bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoantigenic epitopes on eukaryotic L7
Authors: VON MIKECZ A H , HEMMERICH P H , PETER H-H , KRAWINKEL U
Published: July 2010

Ribosomal protein L7 has been established recently as a novel autoantigen representing a frequent target for autoantibodies from patients with systemic autoimmune diseases. Up to 75% of systemic lupus erythematosus (SLE) patients and 50% of mixed connective tissue disease (MCTD) and progressive systemic sclerosis (PSS) patients produce antibodies against in vitro translated L7 and form immunoprecipitable complexes. In this study the B cell response to protein L7 was investigated with respect to the immunogenic determinants recognized by autoantibodies. Eighteen truncated fragments of protein L7 were generated as recombinant fusions with glutathione-S-transferase and examined by immunoblotting for their reactivity with sera from patients suffering from systemic rheumatic diseases. Anti-L7 antibodies target three major non-overlapping autoepitopes. Two epitopes reside in the highly conserved C-terminal part of the protein, whereas the N-terminal autoepitope is not conserved during evolution. The N-terminal epitope comprises 24 amino acid residues. Ten amino acid resides of this epitope are shared with the BZIP-like RNA binding domain of protein L7. Autoantibodies recognizing this epitope cross-react with the corresponding region of a L7 homologue, namely ribosomal protein L7 (RPL7) from Dictyostelium discoideum. This indicates that amino acid residues 14VPE…KKR22, which are conserved between humans and fungi, contribute essentially to the formation of autoantibody-autoantigen complexes.
Detection of anti-topoisomerase I antibodies using a full length human topoisomerase I recombinant protein purified from a baculovirus expression system
Authors: WHYTE J , EARNSHAW W C , CHAMPOUX J J , PARKER L H , STEWART L , HALL N D , MCHUGH N J
Published: July 2010

Topoisomerase I (topo I) is a major systemic sclerosis (SSc)-associated autoantigen. A cDNA construct encoding full length human topo I in a recombinant baculovirus transfer vector was used to infect insect cells in culture from which recombinant protein was purified. An ELISA using recombinant protein was evaluated in 340 sera including sera from 134 patients with SSc, of whom 33 had anti-topo I antibodies detected by immunodiffusion. A high yield of pure topo I of expected molecular mass and catalytic activity was obtained. The recombinant topo I ELISA was 92% sensitive and 98% specific in detecting anti-topo I antibodies which were present almost exclusively in patients with SSc. Therefore, the potential advantages of expressing human autoantigens in eukaryotic systems for diagnostic purposes were confirmed.
Increased expression of intercellular adhesion molecule-1 (ICAM-1) in a murine model of pulmonary eosinophilia and high IgE level
Authors: CHU H W , WANG J M , BOUTET M , BOULET L-P , LAVIOLETTE M
Published: July 2010

T lymphocytes and eosinophils are probably involved in the pathogenesis of allergic broncho-pulmonary aspergillosis (ABPA), a disease characterized by pulmonary eosinophilia and high serum and lavage IgE levels. We recently developed a murine model of ABPA. To investigate the mechanisms of T lymphocyte and eosinophil recruitment to the lung in this disease, we examined the expression of ICAM-1 In the lung tissue of mouse challenged with Aspergillus fumigatus (Af) antigen. C57B1/6 mice were intranasally exposed to Af (Af group) or saline (control group) three times a week for 1, 2 or 3 weeks. On days 4, 7, 14 and 21, mice were killed and lung tissue was fixed in acetone and embedded in glycol methacrylate. Serial μm sections were stained with chromotrope 2R and MoAbs against ICAM-I. CD11a/CD18 (LFA-1) and CD3. Af-challenged mice presented significant increases in eosinophil, T lymphocyte and LFA-1-positive cell count and up-regulated expression of ICAM-1 in the lung tissue at all the time points examined. ICAM-1 expression intensity correlated with the number of T lymphocytes (r= 0·59, P <0·01), LFA-1-positive cells (r= 0·68, P < 0·001), but not of eosinophils (r=−0·24, P > 0·05). These findings suggest that up-regulation of ICAM-1 expression is involved in the inflammatory process of this murine model of ABPA, and that this up-regulation may be more relevant to the T lymphocyte accumulation in the lung.
Antibodies to acetylcholinesterase cross-reacting with thyroglobulin in myasthenia gravis and Graves' disease
Authors: MAPPOURAS D G , PHILIPPOU G , HARALAMBOUS S , TZARTOS S J , BALAFAS A , SOUVATZOGLOU A , LYMBERI P
Published: July 2010

In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimoto's thyroiditis (HT), 53 with Graves’ disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti-AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti-Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti-AChE and anti-Tg activities (MG, 18%; P<0·001; and GD, 15%; P<0·001, versus CR, 0%). This bi-reactivity was exhibited by anti-AChE antibodies cross-reacting with Tg (anti-AChE/Tg activity); (i) serum anti-AChE activity was effectively inhibited by soluble Tg, and (ii) affinity-purified anti-Tg antibodies cross-reacted with AChE. Cross-reactivity seems to be a property of pathological (auto) antibodies; induced (rabbit) antibodies to AChE or Tg were highly mono-specific. Analysis of clinical data showed that increased IgG anti-AChE/Tg activity was well associated with: (i) overlapping GD in MG (P<0·02), and (ii) ophthalmopathy in GD (P<0·01). In contrast, no correlation was noted in MG between anti-AChE activity units and anti-Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti-AChE/Tg antibodies remains to be elucidated.
The influence of allo-class II MHC-specific Th2 cells on the generation of CD4 and CD8 cytotoxic T cells to associated class I and class II MHC alloantigen
Authors: WOOD P J , COSSENS I A
Published: July 2010

There is considerable interest in whether CD4 T cell function can affect the outcome of allogeneic transplants. In mice tolerant to an isolated class II MHC disparity, the normal Th1 activity in vitro associated with graft rejection is switched to Th2 in tolerant animals. Because clinical transplants involve multiple class I and II MHC disparities we tested how the switch to Th2 activity of tolerant mice would affect the generation of CD4 and CD8 cytotoxic T cells (CTL) against MHC alloantigens to which the mice were not tolerant. A.TH mice (KkIsDd) were rendered neonatally tolerant of A.TL (KkIkDd) and the generation of CD4 or CD8 CTL measured in a mixed lymphocyte reaction (MLR) against (A.TLxB6)F1 stimulators. Normal mice generated CD4 CTL against both A.TL and B6 (KbIbDb), but tolerant mice were unable to generate cytotoxicity against either A.TL or B6. However, tolerant cells were able to generate CD8 CTL against B6. IL-4 inhibited the generation of CD4, but not CD8, CTL by normal cells and anti-IL-4 antibody was shown to increase the generation of CD4 CTL against B6 in F1 stimulated cultures. Overall the results showed that a Th2 response could inhibit the generation of CD4 CTL against concomitant alloantigen in a process at least partially involving IL-4, but that, conversely, tolerant Th2 cells could help in the generation of CD8 CTL. The results suggest that with whole MHC disparities a simple change of CD4 T cells to Th2 would not be enough to procure graft acceptance.
Two novel cationic staphylococcal proteins induce IL-2 secretion, proliferation and immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC) of both healthy controls and patients with common variable immunodeficiency (CVID)
Authors: JAHREIS A , YOUSIF Y , RUMP J A , DRÄGER R , VOGT A , PETER H H , SCHLESIER M
Published: July 2010

Two cationic proteins, a neutral phosphatase (NP-tase) and a 70-kD protein (p70) were isolated from Staphylococcus aureus by ion exchange chromatography. We compared their properties to those of the well established B cell mitogen of whole, fixed Staph. aureus strain Cowan I cells (SAC). Both purified proteins were able to induce immunoglobulin synthesis in PBMC cultures of healthy donors. NP-tase and p70 also induced immunoglobulin synthesis of PBMC from those patienis with CVID who were also responsive to SAC plus IL-2 stimulation. Immunoglobulin synthesis in response to NP-tase and to p70 was time- and dose-dependent and could be inhibited by addition of specific antibodies against the proteins. In contrast to SAC, no addition of exogenous IL-2 was necessary to obtain maximal immunoglobulin synthesis induced by NP-tase or p70. However, neither protein was able to induce immunoglobulin synthesis in B cell-enriched cultures. High amounts of IL-2 were found in supernatants of PBMC from healthy donors following stimulation with low concentrations of NP-tase or p70, and this was associated with vigorous lymphocyte proliferation. Both proteins behave like typical antigens, and not like lectins or superantigens, since an NP-tase-stimulated T cell line showed an antigen-specific, MHC-restricted secondary response. In addition, no preferential T cell receptor Vβ chain usage was found with eight Vβ-specific MoAb. It is likely that the two proteins induce antigen-specific T cell activation, which is then followed by polyclonal activation of B cells via CD40 receptors and cytokine release
Alterations in interleukin secretion (IL-2 and IL-4) by CD4 and CD4 CD45RO cells from common variable immunodeficiency (CVI) patients
Authors: Ferrer J M , Iglesias J , HernÁNdez M , Matamoros N
Published: July 2010

The failure of B cells from CVI patients to secrete normal amounts of antibodies has been attributed either to an intrinsic B cell defect or to a lack of cooperation from T cells. In an attempt to improve the definition of the origin of this defect in one of the main cellular compartments, we studied the ability of helper CD4 cells and their CD4 CD45RO subpopulation from CVI patients to secrete interleukins (IL-2 and IL-4) in response to mitogen stimulation. We found that CD4 and CD4 CD45RO cells from some patients secrete abnormal amounts of interleukins (in general low levels of IL-2 and high levels of IL-4) upon stimulation with pokeweed mitogen (PWM). These irregularities may contribute to the defective differentiation of B cells in these patients.
Expression of vascular adhesion molecules on human endothelia in autoimmune thyroid disorders
Authors: Marazuela M , SÁNchez-Madrid F , Acevedo A , LarraÑAga E , De LandÁZuri M O
Published: July 2010

Cellular activation and expression of certain adhesion molecules within vascular endothelium is a critical event in leucocyte recruitment and emigration. A wide array of different adhesion receptors has been identified to mediate the interaction between endothelial cells (EC) and leucocyte subpopulations. In this study, the tissue expression of E-selectin, P-selectin, CD31, and endoglin endothelial cell adhesion molecules was studied on thyroid tissue from patients with Graves’ disease (GD) and Hashimoto's thyroiditis (HT), We found an up-regulated expression of E-selectin in EC in GD and HT thyroids, specifically in those areas more severely inflamed, with no reactivity in control thyroids, P-selectin was basally expressed in postcapillary venules in control glands, with an increased expression in HT and GD glands. On the other hand, increased CD31 expression was found on perifollicular, small and large venule EC from GD and HT glands, that correlated with the severity of mononuclear infiltration. In addition, CD31 expression was observed in some intrathyroidal macrophages and T cells in close proximity to CD31+ EC. Furthermore, a markedly enhanced expression of endoglin, a transforming growth factor-beta binding protein, was mainly located on perifollicular EC and EC from small venules as well as In adjacent macrophages from GD and HT thyroid glands. This enhanced expresion of E- and P-selectins, CD31 and endoglin by thyroid EC in GD and HT may reflect their ability to regulate leucocyte trafficking and activation.
Characterization and specificity of anti-endothelial cell membrane antibodies and their relationship to thrombosis in primary antiphospholipid syndrome (APS)
Authors: Hill M B , Phipps J L , Malia R G , Greaves M , Hughes P
Published: July 2010

Immunoblotting was used to detect antibodies reacting with membrane and cytosol preparations of human umbilical vein endothelial cells (HUVEC), fibroblasts and a T lymphoma line HUT78 in 18 patients with anticardiolipin antibodies (ACA) (14 of whom had had a thrombotic event), 11 patients with a recent myocardial infarction and 17 controls. Multiple membrane-specific anti-bodies to HUVEC were found in 10 of the patients with ACA (28 bands) and in nine of the patients with thromboses (27 bands) in contrast to only three of the patients with myocardial infarction (four bands) and one control (one band). The most frequently recognized HUVEC membrane epitopes were at 33 kD (four sera), 61–63 kD (five sera) and 76–79 kD (four sera). Although cross-reactivity with fibroblast and/or HUT78 membranes was seen at 33 kD, binding at 61–63 kD and 76–79 kD was specific for endothelial membranes. Although no correlations with the presence and titre of ACA were seen, HUVEC membrane-specific antibodies showed a correlation with venous thrombotic events.
Peptide phage libraries can be an efficient tool for identifying antibody ligands for polyclonal antisera
Authors: Dybwad A , Bogen B , Natvig JB , FØRre Ø , Sioud M
Published: July 2010

We have examined the potential of isolating ligands for polyclonal antibodies from a nanopeptide phage library. The library was screened with a rabbit polyclonal antiserum raised against a synthetic peptide (ALWFRNHFVFGGGTKVT). Following screening, the positive phages were tested in an ELISA for their reactivity with the antiserum. Phages that showed positive reactivity with the antiserum compared with a normal rabbit serum were selected and their displayed peptides were determined. Among the 36 random positive clones, 31 clones carried the sequence AVFGGGTKL, PFFGGGSRA or APTGGSKRT that have a significant homology to the immunizing peptide. Five positive phages displayed the ATNIFIEGT sequence, which has no obvious linear homology with either the other selected peptides or with the peptide used for immunization. In contrast to the control peptide, the immunizing peptide inhibited binding of the antiserum to the peptide-displaying phages in a dose-dependent manner, thus demonstrating the specificity of the interaction. Furthermore, the rabbit B cell response to the peptide was found to be limited and focused on its C-terminal. Taken together, our data demonstrate the potential of random peptide phage libraries for defining epitopes for polyclonal antisera as well as for investigation of the nature of B cell responses to any given antigen.
Relationships between humoral factors in HIV-1-infected mothers and the occurrence of HIV infection in their infants
Authors: MABONDZO A , ROUVIER P , RAOUL H , LE NAOUR R , COURPOTIN C , HERVÉ F , PARNET-MATHIEU F , LASFARGUES G , DORMONT D
Published: July 2010

Based on what is known about the biology of HIV-1 vertical transmission, the HIV burden of the mother, maternal immune factors and the integrity of the placental barrier are likely to play major roles. We therefore sought to determine whether the presence of antibodies in sera from 47 HI V-1- infected mothers, including 30 non-transmitting and 17 transmitting mothers, affected the risk of HIV-1 transmission to infants. Our findings showed no significant correlation between the capacity of antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and their capacity to induce protection of the child from HIV-I infection (p=0.14). Furthermore, no correlation was found between the capacity of maternal antibodies to neutralize in vitro lymphocyte or macrophage heterologous viral infection and the occurrence of in vivo HIV-1 infection in the infant. Sera recovered from five of 12 transmitting mothers and from five of 11 non-transmitting mothers were compared in their capacity to neutralize the viruses drawn from the same individuals. Four out of five maternal isolates from transmitting mothers and all maternal isolates from non-transmitting mothers were sensitive to enhancement of infection mediated by the maternal serum.
High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints
Authors: Gerli R , Muscat C , Bistoni O , Falini B , Tomassini C , Agea E , Tognelllni R , Biagini P , Bertotto A
Published: July 2010

The CD30 is a surface molecule expressed by Th2-type lymphokine-producitig T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA.
Complement activation by malignant B cells from patients with chronic lymphocytic leukaemia (CLL)
Authors: Marquart H V , GrØNbÆEk K , Christensen B E , Svehag S-E , Leslie R G Q
Published: July 2010

It has previously been reported that the expression of the complement receptors CRI (CD35) and CR2 (CD21) on malignant B cells in CLL is reduced compared with the expression on normal B cells, while deposition of complement C3 fragments, as a consequence of alternative pathway (AP) activation of complement, is observed on mononuclear cells from patients with B CLL. Following our demonstration that normal B cells are capable of activating the AP of complement in a CR2- dependent fashion, we have chosen to re-examine the complement-activating ability of B CLL cells in relation to their altered phenotype with respect to CR2 and the complement regulatory membrane proteins. CRI. Decay accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46). Flow cytometry was used to measure expression of complement receptors and regulatory proteins on CDS+ B cells from CLL patients, as well as the deposition of C3 fragments occurring both in vivo and after in vitro AP activation. We have confirmed the reduced expression of CRI and CR2 on CLL cells and have shown that AP activation in the presence of homologous, normal serum was reduced on B CLL cells compared with normal B cells. The degree of AP activation correlated directly with CR2 expression. In addition, we observed that CLL cells bear in vivo-deposited C3d, g, although at a significantly lower level than normal B cells.
Decreased release of IL-10 by monocytes from patients with lipoid nephrosis
Authors: Matsumoto K
Published: July 2010

TL-10, a cross-regulatory cytokine produced by several cell types. Including monocytes, is known to stimulate B cell growth and maturation and to inhibit cytokine production, In order to characterize further monocyte function in patients with lipoid nephrosis (LN). the release of IL-10 was measured in supernatants of cultured peripheral blood monocytes (PBM) that were obtained from LN patients and healthy controls. Spontaneous and lipopolysaccharide (LPS)-induced IL-IO release was decreased in patients with LN compared with those in normal controls and lower in LN patients with the nephrotic syndrome (NS) than in those without NS. In contrast, the values in IgA nephropathy (IgAN) patients with or without NS did not differ from normal subjects. There was a negative correlation between IL-IO concentration and the quantity of vascular permeability factor (VPF) released in LN patients. These imply that there is a relative deficit in IL-10 release in active LN, which suggests the possibility that inadequate release of IL-IO may lead to increased VPF activity in active LN patients and the measurements of IL-10 may be of value for monitoring kidney disease. The data provide the first detailed analysis of IL-10 in a group of patients with LN.
The influence of tetracyclines on T cell activation
Authors: Kloppenburg M , Verweij C L , Miltenburg A M M , Verhoeven A J , Daha M R , Dijkmans B A C , Breedveld F C
Published: July 2010

Minocycline has been shown to have an anti-inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell-mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose-dependent inhibition of T cell proliferation and reduction in production of lL-2. interferon-gamma (IFN-γ) and tumour necrosis faetor-alpha (TNF-α). Besides an inhibition of IL-2 production, mitiocycline exerted its effect on T cell proliferation by induction of a decreased IL-2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3-induced intracellular Ca2+ mobilization. Taken together. the results clearly indicate that minocycline has immunomodulating effects on human T cells.
Efficacy of HIV-specific and ‘antibody-independent’ mechanisms for complement activation by HIV-infected cells
Authors: Saarloos M-N , Lint TF , Spear GT
Published: July 2010

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed ‘antibody-independent’ C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the ‘antibody-independent’ C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally. MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore,‘antibody-independent’ C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.
A prospective study of CD45 isoform expression in haemophagocytic lymphohistiocytosis; an abnormal inherited immunophenotype in one family
Authors: Wagner R , Morgan G , Strobel S
Published: July 2010

In a prospective study, CD45 isoform expression on T lymphocytes was analysed in seven patients with haemophagocytic lymphohistiocytosis (HLH) and their family members. Six patients, their parents and seven healthy age-matched controls showed the normal pattern of three subpopulations of CD45R expression. In one patient, his mother and one of his healthy brothers only two subpopulations could be identified, which might indicate that the maturation from a CD45RA/RO double-positive to a CD45RO single-positive phenotype does not occur in these individuals. It is not clear if this finding had any impact on the development of HLH in this patient and if the pattern of CD45RA and RO expression observed in this family might represent a predisposition to HLH. In another patient a marked increase of the CD45RO single-positive T cell population was observed during a period of increased disease activity. The clinical relevance of this observation and whether CD45RO expression could serve as a marker for disease activity, or in selected individuals as a susceptibility marker for HLH, remain unknown.
Expression of MHC antigens by intestinal epithelial cells. Effect of transforming growth factor-beta 2 (TGF-β2)
Authors: Donnet-Hughes A , Schiffrin E J , Huggett A C
Published: July 2010

The effect of interferon-gamma (IFN-γ) and TGF-β2 on expression of MHC antigens by the human intestinal epithelial cell line HT-29 was examined by immunohistochemistry and flow cytometry. Untreated HT-29 cells constitutively expressed HLA-ABC but little HLA-DR. Expression of both molecules was increased by IFN-γ(100 U/ml, 24 h). TGF-β2 at concentrations × 0·5 ng/ml given before or simultaneously with IFN-γ, inhibited the IFN-induced expression of HLA-DR. Small increases in HLA-ABC expression by IFN-γ were further increased by pretreatment with TGF-β2, while a strong induction of HLA-ABC was inhibited by the TGF-β2 pretreatment. Our results suggest that the inhibitory action of the TGF-β2 on both HLA-ABC and HLA-DR correlates with the degree of induction following IFN-γ treatment. Since TGY-β2 is present in milk and is produced by gut epithelial cells, one of its possible functions may be to regulate expression of HLA antigens in the neonatal intestine and/or diseased intestine.
Up-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erythematosus (SLE): a possible mechanism for lymphopenia
Authors: Amasaki Y , Kobayashi S , Takeda T , Ogura N , Jodo S , Nakabayashi T , Tsutsumi A , Fujisaku A , Koike T
Published: July 2010

Fas antigen (CD95) is a membrane-associated molecule that mediates apoptotic cell death and may play a role in the induction and maintenance of T cell tolerance. To elucidate the involvement of Fas antigen in human autoimmune diseases, we analysed Fas antigen expression by peripheral T cells from patients with SLE and rheumatoid arthritis (RA), using three-colour flow cytometry. Both CD4+ and CD8+ T cell from SLE patients expressed Fas antigen in a higher density than did these cell from healthy donors and from RA patients. Enhancement of Fas antigen density was noted in Fas+CD45RO+ memory T cell from SLE patients. More remarkably, a significant expression of Fas antigen was observed in CD45RO− naive T cells from SLE patients CD4+CD45RO− T cells from SLE patients co-expressed Fas antigen and early to intermediate activation antigens such as CD25 and CD71, and late activation antigen HLA-DR in only FashiCD4+ naive T tells. Such up-regulation of Fas antigen expression in SLE patients seems to be clinically meaningful, because mean fluorescence intensity (MFI) of Fas antigen on CD4+ T cell subsets inversely correlates with the absolute size of CD4+ T tell subsets in peripheral blood of SLE patients. These results suggest that T cells with increased Fas antigen expression may be highly susceptible to apoptotic cell death, in vivo. A putative mechanism for lymphopenia in SLE patients is discussed.
The effect of free and liposome-encapsulated clodronate on the hepatic mononuclear phagocyte system in the rat
Authors: Camilleri J P , Williams A S , Amos N , Douglas-Jones A G , Love W G , Williams B D
Published: July 2010

Clodronate, encapsulated within small unilamellar vesicles (SUVc) will deplete hepatic macrophages after intravenous injection. Functional studies, using probes to evaluate hepatic Fc and C3b uptake, showed a close correlation between the inhibition of receptor-mediated uptake and the depletion of hepatic macrophages. Twenty milligrams of clodronate encapsulated within SUVc produced · 90% inhibition of uptake and clearance of Fc- and C3b-coated erythrocytes and a comparable reduction of hepatic macrophage numbers. Inhibition of macrophage receptor-mediated uptake of these erythroctyes was closely related to the reduction in macrophage numbers. Repopulation of macrophages within the liver took place over 2 weeks. At 1 week after depletion, although repopulation was taking place, receptor-mediated function remained suppressed. In a preliminary experiment, treatment of rats with adjuvant arthritis with 20 mg clodronate encapsulated in SUV suppressed the inflammation and reversed the course of the disease, while treatment with 20 mg free clodronate in saline or 20 mg clodronate in multilamellar vesicles (MLVc) did not.
Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves’ disease
Authors: Carayanniotis G , Huang G C , Nicholson L B , Scott T , Allain P , Mcgregor A M , Banga J P
Published: July 2010

Grave's disease (GD) is a common disorder characterized by the presence of autoantibodies to the thyrotropin receptor. In the past, the exceedingly low expression of the thyrotropin receptor on thyrocytes has not allowed its purification in quantities sufficient to investigate the establishment of an animal model for this disease. In this study, we have purified the 398-amino acid, extracellular region of the human thyrotropin receptor (TSH-R.E) from insect cells using recombinant baculovirus, and explored its immunopathogenic properties in H-2b,d,q,k,s strains of mice. The receptor preparation was highly immunogenic since it elicited strong specific proliferative T cell responses as well as IgG responses in all strains tested. In addition, hyperimmunization with TSH-R.E induced (i) serum antibodies that blocked the binding of 125I-TSH to its receptor, a common feature of GD autoantibodies; and (ii) IgG that reacted with a synthetic peptide (residues 32–54) from the N-terminus of the receptor, a region implicated in the binding of thyroid stimulating antibodies. In SJL animals only, a weak antibody response to two other thyroid antigens, thyroglobulin and thyroid peroxidase, was also observed. The presence of these antibodies, however, was not accompanied by a detectable alteration in thyroid function as assessed by the measurement of serum TSH, T4 and iodine levels. Also mononuclear infiltration of the thyroid gland or morphological changes compatible with an activation state of thyrocytes were not apparent in TSH-R-challenged mice. In contrast, mice treated with the anti-oxidant aminotriazole showed a dramatic increase in serum TSH levels and an activated follicular epithelium. These data demonstrate that a highly immunogenic TSH-R.E in mice does not necessarily provide a proper stimulus for the induction of a hyper- or hypothyroid status as defined by hormonal or histological criteria. Main reasons for the inability to induce receptor-specific antibodies that affect thyroid function such as those generated in GD are likely to be the inappropriate folding of the recombinant extracellular domain of the receptor, or the xenogeneic nature of the autoantigen.
Expression of endogenous retroviruses, ERV3 and λ4–1, in synovial tissues from patients with rheumatoid arthritis
Authors: Takeuchi K , Katsumata K , Ikeda H , Minami M , Wakisaka A , Yoshiki T
Published: July 2010

We addressed the question of whether or not expression of human endogenous retroviruses (ERV). ERV3 and λ4–1, is related to the pathogenesis of rheumatoid arthritis (RA). In genomic Southern hybridization, there were no significant differences between RA patients and healthy volunteers with regard to frequencies of restriction fragment length polymorphism (RFLP) patterns, for either ERV3 or λ4–1. By Northern blot analysis using fresh synovial tissues, cultured synovial cells, and peripheral blood mononuclear cells (PBMC) from patients with RA, we noted two molecular species of ERV3 mRNAs of 3·5 kb and 9·0 kb sizes, and one single molecular species of λ4–1 mRNAs of 4·2 kb size. The expression was detected not only in RA patients but also in synovial cells from osteoarthritis (OA) as a non-RA control and PBMC from healthy volunteers, and was not related to RA activities or treatments. Although ERV3 and λ4–1 expression may not be directly associated with the pathogenic pathway of RA, the possibility exists that human ERV may have a causative role in autoimmune diseases, including RA. We also examined the effect of cytokines on the transcriptional regulation of ERV3. Although the level of ERV3 expression in cultured synovial cells did not change with IL-1β treatment, the level for cultured proximal tubular epithelial cells (hKEC) was up-regulated.
Synovial cells are potent antigen-presenting cells for superantigen, staphylococcal enterotoxin B (SEB)
Authors: Origuchi T , Eguchi K , Kawabe Y , Mizokami A , Ida H , Nagataki S
Published: July 2010

There is ample evidence suggesting that superantigens may act as a triggering factor in the pathogenesis of rheumatoid arthritis (RA). We investigated whether superantigen could activate T cells in the presence of synovial cells. T cells were cultured with SEB in the presence of interferongamma (IFN-γ)-treated synovial cells. T cell proliferation and activation were assessed by 3H-thymidine incorporation and IL-2 production. The expression of HLA class II antigens and adhesion molecules on synovial ceils was detected by flow cytometer. In the presence of IFN-γ-treated synovial cells, T cells proliferated vigorously and produced IL-2 in response to SEB. A low SEB-induced T cell response was noticed in the presence of untreated synovial cells. Allogeneic as well as autologous IFN-γ-treated synovial cells markedly enhanced SEB-induced T cell proliferation. IFN-γ-treated synovial cells had increased expression of HLA class II antigens and intercellular adhesion molecule-1 (ICAM-1) adhesion molecules. MoAbs towards these antigens markedly inhibited the SEB-induced T cell response. These results indicate that activated synovial cells are potent antigen-presenting cells for SEB to T cells, and that superantigens may play a critical role in the pathogenesis of RA through activated synovial cells.
Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene
Authors: Bohn J , Niemann B , Roggenbuck D , DÖCke W , Jahn S
Published: July 2010

Recently we described the occurrence of B cells producing polyspecific natural IgM with anti-tumour specificity in the spleen of non-tumour-bearing individuals as well as in fetal organisms. Immunoprecipitation and 2-D electrophoresis showed the binding of such antibodies to a 55-kD (pI 60) membrane surface glycoprotein. In vitro cultivation of human cancer cell lines in the presence of the purified IgM antibodies resulted in growth inhibition and complement-mediated cell lysis. Furthermore, the antibodies were shown to be able to induce MHC class I molecule expression on tumour cells. Because of this, a role for naturally occurring antibodies with anti-tumour specificity in preventing neoplasias had been suggested. We have constructed and expressed in Escherichia coli single-chain fragments (scFv: VH-linker-VL) derived from a polyspecific human monoclonal IgM autoantibody produced by a human × mouse heterohybridoma which was obtained from the spleen of an autoimmune patient. The mutated complementarity determining region (CDR) gene segments were replaced by the equivalent germ-line sequences and the CDR3 region was swapped for that from another polyspecific human natural antibody with no binding to tumours. Using these four scFv constructs for binding analyses and in vitro cultivation experiments we found: (i) scFv containing the mutated VH region of the original antibody were able to bind to tumour cells, to induce MHC class I molecule expression, and to inhibit tumour growth in a way similar to what had been described for the complete antibody; (ii) replacement of the mutated by the germ-line VH gene independently of the CDR3 to which it had been recombined, resulted in failure to bind to tumour cells. Nevertheless, other antigens (ssDNA, tetanus toxin) were still recognized, although with lower affinity. We discuss the significance of the replacement mutations in the VH gene CDRs. selected probably by B cell contact to an (auto)antigen, for generating a tumour binding capacity, not encoded by the germ-line gene.
In vitroproduction of cytokines by peripheral blood mononuclear cells from hydatid patients
Authors: RiganÒ R , Profumo E , Di Felice O , Ortona E , Teggi A , Siracusano A
Published: July 2010

The role of cytokines in human hydatidosJs (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-γ) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In ceil cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P·0·005). Spontaneous and milogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-γ production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-γ concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-γ production, but high IgG4 responders had IFN-γ levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-γ production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.
HCV infection in patients with primary defects of immunoglobulin production
Authors: Quinti I , Pandolfi F , Paganelli R , Salman D EL , Giovannetti A , Rosso R , Oliva A , Rainaldi L , Aiuti F
Published: July 2010

We tested for infection with hepatitis C virus (HCV) in 58 patients affected by humoral immunodeficiencies: 43 common variable immunodeficiency (CVI), two hyper IgM syndrome (HIM), two IgG subclass deficiency, four ataxia-telangiectasia (AT), and seven X-linked agamma-globulinaemia (XLA). While the assessment of serum specific HCV antibodies in some of these patients was not informative because of the impairment in specific antibody production, the reverse transcriptase polymerase chain reaction (RT-PCR) assay used to detect serum HCV RNA was a useful method for diagnosing infection. We found that 38% of late onset hypogamma-globulinaemic patients (CVI, HIM or IgG subclass deficiency) had evidence of HCV infection. HCV infection was not detectable in patients with XLA or AT. The majority of our pa hems had persistent viraemia. and those who underwent liver biopsy showed histological findings of chronic hepatitis. Moreover, we could demonstrate in vitro that eight of 18 HCV-infected patients were actively producing anti-HCV antibodies, despite their impaired antibody production. The high rate of HCV infection in hypogammaglobulinaemic patients could be related to several nosoeomial routes of transmission, including intravenous immune globulin administration. Despite the persistent viremia only two patients had cirrhosis and none had hepatocarcinoma.
Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages forHistoplasma capsulatum
Authors: Brummer E , Stevens D A
Published: July 2010

The first line of defence against natural infection by Histoplasma capsulatum (Hc) consists of bronchoalveolar macrophages (BAM) and an early inflammatory response in the lungs. Little is known about the interaction of BAM and He, consequently we studied murine BAM in vitro to assess their role in the pulmonary defence in histoplasmosis. A short-term 3-h assay was used to measure fungicidal activity of control BAM and interferon-gamma (IFN-γ) plus lipopolysacchar-ide (LPS)-activated BAM. Fungistatic activity of BAM was determined with a 24-h assay. A method devised for measuring colony-forming units (CFU) of non-ingested non-adherent and adherent ingested yeast cells of Hc in BAM cocultures was used. Activated BAM killed He (reduced inoculum CFU by 25 ± 12%; n = 4). The fungicidal activity of BAM was abrogated by 02 MM NG-monomethyl-L-arginine (NMMA) or catalase but not by superoxide dismutase. In fungistatic assays activated BAM inhibited multiplication of He by 61 ± 4% n = 3) compared with cocultures with control BAM. However, He multiplied 100% more in control BAM cocultures than in medium alone. Data indicated that this was due to advantages that He has in the intracellular environment. Only NMMA inhibited fungistatic activity of activated BAM. In experiments with peritoneal macrophages (PM), results similar to those with BAM were obtained. In conclusion, activated BAM and PM kill yeast cells of He by a mechanism dependent on hydrogen peroxide and products of the nitric oxide synthase (NOS) pathway, whereas fungistasis depends only on products of the NOS pathway.
Potentiation ofin vitrosynthesis of human IgE by cyclosporin A (CsA)
Authors: Wheeler D J , Robins A , Pritchard D I , Bundick R V , Shakib F
Published: July 2010

In this study, we investigated the modulatory effects of CsA on in vitro synthesis of IgE. IgG1 and lgG4 by human peripheral blood mononuclear cells (PBMC). In contrast to its known immunosuppressive effect, we have demonstrated that a low dose of CsA (10−7 M, 120ng/ml) potentiated IgE production by up to 40-fold (i.e. from 33 ± 4·5 to 1346 ± 290 ng/ml). This potentiation was specific for IgE, since no such effect was demonstrable with IgG1 and IgG4. Potentiation of IgE synthesis by CsA in the PBMC cultures was partly due to CsA acting on T cells, as demonstrated by the addition of CsA-treated T cells to T cell-depleted cultures. However, potentiation was also demonstrable in a T cell-depleted. anti-CD40-stimulated culture (four-fold increase from 400 ± 48 to 1606 ± 127 ng/ml). Our data therefore suggest that there are at least two mechanisms for CsA-induced potentiation of IgE synthesis, one T cell-dependent and the other T cell-independent. The clinical implications of these findings are discussed with regard to the use of CsA in the treatment of Th2-mcdiated diseases.
Autoantibodies against oxidized low density lipoproteins (oxLDL): characterization of antibody isotype, subclass, affinity and effect on the macrophage uptake of oxLDL
Authors: Wu R , Lefvert A K
Published: July 2010

Circulating antibodies against oxLDL are present in several inflammatory and autoimmune conditions. Such antibodies are also present in patients with atherosclerosis, although the pathogenic significance of the antibodies is still not known. We have characterized the antibodies with regard to isotype, subclass, affinity and effect on macrophage uptake of oxLDL. Antibodies of IgG and IgM isotype were most common and found both in patients with atherosclerosis and in normal individuals. The subclass of IgG antibodies was mainly IgG2 and IgG3. Scatchard analyses of IgG and IgM antibodies showed that IgG antibodies were heterogeneous with regard to affinity, whereas only one population of high-affinity antibodies was found in the IgM antibody population. The high-affinity populations had an average equilibrium constant (KO) of 8.84 × 109 M−1 for IgG antibodies and of 1.65 × 109 M−1 for IgM antibodies. Incubation of 125I-oxLDL with purified IgG and IgM from sera with high amounts of antibodies enhanced the uptake of 125I-oxLDL in the monocyte-like U937 cell line. Antibody preparations from sera containing no anti-oxLDL antibodies and from sera with antibodies against LDL had less effect on this uptake. The increased uptake was competitively decreased by adding unlabelled oxLDL. This study shows that antibodies against oxLDL are mainly IgG2. IgG3 and IgM. Both IgG and IgM antibodies have a high affinity for the antigen and increase the uptake of oxLDL in a monocyte-like ceil line.
Down-regulation of CD55 and CD46 expression by anti-sense phosphorothioate oligonucleotides (S-ODNs) sensitizes tumour cells to complement attack
Authors: Zell S , Geis N , Rutz R , Schultz S , Giese T , Kirschfink M
Published: July 2010

Overexpression of one or more membrane-bound complement regulatory proteins (mCRPs) protects tumour cells against complement-mediated clearance by the autologous humoral immune response and is also considered as a barrier for successful immunotherapy with monoclonal anti-tumour antibodies. Neutralization of mCRPs by blocking antibodies, enzymatic removal or cytokine-mediated down-regulation has been shown to sensitize tumour cells to complement attack. In our study we applied, for the first time, anti-sense phosphorothioate oligonucleotides (S-ODNs) to knock down the expression of the mCRPs CD55 and CD46 with the aim of exploiting complement more effectively for tumour cell damage. Potent anti-sense oligonucleotides against CD55 and CD46 were identified by screening various target sequences (n = 10) for each regulator. S-ODN anti-CD55(687) reduced CD55 protein expression up to 84% and CD46 protein expression was inhibited up to 76% by S-ODN anti-CD46(85). Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed a similar reduction of the CD55 and CD46 mRNA levels, which argues for an RNAse H-dependent anti-sense mechanism. T47D, A549 and PC3 cells, representing breast, lung and prostate carcinoma, were used for functional studies. Dependent on the particular cell line, anti-sense-based inhibition of mCRP expression enhanced complement-dependent cytolysis (CDC) up to 42% for CD55 and up to 40% for CD46, and the combined inhibition of both regulators yielded further additive effects in T47D cells. C3 opsonization of CD55/CD46-deficient tumour cells was also clearly enhanced upon mCRP suppression. Due to the clinical applicability of S-ODNs, the anti-sense approach described in this study may offer an additional alternative to improve the efficacy of antibody- and complement-based cancer immunotherapy.