 2003

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against β2-glycoprotein I (β2GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and β2GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with β2GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0·05) and at lower antibody concentrations (12·5 µg/ml versus 100 µg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to β2GPI (r = 0·90; P < 0·001) but not to CL (r = 0·06; P = 0·76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-β2GPI or antiprothrombin antibodies. All MoAbs binding β2GPI showed inhibition of thrombin generation, while MoAbs binding domain I of β2GPI had more LA effect.
Enhanced T cell apoptosis in common variable immunodeficiency: negative role of the fas/fasligand system and of the Bcl-2 family proteins and possible role of TNF-RS
Authors: Di Renzo M , Serrano D , Zhou Z , George I , Becker K , Cunningham-Rundles C
Published: March 2003

CVI is a primary immunodeficiency characterized by a failure of B cell differentiation associated with an array of T cell defects, such as enhanced T cell apoptosis. In this study we investigated the mechanisms underlying CVI enhanced T cell death. We analysed both the expression of Fas using flow cytometry techniques and the expression of FasL mRNA using RT-PCR in CVI T cells. We could not find any significant differences between CVI and normal subjects with regard to Fas expression, although there was a subgroup of CVI patients with very high Fas expression which was accompanied by an up-regulation of FasL mRNA. However, attemps to induce Fas-mediated apoptosis in these high Fas expressing cells, as evaluated by propidium iodide staining and APO2·7 staining, were unsuccessful. We also investigated intracellular levels of Bcl-2, bcl-xl and bax in CD4+ and CD8+ CVI T cells, as well as the bax/Bcl-2 ratio, using flow cytometry techniques but could not detect any differences between CVI and normal subjects. Finally we analysed TNF-RI and TNF-RII mRNA expression in CD4+ and CD8+ CVI T cells using semiquantitative RT-PCR and found a significant increase in expression of both TNF-Rs in CD4+ T cells from CVI patients. Our data suggest that the increased expression of both TNF-Rs on T cells may be one of the mechanisms responsible for the accelerated T cell apoptosis in CVI.
Increased expression of CTLA-4 (CD152) by T and B lymphocytes in Wegener's granulomatosis
Authors: Steiner K , Moosig F , Csernok E , Selleng K , Gross W L , Fleischer B , Bröker B M
Published: March 2003

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA-4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegener's granulomatosis. The balance between CD28 and CTLA-4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+ T cells has been described in Wegner's granulomatosis; however, analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on PBMC in patients with Wegener's granulomatosis (n = 25) in comparison with healthy controls (n = 19). Expression levels of CTLA-4 were significantly increased selectively on CD4+ and possibly also on CD4−/CD8− T cells in Wegener's granulomatosis. High CTLA-4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA-4 levels were strongly increased on T cells from controls but in T cells from Wegener's granulomatosis patients this response was severely impaired. Interestingly, while CTLA-4 was seen exclusively on T cells in control individuals, about half of the Wegener's patients showed CTLA-4 expression by a fraction of peripheral B lymphocytes. CTLA-4 positive B cells in the periphery were associated with less acute disease.
An abnormal adherence of monocytes to fibronectin in thyroid autoimmunity has consequences for cell polarization and the development of veiled cells
Authors: Canning M O , Grotenhuis K , De Haan-Meulman M , De Wit H J , Berghout A , Drexhage H A
Published: March 2003

Blood monocytes of patients with thyroid autoimmune disease (TAID) display defects in rearranging their cortical actomyosin cytoskeleton (‘polarize’) in response to chemoattractants. Such rearrangements also take place after the adherence of monocytes to the extracellular matrix (ECM). It is therefore not surprising that monocytes are primed after fibronectin (FN) adherence, displaying an enhanced polarization toward chemoattractants.

We investigated the integrin expression and chemoattractant-induced polarization of monocytes of TAID patients before and after FN adherence. Since cytoskeletal rearrangements are also required during the transition of monocytes into veiled antigen-presenting cells (VCs), we investigated such transition of FN-adherent monocytes of TAID patients.

Adherent and nonadherent monocyte populations from TAID patients and healthy controls were subjected to a polarization test with the chemoattractant fMLP (or MCP-1), FACS analyses (FITC-labelled FN, CD29, CD49e, d, b and a) and tested for their capability to develop into veiled APC.

Monocytes of healthy individuals showed an improved chemoattractant-induced cell polarization after FN adherence, not reflected by TAID monocytes, in which chemoattractant-induced polarization worsened. Monocytes of healthy individuals up-regulated CD49e and d integrins and their capability to bind FITC-labelled FN after adherence to a FN-coated plate, as well as enhancing their capability to generate T cell-stimulatory VCs. Monocytes of TAID patients did not.

These data indicate that integrin- (and chemokine-) mediated functions are hampered in monocytes of TAID patients. Because integrin action is pivotal to processes such as monocyte adherence to endothelial cells, uropod formation, migration into tissues and differentiation into APC and macrophages, these defects might underly immune dysbalances important in thyroid autoimmune development.
Circulatingγ/δT lymphocytes from systemic sclerosis (SSc) patients display a T helper (Th) 1 polarization
Authors: Giacomelli R , Cipriani P , Fulminis A , Barattelli G , Matucci-Cerinic M , D'Alò S , Cifone G , Tonietti G
Published: March 2003

Systemic sclerosis (SSc) is a connective tissue disease in which immune system activation is evidenced by high levels of different cytokines in the sera and/or in the supernatants of cultured peripheral blood mononuclear cells (PBMC) and by the presence of specific autoantibodies. γ/δ T cells accumulate in the lung and the skin of SSc patients suggesting their potential role in the development and maintenance of the disease. The aim of this study was to assess cytokine production and cytotoxic activity of circulating γ/δ T lymphocytes obtained from SSc patients and to evaluate their potential role during this disorder. Our results showed that both the proportion and the absolute number of IFN-γγ/δ-producing cells (i.e. displaying a Th1 polarization) in SSc was significantly higher than either the proportion and the absolute number of IL-4 γ/δ-producing cells in SSc or the proportion and the absolute number of IFN-γγ/δ-producing cells in healthy controls (P < 0·05 for both groups). Furthermore, the cytotoxic activity of enriched γ/δ T cells was significantly increased in SSc patients compared with controls. The results concerning the Vδ1+ T cell subset paralleled those of total γ/δ T lymphocytes. In contrast, α/β T cells from SSc and control subjects displayed Th2 cytokine production. All these findings were independent of both disease subset and clinical status. Our data demonstrate that, although SSc is generally considered a Th2 autoimmune disease, Th1 polarization of γ/δ T cells and an increase in their cytotoxic activity is observed in SSc, suggesting that γ/δ T cells could have a relatively autonomous role in the pathogenesis in this disease.
Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus
Authors: Trigwell S M , Radford P M , Page S R , Loweth A C , James R F L , Morgan N G , Todd I
Published: March 2003

The generation of an autoimmune response against islet beta-cells is central to the pathogenesis of type 1 diabetes mellitus, and this response is driven by the stimulation of autoreactive lymphocytes by components of the beta-cells themselves. Reactive oxygen species (ROS) have been implicated in the beta-cell destruction which leads to type 1 diabetes and may modify beta-cell components so as to enhance their immunogenicity. We investigated the effects of oxidation reactions catalysed by copper or iron on the major beta-cell autoantigen glutamic acid decarboxylase (GAD). Lysates of purified rat islets were exposed to copper or iron sulphate with or without hydrogen peroxide or ascorbic acid. Immunostaining showed that these treatments generated high molecular weight covalently linked aggregates containing GAD. These are not formed by intermolecular disulphide bonds between cysteine residues since they cannot be resolved into monomeric form when electrophoresed under extreme reducing conditions. There was no modification of insulin or pro-insulin by ROS. The same oxidative changes to GAD could be induced in viable islet cells treated with copper sulphate and hydrogen peroxide, and thus the modifications are not an artefact of the catalysed oxidation of cell-free lysates. Sera from patients with type 1 diabetes and stiffman syndrome containing GAD antibodies reacted predominantly with the highest molecular weight modified protein band of GAD: normal human sera did not precipitate GAD. Thus, oxidatively modified aggregates of GAD react with serum antibodies of type 1 diabetes patients and some SMS patients: this is consistent with oxidative modifications of autoantigens being relevant to the pathogenesis of type 1 diabetes.
Differential expression ofβ1 andβ2 integrins and L-selectin on CD4+ and CD8+ T lymphocytes in human blood: comparative analysis between isolated cells, whole blood samples and cryopreserved preparations
Authors: Cavers M , Afzali Khoshkbijari B , Macey M , Mccarthy D A , Irshad S , Brown K A
Published: March 2003

Flow cytometric analysis was used to compare the expression of adhesion molecules on human CD4+ and CD8+ T lymphocytes in isolated blood mononuclear cells (MNCs) in whole blood samples and in cryopreserved MNC preparations. Examination of MNCs revealed that the CD11b and CD11c components of the β2 integrins were preferentially expressed on CD8+ T cells, whereas CD62L was present on more CD4+ T cells. All CD4+ and CD8+ T lymphocytes were positive for CD11a but the CD8+ population had a higher intensity of expression of CD11a and also CD11b. Virtually identical results were obtained with T cells in whole blood samples. In relation to the β1 integrins, the only difference between isolated CD4+ and CD8+ T cells was that the latter subset had a greater proportion of cells bearing CD49d. The naive cell marker CD45RA was present on the majority of CD8+ T cells whereas CD45RA and the memory marker CD45RO were evenly distributed within the CD4+ T cell subset. Although cryopreservation of lymphocytes did not modify the expression of β1 and β2 integrins it produced a marked reduction in the percentage of CD4+ and CD8+ T cells bearing CD62L. With regard to endothelial interactions, it appears that cryopreserved lymphocytes are suitable for inclusion in studies of integrin-mediated adhesion but not for those relating to tethering or recognition of addressins on high endothelial venules. Differences in adhesion molecule expression between CD4+ and CD8+ T lymphocytes could underlie the selective extravasation of these subsets into sites of infection and inflammation.
Autoantibodies to human endogenous retrovirus-K are frequently detected in health and disease and react with multiple epitopes
Authors: HERVÉ C A , LUGLI E B , BRAND A , GRIFFITHS D J , VENABLES P J W
Published: March 2003

A number of studies have found increased levels of antibodies to human endogenous retroviruses (HERVs) in autoimmune rheumatic diseases. It is not clear whether this immune response is driven by the HERV itself or by cross-reactions with an exogenous virus or an autoantigen. To address this question, we examined the antibody response to the Env protein of two closely related members of the HERV-K family, HERV-K10 and IDDMK1,222. By immunoblotting of recombinant proteins, antibodies were found in 32–47% of 84 sera from patients with autoimmune rheumatic disease, and 29% of 35 normal controls. Epitope mapping with overlapping 15mers identified multiple reactive peptides on both antigens, with one (GKTCPKEIPKGSKNT) containing immunodominant epitope(s). By ELISA, the median titre of antibody to this peptide was significantly increased in 39 patients with SLE compared to 39 healthy controls and 86 patients with other rheumatic diseases (P < 0·003).

We have shown that there is a high frequency of IgG antibodies to HERV-K env sequences in human sera, both in health and autoimmune rheumatic disease, and that the response is to multiple epitopes. This supports the hypothesis that the autoimmune response to HERV-K is antigen-driven and may be an early stage in the chain of events that leads to tolerance breakdown to other autoantigens.
Activity of matrix metalloproteinase-9 is elevated in sera of patients with systemic lupus erythematosus
Authors: FABER-ELMANN A , STHOEGER Z , TCHERNIACK A , DAYAN M , MOZES E
Published: March 2003

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)-9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP-9 and MMP-2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP-9 activity, but not MMP-2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP-9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP-9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti-phospholipid antibodies. Changes in activity levels of MMP-9, but not of MMP-2, were observed in sera of the same patient at different periods of the disease course. High levels of MMP-9 did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients. The results of the present study suggest that MMP-9 plays a role in the pathogenesis of SLE.
Immune response modulation by recombinant peptides expressed in virus-like particles
Authors: SVIRSHCHEVSKAYA E V , ALEKSEEVA L , MARCHENKO A , VISKOVA N , ANDRONOVA T M , BENEVOLENSKII S V , KURUP V P
Published: March 2003

Aspergillus fumigatus, a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases in man, including allergic broncopulmonary aspergillosis. Peptide-based immunotherapy may offer an alternative treatment strategy for the management of allergic disease. The objective of this study was to alter the allergen-specific immune response using dominant T cell epitopes of a major A. fumigatus allergen, Asp f2, expressed in yeast as virus-like particles (VLP). The T cell epitopes of Asp f2, recognized in mice with an H-2d background, were determined by producing T-cell hybridomas. Two dominant T cell epitopes, aa60–71 and aa235–249, were identified and expressed in a yeast VLP system. To induce tolerance VLP-peptides were injected subcutaneously into mice previously immunized with recombinant Asp f2. The T cell immune response was abrogated totally in 3 weeks following a single injection of VLP but was restored 2 months later following intranasal antigen exposure. T-cell depletion resulted in the reduction of 20–30% of all antigen-specific immunoglobulin classes. Thus, recombinant peptides expressed in the VLP system can be used successfully in the modulation of Asp f2-induced immune response in mice, although a single administration is not sufficient to maintain a state of tolerance for a long period of time.
Vascular cell adhesion molecule-1 (VCAM-1) blockade in collagen-induced arthritis reduces joint involvement and alters B cell trafficking
Authors: CARTER R A , CAMPBELL I K , O'DONNEL K L , WICKS I P
Published: March 2003

Vascular cell adhesion molecule-1 (VCAM-1 or CD106) is important in leucocyte trafficking and its increased expression is associated with a number of chronic inflammatory diseases, including rheumatoid arthritis (RA). We used a neutralizing monoclonal antibody (M/K-2·7) to investigate the role of VCAM-1 in collagen-induced arthritis (CIA), an autoimmune model of RA. A single injection of M/K-2·7 (0·5 mg) into naive mice caused leucocytosis within 20 h, due to increased numbers of circulating B cells and macrophages, as well as neutrophils. The most marked effect was on the numbers of immature B cells (B220loIgM+) which were increased approximately fourfold. CIA was elicited in DBA/1 mice by immunization with chick type II collagen (CII) in Freund's complete adjuvant, followed by a repeat injection 21 days later. Repeated M/K-2·7 administration from the time of primary CII immunization reduced the clinical severity, but not the incidence, of CIA compared to isotype-control monoclonal antibody-treated mice. Histological assessment showed fewer arthritic joints in M/K-2·7-treated mice; however, affected joints showed the same range of severity as those of control mice. Anti-CII IgG1 levels were reduced in anti-VCAM-1-treated mice but the cellular immune response to CII was unaffected. In contrast, VCAM-1 blockade from the onset of clinical features of CIA did not prevent disease progression. These results establish a role for VCAM-1 in promoting polyarticular involvement in CIA, most probably via an effect on B cells.
Preservation of clonal heterogeneity of thePneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals
Authors: LI PIRA G , FENOGLIO D , BOTTONE L , TERRANOVA P , PONTALI E , CAROLI F , SERI M , CAILLIEZ J-C , KOOPMAN G , ACCOLLA R , DEL GALDO F , ABBATE G , DE PALMA R , MANCA F
Published: March 2003

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.